Plant Cold Hardiness (Cabi)

Plant Cold Hardiness

From the Laboratory to the Field

FRIENDS IN SUNNY SASKATCHEWAN 8th International Plant Cold Hardiness Seminar, Saskatoon, Saskatchewan, Canada, August 3–9, 2007

Plant Cold Hardiness From the Laboratory to the Field

Edited by

Lawrence V. Gusta Department of Plant Sciences, University of Saskatchewan, Canada

Michael E. Wisniewski USDA-ARS, Appalachian Fruit Research Station, USA and

Karen K. Tanino Department of Plant Sciences, University of Saskatchewan, Canada

CABI is a trading name of CAB International CABI Head Office Nosworthy Way Wallingford Oxfordshire OX10 8DE UK Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 E-mail: [email protected] Website: www.cabi.org

CABI North American Office 875 Massachusetts Avenue 7th Floor Cambridge, MA 02139 USA Tel: +1 617 395 4056 Fax: +1 617 354 6875 E-mail: [email protected]

© CAB International 2009. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Plant cold hardiness : from the laboratory to the field / edited by Lawrence V. Gusta, Karen K. Tanino, and Michael E. Wisniewski. p. cm. Includes bibliographical references and index. ISBN 978-1-84593-513-9 (alk. paper) 1. Plants–Effect of cold on. 2. Plant physiology. I. Gusta, Lawrence V. II. Tanino, Karen K. III. Wisniewski, Michael E. IV. Title. QK756.P536 2009 632'.11–dc22 2008046606 ISBN-13: 978 1 84593 513 9 Typeset by SPi, Pondicherry, India. Printed and bound in the UK by the MPG Books Group. The paper used for the text pages in this book is FSC certified. The FSC (Forest Stewardship Council) is an international network to promote responsible management of the world’s forests.

Contents

Contributors

ix

Preface

xv

PART 1: THE FREEZING PROCESS 1

Ice Nucleation, Propagation and Deep Supercooling: the Lost Tribes of Freezing Studies M.E. Wisniewski, L.V. Gusta, M.P. Fuller and D. Karlson

2

Low-temperature Damage to Wheat in Head – Matching Perceptions with Reality M.P. Fuller, J. Christopher and T. Fredericks

12

3

Freezing Behaviours in Plant Tissues: Visualization using NMR Micro-imaging and Biochemical Regulatory Factors Involved M. Ishikawa, H. Ide, W.S. Price, Y. Arata, T. Nakamura and T. Kishimoto

19

4

Factors Related to Change of Deep Supercooling Capability in Xylem Parenchyma Cells of Trees S. Fujikawa, J. Kasuga, N. Takata and K. Arakawa

29

PART 2: MOLECULAR BASIS FOR FREEZING TOLERANCE 5

THE

ACQUISITION

1

OF

Plant Cold-shock Domain Proteins: on the Tip of an Iceberg D. Karlson, K. Nakaminami, K. Thompson, Y. Yang, V. Chaikam and P. Mulinti

43

v

vi

Contents

6

Expressional and Functional Characterization of Arabidopsis Cold-shock Domain Proteins K. Sasaki, M.-H. Kim and R. Imai

55

7

Plasma Membrane and Plant Freezing Tolerance: Possible Involvement of Plasma Membrane Microdomains in Cold Acclimation A. Minami, Y. Kawamura, T. Yamazaki, A. Furuto and M. Uemura

62

8

Global Expression of Cold-responsive Genes in Fruit Trees C.L. Bassett and M.E. Wisniewski

72

9

Could Ethanolic Fermentation During Cold Shock Be a Novel Plant Cold Stress Coping Strategy? F. Kaplan, D.Y. Sung, D. Haskell, G.S. Riad, M. Popp, M. Amaya, A. LaBoon, Y. Kawamura, Y. Tominaga, J. Kopka, M. Uemura, K.-J. Lee, J.K. Brecht and C.L. Guy

80

PART 3:

LINKAGE BETWEEN DEVELOPMENTAL ARREST AND COLD HARDINESS

10

Bud Set – A Landmark of the Seasonal Growth Cycle in Poplar A. Rohde

91

11

An Epigenetic Memory from Time of Embryo Development Affects Climatic Adaptation in Norway Spruce Ø. Johnsen, H. Kvaalen, I. Yakovlev, O.G. Dæhlen, C.G. Fossdal and T. Skrøppa

99

12

The Influence of Temperature on Dormancy Induction and Plant Survival in Woody Plants L. Kalcsits, S. Silim and K. Tanino

108

PART 4: GENETIC BASIS

OF

SUPERIOR COLD TOLERANCE

13

Winter Hardiness and the CBF Genes in the Triticeae E.J. Stockinger

119

14

Regulation of Stress-responsive Signalling Pathways by Eudicot CBF/DREB1 Genes A. Nassuth and M. Siddiqua

131

PART 5: IMPACT 15

OF

GLOBAL CLIMATE CHANGE

ON

PLANTS

Evolution of Plant Cold Hardiness and its Manifestation along the Latitudinal Gradient in the Canadian Arctic J. Svoboda

140

Contents

vii

16

Ice Encasement Damage on Grass Crops and Alpine Plants in Iceland – Impact of Climate Change B.E. Gudleifsson

163

17

Impact of Simulated Acid Snow Stress on Leaves of Cold-acclimated Winter Wheat K. Arakawa, H. Inada and S. Fujikawa

173

18

Elevated Atmospheric CO2 Concentrations Enhance Vulnerability to Frost Damage in a Warming World M.C. Ball and M.J. Hill

183

19

The Occurrence of Winter-freeze Events in Fruit Crops Grown in 190 the Okanagan Valley and the Potential Impact of Climate Change H.A. Quamme, A.J. Cannon, D. Neilsen, J.M. Caprio and W.G. Taylor

20

Cold Hardiness in Antarctic Vascular Plants L.A. Bravo, L. Bascuñán-Godoy, E. Pérez-Torres and L.J. Corcuera

PART 6:

FROM THE LABORATORY BRIDGING THE GAP

TO

198

THE FIELD:

21

Patterns of Freezing in Plants: the Influence of Species, Environment and Experiential Procedures L.V. Gusta, M.E. Wisniewski and R.G. Trischuk

214

22

Going to Extremes: Low-temperature Tolerance and Acclimation in Temperate and Boreal Conifers G.R. Strimbeck and P.G. Schaberg

226

23

The Rapid Cold-hardening Response in Insects: Ecological Significance and Physiological Mechanisms M.A. Elnitsky and R.E. Lee, Jr

240

PART 7: PHOTOSYNTHESIS

AND

SIGNALLING

24

Conifer Cold Hardiness, Climate Change and the Likely Effects of Warmer Temperatures on Photosynthesis I. Ensminger, N.P.A. Hüner and F. Busch

249

25

Chemical Genetics Identifies New Chilling Stress Determinants in Arabidopsis J. Einset

262

26

Analysis of the Ascorbate Antioxidant Pathway in Overwintering Populations of Lucerne (Medicago sativa L.) of Contrasting Freezing Tolerance A. Bertrand, Y. Castonguay, S. Laberge, J. Cloutier and R. Michaud

271

viii

Contents

PART 8: SYSTEMS BIOLOGY 27

Identification of Proteins from Potato Leaves Submitted to Chilling Temperature J. Renaut, S. Planchon, M. Oufir, J.-F. Hausman, L. Hoffmann and D. Evers

279

28

Genomics of Cold Hardiness in Forest Trees J. Holliday

293

Index

The colour plate section can be found following p. 160

305

Contributors

Maria Amaya, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Keita Arakawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. E-mail: [email protected] Yoji Arata, Water Research Institute, Sengen, Tsukuba, Ibaraki 305-0047, Japan Marilyn C. Ball, Functional Ecology Group, Building 46, Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia. E-mail: marilyn.ball@anu. edu.au Luisa Bascuñán-Godoy, Laboratorio de Fisiología Vegetal, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Barrio Universitario, Casilla 160 C, Concepción, Chile Carole L. Bassett, US Department of Agriculture Agricultural Research Service (USDA-ARS), Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV 25430, USA. E-mail: [email protected] Annick Bertrand, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3. E-mail: [email protected] León A. Bravo, Laboratorio de Fisiología Vegetal, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Barrio Universitario, Casilla 160 C, Concepción, Chile. E-mail: [email protected] Jeffrey K. Brecht, Horticultural Sciences Department, University of Florida, Box 110690, Gainesville, FL 32611, USA Florian Busch, Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada, M5S 3B2 (previous address: Department of Biology and Biotron, University of Western Ontario, London, Ontario, Canada, N6A 5B7) Alex J. Cannon, Environment Canada, Pacific and Yukon Region, Vancouver, British Columbia, Canada, V6C 3S5. E-mail: [email protected] Joe M. Caprio, 1801 Willow Way Drive, Boseman, MT 59715, USA. E-mail: mjcaprio@juno. com Yves Castonguay, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3

ix

x

Contributors

Vijay Chaikam, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA Jack Christopher, The Leslie Research Centre, Department for Primary Industries and Fisheries, PO Box 2282, Toowoomba, Queensland 4350, Australia. E-mail: Jack.Christopher@dpi. qld.gov.au Jean Cloutier, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3 Luis J. Corcuera, Laboratorio de Fisiología Vegetal, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Barrio Universitario, Casilla 160 C, Concepción, Chile Ola Gram Dæhlen, Oppland Forest Society, Biri Nursery and Seed Improvement Centre, N-2836 Biri, Norway John Einset, Norwegian University of Life Sciences, N-1430 Ås, Norway. E-mail: john.einset@ umb.no Michael A. Elnitsky, Department of Biology, Mercyhurst College, Erie, PA 16546, USA (previous address: Department of Zoology, Miami University, Oxford, OH 45056, USA). E-mail: [email protected] Ingo Ensminger, Department of Forest Ecology, Forest Research Institute of Baden-Württemberg, D-79100 Freiburg, Germany; Department of Biology and Biotron, University of Western Ontario, London, Ontario, Canada, N6A 5B7. E-mail: [email protected] Daniele Evers, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Carl Gunnar Fossdal, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Troy Fredericks, The Leslie Research Centre, Department for Primary Industries and Fisheries, PO Box 2282, Toowoomba, Queensland 4350, Australia. E-mail: [email protected]. gov.au Seizo Fujikawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. E-mail: [email protected] Michael P. Fuller, University of Plymouth, Plymouth PL4 8AA, UK. E-mail: mfuller@plymouth. ac.uk Akari Furuto, Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan Bjarni E. Gudleifsson, Agricultural University of Iceland, Modruvellir, 601 Akureyri, Iceland. E-mail: [email protected] Lawrence V. Gusta, Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada, S7N 5A8. E-mail: [email protected] Charles L. Guy, Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA. E-mail: [email protected] Dale Haskell, Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA Jean-François Hausman, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Michael J. Hill, Earth System Science and Policy, University of North Dakota, Clifford Hall, Stop 9011, 4149 Campus Drive, Grand Forks, ND 58202, USA Lucien Hoffmann, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Jason Holliday, Department of Forest Sciences, University of British Columbia, 3041-2424 Main Mall, Vancouver, British Columbia, Canada, V6T 1Z4. E-mail: [email protected] Norman P.A. Hüner, Department of Biology and Biotron, University of Western Ontario, London, Ontario, Canada, N6A 5B7 Hiroyuki Ide, Water Research Institute, Sengen, Tsukuba, Ibaraki 305-0047, Japan

Contributors

xi

Ryozo Imai, Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan. E-mail: rzi@ affrc.go.jp Hidetoshi Inada, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Masaya Ishikawa, Environmental Stress Research Unit, National Institute of Agrobiological Sciences, Kan’nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. E-mail: isikawam@affrc. go.jp Øystein Johnsen, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway. E-mail: [email protected] Lee Kalcsits, PFRA Shelterbelt Centre, Box 940, Agriculture and Agri-Food Canada, Indian Head, Saskatchewan, Canada, S0G 2K0; Department of Plant Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. E-mail: [email protected] Fatma Kaplan, Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Dale Karlson, Monsanto Company, Research Triangle Park, NC 27709, USA (previous address: Division of Plant and Soil Sciences, West Virginia University, PO Box 6108, Morgantown, WV 26506-6108, USA). E-mail: [email protected] Jun Kasuga, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Yukio Kawamura, The 21st Century Center of Excellence Program, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan Myung-Hee Kim, Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan Tadashi Kishimoto, Environmental Stress Research Unit, National Institute of Agrobiological Sciences, Kan’nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan Joachim Kopka, Max Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1, D-14476 Golm, Germany Harald Kvaalen, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Serge Laberge, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3 Allison LaBoon, University of Miami Miller School of Medicine, 1600 NW 10th Avenue Suite 2099 Miami, FL 33136, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Kil-Jae Lee, Department of Biology, Korea National University of Education, Chung-buk 363791, Korea (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Richard E. Lee, Department of Zoology, Miami University, Oxford, OH 45056, USA. E-mail: [email protected] Réal Michaud, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, Quebec, Canada, G1V 2J3 Anzu Minami, The 21st Century Center of Excellence Program, Iwate University, Morioka, Iwate 020-8550, Japan Prashanthi Mulinti, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA Kentaro Nakaminami, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA Toshihide Nakamura, Environmental Stress Research Unit, National Institute of Agrobiological Sciences, Kan’nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

xii

Contributors

Annette Nassuth, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W.1E-mail: [email protected] Denise Neilsen, Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada, V0H 1Z0. E-mail: [email protected] Mouhssin Oufir, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Eduardo Pérez-Torres, Laboratorio de Biotecnología, INIA Quilamapu, Av. Vicente Méndez 515, Chillán, Chile Sébastien Planchon, Centre De Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg Michael Popp, Interdisciplinary Center for Biotechnology Research, Box 100156, Gainesville, FL 32610, USA William S. Price, Water Research Institute, Sengen, Tsukuba, Ibaraki 305-0047, Japan Harvey A. Quamme, Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada, V0H 1Z0. E-mail: [email protected] Jenny Renaut, Centre de Recherche Public – Gabriel Lippmann, Department of Environment and Agrobiotechnologies, 41 rue du Brill, L-4422 Belvaux, Luxembourg. E-mail: renaut@ lippmann.lu Gamal S. Riad, Vegetable Research Department, National Research Center, Cairo 12622, Egypt (previous address: Horticultural Sciences Department, University of Florida, Box 110690, Gainesville, FL 32611, USA) Antje Rohde, Department Plant Growth and Development, Institute for Agriculture and Fisheries Research, Caritasstraat 21, B-9090 Melle, Belgium. E-mail: [email protected] Kentaro Sasaki, Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan Paul G. Schaberg, US Department of Agriculture Forest Service, Northern Research Station, South Burlington, VT 05403, USA Mahbuba Siddiqua, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 Salim Silim, PFRA Shelterbelt Centre, Box 940, Agriculture and Agri-Food Canada, Indian Head, Saskatchewan, Canada, S0G 2K0 Tore Skrøppa, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Eric J. Stockinger, Department of Horticulture and Crop Science, The Ohio State University/ Ohio Agricultural Research and Development Center (OARDC), 1680 Madison Ave, Wooster, OH 44691, USA. E-mail: [email protected] G. Richard Strimbeck, Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway. E-mail: [email protected] Dong Yul Sung, Cell and Developmental Biology Section, Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, CA 92093, USA (previous address: Plant Molecular Cellular Biology Program, Environmental Horticulture, University of Florida, Box 110675, Gainesville, FL 32611, USA) Josef Svoboda, Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada, L5L 1C6. E-mail: [email protected] Naoki Takata, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Karen Tanino, Department of Plant Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5A8. E-mail: karen.tanino@ usask.ca Bill G. Taylor, Environment Canada, Pacific and Yukon Region, Vancouver, British Columbia, Canada, V6C 3S5. E-mail: [email protected] Kari Thompson, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA

Contributors

xiii

Yoko Tominaga, Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Québec, Canada, H3C 3P8 (previous address: Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan) Russell G. Trischuk, Dow Agrosciences Canada Inc., Saskatoon, Saskatchewan, Canada, S7N 4L8. E-mail: [email protected] Matsuo Uemura, The 21st Century Center of Excellence Program, Iwate University, Morioka, Iwate 020-8550, Japan; Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan. E-mail: [email protected] Michael Wisniewski, US Department of Agriculture, Agricultural Research Service (USDAARS), Appalachian Fruit Research Station, 2217 Wiltshire Road, Kearneysville, WV 25430, USA. E-mail: [email protected] Igor Yakovlev, Norwegian Forest and Landscape Institute, PO Box 115, N-1431 Ås, Norway Tomokazu Yamazaki, The 21st Century Center of Excellence Program, Iwate University, Morioka, Iwate 020-8550, Japan Yongil Yang, Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA

This page intentionally left blank

Preface

This book is a collection of invited and selected papers on plant cold hardiness that were presented at the 8th International Plant Cold Hardiness Seminar (8IPCHS) hosted by the University of Saskatchewan (U of S) in August 3–9, 2007. It began at the U of S in time for our campus’ 100th anniversary. On the third day, the entire conference moved northward to the edge of the boreal forest at Elk Ridge Resort, Waskesiu, Saskatchewan. There were over 105 attendees representing 22 countries. The theme of the conference was: ‘From the Laboratory to the Field’. The collection of chapters in this book represent many of the topics that were presented during the conference. The conference and the book sought to integrate the most up to date basic and applied research on plant cold hardiness. Attendees at the conference included molecular biologists, plant physiologists, plant breeders, plant ecologists, microbiologists, agronomists, administrators, policy makers, and representatives from multinational companies. Due to the structure of the conference, scientists and students had ample time to personally discuss their research with colleagues and in many cases formulate collaborative research plans. The conference provided a better understanding of the stresses plants experience under field conditions compared to analyzing plants in a greenhouse, laboratory, or growth chamber. It also made applied researchers aware of new technologies to study freezing in plants. We sincerely thank the chapter authors for their important contributions. This conference and book would not have been possible without the generous support of: the University of Saskatchewan, the College of Agriculture and Bioresources and the Dept. of Plant Sciences; the Alberta Government; Alberta Agricultural Research Institute; Genome Prairie; Saskatchewan Agriculture and Food; Pioneer; Monsanto; BASF; Cargill; Performance Plants; Flir Systems; National Resources Canada; Conviron; Sasktel; and the National Research Council. We appreciate the work of our International Core Organizing Committee: Rajeev Arora, Yves Castonguay,

xv

xvi

Preface

Tapio Palva and Mat Uemura. Our International Advisory Committee: Leon Bravo, Geoffrey Fincher, Olavi Junttila, Alina Kacperska, Jenny Renaut, Fedora Sutton, Victor Voinikov. Many local organizing committee members devoted much time to making this conference a success: Kyla Shea, Markel Chernenkoff, Brian Fowler, Gloria Gingera, Gord Gray, Elaine Gusta, Lee Kalcsits, Wilf Keller, Ron Mantyka, M.P.M. Nair, Jie Qiu, Kerry Sproule, Russ Trischuk and Ruojing Wang. Our Local Advisory Committee consisted of: Yuguang Bai, Kirstin Bett, Peta Bonham-Smith, Ravi Chibbar, Degi Chuluunbaatar, Natalie Coetzee, Michelle Gallucci, Edward Kendall, Pramod Kumar, Rob Norris, Elaine Qualtiere, Martin Reaney, Steve Robinson, Isobel Parkin, Nirmala Sharma, Brian Sim, Perumal Vijayan, Susan Varughese, Grant Wood and Scott Wright. Special thanks to Randy Whitter of Elk Ridge. Our conference logo was the Inuksuk (‘In-ook-shook’, meaning ‘likeness of a person’). Built by the Inuit of the Canadian Arctic, it is a stone figure which was used for various purposes: to act as a guide for a safe journey, to warn of imminent danger, to mark a place of respect, to show the path for caribou hunting. The Inuksuk has grown to symbolize leadership, friendship and the spirit of sharing knowledge and wisdom. L.V. Gusta, M.E. Wisniewski and K.K. Tanino

1

Ice Nucleation, Propagation and Deep Supercooling: the Lost Tribes of Freezing Studies M.E. Wisniewski, L.V. Gusta, M.P. Fuller and D. Karlson

Introduction

Ice nucleation and propagation

Prior to the emphasis on the molecular biology of cold acclimation, considerable research was conducted on the processes of ice nucleation and deep supercooling. In many species, these two processes are critical to winter hardiness or surviving episodes of frost. Over the past two decades, however, research on these topics has diminished drastically. Research on these topics in major journals in some ways is much like the lost tribes of Israel, which were sent into exile in 722 BC. Where did the tribes go? So it may be asked of the topics of ice formation and deep supercooling in plants. The objectives of the current report are to review these topics and identify critical questions that still need answers, and to indicate how a greater understanding of these topics may lead to new strategies for improving cold hardiness in plants or new technologies for improving frost protection. The ideas and concepts presented have been developed over the past two decades through experimentation and conversations with a host of plant physiologists, horticulturists and microbiologists who have devoted significant time and thought to the process of ice formation in plants.

Beginning in the late 1970s, a considerable amount of research focused on ice-nucleating agents and their role in inducing plants to freeze at warm, subzero temperatures. Research focused on the identification of extrinsic, especially ice-nucleation-active (INA) bacteria, and intrinsic nucleation agents and their role in the freezing process. While published research in this area diminished greatly in the 1990s, new insights were gained when high-resolution IR thermography was employed to study the freezing process. Additionally, published reports on antifreeze proteins (exhibit hysteresis, bind to ice crystals and affect their morphology, and have the ability to inhibit nucleating compounds) and anti-nucleators (compounds that inhibit the activity of nucleating agents but do not exhibit hysteresis) have added to the complexity of our understanding of what induces a plant to freeze. A review of these findings is presented.

Deep supercooling Of the many aspects of biological ice nucleation and cold hardiness of plants, deep supercooling is perhaps the most problematic and

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

1

2

M.E. Wisniewski et al.

difficult to study. The ability of some plants to maintain symplastic water in an unfrozen condition and without movement of water into the apoplast is a remarkable adaptation that has impressed both biophysicists and plant physiologists. Although the ability of woody plant tissues to avoid freezing by deep supercooling was first documented in the 1960s, the mechanism that allows small domains of water to avoid freezing, despite the presence of extracellular ice, remains little understood. While a great amount of attention has been placed on identifying genes responsible for cold acclimation and understanding their regulation, a similar effort on deep supercooling has been absent. While deep supercooling is considered largely a biophysical trait related to the composition of cell walls, evidence suggests this contention needs further evaluation and that even though cell wall composition and tissue structure play a critical role in deep supercooling, there are many aspects that must be genetically regulated either during development or even on an annual basis.

Ice Nucleation and Propagation In April 2007, the midwest, central and southern plains and south-east portions of the USA experienced a record-breaking freezing event that caused unprecedented damage to many economically important crops (Gu et al., 2008; Warmund, 2008). While heroic efforts were made to provide frost protection, most efforts were futile. Partially to blame for this failure is our continuing lack of knowledge about what makes plants freeze at a particular temperature and what frost protection methods should be used for different freezing weather conditions (Poling, 2008; Wisniewski et al., 2008). The need for a better understanding of ice nucleation and propagation in plants has been noted (Ball et al., 2002; Wisniewski et al., 2002a; Hacker and Neuner, 2007, 2008; Wisniewski et al., 2008). Podcasts of an overview of the subject of ice nucleation in plants and other topics associated with the 2007 US freeze are available at http://ashs.org/db/horttalks/detail. lasso?id=103. The temperature at which ice melts (0°C) is well defined but the temperature at which

water will freeze is not predetermined. Pure water has the ability to supercool to temperatures as low as −40°C (homogeneous nucleation temperature) and perhaps even to temperatures as low as −100°C (Franks, 1985; Chen et al., 2006) but freezes at much warmer, subzero temperatures due to the presence of heterogeneous nucleators that are very effective at inducing ice crystal formation (Franks, 1985). Stated in a simple manner, heterogeneous nucleators act as a template that make it easier for water molecules to begin to take on a crystalline arrangement. Once a core of water molecules has assumed this crystalline arrangement (ice nucleus), the ice nucleus acts as a catalyst to induce the freezing of the surrounding water molecules. Heterogeneous nucleators related to plant freezing can be of two sources, extrinsic and intrinsic, the former representing a foreign substance while the latter representing a natural component of the plant. Understanding the role and source of heterogeneous nucleators in ice nucleation of plants is extremely important because if methods can be developed for regulating their activity, significant advances could be made in limiting frost injury to freezing-sensitive plants (Wisniewski and Fuller, 1999). Part of the problem in trying to resolve how plants freeze has been the difficulty in determining where freezing is initiated in a plant and how the freezing process is propagated throughout the plant. Questions such as: can plants supercool in the absence of extrinsic nucleators, how many nucleation events are needed for a whole plant to freeze, do barriers exist within plants that influence the rates or avenues of ice propagation, how do cold acclimation, antifreeze proteins, anti-nucleators and elevated CO2 affect ice nucleation, and what determines the natural patterns of frost injury present in a field after a freezing event, remain unanswered. Part of the problem in trying to address these questions has been due to the available technology. Until recently, the main approach to monitoring freezing in plants has been through the use of thermocouples. Thermocouples provide localized information on the temperature at which a freezing event occurs, however they do not provide information on the initial site of nucleation or the rate of ice propagation. Additionally, there is

Ice Nucleation, Propagation and Deep Supercooling

evidence that the electrical field associated with thermcouples and/or the insertion of thermocouples into a plant can itself promote nucleation events that would otherwise not occur (Wisniewski et al., 1997; Weissbuch et al., 2003; Lahav and Leiserowitz, 2007). Wisniewski et al. (1997) demonstrated the advantages of using high-resolution IR thermography and this technology is now being used more routinely to study the freezing process in plants (Ball et al., 2002; Lutze et al., 1998; Sekozawa et al., 2004; Hacker and Neuner, 2007, 2008). NMR micro-imaging has also been used to better understand the freezing process in plants (Ishikawa et al., 1997; Ishikawa et al., Chapter 3, this volume). Using infrared technology, it has been possible to determine the temperature at which ice is initiated in a wide variety of plants, how plant structure affects ice nucleation and propagation, how specific patterns of freezing relate to patterns of freezing injury and the role of extrinsic nucleators in the freezing process (see review by Wisniewski et al., 2008). The sensitivity and versatility of this technology is shown in Fig. 1.1 in which droplets of water containing the INA bacterium, Pseudomonas syringae, are seen to have frozen on the petals of

3

an apple flower. More recently, Hacker and Neuner (2007, 2008) have increased the ease of visualizing the freezing process by using the infrared camera in a differential thermal imaging mode.

Factors Affecting Ice Nucleation Moisture and extrinsic ice-nucleating agents Two critical elements that greatly contribute to determining the temperature at which plants will freeze are the presence of moisture and extrinsic nucleating agents (Lindow, 1983; 1995; Ashworth, 1992; Wisniewski and Fuller, 1999; Wisniewski et al., 2002a). Dry plants will supercool to a lower temperature than wet plants, although it is not clear whether the ice nucleation activity at warmer temperatures is due to the presence of moisture itself or the result of the moisture activating extrinsic icenucleating agents present on the plant surface. Leaves having drops of water on their surface containing INA bacteria will freeze at a warmer, subzero temperature than leaves with drops plain water (Wisniewski et al., 1997).

2 min 22 sec

Fig. 1.1. Frozen droplets of water containing ice-nucleation-active bacteria (Pseudomonas syringae) on the petals of an apple flower as seen with high-resolution IR thermography.

4

M.E. Wisniewski et al.

The uniform response of plants sprayed with water freezing at warm, subzero temperatures compared with dry plants would suggest that moisture itself is sufficient to trigger freezing events at temperatures just below 0°C. However, data also indicate that if leaves can be kept dry a significant level of freezing avoidance, and hence frost protection, can be provided. During the major freeze of 2007 in the USA, it was reported (B. Poling, Raleigh, North Carolina, 2008, personal communication) that strawberries that were under row covers survived an episodic frost of −6.1°C even though flower tissues had a hardiness value of approximately −2.0°C. A plausible explanation is that the plants remained dry under the row covers and hence were able to supercool rather than freeze at warmer temperatures due to the activity of extrinsic ice-nucleating agents. The row covers themselves did not afford enough thermal protection to prevent freezing. Inherent in the argument that dry plants supercool more than wet plants is the premise that the plant in question does not contain intrinsic ice-nucleating agents that are active at warm temperatures (Ashworth and Kieft, 1995; Wisniewski and Fuller, 1999). This has been a controversial point of discussion in the literature (see review by Wisniewski et al., 2002a; Ishikawa et al., Chapter 3, this volume). While it appears that many herbaceous species are free of intrinsic ice-nucleating agents that are active in the same temperature range as INA bacteria, woody plants appear to be rich in active ice-nucleating agents that appear to play a role in establishing preferred sites of ice formation (see discussion below).

Hydrophobic barriers to ice propagation While the dynamics of intrinsic ice nucleation events has been examined by several researchers using high-resolution IR thermography (Ball et al., 2002; Gusta et al., 2004; Hacker and Neuner, 2007, 2008; Fuller et al., Chapter 2, this volume), the process by which ice on the surface of a plant induces the plant to freeze has been less studied. Using bean (Phaseolus vulgaris) plants, Wisniewski and Fuller (1999) demonstrated that ice crystals must physically grow through a crack in the cuticle, a broken epider-

mal hair or a stoma to induce ice nucleation within the plant. Furthermore, corroborating earlier work by Wisniewski et al. (1997) on rhododendron, they also demonstrated that the thick cuticle present on azalea leaves (Rhododendron spp.) was sufficient to block external ice from inducing an internal nucleation event. Workmaster et al. (1999) also reported on the ability of a thick cuticle to block ice nucleation in cranberry (Vaccinium macrocarpon). These results suggested that hydrophobic barriers could prevent external freezing events from inducing a plant to freeze. Tomato (Lycopersicon esculentum) plants coated with a hydrophobic particle film supercooled to as low as −6°C, despite having been sprayed with water containing INA bacteria (Wisniewski et al., 2002b). In contrast, control plants (uncoated and sprayed) froze at −2.5°C. Similar results were obtained by Fuller et al. (2003), who demonstrated potato (Solanum tuberosum), grape (Vitis vinifera) and citrus (Citrus limon) coated with a hydrophobic particle film supercooled more than control plants.

Plant structure and its role in ice formation and propagation It has commonly been observed that the formation of ice within a plant does not occur in a uniform manner but rather at select sites where ice preferentially accumulates (reviewed by Ashworth, 1992; Pearce, 2001; McCully et al., 2004; Ishikawa et al., Chapter 3, this volume). The factors associated with determining where ice forms and accumulates are not clearly understood but clearly must involve aspects of plant structure that have evolved to deal with ice formation within tissues and the activity of intrinsic nucleating agents, presumably of plant origin. The mid-rib of leaves, the base of petioles, areas near vascular bundles and phloem fibres have all been identified using infrared technology as common sites for initial nucleation events (Ball et al., 2002, 2004; Hacker and Neuner, 2008). McCully et al. (2004) conducted a detailed analysis of ice formation in the petioles of two frost-tolerant herbaceous plants (Trifolium repens and Eschscholzia californica). It was concluded that these plants have evolved a complicated arrangement of

Ice Nucleation, Propagation and Deep Supercooling

structural strengths and weaknesses within petiole tissues which enables them to accommodate large volumes of intercellular ice during freezing events. Upon thawing, the previously frozen tissue returned to its original structural organization. These findings raised several questions regarding the composition and quality of the cell walls associated with the points of strengths and weaknesses, and the mechanism of water movement to and from sites of ice for-

5

mation. An example of selective locations of ice formation is presented in Fig. 1.2, where voids created by the formation of ice can be seen in bud scale tissues and in pith tissues subtending the floral bud of peach (Prunus persica). Wisniewski et al. (1997), Workmaster et al. (1999), Carter et al. (2001), Hacker and Neuner (2007) and Fuller et al. (see Chapter 2, this volume) have all noted the existence of barriers within a plant that influence the direction

Fig. 1.2. Longitudinal section through a peach flower and its subtending axis after being exposed to a freezing event. Voids in the pith tissue subtending the flower and within the surrounding bud scales are evident and result from the formation of ice during the freezing event.

6

M.E. Wisniewski et al.

and rate of ice propagation. In some cases, this may directly affect the resulting pattern of injury observed after a potentially lethal freezing event has occurred. It appears that where ice forms, how it propagates and how it is accommodated are all important factors that affect the ability of a plant to survive freezing and may be as important as the ability to withstand the dehydrative stresses associated with ice formation. Unfortunately, while the latter topic has received much attention in the literature, the former topic has largely been neglected (Gusta et al., Chapter 21, this volume).

Ice nucleators, antifreeze proteins, sugars and anti-nucleators The ice nucleation activity of bacteria has been well characterized (Lindow, 1995) and the protein, and corresponding gene, have been isolated and identified (Lindow et al., 1989). In contrast, while ice nucleation activity of plant origin has been commonly observed, especially in woody plants (Ashworth and Kieft, 1995), the compounds responsible for the ice nucleation activity have not been identified. Reports on the composition of plant INA compounds have ranged from a soluble (Krog et al., 1979; Embuscado et al. 1996) or structural (cell wall) polysaccharide (Gross et al., 1988) to either a protein (Constantinidou and Menkissoglu, 1992) or a complex molecule such as a phospholipid (Brush et al., 1994). So, while there is strong evidence for the existence of plant INA compounds, their identity remains ambiguous, as does an understanding of their origin, development, distribution, turnover and role in adaptation to freezing temperatures. The temperature at which extrinsic nucleation occurs in plants appears to be an adaptive process requiring de novo synthesis, as it is influenced by cold acclimation which in turn is affected by ambient levels of CO2 (Lutze et al., 1998; Beerling et al., 2001; Ball and Hill, Chapter 18, this volume; Ishikawa et al., Chapter 3, this volume). Antifreeze proteins (AFPs), also known as hysteresis proteins (THPs), inhibit ice crystal growth in a non-colligative mechanism, lowering the freezing point of water below the melting point, thereby producing a thermal hysteresis

(DeVries, 1971; Duman and Olsen, 1993). First described in fish, AFPs have also been reported in insects (Duman, 2001) and plants (Griffith and Yaish, 2004). Thermal hysteresis activity of plant AFPs is low (0.2–0.5°C) compared with fish (0.7–1.5°C) and insects (3–6°C). Because of the low activity of plant AFPs, questions have been raised regarding their role in the survival of plants exposed to freezing temperatures. Griffith et al. (2005) demonstrated that rye (Secale cereale) AFPs did not exhibit any cryoprotective activity but rather interacted directly with ice in planta and reduced freezing injury by slowing the growth and recrystallization of ice. Gusta et al. (2004) reported that sugars had a much greater effect than proteins on determining rates of ice propagation in strips of filter paper. Wisniewski et al. (1999) reported that a dehydrin (PCA60) obtained from peach bark tissues exhibited both cryoprotective and antifreeze activity. The antifreeze activity was surprising since PCA60 is an intracellular protein and AFPs are generally secreted proteins. Postulating on the role of PCA60, they suggested that perhaps the main function of PCA60 was to act as an anti-nucleator, inhibiting the activity of ice-nucleating agents as had been described for other AFPs (Parody-Morreale et al., 1988; Zamecnik and Janacek, 1992). Interestingly, when Huang et al. (2002) expressed an insect AFP gene from the beetle, Dendroides canadensis, in Arabidopsis, supercooling of dry plants was enhanced. The increase in supercooling was much greater than the hysteresis activity, suggesting that the insect AFP blocked the activity of intrinsic ice-nucleating agents. Duman (2002) also reported that the inhibition of ice nucleators by insect AFPs could be enhanced by glycerol and citrate. Anti-nucleators (compounds that inhibit ice nucleation activity but do not exhibit hysteresis) have been identified from a variety of sources including microorganisms, insects, plants and synthetic polymers (see review by Holt, 2003; Fujikawa et al., Chapter 4, this volume). While they have been shown to inhibit ice nucleation activity in vitro, their role in defining the temperature at which a plant will freeze has not been definitely demonstrated. The practical application of these compounds in promoting supercooling has yet to be explored.

Ice Nucleation, Propagation and Deep Supercooling

Deep Supercooling in Buds and Xylem Tissues of Woody Plants Deep supercooling of bud (reviewed by Quamme, 1995) and xylem parenchyma tissues (reviewed by Wisniewski, 1995; Fujikawa et al., Chapter 4, this volume) of woody plants is one of the most enigmatic aspects of biological ice nucleation and cold hardiness. Water in these tissues exists in the liquid phase to temperatures as low as −50°C by being isolated from internal, heterogeneous ice nucleators including extracellular ice (Burke, 1979). Upon nucleation freezing occurs intracellularly, which is a lethal event. There is a strong correlation between the temperature range of deep supercooling and tissue injury (see review by Wisniewski, 1995). Deep supercooling in plant tissues can be monitored using differential thermal analysis (DTA) as outlined by Quamme et al. (1982), which relies on the use of thermocouples to detect the latent heat released by water in tissues as it undergoes a liquid to solid phase change. Despite deep supercooling being so integral to the cold hardiness of many woody plants, especially economically important tree fruit crops, it has received only minor research attention in the last 20 years. The majority of the research effort to understand deep supercooling in woody plants has been conducted by Fujikawa and colleagues (Fujikawa et al., Chapter 4, this volume). Early work indicated that deep supercooling of xylem tissues predominated in northern hardwood species, especially those having ring porous xylem structure (George et al., 1974; Becwar et al., 1981). Due to the homogeneous nucleation point of water (−38°C), it was suggested that species exhibiting deep supercooling would be limited to below the −40°C isotherm. Gusta et al. (1983) reported several exceptions to the −40°C isotherm limit and suggested that under prolonged exposure to freezing temperature (several weeks), xylem parenchyma cells could slowly dehydrate which resulted in depression of the freezing point. Additionally, Kuroda et al. (1997, 2003) have suggested that tropical, subtropical and boreal tree species also supercool (see review by Fujikawa et al., Chapter 4, this volume), although this has not yet been corroborated by

7

other researchers. Karlson et al. (2004) indicated that within the genus Cornus, deep supercooling is an ancestral trait that was present prior to the development of freeze tolerance mechanisms that rely on the accumulation of cryoprotective proteins such as dehydrins. As stated by George and Burke (1977), in order for tissues to supercool, the cells within the tissue must: (i) be free of heterogeneous nucleating agents that are active at warm temperatures; (ii) have a barrier the excludes the growth of ice crystals into the supercooled cells from the surrounding apoplast; (iii) have a barrier that prevents the rapid loss of cellular water to sites of extracellular ice despite the presence of a large vapour pressure gradient; and (iv) have cell walls with sufficient tensile strength to counteract the negative hydrostatic pressures that result from a large vapour pressure gradient. Current theory suggests that the ability to deep supercool is largely a biophysical trait and defined by the physical properties of the apoplast rather than the symplast in which the capillary structure (porosity) of the cell wall plays an essential role (Wisniewski, 1995). While this is an attractive hypothesis it does not explain how the ability to deep supercool changes on a seasonal basis or how different species of temperate tree species that exhibit similar wood properties are either deep supercooling or not. Figure 1.3 is an electron micrograph of a xylem ray parenchyma cell in midwinter when supercooling is at its maximum. As reviewed by Wisniewski (1995), rather than the entire cell wall, the properties of the pit membrane and underlying protective layer may define whether or not a xylem cell would supercool. Removal of pectin from these portions of the cell wall dramatically decreased the ability of the cells to supercool, which suggests that seasonal changes in pit membrane structure could account for seasonal changes in deep supercooling. Wisniewski et al. (1999) also suggested that AFPs within the xylem parenchyma cells of peach xylem ray parenchyma may block the activity of intrinsic nucleators and hence allow the cells to supercool. Kasuga et al. (2006) have identified anti-ice nucleation activity in xylem extracts in woody plants. The anti-ice nucleators are mainly

8

M.E. Wisniewski et al.

Fig. 1.3. Electron micrograph of a cross-section through xylem tissues of peach (Prunus persica L. Batsch) showing a xylem ray parenchyma cell that is located next to a vessel element. Note the complexity of the wall structure of the pit membrane, which is composed of a primary wall from adjacent cells, a middle lamella and a tertiary wall (protective layer) that is formed after completion and lignification of the secondary cell wall. Pit membranes are often also covered with a ‘black cap’ of carbohydrate material. Wisniewski (1995) proposed that it is the structure of the pit membrane that determines the degree of deep supercooling exhibited by the living cells in the xylem tissues of woody plants.

secondary metabolites and may play a key role in defining the supercooling ability of xylem parenchyma cells (Fujikawa et al., Chapter 4, this volume). These findings provide a new mechanism of regulating deep supercooling in woody plants.

Conclusions The past 20 years have seen an explosion of research in trying to identify genes involved in cold acclimation. Hundreds of genes are affected by exposure to low temperature but studies have mainly focused on genes that provide cryoprotection or tolerance to dehydrative stress. Some of the genes identified,

however, may also be involved in other aspects of adaption to low temperature. As Gusta et al. (see Chapter 21, this volume) have indicated, the freezing process, as well as a plant’s response to the presence of ice within its tissues, is complex and quite diverse. Factors such as ice nucleation activity, antifreeze proteins, anti-nucleators and plant structure should be considered to develop a holistic understanding of cold hardiness within any given species. Ice nucleation, ice propagation and deep supercooling are integral aspects of plant adaptation to freezing temperatures. A deeper understanding of the genetic regulation and inheritance of these traits will lead to new strategies and technologies for improving plant cold hardiness.

References Ashworth, E.N. (1992) Formation and spread of ice in plant tissues. Horticultural Reviews 13, 215–255. Ashworth, E.N. and Kieft, T.L. (1995) Ice nucleation activity associated with plants and fungi. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 137–162.

Ice Nucleation, Propagation and Deep Supercooling

9

Ball, M.C., Woldfe, J., Canny, M., Hofmann, M., Nicotra, A.B. and Hughes, D. (2002) Space and time dependence of temperature and freezing in evergreen leaves. Functional Plant Biology 29, 1259–1272. Ball, M.C., Canny, M.J., Huang, C.X. and Heady, R. (2004) Structural changes in acclimated and unacclimated leaves during freezing and thawing. Functional Plant Biology 31, 29–40. Becwar, M.R., Rajashekar, C., Hansen-Bristow, K.J. and Burke, M.J. (1981) Deep supercooling of tissue water and winter hardiness limitations in timberline flora. Plant Physiology 68, 111–114. Beerling, D., Terry, A., Mitchell, P., Callaghan, T., Gwynn-Jones, D. and Lee, J. (2001) Time to chill: effects of simulated global change on leaf ice nucleation temperatures of subarctic vegetation. American Journal of Botany 4, 628–633. Brush, R.A., Griffith, M. and Mlynarz, A. (1994) Characterization and quantification of intrinsic ice nucleators in winter rye (Secale cereale) leaves. Plant Physiology 104, 725–735. Burke, M.J. (1979) Discussion. Water in plants: the phenomenon of frost survival. In: Underwood, L.S., Tieszen, L.L., Callahan, A.B. and Folk, G.E. (eds) Comparative Mechanisms of Cold Adaptations. Academic Press, New York, New York, pp. 259–281. Carter, J., Brennan, R. and Wisniewski, M. (2001) Patterns of ice formation and movement in blackcurrant. HortScience 36, 1027–1032. Chen, S.-H., Mallamace, F., Mou, C.-Y., Brocdo, M., Corsavo, C., Faraone, A. and Liu, L. (2006) The violation of the Stokes–Einstein relation in supercooled water. Proceedings of the National Academy of Sciences USA 103, 12974–12978. Constantinidou, H.A. and Menkissoglu, O. (1992) Characteristics and importance of heterogeneous ice nuclei associated with Citrus fruits. Journal of Experimental Botany 43, 585–591. DeVries, A.L. (1971) Glycoproteins as biological antifreeze agents in Antarctic fishes. Science 172, 1152–1155. Duman, J.G. (2001) Antifreeze and ice nucleator proteins in terrestrial arthropods. Annual Review of Physiology 63, 327–357. Duman, J.G. (2002) The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate. Journal of Comparative Physiology 172, 163–168. Duman, J.G. and Olsen, T.M. (1993) Thermal hysteresis protein activity in bacteria, fungi, and phylogenetically diverse plants. Cryobiology 30, 322–328. Embuscado, M.E., BeMiller, J.N. and Knox, E.B. (1996) A survey and partial characterization of ice-nucleating fluids secreted by giant-rosette (Lobelia and Dendrosenecio) plants of the mountains of eastern Africa. Carbohydrate Polymers 31, 1–9. Fuller, M.P., Hamed, M., Wisniewski, M. and Glenn, D.M. (2003) Protection of plants from frost using hydrophobic particle film and acrylic polymer. Annals of Applied Biology 143, 93–97. Franks, F. (1985) Biophysics and Biochemistry at Low Temperatures. Cambridge University Press, Cambridge, UK. George, M.F. and Burke, M.J. (1977) Cold hardiness and deep supercooling in xylem of shagbark hickory. Plant Physiology 59, 319–325. George, M.F., Burke, M.J., Pellet, H.M. and Johnson, A.G. (1974) Low temperature exotherms and woody plant distribution. HortScience 9, 519–522. Griffith, M. and Yaish, M.W.F. (2004) Antifreeze proteins in overwintering plants. Trends in Plant Science 9, 399–405. Griffith, M., Lumb, C., Wiseman, S.B., Wisniewski, M., Johnson, R.W. and Marangoni, A.G. (2005) Antifreeze proteins modify the freezing process in planta. Plant Physiology 138, 330–340. Gross, D.C., Proebsting, E.L. and MacCrindle-Zimmerman, H. (1988) Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in Prunus wood. Plant Physiology 88, 915–922. Gu, L., Hanson, P.J., Post, W.M., Kaiser, D.P., Yang, B., Nemani, R., Pallardy, S.G. and Meyers, T. (2008) The 2007 eastern US spring freeze: increased cold damage in a warming world? Bioscience 58, 253–262. Gusta, L.V., Tyler, M.J. and Chen, T.H. (1983) Deep undercooling in woody taxa growing north of the −40°C isotherm. Plant Physiology 72, 122–128. Gusta, L.V., Wisniewski, M., Nestbitt, N.T. and Gusta, M.L. (2004) The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves. Plant Physiology 135, 1641–1653. Hacker, J. and Neuner, G. (2007) Ice propagation in plants visualized at the tissue level by infrared differential thermal analysis (IDTA). Tree Physiology 27, 1661–1670. Hacker, J. and Neuner, G. (2008) Ice propagation in dehardened alpine plant species studied by infrared differential thermal analysis (IDTA). Arctic Antarctic and Alpine Research 40, 660–670.

10

M.E. Wisniewski et al.

Holt, C.B. (2003) substances which inhibit ice nucleation: a review. CryoLetters 24, 269–274. Huang, T., Nicodemus, J., Zarka, D.G., Thomashow, M.F., Wisniewski, M. and Duman, J.G. (2002) Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature. Plant Molecular Biology 50, 333–344. Ishikawa, M., Price, W.S., Ide, H. and Arata, Y. (1997) Visualization of freezing behaviors in leaf and flower buds of full-moon maple by nuclear magnetic resonance microscopy. Plant Physiology 115, 1515–1524. Karlson, D.T., Xiang, Q.-Y., Stirm, V.E., Shirazi, A.M. and Ashworth, E.N. (2004) Phylogenetic analyses in Cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins. Plant Physiology 135, 1654–1665. Kasuga, J., Mizuno, K., Miyaji, N., Arakawa, K. and Fujisawa, S. (2006) Role of intracellular contents to facilitate supercooling capability in beech (Fagus crenata) xylem parenchyma cells. CryoLetters 27, 305–310. Krog, J.O., Zachariassen, K.E., Larson, B. and Smidsrod, O. (1979) Thermal buffering in afro-alpine plants due to nucleating agent-induced water freezing. Nature 282, 300–301. Kuroda, K., Ohatani, J. and Fujikawa, S. (1997) Supercooling of xylem parenchyma cells in tropical and subtropical hardwood species. Trees 12, 97–106. Kuroda, K., Kasuga, J., Arakawa, K, and Fujikawa, S. (2003) Xylem ray parenchyma cells in boreal hardwood species respond to subfreezing temperatures by deep supercooling that is accompanied by incomplete desiccation. Plant Physiology 131, 736–744. Lahav, M. and Leiserowitz, L. (2007) Ice freezing induced by amphiphilic alcohols and local electric fields of polar crystals. In: Proceedings of the 2nd Annual Conference on the Physics, Chemistry, and Biology of Water, 18–21 October 2007, Brattleboro, Vermont; available at http://vermontphotonics.net/water_2007/ abstracts.html Lindow, S.E. (1983) The role of bacterial ice nucleation in frost injury to plants. Annual Review of Phytopathology 21, 363–384. Lindow, S.E. (1995) Control of epiphytic ice-nucleation-active bacteria for management of plant frost injury. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 239–256. Lindow, S.W., Lahue, E., Govindarajan, A.G., Panopoulos, N.J. and Gies, D. (1989) Localization of ice nucleation activity and the iceC gene product in Pseudomonas syringae and Escherichia coli. Molecular Plant– Microbe Interactions 2, 262–272. Lutze, J.L., Roden, J.S., Holly, C., Wolfe, J., Egerton, J.J.G. and Ball, M.C. (1998) Elevated atmospheric CO2 promotes frost damage in evergreen tree seedlings. Plant, Cell and Environment 21, 631–635. McCully, M.E., Canny, M.J. and Huang, C.X. (2004) The management of extracellular ice by petioles of frostresistant herbaceous plants. Annals of Botany 94, 665–674. Parody-Morreale, A., Murphy, K.P., DiCera, E., Fall, R., DeVrie, A.L. and Gill, S.J. (1988) Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins. Nature 333, 782–783. Pearce, R.S. (2001) Plant freezing and damage. Annals of Botany 87, 417–424. Poling, B. (2008) Spring cold injury to winegrapes and protection strategies and methods. HortScience 43, 1652–1662. Quamme, H.A. (1995) Deep supercooling in buds of woody plants. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 183–199. Quamme, H.A., Chen, P.M. and Gusta, L.V. (1982). Relationship of deep supercooling and dehydrative resistance to freezing injury in dormant stems of ‘Starkcrimson Delicious’ apple and ‘Siberian C’ peach. Journal of the American Society of Horticultural Science 107, 299–304. Sekozawa, Y., Sugaya, S. and Gemma, H. (2004) Observations of ice nucleation and propagation in flowers of Japanes pear (Pyrus pyrifolia Nakai) using infrared video thermography. Journal of the Japanese Society of Horticultural Science 73, 1–6. Warmund, M. (2008) Temperatures and cold damage to small fruit crops across the eastern US associated with the April 2007 freeze. HortScience 43, 1643–1647. Weissbuch, I., Lahav, M. and Leiserowitz, L. (2003) Toward stereochemical control monitoring, and understanding of crystal nucleation. Crystal Growth and Design 3, 125–150. Wisniewski, M. (1995) Deep supercooling in woody plants and the role of plant structure. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 163–181.

Ice Nucleation, Propagation and Deep Supercooling

11

Wisniewski, M., and Fuller, M.P. (1999) Ice nucleation and deep supercooling in plants: new insights using infrared thermography. In: Maregesin, R. and Schinner, F. (eds) Cold-Adapted Organisms: Ecology, Physiology, Enzymology and Molecular Biology. Springer Verlag, Berlin, pp. 105–118. Wisniewski, M., Lindow, S. and Ashworth, E. (1997) Observations of ice nucleation and propagation in plants using infrared thermography, Plant Physiology 113, 327–334. Wisniewski, M., Webb, R., Balsamo, R., Close, T.J., Yu, X.-M. and Griffith, M. (1999) Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiologia Plantarum 105, 600–608. Wisniewski, M., Fuller, M., Glenn, D.M., Gusta, L., Duman, J., and Griffith, M. (2002a) Extrinsic ice nucleation in plants: What are the factors involved and can they be manipulated. In: Li, P.H. and Palva, E.T. (eds) Plant Cold Hardiness: Gene Regulation and Genetic Engineering. Kluwer Academic/Plenum Publishers, New York, New York, pp. 211–221. Wisniewski, M., Glenn, D.M. and Fuller, M.P. (2002b) Use of a hydrophobic particle film as a barrier to extrinsic ice nucleation in tomato plants. Journal of the American Society of Horticultural Science 127, 358–364. Wisniewski, M., Glenn, D.M., Gusta, L. and Fuller, M.P. (2008) Using infrared thermography to study freezing in plants. HortScience 43, 1648–1651. Workmaster, B.A., Palta, J.P. and Wisniewski, M. (1999) Ice nucleation and propagation in cranberry uprights and fruit using infrared thermography. Journal of the American Society of Horticultural Science 124, 619–625. Zamecnik, J. and Janacek, J. (1992) Interaction of antifreeze proteins from cold hardened cereals with ice nucleation active bacteria. Cryobiology 29, 718–719.

2

Low-temperature Damage to Wheat in Head – Matching Perceptions with Reality M.P. Fuller, J. Christopher and T. Fredericks

Abstract Wheat grown in eastern Australia can suffer severe frost damage during radiation frosts at ear emergence. Few studies have attempted to understand the characteristics of freezing and frost damage to wheat during late developmental stages. While the cultivars used in this region have adequate frost tolerance during their vegetative development, this is not maintained during ear emergence. It is perceived that this lack of head resistance is genetically controlled and that germplasm may be available for specific radiation frost resistance. Recent work has shown, however, that cold tolerance cannot be up-regulated at these developmental stages and that conventional screening of germplasm has little chance of producing field resistant material. However, wheat in head can tolerate a limited amount of freezing without damage and furthermore can supercool substantially in both controlled environments and in the field. The present chapter demonstrates the evidence for these findings and suggests new approaches for screening for resistance in an attempt to make headway in this recalcitrant phenomenon.

Introduction In subtropical areas and some Mediterranean regions, spring wheat is sown in the late summer and autumn to allow flowering in midwinter to avoid water stress during the summer months (Woodruff and Tonks, 1983). Such cropping strategies expose the flowering crop to episodic frost risk. Significant crop losses in eastern Australia due to frost damage have resulted in economic losses in excess of $AU100 million per annum. Occasional late frosts also cause frost damage to wheat in head in several other regions including the Mediterranean, South America and in continental Canada, Russia and the USA (Shroyer et al., 1995). In Queensland and northern New South Wales (NSW), Australia, yield losses associated with frost damage are significant and gross yield reductions of 10% as a direct result of frost injury are common. Record losses occurred in the 1965 season when 50% of

12

the NSW wheat crop was lost due to heavy frosts (Boer et al., 1993). Individual regions can suffer losses in excess of 85% (Paulsen and Heyne, 1983) and individual growers risk total crop loss. Growers frequently minimize frost risk by delayed planting and using longer-season varieties which delays flowering beyond the most significant frost risk periods (Gomezmacpherson and Richards, 1995; Shackley and Anderson, 1995). These measures, however, result in delayed maturity and can in themselves lead to even greater yield and quality losses if grain-filling is pushed into a significant drought period or the beginning of the rainy season (Woodruff and Tonks, 1983; Woodruff, 1992). Thus frost damage limits yield not only by causing actual damage but also by restricting use of the most effective flowering period. A compromise between the effects of frost and drought has been sought using computer-aided decision-making systems such as ‘WHEATMAN’ (Woodruff and Tonks, 1983).

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Low-temperature Damage to Wheat

Tolerance to freezing damage during the vegetative stage does not appear to confer tolerance in the reproductive stages in wheat (Fuller et al., 2007). Many elite winter wheats are tolerant to temperatures of −20°C in the vegetative stages (LT50) but suffer severe damage at much more moderate temperatures (−5 to −7°C) during the reproductive stages. Despite the apparent high levels of freezing tolerance in winter wheats, there appears to be little or no additional protection from frost injury in the reproductive stages compared with spring varieties (Paulsen and Heyne, 1983; Marcellos and Single, 1984; Cromey et al., 1998; Fuller et al., 2007). Vegetative freezing tolerance requires a period of 7 to 14 days’ exposure to temperatures less than 8°C before there is significant increase in frost tolerance in winter wheat. Unfortunately, the temperatures to confer acclimation in winter wheat are rarely experienced for prolonged periods during the flowering stages of either winter or spring wheats in eastern Australia; therefore it is possible that the frost damage during flowering in wheat is a result of a lack of expression of acclimation. The physical processes occurring during freezing temperatures in wheat plants during the reproductive stages are yet to be fully characterized and this work is complicated by the difficulty of working with natural frost events. Screening using natural frosts in the field can be problematic due to the unpredictable nature of frost events in terms of both timing and severity. The effect of natural frosts on winter cereals post ear emergence is difficult to simulate, but it may not be necessary to faithfully reproduce the conditions of a natural frost to successfully identify plants with increased resistance. Unfortunately, there are no conclusive data to indicate that this can be achieved with any of the frost cabinets tested to date (Fredericks et al., 2004). Despite more than a century of interest in improving in-head frost resistance in Australian wheats (Farrer, 1900 cited in Single, 1985), very little genetic gain has been made. In the current chapter we present some initial findings on freezing of in-head wheat aimed at examining the effect of simulated freezing in an attempt to characterize ice

13

nucleation and frost resistance and to help develop sensible research strategies.

Materials and Methods A number of experiments were undertaken to investigate the freezing behaviour of wheat in ear. Results presented are observations taken from a range of these experiments. All plant material was spring habit wheat including the cultivars Hartog, SeriM82 and a number of experimental genotypes. Hartog is a cultivar widely grown in the Australian subtropical grain belt.

Frost testing Three freezing cabinets were used. Convective freezing was conducted in a Sanyo M533 incubator where the set temperatures were preprogrammed according to the desired regime. Radiative freezing was conducted in a custombuilt radiation freezing chamber (Fuller and Le Grice, 1998) and in a custom-built radiation chamber at the Australian Genome Research Facility (AGRF), Adelaide (www.agrf.org.au) (Long et al., 2005). The AGRF radiation frost chamber is a recently built chamber which provides a unique facility to address the Australian cereal frost problem. It utilizes three ceilingmounted freezing batteries cooled to approximately −20°C in a walk-in chamber with no air movement. It is being used to routinely screen genetic material in the Australian legume breeding programmes and is being tested for use with wheat and barley. Typically, plants were allowed to equilibrate to a pre-set chamber temperature of 2 to 4°C for 1 to 2 h prior to being subjected to subzero temperatures. Following exposure to subzero temperatures, the plants were held at 2 to 4°C for several hours to defrost. Monitoring of plants in the field at Kingsthorpe, Queensland was also undertaken during natural radiative freezing conditions. An IR camera (Inframetrics model 760) was used to observe the plants’ exposure to subzero temperatures in order to determine the temperature and location of ice nucleation

14

M.P. Fuller et al.

and the freezing pattern of plants and plant parts, as described previously by Wisniewski et al. (1997), Fuller and Wisniewski (1998) and Pearce and Fuller (2001).

Results In many of the experiments undertaken it was apparent that plants or isolated ears of wheat demonstrated supercooling, i.e. ice did not occur in their tissues. Although plants were misted with water and droplets of water on the leaves froze, freezing was not detected in up to 90% of the culms (Fig. 2.1). In the experiment illustrated in Fig. 2.1, only one culm showed damage and this was the culm identified to have frozen and visualized by IR thermography. When analysed using Thermotechnix™ software, the culm that froze in this experiment showed a characteristic two-phase pattern of freezing (Fig. 2.2) with an initial quick freeze, interpreted as the freezing of the apoplastic water, followed by a second slower freeze interpreted as freeze dehydration. Symptoms of frost damage included bleaching of the head, which only occurred if

freezing in the tissues also occurred (Fig. 2.3). It was not possible to reproduce partial floret damage to the heads in controlled frost tests although partial floret damage is readily observable in the field (Fig. 2.3). Following two nights of field observation when the air temperature fell to −7°C, not a single ear of wheat was observed to freeze and detected by IR thermography; neighbouring vegetative wheat plants however were observed to freeze (Fig. 2.4). A standard freezing programme utilized for screening the Australian wheat and barley germplasm (2°C for 2 h, slow freeze to −4.5°C for 8 h, slow defrost to 2°C) was conducted on four pots of wheat at ear emergence. Analysis of this experiment by IR thermography revealed that only 15% of the plant material and only one ear froze while the remaining material supercooled (Fig. 2.5). It was possible to discern frozen tissue from unfrozen tissue during rewarming the chamber to −0.5°C (Fig. 2.5). Plants that had frozen are depicted as hatching in Fig. 2.5 in contrast to those which supercooled, which are circled (see Plate 1 where green represents plants that had frozen and red those

Fig. 2.1. Greyscale IR image of wheat plants at ear emergence during radiative freezing. Structures which appear white are undergoing freezing (exhibiting exotherms) and include water droplets and one culm (far left of picture).

Low-temperature Damage to Wheat

15

0.35 0.30

Temperature (d, °C)

0.25 0.20 0.15 0.10 0.05 0.00

-0.05 -0.10 -0.15 0.00

10.00

20.00

30.00

40.00 50.00 Time (min)

60.00

70.00

80.00

90.00

Fig. 2.2. Temperature (d, °C) trace taken from a freezing wheat ear showing two phases of freezing.

Fig. 2.3. Observed frost damage to heads of wheat following freezing. Left: following frost testing, the head on the left froze and the three heads on the right supercooled; right, typical head damage observed in the field during head development. NB: see Plate 1 for colour representation.

which supercooled). When these plants were thawed, only the ear that was observed to freeze showed frost damage as illustrated in Fig. 2.3. Vegetative material that was observed to freeze showed no frost damage. Follow-up observations on the plants at grainfill showed that both control (unfrozen but with a moderate baseline levels of sterility) and treated (supercooled) plants had floret infertility and this was significantly higher (P<0.05) in the treated plants (the frozen head data were excluded from this analysis as they showed complete death and therefore 100% infertility).

Discussion It is a common fallacy when conducting frost hardiness experiments to assume that all plants subjected to subzero temperatures have frozen and that they all freeze at the same time and therefore have experienced the same level of stress. However, experiments conducted by Ashworth and Kieft (1995) clearly showed that plants are capable of supercooling during artificial frost tests. This observation has led to speculation by several workers that if ice nucleation can be delayed or lowered to a temperature below the lowest temperature of the frost,

16

M.P. Fuller et al.

Fig. 2.4. Heads of wheat which supercooled and survived a −7°C field frost (left) despite observed freezing of a vegetative canopy of wheat (right).

Fig. 2.5. IR images taken during freezing and thawing of wheat plants at ear emergence. Top left: during freezing, showing a single exothermic leaf freezing event (white leaf, centre of picture); top right: during freezing, showing a single exothermic ear freezing event (white ear, top right of picture); bottom left: defrosting plants, only material appearing hatched in the image is frozen, the remainder supercooled throughout the test; bottom right: photograph of plants for reference. NB: see Plate 2 for colour representation.

Low-temperature Damage to Wheat

then susceptible plants can supercool and escape frost damage (Lindow, 1995; Wisniewski et al., 2002; Fuller et al., 2003). There is very little information in the literature, however, on the freezing characteristics of wheat during ear emergence and there is limited information as to how freezing occurs in wheat (Gusta et al., 2000; Fuller et al., 2007). Despite considerable genetic ability in wheat to tolerate freezing to low subzero temperatures in a vegetative state, emerged ears possess little or no freezing tolerance. Isolated ears of wheat can supercool readily in frost tests but if freezing does occur then damage is complete. The observations from the experiments reported herein confirm these findings and suggest that intact wheat plants in ear demonstrate considerable supercooling ability either in frost tests or in the field. The findings here and in Fuller et al. (2007) raise a big query over how to assess the frost resistance of wheat during ear emergence. Screening of germplasm at a vegetative stage appears completely inappropriate as this resistance cannot apparently be expressed in the later development stages of the plant. Use of standard screening frost programmes such as that demonstrated here using the Adelaide chamber clearly demonstrate how artefacts such as supercooling can confound results and lead to misleading conclusions. The failure to reproduce partial ear damage as observed in the field in artificial frost tests also suggests that field damaging conditions have yet to be satisfactorily recreated in cabinets and chambers and demonstrates a need for more detailed and definitive experiments. A limited amount of work is presented here which suggests that plants which supercooled had higher levels of floret infertility than control (unfrozen) plants. This suggests an effect of cooling that is different

17

to freezing, possibly similar to chill injury. This hypothesis is supported by work carried out at Sydney University where wheat plants grown at chilling temperatures just above freezing have been shown to lead to infertility in wheat florets (Cantrill et al., 2006). Such chill-induced floret damage is commonly observed in other warm temperate crops (Wood et al., 2006).

Conclusion It is suggested that low-temperature damage to wheat during ear development is a reality but damage due to freezing may differ from chilling injury of supercooled plants. In controlled environments if wheat in ear freezes then catastrophic damage can be expected, but if it supercools then some floret infertility could be expected. However, chill-induced sterility of economic levels is not typically observed in commercial wheat crops following mild subzero frost events. Given these findings, it is suggested that a completely new strategy is needed to reveal the causes of the effects observed in the field, and that observations made in controlled environments need to be confirmed in the field. Possible new avenues for investigation include: screening for variation in supercooling ability and ice nucleation resistance or interruption of ice nucleation using externally applied agents; resistance to chilling damage; constitutive expression of frost resistance; and unlocking the potential for expression of cold resistance genes during late stages of development. The use of IR thermography in such investigations can assist greatly in distinguishing between supercooling and freezing effects and should be used routinely to avoid artefacts confounding experimental interpretation.

References Ashworth, E.N. and Kieft, T.L. (1995) Ice nucleation activity associated with plants and fungi. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 137–162. Boer, R., Campbell, L. and Fletcher, D. (1993) Characteristics of frost in a major wheat-growing region of Australia. Australian Journal of Agricultural Research 44, 1731–1743. Cantrill, L.C., Sutton, B.C. and Overall, R.L. (2006) Low temperature and reproductive development in wheat. Presented at Frost and Chilling Tolerance Workshop, Adelaide University, Adelaide, Australia, 23 November 2006.

18

M.P. Fuller et al.

Cromey, M., Wright, D. and Boddington, H. (1998) Effects of frost during grain filling on wheat yield and grain structure. New Zealand Journal of Crop and Horticultural Science 26, 279–290. Fredericks, T.M., Christopher, J.T. and Borrell, A.K.B (2004) Simulation of radiant frost as pertaining to winter cereals post head-emergence. Presented at International Controlled Environment Meeting, Brisbane, Australia, 16 March 2004; available at http://ncr101.montana.edu/meetings_past.htm Fuller, M.P. and Le Grice, P. (1998) A chamber for the simulation of radiation freezing of plants. Annals of Applied Biology 133, 111–121. Fuller, M.P. and Wisniewski, M. (1998) The use of infrared thermal imaging in the study of ice nucleation and freezing of plants. Journal of Thermal Biology 23, 81–89. Fuller, M.P., Hamed, F., Wisniewski, M. and Glenn, D.G. (2003) Protection of crops from frost using a hydrophobic particle film and an acrylic polymer. Annals of Applied Biology 143, 93–97. Fuller, M.P., Fuller, A.M., Kaniouras, S., Christophers, J. and Fredericks, T. (2007) The freezing characteristics of wheat at ear emergence. European Journal of Agronomy 26, 435–441. Gomezmacpherson, H. and Richards, R.A. (1995) Effect of sowing time on yield and agronomic characteristics of wheat in south-eastern Australia. Australian Journal of Agricultural Research 46, 1381–1399. Gusta, L.V., Wisniewski, M., Nesbitt, N.T. and Gusta, M.L. (2000) Freezing injury in cereals: an overview and new approaches on increasing the frost tolerance of cereals. In: Proceedings of the 8th International Barley Genetics Symposium, Adelaide, Australia, 22–27 October 2003. Festival City Conventions Pty Ltd, Kent Town, Australia, pp. 260–264. Lindow, S.E. (1995) Control of epiphytic ice-nucleating bacteria for management of frost injury. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 239–256. Long, N.R., Reinheimer, J.L., Laws, M.R., Barr, A.R. and Eglington, J. (2005) Delivering improved varieties for hostile environments: potential of frost simulation in frost tolerance breeding. In: Proceedings of the 12th Australian Barley Technical Symposium, Hobart, Australia, 11–14 September 2005 [CD-ROM]. Australian Barley Association, Australia. Marcellos, H. and Single, W.V. (1984) Frost injury in wheat ears after ear emergence. Australian Journal of Plant Physiology 11, 7–15. Paulsen, G. and Heyne, E. (1983) Grain production of winter wheat after spring freeze injury. Agronomy Journal 75, 705–707. Pearce, R.S. and Fuller, M.P. (2001) Freezing of barley studied by infrared video thermography. Plant Physiology 125, 227–240. Shackley, B.J. and Anderson, W.K. (1995) Responses of wheat cultivars to time of sowing in the southern wheat-belt of western Australia. Australian Journal of Experimental Agriculture 35, 579–587. Shroyer, J.P., Mikesell, M.E. and Paulsen, G.M. (1995) Spring Freeze Injury to Kansas Wheat. Kansas State University, Manhattan, Kansas. Single, W.V. (1985) Frost injury and the physiology of the wheat plant. Journal of the Australian Institute of Agricultural Science 51, 128–134. Wisniewski, M., Lindow, S.E. and Ashworth, E.N. (1997) Observations of ice nucleation and propagation in plants using infrared thermography. Plant Physiology 113, 327–334. Wisniewski, M., Glenn, D.M. and Fuller, M.P. (2002) The use of hydrophobic particle films as a barrier to extrinsic ice nucleation in plants. Journal of the American Society of Horticultural Science 127, 358–364. Wood, A.W., Tan, D.K.Y., Mamun, E.A. and Sutton, B.G. (2006) Sorghum can compensate for chilling-induced grain loss. Journal of Agronomy and Crop Science 192, 445–451. Woodruff, D. (1992) ‘WHEATMAN’, a decision support system for wheat management in subtropical Australia. Australian Journal of Agricultural Research 43, 1483–1499. Woodruff, D. and Tonks, J. (1983) Relationship between time of anthesis and grain yield of winter genotypes with differing developmental patterns. Australian Journal of Agricultural Research 34, 1–11.

3

Freezing Behaviours in Plant Tissues: Visualization using NMR Micro-imaging and Biochemical Regulatory Factors Involved M. Ishikawa, H. Ide, W.S. Price, Y. Arata, T. Nakamura and T. Kishimoto

Introduction Freezing is one of the severest stresses that plants encounter in the environment because it causes ice formation, dehydration and cell deformation that can result in death. Since plant tissues contain a large amount of water, the behaviour of cell water under subzero temperatures is key to tissue survival. To survive freezing, cold-hardy plants have developed various mechanisms to regulate the formation of ice in their tissues, which results in diverse but tissue- and species-specific freezing behaviours. For instance, wintering temperate woody plant species display diverse freezing behaviours. Bark tissues undergo extracellular freezing and xylem ray parenchyma cells of many hard wood species deep supercool, while flower and leaf buds of several genera undergo extra-organ freezing (Ishikawa and Sakai, 1982). On the other hand, most of the tissues of cold-hardy herbaceous plants undergo extracellular freezing. The type of freezing behaviour is an important mechanism of cold hardiness. Elucidation of freezing behaviours in complex tissues and their regulatory mechanisms provides an aid to improve plant cold hardiness to avoid freezing injuries. Conventionally, studies on freezing behaviours in plant tissues have been done

by differential thermal analysis (DTA), NMR spectrometry, scanning electron microscopy and visual observation. However, these methods only provide averaged information from a bulk sample or are destructive and liable to cause artefacts. We have developed a novel non-invasive method to visualize the localization of unfrozen water in plant tissues at subzero temperatures using NMR micro-imaging. We also considered the theoretical background for interpreting the images acquired at freezing temperatures (Ishikawa et al., 1997; Price et al., 1997b). Using this method, we successfully visualized various freezing behaviours in cold-hardy woody plant tissues in detail, a part of which has been published (Ishikawa et al., 1997; Price et al., 1997a; Ide et al., 1998). NMR micro-imaging clearly shows that freezing behaviours in cold-hardy plant tissues are finely regulated. As yet, mechanisms involved in the freezing behaviours are poorly understood: why do some tissues remain deep supercooled while other tissues freeze just below 0°C and can undergo extracellular freezing? In the present chapter we review the use of NMR micro-imaging to study the freezing behaviours in plants and biochemical regulatory factors involved in the freezing behaviours.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

19

20

M. Ishikawa et al.

NMR Micro-imaging: Experimental Details NMR microscopy (also known as high-resolution NMR imaging or NMR micro-imaging) is essentially the same as MRI used in the medical sciences, except that a much higher resolution is used (typically three or more orders of magnitude higher). This allows localized information at the tissue–cellular level (practical resolution of 25 to 100 µm) to be obtained. 1 H-NMR micro-imaging was conducted on a Bruker DRX 300 NMR spectrometer operating at 300 MHz as detailed elsewhere (Ishikawa et al., 1997; Price et al., 1997a). Briefly, a 10 mm 1H-imaging insert was used in the imaging probe. Temperature of the sample tube was controlled using a cold nitrogen gas heating system provided by the manufacturer. Images were acquired using specimens wrapped with Parafilm to reduce evaporation during the imaging process. The images were acquired using a multi-slice multi-echo pulse sequence to ensure that all relevant parts of the specimen were imaged in reasonable time. Normally a field of view of 10 mm×10 mm digitized into 128 pixels in each direction (i.e. the in-plane resolution was typically 78 µm) and a slice thickness of 500 µm were used. This degree of resolution was deemed to be a suitable compromise between resolution and acquisition time (note that the signal-to-noise ratio is proportional to the volume element). Typical image acquisition parameters were a recycle delay (TR) of 1.2 s and an echo time (TE) of about 7 ms (the minimum possible echo time given other acquisition parameters). Using these experimental parameters, each set of four multislice images required about 20 min to acquire. The contrast in the images reflects mainly the spin–spin relaxation (T2) and spin–lattice relaxation (T1) of the studied nuclei (protons in the present case). We used a long recycle delay and a short echo time and thus the intensity in the images predominantly reflected the density of the mobile protons (mainly from liquid water). In winter buds, the (liquid) water has a sufficiently long T2 such that its signal is still measurable at the end of the echo period in the imaging pulse sequence. However, water in the ice state has a sub-

millisecond T2 and consequently is not detected at the end of the echo period. Thus, in the figures the light areas represent liquid water while the dark areas represent frozen water (or very low proton density).

Typical NMR Images of Freezing Behaviours in Wintering Woody Plant Tissues Here we present only a few but typical examples from diverse freezing behaviours that are specific to plant tissues and species.

Extracellular freezing Typical images of extracellular freezing are seen in the scales and bark tissues of Japanese azalea (Rhododendron japonicum) (Fig. 3.1A–D, H) and full-moon maple (Acer japonicum) flower buds (Fig. 3.2A–C). The intensity of the NMR signals from these tissues decreased greatly upon cooling to −7°C (Figs 3.1B and 3.2B). This indicates the spontaneous freezing of these tissues, most probably extracellular freezing, which corresponds to the high-temperature exotherm (HTE) in the DTA profile (Fig. 3.1I ). When the buds were further cooled to −14°C, the signal intensity of these tissues decreased further, implying that the tissues had little unfrozen water (Figs 3.1C and 3.2C). These decreases in signal intensity are presented schematically in Fig. 3.1E. This is consistent with the report by Gusta et al. (1975) that showed 70–80% of the total tissue water freezes during cooling to −14°C during extracellular freezing.

Extra-organ freezing in shoot and flower primordia In Japanese azalea flower buds, all of the florets remained unfrozen at −14°C and some florets even at −21°C (Fig. 3.1C and D). This was consistent with the low-temperature exotherm (LTE) recorded between −17 and −27°C in the DTA profile (Fig. 3.1I, trace a). During cooling to lower temperatures (−7 and −14°C), a gradual reduction in floret size was also noted in the NMR images of this species when the

Freezing Behaviours in Plant Tissues

21

Extracellular freezing

Sc X

A Bark, scales

P

B Sc

A

Relative signal intensity

E

B

Florets, pith

Axis Bud scales

F

Deep supercooled Florets

C

2 mm

D

Temperature

scale

floret Ice scale -15°C

Exothermic response

Ice

H

Intracellular freezing

G

HTE

LTE

Low I

a

b -5

-10

-15 -20 -25 -30 Temperature (°C)

-35 -40

Fig. 3.1. (A–D) 1H-NMR images of a wintering Japanese azalea (Rhododendron japonicum) flower bud cooled at 5°C/h to various subzero temperatures (adapted from Price et al., 1997a). White portions in the images represent the density of unfrozen water. A, axis; B, bark; F, floret; P, pith; Sc, scales; X, xylem. (E–G) Changes in the relative signal intensity of each tissue or each type of freezing behaviour are shown schematically. (H) A longitudinal section of a Japanese azalea flower bud cooled similarly and observed at −15°C. Note the accumulation of ice crystals (arrowed) in the bud scales. (I) Typical differential thermal analysis profiles of a flower bud of Japanese azalea (trace a) and a twig piece of full-moon maple (Acer japonicum) (trace b) cooled at 5°C/h. HTE and LTE represent high- and low-temperature exotherms, respectively.

areas of individual floret images (white portions) were compared using image analyses (Fig. 3.1A–C). There was about a 20% decrease in the area of the image of florets that remained supercooled during cooling from 1°C to −21°C (details not shown). The slow dehydration of florets is consistent with the previous observation of flower buds subjected to subzero temperatures (Ishikawa and Sakai, 1981, 1982). Ice formation in the bud scales creates a vapour pressure deficit which results in slow dehydration of the florets due to the migration of floret water to the frozen scales. This slow dehydration of the supercooled florets resulted in an enhancement of deep supercooling while water

migrated from florets accumulated in the bud scales, resulting in the formation of massive ice crystals (Fig. 3.1H) under naturally occurring cooling rates (Ishikawa and Sakai, 1982). This complex freezing event (i.e. extra-organ freezing) is easily observed in the NMR images of flower buds of Japanese azalea (Fig. 3.1) and full-moon maple (Fig. 3.2). Typical changes in the signal intensity of the tissues during extraorgan freezing are summarized in Fig. 3.1F. Freezing of supercooled florets is lethal (data not shown) probably due to intracellular freezing. This event was seen in the NMR images as a sudden decrease in the signal intensity (Fig. 3.1C, D and G).

22

M. Ishikawa et al.

Fig. 3.2. 1H-NMR images showing extra-organ freezing of a flower bud of full-moon maple (Acer japonicum) cooled at 5°C/h; (A–C) are images taken at different temperatures of the same bud. There was a clear boundary (indicated by the arrow in (B)) between the frozen immature stem (IS) and the supercooled inflorescence (infl).

In full-moon maple flower buds, the entire flower primordia (inflorescence with two surrounding leaves) remained supercooled to −20°C and the size of the primordia was gradually reduced during this cooling step (compare images taken between 0 and −14°C in Fig. 3.2). Freezing of the primordia occurred at −21°C, which resulted in death (data not shown). As shown in the NMR images (Figs 3.1 and 3.2), the frozen tissues (scales, bark, etc.) and the supercooled tissues (florets, inflorescence) are in close vicinity. However, ice formation in the bud scales and bark tissues does not necessarily propagate into the unfrozen flower primordia. The mechanisms that prevent propagation from the frozen to the unfrozen tissues (e.g. bud axis in Japanese azalea, immature stem in full-moon maple) are still unknown. NMR micro-imaging clearly allows the visualization of such boundaries; for example, between the frozen immature stem and supercooled inflorescence (Fig. 3.2B, arrow) and the gradual dehydration (arrowhead in Fig. 3.2C) from the basal part of the inflorescence during cooling to the adjacent already frozen tissues, which otherwise are not detectable with traditional methods.

Deep supercooling in the xylem In many temperate deciduous trees, the xylem ray parenchyma cells are generally believed to

deep supercool and thereby remain viable, whereas freezing of these cells is lethal. NMR images revealed that the mature xylem tissues of full-moon maple (Fig. 3.2B and C) remained unfrozen (showing high signal intensity) between −7 and −21°C, which is consistent with the DTA profile of the twig piece, which had a broad LTE starting around −30°C (Fig. 3.1I, trace b). However, with the resolution employed, it is not possible to confirm whether only the xylem ray parenchyma supercool as the images appear as if the entire xylem remained unfrozen (Fig. 3.2). In contrast, in full-moon maple flower buds, deep supercooling was detected between −7 and −21°C in the NMR images of the mature pith and not in the xylem (Fig. 3.1).

Technical and Interpretation Problems In comparing the images taken at different temperatures, it is important to consider the factors that influence signal intensity other than those arising from the transition of water to ice. Many factors affect the image intensity as the temperature decreases, which have been considered in detail elsewhere (Ishikawa et al., 1997; Price et al., 1997b). As the temperature decreases, the T1 of the tissue water decreases which results in an increase in image intensity because the water comes closer to a full thermal equilibrium before the start of the next scan in the imaging sequence. The repeti-

Freezing Behaviours in Plant Tissues

tion delay of the imaging sequence is normally too short to allow the protons to fully relax at ambient temperatures, resulting in partial saturation. In contrast, the T2 of the water resonance decreases resulting in a loss of signal intensity. These two competing effects largely compensate for each other except when freezing occurs, which results in a loss of signal intensity due to the drastic shortening of T2. Nevertheless, in some tissue such as supercooled florets (Fig. 3.1), the increase in intensity due to the decrease in the T1 saturation effect with decreasing temperature is observed (Ishikawa et al., 1997; Price et al., 1997b). Other factors that need to be accounted for when interpreting NMR images include signals from non-water protons, local distortions caused by magnetic susceptibility differences due to air bubbles and ghosting of images (Ishikawa et al., 1997; Price et al., 1997a,b).

Advantages of NMR Micro-imaging for Freezing Behaviour Studies NMR imaging provides spatial information; in particular, it allows the identification of the tissues that produce the exotherms observed using DTA and where the barriers between the frozen tissues and supercooled tissues occur. The high resolution and non-invasive nature of NMR imaging allows the organized fine freezing behaviours to be observed in complex tissues and difficult (or impossible) to excise tissue pieces (e.g. bud axis). Apart from the non-invasive nature which allows visualization of ongoing physiological processes in live samples, another advantage of NMR imaging is its sensitivity to a wide range of chemical (i.e. chemical shift) (Pope et al., 1993; Ishida et al., 1996) and physical contrast mechanisms such as diffusion, flow and relaxation (Köckenberger et al., 1997; Price, 1998). These contrast mechanisms cannot be observed by other methods. In the nonfrozen examples shown, the intensity in the images represents mostly the density of liquid water as a long recycle delay and a short echo time were used (density-weighted images). As noted earlier, water in different parts of the buds has different characteristics (e.g. different

23

T1 and T2 relaxation times and activation energies). By using different imaging sequences and appropriate parameters, these differences provide additional sources of contrast (e.g. T1- and T2-weighted images). Thus, the imaging technique and parameters can be ‘tuned’ to be sensitive to the different states of water in buds and the resulting information is ultimately useful in helping to understand the mechanisms involved in freezing behaviours and cold acclimation (Millard et al., 1995).

Biochemical Regulatory Mechanisms Involved in Freezing Behaviours As seen in the NMR micro-images, various fractions of water in cold-hardy plant tissues remain stably supercooled to temperatures as low as −20°C or lower in spite of adjacent tissues freezing spontaneously at temperatures higher than −7°C (Figs 3.1 and 3.2). It is of interest to know how the plants regulate the freezing of their tissues. There are at least structural and biochemical factors involved, but here we focus on only biochemical factors.

Anti-nucleating activities and supercooling stabilizing factors Since deep supercooled tissues such as florets and xylem ray parenchyma are either minute or dispersed, it is difficult to obtain a sufficient amount of these tissues (without contamination from other tissues) for biochemical analyses. To overcome this problem, we selected Trachycarpus fortunei, which is probably the most cold-hardy palm species and employs deep supercooling as the mechanism of cold hardiness in most of its leaf tissues (Larcher et al., 1991). The leaf tissues of this species tolerated −14°C without any injury. NMR micro-imaging with a resolution of 31 µm successfully visualized non-invasively the freezing behaviour of Trachycarpus leaves (Fig. 3.3C). Water in the vascular bundles and epidermis froze at −10°C, which corresponds to the hightemperature exotherm (HTE) (Fig. 3.3B, trace a); however, the mesophyll cells remained deep supercooled to −14°C. The cells on the adaxial

24

M. Ishikawa et al.

side froze first, mostly between −16 and −19°C, followed by freezing of cells on the abaxial side (−19 and −21°C). The hypodermis and vascular sclerenchyma remained deep supercooled to −22°C or lower (Fig. 3.3A and C). These freezing events altogether are represented by the large LTE in DTA profiles (Fig. 3.3B, trace a). NMR micro-images suggest that these tis-

A

Hpd

sues employ deep supercooling as a mechanism of cold hardiness. Moreover, the HTE of Trachycarpus leaves started at −10°C, which is unusually low compared with HTE arising from tissues of other cold-hardy species (e.g. Fig. 3.1I). This implies that Trachycarpus leaves may have mechanisms to remain supercooled in a stable manner and/or avoid ice nucleation.

Adaxial

Epd

LTE

Exotherm

HTE F P

VT

Sp Sc

Hpd

Epd

a

b Abaxial

-5

-10 -15 -20 Temperature (°C)

C -18°C

Cumulative % of frozen samples

MesophyII cells remain unfrozen

Adaxial side froze first

100 D Water 50

+Palm extracts 0

0

10

20 Days

30

-25

-22°C

40

Cumulative % of frozen samples

-14°C

B

Most cells frozen

100 E

50

Control +Palm extracts

0 -12

-10

-8 -6 -4 -2 Temperature (°C)

0

Fig. 3.3. (A) Light microscopic view of a transverse section of Trachycarpus fortunei leaves, (B, trace a) a typical differential thermal analysis (DTA) profile and (C) NMR micro-imaging of the leaves cooled at 5°C/h. Epd, epidermis; Hpd, hypodermis; Sc, schlerenchyma; Sp, spongy tissues; P, palisade tissues, F, fibres; VT, vascular tissues. (B, trace b) Following extraction with methanol and rehydration to the same water content, the high-temperature exotherm (HTE) and low-temperature exotherm (LTE) of the leaf were shifted to higher temperatures in the DTA profile. (D) Cumulative percentage of tubes frozen during 40 days of exposure to −15°C, where each tube contained 2 ml of sterilized Milli-Q water with or without the extract of T. fortunei leaves (40 replicates). (E) Ice nucleation temperature of ice-nucleating bacteria (control) was shifted to lower temperatures by the addition of the leaf extract.

Freezing Behaviours in Plant Tissues

Therefore, we used this as a model system to study the biochemical mechanisms involved in deep supercooling. To test this hypothesis, Trachycarpus leaves were extracted with methanol or hot water and rehydrated to the same water content by imbibition in water or air-drying prior to DTA. Interestingly, DTA profiles of these samples had both the HTE and the LTE shifted to higher temperatures compared with those of intact leaves (Fig. 3.3B, trace b). The methanol extract of the leaves was examined for antinucleating activity (Fig. 3.3E) and for supercooling stabilizing capability (Fig. 3.3D). We developed a method to determine anti-nucleation activity using PCR plates placed on a precision-controlled cold bath. Mixture (10 µl+10 µl) of ice nucleator solution and the extract cooled at 2°C/h allowed anti-nucleating activities to be reproducibly determined (20 wells or more for each sample). The extract inhibited the ice nucleation by several known ice nucleators, but most pronouncedly ice-nucleating bacteria in a concentration-dependent manner (Fig. 3.3E) (Sawada and Ishikawa, 2001). The extract had the ability to stabilize deep supercooling of water (2 ml) during 40 days of exposure to −10°C or following vigorous shaking at −10°C (Fig. 3.3D). We are currently analysing substances responsible for anti-nucleating activity against ice-nucleating bacteria using HPLC. At least several compounds are involved in the anti-nucleating activity but it still remains unknown whether they also confer supercooling stabilizing activity or not.

Ice-nucleating activities Subzero temperatures experienced by plants in field conditions do not necessarily accompany the occurrence of external ice nucleators such as frozen dews, snowflakes or epidemic ice-nucleating bacteria on plants. Even in the absence of extrinsic ice nucleators, tissues of cold-hardy plants that undergo extracellular freezing readily freeze at high subzero temperatures and avoid excessive supercooling of the tissues. Such tissues appear to positively control the initiation of freezing by producing ice-nucleating substances. To determine why the scale tissues of Japanese azalea freeze spontaneously during

25

cooling to −7°C (Fig. 3.1), the ice-nucleating activity of detached scales and other tissues was determined using an improved test-tube method (2 ml plus sample cooled at 2°C/h) based on the method developed by Hirano et al. (1985). The outer and inner bud scales which freeze first and act as ice sinks had the highest icenucleating activity (INA) (−5.9 and −6.8°C, respectively), while the florets which remain supercooled lacked effective INA (−13 to −16°C). In the twigs, the bark tissues which undergo extracellular freezing had a high INA of −6.3°C while the xylem and pith tissues which undergo deep supercooling had low INA (−12.8 and −12.4°C, respectively). The INA of each tissue closely corresponded with its freezing behaviour. These INA values were affected only slightly by a fourfold increase in the tissue amount and also by an increase in the incubation time at subzero temperatures (data not shown). This suggests that these INA are specific to the tissues. The high INA in the outer scales and bark tissues was unaffected by homogenization of the tissues into fine powders and subsequent extensive washings (data not shown). Subcellular fractionation of the INA in the bark by differential centrifugation revealed that the INA was associated with the cell wallrich fraction (M. Ishikawa, unpublished results). These results imply that this INA does not arise from macro structures such as the presence of trichomes or the outer scales or bark situated in the outermost part of the buds or stems, but from substances tightly bound to the cell walls. The substances responsible for the INA in each tissue have different sensitivities to heat treatment: the high INA in the bud scale tissues was unaffected by autoclaving (121°C for 15 min) while the high INA in the bark tissues was sensitive to autoclaving (decreased to −11 to −12°C) (data not shown). Flower bud scales of Japanese azalea showed seasonal changes in INA. The outer and inner scales of late August flower buds (just completed morphological development) had low INA, from there on INA increased and attained the maximum activity in late October or early November at the time of the first frost (data not shown). In wintertime, INA was maintained at high levels. Interestingly, flower bud scales of tropical Rhododendron species had very low INA irrespective of the season

26

M. Ishikawa et al.

(M. Ishikawa, unpublished results). We surveyed the INA of various tissues of nearly 500 species ranging from tropical to boreal plants and found that cold-hardy plants from temperate to boreal regions in general showed higher INA in some tissues compared with tropical plants (M. Ishikawa, unpublished results). These results imply that when cold-hardy plants cold acclimate, they regulate freezing by producing intrinsic ice nucleators in tissues that undergo extracellular freezing or tissues that act as an ice sink in extra-organ freezing. It appears that tropical plants (sensitive to freezing) lack such intrinsic ice nucleators and tend to supercool extensively (lower than −7°C). When freezing does occur, it tends to be lethal presumably due to intracellular freezing. Isolation and identification of such intrinsic ice-nucleating substances to date have not been successful to our knowledge. Studies are underway in our laboratory and will be published elsewhere.

Antifreeze activities Antifreeze activity has been detected in the apoplast of some cold-hardy herbaceous plant tissues. Griffith and co-workers (Hon et al., 1995) first reported that three pathogenesisrelated (PR) proteins in the apoplast of coldhardened winter rye have antifreeze activity. Later, a polygalacturonase inhibitor homologue (Worrall et al., 1998; Meyer et al., 1999) in carrot, a heat-stable protein (Kuiper et al., 2001) in perennial ryegrass, a leucine-rich repeat-containing protein in wheat (Tremblay et al., 2005) and a WRKY protein in Solanum dulcamara (Huang and Duman, 2002) were reported to have antifreeze activity. The function of these antifreeze proteins in the apoplast of herbaceous plants which undergo extracellular freezing is not very clear, since the degree of thermal hysteresis (i.e. actual prevention of initiation of freezing) generated by these proteins is small. It is generally thought that they also inhibit the recrystallization of ice and thus maintain ice crystals in non-injurious small sizes during prolonged exposure to subzero temperatures, which may contribute to winter survival. Wang et al. (2002) found carrot antifreeze protein lost half of its recrystallization inhibition activity within 10–20 weeks at −80°C.

The isolated or recombinant antifreeze proteins may not necessarily be in the same form or environment as in planta. Stressmann et al. (2004) reported that the crude antifreeze activity was affected by many factors such as Ca ions. The antifreeze activity assays employed do not necessarily represent the conditions in planta. For example, the recrystallization inhibition assay (splat assay) developed by Worrall et al. (1998) contains high levels of sucrose compared with the levels measured in planta. The presence of rye antifreeze activity has been shown to improve freeze survival of nonacclimated rye suspension cells (PihakaskiMaunsbach et al., 2003) and to influence freezing patterns of rye plants (Griffith et al., 2005). It is suggested that similar approaches be taken to elucidate the function of antifreeze activity in plants in comparison with proper control experiments. Many people have been wondering why Griffith used crude rye antifreeze activity fractions in her experiments even after she isolated the responsible genes. While she detected antifreeze activities with rye thaumatin-like proteins (Hon et al., 1995), others (C. Kuwabara, Sapporo, Japan, 2006, personal communications) were unable to validate this finding when analysing products of all the thaumatin-like protein genes. Also, a coldinduced extracellular chitinase gene (BiCHT1) isolated from bromegrass, which is highly homologous to rye antifreeze protein gene CHT9, did not display antifreeze activity (Nakamura et al., 2008). However, a rye glucanase has been shown to have antifreeze activity (Yaish et al., 2006). From the authors’ experience, isolation of antifreeze proteins is difficult due to their low abundance in the apoplast in contrast to arctic fish. In spite of the difficulties in the assay and isolation, effort should be made to isolate new classes of antifreeze proteins from various cold-hardy plant tissues and to elucidate the conditions of their occurrence per se and their functions in the freeze regulation of tissues.

Conclusion NMR micro-imaging is a powerful tool to study freezing behaviours in cold-hardy plant tissues since it can non-invasively provide spatially

Freezing Behaviours in Plant Tissues

specific information. Cold-hardy plant tissues appear to have developed various mechanisms to control freezing, such as anti-nucleation activity, ice-nucleating activity and antifreeze activity. Studies are underway to isolate compounds and/or genes responsible for these

27

activities and to elucidate their functions in vitro and in vivo. As yet, only a fraction of the whole picture of freeze-regulation mechanisms in plants has been elucidated, including structural mechanisms, which are not covered in the present review.

References Griffith, M., Lumb, C., Wiseman, S.B., Wisniewski, M., Johnson, R.W. and Marangoni, A.G. (2005) Antifreeze proteins modify the freezing process in planta. Plant Physiology 138, 330–340. Gusta, L.V., Burke, M.J. and Kapoor, A.C. (1975) Determination of unfrozen water in winter cereals at subfreezing temperatures. Plant Physiology 56, 707–709. Hirano, S.S., Baker, L.S. and Upper, C.D. (1985) Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury. Plant Physiology 77, 259–265. Hon, W.-C., Griffith, M., Mlynarz, A., Kwok, Y.C. and Yang, D.S.C. (1995) Antifreeze proteins in winter rye are similar to pathogenesis-related proteins. Plant Physiology 109, 879–889. Huang, T. and Duman, J.G. (2002) Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Molecular Biology 48, 339–350. Ide, H., Price, W.S., Arata, Y. and Ishikawa, M. (1998) Freezing behaviors in leaf buds of cold hardy conifers visualized by NMR microscopy. Tree Physiology 18, 451–458. Ishida, N., Koizumi, M. and Kano, H. (1996) Location of sugars in barley seeds during germination by NMR microscopy. Plant, Cell and Environment 19, 1415–1422. Ishikawa, M. and Sakai, A. (1981) Freezing avoidance mechanisms by supercooling in some Rhododendron flower buds with reference to water relations. Plant & Cell Physiology 22, 953–967. Ishikawa, M. and Sakai, A. (1982) Characteristics of freezing avoidance in comparison with freezing tolerance: a demonstration of extraorgan freezing. In: Li, P.H. and Sakai, A. (eds) Plant Cold Hardiness and Freezing Stress. Academic Press, New York, New York, pp. 325–340. Ishikawa, M., Price, W.S., Ide, H. and Arata, Y. (1997) Visualization of freezing behaviors in leaf and flower buds of full-moon maple by nuclear magnetic resonance microscopy. Plant Physiology 115, 1515–1524. Köckenberger, W., Pope, J.M., Xia, Y., Jeffrey, K.R., Komor, E. and Callaghan, P.T. (1997) A non-invasive measurement of phloem and xylem water flow in castor bean seedlings by nuclear magnetic resonance microimaging. Planta 201, 53–63. Kuiper, M.J., Davies, P.L. and Walker, V.K. (2001) A theoretical model of a plant antifreeze protein from Lolium perenne. Biophysical Journal 81, 3560–3565. Larcher, W., Meindl, U., Ralser, E. and Ishikawa, M. (1991) Persistent supercooling and silica deposition in cell walls of palm leaves. Journal of Plant Physiology 139, 146–154. Meyer, K., Keil, M. and Naldrett, M.J. (1999) A leucine rich repeat protein of carrot that exhibits antifreeze activity. FEBS Letters 447, 171–178. Millard, M.M., Veisz, O.B., Krizek, D.T. and Line, M. (1995) Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat. Plant, Cell and Environment 18, 535–544. Nakamura, T., Ishikawa, M., Nakatani, H. and Oda, A. (2008) Characterization of cold-responsive extracellular chitinase in bromegrass cell cultures and its relationship to antifreeze activity. Plant Physiology 147, 391–401. Pihakaski-Maunsbach, K., Tamminen, I., Pietianinen, M. and Griffith, M. (2003) Antifreeze proteins are secreted by winter rye cells in suspension culture. Physiologia Plantarum 118, 390–398. Pope, J.M., Jonas, D. and Walker, R.R. (1993) Applications of NMR micro-imaging to the study of water, lipid, and carbohydrate distribution in grape berries. Protoplasma 173, 177–186. Price, W.S. (1998) NMR imaging. In: Webb, G.A. (ed.) Annual Reports on NMR Spectroscopy. Academic Press, New York, New York, pp. 139–216. Price, W.S., Ide, H., Arata, Y. and Ishikawa, M. (1997a) Visualization of freezing behaviours in flower bud tissues of cold hardy Rhododendron japonicum by nuclear magnetic resonance micro-imaging. Australian Journal of Plant Physiology 24, 599–605. Price, W.S., Ide, H., Ishikawa, M. and Arata Y. (1997b) Intensity changes in 1H-NMR micro-images of plant materials exposed to subfreezing temperatures. Bioimages 5, 91–99.

28

M. Ishikawa et al.

Sawada, K. and Ishikawa, M. (2001) Anti-nucleating activity in wintering cold hardy plant tissues. Journal of Plant Research 114, Supplement, 85–86. Stressmann, M., Kitao, S., Griffith, M., Moresoli, C., Bravo, L.A. and Marangoni, A.G. (2004) Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye. Plant Physiology 135, 364–376. Tremblay, K., Ouellet, F., Fournier, J., Danyluk, J. and Sarhan, F. (2005) Molecular characterization and origin of novel bipartite cold-regulated ice recrystallization inhibition proteins from cereals. Plant & Cell Physiology 46, 884–891. Wang, L.H., Wusteman, M.C., Smallwood, M. and Pegg, D.E. (2002) The stability during low-temperature storage of an antifreeze protein isolated from the roots of cold-acclimated carrots. Cryobiology 44, 307–310. Worrall, D., Elias, L., Ashford, D., Smallwood, M., Sidebottom, C., Lillford, P., Telford, J., Holt, C. and Bowles, D. (1998) A carrot leucine-rich-repeat protein that inhibits ice recrystallization. Science 282, 115–117. Yaish, M.W.F., Doxey, A.C., McConkey, B.J., Moffatt, B.A. and Griffith, M. (2006) Cold-active winter rye glucanases with ice-binding capacity. Plant Physiology 141, 1459–1472.

4

Factors Related to Change of Deep Supercooling Capability in Xylem Parenchyma Cells of Trees S. Fujikawa, J. Kasuga, N. Takata and K. Arakawa

Introduction Most plant cells, including the cortical parenchyma cells (CPCs) of trees, can adapt to subfreezing temperatures, which result in freezing of apoplast water, by extracellular freezing (Sakai and Larcher, 1987). On the other hand, xylem parenchyma cells (XPCs) in trees adapt to such subfreezing temperatures by so-called deep supercooling (Quamme et al., 1973; George and Burke, 1977; Quamme et al., 1982; Ashworth et al., 1988; Malone and Ashworth, 1991). Recent studies have confirmed that XPCs in almost all tree species, including tropical (Kuroda et al., 1997), temperate (Fujikawa and Kuroda, 2000) and even boreal trees (Kuroda et al., 2003), adapt to subfreezing temperatures by deep supercooling. Both adaptation mechanisms operate to avoid occurrence of lethal intracellular freezing, but the two mechanisms are completely different. By extracellular freezing, cells avoid intracellular freezing by dehydration. In extracellular freezing, when apoplastic water has frozen, the difference between the vapour pressures of extracellular ice and intracellular water results in dehydration of cellular water towards extracellular ice in direct parallel to temperature reductions (Steponkus, 1984). Because of this equilibrium dehydration, cells are able to avoid the occurrence of intracellular freez-

ing. Extracellular freezing cells have thin, soft walls generally consisting of unlignified primary walls, and extracellular freezing therefore results in both shrinkage and deformation of the cells in response to dehydration as well as due to the formation of large extracellular ice crystals (Pearce, 1988; Fujikawa, 1994; Nagao et al., 2008). In extracellular freezing cells, survival depends mainly on the degree of tolerance to freezing-induced dehydration, and the temperatures at which cells can survive range from just below zero to liquid nitrogen temperatures depending on plant species and the conditions of cold acclimation and deacclimation (Sakai and Larcher, 1987). On the other hand, with deep supercooling, cells avoid intracellular freezing without experiencing dehydration by supercooling. XPCs have developed a unique mechanism for maintaining a supercooled state in a metastable equilibrium for a long period of time spanning several weeks, under exposure to low subfreezing temperatures (Quamme et al., 1982). This adaptive mechanism is thus referred to as deep supercooling to distinguish it from temporal supercooling (Quamme, 1991). Deep supercooling XPCs have thick, rigid walls consisting of lignified secondary walls (Fujikawa et al., 1999; Fujikawa and Kuroda, 2000). Cells undergoing deep supercooling retain their original morphology at subfreezing temperatures because no dehydration occurs (Fujikawa et al.,

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

29

30

S. Fujikawa et al.

1994, 1997). XPCs can survive freezing events under supercooling, but supercooling has a physical temperature limit. When the temperature falls below the limit, the capacity for maintaining a metastable equilibrium is exceeded and lethal intracellular freezing occurs (Burke et al., 1976; Fujikawa and Kuroda, 2000). The freezing resistance of deep supercooling XPCs is generally lower than that of extracellular freezing cells, especially in boreal trees (Sakai and Larcher, 1987). Death of XPCs caused by intracellular freezing due to the breakdown of supercooling results in death of the entire tree (Quamme et al., 1982). Therefore, the temperature limit of deep supercooling in XPCs determines the freezing resistance of an entire tree and is the most important factor for the distribution of trees in cold areas (George et al., 1974, 1982; Burke and Stushnoff, 1979; Becwar et al., 1981; Fujikawa and Kuroda, 2000). The maximum temperature limit of supercooling in XPCs changes with latitude and ranges from −10°C in tropical trees to −40°C in temperate trees and to almost −70°C in boreal trees, possibly as a result of evolutionary adaptation (Kuroda et al., 1997; Fujikawa and Kuroda, 2000; Kuroda et al., 2003). The temperature limit of supercooling is also changed greatly by seasonal cold acclimation and deacclimation (Quamme et al., 1982; Fujikawa and Kuroda, 2000). The temperature limit of supercooling in XPCs of boreal trees, for example, is about −20°C in summer but is reduced to near −70°C during winter (Gusta et al., 1983; Fujikawa and Kuroda, 2000; Kuroda et al., 2003). In addition, the supercooling capability of XPCs is changed by artificial cold acclimation and deacclimation of tree twigs (Hong and Sucoff, 1982a,b; Takata et al., 2007). Therefore, trees adapt to environmental temperature changes by changing the temperature limit of supercooling in XPCs, an adaptation that depends on environmental temperature changes experienced where the trees are growing. Taken together, results of previous studies clearly indicate that the supercooling capability of XPCs is controlled by cold acclimation and deacclimation. Many studies have revealed molecular mechanisms underlying changes in freezing tolerance as a result of cold acclima-

tion and deacclimation in cells that exhibit extracellular freezing (Guy, 1990; Hughes and Dunn, 1996; Thomashow, 1999). However, there have been few studies on the mechanisms responsible for deep supercooling in XPCs. One reason for this may be that until recently the deep supercooling of XPCs has been explained solely by the physical state of water as an isolated droplet (Ashworth and Abeles, 1984; George et al., 1982). However, recent studies suggest that deep supercooling of XPCs may be regulated by more complex mechanisms associated with cold acclimation and deacclimation. In the current chapter, we first describe our view that the mechanisms of deep supercooling in XPCs is difficult to explain only by an isolated water droplet theory. We then present the results of a few recent studies suggesting that deep supercooling of XPCs is associated with a more complex phenomenon in relation to diverse intracellular changes as a result of cold acclimation and deacclimation.

Isolated Water Droplet Theory Explains Only Part of the Deep Supercooling Phenomenon in Xylem Parenchyma Cells Until recently, based on the results of in vitro experiments on the supercooling of small isolated water droplets (Fletcher, 1970; MacKenzie, 1977), the mechanism of deep supercooling in XPCs has been explained solely by such physical isolation of water (Ashworth and Abeles, 1984). It has been suggested that protoplast water in XPCs is isolated from the effects of extracellular ice crystals due to cell walls that do not allow dehydration and the penetration of extracellular ice into protoplasts (George and Burke, 1977; Quamme et al., 1982; George, 1983; Ashworth and Abeles, 1984). It was also speculated that protoplasts of XPCs may not contain heterogeneous ice nucleators (Quamme et al., 1982). Thus, it has been thought that the protoplasts of XPCs as isolated water droplets can supercool to the homogenous ice nucleation temperature of water, which is about −40°C (George and Burke, 1977; Quamme et al.,

Changes in Deep Supercooling in Trees

1982; George, 1983; Ashworth and Abeles, 1984). The isolation of protoplasts of XPCs from the effects of extracellular ice is undoubtedly a prerequisite for supercooling of XPCs. However, it is difficult to explain the deep supercooling phenomenon of XPCs, especially the long-term stability of supercooling, only by such a physical state of water. It has been indicated that the incidence of nucleation of isolated water droplets in a metastable equilibrium depends on the size of water droplets and time periods of cooling; smaller water droplets and higher cooling rates yield lower nucleation temperatures because of the lower incidence in development of ice clusters, which become homogeneous ice nucleators (Fletcher, 1970; MacKenzie, 1977). In nature, protoplasts of XPCs can continue supercooling for several weeks, a time frame that is overwhelmingly longer than the time frame of experimental conditions. Furthermore, the process of supercooling is easily disrupted by vibration. Although trees and their XPCs are subjected to strong vibration due to wind, especially during winter, XPCs continue to undergo deep supercooling under such conditions. Therefore, it is thought that XPCs may have mechanisms to stabilize supercooling for a long time. In the isolated water droplet theory, seasonal and other fluctuations in the deep supercooling capacity of XPCs are difficult to explain. Based on this theory, the temperature limit of supercooling corresponds to the temperatures at which cell walls lose their barrier property against penetration by extracellular ice. Loss of the barrier results in seeding of ice to protoplasts, inducing intracellular freezing (Burke and Stushnoff, 1979; Ashworth and Abeles, 1984). In a series of articles (see review by Wisniewski, 1995), Wisniewski and co-authors suggested that seasonal changes in the structure of specific areas of the cell wall (pit membrane) of XPCs might account for changes in supercooling capacity. They postulated that changes in pectins (which line cell wall pores) could affect pore size and thus the ability for ice crystals to penetrate XPCs. A recent study, however, showed that even in extracellular freezing cells in tree and herbaceous plant species, plant cell walls have an inherent property to inhibit penetration of extracellular ice into

31

cells (Yamada et al., 2002). Furthermore, extracellular freezing results in cytorrhysis, which causes collapse of protoplasts together with their cell walls, but not in plasmolysis, which produces a collapse of protoplasts with a separation from cell walls (Carpita et al., 1979), suggesting also the inhibitory effects of cell walls against penetration of extracellular ice. It is also confirmed that extracellular ice crystals do not penetrate the walls of XPCs in several tree species, even though large macromolecules such as PEG6000 can penetrate cell walls freely (Fujikawa et al., 1997; Tanaka et al., 2003). Finally, almost all plasma membranes may function as a barrier to extracellular ice, even though ice crystals may come into contact with the plasma membranes after penetration through the cell walls (Steponkus, 1984). All of these results may throw doubt on the cause of fluctuation in deep supercooling capability by seeding of extracellular ice through cell walls and suggest the involvement of more complex factors in the deep supercooling phenomenon of XPCs.

Gene Expression is Associated with Fluctuation of Deep Supercooling Capability in Xylem Parenchyma Cells There have been limited studies on gene expression in association with the fluctuation of deep supercooling capability in XPCs. Recently, however, Takata et al. (2007) examined the change of gene expression in xylem and found a close association with the change of deep supercooling capability in XPCs of larch (Larix kaempferi). Wisniewski et al. (2008) also presented a comprehensive expressed sequence tag (EST) analysis of the response of apple tissues, including XPCs, in response to cold and drought. According to the study by Takata et al. (2007), the supercooling capability of XPCs in larch changes seasonally from −30°C during summer to −60°C during winter as a result of seasonal cold acclimation and deacclimation. Takata et al. (2007) also found that deacclimation of winter twigs for 7 days reduced the supercooling capability of XPCs from −60°C to −30°C. By differential screening between winter

32

S. Fujikawa et al.

xylem and deacclimated xylem, Takata et al. (2007) found 46 genes that were expressed specifically in winter xylem but for which the expression disappeared or was significantly reduced in deacclimated xylem. Sixteen of those 46 genes were novel genes that had not been recorded in databases, while the other 30 genes had been recorded in databases. These 30 recorded genes, which were specifically expressed during winter when the supercooling capability was maximum, were named WXL (Winter accumulated Xylem genes in Larch, which were detected by differential display analysis) or SXL (Supercooling related Xylem genes in Larch, which were detected by cDNA filter array). These 30 genes were categorized into several groups according to their function: signal transduction factors, metabolic enzymes, late embryogenesis-abundant (LEA) proteins, heat-shock proteins (HSPs), protein synthesis proteins and chromatin constructed-proteins, defence response proteins, membrane transporters, metal-binding proteins and functionally unknown proteins. Northern blot analysis showed that all of these 30 genes were expressed abundantly during winter and that their expression was reduced or disappeared during summer in parallel with the reduction of supercooling capability of XPCs by seasonal cold acclimation and deacclimation. The expression of all but two of these genes was significantly reduced or disappeared with deacclimation of winter twigs, when supercooling capability was significantly reduced. Interestingly, all but one of the genes were expressed more abundantly in the xylem with deep supercooling XPCs than in the cortex with extracellular freezing CPCs despite the fact that many dehydration-related genes, such as genes encoding LEA proteins, were included in these genes. About two-thirds of the 30 genes are known cold-induced genes that have been found in extracellular freezing plant cells, but the remaining genes are novel cold-induced genes. The known cold-induced genes have been shown to have functions in low-temperature signal transduction by ABA (abscisic acid), stress and ripening (ASR) proteins and by protein phosphatase 2A regulatory subunit (PP2A) (SXL2, 8, 9 and 10) (Schneider et al., 1997;

Monroy, 1998; Maskin et al., 2001; Kim et al., 2002; Yang et al., 2005); functions in metabolism changes under low-temperature conditions, including biosynthesis of raffinose family oligosaccharides (RFOs) by galactinol synthase (SXL3) (Taji et al., 2002; Pennycooke et al., 2003), phospholipid synthesis by aminoalcoholphosphotransferase (WXL4) (Qi et al., 2003) and glucosylation of flavonoids by flavonol-3-O-glucosyltransferase (WXL5) (Christie et al., 1994; Chalker-Scott, 1999; WinkelShirley, 2002; Lo Piero et al., 2005); functions in protection of macromolecules from freezing-induced dehydration by LEA proteins (SXL 1, 5, 4, 7, 12, 13 and 14) (Houde et al., 1995; Close, 1997; Danyluk et al., 1998; Wisniewski et al., 1999; Fujikawa et al., 2006); functions as membrane stabilizers, molecular chaperones and cryoprotectants under freezing by HSPs (SXL15) (van Berkel et al., 1994; Sabehat et al., 1998; Ukaji et al., 1999; Török et al., 2001; van Montfort et al., 2001; Fujikawa et al., 2006); functions in regulation of protein synthesis and chromatin construction under low-temperature conditions by peptide chain release factor subunit 1 (eRF1) and by histone H2A proteins (SXL17 and WXL6) (Seki et al., 2002); and a function in maintenance of pH gradient between cytoplasm and vacuole under low temperature conditions by H+-pyrophosphatase (WXL3) (Carystinos et al., 1995). Additionally, metallothionein genes, encoding metal-binding protein (SXL22), are induced by cold, although their functional roles under cold stress are unknown (Reid and Ross, 1997; Cho et al., 2006). Eleven of the 30 genes are novel cold-induced genes. The majority of the novel coldinduced genes are genes with unknown functions (WXL9, 10, 11; SXL6, 23, 24, 25) and the other genes are involved in signal transduction (WXL1 and 7) (Meyers et al., 2002; Grant et al., 2003; Chini et al., 2004), defence response (SXL20) (Nadimpalli et al., 2000; Borner et al., 2005) and membrane transport (SXL21) (Lawand et al., 2002). The results of the study by Takata et al. (2007) described above suggest that the change in supercooling capability of XPCs is closely associated with the expression of specific genes,

Changes in Deep Supercooling in Trees

including genes whose functions have not been determined. Their results also indicate that gene products that had been thought to play a role in dehydration tolerance by extracellular freezing, such as various LEA proteins, also have a function in adaptation to subfreezing temperatures by deep supercooling. The presence of both already known and novel cold-induced genes in deep supercooling XPCs suggests both similarities and differences in adaptation to subfreezing temperatures by supercooling and extracellular freezing. However, the functions of the majority of known cold-induced genes in deep supercooling of XPCs are unclear. Furthermore, the functions of the novel cold-induced genes, not only in low temperatures but also in deep supercooling, are not known. It is necessary to identify more genes that are associated with change in deep supercooling capability and also to elucidate the role of each gene in the deep supercooling capability of XPCs.

Intracellular Substances Affect Deep Supercooling Capability of Xylem Parenchyma Cells While the exact role of genes in deep supercooling capability of XPCs is unclear, the involvement of some intracellular substances in deep supercooling capability has been indicated. An early study (Quamme et al., 1973) showed that after breakdown of protoplasts in apple (Pyrus malus) XPCs by intracellular freezing, resulting from slow cooling to a temperature exceeding the temperature limit of supercooling, there was little change in the supercooling capability of XPCs when samples were re-cooled after thawing. This observation provides strong supports that deep supercooling of XPCs occurs due to a physical isolation of water confined within the cell walls of XPCs. However, a recent study showed that supercooling capability of XPCs was significantly changed when intracellular substances were released from XPCs (Kasuga et al., 2006). In that study, it was found that XPCs in freshly collected Siebold’s beech (Fagus crenata) harvested during winter supercooled to −40°C but that the supercooling capability of XPCs after complete

33

release of intracellular substances by boiling was reduced to −20°C. It was also confirmed in that study that boiling did not cause detectable ultrastructural changes in cell walls. Furthermore, it was shown in that study that when xylem prefrozen by a procedure similar to that used in a previous study (Quamme et al., 1973) was washed well after thawing, the supercooling capability of XPCs in the re-cooled xylem was significantly reduced (Kasuga et al., 2006). It was suggested that in the previous study (Quamme et al., 1973) release of intracellular substances from XPCs after intracellular freezing without thawing may have been insufficient, resulting in little change in supercooling capability (Kasuga et al., 2006). These results suggest that physical isolation of water within walls of XPCs induces supercooling to some speciesspecific degree, but that intracellular substances may also play an important role in supercooling of XPCs. In the case of Siebold’s beech XPCs with supercooling capability of −40°C, it is thought that physical isolation of water induces supercooling to −20°C and that released intracellular substances may be involved in the remaining supercooling capability of −20°C.

Effects of intracellular soluble sugars Soluble sugars may be intracellular substances that affect the deep supercooling capability of XPCs. Although the seasonal changes in accumulation of soluble sugars in trees have been investigated in many studies, almost all of those studies focused on cortical tissues with extracellular freezing CPCs (Sakai and Larcher, 1987). There have been only a few studies on seasonal changes in soluble sugars in xylem tissues of boreal hardwood species, including red osier dogwood (Cornus sericea) (Ashworth et al., 1993), poplar (Populus×canadensis) (Sauter and Kloth, 1987; Sauter et al., 1996), willow (Salix caprea) (Sauter and Wellenkamp, 1998) and silver birch (Betula pendula) (Piispanen and Saranpää, 2001). However, none of those previous studies examined soluble sugars in xylem in relationship to deep supercooling of XPCs because it had been thought until recently that XPCs of these boreal hardwood species adapt to subfreezing

34

S. Fujikawa et al.

temperatures by extracellular freezing (Sakai and Larcher, 1987; Fujikawa and Kuroda, 2000). However, a recent cryo-scanning electron microscopic study confirmed that XPCs in all of these boreal hardwood species responded to subfreezing temperature by deep supercooling (Kuroda et al., 2003). With the premise that both tissue cells have contrasting freezing adaptation mechanisms, the first study in which soluble sugars in extracellular freezing CPCs and deep supercooling XPCs were compared was carried out by Kasuga et al. (2007a) in order to determine the roles of soluble sugars in deep supercooling of XPCs in Japanese white birch (Betura platyphylla). That study confirmed that in both CPCs and XPCs with contrasting freezing adaptation mechanisms, total amounts of soluble sugars were larger during winter than during summer, both at the entire tissue level and the intracellular level after removal of apoplastic sugars (Kasuga et al., 2007a). Previous studies also showed that total amounts of soluble sugars were larger in winter than in summer in both cortical and xylem tissues in poplar and willow (Sauter et al., 1996; Sauter and Wellenkamp, 1998). The study by Kasuga et al. (2007a) also confirmed that the amounts of sucrose, raffinose and stachyose increased in both CPCs and XPCs during winter but that the amounts of glucose and fructose did not change in either CPCs or XPCs throughout the entire season. Thus, seasonal changes in accumulation of soluble sugars are the same in CPCs and XPCs with contrasting freezing adaptation mechanisms in Japanese white birch (Kasuga et al., 2007a). Similar results were also obtained in red osier dogwood (Ashworth et al., 1993). However, the study by Kasuga et al. (2007a) specifically related sugar levels to contrasting mechanisms of freeze adaptation. Kasuga et al. (2007a) found that concentrations of soluble sugars were clearly different in deep supercooling XPCs versus extracellular freezing CPCs in Japanese white birch. Although the total amounts of soluble sugars at the tissue level were higher in cortical tissues than in xylem tissues, combined estimation based on measurement of total amounts of soluble sugars in cells of each tissue (after washing of apoplastic sugars) and taking into account the volume of parenchyma cells in

each tissue indicated that deep supercooling XPCs contained a much higher concentration of soluble sugars than did extracellular freezing CPCs throughout a year. It was shown at the cellular level that XPCs contained sevenfold and fourfold higher concentrations of total soluble sugars than those in CPCs during winter and summer, respectively, and that the concentration of soluble sugars in XPCs during winter was fourfold higher than that during summer. Similar results were also obtained in XPCs of Siebold’s beech and larch (J. Kasuga and S. Fujikawa, unpublished results). It has been reported that the total amount of soluble sugars in cortical tissues was twofold larger than that in xylem tissues of red osier dogwood (Ashworth et al., 1993). Considering the difference in the volume of parenchyma cells in each tissue, however, deep supercooling XPCs of red osier dogwood may also contain a much higher concentration of soluble sugars than that in extracellular freezing CPCs. Thus, it is suggested that deep supercooling XPCs of many tree species may contain high concentrations of soluble sugars. When high concentration of solutes depressed the equilibrium melting point of water as a function of a colligative effect, the ice nucleation temperature was depressed by nearly two times for common solutes including sugars (Rasmussen and MacKenzie, 1972). Thus, a high intracellular osmotic concentration in XPCs due to a large accumulation of soluble sugars may enhance the capacity for supercooling by equilibrium melting point depression and consequent nucleation temperature depression (Gusta et al., 1983). Cryoscanning electron microscopic observations have been shown that the equilibrium melting point of XPCs in Japanese white birch during winter was −4°C (Kasuga et al., 2007a). Thus, the melting point depression may reduce the freezing point to −8°C. This shows the importance of soluble sugars in facilitating and changing the supercooling capacity of XPCs by a change in the sugar concentration.

Effect of proteins It is thought that the presence of proteins, such as antifreeze proteins, promotes the deep

Changes in Deep Supercooling in Trees

supercooling capability of XPCs because some antifreeze proteins and antifreeze glycoproteins have been shown to have an anti-ice nucleation activity that facilitates supercooling of water (Parody-Morreale et al., 1988; Wilson and Leader, 1995; Duman, 2002; Holt, 2003). Promotion of supercooling by the presence of polypeptides has also been reported (Kawahara et al., 1996). However, there have been few studies on the role of proteins in deep supercooling of XPCs. Arora et al. (1992) showed that SDSPAGE protein profiles of xylem tissues in deciduous and evergreen peach trees (Prunus persica) changed seasonally, with a close relationship to the cold hardiness (corresponding to the temperature limit of supercooling in XPCs). Such seasonal changes in protein profiles in xylem have also been reported in Japanese white birch (Takahashi et al., 2005) and larch (K. Morimoto, S. Fujikawa and K. Arakawa, unpublished results). All of these studies suggest that there is a close association between protein accumulation and fluctuation of deep supercooling capability in XPCs. Arora and Wisniewski (1996) showed specific accumulation of a 60-kDa dehydrin protein (PCA60) in peach xylem during winter. PCA60 is distributed in the cytoplasm, plastids and nucleus in deep supercooling XPCs and also in extracellular freezing CPCs (Wisniewski et al., 1999). They also showed that purified PCA60 preserved the in vitro enzymatic activity of freezing-sensitive lactate dehydrogenase after several freeze–thaw cycles in liquid nitrogen (Wisniewski et al., 1999). PCA60 exhibited antifreeze protein activity as evidenced by morphology of ice crystal growth (Wisniewski et al., 1999). PCA60 also exhibited thermal hysteresis activity, although the activity is only 0.06±0.03°C (Wisniewski et al., 1999). However, these authors did suggest that the main role of the dehydrin (PCA60) may be to inhibit intracellular ice nucleation and promote supercooling in XPCs. In red osier dogwood, 24-kDa dehydrin-like proteins accumulate in the xylem during winter and disappear during summer in close association with change of freezing resistance (temperature limit of supercooling) in XPCs (Sarnighausen et al., 2002). It was shown that the expression of these 24-kDa dehydrin-like proteins was induced by

35

dehydration stress caused by short day length in autumn during seasonal cold acclimation (Sarnighausen et al., 2002). Seasonal dehydrin fluctuations have also been observed in the xylem of multiple Populus species and willow (Sauter et al., 1999). It is also notable that many clones of genes encoding dehydrin-like proteins in larch are expressed in the xylem rather than in cortex in association with increased supercooling capability of XPCs during winter (Takata et al., 2007; see also this chapter). Except for PCA60, however, the function of dehydrins as antifreeze proteins, which might have anti-ice nucleation activity or thermal hysteresis, was not examined. Therefore, the roles of many proteins to deep supercooling of XPCs are unknown.

Effects of secondary metabolites as anti-ice nucleation substances Anti-ice nucleation substances may facilitate supercooling of water at very low concentrations by a non-colligative effect (Hoshino et al., 1999) in a different way from supercooling in association with equilibrium melting point depression in general solutes by a colligative effect. Although the number is very few, such anti-ice nucleation substances have been reported (for a list, see Kasuga et al., 2007b, 2008). These substances include antifreeze proteins from insects (Duman, 2002), antifreeze proteins and antifreeze glycoproteins from fish (ParodyMorreale et al., 1988; Wilson and Leader, 1995; Holt, 2003), anti-nucleating proteins from bacteria (Kawahara et al., 1996) and polysaccharides from bacteria (Yamashita et al., 2002). As substances originating from plants, hinokitiol from the needles of Taiwan yellow cypress (Chamaecyparis taiwanensis) (Kawahara et al., 2000) and eugenol from clove (Kawahara and Obata, 1996; Kawahara et al., 1996) are known as anti-ice nucleation substances. Crude extracts from seeds of trees and supernatant liquid from germinating legume seeds also have high levels of activity (Caple et al., 1983). As chemical substances, polyvinyl alcohol and polyglycerol are also known to have anti-ice nucleation activity (Wowk and Fahy, 2002; Holt, 2003).

36

S. Fujikawa et al.

The possibility of the existence of such anti-ice nucleation substances in deep supercooling XPCs in trees has recently been reported. Kasuga et al. (2007b) showed that crude ethanol extracts from the xylem of several boreal hardwood species that contained deep supercooling XPCs exhibited anti-ice nucleation activity. Crude xylem extracts from Japanese white birch, Japanese chestnut (Castanea crenata), katsura tree (Cercidiphyllum japonicum), Siebold’s beech, mulberry (Morus bombycis) and Japanese rowan (Sorbus commixta) promoted supercooling capability of water to 1.0, 1.2, 1.9, 1.3, 0.3 and 1.0°C, respectively, as compared with effects by the same concentration of glucose (Kasuga et al., 2007b). Furthermore, crude xylem extracts from several softwood species, including larch, that also contained deep supercooling XPCs, exhibited anti-ice nucleation activity (Mizuno et al., 2005). The anti-ice nucleation activities were not changed by boiling and/or by proteinase treatment, suggesting that the causative substances of supercooling were not proteinaceous in nature. Among these crude xylem extracts, the anti-ice nucleation activity of crude xylem extracts from katsura tree was further evaluated (Kasuga et al., 2007b). The crude xylem extracts exhibited anti-ice nucleation activity to diverse kinds of heterogeneous ice nucleators, including ice-nucleating bacteria such as Pseudomonas syringae, Xanthomonas campestris and Erwinia ananas, silver iodide and airborne impurities, but the crude xylem extracts did not affect homogeneous ice nucleation (Kasuga et al., 2007b). Antifreeze glycoproteins from fish also have the same effect, showing the presence of anti-ice nucleation activity towards heterogeneous ice nucleators (Parody-Morreale et al., 1988; Wilson and Leader, 1995) but the absence of an effect on homogeneous ice nucleation (Franks et al., 1987). Our current understanding of supercooling in XPCs neglects the presence of heterogeneous ice nucleators in the protoplasts of XPCs (Burke et al., 1976). If this is true, anti-ice nucleation activity in xylem extracts should not influence the supercooling capability of XPCs. However, there is no direct evi-

dence showing the absence of heterogeneous ice nucleators in deep supercooling XPCs. Rather, George and Burke (1977) showed that ice nucleation activity in supercooling XPCs of shagbark hickory (Carya ovata) was intermediate between homogeneous ice nucleation for pure water (Fletcher, 1970) and weak heterogeneous ice nucleation in yeast cells (Rasmussen and MacKenzie, 1972). This finding indicates that although the activity level is very low, some heterogeneous ice nucleators might be present in XPCs. Thus, it is thought that antiice nucleation substances in xylem extracts that inhibit a wide variety of heterogeneous ice nucleators may serve to inhibit intracellular freezing due to heterogeneous ice nucleators in protoplasts of XPCs and may have a function to enhance and/or stabilize supercooling of XPCs. Kasuga et al. (2008) further tried to identify anti-ice nucleation substances from crude xylem extracts of katsura tree. They suggested that the xylem of katsura tree may contain diverse kinds of anti-ice nucleation substances, a surprising result considering the small number of known anti-ice nucleation substances. Separation of the crude xylem extract by EtOAc–water showed a higher level of anti-ice nucleation activity of 2.3°C in the EtOAc fraction, but the water fraction also showed weak activity of 0.9°C, suggesting the presence of different types of anti-ice nucleation substances in the two fractions. Further separation of the EtOAc fraction by silica gel column chromatography showed that all of the fractions (17 fractions) had high levels of anti-ice nucleation activity in the range of 1.4 to 5.8°C. These results also strongly suggested the presence of many kinds of anti-ice nucleation substances in xylem of katsura tree. In order to identify chemical structures of anti-ice nucleation substances with high levels of activity, the fraction with the highest level of activity among the 17 fractions separated by a silica gel column was further analysed by preparative HPLC. HPLC profiles showed numerous peaks corresponding to individual compounds, and four of these peaks showed high levels of anti-ice nucleation activity. By analysis of those four peaks with UV, MS and NMR spectroscopy, four kinds of flavonol

Changes in Deep Supercooling in Trees

glycosides were finally identified as anti-ice nucleation substances. These flavonol glycosides were quercetin-3-O-b-glucoside, kaempferol-7 -O-b-glucoside, 8-methoxykaempferol-3-O-bglucoside and kaempferol-3-O-b-glucoside and they had anti-ice nucleation activity levels in the range of 2.8 to 9.0°C. This is the first attempt to identify anti-ice nucleation substances in tree xylem, especially in xylem containing deep supercooling XPCs. This was also the first demonstration that flavonol glycosides have anti-ice nucleation activity. Compared with activity levels of known antiice nucleation substances, the activity levels of the flavonol glycosides from katsura tree are very high, and the activity level of kaempferol7-O-b-glycoside is the highest among known anti-ice nucleation substances. The presence of flavonoids in the cytoplasm of deep supercooling XPCs in katsura tree was also confirmed by fluorescence staining of flavones and flavonols (Kasuga et al., 2008). From these results, it is thought that the presence of flavonol glycosides with anti-ice nucleation activity in XPCs may have an important role in the deep supercooling phenomenon. In support of this assumption, it has been reported that the amounts of anthocyanins, a kind of flavonoids, increase in xylem tissue of European beech (Fagus sylvatica) during winter (Schmucker, 1947). It has also been shown that the expression level of WXL5 encoding flavonol-3-O-glucosyltransferase in xylem of larch increases significantly during winter, when the supercooling capability of XPCs becomes a maximum, and decreases significantly during summer or by deacclimation of winter twigs, in parallel with the significant reduction of supercooling capability in XPCs (Takata et al., 2007). Furthermore, the expression level of WXL5 was much higher in xylem than in cortical tissues of larch (Takata et al., 2007), suggesting specific accumulation of flavonol glycosides in XPCs. Additional studies will be needed to identify other kinds of anti-ice nucleation substances that may exist in XPCs, and also to analyse seasonal changes in the accumulation of anti-ice nucleation substances as well as their tissue-specific accumulation in order to find their association to deep supercooling of XPCs.

37

Conclusion The temperature limit of supercooling in XPCs is a key factor for the distribution of trees to cold areas. The temperature limit of supercooling of XPCs changes depending on cold acclimation and deacclimation. Isolation of protoplasts in XPCs from the effect of extracellular ice as an isolated water droplet is a prerequisite for deep supercooling of XPCs, but it is suggested that the deep supercooling phenomenon in XPCs is regulated by more complex factors. Although direct evidence has not yet been obtained, change in deep supercooling capability is thought to be closely associated with gene expression as well as protein accumulation as a result of cold acclimation and deacclimation. Intracellular substances are thought to have significant effects on deep supercooling of XPCs. Among intracellular substances, the concentration of soluble sugars is closely related to the supercooling capability in XPCs. Additionally, XPCs contain diverse kinds of anti-ice nucleation substances. The presence of such anti-ice nucleation substances may also have an important role in deep supercooling of XPCs. Studies on molecular mechanisms of deep supercooling of XPCs in trees have just begun. It is expected that the mechanisms responsible for the regulation of deep supercooling of XPCs will be elucidated at the molecular level and that this information will be used for expanding the cultivation areas of trees to colder areas.

Acknowledgements The authors thank Dr E. Fukushi, Mr K. Watanabe and Mr T. Ito for their excellent technical assistance. The present study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (19880002 to J.K.), by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan (17380101 and 20380099 to S.F.) and by a Special Science Grant from Sekisui Integrated Research Co. Ltd. (to S.F.).

38

S. Fujikawa et al.

References Arora, R. and Wisniewski, M.E. (1996) Accumulation of a 60-kD dehydrin protein in peach xylem tissues and its relationship to cold acclimation. HortScience 31, 923–925. Arora, R., Wisniewski, M.E. and Scorza, R. (1992) Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch): I. Seasonal changes in cold hardiness and polypeptides of bark and xylem tissues. Plant Physiology 99, 1562–1568. Ashworth, E.N. and Abeles, F.B. (1984) Freezing behavior of water in small pores and the possible role in the freezing of plant tissues. Plant Physiology 76, 201–204. Ashworth, E.N., Echlin, P., Pearce, R.S. and Hayes, T.L. (1988) Ice formation and tissue response in apple twigs. Plant, Cell and Environment 11, 703–710. Ashworth, E.N., Stirm, V.E. and Volenec, J.J. (1993) Seasonal variations in soluble sugars and starch within woody stems of Cornus sericea L. Tree Physiology 13, 379–388. Becwar, M.R., Rajashekar, C., Bristow, K.J.H. and Burke, M.J. (1981) Deep undercooling of tissue water and winter hardiness limitations in timberline flora. Plant Physiology 68, 111–114. Borner, G.H., Sherrier, D.J., Weimar, T., Michaelson, L.V., Hawkins, N.D., Macaskill, A., Napier, J.A., Beale, M.H., Lilley, K.S. and Dupree, P. (2005) Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiology 137, 104–116. Burke, M.J. and Stushnoff, C. (1979) Frost hardiness: a discussion of possible molecular causes of injury with particular reference to deep supercooling of water. In: Musell, H. and Staples, R.C. (eds) Stress Physiology in Crop Plants. Wiley, New York, New York, pp. 197–225. Burke, M.J., Gusta, L.V., Quamme, H.A., Weiser, C.J. and Li, P.H. (1976) Freezing and injury in plants. Annual Review of Plant Physiology 27, 507–528. Caple, G., Layton, R.G. and McCurdy, S.N. (1983) Biogenic effects in heterogeneous ice nucleation. CryoLetters 4, 59–64. Carpita, N., Sabularse, D., Montezinos, D. and Delmer, D.P. (1979) Determination of the pore size of cell walls of living plant cells. Science 205, 1144–1147. Carystinos, G.D., MacDonald, H.R., Monroy, A.F., Dhindsa, R.S. and Poole R.J. (1995) Vacuolar H+-translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice. Plant Physiology 108, 641–649. Chalker-Scott, L. (1999) Environmental significance of anthocyanins in plant stress responses. Photochemistry and Photobiology 70, 1–9. Chini, A., Grant, J.J., Seki, M., Shinozaki, K. and Loake, G.J. (2004) Drought tolerance established by enhanced expression of the CC-NBS-LRR gene, ADR1, requires salicylic acid, EDS1 and ABI1. The Plant Journal 38, 810–822. Cho, S.H., Hoang, Q.T., Kim, Y.Y., Shin, H.Y., Ok, S.H., Bae, J.M. and Shin, J.S. (2006) Proteome analysis of gametophores identified a metallothionein involved in various abiotic stress responses in Physcomitrella patens. Plant Cell Reports 25, 475–488. Christie, P.J., Alfenito, M.R. and Walbot, V. (1994) Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194, 541–549. Close, T.J. (1997) Dehydrins: a commonality in the response of plants to dehydration and low temperature. Physiologia Plantarum 100, 291–296. Danyluk, J., Perron, A., Houde, M., Limin, A., Fowler, B., Benhamou, N. and Sarhan, F. (1998) Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. The Plant Cell 10, 623–638. Duman, J.G. (2002) The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate. Journal of Comparative Physiology B 172, 163–168. Fletcher, N.H. (1970) The Chemical Physics of Ice. Cambridge University Press, London, pp. 73–103. Franks, J.F., Darlington, J., Schenz, T., Mathias, S.F., Slade, L. and Levine, H. (1987) Antifreeze activity of Antarctic fish glycoprotein and a synthetic polymer. Nature 325, 146–147. Fujikawa, S. (1994) Seasonal ultrastructural alterations in the plasma membrane produced by slow freezing in cortical tissues of mulberry (Morus bombycis Koidz. cv. Goroji). Trees 8, 288–296. Fujikawa, S. and Kuroda, K. (2000) Cryo-scanning electron microscopic study on freezing behavior of xylem ray parenchyma cells in hardwood species. Micron 31, 669–686. Fujikawa, S., Kuroda, K. and Fukazawa, K. (1994) Ultrastructural study of deep supercooling of xylem ray parenchyma cells from Styrax obassia. Micron 25, 241–252.

Changes in Deep Supercooling in Trees

39

Fujikawa, S., Kuroda, K. and Ohtani, J. (1997) Seasonal changes in dehydration tolerance of xylem ray parenchyma cells of Stylax obassia twigs that survive freezing temperatures by deep supercooling. Protoplasma 197, 34–44. Fujikawa, S., Kuroda, K., Jitsuyama, Y., Sano, Y. and Ohtani, J. (1999) Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell walls. Protoplasma 206, 31–40. Fujikawa, S., Ukaji, N., Yamane, K., Nagao, M., Takezawa, D. and Arakawa, K. (2006) Functional role of winter-accumulating proteins from mulberry tree in adaptation to winter-induced stresses. In: Chen, T., Uemura, M. and Fujikawa, S. (eds) Cold Hardiness in Plants: Molecular Genetics, Cell Biology and Physiology. CAB International, Wallingford, UK, pp. 181–202. George, M.F. (1983) Freezing avoidance by deep supercooling in woody plant xylem: preliminary data on the importance of cell wall porosity. In: Randall, D.D., Blevins, D.G., Larson, R.L. and Rapp, B.J. (eds) Current Topics in Plant Biochemistry and Physiology. University of Missouri Press, Columbia, Missouri, pp. 84–95. George, M.F. and Burke, M.J. (1977) Cold hardiness and deep supercooling in xylem of shagbark hickory. Plant Physiology 59, 319–325. George, M.F., Burke, M.J., Pellett, H.M. and Johnson, A.G. (1974) Low temperature exotherms and woody plant distribution. HortScience 9, 519–522. George, M.F., Becwar, M.R. and Burke, M.J. (1982) Freezing avoidance by deep undercooling of tissue water in winter-hardy plants. Cryobiology 19, 628–639. Grant, J.J., Chini, A., Basu, D. and Loake, G.J. (2003) Targeted activation tagging of the Arabidopsis NBS-LRR gene, ADR1, conveys resistance to virulent pathogens. Molecular Plant–Microbe Interactions 16, 669–680. Gusta, L.V., Tyler, N.J. and Chen, T.H.H. (1983) Deep undercooling in woody taxa growing north of the –40°C isotherm. Plant Physiology 72, 122–128. Guy, C.L. (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 41, 187–223. Holt, C.B. (2003) The effect of antifreeze proteins and poly(vinyl alcohol) on the nucleation of ice: a preliminary study. CryoLetters 24, 323–330. Hong, S.G. and Sucoff, E. (1982a) Rapid increase in deep supercooling of xylem parenchyma. Plant Physiology 69, 697–700. Hong, S.G. and Sucoff, E. (1982b) Temperature effects on acclimation and deacclimation of supercooling in apple xylem. In: Li, P.H. and Sakai, A. (eds) Plant Cold Hardiness and Freezing Stress: Mechanisms and Crop Implications, Vol. 2. Academic Press, New York, New York, pp. 341–356. Hoshino, T., Odaira, M., Yoshida, M. and Tsuda, S. (1999) Physiological and biochemical significance of antifreeze substances in plants. Journal of Plant Research 112, 255–261. Houde, M., Daniel, C., Lachapelle, M., Allard, F., Laliberte, S. and Sarhan, F. (1995) Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. The Plant Journal 8, 583–593. Hughes, M.A. and Dunn, M.A. (1996) The molecular biology of plant acclimation to low temperature. Journal of Experimental Botany 47, 291–305. Kasuga, J., Mizuno, K., Miyaji, N., Arakawa, K. and Fujikawa, S. (2006) Role of intracellular contents to facilitate supercooling capability in beech (Fagus crenata) xylem parenchyma cells. CryoLetters 27, 305–310. Kasuga, J., Arakawa, K. and Fujikawa, S. (2007a) High accumulation of soluble sugars in deep supercooling Japanese white birch xylem parenchyma cells. New Phytologist 174, 569–579. Kasuga, J., Mizuno, K., Arakawa, K. and Fujikawa, S. (2007b) Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells. Cryobiology 55, 305–314. Kasuga, J., Hashidoko, Y., Nishioka, A., Yoshiba, M., Arakawa, K. and Fujikawa, S. (2008) Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity. Plant, Cell and Environment 31, 1335–1348. Kawahara, H. and Obata, H. (1996) Identification of a compound in spices inhibiting the ice-nucleating activity of Erwinia uredovora KUIN-3. Journal of Antibacterial and Antifungal Agents 24, 95–100. Kawahara, H., Nagae, I. and Obata, H. (1996) Purification and characterization of a new anti-nucleating protein isolated from Acinetobacter calcoaceticus KINI-1. Biocontrol Science 1, 11–17. Kawahara, H., Masuda, K. and Obata, H. (2000) Identification of a compound in Chamaecyparis taiwanensis inhibiting the ice-nucleation activity of Pseudomonas fluorescens KUIN-1. Bioscience, Biotechnology and Biochemistry 64, 2651–2656.

40

S. Fujikawa et al.

Kim, H.J., Kim, Y.K., Park, J.Y. and Kim, J. (2002) Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. The Plant Journal 29, 693–704. Kuroda, K., Ohtani, J. and Fujikawa, S. (1997) Supercooling of xylem parenchyma cells in tropical and subtropical hardwood species. Trees 12, 97–106. Kuroda, K., Kasuga, J., Arakawa, K. and Fujikawa, S. (2003) Xylem ray parenchyma cells in boreal hardwood species respond to subfreezing temperatures by deep supercooling that is accompanied by incomplete desiccation. Plant Physiology 131, 736–744. Lawand, S., Dorne, A.J., Long, D., Coupland, G., Mache, R. and Carol, P. (2002) Arabidopsis A BOUT DE SOUFFLE, which is homologous with mammalian carnitine acyl carrier, is required for postembryonic growth in the light. The Plant Cell 14, 2161–2173. Lo Piero, A.R., Puglisi, I., Rapisarda, P. and Petrone, G. (2005) Anthocyanins accumulation and related gene expression in red orange fruit induced by low temperature storage. Journal of Agricultural and Food Chemistry 53, 9083–9088. MacKenzie, A.P. (1977) Non-equilibrium freezing behaviour of aqueous systems. Philosophical Transactions of the Royal Society of London B 278, 167–189. Malone, S.R. and Ashworth, E.N. (1991) Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques. Plant Physiology 95, 871–881. Maskin, L., Gubesblat, G.E., Moreno, J.E., Carrari, F.O., Frankel, N., Sambade, A., Rossi, M. and Iusem, N.D. (2001) Differential expression of the members of the Asr gene family in tomato (Lycopersicon esculentum). Plant Science 161, 739–746. Meyers, B.C., Morgante, M. and Michelmore, R.W. (2002) TIR-X and TIR-NBS proteins: two new families related to disease resistance TIR-NBS-LRR proteins encoded in Arabidopsis and other plant genomes. The Plant Journal 32, 77–92. Mizuno, K., Kasuga, J., Arakawa, K. and Fujikawa, S. (2005) Accumulation of anti-ice-nucleating substances in conifer xylem. Cryobiology and Cryotechnology 51, 111–114. Monroy, A.F., Sangwan, V. and Dhindsa, R.S. (1998) Low temperature signal transduction during cold acclimation: protein phosphatase 2A as an early target for cold-inactivation. The Plant Journal 13, 653–660. Nadimpalli, R., Yalpani, N., Johal, G.S. and Simmons, C.R. (2000) Prohibitins, stomatins, and plant disease response genes compose a protein superfamily that controls cell proliferation, ion channel regulation, and death. Journal of Biological Chemistry 275, 29579–29586. Nagao, M., Arakawa, K., Takezawa, D. and Fujikawa, S. (2008) Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells. Planta 227, 477–489. Parody-Morreale, A., Murphy, K.P., Di Cera, E., Fall, R., DeVries, A.L. and Gill, S.J. (1988) Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins. Nature 333, 782–783. Pearce, R.S. (1988) Extracellular ice and cell shape in frost-stressed cereal leaves: a low temperature scanningelectron-microscopy study. Planta 175, 313–324. Pennycooke, J.C., Jones, M.L. and Stushnoff, C. (2003) Down-regulating a-galactosidase enhances freezing tolerance in transgenic petunia. Plant Physiology 133, 901–909. Piispanen, R. and Saranpää, P. (2001) Variation of non-structural carbohydrates in silver birch (Betula pendula Roth) wood. Trees 15, 444–451. Qi, Q., Huang, Y.F., Cutler, A.J., Abrams, S.R. and Taylor, D.C. (2003) Molecular and biochemical characterization of an aminoalcoholphosphotransferase (AAPT1) from Brassica napus: effects of low temperature and abscisic acid treatments on AAPT expression in Arabidopsis plants and effects of over-expression of BnAAPT1 in transgenic Arabidopsis. Planta 217, 547–558. Quamme, H.A. (1991) Application of thermal analysis to breeding fruit crops for increased cold hardiness. HortScience 26, 513–517. Quamme, H., Weiser, C.J. and Stushnoff, C. (1973) The mechanism of freezing injury in xylem of winter apple twigs. Plant Physiology 51, 273–277. Quamme, H.A., Chen, P.M. and Gusta, L.V. (1982) Relationship of deep supercooling and dehydration resistance to freezing injury in dormant stem tissues of ‘Starkrimson Delicious’ apple and ‘Siberian C’ peach. Journal of the American Society for Horticulture Science 107, 299–304. Rasmussen, D.H. and MacKenzie, A.P. (1972) Effect of solute on ice–solution interfacial free energy: calculation from measured homogeneous nucleation temperatures. In: Jellnek, H.H.G. (ed) Water Structure at the Water Polymer Interface. Plenum Press, New York, New York, pp. 126–145.

Changes in Deep Supercooling in Trees

41

Reid, S.J. and Ross, G.S. (1997) Up-regulation of two cDNA clones encoding metallothionein-like proteins in apple fruit during cool storage. Physiologia Plantarum 100, 183–189. Sabehat, A., Lurie, S. and Weiss, D. (1998) Expression of small heat-shock proteins at low temperatures. A possible role in protecting against chilling injuries. Plant Physiology 117, 651–658. Sakai, A. and Larcher, W. (1987) Frost Survival of Plants: Responses and Adaptation to Freezing Stress. Springer-Verlag, Berlin. Sarnighausen, E., Karlson, D. and Ashworth, E. (2002) Seasonal regulation of a 24-kDa protein from red-osier dogwood (Cornus sericea) xylem. Tree Physiology 22, 423–430. Sauter, J.J. and Kloth, S. (1987) Changes in carbohydrates and ultrastructure in xylem ray cells of Populus in response to chilling. Protoplasma 137, 45–55. Sauter, J.J. and Wellenkamp, S. (1998) Seasonal changes in content of starch, protein and sugars in the twig wood of Salix caprea L. Horzforschung 52, 255–262. Sauter, J.J., Wisniewski, M. and Witt, W. (1996) Interrelationships between ultrastructure, sugar levels, and frost hardiness of ray parenchyma cells during frost acclimation and deacclimation in poplar (Poplus×canadensis Moench ) wood. Journal of Plant Physiology 149, 451–461. Sauter, J.J., Westphal, S. and Wisniewski, M. (1999) Immunological identification of dehydrin-related proteins in the wood of five species of Populus and in Salix caprea L. Journal of Plant Physiology 154, 781–788. Schmucker, T. (1947) Anthocyan im Holz der Rotbuche. Naturwissenschaften 34, 91. Schneider, A., Salamini, F. and Gebhardt, C. (1997) Expression patterns and promoter activity of the coldregulated gene ci21A of potato. Plant Physiology 113, 335–345. Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., Kamiya, A., Nakajima, M., Enju, A., Sakurai, T., Satou, M., Akiyama, K., Taji, T., Yamaguchi-Shinozaki, K., Carninci, P., Kawai, J., Hayashizaki, Y. and Shinozaki, K. (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal 31, 279–292. Steponkus, P.L. (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology 35, 543–584. Taji, T., Ohsumi, C., Iuchi, S., Seki, M., Kasuga, M., Kobayashi, M., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. The Plant Journal 29, 417–426. Takahashi, H., Arakawa, K. and Fujikawa S. (2005) Study on proteins related to deep supercooling ability of xylem tissues of Fagus crenata L. Cryobiology and Cryotechnology 51, 105–109. Takata, N., Kasuga, J., Takezawa, D., Arakawa, K. and Fujikawa, S. (2007) Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi). Journal of Experimental Botany 58, 3731–3742. Tanaka, S., Nagao, M., Funada, R., Fujikawa, S. and Arakawa, K. (2003) The relation between small diameter capillaries in cell wall and deep supercooling in xylem ray parenchyma cells of woody plants. Cryobiology and Cryotechnology 49, 209–213. Thomashow, M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50, 571–599. Török, Z., Goloubinoff, P., Horváth, I., Tsvetkova, N.M., Glatz, A., Balogh, G., Varvasovszki, V., Los, D.A., Vierling, E., Crowe, J.H. and Vígh, L. (2001) Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding. Proceedings of the National Academy of Sciences USA 98, 3098–3103. Ukaji, N., Kuwabara, C., Takezawa, D., Arakawa, K., Yoshida, S. and Fujikawa, S. (1999) Accumulation of small heat-shock protein homologs in the endoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation. Plant Physiology 120, 481–490. van Berkel, J., Salamini, F. and Gebhardt, C. (1994) Transcripts accumulating during cold storage of potato (Solanum tuberosum L.) tubers are sequence related to stress-responsive genes. Plant Physiology 104, 445–452. van Montfort, R., Slingsby, C. and Vierling, E. (2001) Structure and function of the small heat shock protein/acrystallin family of molecular chaperones. Advances in Protein Chemistry 59, 105–156. Wilson, P.W. and Leader, J.P. (1995) Stabilization of supercooled fluids by thermal hysteresis proteins. Biophysical Journal 68, 2098–2107. Winkel-Shirley, B. (2002) Biosynthesis of flavonoids and effects of stress. Current Opinion in Plant Biology 5, 218–223.

42

S. Fujikawa et al.

Wisniewski, M. (1995) Deep supercooling and the role of cell wall structure. In: Lee, R.L. Jr, Warren, G..J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 163–182. Wisniewski, M., Webb, R., Balsamo, R., Close, T.J., Yu, X.M. and Griffith, M. (1999) Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiologia Plantarum 105, 600–608. Wisniewski, M., Bassett, C., Norelli, J., Macarisin, D., Artlip, T., Gasic, K. and Korban, S. (2008) Expressed sequence tag analysis of the response of apple (Malus×domestica ‘Royal Gala’) to low temperature and water stress. Physiologia Plantarum 133, 298–317. Wowk, B. and Fahy, G.M. (2002) Inhibition of bacterial ice nucleation by polyglycerol polymers. Cryobiology 44, 14–23. Yamada, T., Kuroda, K., Jitsuyama, Y., Takezawa, D., Arakawa, K. and Fujikawa, S. (2002) Roles of the plasma membrane and cell wall in the responses of plant cells to freezing. Planta 215, 770–778. Yamashita, Y., Kawahara, H. and Obata, H. (2002) Identification of a novel anti-ice-nucleating polysaccharide from Bacillus thuringiensis YY529. Bioscience, Biotechnology and Biochemistry 66, 948–954. Yang, C.Y., Chen, Y.C., Jauh, G.Y. and Wang, C.S. (2005) A lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis. Plant Physiology 139, 836–846.

5

Plant Cold-shock Domain Proteins: on the Tip of an Iceberg

D. Karlson, K. Nakaminami, K. Thompson, Y. Yang, V. Chaikam and P. Mulinti

Bacterial Cold-shock Proteins The cold-shock domain (CSD) is an ancient molecule proposed to be present prior to the origin of single-cell life (Graumann and Marahiel, 1998) and is one of the most evolutionarily conserved nucleic acid-binding domains within prokaryotes and eukaryotes (Wolffe et al., 1992; Wolffe, 1994; Graumann and Marahiel, 1998). Specifically, the CSD has been identified in organisms ranging from Archaea to higher eukaryotes, including man (Sommerville, 1999; Saunders et al., 2003). Despite the high conservation of the CSD among prokaryotes and eukaryotes, the CSD has evolved multiple and diverse functions in higher organisms. In prokaryotes, cold-shock proteins (Csps) are small proteins that consist solely of a single CSD and they have been extensively described in more than 50 Gram-negative and Gram-positive bacterial species (Graumann and Marahiel, 1998). Bacterial Csps are often present in multiple copies comprising small gene families (Phadtare and Inouye, 1999). Bacterial Csps were named accordingly for their up-regulation in response to lowtemperature downshifts. Escherichia coli contains nine Csps (Yamanaka et al., 1998; Wang et al., 1999); however, its individual members are differentially regulated in response to lowtemperature stress (Yamanaka et al., 1998).

The cold-induced Csps include CspA, CspB, CspG and CspI. The other five members are involved in various physiological activities such as cell division and response to low nutrient levels (Graumann and Marahiel, 1998). CspA, the most predominant Csp, accumulates up to 10% of total proteins subsequent to low-temperature exposure (Goldstein et al., 1990). As described below, the functional significance of bacterial Csps to low-temperature stress is directly related to RNA behaviour at low temperature and their ability to bind and melt nucleic acids and maintain them in single-stranded confirmation. RNA molecules typically form stable secondary structures in response to low temperature (Polissi et al., 2003). Due to the design of prokaryotic transcriptional machinery, coldinduced RNA secondary structure may impose premature transcription termination. In 1997, Jiang et al. determined that CspA functions as an RNA chaperone inhibiting RNA secondary structures at low temperatures. At colder temperatures, RNA tends towards secondary structures because they are more thermodynamically favoured (Graumann et al., 1996). Thus, CspA enhances translation at low temperature through the elimination of stabilized RNA secondary structures (Jiang et al., 1997). Bae et al. (2000) subsequently demonstrated that CspC and CspE also possess in vitro and in vivo transcription anti-termination activity.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

43

44

D. Karlson et al.

Eukaryotic Cold-shock Domain Proteins Eukaryotic cold-shock domain proteins were initially named ‘Y-box proteins’ for their preferential binding to the Y-box sequence (CTGATTGGYYAA) of major histocompatibility complex (MHC) class II promoters through their CSD (Didier et al., 1988). The Y-box structure is characterized as having a variable N-terminal domain, a CSD and a C-terminal tail domain with interspersed basic and aromatic islands (Kohno et al., 2003). Human Y-box proteins are involved in many steps of RNA metabolism such as transcription (Ladomery and Sommerville, 1995), pre-mRNA splicing (Stickeler et al., 2001), translational control (Evdokimova et al., 1998; Sommerville, 1999), mRNA stability (Evdokimova et al., 2001) and detection of RNA damage (Hayakawa et al., 2002), among others. In Xenopus oocytes, FRGY2 complexes with RNA and functions as a core component of mRNPs (messenger ribonucleoprotein complexes) and acts to control translation by masking RNA as a precise means to control developmental timing via translational repression (Ranjan et al., 1993; Bouvet and Wolffe, 1994). LIN-28 is another well-studied cold-shock domain protein from the soil nematode Caenorhabditis elegans that is involved with post-transcriptional regulation and is critically important for timing of developmental transitions (Moss and Tang, 2003). Unlike bacteria, functional studies of animal Y-box proteins relating to low-temperature stress are lacking. To our knowledge, only one study has linked a eukaryotic animal cold-shock domain protein to cold stress. Specifically, chicken YB-1 has been shown to reinstate the growth of cells at low temperature (Matsumoto et al., 2005). Despite this study, the vast majority of these aforementioned functions are not directly related to cold-shock response or cold adaptation.

Plant Cold-shock Domain Proteins A previous report documented the widespread occurrence of plant cold-shock domain proteins (CSPs) across multiple genera representing lower plants, monocots and dicots (Karlson and

Imai, 2003). In our current studies, we used current publicly available EST sequence data and documented more than 121 unique CSD sequences from 74 different organisms representing 56 genera. A major difference between plant and bacterial cold-shock proteins is the presence of various auxiliary C-terminal domains in plant CSPs. The domains present in the C-terminus include glycine-rich regions, retroviral-like zinc fingers (Kingsley and Palis, 1994; Karlson and Imai, 2003), RG-like repeats, RRM (RNA recognition motif) domains (Mussgnug et al., 2005) and additional CSDs. In the phylogenetic analysis, CSPs were found in all major evolutionary clades ranging from single-cell photosynthetic organisms, ferns, mosses, monocots, angiosperms and woody plants. Thus, we hypothesize that CSPs have been conserved in all plants. In contrast to the vast amount of information known about prokaryotic and other eukaryotic cold-shock domain proteins, very little is known regarding their function in plants. In the first functional characterization of a plant CSP, a gene from wheat (WCSP1), was shown to be up-regulated in response to low temperature (Karlson et al., 2002). Similar observations have subsequently been described in plants capable of cold acclimation: Arabidopsis (Karlson and Imai, 2003; Fusaro et al., 2007; Kim et al., 2007) and the moss Physcomitrella patens (K. Thompson, Y. Hiwatashi, M. Hasebe and D. Karlson, unpublished results). Interestingly, two rice CSPs show a very rapid, marginal and transient response to low temperature. Western blot analysis confirmed that both proteins remain constitutively expressed during low-temperature treatments. These data contrast sharply with those obtained from previous studies performed in winter wheat (Chaikam and Karlson, 2008). On the protein level, WCSP1 accumulates to very high levels subsequent to cold treatment (Karlson et al., 2002). Studies with recombinant WCSP1 confirmed that it binds ssDNA, dsDNA and RNA ribohomopolymers (Karlson et al., 2002) and this activity is stable to boiling (Nakaminami et al., 2005). Collective observations gained from in vitro DNA binding and DNA melting assays, in vivo functional complementation of bacterial mutants and subcellular localization suggest that WCSP1 is

Plant Cold-shock Domain Proteins

45

capable of functioning in a similar manner to bacterial Csps (Nakaminami et al., 2006). Like most plant CSPs, WCSP1 has an N-terminal CSD and three zinc fingers that are interspersed between glycine-rich regions (Karlson et al., 2002). The quadruple deletion mutant (cspA, cspB, cspE and cspG) BX04 line of E. coli is sensitive to low temperature and serves as a useful in vivo system for identifying proteins involved in improving growth under cold stress (Xia et al., 2001). Using this mutant, WCSP1 complemented the cold-sensitive phenotype of these cells. Usage of another bacterial strain (RL211) confirmed in vivo transcription anti-termination activity for WCSP1 (Karlson et al., 2002). Like WCSP1, Arabidopsis AtCSP2/AtGRP2 also binds nucleic acids (Fusaro et al., 2007). Arabidopsis AtCSP1/CSDP1 (At4g36020) and AtCSP2/AtGRP2 are both capable of improving the growth of BX04 E. coli cells at cold temperatures and exhibit RNA chaperone activity (Kim et al., 2007; Sasaki et al., 2007). Transient expression analysis of WCSP1 confirmed its localization to the nucleus and endoplasmic reticulum (Nakaminami et al., 2006). In Arabidopsis, nucleolar and cytosolic localization was observed for AtCSP2/AtGRP2 (Sasaki et al., 2007). Using in situ hybridization, AtCSP2/AtGRP2 transcripts were shown to accumulate in apical meristems and root tips in Arabidopsis plants (Fusaro et al., 2007). In later stages of development, localization was observed in reproductive tissues such as ovules and pollen, and finally in the embryos of Arabidopsis seeds (Fusaro et al., 2007; Nakaminami et al., 2009). Usage of RNAi lines confirmed that AtCSP2/ AtGRP2 has roles in flowering time as well as flower and seed development (Fusaro et al., 2007). NAB1, a CSP from Chlamydomonas reinhardtii, localizes to the cytoplasm and masks RNA similar to the FRGY2 protein from Xenopus (Mussgnug et al., 2005).

function in plants. In 2003, cold-shock domain proteins were first described to be widespread and conserved within plants (Karlson and Imai, 2003). Since this initial study, we have characterized 121 unique CSPs from lower plants through higher plants and identified various combinations of domain structures within them. Specifically, we have analysed CSPs from Haptophyta, Bacillariophyta, Chlorophyta, Charophyta, Bryophyta, Lycophyta, Pterophyta and higher plants. One of the most striking findings was the diversity of C-terminal auxiliary domain structures. As previously mentioned, bacterial Csps consist solely of a single CSD. In Haptophytes, we identified cold-shock domain proteins consisting of two tandem CSDs. Interestingly, a Chlorophyte cold-shock domain protein consists of an N-terminal CSD and a C-terminal RRM (Mussgnug et al., 2005). The most striking observation was the occurrence of retroviral-like CCHC zinc fingers (CX2CX4HX4C) in CSPs from land plants. This conserved region is identical to those found within retroviral nucleocapsid proteins (Coleman, 1992; Dannull et al., 1994). The well-studied C. elegans (LIN-28) protein also contains the unique combination of an N-terminal CSD and retroviral-like zinc fingers in its C-terminus. In known plant CSPs, the number of C-terminal CCHC zinc fingers is highly variable and ranges from 1 to as many as 7. The occurrence of a single C-terminal CCHC zinc finger in Mesostigma viride is very intriguing. This aquatic organism is argued to be the closest living relative to land plants (Lemieux et al., 2007). Since this was the only aquatic photosynthetic organism which we found to contain a C-terminal zinc finger, it is tempting to speculate that various combinations of zinc fingers have facilitated diversified function of land plant CSPs and may affect their preference for binding and interacting with specific nucleic acids.

Evolution of Plant Cold-shock Domain Proteins

Post-translational Modification of Plant Cold-shock Domain Proteins

Despite the vast amount of research on coldshock proteins in bacteria and other eukaryotes, very little is known about their evolution and

In a recent multi-sequence analysis of plant CSPs, we noticed conservation of a motif (FRSL) located between β-sheets 3 and 4 of the

46

D. Karlson et al.

consensus plant CSD sequence. This is of outstanding interest since a similar location within the CSD of human YB-1 protein contains a critical serine residue which is a target site of the well-studied AKT kinase. YB-1 has been broadly studied in relation to human cancer and mutation of this single residue suppresses cancer tumour cell growth (Sutherland et al., 2005). Out of 121 unique plant CSD sequences, only two exceptions occurred with a nonphosphorylatable residue in this motif. Two other conserved regions within the CSD were identified as putative phosphorylation targets. In silico analysis with the NETPHOS program confirmed with high probability that the FRSL regions exhibit the greatest probability for phosphorylation. Using purified recombinant AtCSPs and an in vitro phosphorylation assay with whole cell extracts, we confirmed that all AtCSPs are phosphorylated (data not shown). In the future, it will be of great interest to identify the protein kinase(s) and their preferential target residues within the CSD. Once the critical residues are confirmed, future site-directed mutagenesis of these residues may enable us to decipher the in vivo functional role for the phosphorylation of the CSD in planta. Conserved sites for sumoylation were also identified within our multi-sequence alignment. Sumoylation is a post-translation modification in which a SUMO (small ubiquitin-like modifier) protein is covalently conjugated to proteins by an E3 ligase. Given the recently described functional importance of sumoylation in the adaptation of plants to stress and plant development (Kurepa et al., 2003; Lois et al., 2003; Murtas et al., 2003; Catala et al., 2007; Miura et al., 2007; Jin et al., 2008), it will be important to understand the role of sumoylation in the modification and activity of plant CSPs. Using rice as a model system, we confirmed that both of its CSPs are capable of being sumoylated. Recombinant rice CSPs were purified and added to an in vitro sumoylation reaction as previously described (Miura et al., 2007) and sumoylated products were detected only in reactions containing SUMO components in the reaction mixture (Fig. 5.1). Future functional investigations focused on sumoylation and phosphorylation will increase our understanding of the overall biological role and function of plant CSPs.

SUMO components

OsCSP1 + –

OsCSP2 – +

*

*

OsCSPs

Fig. 5.1. In vitro sumoylation assay of Oryza sativa (rice) cold-shock domain proteins (OsCSPs). Recombinant OsCSPs were purified as GST (glutathione S-transferase) fusion proteins and added to an in vitro sumoylation assay as previously described (Miura et al., 2007). Sumoylated proteins were detected for both rice CSP proteins and are indicated by asterisks. Note that sumoylated products were not detected in negative controls lacking SUMO (small ubiquitin-like modifier) components within the in vitro reaction mixture.

Plant Cold-shock Domain Proteins – Gene Expression Analyses We characterized the expression of the Arabidopsis thaliana CSP gene family (AtCSPs) in response to low-temperature stress and stages of plant development. The four genes are described as: AtCSP1/CSDP1 (At4g36020), AtCSP2/CSDP2/AtGRP2 (At4g38680), AtCSP3 (At2g17870) and AtCSP4/AtGRP2b (At 2g21060). On an amino acid sequence level, AtCSP2 and AtCSP4 share 72% amino acid identity. Both contain an N-terminal CSD and two C-terminal CCHC retroviral-like zinc fingers interspersed within glycine-rich regions. AtCSP1 and AtCSP3 share 59% identity on the amino acid level. These proteins differ from AtCSP2 and AtCSP4 in that they contain seven C-terminal CCHC retroviral-like zinc fingers.

Stress-related expression of AtCSPs Quantitative real-time PCR (qRT-PCR) confirmed that all AtCSPs respond positively to low-temperature stress within a 48 h testing period. In a recent study, we confirmed that two CSP genes from the chilling-sensitive rice model system exhibit a marginal and very transient cold response which peaks within 30 min and 1 h subsequent to low-temperature exposure in roots and shoots, respectively (Chaikam and Karlson, 2008). These data contrast

Plant Cold-shock Domain Proteins

sharply with those obtained from winter wheat (Karlson et al., 2002) and those previously described for Arabidopsis (Karlson and Imai, 2003; Fusaro et al., 2007; Kim et al., 2007). Using the extremely freeze-tolerant red osier dogwood model system, we confirmed for the first time that woody plant CSPs are seasonally regulated (Fig. 5.2) and accumulate during periods of maximum freezing tolerance (Karlson et al., 2003). We performed the first study aimed to qualify the relationship of plant CSP gene regulation to the well-studied CBF/DREB transcription factors (Stockinger et al., 1997; Gilmour et al., 1998; Liu et al., 1998) by using transgenic Arabidopsis lines ectopically overexpressing each individual CBF/DREB gene. In wild-type and vector control backgrounds, AtCSP3 gene expression was maximally induced 12 h subsequent to low-temperature stress. Interestingly, in the CBF3/DREB1A overexpression background, its highest gene expression level was detected prior to the shift to low temperature. These data strongly suggest that AtCSP3 transcription is affected by the CBF3/DREB1A transcription factor. Due to the functional data correlating CBF3/DREB1A to low-temperature stress adaptation (Gilmour et al., 2000; Kasuga et al., 2004; Novillo et al., 2004; Oh et al., 2005), these correlative data suggest that AtCSP3 may function in the adaptation to lowtemperature stress. Analysis of abscisic acid (ABA) mutants (aba1–4, abi1–1 and abi1–2) indicated that both AtCSP3 and AtCSP4 have an altered

15 14 13 12 11 10 9 10 11 12 13 14

Fig. 5.2. Seasonal regulation of a cold-shock domain protein (CSP)-like protein in red osier dogwood. Total proteins were extracted from xylem tissue of field-grown red osier dogwood at each incremental change in day length as previously described (Karlson et al., 2003). Western blot analysis was performed to detect CSP-like proteins in dogwood. A prominent single band was detected and found to accumulate during periods of the year when red osier dogwood exhibits maximum freezing tolerance. These data represent the first reported seasonal regulation of a CSP-like protein in woody plants.

47

expression pattern in the ABA-deficient mutant after exposure to 4°C for 24 h. In contrast, repression was not observed in ABA-sensitive lines. These data suggest that gene expression for both AtCSP3 and AtCSP4 is affected by an ABA-dependent signal transduction pathway. Since AtCSP3 expression is affected by CBF3/DREB1A, which regulates gene expression in an ABA-independent manner (Medina et al., 1999; Oh et al., 2005), these data suggest that AtCSP3 expression is at least partially affected by both ABA-dependent and ABA-independent pathways.

Developmental-related expression of AtCSPs We have been interested to qualify the relationship of AtCSPs to plant development and placed particular emphasis on flowering and embryogenesis. Multiple RNA-binding proteins play an important role in floral development and affect flowering by functioning on the post-transcriptional level by affecting transcripts of floweringspecific genes (Macknight et al., 1997; Page et al., 1999; Lim et al., 2004; Simpson et al., 2004; Quesada et al., 2005). We performed genome-wide expression analyses for AtCSPs with the GENEVESTIGATOR microarray analysis software (Zimmermann et al., 2004). Expression patterns revealed tissue specificity and relationships to developmental transitions. We performed subsequent qRT-PCR analyses to validate the microarray observations. AtCSP expression was characterized from cotyledons, shoots, roots, stems, rosette and cauline leaves, shoot apices, floral buds, open flowers and siliques. All AtCSPs were enriched in shoot apices and siliques. Thus, we performed focused experiments designed to further characterize the relationship of AtCSPs to flowering and embryogenesis on both the transcript and protein level (Nakaminami et al., 2009). We performed a floral transition study where shoot apices were harvested from various stages of vegetative development. Specifically, shoot apices were harvested from 1- and 2-week-old seedlings and from 3-week-old seedlings, before and after bolting. Rosette leaves were harvested from identical plants to provide a relative comparison for transcript

48

D. Karlson et al.

and protein levels in leaf tissue. On both the protein and transcript levels, AtCSP transcripts were clearly enriched in shoot apices. On the protein level, their accumulation peaked just prior to bolting and reduced slightly thereafter. To investigate an association to floral induction, wild-type plants of the Ler (Landsberg erecta) ecotype were grown under short days for 30 days and were subsequently shifted to inductive long day conditions. Analysis with qRT-PCR revealed a clear and progressive induction of gene expression 3, 5 and 7 days subsequent to the shift to long days. Similar studies were performed with two flowering time mutants (co-2 and ft-2) in the Ler background. In both cases, expression of AtCSPs was suppressed and was never induced subsequent to the shift to inductive long days. Collectively, these data strongly suggest that AtCSPs are related to the induction of flowering in Arabidopsis. We next investigated the expression patterns of AtCSPs throughout the stages of embryo development in the Col (Columbia ecotype) background. AtCSP gene expression varied throughout the various developmental stages. Gene expression levels tended to be highest in the earliest stages of silique development. These data were corroborated with subsequent protein blot analyses. In situ hybridization for the highly expressed AtCSP2 and AtCSP4 confirmed specific localization to developing embryos. Due to the clear association to developing embryos, we were interested to pursue potential embryo-related mechanisms of gene regulation for AtCSPs.

Transcriptional regulation of AtCSPs We identified a cis-element matching the preferential binding site for AGL15 (CArG motif with a longer A/T-rich core) within AtCSP promoter regions. AGL15 is a well-studied MADS-domain transcription factor that is predominantly expressed in developing embryos (Heck et al., 1995) and has a postembryonic accumulation in shoot apices of Arabidopsis (Fernandez et al., 2000). We employed chromatin immunoprecipitation with AGL15specific antibodies to confirm if the promoter regions of both AtCSP2 and AtCSP4 contain-

ing the consensus CArG motifs are bound by AGL15. To substantiate the biological significance of this interaction, we performed a series of qRT-PCR analyses and monitored AtCSP2 and AtCSP4 transcripts in both knockout and overexpression mutant backgrounds of AGL15. Relative to wild-type controls, AtCSP2 and AtCSP4 transcripts were altered in the mutant backgrounds. Thus, AGL15 appears to affect the transcription of AtCSP2 and AtCSP4 in planta. Publicly available microarray data from GENEVESTIGATOR (https://www.genevestigator. ethz.ch/) are in good accordance with this hypothesis. Given that AGL15 functions as a transcriptional repressor, the inverse relationship of AtCSP4 expression relative to AGL15 strongly supports this interaction (Fig. 5.3).

DNA polymorphic variation of promoter region for AtCSP4 between Arabidopsis ecotypes In a previous comparative genomic analysis, Ler and Col ecotypes were studied with microarray analysis as a means to assess DNA polymorphisms (Schmid et al., 2003). Hybridization of labelled genomic DNA on to AtGenome 1 arrays confirmed that less than 1% of all loci contained six or more single-feature polymorphisms and these were predicted to be highly polymorphic or deleted in Ler. Interestingly, AtCSP4 (At2g21060) was identified among the highly polymorphic loci. In relative comparison to the Col ecotype, we used genespecific qRT-PCR analysis to confirm that Ler AtCSP4 expression is lower by 1000-fold. We subsequently cloned the entire Ler AtCSP4 genomic locus and performed comparative sequence analysis to the Col ecotype. Interestingly, several nucleotide discrepancies were found within the AtCSP4 exon; however, none of these codon changes resulted in a different translatable amino acid. The 5′ and 3′ UTR regions of AtCSP4 contained only six single altered nucleotides, whereas a total of 47 polymorphisms were detected within its promoter region. Several interesting putative regulatory cis-elements differed between Ler and Col. Two specific cis-element regions of high interest that are not present in the Ler promoter sequence are the site II element

Plant Cold-shock Domain Proteins

49

7000

Relative expression level

6000 5000 4000 3000 2000 1000 0 3

4

5 6 7 8 Embryo development stag e

9

10

Fig. 5.3. Inverse relationship of AGL15 ( ) and AtCSP4 ( ) gene expression. Using chromatin immunoprecipitation, we confirmed an in vivo interaction of AGL15 on the promoter of AtCSP4. We performed comparative expression analysis of these two genes in order to gain insight into the biological relevance of this interaction. Analysis of publicly available microarray data from GENEVESTIGATOR revealed a striking inverse-related expression pattern for both genes of interest through stages of embryo development. Note the dramatic reduction of AtCSP4 transcript when AGL15 expression was highest. Since AGL15 can function as a repressor, these correlative data suggest that AGL15 can alter AtCSP4 gene expression in planta.

(target for TCP transcription factors) and a sugar-related sequence (TATCCAOSAMY). TCP transcription factors are broadly implicated in various aspects of plant growth and development (Cubas et al., 1999; Corley et al., 2005; Li et al., 2005) and sugar-sensing and related signal transduction networks have broad functional importance spanning abiotic stress and growth/development (Koch, 1996, 2004; Rolland et al., 2002, 2006; Gibson, 2005; Smeekens, 2000) and regulation of the cell cycle (Riou-Khamlichi et al., 2000). Further characterization of these cis-elements is of great interest since they may eventually enable us to identify the mechanism(s) controlling AtCSP4 transcription.

Localization of Plant Cold-shock Domain Proteins Subcellular localization Consistent with a putative function as RNAbinding proteins, plant CSPs have been identified in cytosol (Nakaminami et al., 2006;

Fusaro et al., 2007) and nucleolar regions (Sasaki et al., 2007). We utilized the P. patens system to understand the temporal and spatial regulation of two CSPs (PpCSP1 and PpCSP2) under the control of their respective native promoters. Using homologous recombination, we created YFP (yellow fluorescent protein) and GUS (b-glucuronidase) fusions for PpCSP1 and PpCSP2 and homologously recombined them into their respective native gene loci. Consistent to previous studies, we observed a predominant cytosolic localization for both PpCSP1 and PpCSP2. Interestingly, localization was observed within discrete cytoplasmic foci, bearing resemblance to cytoplasmic processing bodies (P-bodies) (Fig. 5.4). Since decapping proteins have been shown to localize and function within Arabidopsis P-bodies (Xu et al., 2006; Iwasaki et al., 2007), we cloned a moss DCP homologue as a positive control for co-localization studies with our PpCSP YFP-tagged lines. Future co-localization studies are underway and will provide conclusive evidence to determine if PpCSPs localize to cytoplasmic P-bodies. Cytoplasmic P-bodies are sites for the processing of microRNAs (miRNAs) (Balzer

50

D. Karlson et al.

Fig. 5.4. Localization of a Physcomitrella patens (moss) cold-shock domain protein (PpCSP1) to putative cytoplasmic processing bodies. Using stably transformed moss lines as fusions of PpCSPs to YFP (yellow fluorescent protein), we observed discrete cytoplasmic foci in protonemal tissue. Large chloroplasts (cp) are evident in the light micrograph and discrete fluorescent dots were visible in areas of compressed cytoplasm (c). Consistent to observations from human and worm, these data represent the first documented report of a putative P-body localization for a plant CSP.

and Moss, 2007) and the degradation of mRNAs (Decker et al., 2007; Teixeira and Parker, 2007). Several studies have described functional roles for cytoplasmic foci in the miRNA-mediated repression of gene expression (Jakymiw et al., 2005; Liu et al., 2005; Pillai et al., 2005, 2007). Our data represent the first putative localization of plant CSPs to P-bodies and are in good accordance with the recently described localization pattern for two eukaryotic cold-shock domain proteins: LIN-28 protein from C. elegans (Balzer and Moss, 2007) and human Y-box protein 1 (Yang and Bloch, 2007). These exciting data provide correlative evidence suggesting that the in vivo functional role of plant CSPs may relate to mRNA stability, a function that is well established for the CSP YB-1 (Evdokimova et al., 2001, 2006). Given their proven ability to bind/melt double-stranded nucleic acids (Nakaminami et al., 2006; Kim et al., 2007), it is intriguing to consider that plant CSPs may be involved in either the processing of miRNAs or miRNA-mediated gene repression. In the future, functional studies with PpCSP knockouts will enable us to confirm these putative functional roles.

Tissue-specific expression patterns Our stably transformed PpCSP1 and PpCSP2 YFP lines enabled us to characterize their temporal and spatial expression patterns through-

out all stages of moss development on a protein level. PpCSPs were highly expressed in young protonemata. A striking and specific enrichment of PpCSPs was detected in budding protonemata tissue. PpCSPs were highly specific and accumulated within developing gametophore buds. During the formation of sexual reproductive organs, PpCSPs exhibited a clear accumulation to both antheridia and archegonia. Specifically, within archegonia, egg cells displayed a pronounced accumulation of PpCSPs. Consistent with our protein blot analyses in both Arabidopsis and rice (Chaikam and Karlson, 2008), PpCSPs also exhibited preferential accumulation to meristematic tissues undergoing high cell division and those tissues undergoing developmental transitions and/or the formation of reproductive organs.

Acknowledgements The authors would like to thank Dr Michael Thomashow for graciously providing seeds for the CBF overexpression plant lines. We would also like to thank the Arabidopsis Biological Resource Center for providing seeds for ABA and flowering time mutants. The authors would like to thank Drs Sharyn Perry and Kristine Hill for AGL15 chromatin immunoprecipitation studies and Dr Donna Fernandez for donating AGL15 overexpression and knockout seed lines. We also thank Dr Imara Perera (North Carolina State University) for in vitro

Plant Cold-shock Domain Proteins

phosphorylation assays and Drs Kenji Miura and Michael Hasegawa for in vitro sumoylation assays. We also thank Drs Mitsuyasu Hasebe and Yuji Hiwatashi for extensive assistance and guidance for Physcomitrella patens

51

work. This research was funded by a National Science Foundation grant (#IBN-0416945) to D.K. and a National Science Foundation Summer Research Fellowship to K.T. in the laboratory of Dr Mitsuyasu Hasebe.

References Bae, W., Xia, B., Inouye, M. and Severinov, K. (2000) Escherichia coli CspA-family RNA chaperones are transcription antiterminators. Proceedings of the National Academy of Sciences USA, 97, 7784–7789. Balzer, E. and Moss, E.G. (2007) Localization of the developmental timing regulator Lin28 to mRNP complexes, P-bodies and stress granules. RNA Biology 4, 16–25. Bouvet, P. and Wolffe, A.P. (1994) A role for transcription and FRGY2 in masking maternal messenger-RNA within Xenopus-oocytes. Cell 77, 931–941. Catala, R., Ouyang, J., Abreu, I.A., Hu, Y., Seo, H., Zhang, X. and Chua, N.H. (2007) The Arabidopsis E3 SUMO ligase SIZ1 regulates plant growth and drought responses. The Plant Cell 19, 2952–2966. Chaikam, V. and Karlson, D. (2008) Functional characterization of two cold shock domain proteins from Oryza sativa. Plant, Cell and Environment 31, 995–1006. Coleman, J.E. (1992) Zinc proteins – enzymes, storage proteins, transcription factors, and replication proteins. Annual Review of Biochemistry 61, 897–946. Corley, S.B., Carpenter, R., Copsey, L. and Coen, E. (2005) Floral asymmetry involves an interplay between TO and MYB transcription factors in Antirrhinum. Proceedings of the National Academy of Sciences USA 102, 5068–5073. Cubas, P., Lauter, N., Doebley, J. and Coen, E. (1999) The TCP domain: a motif found in proteins regulating plant growth and development. The Plant Journal 18, 215–222. Dannull, J., Surovoy, A., Jung, G. and Moelling, K. (1994) Specific binding of HIV-1 nucleocapsid protein to psi-RNA in-vitro requires N-terminal zinc-finger and flanking basic-amino-acid residues. EMBO Journal 13, 1525–1533. Decker, C.J., Teixeira, D. and Parker, R. (2007) Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. Journal of Cell Biology 179, 437–449. Didier, D.K., Schiffenbauer, J., Woulfe, S.L., Zacheis, M. and Schwartz, B.D. (1988) Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box. Proceedings of the National Academy of Sciences USA 85, 7322–7326. Evdokimova, V.M., Kovrigina, E.A., Nashchekin, D.V., Davydova, E.K., Hershey, J.W.B. and Ovchinnikov, L.P. (1998) The major core protein of messenger ribonucleoprotein particles (p50) promotes initiation of protein biosynthesis in vitro. Journal of Biological Chemistry 273, 3574–3581. Evdokimova, V., Ruzanov, P., Imataka, H., Raught, B., Svitkin, Y., Ovchinnikov, L.P. and Sonenberg, N. (2001) The major mRNA-associated protein YB-1 is a potent 5? cap-dependent mRNA stabilizer. EMBO Journal 20, 5491–5502. Evdokimova, V., Ruzanov, P., Anglesio, M.S., Sorokin, A.V., Ovchinnikov, L.P., Buckley, J., Triche, T.J., Sonenberg, N. and Sorensen, P.H. (2006) Akt-mediated YB-1 phosphorylation activates translation of silent mRNA species. Molecular and Cellular Biology 26, 277–292. Fernandez, D.E., Heck, G.R., Perry, S.E., Patterson, S.E., Bleecker, A.B. and Fang, S.C. (2000) The embryo MADS domain factor AGL15 acts postembryonically. Inhibition of perianth senescence and abscission via constitutive expression. The Plant Cell 12, 183–198. Fusaro, A.F., Bocca, S.N., Ramos, R.L.B., Barroco, R.M., Magioli, C., Jorge, V.C., Coutinho, T.C., RangelLima, C.M., de Rycke, R., Inze, D., Engler, G. and Sachetto-Martins, G. (2007) AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development. Planta 225, 1339–1351. Gibson, S.I. (2005) Control of plant development and gene expression by sugar signaling. Current Opinion in Plant Biology 8, 93–102. Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M. and Thomashow, M.F. (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. The Plant Journal 16, 433–442.

52

D. Karlson et al.

Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D. and Thomashow, M.F. (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology 124, 1854–1865. Goldstein, J., Pollitt, N.S. and Inouye, M. (1990) Major cold shock protein of Escherichia coli. Proceedings of the National Academy of Sciences USA 87, 283–287. Graumann, P.L. and Marahiel, M.A. (1998) A superfamily of proteins that contain the cold-shock domain. Trends in Biochemical Sciences 23, 286–290. Graumann, P., Schroder, K., Schmid, R. and Marahiel, M.A. (1996) Cold shock stress-induced proteins in Bacillus subtilis. Journal of Bacteriology 178, 4611–4619. Hayakawa, H., Uchiumi, T., Fukuda, T., Ashizuka, M., Kohno, K., Kuwano, M. and Sekiguchi, M. (2002) Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine. Biochemistry 41, 12739–12744. Heck, G.R., Perry, S.E., Nichols, K.W. and Fernandez, D.E. (1995) AGL15, a MADS domain protein expressed in developing embryos. The Plant Cell 7, 1271–1282. Iwasaki, S., Takeda, A., Motose, H. and Watanabe, Y. (2007) Characterization of Arabidopsis decapping proteins AtDCPI and AtDCP2, which are essential for post-embryonic development. FEBS Letters 581, 2455–2459. Jakymiw, A., Lian, S.L., Eystathioy, T., Li, S.Q., Satoh, M., Hamel, J.C., Fritzler, M.J. and Chan, E.K.L. (2005) Disruption of GW bodies impairs mammalian RNA interference. Nature Cell Biology 7, 1267–1274. Jiang, W., Hou, Y. and Inouye, M. (1997) CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. Journal of Biological Chemistry 272, 196–202. Jin, J.B., Jin, Y.H., Lee, J., Miura, K., Yoo, C.Y., Kim, W.Y., van Oosten, M., Hyun, Y., Somers, D.E., Lee, I., Yun, D.J., Bressan, R.A. and Hasegawa, P.M. (2008) The SUMO E3 ligase, AtS1Z1, regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on FLC chromatin structure. The Plant Journal 53, 530–540. Karlson, D. and Imai, R. (2003) Conservation of the cold shock domain protein family in plants. Plant Physiology 131, 12–15. Karlson, D., Nakaminami, K., Toyomasu, T. and Imai, R. (2002) A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins. Journal of Biological Chemistry 277, 35248–35256. Karlson, D.T., Zeng, Y., Stirm, V.E., Joly, R.J. and Ashworth, E.N. (2003) Photoperiodic regulation of a 24-kD dehydrin-like protein in red-osier dogwood (Cornus sericea L.) in relation to freeze-tolerance. Plant & Cell Physiology 44, 25–34. Kasuga, M., Miura, S., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer. Plant & Cell Physiology 45, 346–350. Kim, J.S., Park, S.J., Kwak, K.J., Kim, Y.O., Kim, J.Y., Song, J., Jang, B., Jung, C.H. and Kang, H. (2007) Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli. Nucleic Acids Research 35, 506–516. Kingsley, P.D. and Palis, J. (1994) GRP2 proteins contain both CCHC zinc fingers and a cold shock domain. The Plant Cell 6, 1522–1523. Koch, K. (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Current Opinion in Plant Biology 7, 235–246. Koch, K.E. (1996) Carbohydrate-modulated gene expression in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 509–540. Kohno, K., Izumi, H., Uchiumi, T., Ashizuka, M. and Kuwano, M. (2003) The pleiotropic functions of the Y-box-binding protein, YB-1. BioEssays 25, 691–698. Kurepa, J., Walker, J.M., Smalle, J., Gosink, M.M., Davis, S.J., Durham, T.L., Sung, D.Y. and Vierstra, R.D. (2003) The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis – accumulation of SUMO1 and -2 conjugates is increased by stress. Journal of Biological Chemistry 278, 6862–6872. Ladomery, M. and Sommerville, J. (1995) A role for Y-box proteins in cell-proliferation. BioEssays 17, 9–11. Lemieux, C., Otis, C. and Turmel, M. (2007) A clade uniting the green algae Mesostigma viride and Chlorokybus atmophyticus represents the deepest branch of the Streptophyta in chloroplast genome-based phylogenies. BMC Biology 5, 2. Li, C.X., Potuschak, T., Colon-Carmona, A., Gutierrez, R.A. and Doerner, P. (2005) Arabidopsis TCP20 links regulation of growth and cell division control pathways. Proceedings of the National Academy of Sciences USA 102, 12978–12983. Lim, M.H., Kim, J., Kim, Y.S., Chung, K.S., Seo, Y.H., Lee, I., Kim, J., Hong, C.B., Kim, H.J. and Park, C.M. (2004) A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C. The Plant Cell 16, 731–740.

Plant Cold-shock Domain Proteins

53

Liu, J.D., Valencia-Sanchez, M.A., Hannon, G.J. and Parker, R. (2005) MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nature Cell Biology 7, 719-U118. Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. The Plant Cell 10, 1391–1406. Lois, L.M., Lima, C.D. and Chua, N.H. (2003) Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis. The Plant Cell 15, 1347–1359. Macknight, R., Bancroft, I., Page, T., Lister, C., Schmidt, R., Love, K., Westphal, L., Murphy, G., Sherson, S., Cobbett, C. and Dean, C. (1997) FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains. Cell 89, 737–745. Matsumoto, K., Tanaka, K.J. and Tsujimoto, M. (2005) An acidic protein, YBAP1, mediates the release of YB-1 from mRNA and relieves the translational repression activity of YB-1. Molecular and Cellular Biology 25, 1779–1792. Medina, J., Bargues, M., Terol, J., Perez-Alonso, M. and Salinas, J. (1999) The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiology 119, 463–470. Miura, K., Jin, J.B., Lee, J., Yoo, C.Y., Stirm, V., Miura, T., Ashworth, E.N., Bressan, R.A., Yun, D.J. and Hasegawa, P.M. (2007) SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. The Plant Cell 19, 1403–1414. Moss, E.G. and Tang, L.J. (2003) Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites. Developmental Biology 258, 432–442. Murtas, G., Reeves, P.H., Fu, Y.F., Bancroft, I., Dean, C. and Coupland, G. (2003) A nuclear protease required for flowering-time regulation in Arabidopsis reduces the abundance of SMALL UBIQUITIN-RELATED MODIFIER conjugates. The Plant Cell 15, 2308–2319. Mussgnug, J.H., Wobbe, L., Elles, I., Claus, C., Hamilton, M., Fink, A., Kahmann, U., Kapazoglou, A., Mullineaux, C.W., Hippler, M., Nickelsen, J., Nixon, P.J. and Kruse, O. (2005) NAB1 is an RNA binding protein involved in the light-regulated differential expression of the light-harvesting antenna of Chlamydomonas reinhardtii. The Plant Cell 17, 3409–3421. Nakaminami, K., Sasaki, K., Kajita, S., Takeda, H., Karlson, D., Ohgi, K. and Imai, R. (2005) Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein. FEBS Letters 579, 4887–4891. Nakaminami, K., Karlson, D.T. and Imai, R. (2006) Functional conservation of cold shock domains in bacteria and higher plants. Proceedings of the National Academy of Sciences USA 103, 10122–10127. Nakaminami, K., Hill, K., Perry, S.E., Sentogu, N., Long, J.A. and Carlson, D.T. (2009) Arabidopsis cold shock domain proteins: relationships to floral and silique development. Journal of Experimental Botany 60, 1047–1062. Novillo, F., Alonso, J.M., Ecker, J.R. and Salinas, J. (2004) CBF2/DREB1C is a negative regulator of CBF1/ DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proceedings of the National Academy of Sciences USA 101, 3985–3990. Oh, S.J., Song, S.I., Kim, Y.S., Jang, H.J., Kim, S.Y., Kim, M., Kim, Y.K., Nahm, B.H. and Kim, J.K. (2005) Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiology 138, 341–351. Page, T., Macknight, R., Yang, C.H. and DEAN, C. (1999) Genetic interactions of the Arabidopsis flowering time gene FCA, with genes regulating floral initiation. The Plant Journal 17, 231–239. Phadtare, S. and Inouye, M. (1999) Sequence-selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli. Molecular Microbiology 33, 1004–1014. Pillai, R.S., Bhattacharyya, S.N., Artus, C.G., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E. and Filipowicz, W. (2005) Inhibition of translational initiation by Let-7 microRNA in human cells. Science 309, 1573–1576. Pillai, R.S., Bhattacharyya, S.N. and Filipowicz, W. (2007) Repression of protein synthesis by miRNAs: how many mechanisms? Trends in Cell Biology 17, 118–126. Polissi, A., de Laurentis, W., Zangrossi, S., Briani, F., Longhi, V., Pesole, G. and Deho, G. (2003) Changes in Escherichia coli transcriptome during acclimatization at low temperature. Research in Microbiology 154, 573–580. Quesada, V., Dean, C. and Simpson, G.G. (2005) Regulated RNA processing in the control of Arabidopsis flowering. International Journal of Developmental Biology 49, 773–780. Ranjan, M., Tafuri, S.R. and Wolffe, A.P. (1993) Masking messenger-RNA from translation in somatic-cells. Genes and Development 7, 1725–1736.

54

D. Karlson et al.

Riou-Khamlichi, C., Menges, M., Healy, J.M.S. and Murray, J.A.H. (2000) Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Molecular and Cellular Biology 20, 4513–4521. Rolland, F., Moore, B. and Sheen, J. (2002) Sugar sensing and signaling in plants. The Plant Cell 14, S185–S205. Rolland, F., Baena-Gonzalez, E. and Sheen, J. (2006) Sugar sensing and signaling in plants: conserved and novel mechanisms. Annual Review of Plant Biology 57, 675–709. Sasaki, K., Kim, M.H. and Imai, R. (2007) Arabidopsis COLD SHOCK DOMAIN PROTEIN2 is a RNA chaperone that is regulated by cold and developmental signals. Biochemical and Biophysical Research Communications 364, 633–638. Saunders, N.F.W., Thomas, T., Curmi, P.M.G., Mattick, J.S., Kuczek, E., Slade, R., Davis, J., Franzmann, P.D., Boone, D., Rusterholtz, K., Feldman, R., Gates, C., Bench, S., Sowers, K., Kadner, K., Aerts, A., Dehal, P., Detter, C., Glavina, T., Lucas, S., Richardson, P., Larimer, F., Hauser, L., Land, M. and Cavicchioli, R. (2003) Mechanisms of thermal adaptation revealed from the genomes of the Antarctic Archaea Methanogenium frigidum and Methanococcoides burtonii. Genome Research 13, 1580–1588. Schmid, M., Uhlenhaut, N.H., Godard, F., Demar, M., Bressan, R., Weigel, D. and Lohmann, J.U. (2003) Dissection of floral induction pathways using global expression analysis. Development 130, 6001–6012. Simpson, G.G., Quesada, V., Henderson, I.R., Dijkwel, P.P., Macknight, R. and Dean, C. (2004) RNA processing and Arabidopsis flowering time control. Biochemical Society Transactions 32, 565–566. Smeekens, S. (2000) Sugar-induced signal transduction in plants. Annual Review of Plant Physiology and Plant Molecular Biology 51, 49–81. Sommerville, J. (1999) Activities of cold-shock domain proteins in translation control. BioEssays 21, 319–325. Stickeler, E., Fraser, S.D., Honig, A., Chen, A.L., Berget, S.M. and Cooper, T.A. (2001) The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4. EMBO Journal 20, 3821–3830. Stockinger, E.J., Gilmour, S.J. and Thomashow, M.F. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proceedings of the National Academy of Sciences USA 94, 1035–1040. Sutherland, B.W., Kucab, J., Wu, J., Lee, C., Cheang, M.C., Yorida, E., Turbin, D., Dedhar, S., Nelson, C., Pollak, M., Leighton Grimes, H., Miller, K., Badve, S., Huntsman, D., Blake-Gilks, C., Chen, M., Pallen, C.J. and Dunn, S E. (2005) Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells. Oncogene 24, 4281–4292. Teixeira, D. and Parker, R. (2007) Analysis of P-body assembly in Saccharomyces cerevisiae. Molecular Biology of the Cell 18, 2274–2287. Wang, N., Yamanaka, K. and Inouye, M. (1999) CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock. Journal of Bacteriology 181, 1603–1609. Wolffe, A.P. (1994) Structural and functional-properties of the evolutionarily ancient Y-box family of nucleicacid binding-proteins. BioEssays 16, 245–251. Wolffe, A.P., Tafuri, S., Ranjan, M. and Familari, M. (1992) The Y-box factors: a family of nucleic acid binding proteins conserved from Escherichia coli to man. New Biology 4, 290–298. Xia, B., Ke, H. and Inouye, M. (2001) Acquirement of cold sensitivity by quadruple deletion of the cspA family and its suppression by PNPase S1 domain in Escherichia coli. Molecular Microbiology 40, 179–188. Xu, J., Yang, J.Y., Niu, Q.W. and Chua, N. H. (2006) Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development. The Plant Cell 18, 3386–3398. Yamanaka, K., Fang, L. and Inouye, M. (1998) The CspA family in Escherichia coli: multiple gene duplication for stress adaptation. Molecular Microbiology 27, 247–255. Yang, W.H. and Bloch, D.B. (2007) Probing the mRNA processing body using protein macroarrays and ‘autoantigenomics’. RNA 13, 704–712. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L. and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiology 136, 2621–2632.

6 Expressional and Functional Characterization of Arabidopsis Cold-shock Domain Proteins K. Sasaki, M.-H. Kim and R. Imai

Introduction Temperature is one of the most critical environmental factors that limits the growth and distribution of living organisms. In response to high temperature or heat stress, organisms have to protect themselves from cellular dysfunctions such as protein denaturation. The mechanisms that recruit heat-shock proteins with molecular chaperone activities to renature protein structure and function are highly conserved from bacteria to mammals (Becker and Craig, 1994). Low temperature also limits growth and survival in diverse organisms. Therefore, it is reasonable to think that adaptation to low temperature or cold stress is also conserved through evolution. However, it is not known if there is a highly conserved response to low temperature which is similar to the heat-shock response. In Escherichia coli, cold-shock proteins are induced in response to cold. CspA, the major cold-shock protein of E. coli, accumulates up to 10% of total proteins during cold stress (Goldstein et al., 1990) and its transcript level also increases in response to cold (Tanabe et al., 1992). Nine genes encoding Csp family members are identified in E. coli (cspA to cspI) (Wang et al., 1999; Yamanaka et al., 1998). Expression of cspA, cspB, cspG and cspI is induced by cold (Wang et al., 1999). CspA can bind ssDNA/ RNA through two RNA recognition motifs

(RNP1 and RNP2), while it lacks ability to bind dsDNA (Jiang et al., 1997). It was suggested that CspA binds to RNA to destabilize secondary structure and facilitate translation at low temperature (Jiang et al., 1997). It was also demonstrated that CspA functions as a transcriptional anti-terminator to regulate expression of metY-rpsO operon genes at the transcriptional level (Bae et al., 2000). Similar sequences to the bacterial coldshock proteins were found in eukaryotic proteins as a functional domain. The most widely studied cold-shock domain protein is the Y-box protein family. The Y-box proteins were initially isolated as a transcriptional factor that has an ability to bind the Y-box sequence (CTGATTGGYYAA) of major histocompatability complex (MHC) class II promoters through their cold-shock domain (CSD) (Didier et al., 1988). The Y-box proteins bind to ssDNA, dsDNA and RNA (Wolffe, 1994). Y-box binding protein 1 (YB-1) binds to promoters containing the Y-box sequence and either activates or represses gene expression (Ting et al., 1994; Mertens et al., 1997; Norman et al., 2001; Fukada and Tonks, 2003). YB-1 is the most abundant protein of messenger ribonucleoprotein particles (mRNPs) and controls translation (Minich and Ovchinnikov, 1992). It has been proposed that masking of mRNA by YB-1 inhibits translation (Ranjan et al., 1993; Sommerville and Ladomery, 1996; Evdokimova

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

55

56

K. Sasaki et al.

and Ovchinnikov, 1999; Bader and Vogt, 2004). LIN28, a cold-shock domain protein of Caenorhabditis elegans, regulates the cell fate from the second larval stage (L2) to the third larval stage (L3) in larval development (Moss et al., 1997). However, functions of these eukaryotic cold-shock domain proteins are not directly related in response to cold. Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through cold acclimation. We have identified a novel cold-induced plant cold-shock domain protein (CSP), WCSP1, from winter wheat (Karlson et al., 2002). WCSP1 contains three glycinerich regions and three CCHC zinc fingers (Fig. 6.1). WCSP1 mRNA is up-regulated by cold and the corresponding protein accumulates to high levels during cold acclimation in crown tissues and young seedlings (Karlson et al., 2002). WCSP1 mRNA, however, is not induced by drought, salt, high temperature or treatment with abscisic acid (Karlson et al., 2002). It is thought that the function of WCSP1 is specific to the cold response. WCSP1 binds to ssDNA, dsDNA and RNA (Nakaminami et al., 2005). In addition, in vitro melting assays clearly demonstrated that WCSP1 unwinds dsDNA (Nakaminami et al., 2006). Constitutive expres-

sion of WCSP1 in the E. coli csp quadruple deletion mutant complemented its cold-sensitive phenotype (Nakaminami et al., 2006). WCSP1 has a transcription anti-termination activity demonstrated by using an E. coli strain that has a hairpin loop upstream of a chloramphenicol resistance gene (Nakaminami et al., 2006). These data suggested that WCSP1 shares a function with E. coli cold-shock proteins during the process of cold adaptation. In Arabidopsis thaliana (At), genes four encoding CSPs, AtCSP1 to AtCSP4, were identified in the genome sequence (Karlson and Imai, 2003). AtCSP2 (At4g38680) and AtCSP4 (At2g21060) contain two glycine-rich regions and two CCHC zinc fingers; however, AtCSP1 (At4g36020) and AtCSP3 (At2g17870) contain seven glycine-rich regions and seven CCHC zinc fingers (Fig. 6.1A). RT-PCR analysis demonstrated that AtCSP1–AtCSP3 mRNA was induced by cold, but AtCSP4 mRNA was decreased (Karlson and Imai, 2003). These data suggested that the WCSP1like protein family in Arabidopsis is also related to the cold acclimation process. In the present chapter we describe expressional and functional characterization of Arabidopsis CSPs, focusing on AtCSP2.

A CspA

B

WCSP1 AtCSP1

AtCSP1

AtCSP2

AtCSP2

AtCSP3

AtCSP3

AtCSP4

AtCSP4

YB-1 LIN28

Tubulin

Shoot, 4°C 0

1

3

0

1

3

GR

7

10 14 21 (days)

Root, 4°C

20 aa CSD

5

ZnF

5

7

10 14 21 (days)

AtCSP1

C

AtCSP2

Cold (4°C) 0

1

3

5

7 10

14

21 (days)

AtCSP3

Shoot

AtCSP4

Root

Tubulin

Fig. 6.1. (A) Structural features of cold-shock domain proteins (CspA from Escherichia coli, WCSP1 from wheat, AtCSP1–AtCSP4 from Arabidopsis thaliana, YB-1 from man, LIN28 from Caenorhabditis elegans). GR, glycine-rich region; ZnF, zinc finger motif; aa, amino acids. (B) Semi-quantitative RT-PCR analysis of four Arabidopsis genes encoding cold-shock domain proteins in 10-day-old cold-treated wild-type seedlings. Total RNA was extracted from shoot and root tissues held at 4°C. (C) Western blot analyses of AtCSP2 expression. Total protein (15 µg) extracted from shoot and root tissues of an AtCSP4 knockout mutant, atcsp4–1, which was treated at 4°C for 0–21 days.

Characterization of Arabidopsis Cold-shock Domain Proteins

Arabidopsis Cold-shock Domain Proteins Expression of AtCSP genes in response to cold Expression of the four Arabidopsis CSP genes, AtCSP1-AtCSP4, in response to a 21-day cold treatment was examined using 10-day-old seedlings. RT-PCR analysis indicated that AtCSP1, AtCSP2 and AtCSP3 were up-regulated in shoots during the cold treatment whereas expression of AtCSP4 remained unchanged (Fig. 6.1B). In roots, expression of AtCSP1, AtCSP2 and AtCSP3 was induced after 1 day of cold treatment and then declined after 14 days (Fig. 6.1B). As observed in shoots, expression of AtCSP4 did not change during the cold treatment (Fig. 6.1B). These expression analyses indicated that expression of AtCSP1, AtCSP2 and AtCSP3 is differentially regulated during a long-term cold exposure. Of all the Arabidopsis CSP genes, AtCSP2 is up-regulated the highest after cold treatment in both shoot and root. Expression of AtCSP2 in response to cold was further examined at the protein level using 10-day-old seedlings. Since AtCSP2 and AtCSP4 are similar in size, a knockout line for AtCSP4 (atcsp4–1; K. Sasaki, M.-H. Kim and R. Imai, unpublished results) was utilized to allow specific detection of AtCSP2 with a polyclonal antibody against WCSP1. Western blot analysis revealed accumulation of AtCSP2 in the shoots and roots of atcsp4–1 seedlings during a 21-day period of cold acclimation (Fig. 6.1C). Protein accumulation occurred during cold acclimation with the highest levels attained between 10 and 14 days in shoots, and after 21 days in roots. It is interesting to note that AtCSP2 protein and mRNA accumulation patterns are not in parallel. Protein accumulation is delayed and more pronounced, which suggests that AtCSP2 is under post-transcriptional regulation.

RNA chaperone activity of AtCSP2 To identify the biochemical activity of AtCSP2, we performed a nucleic acid binding assay, a

57

nucleic acid melting assay as well as an in vivo complementation assay with the cold-sensitive E. coli csp mutant. Nucleic acid-binding activities using recombinant AtCSP2 protein in a gel shift assay revealed that AtCSP2 is capable of binding ssDNA, dsDNA and mRNA (Fig. 6.2A, B and C). In contrast, glutathione S-transferase (GST), a negative control, did not bind to ssDNA, dsDNA or mRNA (Fig. 6.2A, B and C). Fusaro et al. (2007) also reported that the AtGRP2/AtCSP2 fused with a maltose-binding protein (MBP) binds to ssDNA, dsDNA and RNA ribohomopolymers. This preference was conserved with plant and animal coldshock domain but not with bacterial cold-shock proteins, which only bind single-stranded nucleic acids. Nucleic acid melting assay has been utilized to demonstrate RNA chaperone activity of bacterial cold-shock proteins. It was therefore determined if AtCSP2 displays a similar activity. Two partially complementing oligonucleotides, one of which was labelled with fluorescein isothiocyanate (FITC) at the 5′ terminus and the other labelled with a quencher BHQ1 at the 3′ terminus, were used as a molecular beacon. Under the conditions utilized, the annealed substrate emitted only 16.1% relative FITC intensity of the denatured substrate (Fig. 6.2D). When the substrate was incubated with AtCSP2, the FITC intensity increased to 40.1%, a level similar to that observed with WCSP1 (39.5%) but lower than that of CspA (81.0%) (Fig. 6.2D). Addition of GST or buffer alone resulted in no significant changes (Fig. 6.2D). Thus the in vitro DNA melting assay revealed an activity similar to WCSP1 but lower than CspA. AtCSP2 contains a C-terminal extension that can be involved in protein–protein interaction. Therefore, it is possible to speculate that AtCSP2 becomes fully functional or stabilized when it is involved in a complex. It was examined if AtCSP2 could complement the cold-sensitive phenotype of an E. coli csp quadruple mutant, BX04 (cspA, cspB, cspE, cspG). Growth at 17°C of BX04 containing the vector pINIII was unaffected. However, vigorous growth was observed for BX04 containing pINIII-CspA and pINIIIWCSP1 (Fig. 6.2E). Growth at 17°C of

58

K. Sasaki et al.

A

AtCSP2 GST 70 350 700 0

pmol

ssDNA

B

AtCSP2 GST 700

0

7

70 350 700 0

pmol

D

100

dsDNA

80 60 40 20 0 d

C

a

b

C sp A

7

III

0

Relative intensity of FITC (%)

700

G

W

2

A

E

mRNA

N

pmol

pI

1000 0 250 500 750 1000 0

W C SP 1 At C SP 2

AtCSP2 GST

37°C

17°C

Fig. 6.2. (A–C) Nucleic acid-binding activity of AtCSP2 revealed by a gel shift assay. Recombinant AtCSP2 protein was incubated with ssDNA (A), dsDNA (B) or in vitro-transcribed luciferase mRNA (C) and separated by agarose gel electrophoresis. Gel shifts were subsequently visualized by EtBr staining. (D) Nucleic acidmelting activity of AtCSP2. Relative fluorescence with each protein is shown in comparison to completely heat-denatured molecular beacons (100% relative intensity). d, denatured; a, annealed; b, buffer; G, glutathione S-transferase; W, WCSP1; 2, AtCSP2; A, CspA. (E) Complementation of a low-temperature sensitivity of an Esecherichia coli quadruple mutant, BX04 (cspA, cspB, cspE, cspG), with AtCSP2. Overnight liquid cultures were adjusted to the same cell density, uniformly spotted on to LB-ampicilin plates and grown at either 37°C overnight or 17°C for 5 days.

BX04 containing pINIII-AtCSP2 was slower compared with the growth of BX04 with pINIII-CspA or pINIII-WCSP1 (Fig. 6.2E). Kim et al. (2007) reported recently that the CSDP2/AtCSP2 protein was unable to complement E. coli BX04 mutant and was defective in a dsDNA melting activity in vitro. However, under the conditions utilized, we were able to detect similar but lower activity compared with WCSP1. We therefore concluded that AtCSP2 is an RNA chaperone that shares biochemical functions with bacterial cold-shock proteins.

Expression of AtCSP2 is developmentally controlled and regulated by cold The spatial pattern of AtCSP2 expression was examined with transgenic Arabidopsis plants expressing the AtCSP2 promoter– b-glucuronidase (GUS) fusion gene (ProCSP2::GUS). In the ProCSP2::GUS plants, GUS activity was detectable in the embryo of dry seeds and after 2-day stratification (Fig 6.3A, a). Fusaro et al. (2007) also reported that GUS activity was detectable during seed development. GUS activity declined thereafter

Characterization of Arabidopsis Cold-shock Domain Proteins

and was detectable only in the shoot apex and root tip regions 5 days post germination (Fig. 6.3A, b–f). If 10-day-old ProCSP2::GUS plants were exposed to cold for 3 days, GUS activity was detected in a wider region of the root (Fig. 6.3A, j–m). GUS activity was also detected in the pollen, ovule and transmitting tissues (Fig. 6.3A, g–i). It is interesting to note that the shoot and root apices are the regions in which several genes associated with flowering time and vernalization such as VIN3 and FLC are expressed (Sung and Amasino, 2004). FLC expression is

A a

b

59

suppressed after 20 days of cold treatment when VIN3 mRNA starts to accumulate (Sung and Amasino, 2004). Since high levels of AtCSP2 protein accumulate after 21 days of cold treatment (Fig. 6.1C), AtCSP2 may be associated with the vernalization response. Interestingly, it was reported recently that RNAi suppression of AtGRP2/AtCSP2 results in an early flowering phenotype and AtGRP2/ AtCSP2 overexpression delays flowering in the C24 ecotype (Fusaro et al., 2007). In this case, it was suggested that AtCSP2 may function as a negative regulator of flowering in non-vernalized conditions.

c

Subcellular localization of AtCSP2 d

e

f

g

h

i

j

k

B

a

22°C

22°C

m

b

d

g

l

h

4°C, 3 days

4°C, 3 days

c

e

f

i

It was reported that wheat WCSP1 localized in the nucleus and endoplasmic reticulum, where it is considered to have a function in RNA binding (Nakaminami et al., 2006). Transgenic plants expressing an AtCSP2::GFP fusion construct were utilized for AtCSP2 subcellular localization. GFP (green fluorescent protein) fluorescence in the transgenic plants revealed

Fig. 6.3. (A) Histochemical localization of b-glucuronidase (GUS) activity in transgenic Arabidopsis plants expressing ProCSP2::GUS. Embryo after 2 days of stratification at 4°C (a). Seedlings at 1 day (b), 2 days (c), 3 days (d), 4 days (e) and 5 days (f) post germination. Floral tissues of a pre-anthesis flower (g). Close-up images of a pistil (h) and a carpel (i). Seedlings at 10 days post germination (j) were subjected to 4°C for 3 days (l). Close-up images of (j) and (l) (k, m). Bar=2.5 mm. (B) Subcellular localization of AtCSP2 in Arabidopsis. (a) Fluorescent image of a nucleolus from root cells overexpressing AtCSP2::GFP. (b) Cells in (a) were stained with DAPI (4¢,6-diamidino2-phenylindole). (c) Fluorescent image of a root cell transiently expressing AtCSP2::GFP. (d,e) Images of cells co-bombarded with AtCSP2::GFP (d) and a nucleolus marker, AtFbr1::RFP (e). (f) A merged image of (d) and (e). (g, h) Fluorescent image of a nucleus from the root cells co-bombarded with AtCSP2–ZnF1::GFP (g) and AtFbr1::RFP (h). (i) Fluorescent image of a root cell of Arabidopsis transiently expressing AtCSP2–CSD::GFP. Arrows indicate nucleus. Bar=2 mm.

60

K. Sasaki et al.

localization of AtCSP2::GFP in the cytoplasm and nucleus (Fig. 6.3B, a, b and c). Within the nucleus a strong signal was detected in a nucleolus-like compartment (Fig. 6.3B, a and c). To confirm the nucleolus localization, the AtCSP2::GFP construct was transiently expressed in Arabidopsis root tissue with a fibrillarin::RFP gene as a nucleolus marker. This revealed the GFP and RFP (red fluorescent protein) signals merged, indicating co-localization of AtCSP2 and fibrillarin in the nucleolus (Fig. 6.3B, d–f ). Although it was reported that AtGRP2/ AtCSP2 is localized in the nucleus and cytoplasm ( Fusaro et al., 2007), our data indicated that AtCSP2 is localized in the cytoplasm, nucleoplasm and nucleolus. If one C-terminal zinc finger motif (ZnF ) and one glycine-rich region (GR) were eliminated (AtCSP2–ZnF1:: GFP), labelling was not detected in the nucleolus ( Fig. 6.3B, g and h). Cytoplasm and nucleus labelling was maintained if two C-terminal GR/ ZnF regions were deleted (AtCSP2–CSD::GFP) (Fig. 6.3B, i). One possible functional model is that AtCSP2 targets cytoplasmic free mRNA and modulates its translation or degradation. The nucleolar localization of AtCSP2 suggests that AtCSP2 may be involved in nuclear RNA-

processing events as well. We found that one of the C-terminal GR and ZnF motifs is necessary for nucleolus localization, suggesting AtCSP2 may be localized to the nucleolus through a protein–protein interaction.

Conclusion The function of cold-shock domain proteins was studied in Arabidopsis. Expression and functional analyses suggest that AtCSP2 is an RNA chaperone involved in multiple steps of plant development and in adaptation to cold. Evolutionary conserved RNA chaperone activity of cold-inducible cold-shock domain proteins suggests that this activity is necessary for cold acclimation in both bacteria and higher plants. Further physiological studies employing transgenic overexpressors and knockout mutants will be needed to elucidate the function of individual cold-shock domain proteins. We speculate that all AtCSPs have a common RNA chaperone activity and are possibly involved in different processes such as cold acclimation, vernalization and development.

References Bader, A.G. and Vogt, P. K. (2004) An essential role for protein synthesis in oncogenic cellular transformation. Oncogene 23, 3145–3150. Bae, W., Xia, B., Inouye, M. and Severinov, K. (2000) Escherichia coli CspA-family RNA chaperones are transcription antiterminators. Proceedings of the National Academy of Sciences USA 97, 7784–7789. Becker, J. and Craig, E.A. (1994) Heat-shock proteins as molecular chaperones. European Journal of Biochemistry 219, 11–23. Didier, D.K., Schiffenbauer, J., Woulfe, S.L., Zacheis, M. and Schwartz, B.D. (1988) Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box. Proceedings of the National Academy of Sciences USA 85, 7322–7326. Evdokimova, V.M. and Ovchinnikov, L.P. (1999) Translational regulation by Y-box transcription factor: involvement of the major mRNA-associated protein, p50. International Journal of Biochemistry & Cell Biology 31, 139–149. Fukada, T. and Tonks, N.K. (2003) Identification of YB-1 as a regulator of PTP1B expression: implications for regulation of insulin and cytokine signaling. EMBO Journal 22, 479–493. Fusaro, A.F., Bocca, S.N., Ramos, R.L., Barroco, R.M., Magioli, C., Jorge, V.C., Coutinho, T.C., Rangel-Lima, C.M., De Rycke, R., Inze, D., Engler, G. and Sachetto-Martins, G. (2007) AtGRP2, a cold-induced nucleocytoplasmic RNA-binding protein, has a role in flower and seed development. Planta 225, 1339–1351. Goldstein, J., Pollitt, N.S. and Inouye, M. (1990) Major cold shock protein of Escherichia coli. Proceedings of the National Academy of Sciences USA 87, 283–287. Jiang, W., Hou, Y. and Inouye, M. (1997) CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. Journal of Biological Chemistry 272, 196–202. Karlson, D. and Imai, R. (2003) Conservation of the cold shock domain protein family in plants. Plant Physiology 131, 12–15.

Characterization of Arabidopsis Cold-shock Domain Proteins

61

Karlson, D., Nakaminami, K., Toyomasu, T. and Imai, R. (2002) A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins. Journal of Biological Chemistry 277, 35248–35256. Kim, J.S., Park, S.J., Kwak, K.J., Kim, Y.O., Kim, J.Y., Song, J., Jang, B., Jung, C.H. and Kang, H. (2007) Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli. Nucleic Acids Research 35, 506–516. Mertens, P.R., Harendza, S., Pollock, A.S. and Lovett, D.H. (1997) Glomerular mesangial cell-specific transactivation of matrix metalloproteinase 2 transcription is mediated by YB-1. Journal of Biological Chemistry 272, 22905–22912. Minich, W.B. and Ovchinnikov, L.P. (1992) Role of cytoplasmic mRNP proteins in translation. Biochimie 74, 477–483. Moss, E.G., Lee, R.C. and Ambros, V. (1997) The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA. Cell 88, 637–646. Nakaminami, K., Sasaki, K., Kajita, S., Takeda, H., Karlson, D., Ohgi, K. and Imai, R. (2005) Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein. FEBS Letters 579, 4887–4891. Nakaminami, K., Karlson, D.T. and Imai, R. (2006) Functional conservation of cold shock domains in bacteria and higher plants. Proceedings of National Academy of Sciences USA 103, 10122–10127. Norman, J.T., Lindahl, G.E., Shakib, K., En-Nia, A., Yilmaz, E. and Mertens, P.R. (2001) The Y-box binding protein YB-1 suppresses collagen a 1(I) gene transcription via an evolutionarily conserved regulatory element in the proximal promoter. Journal of Biological Chemistry 276, 29880–29890. Ranjan, M., Tafuri, S.R. and Wolffe, A.P. (1993) Masking mRNA from translation in somatic cells. Genes & Development 7, 1725–1736. Sommerville, J. and Ladomery, M. (1996) Masking of mRNA by Y-box proteins. FASEB Journal 10, 435–443. Sung, S. and Amasino, R.M. (2004) Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 427, 159–164. Tanabe, H., Goldstein, J., Yang, M. and Inouye, M. (1992) Identification of the promoter region of the Escherichia coli major cold shock gene, cspA. Journal of Bacteriology 174, 3867–3873. Ting, J.P., Painter, A., Zeleznik-Le, N.J., Macdonald, G., Moore, T.M., Brown, A. and Schwartz, B.D. (1994) YB-1 DNA-binding protein represses interferon g activation of class II major histocompatibility complex genes. Journal of Experimental Medicine 179, 1605–1611. Wang, N., Yamanaka, K. and Inouye, M. (1999) CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock. Journal of Bacteriology 181, 1603–1609. Wolffe, A.P. (1994) Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins. BioEssays 16, 245–251. Yamanaka, K., Fang, L. and Inouye, M. (1998) The CspA family in Escherichia coli: multiple gene duplication for stress adaptation. Molecular Microbiology 27, 247–255.

7

Plasma Membrane and Plant Freezing Tolerance: Possible Involvement of Plasma Membrane Microdomains in Cold Acclimation A. Minami, Y. Kawamura, T. Yamazaki, A. Furuto and M. Uemura

Abstract Cold acclimation-induced increase in freezing tolerance is associated with diverse changes in the plasma membrane, which ultimately results in an increase in the cryostability of the plasma membrane during a freeze–thaw excursion and an acceleration of the recovery process after thawing. Many reports clearly show the occurrence of alterations in the plasma membrane during cold acclimation and/or a freeze–thaw cycle. Recently, there is accumulating evidence supporting the presence of plasma membrane microdomains in both animal and plant cells. These microdomains contain lipid and protein composition distinct from other parts of the plasma membrane. Thus, we revisit our past findings of the plasma membrane alterations associated with freezing tolerance by introducing the concept of plasma membrane microdomains. The microdomains in the plasma membrane, which are isolated as detergent-resistant membrane (DRM) fractions, responded to cold acclimation: protein composition in the DRM was altered considerably after cold acclimation. The DRM contained proteins of many important functions including membrane transport, plasma membrane–cell wall interactions (via the cytoskeleton) and membrane trafficking. Based on protein changes in DRMs during cold acclimation, we discuss the function of DRM-localized proteins in cold acclimation and/or freezing tolerance in plants.

Introduction The ability of many temperate plants to tolerate abiotic stresses determines their geographical distribution and maintains productivity. Among these abiotic stresses, low temperatures and freezing are the most critical factors to affect plant performance (Levitt, 1980; Boyer, 1982). Many temperate plants, however, are able to increase their freezing tolerance when exposed to low but non-freezing temperatures, which is known as cold acclimation. During cold acclimation, there are many seemingly disparate responses occurring in many different aspects, including alterations in membrane composition and accumulation of compatible solutes, many of which require new or altered gene expression (Guy, 1990; Thomashow, 1999). Because 62

much evidence supports a consensus that irreversible dysfunction (both structural and functional) of the plasma membrane as a consequence of freeze-induced dehydration is the primary cause of freezing injury (Steponkus, 1984; Steponkus et al., 1993b), cold acclimationinduced changes must contribute to the increase in the cryostability of the plasma membrane during a freeze–thaw excursion. The plasma membrane consists of lipids and proteins. It is well known that plasma membrane lipid composition changes considerably after cold acclimation in many aspects (Steponkus et al., 1993a). These changes include those at the lipid class level (such as phospholipids, sterols and glucocerebrosides) and at the molecular species level in two major phospholipids, phosphatidylcholine and phos-

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Plasma Membrane and Plant Freezing Tolerance

phatidylethanolamine (Lynch and Steponkus, 1987; Uemura et al., 1995). In some cases, the proportion of sterol species changes considerably (Uemura and Steponkus, 1994, 1999). In fact, lipid alterations in the plasma membrane during cold acclimation result in an increase in the cryostability of the plasma membrane and, hence, an increase in survival of plant cells. This is demonstrated by membrane-engineering studies in which protoplasts isolated from non-acclimated plants (rye and Arabidopsis) were fused with liposomes containing unsaturated species of phosphatidylcholine, resulting in an enrichment of unsaturated phosphatidylcholine species in the plasma membrane (in other words, the plasma membrane becomes similar to that of protoplasts isolated from cold-acclimated plants), leading to increased survival of the protoplasts after a freeze–thaw cycle (Steponkus et al., 1988; Uemura and Steponkus 1989; Uemura et al., 1995). Therefore, the behaviour and more importantly the functional stability of the plasma membrane under freeze-induced dehydration are clearly affected by the lipid composition in the plasma membrane. For plasma membrane proteins, there are only a few reports showing a clear vision of changes induced during cold acclimation. Kawamura and Uemura (2003) carried out the proteome analysis of plasma membrane proteins with non-acclimated and coldacclimated Arabidopsis leaves using a peptide mass fingerprinting method coupled with MS. They found approximately 30 proteins that alter in quantity during cold acclimation, and named them as cold-responsive plasma membrane proteins. Except a few, it is still to be determined whether these cold-responsive plasma membrane proteins are functionally associated with an increase in freezing tolerance during cold acclimation. Recently, a new structure of the plasma membrane, ‘membrane microdomains’ (also known as ‘lipid rafts’ in some cases), has been proposed in animals and microorganisms (Kusumi et al., 2005; Lillemeier et al., 2006). Although the plasma membrane has been described as a fluid mosaic of lipids and proteins (Singer and Nicolson, 1972), the membrane microdomain concept assumes that membrane lipids and proteins move in specific ways and

63

form various regions with distinct lipid and protein compositions. The formation of microdomains is considered to be dependent on sphingolipids and sterols. Because sphingolipids and sterols are more resistant to detergent extraction than phospholipids (Schroeder et al., 1994), sphingolipid/sterol-enriched microdomains, which are believed to be about 50 to 200 nm in diameter (Anderson and Jacobson, 2002), can be biochemically isolated as detergent-resistant membrane (DRM) fractions from plasma membrane preparations. The protein composition of the DRM fraction in animal cells is different from that of bulk plasma membrane preparations and contains receptor proteins, kinases, G-proteins, actins, flotillins and GPIanchored proteins, suggesting that the microdomains contribute to membrane trafficking, signal transduction and pathogen infection in animal cells (Brown and London, 1998). There is accumulating evidence that indicates the presence of microdomains in the plant plasma membrane. Plant DRMs were isolated and their protein and lipid compositions were found to be consistent with those in animal and microorganism cells (Berczi and Horvath, 2003; Mongrand et al., 2004; Laloi et al., 2007; Lefebvre et al., 2007). Nevertheless, the function of microdomains in the plant plasma membrane has yet to be determined in abiotic stress responses. The present chapter describes work undertaken as a first step to elucidate the function of plasma membrane microdomains in plant cold acclimation/freezing tolerance. We tried to isolate DRMs from plasma membrane fractions prepared from Arabidopsis seedlings and to elucidate the effect of cold acclimation on their biochemical characteristics.

Results and Discussion Preparation of microdomains from isolated plasma membrane fractions Figure 7.1 outlines the protocol for obtaining plasma membrane microdomains as DRM fractions. First, we prepared plasma membrane fractions from Arabidopsis seedlings using an aqueous two-polymer phase partition system (Uemura et al., 1995; Kawamura and Uemura,

64

A. Minami et al.

1) Arabidopsis thaliana L. (Columbia) seedlings Cold acclimation (2°C, 12 h light/12 h dark) Aqueous two-polymer phase system

2) Plasma membrane (PM) enriched fractions 1% Triton X-100 (4°C, 30 min) PM fraction

5% 30% 35% 52%

Sucrose density gradient centrifugation

28,000 rpm for 20 h at 4°C

3) Detergent-resistant plasma membrane (DRM) fractions (≈Sphingolipid-enriched microdomains) Fig. 7.1. Protocol for obtaining detergent-resistant plasma membrane (DRM) fractions from Arabidopsis seedlings. We used Arabidopsis seedlings before or after cold acclimation as experimental materials. Briefly, after highly purified plasma membrane fractions were prepared with an aqueous two-polymer phase partition system (Kawamura and Uemura, 2003), the plasma membrane fractions were subjected to treatment with a non-ionic detergent (e.g. Triton X-100) at 4°C and then to a floating sucrose density gradient centrifugation. After centrifugation, there were two white bands at positions near the interface of 35 and 52% sucrose layers. These two bands showed similar protein profiles and, hence, were combined and designated as DRM fractions.

2003). The two-polymer phase partition system allows us to obtain a highly purified plasma membrane preparation (Widell et al., 1982; Uemura and Yoshida, 1983; Yoshida et al., 1983; Uemura et al., 1995), essential to proceed further in the isolation of microdomains as DRM fractions. Plasma membrane fractions were then treated with 1% (w/v) Triton X-100 at 4°C for 30 min and subsequently subjected to a floating sucrose density gradient centrifugation. After centrifugation, two white bands at the positions near the interface of 35/52% sucrose layers were collected. Because protein profiles after two-dimensional (2D) PAGE showed similar protein patterns in these two bands (data not shown), we designated these bands as DRM. When protein amount in the DRM was determined and compared with the total protein content in the isolated plasma membrane fraction, we found that the recovery of DRM was 10% or less in samples throughout cold acclimation. The recovery of the DRM protein was less in samples prepared after cold acclimation than in samples before cold

acclimation. Because the proportion of a sphingolipid (specifically glucocerebroside), which is known to be an essential lipid component in plasma membrane microdomains (Borner et al., 2005), decreases after cold acclimation in Arabidopsis plasma membrane, the decrease in DRM recovery may be associated with the decrease in glucocerebrosides in the plasma membrane. Alternatively, protein concentrations in microdomains in the plasma membrane may decrease after cold acclimation.

Protein composition of the detergent-resistant membrane and the effect of cold acclimation When we compared protein profiles of the DRM and total plasma membrane fractions, there were distinct differences between the two samples and the number of protein bands in the DRM seemed to be much less than that of the plasma membrane (Fig. 7.2). For example, the band intensities of proteins at 102,

Plasma Membrane and Plant Freezing Tolerance

79, 70, 52, 50, 30, 29 and 25 kDa (with arrowheads in Fig. 7.2) were apparently stronger in DRM than in the plasma membrane. We have identified these DRM-enriched proteins using MS techniques and have so far successfully found that the 102- and 25-kDa proteins are P-type H+-ATPase and aquaporins, respectively. Western blot analyses further confirmed that DRMs were enriched in specific protein sets (Fig. 7.3). The P-type H+-ATPase is reported as a protein enriched in plasma membrane microdomains in yeast (Bagnat et al., 2001; Lee et al., 2002). In fact, we concluded that Arabidopsis microdomains are

65

enriched in P-type H+-ATPase. SKU5 protein, a DRM-enriched protein (Sedbrook et al., 2002; Borner et al., 2005), was eventually detected in DRM but also in plasma membrane preparations. Thus, we could not confirm SKU5 as a DRM-enriched protein. β-Tubulin (Mongrand et al., 2004), band-7 protein (Shahollari et al., 2005) and aquaporins (Borner et al., 2005) are all DRM-enriched proteins. Using Western blotting, we confirmed that these proteins were in fact enriched in (or almost exclusively in the case of band-7 protein) DRM. The results shown in

NA (kDa) 200 150 120 100 85

PM

DRM

PM

DRM P-type H+-ATPase

SKU5 70 60

β-TUB

50

Band-7

40

Aquaporin

30

25

Fig. 7.2. Protein composition of total plasma membrane (PM) and detergent-resistant membrane (DRM) fractions prepared from non-acclimated Arabidopsis seedlings. Non-acclimated seedlings were maintained at 23°C under continuous illumination at 50 mmol/m2/s for about 3 weeks. After PM and DRM fractions were prepared according to the protocol shown in Fig. 7.1, the same amount of protein samples were subjected to one-dimensional SDS gel electrophoresis and protein bands were then visualized by the silverstaining method. Protein bands that are enriched in the DRM sample are indicated by arrowheads.

Fig. 7.3. Western blot analysis of proteins enriched in the detergent-resistant plasma membrane (DRM) fraction obtained from Arabidopsis plasma membrane (PM) fraction. After PM and DRM proteins prepared from non-acclimated (NA) seedlings were subjected to gel electrophoresis and then electron transfer to a PVDF membrane, antibodies specific for each protein were applied and detected. Antibodies employed (top to bottom) are tobacco P-type H+-ATPase (Morsomme et al., 1998), SKU5 (Sedbrook et al., 2002), b-tubulin T5201 (Sigma-Aldrich, USA), band-7 protein (Minami et al., 2009) and aquaporin (Ohshima et al., 2001). After reaction with antibodies, the PVDF membrane was washed, incubated with HRPconjugated goat anti-rabbit or anti-mouse IgG (H+L) secondary antibodies (1:2,000 dilution; PIERCE, USA) in blocking buffer, and then detected using SuperSignal West Femto Maximum Sensitivity Substrate (PIERCE, USA). Images were captured with a LightCapture system (ATTO, Japan).

66

A. Minami et al.

Figs 7.2 and 7.3 indicate that DRMs contain specific sets of proteins, which apparently are different from protein profiles of the total plasma membrane preparations. Protein profiles of DRM fractions changed significantly during cold acclimation (Fig. 7.4). One-dimensional SDS gel electrophoresis patterns easily allowed us to identify several proteins either induced or decreased by cold acclimation (see arrowheads in Fig. 7.4). However, it is not so easy to isolate coldresponsive proteins on the gels due to molecular weight similarity.

DRM NA

CA2

(kDa) 200 150 120 100 85 70 60 50 40

30

To further compare protein profiles of the plasma membrane microdomains before and after cold acclimation, we separated proteins in DRM fractions using 2D-PAGE, stained with silver and then analysed spot patterns on gels of non-acclimated and 2-day-acclimated samples (Fig. 7.5). We could identify a few hundred spots on each gel, among which about 200 spots were always detected on gels regardless of temperature treatments. In Fig. 7.5, we have marked protein spots which either increased or decreased after cold acclimation. In addition, we carried out 2D fluorescence differential gel electrophoresis to accurately analyse quantitative differences in expression patterns of DRM proteins during cold acclimation (Minami et al., 2009). Two-dimensional fluorescence difference gel electrophoresis is a technique that allows us to compare protein profiles of several different samples separated on the same gel using three different fluorescent dyes and has been successfully applied for plant DRMs (Borner et al., 2005). The 2D fluorescence difference gel electrophoresis further confirmed that proteins in DRMs changed dynamically both in quantitative and qualitative ways during cold acclimation. After 2D-PAGE separation, we identified the cold-responsive proteins using MS methods as described above (Minami et al., 2009). Preliminarily, we identified about 30 proteins and then classified these proteins according to function deduced from a database search. The categories included membrane transport, cytoskeleton structure, intracellular vesicular trafficking, plasma membrane and cell–wall reconstruction, and undetermined but reported as those associated with plasma membrane microdomain.

25 Fig. 7.4. Alterations in the protein composition of detergent-resistant plasma membrane (DRM) fractions prepared from Arabidopsis seedlings before (NA) and after 2 days of cold acclimation (CA2; at 2°C under an 8 h photoperiod condition at 75 µmol/m2/s). After DRM fractions were prepared from seedlings according to the protocol shown in Fig. 7.1, the same amount of protein samples were subjected to one-dimensional SDS gel electrophoresis and protein bands were then visualized by the silver-staining method. Major protein bands that changed during cold acclimation are indicated by arrowheads.

Microdomain protein function in plant cold acclimation Because DRM fractions from animal cells contain functionally important proteins, such as receptor proteins, kinases, G-proteins, actins and others, it is believed that the plasma membrane microdomains are functionally associated with several key processes in cellular systems including membrane trafficking, signal transduction and pathogen infection (Brown

Plasma Membrane and Plant Freezing Tolerance

(kDa) 200 150 120

NA pI

3

67

CA2 11

pI

3

11

100 85 70 60 50 40

30 25

Fig. 7.5. Two-dimensional (2D) gel electrophoresis patterns of proteins in the detergent-resistant plasma membrane (DRM) fractions prepared from Arabidopsis seedlings before (NA) and after cold acclimation for 2 days (CA2). After DRM fractions were prepared from seedlings according to the protocol shown in Fig. 7.1, the same amount of protein samples were subjected to 2D-PAGE and protein spots were then visualized by the silver-staining method. Protein spots that decreased (on NA gel) or increased (on CA2 gel) are indicated by arrows.

and London, 1998). DRM fractions have been also isolated from plants and their protein and lipid compositions have been reported. Sphingolipids (primarily glucocerebrosides) and sterols (primarily free sterols) are the major lipid components in plant DRMs (Berczi and Horvath, 2003; Mongrand et al., 2004; Borner et al., 2005; Laloi et al., 2007; Lefebvre et al., 2007). Our preliminary analysis of the lipid composition of Arabidopsis DRMs is consistent with results in the literature. That is, the proportions of glucocerebrosides and free sterols on the protein basis were greater in the DRM than in the total plasma membrane fraction (Minami et al., 2009). Thus, our DRM preparations were suitable for further analysis of functional/compositional aspects of microdomains in plant cold acclimation. Proteomic analysis of plant DRMs has suggested that plant microdomains actively play a role in signal transduction, membrane trafficking and cell wall metabolism (Peskan et al., 2000; Mongrand et al., 2004; Shahollari et al., 2004; Bhat and Panstruga, 2005; Borner et al., 2005; Morel et al., 2006; Laloi

et al., 2007; Lefebvre et al., 2007). The results of protein composition of DRMs presented here agreed with this notion. Furthermore, during cold acclimation, we observed dynamic changes in protein compositions in DRM fractions (Figs 7.2 to 7.5). For example, P-type H+-ATPase activity has been reported to increase after cold acclimation although the amount of P-type H+-ATPase protein per se was not determined (Puhakainen et al., 1999; Martz et al., 2006). Here, we showed that the ATPase protein increased in the DRM fractions, meaning that either ATPase is up-regulated by cold or the distribution of ATPase in the plasma membrane (i.e., within microdomains or in the extra-microdomain region) is modified by cold. We are currently investigating this question. Another notable membrane transport protein that is affected by cold acclimation is aquaporin. It is well known that cold treatment considerably affects the water status of plants, which is believed to be associated, at least in part, with aquaporin activity on the plasma membrane. Expression of aquaporin genes under cold conditions in general decreases under low temperatures

68

A. Minami et al.

except for a few aquaporin family members (Jang et al., 2004; Sakurai et al., 2005). However, our results indicate that aquaporins eventually increased in the DRM fraction after cold acclimation. This is probably due to the distributional changes of aquaporins in the plasma membrane after cold acclimation. Although it is not clearly understood why aquaporin distribution in the plasma membrane is altered by cold, an increase in the proportion of aquaporins in plant plasma membrane microdomains may efficiently lead to improved osmotic water permeability at low temperatures and under freezing conditions, and hence increase survival under these conditions. Plant cold acclimation is closely associated with reconstitution of membrane components both in lipid and protein populations (Steponkus, 1984; Uemura and Steponkus, 1999; Uemura et al., 2006). This process must be accompanied with membrane cycling pathways that actively occur at low temperature. It is well known that plasma membrane cycling is in part governed by exocytotic and endocytotic membrane trafficking systems (Battey et al., 1999). It has been reported that membrane trafficking-associated proteins are found in DRMs in plants (Low and Chandra, 1994; Mongrand et al., 2004; Morel et al., 2006). In fact, the present work also revealed that there are several membrane traffickingassociated proteins in Arabidopsis DRMs, including clathrin and dynamines, the amounts of which are altered by exposure to cold (Minami et al., 2009). From these results, we think that cold treatment regulates the endocytosis pathway through the activity of membrane trafficking-associated proteins and, hence, alters lipid and protein compositions in the plasma membrane, which is essential to cell survival under freezing conditions. The cytoskeleton is one of the important factors that determine the cryobehaviour of plant cells. Cytoskeleton consists of microtubules that are composed of α- and β-tubulin heterodimers and actin filaments and plays an important role in the development of freezing tolerance. The role of cytoskeleton modifications in low-temperature tolerance in plants (Kerr and Carter, 1990; Chu et al., 1993; Ishikawa, 1996) has been studied. These reports suggest that the degree of depolymeri-

zation/polymerization of microtubules affects low-temperature tolerance in plants. In addition, cytoskeleton reorganization has been reported to be an integral component in lowtemperature signal transduction in plant cells, which may transfer signals from membrane rigidification to Ca2+ influx during cold acclimation (Orvar et al., 2000). Taken together with the present results, reorganization of the plasma membrane and/or regulation of plasma membrane fluidity through cytoskeleton alterations occur in microdomains in the plasma membrane at low temperatures, which is necessary to maintain the survival of plant cells.

Conclusions We successfully demonstrated that plasma membrane microdomains of Arabidopsis cells respond to cold treatment and change in their protein compositions. As with plasma membrane microdomains in animal cells, plant microdomains (isolated as detergent-resistant membrane fraction, DRM fraction) are considered to have important roles in essential cellular functions such as membrane transport, cell wall–plasma membrane interactions through the cytoskeleton, intracellular membrane trafficking, and others. Protein alterations in microdomains during cold acclimation suggest that microdomains are closely associated with alterations in cellular responses to and acclimation of cells at low temperatures. We are in progress to investigate the functions of microdomain-associated proteins in cold acclimation and freezing tolerance in planta via various approaches in molecular biology. These studies, together with the present results, will lead us into a new research area in functional and structural aspects of the plasma membrane in plant cold acclimation.

Acknowledgements This study was supported in part by Grantsin-Aid from the Ministry of Education, Sports, Science and Technology of Japan and Iwate University. We thank laboratory members who helped us throughout the course of study.

Plasma Membrane and Plant Freezing Tolerance

69

References Anderson, R.G. and Jacobson, K. (2002) A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296, 1821–1825. Bagnat, M., Chang, A. and Simons, K. (2001) Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast. Molecular Biology of the Cell 12, 4129–4138. Battey, N.H., James, N.C., Greenland, A.J. and Brownlee, C. (1999) Exocytosis and endocytosis. The Plant Cell 11, 643–660. Bhat, R.A. and Panstruga, R. (2005) Lipid rafts in plants. Planta 223, 5–19. Berczi, A. and Horvath, G. (2003) Lipid rafts in the plant plasma membrane? Acta Biologica Szeged 47, 7–10. Borner, G.H., Sherrier, D.J., Weimar, T., Michaelson, L.V., Hawkins, N.D., Macaskill, A., Napier, J.A., Beale, M.H., Lilley, K.S. and Dupree, P. (2005) Analysis of detergent-resistant membranes in Arabidopsis: evidence for plasma membrane lipid rafts. Plant Physiology 137, 104–116. Boyer, J.S. (1982) Plant productivity and environment. Science 218, 443–448. Brown, D.A. and London, E. (1998) Structure and origin of ordered lipid domains in biological membranes. Journal of Membrane Biology 164, 103–114. Chu, B., Snustad, D.P. and Carter, J.V. (1993) Alteration of b-tubulin gene expression during low-temperature exposure in leaves of Arabidopsis thaliana. Plant Physiology 103, 371–377. Guy, C.L. (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 41, 187–223. Ishikawa, H.A. (1996) Ultrastructural features of chilling injury: injured cells and the early events during chilling of suspension-cultured mung bean cells. American Journal of Botany 83, 825–835. Jang, J.Y., Kim, D.G., Kim, Y.O., Kim, J.S. and Kang, H.S. (2004) An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Molecular Biology 54, 713–725. Kawamura, Y. and Uemura, M. (2003) Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation. The Plant Journal 36, 141–154. Kerr, G.P. and Carter, J.V. (1990) Relationship between freezing tolerance of root-tip cells and cold stability of microtubules in rye (Secale cereale L. cv. Puma). Plant Physiology 93, 77–82. Kusumi, A., Nakada, C., Ritchie, K., Murase, K., Suzuki, K., Murakoshi, H., Kasai, R.S., Kondo, J. and Fujiwara, T. (2005) Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annual Review of Biophysics and Biomolecular Structure 34, 351–378. Laloi, M., Perret, A.M., Chatre, L., Melser, S., Cantrel, C., Vaultier, M.N., Zachowski, A., Bathany, K., Schmitter, J.M., Vallet, M., Lessire, R., Hartmann, M.A. and Moreau, P. (2007) Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells. Plant Physiology 143, 461–472. Lee, M.C., Hamamoto, S. and Schekman, R. (2002) Ceramide biosynthesis is required for the formation of the oligomeric H+-ATPase Pma1p in the yeast endoplasmic reticulum. Journal of Biological Chemistry 277, 22395–22401. Lefebvre, B., Furt, F., Hartmann, M.A., Michaelson, L.V., Carde, J.P., Sargueil-Boiron, F., Rossignol, M., Napier, J.A., Cullimore, J., Bessoule, J.J. and Mongrand, S. (2007) Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system. Plant Physiology 144, 402–418. Levitt, J. (1980) Responses of Plants to Environmental Stresses, 2nd edn. Academic Press, New York, New York. Lillemeier, B.F., Pfeiffer, J.R., Surviladze, Z., Wilson, B.S. and Davis, M.M. (2006) Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proceedings of the National Academy of Sciences USA 103, 18992–18997. Low, P.S. and Chandra, S. (1994) Endocytosis in plants. Annual Review of Plant Physiology and Plant Molecular Biology 45, 609–631. Lynch, D.V. and Steponkus, P.L. (1987) Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv. Puma). Plant Physiology 83, 761–767. Martz, F., Sutinen, M.L., Kivineemi, S. and Palta, J.P. (2006) Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation. Tree Physiology 26, 783–790.

70

A. Minami et al.

Minami, A., Fujiwara, M., Furuto, A., Fukao, Y., Yamashita, T., Kamo, M., Kawamura, Y. and Uemura, M. (2009) Alterations in detergent-resistant plasma membrane microdomains in Arabidopsis thaliana during cold acclimation. Plant & Cell Physiology 50, 341–359. Mongrand, S., Morel, J., Laroche, J., Claverol, S., Carde, J.P., Hartmann, M.A., Bonneu, M., Simon-Plas, F., Lessire, R. and Bessoule, J.J. (2004) Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane. Journal of Biological Chemistry 279, 36277–36286. Morel, J., Claverol, S., Mongrand, S., Furt, F., Fromentin, J., Bessoule, J.J., Blein, J.P. and Simon-Plas, F. (2006) Proteomics of plant detergent-resistant membranes. Molecular & Cellular Proteomics 5, 1396–1411. Morsomme, P., Dambly, S., Maudoux, O. and Boutry, M. (1998) Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region. Journal of Biological Chemistry 273, 34837–34842. Ohshima, Y., Iwasaki, I., Suga, S., Murakami, M., Inoue, K. and Maeshima, M. (2001) Low aquaporin content and low osmotic water permeability of the plasma and vacuolar membranes of a CAM plant Graptopetalum paraguayense: comparison with radish. Plant & Cell Physiology 42, 1119–1129. Orvar, B.L., Sangwan, V., Omann, F. and Dhindsa, R.S. (2000) Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity. The Plant Journal 23, 785–794. Peskan, T., Westermann, M. and Oelmuller, R. (2000) Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants. European Journal of Biochemistry 267, 6989–6995. Puhakainen, T., Pihakaski-Maunsbach, K., Widell, S. and Sommarin, M. (1999) Cold acclimation enhances the activity of plasma membrane Ca2+ ATPase in winter rye leaves. Plant Physiology and Biochemistry 37, 231–239. Sakurai, J., Ishikawa, F., Yamaguchi, T., Uemura, M. and Maeshima, M. (2005) Identification of 33 rice aquaporin genes and analysis of their expression and function. Plant & Cell Physiology 46, 1568–1577. Schroeder, R., London, E. and Brown, D. (1994) Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proceedings of the National Academy of Sciences USA 91, 12130–12134. Sedbrook, J.C., Carroll, K.L., Hung, K.F., Masson, P.H. and Somerville, C.R. (2002) The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth. The Plant Cell 14, 1635–1648. Shahollari, B., Peskan-Berghofer, T. and Oelmuller, R. (2004) Receptor kinases with leucine-rich repeats are enriched in Triton X-100 insoluble plasma membrane microdomains from plants. Physiologia Plantarum 122, 394–403. Shahollaria, B., Varmab, A. and Oelmüller, R. (2005) Expression of a receptor kinase in Arabidopsis roots is stimulated by the basidiomycete Piriformospora indica and the protein accumulates in Triton X-100 insoluble plasma membrane microdomains. Journal of Plant Physiology 162, 945–958. Singer, S.J. and Nicolson, G.L. (1972) The fluid mosaic model of the structure of cell membranes. Science 175, 720–731. Steponkus, P.L. (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology 35, 543–584. Steponkus, P.L., Uemura, M., Balsamo, R.A., Arvinte, T. and Lynch, D.V. (1988) Transformation of the cryobehavior of rye protoplasts by modification of the plasma membrane lipid composition. Proceedings of the National Academy of Sciences USA 85, 9026–9030. Steponkus, P.L., Uemura, M. and Webb, M.S. (1993a) A contrast of the cryostability of the plasma membrane of winter rye and spring oat – two species that widely differ in their freezing tolerance and plasma membrane lipid composition. In: Steponkus, P.L. (ed.) Advances in Low Temperature Biology, Vol. 2. JAI Press, London, pp. 211–312. Steponkus, P.L., Uemura, M. and Webb, M.S. (1993b) Membrane destabilization during freeze-induced dehydration. In: Close, T.J. and Bray, E.A. (eds) Plant Responses to Cellular Dehydration during Environmental Stress. American Society of Plant Physiologists, Rockville, Maryland, pp. 34–47. Thomashow, M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50, 571–599. Uemura, M. and Steponkus, P.L. (1989) Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves. Plant Physiology 91, 1131–1137.

Plasma Membrane and Plant Freezing Tolerance

71

Uemura, M. and Steponkus, P.L. (1994) A contrast of the plasma membrane lipid composition of oat and rye leaves in relation to freezing tolerance. Plant Physiology 104, 479–496. Uemura, M. and Steponkus, P.L. (1999) Cold acclimation in plants: relationship between the lipid composition and the cryostability of the plasma membrane. Journal of Plant Research 112, 245–254. Uemura, M. and Yoshida, S. (1983) Isolation and identification of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv. Puma). Plant Physiology 73, 586–597. Uemura, M., Joseph, R.A. and Steponkus, P.L. (1995) Cold acclimation of Arabidopsis thaliana: effect on plasma membrane lipid composition and freeze-induced lesions. Plant Physiology 109, 15–30. Uemura, M., Tominaga, Y., Nakagawara, C., Shigematsu, S., Minami, A. and Kawamura, Y. (2006) Responses of the plasma membrane to low temperatures. Physiologia Plantarum 126, 81–89. Widell, S., Lundborg, T. and Larsson, C. (1982) Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system: on the use of light-induced cytochrome b reduction as a marker for the plasma membrane. Plant Physiology 70, 1429–1435. Yoshida, S., Uemura, M., Niki, T., Sakai, A. and Gusta, L.V. (1983) Partition of membrane particles in aqueous two-polymer phase systems and its practical use for purification of plasma membranes from plants. Plant Physiology 72, 105–114.

8

Global Expression of Cold-responsive Genes in Fruit Trees C.L. Bassett and M.E. Wisniewski

Introduction When the techniques of molecular biology were first applied to plant systems, the focus was on genes and genomes of easy access; for example, restriction enzyme mapping of chloroplast genomes and the isolation and characterization of abundant genes, such as those encoding proteins associated with photosynthesis. As new methods became available, researchers exploited those that provided the means to address global aspects of gene regulation. As a result, in plants subjected to various treatments or at different developmental stages, suites of genes with similar regulatory relationships were identified. Such studies, using large expressed sequence tag (EST) libraries, two-dimensional protein separation in combination with matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) MS, microarray analyses, differential display RT-PCR, suppression subtractive hybridization (SSH) and high-throughput sequencing strategies (massively parallel signature sequencing (MPSS) ), have proved invaluable in modelling the expression of many genes simultaneously. The results have provided new insights into the enormous complexity of gene responses to environmental and biotic stresses, to signalling pathways related to hormonal and chemical cues and to developmental progression.

72

The use of global techniques to identify plant genes that respond to low temperature is a relatively recent application, and as expected, initial studies were conducted in herbaceous plants. For example, Fowler and Thomashow (2002) described the Arabidopsis transcriptome during cold acclimation. Similarly, Kreps et al. (2002) reported changes in Arabidopsis gene expression in response to several abiotic stress treatments, including cold. Using fulllength cDNA microarrays, Seki et al. (2002) determined transcriptome differences for 7000 genes in Arabidopsis exposed to drought, cold and salt stresses. Changes in gene expression in Arabidopsis during cold treatment have also been monitored with SAGE (serial analysis of gene expression) technology (Jung et al., 2003). The Institute for Genomic Research (TIGR) maintains databases for global gene expression studies in plants. A list of available plant transcript assemblies (approximately 140 species are represented) and databases can be found at http://www.tigr.org/plantProjects. shtml#. Woody perennials in temperate zones must acclimate to overwinter under often harsh environmental conditions. Exposure to sublethal low temperatures enables plants to survive a freezing event, a phenomenon known as cold acclimation. Although cessation of tree growth in winter (endodormancy) is most often

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Global Expression of Cold-responsive Genes

associated with decreasing day length, some trees apparently rely almost exclusively on cold acclimation to initiate the process (Heide and Prestrud, 2005). In fact, many crop and ornamental species have incorporated a requirement for cold exposure not only to acclimate to overwintering, but also to allow bud break in the spring. If this chilling requirement is not met, bud break will be delayed or sporadic. When the chilling requirement is met, the plant is said to have transitioned from endodormancy to ecodormancy (Lang et al., 1987). As a result, there is considerable interest in understanding how plants acclimate to very low temperatures, as well as how they are released from endodormancy. Dissecting the development of cold acclimation and the achievement of and release from endodormancy are complicated by the frequently observed overlap in gene expression between these processes. To understand these processes requires a global approach to identify the genes associated with each process and to define their patterns of expression in time and space.

Malus Protein profiles An early study of protein profiles in apple fruit was conducted to determine what changes might be associated with low-temperature breakdown (a physiological disorder of apple fruit stored for long periods below 3°C) at 0°C compared with storage at 4°C (Cregoe et al., 1993). Fruit were also held at 20°C for control comparison. Using one-dimensional (1D) SDSPAGE very few changes in proteins were detected as a consequence of exposure to 0 or 4°C. A band at ~68 kDa appeared in the sample exposed to 0°C for 10 weeks, but no other changes were observed. RNA blot analysis of the same stored fruits showed mixed results with clones representing known cold-responsive genes. This was partly due to the difficulty of obtaining high-quality RNA from the fruit. Immunoblot analysis of the expression of dehydrins and the Hsp70 protein family was undertaken in bark collected from eight species

73

of woody plants, including apple and peach (Wisniewski et al., 1996). Some polypeptides immunologically related to dehydrins were observed to be constitutively expressed, while others showed specific seasonal patterns of expression associated with cold acclimation. Hsp70 polypeptides were more variable in their seasonal patterns of expression.

Transcript profiles Although several studies on floral bud and fruit development in apple have employed global approaches to identify genes associated with these processes, few studies documenting global changes in gene expression in response to low temperature have been reported. Wisniewski et al. (2008) examined expression of ESTs in libraries generated from coldexposed or control apple leaf, bark and xylem tissues. As has been previously observed in other plants, more genes were up-regulated by low temperature than down-regulated. A substantial number of different genes were upregulated in bark compared with leaf and xylem tissues. These included defence-related genes like chalcone synthase and dehydrins, as well as a large number of genes associated with protein and nucleic acid metabolism (e.g. translation initiation factors, mRNA-binding proteins and histones). Genes up-regulated by cold in leaves tended to fall into the classes of energy/general metabolism and photosynthesis. Cold-responsive genes in xylem consisted of the general metabolism and cell trafficking classes; some ‘unclassified’ genes were also upregulated in the xylem as opposed to leaf or bark tissues. Some of the expression differences noted in the various apple tissues may reflect the ‘behaviour’ of tissues which are more annual-like (leaves) than perennial (bark and xylem). Genes down-regulated after cold treatment showed a distinct preference for annual or perennial tissues, as there was complete overlap between bark and xylem patterns, but little or no overlap in ESTs between these two tissues and leaf. In leaves, most of the down-regulated genes were in the general

74

C.L. Bassett and M.E. Wisniewski

metabolism and photosynthetic classes; as one might expect, the down-regulated photosynthetic genes in leaves were different from those that were up-regulated. Most of the observations with reference to apple EST abundance in response to cold were similar to studies of other plants as to the identities of genes responding and the direction of the response (Fowler and Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002; Jung et al., 2003).

Prunus Protein profiles Among the earliest global studies on cold exposure in peach were those of Lang and Tao (1990, 1991) who examined changes in flower bud soluble protein profiles related to endodormancy. They observed two proteins (81 and 89 kDa) that increased late in the chilling period and two (18 and 61 kDa) that decreased (Lang and Tao, 1990). Additional studies (Lang and Tao, 1992) revealed a 62-kDa protein that decreased in response to chilling temperatures in peach floral buds and xylem, but was absent in other woody fruit crops, such as grape and apple. The technology for identifying specific polypeptide bands was not readily available at the time these experiments were conducted, making it impossible to identify the polypeptides based solely on molecular size, particularly since post-translational modifications could potentially affect their size and/or electrophoretic mobility. Around the same time, Arora et al. (1992) reported changes in 1D SDS-PAGE polypeptide profiles from the bark and xylem of two sibling peach lines. From a cross between ‘Empress’ dwarf OP and an evergreen peach introduction (P.I. 442380) from Mexico, deciduous and evergreen genotypes were obtained in the F1. The evergreen (also known as evergrowing) genotype exhibited continuous terminal growth, indicating failure to achieve endodormancy. Furthermore, this line did not develop cold hardiness until late October; in comparison, the deciduous sibling developed cold hardiness in mid-September. Examining

the seasonal pattern of protein profiles from both genotypes revealed two polypeptides (19 kDa and 60 kDa) that were maximally expressed during December and January. The authors speculated that these two polypeptides might be associated more with cold acclimation than with endodormancy per se, since their expression pattern was similar in both genotypes. In follow-up studies, the 60-kDa protein (PpDhn1) was identified as a member of the dehydrin family of LEA (late embryogenesisabundant) proteins (Arora and Wisniewski, 1994; Wisniewski et al., 2004). The 19-kDa protein appeared to be an allergen similar to Mald1 associated with pathogenesis-related proteins. No proteins matched a third seasonally regulated protein (16 kDa) which may actually be a storage protein based on its pattern of expression (Wisniewski et al., 2004). Similar results were obtained by Gomez and Faurobert (2002) in a study of seasonal expression of vegetative storage proteins in peach. They identified polypeptides of ~19 kDa and 16.5 kDa that they considered to be storage proteins mobilized during spring growth. However, it is not known whether these proteins are the same as those identified by Wisniewski et al. (2004), since they were identified by molecular size alone. Likewise, polypeptides of 19, 18 and 16.5 kDa were identified from phloem and parenchyma tissues from four varieties of apricot (Prunus armeniaca) and were shown to vary during the annual cycle like the low-molecular-weight polypeptides from peach (Faurobert and Gomez, 2006). Interestingly, one of the varieties showed a substantial increase in a 58-kDa band during winter, but not summer. This band may represent a homologue of the peach PpDhn1. A similar study of floral buds in a high chill-requiring cultivar of the Japanese apricot (Prunus mume) (Tao, 2005) identified a 65-kDa dehydrin that peaked in parallel with increasing chilling accumulation and dropped to pre-chill levels after the chilling requirement was met. A low chill-requiring cultivar expressed the dehydrin at much lower levels and over a longer period of time. Taken together, these results indicate that the ~60-kDa dehydrin is associated more with cold acclimation than with the development of endodormancy in peach and apricot.

Global Expression of Cold-responsive Genes

A recent study using quantitative proteomics of peach bark tissues exposed to low temperature (LT) under different photoperiods documented a number of changes in polypeptide abundance in response to cold or short days (SD). Renaut et al. (2008) used difference in-gel electrophoresis (DIGE) combined with MALDI-TOF to identify polypeptides from peach bark responding to different combinations of temperature and photoperiod. More polypeptides responded to LT than to SD; indeed, the number of polypeptides that increased in response to SD was approximately one-third the number that increased after 3 and 5 weeks of LT exposure. The authors concluded that LT was the most significant single factor affecting polypeptide abundance and that the combination of LT and SD had a synergistic effect. These results are consistent with a study of dehydrin transcript expression in birch (Welling et al., 2004).

Transcript profiles Few studies of global RNA expression in woody plants have been reported to date. Bassett et al. (2006) used SSH to identify peach bark genes whose expression was altered after prolonged exposure to different combinations of temperature (low=5°C; high=25°C) and photoperiod (SD=8 h light/16 h dark; night break=8 h light/8 h dark/15 min light/8 h dark). A number of defence genes were up-regulated by either cold or SD treatments. Among those up-regulated by cold were genes encoding two dehydrins, several chitinases, an mRNA-binding protein and other defence-related proteins. For most of the cold up-regulated sequences, subsequent characterization also indicated that they were seasonally expressed in bark tissues having patterns similar to PpDhn1 (C.L. Bassett, unpublished results). Using SSH, Tao (2005) isolated cDNAs representing genes induced in P. mume floral buds during late endodormancy. Among those identified were genes encoding several cellcycle-related molecules, including a homologue of Cdc20 and a RING (Really Interesting New Gene) finger protein family member that may be associated with degradation of cyclin or cyclin-dependent kinase (Tang et al., 2001).

75

Also found was a Myb family transcription factor; a myb protein is thought to control the level of B-type cyclins (Weston, 1998). It may be that endodormancy is maintained by inhibition of cell-cycle progress in part or whole.

Vaccinium Protein profiles One of the earliest studies of global protein profiles related to chilling in blueberry was reported by Muthalif and Rowland (1994a). Using 1D SDS-PAGE, they identified floral bud protein patterns that changed as a result of chilling exposure. This group of polypeptides (65, 60 and 14 kDa) increased substantially during the first 300 hours of chill unit accumulation and declined when the chilling requirement was met. Interestingly, these polypeptides shared a number of similarities to dehydrins, and a subsequent paper indeed identified them as dehydrins (Muthalif and Rowland, 1994b).

Transcript profiles In order to identify genes linked to cold acclimation in blueberry (Rowland et al., 2003), a cDNA library was prepared from floral buds of fully cold-acclimated plants. Approximately 500 sequences were obtained containing a number of genes previously shown to be expressed in response to cold in other plant systems (Fowler and Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002; Jung et al., 2003). In subsequent studies, EST libraries prepared from cold-acclimated and non-acclimated blueberry floral buds identified nearly 700 ESTs from the cold-acclimated library and 600 from the non-acclimated control (Dhanaraj et al., 2004). Of the approximately 450 unigenes identified in each library, less than 5% were in common between the two treatments. Since the buds were isolated from field grown plants in October (non-acclimated) or December (coldacclimated), it is impossible to separate out genes responding to differences in photoperiod or other environmental parameters in addition

76

C.L. Bassett and M.E. Wisniewski

or in contrast to those responding to cold exposure. However, these differences are more likely to represent actual genes associated with cold acclimation, a conclusion supported by a follow-up study that revealed substantial differences in field-grown versus growth chamberacclimated plants (Dhanaraj et al., 2007). More recently, Naik et al. (2007) used SSH to identify up- and down-regulated genes from dormant flower buds of blueberry compared with buds not exposed to low temperature. In addition to identifying genes from the forward subtraction (up-regulated in response to 400 chill units) that were previously identified in EST libraries, novel genes representing transcription factors and signalling pathway components were found. These results emphasize the power of using multiple approaches to analyse global gene expression.

Citrus Protein profiles An early proteomic study using two-dimensional (2D) PAGE on cold-acclimated (1 week at 5°C) ‘Valencia’ orange (Citrus sinensis) revealed a number of quantitative changes in leaf polypeptide profiles between cold-acclimated and nonacclimated seedlings (Guy et al., 1988). Several polypeptides of low molecular weight increased in response to cold treatment, as did a very large polypeptide spot of about 160 kDa. In addition, low-molecular-weight polypeptides migrating similarly to a small subunit of RuBisCO were observed to decrease in coldacclimated leaves. A subsequent study of translatable RNA in citrus revealed numerous qualitative changes in the 2D SDS-PAGE profiles of polypeptides following cold acclimation (Durham et al., 1991). Comparison between Citrus grandis (pummelo; cold-sensitive) and Poncirus trifoliata (trifoliate orange; cold-hardy) profiles revealed accumulation of a ~160-kDa polypeptide in P. trifoliata, but not in C. grandis. It is not clear if the 160-kDa polypeptide observed in coldacclimated C. sinensis is related to or the same as that seen in P. trifoliata. Vu et al. (1995) examined soluble proteins extracted from leaves of orange seedlings

with different cold-hardy rankings. All varieties examined showed differences in 1D SDSPAGE polypeptide profiles of cold-acclimated (~4°C) compared with heat-acclimated (~32°C) plants. Of note is the observation that all three genotypes showed cold acclimation-enhanced accumulation of the large subunit of RuBisCO, whereas the small subunit was repressed. Likewise, cold-enhanced accumulation of phosphoenolpyruvate carboxylase (PEPCase) was positively associated with the cold-hardy ranking of the genotype, i.e. accumulation was greatest in the cold-hardy P. trifoliata and FF6-7-2 hybrid and least in the moderately cold-hardy C. sinensis.

Transcript profiles Most economically important Citrus species are classified as evergreen fruit trees of tropical/subtropical origin, yet they are frequently grown in regions where freezing temperatures are common. Citrus unshiu is considered to be a relatively cold-hardy commercial species, surviving temperatures of −10°C when fully acclimated. However, its relative, P. trifoliata, can tolerate −26°C. In fact, P. trifoliata is frequently used in citrus breeding programmes to develop cold-hardy lines. Lang et al. (2005) used differential display RT-PCR to identify genes up- and down-regulated by cold in leaves of C. unshiu. Six genes were substantially up-regulated after three weeks of gradual cold treatment (final temperature was one week at 18°C day/7°C night) and two genes were down-regulated. Two ribosomal protein genes increased in response to cold, as well as a 14-3-3 protein implicated in signal transduction. Using the same technique, Zhang et al. (2005) identified genes from P. trifoliata that were both up- and down-regulated by a cold treatment. The genes identified from P. trifoliata as altered in expression were different from those observed for C. unshiu. For example, genes encoding a water channel protein, an early light-inducible protein (ELIP) and a betaine/proline transporter were among those up-regulated by cold in P. trifoliata. The single overlapping gene observed in both species was that encoding the ribosomal protein, L15. Sequences down-regulated by the gradual

Global Expression of Cold-responsive Genes

cold treatment in P. trifoliata were for the most part related to photoprotection and/or photosynthesis (Zhang et al., 2005). Cold-responsive gene expression in P. trifoliata was also monitored using an SSH approach (S¸ahin-Çevik and Moore, 2006). Plants were cold-shocked for 2 days at 4°C and RNA was extracted from leaves for library synthesis. Approximately 67 unigenes were obtained from the up-regulated subtraction; in contrast, fewer genes were obtained from the down-regulated reverse subtraction library and none of them could be identified. Although Zhang et al. (2005) were able to identify downregulated genes from P. trifoliata, their results were based on a gradual cold exposure over a period of weeks. Furthermore, differences in methodology could also account for some of the differences between the two studies. Likewise, there was little or no overlap in the up-regulated genes identified by the two studies. Of interest is the characterization of expression of a bZIP-like transcription factor (TF) isolated from the P. trifoliata up-regulated library. In a timed study following expression of the putative TF after transfer to 4°C, the mRNA encoding this gene became visible on RNA blots after 1 h, and reached maximum expression at around 48 h from the beginning of cold treatment S¸ahin-Çevik and Moore, 2006). This level was maintained for the duration of the experiment (to 7 days). In contrast, the tran-

77

script was barely visible after 8 h in the coldsensitive pummelo (C. grandis) and could not be detected thereafter. Furthermore, in leaves from drought-treated plants of both species, the TF transcript was undetectable, indicating that this gene is associated more with cold stress than with drought.

Summary and Future Research Areas The objective of the present chapter review was to summarize the status of global gene expression analysis in fruit tree research. Literature cited here contains numerous examples of genes up- and down-regulated in response to low temperature, with important overlaps between different fruit tree genera. Table 8.1 summarizes some of the more commonly up-regulated genes identified in response to cold and highlights those found in some or all of the woody plants discussed here. It is interesting to note that while there is some overlap in the results obtained with individual techniques applied to a single variety and stress treatment, it is often the case that each methodology is associated with a unique set(s) of genes. In this way, global studies can be enhanced by the complementarity of different techniques and, as a result, researchers should be encouraged to apply different approaches to address global gene expression whenever possible. Furthermore, we should

Table 8.1. Genes commonly up-regulated in response to low temperature. Gene encodes Early light-inducible protein (ELIP) Hsp70 Water channel proteins LEA proteins (other than dehydrins) Dehydrins Glutathione S-transferase β-Amylase RING zinc finger protein a

Malusa

Prunusb

Vacciniumc

Oranged

+e + + − + − + −

− − − + + − − +

+ − + + + + + +

+ + + + + + − +

Compiled from Wisniewski et al. (2008). Compiled from Bassett et al. (2006) and Tao (2005). c Compiled from Naik et al. (2007) and Dhanaraj et al. (2007). d Compiled from Zhang et al. (2005) and S¸ahin-Çevik and Moore (2006). e + means the gene was found in a study of the indicated fruit tree; – means the gene was not found in any reported study or was down-regulated. b

78

C.L. Bassett and M.E. Wisniewski

always be aware of potential differences between carefully designed field experiments and carefully controlled laboratory experiments. Extrapolation from one to the other may not be realistic given our present state of knowledge. The most promising future research areas in cold acclimation and dormancy in fruit trees will be focused on the detailed regulation of those genes and proteins we have already identified. This will likely include the identification of components of cold-responsive signalling

pathways other than CBF (C-repeat binding factor), as well as additional components that function directly or indirectly with the CBF pathway. In addition, discovering the interacting partners of the products of some of the cold-responsive genes (both protein and RNA) will help us understand some of the more subtle regulatory controls and cross-talk between different signalling pathways. Finally, defining the functional roles of the proteins encoded by cold-responsive genes will be a difficult, but intellectually lucrative undertaking.

References Arora, R. and Wisniewski, M.E. (1994) Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). II. A 60-kilodalton bark protein in cold-acclimated tissues of peach is heat stable and related to the dehydrin family of proteins. Plant Physiology 105, 95–101. Arora, R., Wisniewski, M.E. and Scorza, R. (1992) Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). I. Seasonal changes in cold hardiness and polypeptides of bark and xylem tissues. Plant Physiology 99, 1562–1568. Bassett, C.L., Wisniewski, M.E., Artlip, T.S., Norelli, J.L., Renaut, J. and Farrell, R.E. Jr (2006) Global analysis of genes regulated by low temperature and photoperiod in peach bark. Journal of the American Society for Horticultural Science 131, 551–563. Cregoe, B.A., Ross, G.S. and Watkins, C.B. (1993) Changes in protein and mRNA expression during cold storage of ‘Cox’s Orange Pippin’ apple fruit. Acta Horticulturae (ISHS) 326, 315–324. Dhanaraj, A., Slovin, J.P. and Rowland, L.J. (2004) Analysis of gene expression associated with cold acclimation in blueberry floral buds using expressed sequence tags. Plant Science 166, 863–872. Dhanaraj, A.L., Alkharouf, N.W., Beard, H.S., Chouikha, I.B., Matthews, B.F., Wei, H., Arora, R. and Rowland, L.J. (2007) Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions. Planta 225, 735–751. Durham, R.E., Moore, G.A., Haskell, D. and Guy, C.L. (1991) Cold-acclimation induced changes in freezing tolerance and translatable RNA content in Citrus grandis and Poncirus trifoliata. Physiologia Plantarum 82, 519–522. Faurobert, M. and Gomez, L. (2006) Seasonal protein variations in apricot bark. Acta Horticulturae (ISHS) 701, 113–118. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14, 1675–1690. Gomez, L. and Faurobert, M. (2002) Contribution of vegetative storage proteins to seasonal nitrogen variations in the young shoots of peach trees (Prunus persica L. Batsch). Journal of Experimental Botany 53, 2431–2439. Guy, C.L., Haskell, D. and Yelenosky, G. (1988) Changes in freezing tolerance and polypeptide content of spinach and citrus at 5°C. Cryobiology 25, 264–271. Heide, O.M. and Prestrud, A.K. (2005) Low temperature, but not photoperiod, controls growth cessation and dormancy induction and release in apple and pear. Tree Physiology 25, 109–114. Jung, S-H., Lee, J-Y. and Lee, D-H. (2003) Use of SAGE technology to reveal changes in gene expression in Arabidopsis leaves undergoing cold stress. Plant Molecular Biology 52, 553–567. Kreps, J.A., Wu, Y., Chang, H.-S., Zhu, T., Wang, X. and Harper, J.F. (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic and cold stress. Plant Physiology 130, 2129–2141. Lang, G.A. and Tao, J. (1990) Analysis of fruit bud proteins associated with plant dormancy. HortScience 25, 1068 (abstract). Lang, G.A. and Tao, J. (1991) Dormant peach flower bud proteins associated with chill unit accumulation or negation temperatures. HortScience 26, 733 (abstract).

Global Expression of Cold-responsive Genes

79

Lang, G.A. and Tao, J. (1992) Protein changes in endodormant shoots, spurs, and floral buds of various fruit crops during winter. HortScience 27, 685 (abstract). Lang, G.A., Early, J.D., Martin, G.C. and Darnell, R.L. (1987) Endo-, para-, and ecodormancy: physiological terminology and classification for dormancy research. HortScience 22, 371–377. Lang, P., Zhang, C.-K., Ebel, R.C., Dane, F. and Dozier, W.A. (2005) Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display. Gene 359, 111–118. Muthalif, M.M. and Rowland, L.J. (1994a) Identification of chilling-responsive proteins from floral buds of blueberry. Plant Science 101, 41–49. Muthalif, M.M. and Rowland, L.J. (1994b) Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus). Plant Physiology 104, 1439–1447. Naik, D., Dhanaraj, A.L., Arora, R. and Rowland, L.J. (2007) Identification of genes associated with cold acclimation in blueberry (Vaccinium corymbosum L.) using a subtractive hybridization approach. Plant Science 173, 213–222. Renaut, J., Hausman, J.-F., Bassett, C., Artlip, T., Cauchie, H.-M., Witters, E. and Wisniewski, M. (2008) Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (Prunus persica L. Batsch). Tree Genetics & Genomes (published online doi:10.1007/s11295-0080134-4). Rowland, L.J., Mehra, S., Dhanaraj, A, Ogdan, E.L. and Arora, R. (2003) Identification of molecular markers associated with cold tolerance in blueberry. Acta Horticulturae 625, 59–65. S¸ahin-Çevik, M. and Moore, G.A. (2006) Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf. Plant Molecular Biology 62, 83–97. Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., Kamiya, A., Nakajima, M., Enju, A., Sakurai, T., Satou, M., Akiyama, K., Taji, T., Yamaguchi-Shinozaki, K., Carninci, P., Kawai, J., Hayashizaki, Y. and Shinozaki, K. (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal 31, 279–292. Tang, Z., Li, B., Bharadwaj, R., Zhu, H., Özkan, E., Hakala, K., Deisenhofer, J. and Yu, H. (2001) APC2 cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex. Molecular Biology of the Cell 12, 3839–3851. Tao, R. (2005) Studies on the gene expression of dormant buds of Japanese apricot (Prunus mume). In: George, A.P. and Boonprakob, U. (eds) Production Technologies for Low-chill Temperate Fruits. ACIAR Technical Reports No. 61. Elect Printing, Canberra, pp. 48–53. Vu, J.C.V., Gupta, S.K., Yelenosky, G. and Ku, M.S.B. (1995) Cold-induced changes in ribulose 1,5-bisphosphate carboxylase–oxygenase and phosphoenolpyruvate carboxylase in citrus. Environmental and Experimental Botany 35, 25–31. Welling, A., Rinne, P., Viherä-Aarnio, A., Kontunen-Soppela, S., Heino, P. and Palva, E.T. (2004) Photoperiod and temperature differentially regulate the expression of two dehydrin genes during overwintering of birch (Betula pubescens Ehrh.). Journal of Experimental Botany 55, 507–516. Weston, K. (1998) Myb proteins in life, death and differentiation. Current Opinion in Genetics and Development 8, 76–81. Wisniewski, M., Close, T.J., Artlip, T. and Arora, R. (1996) Seasonal patterns of dehydrins and 70-kDa heatshock proteins in bark tissues of eight species of woody plants. Physiologia Plantarum 96, 496–505. Wisniewski, M., Bassett, C. and Arora, R. (2004) Distribution and partial characterization of seasonally expressed proteins in different aged shoots and roots of ‘Loring’ peach (Prunus persica). Tree Physiology 24, 339–345. Wisniewski, M., Bassett, C., Norelli, J., Macarisin, D., Artlip, T., Gasic, K. and Korban, S. (2008) Expressed sequence tag analysis of the response of apple (Malus×domestica ‘Royal Gala’) to low temperature and water deficit. Physiologia Plantarum 133, 298–317. Zhang, C.-K., Lang, P., Ebel, R.C., Dane, F., Singh, N.K., Locy, R.D. and Dozier, W.A. (2005) Down-regulated gene expression of cold acclimated Poncirus trifoliata. Canadian Journal of Plant Science 85, 417–424.

9

Could Ethanolic Fermentation During Cold Shock Be a Novel Plant Cold Stress Coping Strategy? F. Kaplan, D.Y. Sung, D. Haskell, G.S. Riad, M. Popp, M. Amaya, A. LaBoon, Y. Kawamura, Y. Tominaga, J. Kopka, M. Uemura, K.-J. Lee, J.K. Brecht and C.L. Guy

Abstract Plants possess inducible tolerance mechanisms to temperature extremes that contribute to survival, yet many aspects of stress-inducible responses remain poorly understood. One example is the cold induction of pyruvate decarboxylase and alcohol dehydrogenase gene expression that has long been a mystery. In the present work, comparative transcriptome and metabolite profiling analyses of the cold-shock responses of Arabidopsis reveal the specific coordinated induction of ethanolic fermentation during cold shock. Ethanol, a fluidizing agent of membrane physical structure, appears to be particularly beneficial in helping preserve membrane function and stability during the early stages of cold shock and/or during freezing stress. Brief exposure of Arabidopsis plants or protoplasts to ethanolic solutions enhanced membrane integrity during a freeze–thaw stress. These and other findings provide evidence for a novel functional role of ethanolic fermentation in plant low-temperature stress tolerance.

Introduction Stress resulting from exposure to temperature extremes is unavoidable for most terrestrial plants. Layered upon the daily oscillation of temperature rise and fall are seasonal and unseasonable changes that can lead to high cellular temperatures during warm and/or dry periods or low cellular temperatures during cold periods (Levitt, 1972). To survive in such an uncertain thermal environment where the threat of deleterious conditions is ever present, plants have an evolved array of coping strategies that range from developmental timingbased avoidance mechanisms including dormancy to constitutive and acclimationinducible physiological, molecular, metabolic and biochemical changes linked to acquired freezing tolerance (Levitt, 1972; Sung et al., 2003). Since sessile poikilothermic organisms, which include plants, have externally imposed 80

unregulated temperatures, they are generally considered to possess a more flexible physiology and metabolism than homeotherms (Hochachka and Somero, 2002). For example, Arabidopsis and many other organisms vary membrane fatty acid composition as a function of growth temperature (Sinensky, 1974; Cossins and Prosser, 1978; Uemura et al., 1995). As temperature increases, there is a tendency to incorporate more saturated lipids into membranes, and conversely incorporate more unsaturated lipids when temperature decreases. An outcome of altered saturation levels of membrane fatty acids and membrane lipid composition is to favour greater solidto-liquid-crystalline phase transitions at warmer temperatures and favour reduced solid-to-liquidcrystalline phase transitions at lower temperatures. This evolutionarily conserved ability to adjust membrane lipid and fatty acid composition in a changing temperature environment

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Ethanolic Fermentation During Cold Shock

is known as ‘homeoviscous adaptation’ (Sinensky, 1974; Cossins and Prosser, 1978), and has been associated with membrane stability and function in plants (Moon et al., 1995; Routaboul et al., 2000). Prior to the development of DNA array technology, it was well established that many cold-regulated genes were also differentially regulated in response to high salinity, osmotic and drought stress (Yamaguchi-Shinozaki and Shinozaki, 1994; Seki et al., 2003). Now a number of microarray studies have described the transcriptome responses of Arabidopsis to different forms of environmental extremes (Seki et al., 2001, 2002; Fowler and Thomashow, 2002; Kreps et al., 2002; Kim et al., 2003; Lee and Lee, 2003; Rizhsky et al., 2004). These studies have firmly established the multigenic nature of transcriptome responses to environmental stresses, and have permitted researchers to observe how entire pathways and processes are regulated through changes in steady-state mRNA levels for genes numbering in the thousands (Kreps et al., 2002). Further, these studies have demonstrated that not only are there unique responses to individual stresses, but also there is an interconnectedness of the responses to different stresses. Knowing which genes are differentially regulated by a number of abiotic stresses is beneficial in efforts to understand signalling pathway relationships and common, as well as divergent, elements of tolerance mechanisms (Zhu, 2001). Parallel time-course transcriptome- and metabolite-profiling experiments were conducted with uniform Arabidopsis plants to monitor gene expression and metabolite levels in response to a cold shock (CS) that would permit comparisons and contrasts of the temporal responses of the transcriptome and metabolome.

Results and Discussion The CS responses of Arabidopsis are characterized by the strong induction of benchmark stress-responsive genes, such as rd29A/cor78 (Horvath et al., 1993; Yamaguchi-Shinozaki and Shinozaki, 1994), and this expression pattern demonstrates the effectiveness of the

81

applied temperature treatment in eliciting the expected response (Fig. 9.1A). In the present study, maximum expression of rd29A/cor78 occurred at 12 and 24 h of CS. Analysis of genome-wide gene expression results revealed a striking connection between CS and the ethanolic fermentation pathway (Fig. 9.1). Two of the most strongly induced genes in response to CS were those encoding pyruvate decarboxylase (PDC) (Fig. 9.1C) and alcohol dehydrogenase (ADH) (Fig. 9.1D). Both of these genes were previously shown to be cold-regulated in plants (Christie et al., 1991; Jarillo et al., 1993; Kürsteiner et al., 2003). In addition, at least four members of the 11-member pyruvate kinase (PK) gene family were up-regulated by CS (Fig. 9.1B). PDC is encoded by five genes, and three (PCD1, 2 and 5) were found to be responsive to CS (Fig. 9.1C). ADH is also encoded by a gene family, but only a single member, ADH1, exhibited detectable expression. PK, PDC and ADH constitute the ethanolic fermentation pathway, but genes encoding for other components of the anaerobic response were similarly cold-responsive, in particular, those for lactate dehydrogenase (LDH) and alanine aminotransferase (AAT) (Fig. 9.1E and F). We looked at the expression profiles of genes involved in glycolysis to determine whether the coordinated induction included a wider array of genes for the glycolytic pathway. Certain members of the gene families for a number of glycolytic enzymes (hexokinase (HXK), fructokinase (FK), phosphofructokinase (PFK), pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) and phosphoglycerate mutase (PGM) ) were up-regulated. The parallel metabolite profiling also revealed that CS increased the pool sizes of several glycolytic metabolites (glucose-6-phosphate, fructose-6-phosphate and 3-phosphoglyceric acid) that were also correlated with gene expression profiles (Fig. 9.2). In contrast, similar responses were not observed during heat shock (data not shown) and therefore demonstrate the specificity of this response to CS, showing that it is not a general temperature stress response. Next, we measured acetaldehyde and ethanol levels by GC in extracts prepared from the aerial portions of CS plants (Riad, 2004) to further investigate the connection between

82

F. Kaplan et al.

7000

5000

Hsp70 /[rd29A/cor78 ]

A Signal intensity

Signal intensity

6000 5000 4000 3000 2000

PK

B

E

2000

250

Signal intensity

Signal intensity

LDH

3000

300

1400

1000 800 600 400

200 150 100 50

200

1400

1200

PDC

C

1200

Signal intensity

1000

Signal intensity

D

1000

1000

1200

ADH 4000

800 600 400 200

AAT

F

1000 800 600 400 200 0

0

1

4

12

24

48

96

DA

Time at 4°C (h)

0

1

4

12

24

48

96

DA

Time at 4°C (h)

Fig. 9.1. Time course of expression for the benchmark temperature stress-responsive genes and genes typical of the anaerobic response of 3-week-old Arabidopsis shoots during cold shock. (A) Hsp70 ( , At3g12580) and rd29A/cor78 ( , At5g52310); (B) pyruvate kinase ( , At1g32440; , At2g36580; , At5g08570; , At5g56350); (C) pyruvate decarboxylase ( , At4g33070; , At5g17380; , At5g54960); (D) alcohol dehydrogenase (At1g77120); (E) lactate dehydrogenase (At4g17260); (F) alanine aminotransferase (At1g17290). DA denotes deacclimation, where plants at 4°C for 96 h were returned to 20°C for 24 h. Values are means with their standard deviations represented by vertical bars.

gene expression profiles and metabolism. Acetaldehyde levels were slightly elevated at 24 h of CS compared with plants grown at 20°C (Fig. 9.3A). Ethanol levels during CS appeared slightly elevated compared with those of 20°C grown plants. Since overall metabolism and respiratory processes were significantly depressed during the initial stages of CS, these results suggested that the induction of ethanolic fermentation during CS serves to maintain or slightly enhance ethanol levels during a period of reduced metabolic activity. We therefore hypothesized that the CS induction of ethanolic fermentation permits

the maintenance of ethanol production at low temperature when overall metabolic activity is low and that the maintained ethanol production is an adaptive response that provides a beneficial function during low-temperature stress. Since there is wide agreement in the literature that membranes are primary targets of ethanol interactions (Da Silveira et al., 2003) leading to adjustments in membrane physical properties and lipid composition (Dombek and Ingram, 1984), which are also factors in ethanol tolerance during chronic ethanol exposure (Chin and Goldstein, 1976), it seems reasonable that the observed ethanol

Ethanolic Fermentation During Cold Shock

Chloroplast

83

GLC-1-P PGM

GLC*

HXK

Pentose Phosphate Pathway, RNA, DNA, Proteins

GLC-6-P* PGI

FRU*

FK

FRU-6-P* PFP

PFP

PFK

FBP FRU-1, 6-P ALD

Chloroplast

DHAP TPI

GAP GAPDH 1,3-BPG PGK 3PG* PGM 2PG

Ethanol

ENO ADH

ADH PEP Lactate*

LDH

PK Pyruvate

Alanine*

AAT

Acetaldehyde PDC

ALDH

PDH ACS

Acetate

Acetyl-CoA

Fig. 9.2. Overview of glycolytic and ethanolic fermentation pathway gene expression patterns and metabolite levels. Callout boxes for enzymatic steps with a white background are unchanged, but those with a grey background indicate increased gene expression during the cold shock (CS) time course. Metabolites shown in upright font were unchanged, while those shown in italics increased or decreased during 12 and 24 h of CS compared with the levels from plants grown at 20°C. Metabolites with statistically different signal levels are indicated by asterisks. The expression responses are indicated for the following genes: PGM (At1g23190); HXK (At2g19860); PGI (At5g42740); FK (At5g51830); PFP (At5g56630 and At4g26270); PFK (At5g03300); FBPase (At3g54050); ALD (At2g36460); TPI (At3g55440); GAPDH (At3g04120); PGK (At1g79550); PGM (At2g17280 and At3g08590); ENO (At2g36530); PK (At1g32440, At2g36580, At5g08570 and At5g56350); LDH (At4g17260); AAT (At1g17290); PDC (PDC1/At4g33070, PDC5/At5g17380 and PDC2/At5g54960); ADH (ADH1/At1g77120); ALDH (At3g48000); ACS (At5g36880).

84

F. Kaplan et al.

(Acetaldehyde units/ g fresh weight ⫻(10-1)

6 A

5 b 4 3

b a

2 c 1 0

(Ethanol mmol/ g fresh weight)

B

12 a 8

a

a

4 b

(ADH mmol/ g fresh weight)

0 60 C

40 b 20

0

a 20°C wt

a 4°C wt 4°C KO Temperature treatment

a 40°C wt

Fig. 9.3. Acetaldehyde (A) and ethanol levels (B) and alcohol dehydrogenase (ADH) enzymatic activity (C) in Arabidopsis shoots. Wild-type (wt) plants grown at 20°C were shifted to 4°C for 24 h; KO denotes ADH1 gene knockout. Acetaldehyde and ethanol levels were determined by GC. Values are means with their standard deviations represented by vertical bars. a,b,cMean values with unlike lower case letters were significantly different (P<0.05).

production responses of Arabidopsis during CS could differentially affect cell membranes. Ethanol is a fluidizing agent for membranes (Chin and Goldstein, 1976; Dombek and Ingram, 1984; Da Silveira et al., 2003). During high-temperature exposure membranes become too fluid, and during CS too immobile. Maximov (1912; as cited in Levitt, 1972) first suggested that membranes appear to be a primary site of freezing stress injury in plant cells, and now this view has become experimentally supported and widely accepted (Dowgert and

Steponkus, 1984; Uemura et al., 1995). Therefore, increased interaction of ethanol with membranes might influence plant and cell freezing tolerance. To explore this possibility, we immersed the aerial portions of 20°C grown, 3-week-old Arabidopsis plants in either water or 500 mM ethanol (2.9% v/v) for 10 min at 20°C, then immediately subjected them to a standard freeze–thaw cycle (Sung and Guy, 2003; Kaplan et al., 2004). Brief immersion in 500 mM ethanol had no obvious deleterious effects on plants in regrowth studies or on elec-

Ethanolic Fermentation During Cold Shock

trolyte leakage characteristics of unfrozen plants (Fig. 9.4). However, ethanol immersion prior to freezing dramatically reduced electrolyte leakage following freezing at −5, −6 and −7°C (Fig. 9.4A). This effect was concentration-dependent with 100 mM and 250 mM ethanol treatments yielding smaller reductions in electrolyte leakage versus the control. In

85

contrast, ethanol treatment immediately prior to heat shock slightly increased subsequent membrane leakage (Fig. 9.4B). Since electrolyte leakage assays report on the integrity and functionality of membranes, these results demonstrate the remarkable ability of ethanol to preserve membrane integrity during a period of freeze–thaw stress.

A 90

Electrolyte leakage (%)

80

a a

b

70

a

a

b

60 50 a a

40 b

30 20 10

b

a

a

a

a

0 0

–4

–5

–6 Temperature (°C)

–7

–8

–9

C

B a a

80

a

a a

b 60 40 a 20

a

a a

b

b

0 25

40

42 44 Temperature (°C)

46

100

Survival at -5°C (% of control)

Electrolyte leakage (%)

100

80

c b

60 a

a

40

20

48 0

Fig. 9.4. Influence of exogenous ethanol application on freezing tolerance of plants and protoplasts. (A) Plants were immersed in either water ( ) or 500 mM ethanol ( ) for 10 min at 25°C, then removed from the solutions, blotted dry with tissue paper and immediately subjected to a freeze–thaw stress. (B) Plants were immersed in either water ( ) or 500 mM ethanol ( ) for 10 min at 25°C, then removed from solution, blotted dry with tissue paper and immediately subjected to a heat shock stress at the indicated temperature for 10 min by immersion of the aerial portion of the plants in a temperature-controlled water bath. (C) Survival of Arabidopsis leaf protoplasts treated with water ( ), 100 mM ethanol ( ) or 250 mM ethanol ( ) for 10 min at 25°C and then immediately frozen, or where the ethanol (250 mM) was removed ( ) before the protoplasts were frozen. Values are means with their standard deviations represented by vertical bars. a,b,cMean values with unlike lower case letters were significantly different (P<0.05, Student’s t test).

86

F. Kaplan et al.

We further explored the effects of ethanol on protoplasts subjected to a freeze–thaw stress. Treatment with 100 and 250 mM ethanol for 10 min prior to freezing increased protoplast survival dramatically (Fig. 9.4C). If ethanol was removed prior to freezing, there was no residual effect on survival compared with the controls, suggesting that its effects were the result of a direct interaction and not caused by an alteration of cellular metabolism or modified gene expression. The major forms of cryoinjury in nonacclimated Arabidopsis protoplasts in the range of −2 to −6°C are expansion-induced lysis (EIL) and loss of osmotic responsiveness (LOR). Injury due to EIL occurs at higher freezing temperatures and is a minor determinant of survival, while LOR is the major form of membrane injury and the primary determinant of survival at lower freezing temperatures around the LT50 (Uemura et al., 2003). During cold acclimation, injury caused by EIL is eliminated by changes in membrane lipid composition and LOR is shifted to lower temperatures. Membrane damage by LOR occurs because of dehydration-induced alterations in the ultrastructure of the plasma membrane (Uemura and Steponkus, 1989). Therefore, it seems likely that the ethanol treatment influenced membrane cryobehaviour by reducing injury caused by LOR. It is important to note that in the case of the freezing and heat stress treatments, the only residual ethanolic solution present would be that which was absorbed into the tissue. There was no external ethanol present during the freezing and heat stress treatments. In the freezing stress test, nucleation was achieved at −2°C using ice chips. Observation of the tissue did not detect any difference in tissue nucleation pattern between the treatments. Therefore, any influence of the ethanol treatments on nucleation and tissue freezing was judged to be minimal with respect to the general stress of freezing. We considered a gene knockout approach to further investigate the function of the CS induction of ethanolic fermentation. PDC, being the first committed step of ethanolic fermentation, is highly regulated and thought to control flux in the pathway (Dolferus et al., 1997; Ismond et al., 2003). Because four

PDC genes are expressed and three were responsive to CS, a quadruple knockout would be minimally necessary to assess the biological function of the pathway during CS. In contrast, only one member of the ADH gene family (ADH1) exhibited detectable or increased expression in Arabidopsis shoots during CS (Fig. 9.1D), suggesting that a single gene knockout might be sufficient to block ethanol production. A Salk T-DNA insertion line (SALK_052699) for ADH1 (At1g77120) was obtained (Alonso et al., 2003) and ADH activity was assayed as the ethanol-dependent reduction of NAD+ (Rumpho and Kennedy, 1981). Wild-type plants exhibited very low activity when grown at 20°C (Fig. 9.3C). As expected, ADH activity of wild-type plants was increased markedly after 24 h of CS, while the activity of the homozygous knockout line following 24 h of CS remained just above background (Fig. 9.3C). In the knockout line given a 24 h CS, acetaldehyde levels were elevated by 36%, but ethanol levels were found to be only 15% lower than that of similarly CS-treated wild-type plants (Fig. 9.3A, B). This unexpected result in the absence of major NAD+-dependent ADH activity, while in agreement with findings from another group (Ismond et al., 2003), suggests that there must be an alternative pathway for ethanol synthesis operating in the shoots of knockout plants during CS. One possibility could be a yet unknown, NADH/NADPH-dependent alcohol dehydrogenase in Arabidopsis able to use acetaldehyde as a substrate similar to that found in a number of other organisms. We tested this hypothesis by conducting ADH assays in the forward direction as acetaldehyde-dependent NADH/NADPH oxidation and found enough activity in both wild-type and knockout plants (60–70 units/g fresh weight) to account for the presence of ethanol in the knockout plants. The presence of ethanol in the absence of ADH1 enzymatic activity could explain why an ADH1 null mutation had no effect on Arabidopsis freezing tolerance in an earlier study (Jarillo et al., 1993). It has not escaped our attention that CS might somehow cause an oxygen deficit. However, we view a CS-invoked hypoxia as unlikely for several reasons. First, reduced temperatures, especially in the form of a rapid

Ethanolic Fermentation During Cold Shock

onset of CS, profoundly depress leaf respiratory processes, and dramatically reduce oxygen consumption (Yelenosky and Guy, 1977). Thus, the demand for oxygen during CS is reduced to just a small fraction of that at 20°C, and therefore would be unlikely to result in a reduction of cellular oxygen levels, even if stomatal aperture were severely restricted. Second, oxygen dissolved in water obeys Henry’s Law; meaning the solubility is roughly proportional to the partial pressure of oxygen in the air. In addition, oxygen solubility in water is strongly affected by temperature. At an atmospheric pressure of 760 mmHg, the solubility of oxygen in water is 13.1 mg/l at 4°C, 9.1 mg/l at 20°C and 6.4 mg/l at 40°C; thus CS would be expected to actually enhance cellular oxygen tensions. Finally, if CS were to invoke a hypoxic condition that elicited the induction of ethanolic fermentation, then it would be expected to also induce the hypoxiaresponsive oxygen-binding non-symbiotic haemoglobins found in Arabidopsis as well (Trevaskis et al., 1997). Inspection of expression data showed that CS did not induce either haemoglobin AHB1 or AHB2 in the present study (not shown), thus reinforcing the conclusion that oxygen levels were not reduced or in any way limiting. Therefore, it is clear that induced or maintained ethanol production during CS is a direct temperature response independent of a hypoxic response. The CS induction of ethanolic fermentation and heat shock repression is consistent with the need for ‘homeoviscous adaptation’, where ethanol serves as a membrane-fluidizing agent. Arabidopsis has the ability to adjust plasma membrane lipid composition during long-term low-temperature exposure, a process known as cold acclimation. Changes in lipid composition during cold acclimation have been associated with altered membrane cryobehaviour, and in particular, with a decreased propensity of EIL, LOR and freeze-induced HexII non-bilayer formation (Uemura et al., 1995). However, a change in membrane composition that would be associated with ‘homeoviscous adaptation’ and altered membrane cryobehaviour in Arabidopsis does not occur before 7 days of low-temperature exposure under CS conditions (Uemura et al., 1995; Falcone et al., 2004). Therefore, the

87

rapid induction of ethanolic fermentation during the first 12–24 h of CS could provide a readily accessible alternative biochemical mechanism by which the physical structure of cell membranes can be rapidly adjusted or modified until lipid compositional changes associated with longer-term cold acclimation can occur to restore optimal membrane physical structure and function and provide the physical stability necessary during freeze–thaw stress events. Regarding the overall function for CS induction of ethanolic fermentation, a second role seems plausible. Upon imposition of CS, anabolic metabolism is diminished dramatically, while catabolic processes are similarly affected, but to a lesser degree. The net result is that energy charge (Sobczyk et al., 1985; Strand et al., 1997; Hurry et al., 2000; Savitch et al., 2001) and pyridine nucleotide redox poise increase (Kuraishi et al., 1968; Savitch et al., 2001), especially in photosynthetic organs during illumination (Savitch et al., 2001). If NADH and NADPH levels become too high and NAD+ and NADP+ too low during CS, then perhaps ethanolic fermentation is induced to partially regenerate NAD+ and NADP+ levels. Inspection of expression and metabolite data in the present study revealed a number of CS responses that lend support to the development of high energy charge and pyridine nucleotide redox poise. Among these were the CS induction of ndb2 NADPH dehydrogenase (At4g05020), an alternative oxidase AOX1a (At3g22370), an uncoupling protein (At4g24570), and the eight- and 26-fold increase of malate and γ-aminobutyric acid, respectively (Kaplan et al., 2004). The linkage between ethanolic fermentation and CS is not simply limited to Arabidopsis plants growing in the laboratory, but may be a more general acclimation response in nature. Studies with Douglas fir (Pseudotsuga menziesii) growing in a natural environment along the Oregon coast have revealed a seasonal accumulation of ethanol in the phloem and sapwood near the root collar, where concentrations of ethanol reached high levels during winter and exhibited the lowest levels in summer (Kelsey and Joseph, 1998). Further, when incubated in a nitrogen atmosphere,

88

F. Kaplan et al.

stem segments sampled in December produced 1.8 times more ethanol than segments sampled in May (Joseph and Kelsey, 2004) indicating an enhanced ability for ethanol synthesis during winter. Thus, the seasonal pattern of ethanol accumulation in a forest tree species is entirely consistent with the possibility that ethanol provides a beneficial influence to plants during periods of low temperature by helping to enhance the functionality and stability of cell membranes.

Conclusion The study reported in the present chapter provides evidence for important functional roles for ethanolic fermentation during CS in Arabidopsis. The present findings further highlight that the responses of plants to temperature extremes are multigenic, with tolerance mechanisms emanating from an intricate interplay of a variety of molecular, biochemical and physiological processes. Thus, CS induction of ethanolic fermentation appears to be an important component of an overall set of coping strategies that Arabidopsis has at its

disposal in its bid to survive and flourish in an ever-challenging thermal environment.

Acknowledgements We gratefully acknowledge the gift of the homozygous ADH1 knockout line from W.-H. Cheng, Arabidopsis Biological Resource Center; L. Liu for data software development; and the constructive comments and support of R. Ferl, A.-L. Paul and L. Willmitzer. This research was supported by a grant from the National Aeronautics and Space Administration #NAG10-316 and by the National Research Initiative of the US Department of Agriculture’s Cooperative State Research, Education and Extension Service, grant numbers 200035100-9532, 2002-35100-12110 (C.L.G.) and 2001-35503-10791 (J.K.B.). The Max Planck Society, the Institute of Food and Agricultural Sciences at the University of Florida and MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan) Grant-in-Aid provided additional support for the 21st Century Center of Excellence (COE) Programme to Iwate University (M.U.).

References Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C. and Ecker, J.R. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657. Chin, J.H. and Goldstein, D.B (1976) Drug tolerance in biomembranes: a spin label study of the effects of ethanol. Science 196, 684–685. Christie, P.J., Hahn, M. and Walbot, V. (1991) Low-temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings. Plant Physiology 95, 699–706. Cossins, A.R. and Prosser, C.L. (1978) Evolutionary adaptation of membranes to temperature. Proceeding of the National Academy of Sciences USA 75, 2040–2043. Da Silveira, M.G., Golovina, E.A., Hoekstra, F.A., Rombouts, F.M. and Abe, T. (2003) Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells. Applied Environmental Microbiology 69, 5826–5832. Dolferus, R., Ellis, M., De Bruxelles, G., Trevaskis, B., Hoeren, F., Dennis, E.S. and Peacock, W.J. (1997) Strategies of gene action in Arabidopsis during hypoxia. Annals of Botany 79, Suppl. A, 21–31. Dombek, K.M. and Ingram, L.O. (1984) Effects of ethanol on the Escherichia coli plasma membrane. Journal of Bacteriology 157, 233–239. Dowgert, M.F. and Steponkus, P.L. (1984) Behavior of the plasma membrane of isolated protoplasts during a freeze–thaw cycle. Plant Physiology 75, 1139–1151. Falcone, D.L., Ogas, J.P. and Somerville, C.R. (2004) Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition. BMC Plant Biology 4, 17.

Ethanolic Fermentation During Cold Shock

89

Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14, 1675–1690. Hochachka, P.W. and Somero, G.N. (2002) Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, New York, New York. Horvath, D.P., McLarney, B.K. and Thomashow, M.F. (1993) Regulation of Arabidopsis thaliana L. (Heyn) cor78 in response to low temperature. Plant Physiology 103, 1047–1053. Hurry, V., Strand, A., Furbank, R. and Stitt, M. (2000) The role of inorganic phosphate in the development of freezing tolerance and the acclimatization of photosynthesis to low temperature is revealed by the pho mutants of Arabidopsis thaliana. The Plant Journal 24, 383–396. Ismond, K.P., Dolferus, R., de Pauw, M., Dennis, E.S. and Good, A.G. (2003) Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway. Plant Physiology 132, 1292–1302. Jarillo, J.A., Leyva, A., Salinas, J. and Marinez-Zapater, J.M. (1993) Low temperature induces the accumulation of alcohol dehydrogenase mRNA in Arabidopsis thaliana, a chilling-tolerant plant. Plant Physiology 101, 833–837. Joseph, G. and Kelsey, R.G. (2004) Ethanol synthesis and aerobic respiration in the laboratory by leader segments of Douglas-fir seedlings from winter and spring. Journal of Experimental Botany 55, 1095–1103. Kaplan, F., Kopka, J., Haskell, D.W., Zhao, W. Schiller, K.C., Gatzke, N., Sung, D.Y. and Guy, C.L. (2004) Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiology 136, 4159–4168. Kelsey, R.G. and Joseph, G. (1998) Ethanol in Douglas-fir with black-stain root disease (Leptographium wageneri). Canadian Journal of Forestry Research 28, 1207–1212. Kim, H., Snesrud, E.C., Haas, B., Cheung, F., Town, C.D. and Quackenbush, J. (2003) Gene expression analyses of Arabidopsis chromosome 2 using a genomic DNA amplicon microarray. Genome Research 13, 327–340. Kreps, J.A., Wu, Y., Chang, H.S., Zhu, T., Wang, X. and Harper, J.F. (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiology 130, 2129–2149. Kuraishi, S., Arai, N., Ushijima, T. and Tazaki, T. (1968) Oxidized and reduced nicotinamide adenine dinucleotide phosphate levels of plants hardened and unhardened against chilling injury. Plant Physiology 43, 238–242. Kürsteiner, O., Dupuis, I. and Kuhlemeier, C. (2003) The pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses. Plant Physiology 132, 968–978. Lee, J.Y. and Lee, D.H. (2003) Use of serial analysis of gene expression technology to reveal changes in gene expression in Arabidopsis pollen undergoing cold stress. Plant Physiology 132, 517–529. Levitt, J. (1972) The Responses of Plants to Environmental Stresses. Academic Press, New York, New York. Moon, B.Y., Higashi, S., Gombos, Z. and Murata, N. (1995) Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants. Proceedings of the National Academy of Sciences USA 92, 6219–6223. Riad, G. (2004) Atmosphere modification to control quality deterioration during storage of fresh sweetcorn cobs and fresh-cut kernels. PhD dissertation, University of Florida, Gainesville, Florida. Rizhsky, L., Liang, H., Shuman, J., Shulaev, V., Davletova, S. and Mittler, R. (2004) When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiology 134, 1683–1696. Routaboul, J.M., Fischer, S.F. and Browse, J. (2000) Trienoic fatty acids are required to maintain chloroplast function at low temperatures. Plant Physiology 124, 1697–1705. Rumpho, M.E. and Kennedy, R.A (1981) Anaerobic metabolism in germinating seeds of Echinochloa crus-galli (barnyard grass): metabolite and enzyme studies. Plant Physiology 68, 165–168. Savitch, L.V., Barker-Astrom, J., Ivanov, A.G., Hurry, V., Oquist, G., Huner, N.P. and Gardestrom, P. (2001) Cold acclimation of Arabidopsis thaliana results in incomplete recovery of photosynthetic capacity, associated with an increased reduction of the chloroplast stroma. Planta 214, 295–303. Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P., Hayashizaki, Y. and Shinozaki, K. (2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. The Plant Cell 13, 61–72. Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., Kamiya, A., Nakajima, M., Enju, A., Sakurai, T., Satou, M., Akiyama, K., Taji, T., Yamaguchi-Shinozaki, K., Carninci, P., Kawai, J., Hayashizaki, Y. and Shinozaki, K. (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal 31, 279–292.

90

F. Kaplan et al.

Seki, M., Kamie, A., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2003) Molecular responses to drought, salinity and frost: common and different paths for plant protection. Current Opinion in Biotechnology 14, 194–199. Sinensky, M. (1974) Homeoviscous adaptation – a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proceedings of the National Academy of Sciences USA 71, 522–525. Sobczyk, E.A., Marszalek, A. and Kacperska, A. (1985) ATP involvement in plant tissue responses to low temperature. Physiologia Plantarum 63, 399–405. Strand, A., Hurry, V., Gustafsson, P. and Gardestrom, P. (1997) Development of Arabidopsis thaliana leaves at low temperatures releases the suppression of photosynthesis and photosynthetic gene expression despite the accumulation of soluble carbohydrates. The Plant Journal 12, 605–614. Sung, D.Y. and Guy, C.L. (2003) Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences. Plant Physiology 132, 979–987. Sung, D.Y., Kaplan, F., Lee, K.J. and Guy, C.L. (2003) Acquired tolerance to temperature extremes. Trends in Plant Sciences 8, 179–187. Trevaskis, B., Watts, R.A., Andersson, C.R., Llewellyn, D.J., Hargrove, M.S., Olson, J.S. Dennis, E.S. and Peacock, W.J (1997) Two hemoglobin genes in Arabidopsis thaliana: the evolutionary origins of leghemoglobins. Proceedings of the National Academy of Sciences USA 94, 12230–12234. Uemura, M. and Steponkus, P.L. (1989) Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves. Plant Physiology 91, 1131–1137. Uemura, M., Joseph, R.A. and Steponkus, P.L. (1995) Cold acclimation of Arabidopsis thaliana (effect on plasma membrane lipid composition and freeze-induced lesions). Plant Physiology 109, 15–30. Uemura, M., Warren, G. and Steponkus, P.L. (2003) Freezing sensitivity in the sfr4 mutant of Arabidopsis is due to low sugar content and is manifested by loss of osmotic responsiveness. Plant Physiology 131, 1800–1807. Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress. The Plant Cell 6, 251–264. Yelenosky, G. and Guy, C.L. (1977) Carbohydrate accumulation in leaves and stems of ‘Valencia’ orange at progressively colder temperatures. Botanical Gazette 138, 13–17. Zhu, J.-K. (2001) Cell signaling under salt, water and cold stresses. Current Opinion in Plant Biology 4, 401–406.

10

Bud Set – A Landmark of the Seasonal Growth Cycle in Poplar A. Rohde

The Seasonal Growth Cycle in a Changing Climate Coping with their sessile lifestyle, plants continuously adapt to the environment to improve their chances of survival and reproduction. Within an optimal adaptation, phenology – i.e. the seasonal timing of growth processes – determines to a large extent the geographic distribution of many plants. In trees, events of bud flush and bud set delimit the seasonal growth period and are under strong selection to avoid cold injury. Both traits show particularly strong genetic differentiation along latitudinal and altitudinal clines, typically resulting in locally adapted ecotypes (Howe et al., 2003; Savolainen et al., 2004; Aitken et al., 2008). Of the various environmental factors that are sensed for proper timing of seasonal growth events, temperature is subject to climate change (Menzel and Fabian, 1999). Between 1981 and 1991, the length of the growing season increased by 5 days per 1°C temperature rise on average, or by 12 days at high latitudes (Zhang et al., 2004). The corresponding changes in phenology due to global warming have been noticed during the past decades and challenge a controversial discussion on whether plants, trees in particular, can cope at all with the expected speed of climate change (Saxe et al., 2001; Jump and Penuelas, 2005; Aitken et al., 2008). Moreover, climate will

continue to change significantly over the next 100 years (IPCC, 2007). This timeline offers little opportunity for trees to evolve or to migrate. Thus, populations will need to draw from phenotypic plasticity of these traits for coping with climate change. The extent of phenotypic plasticity is largely unknown for bud flush and bud set, as it is not assessed in traditional provenance trials. The dilemma consists thus of a seasonal growth cycle that is strongly adapted to the current local conditions and changing climate determinants. Seasonal changes in day length, light quality and temperature provide the most important environmental cues for synchronizing growth with seasonality. However, which elements of the annual growth cycle will be primarily affected by the projected increase in temperature? Warmer spring temperatures will result in a generally earlier bud flush (increasing the risk of injury by occasional late frosts). While generally true, particularly cold winters can also reduce the thermal requirement for bud flush (Thompson and Clark, 2008). In autumn, many broadleaved trees cease growth in response to increasing night length or in response to a combination of night length with temperature (i.e. the sensitivity to night length increases with decreasing temperature). The night-length-sensing mechanism has evolved because it is inter-annually stable and ensures the avoidance of shoot damage by frost.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

91

92

A. Rohde

Growth cessation will occur invariably in future climates but earlier than optimal, leaving part of the growing season unexploited for growth. The subsequent autumnal bud development is a composite of bud formation, simultaneous acclimation to dehydration and cold, and acquisition of dormancy (Ruttink et al., 2007). How will climate change affect these three processes? First, bud formation, as with many other growth processes, will benefit from a higher autumn temperature. In Salix, the rate of bud growth, i.e. primordium initiation within a bud, is positively affected by temperature (Junttila, 2007). In addition to warmer temperature, elevated atmospheric CO2 will also support growth processes and delay leaf senescence (Taylor et al., 2008). Second, the hardening to cold and dehydration will not be synchronized to the present extent with growth cessation and bud formation (Saxe et al., 2001). Cold acclimation typically proceeds in two phases, with the first induced by night length and the second by temperatures lower than 5°C. For maximal hardening, cold must impinge at a particular time relative to increasing night length. Hardening will occur more in response to night length and earlier than optimal in warm autumns. Third, the achieved levels of dormancy will vary. Some authors claim that a warmer autumn will induce shallower dormancy (e.g. Saxe et al., 2001), while others have demonstrated a stronger dormancy (Heide, 2003; Søgaard et al., 2008). Fourth, the obligate chilling requirement that most trees have for the release from dormancy might not be met anymore by milder winters and lead to erratic or delayed bud flush. Thus, as illustrated with the foregoing examples, an altered temperature will influence various points in the seasonal trajectory and the outcomes on seasonal growth will be manifold. Each species, and ecotype within a species, has a discrete day length for inducing growth cessation, a specific depth of dormancy and chilling requirement for dormancy release, and a dependence on accumulated warm temperature at bud flush – creating a species-specific developmental trajectory through the season. The notion of this complexity must be met by future research approaches in that effects of environmental factors

need to be assessed over the complete seasonal growth cycle. Moreover, next to the most common environment-sensing mechanisms in temperate-zone trees, other scenarios might apply for trees that set bud in long days (Pinus), in response to temperature (Malus), require long days for bud flush (Fagus) or are determined by a strong internal growth rhythm (Quercus), to just name a few examples. Despite their great importance for productivity and survival, processes related to the transition into and out of dormancy are only poorly understood at the molecular level. Very little is known about the processes that occur during the dormant period in woody perennials, particularly during the release from endodormancy (Rohde et al., 2000; Arora et al., 2003; Horvath et al., 2003; Rohde and Bhalerao, 2007). Put back into the perspective of climate change, however, the diversity in the genes with a functional role during these transitions will determine how fast populations respond to environmental change. Of the two events delimiting the growing season, bud set typically exhibits stronger genetic clines and higher differentiation among populations (Howe et al., 2003; Aitken et al., 2008).

Molecular Approaches to Bud Set Transcriptomic and metabolomic approaches The environmental and hormonal factors controlling the induction of growth cessation and autumnal bud development, such as photoperiod, low temperature and abscisic acid, have long been known (Sylven, 1940; Nitsch, 1957; Eagles and Wareing, 1964; Weiser, 1970). However, their temporal integration into progression of autumnal bud development, their mutual interactions and particularly their molecular targets remained poorly resolved (Horvath et al., 2003; Tanino, 2004; Rohde and Bhalerao, 2007). Moreover, the simultaneous activity of various closely intertwined cellular, physiological and morphological processes confounds the dissection of the underlying developmental programmes and their respec-

Bud Set

tive signals. In a few cases, processes were assigned to the action of a particular signalling route (Welling et al., 2002; Mølmann et al., 2005). Recently, a systems approach with transcript and metabolite profiling of autumnal bud development in poplar has established a timetable of molecular events associated with the transition from a growing apex to a dormant bud in poplar (Ruttink et al., 2007). The molecular changes along autumnal bud development were extensive; as much as 8% of the sampled transcriptome and 17% of the inspected GC-MS peaks were more than fourfold differentially expressed or accumulating, respectively (Ruttink et al., 2007). Yet, short day alone, the only environmental factor considered in this experiment, induced these dramatic changes. An even more complex picture of signal transduction pathways and downstream processes is expected during natural bud development in response to gradually shortening days and declining temperatures. Through a directed search for marker genes, the three components of autumnal bud development – i.e. bud formation, dormancy, and acclimation to dehydration and cold – were dissected and described by specific sets of genes (Fig. 10.1; Ruttink et al., 2007). Light, ethylene and abscisic acid are the major signals that consecutively act to control bud development by setting, modifying or terminating these three processes (Fig. 10.1). Metabolism is inactivated and reconfigured towards the acquisition of cold tolerance and the accumulation of storage compounds (Fig. 10.1). Importantly, transcriptional regulation was found to be a major component in many pathways. The fixation of dormancy, i.e. the final transition from eco- to endodormancy (sensu Lang, 1987), however, might be controlled at a level other than gross transcriptional or metabolic regulation (Ruttink et al., 2007). In a separate experiment, cDNA amplified fragment length polymorphism (AFLP) transcript profiling was used to map differential gene expression during dormancy induction, dormancy, dormancy release by chilling and subsequent bud break in apical buds of poplar (Rohde et al., 2007). Again, although at a lower temporal resolution, processes and potential regulators during different phases of

93

dormancy are described. Novel genes were linked to a crucial transitory step in dormancy induction and to dormancy release through chilling. The chilling requirement through which dormancy is released has remained a central enigma of dormancy research. Nineteen genes expressed mainly during chilling constitute interesting novel candidate genes for functions during the satisfaction of the chilling requirement (Rohde et al., 2007). Although not belonging to a particular pathway, these include six genes of unknown function and a DNA-binding protein with linker histone domains with a potentially regulatory role in dormancy release (Rohde et al., 2007).

Comparing transcriptome data Dormancy can be integrated at diverse periods of the plant life cycle and can become established in meristems of organs as different as bud and seed. It has been known for years that seed and bud dormancy share physiological characteristics, but the similarity was not assessed at the molecular level. A comparison of transcriptome data for growth-to-dormancy transitions in poplar apical buds, poplar vascular cambium and Arabidopsis seeds identified common gene sets that consistently change expression during these transitions (Ruttink et al., 2007). Among these, a set of previously uncharacterized genes has been delineated (Ruttink et al., 2007). Such comparisons help to distinguish processes common to transitions from processes specific for a respective organ and/or its developmental context, and to filter out genes related primarily to cold acclimation. A similar transition from activity to dormancy exists in the developmental gradient of axillary buds along a growing shoot, when paradormancy (sensu Lang, 1987) becomes established. Transcription factors that change significantly in expression at positions where paradormancy is imposed have been identified (Rohde et al., 2007). The same transcription factors are also induced during autumnal bud development. This conserved expression during the transition to dormancy in apical and axillary buds suggests interesting parallels between these two different types of dormancy.

LD/SD

1 week SD

2 weeks SD

3 weeks SD

4 weeks SD

5 weeks SD

6 weeks SD No regrowth

94

Bud scales first visible Elongation growth Bud formation

Signals

Adaptation to dehydration and cold Dormancy Photoperiod

Ethylene

ABA

ETR2↑ ↑ ERS1↑

PP2C ↑

Pathways/processes

PIF4↑ ↑ PIL1↑ SHY2↓

PAT1↑

FKF1↓ LKP 2↓

LHY↑ APRR↑

CTR1↑ ↑ EIN3 ↑ ERN↑ EBF1↑

MYC2↑ HB12 ↑ ABF2 ↑ GBF3 ↑ ATAF1↑NAC072 ↑ DRTY↑ RAV2 ↑

ERFs↑

MPK3↑ ↑ HOS1↑ FIN219↑

ANT↓

KNAT1↑ ↑ KNAT6↑ ↑ AS162↓ REV/KAN ↑ YABBY↓

COL↑↓ ↑↓ [FT ↑ MFT ↑] AREB3↑ ↑ BLH1 ↑ MYB62 ↑

AGL19↑ FD↑ NCED3 ↑ ABA2 ↑

AP1↓ SPL↑↓ ↑↓

A. Rohde

Target/marker genes

Signal transduction and TFs

PKL↑ GAI ↑ FIE ↑

ICL↑ MALS↑ VSP↑ BSP↑ LEA↑ COR↑ ↑ LT1↑ DHN↑ RAB↑

CDKB↓ CYCB2↓ CYCA1↓ CYCD3↓ AMYLASE ↑ SEX1↑

STS ↑

HSP↑ ERD↑ USP ↑ COR ↑ LTI ↑

Hexoses Glyoxylate cycle Starch in young leaves

Starch in subapical domain Early adaptation Late adaptation

Protiferation

Fig. 10.1. Integrative molecular timetable of bud development in poplar. Autumnal bud development is a composite of bud formation (red), acclimation to dehydration and cold (blue) and dormancy (orange). Selected genes or processes that specifically belong to one of these aspects are highlighted accordingly. Simultaneous with bud development, elongation growth (green) gradually ceases in young derivatives that are displaced from the apex. Bud development is characterized by the sequential activation of light, ethylene and abscisic acid signal transduction pathways. The major transcriptional changes at the regulatory and target process levels are depicted at the time that the respective genes show their maximal change in expression. The two major phases of transcriptional and metabolic response are indicated by grey boxes. Below, tentative levels of cellular responses and/or the quantity of major metabolites are indicated with a graded scale. Arrows connect regulators and transcription factors to their putative downstream processes, without implying a genetic or direct molecular interaction. Because of its putative nature, the link between low sugar and ethylene signal transduction is shown with a dashed arrow. Genes shown in grey and within brackets are only found differentially expressed in ABI3-overexpressing poplars. For full explanation, see Ruttink et al. (2008). (Copyright American Society of Plant Biologists; www.plantcell.org). NB: See Plate 4 for colour representation.

Bud Set

Genetic Approaches to Bud Set Covering all phenotypic aspects of bud set Previous phenotypic assessments of bud set have relied on the Julian day of completed bud set (e.g. Frewen et al., 2000). However, apical growth arrest, bud set and bud dormancy are clearly discrete phenotypes and the molecular profiling of autumnal bud development has underscored its sequential nature. From sensing of the critical day length until accomplished bud set, dynamic changes take place and should be dissected at the phenotypic level in field observations (Fig. 10.1). A new scoring system for bud set delineates seven distinct developmental stages for high-resolution data (A. Rohde, unpublished results). Traits derived from temporal measurements address all aspects of the phenotype, including the duration of bud formation and maturation that are probably most affected by climate change. The defined developmental stages were measured in a collection of Populus nigra populations and five poplar breeding pedigrees. Not unexpectedly, the onset of bud development governed the largest part of the phenotype. The P. nigra accessions covering origins from over 11° of latitude showed 27 days difference in onset of bud development, when screened in a common garden in Belgium. This difference is higher than 2 days per degree of latitude that was inferred from remote sensing data of phenological events (Zhang et al., 2004). The period appears longer in P. nigra because clones were displaced south or north into the common garden. Moreover, the total process from the first visible sign of growth arrest until the completed bud lasted about 26 days. Early and late P. nigra populations differed by 6 days at most. Although durationrelated traits were highly significant and had a high heritability, the contribution of duration to the total phenotype is minor as compared with the onset of the process. Irrespective of the bud-set trait assessed, most variation was found between populations. Qst values, a measure for the proportion of total phenotypic variation that is due to differentiation among populations, ranged between 0.6 and 0.7 for all bud-set traits. Individual and genotypic heritabilities of >0.9 were quite high

95

for the onset of bud development (A. Rohde, unpublished results). When characterizing poplar breeding pedigrees, most genetic variation resided in the onset of bud development as compared with relatively little variation for the duration. Together, the results suggest that day length is a safe predictor of the winter hazards in many environments. These experiments did not reveal prominent genetic factors acting on a short or long duration of bud development, although the time from sensing the critical day length until the onset of the first hazardous frost is expected to be different for altitudinal clines and various distances to the sea at the same latitude. One reason for the lack of such differences might be the fact that the majority of clones were screened north of their origin. For southern accessions being screened in the north, sensing of critical day length occurs later in the season and with a steeper change in day length, both leading to a compression of their phenotypic range in duration of bud development. Accessions originating north of the common garden, to the contrary, will have set bud earlier than at their origin and will be allowed to display their phenotypic range. Still, displacement studies can help to characterize the ability to adapt to climate changes. Displacement to the north will indicate the ability to colonize a new region, while ‘impaired’ with a strongly differentiated and too late bud-set trait. On the other hand, movement of clones to the south should indicate their ability to stay in a warmer climate, while the autumnal growth arrest will occur too early.

Towards robust quantitative trait loci for bud set Across the five poplar breeding families, quantitative trait loci (QTLs) were detected for the various bud-set traits (A. Rohde et al., unpublished data). Only a few QTLs for duration were detected, as compared with those for onset of bud development. The average QTL explains less than 10% of the phenotypic variation, confirming the polygenic nature of sequential bud development. Across the various parental maps, QTLs fall on to homologous regions in various instances. In some cases, traits with a

96

A. Rohde

high correlation at the phenotypic level stack in the same region of the genome. In order to investigate whether these tentatively homologous regions indeed overlap physically, the marker density is currently being increased in a few target regions. Common markers not only allow aligning the respective genetic maps, but also anchoring the region in the physical poplar genome sequence. At the same time, if enough markers are shared in a particular region, the data from different pedigrees can be combined, so as to increase the number of informative meioses for refining the QTL intervals (Veyrieras et al., 2007). These data could lay the basis for further dissection of the QTL regions by a targeted genome scan. All genes within the region can be scanned for allelic variation in the collection of P. nigra to pinpoint the gene(s) that underlie(s) the QTL and to identify the relevant alleles for adaptation.

The Way Forward Recently, eco-devo approaches – combining genomics, developmental biology and ecology – have challenged our awareness of how ecologically significant traits originate in the genome and how allelic combinations are maintained in populations and species (Reusch and Wood, 2007). An intense research on flowering in Arabidopsis thaliana has identified over 100 genes and established function for many of them. Only a few of them show diversity that conforms to the differences in flowering time and life strategy of various ecotypes (Stinchcombe et al., 2004; Ehrenreich and Purugganan, 2006). The data and information obtained from gene expression studies of bud set in trees (Druart et al., 2007; Mazzitelli et al., 2007; Ruttink et al., 2007) can similarly be employed in ecological approaches to explain the phenotypic variation in different environments or populations. The functional analysis of genes

involved in bud development will advance our understanding of pathways and regulatory hierarchies in model trees, such as poplar, for which the full genome and associated genomics tools are readily available. To estimate the putative significance for adaptation under climate change scenarios, genes with demonstrated function at bud set will need to be characterized for their diversity, selection and plasticity in ecologically relevant, natural environments. Initial studies have considered a single or a few genes with an assumed function in tree phenology (Hall et al., 2007; Ingvarsson et al., 2008). Population differentiation for bud set and gene expression of five candidate genes for phenology were characterized in a latitudinal cline of Populus tremula (Hall et al., 2007). A detailed analysis of an 80-kb region around one of those candidate genes revealed that two single-nucleotide polymorphisms in the PHYTOCHROME B2 gene are associated with the time of completed bud set. These polymorphisms explain 1.5% and 5% of the observed phenotypic variation in bud set, respectively (Ingvarsson et al., 2008). Certainly, more studies are needed to discern whether adaptation of the annual growth cycle typically relies on the diversity in regulatory or structural genes, and whether both the number and individual effect of genes are large or small. In the end, such an eco-devo approach will provide us with functional mechanisms, at lower levels of biological integration, and ecological/evolutionary significance, at higher levels of biological integration. In the future the research will move towards studying the interaction of genes/alleles and their function in real-world environments, not only in model trees, but also in important forest trees. Combining genomics with functional and developmental approaches and with ecological mechanisms will provide a better understanding of phenotypic adaptation in the context of global warming.

References Aitken, S.N., Yeaman, S., Holliday, J.A., Wang, T. and Curtis-McLane, S. (2008) Adaptation, migration or extirpation: climate change outcomes for tree populations. Evolutionary Applications 1, 95–111. Arora, R., Rowland, L.J. and Tanino, K. (2003) Induction and release of bud dormancy in woody perennials: a science comes of age. HortScience 38, 911–921.

Bud Set

97

Druart, N., Johansson, A., Baba, K., Schrader, J., Sjödin, A., Bhalerao, R.R., Resman, L., Trygg, J., Moritz, T. and Bhalerao, R.P. (2007) Environmental and hormonal regulation of the activity–dormancy cycle in the cambial meristem involves stage-specific modulation of transcriptional and metabolic networks. The Plant Journal 50, 557–573. Eagles, C.F. and Wareing, P.F. (1964) The role of growth substances in the regulation of bud dormancy. Physiologia Plantarum 17, 697–709. Ehrenreich, I.M. and Purugganan, M.D. (2006) The molecular genetic basis of plant adaptation. American Journal of Botany 93, 953–962. Frewen, B.E., Chen, T.H.H., Howe, G.T., Davis, J., Rohde, A., Boerjan, W. and Bradshaw, H.D. (2000) Quantitative trait loci and candidate gene mapping of bud set and bud flush in Populus. Genetics 154, 837–845. Hall, D., Luquez, V., Garcia, V.M., St Onge, K.R., Jansson, S. and Ingvarsson, P.K. (2007) Adaptive population differentiation in phenology across a latitudinal gradient in European aspen (Populus tremula L.): a comparison of neutral markers, candidate genes and phenotypic traits. Evolution 61, 2849–2860. Heide, O.M. (2003) High autumn temperature delays spring bud burst in boreal trees, counterbalancing the effect of climatic warming. Tree Physiology 23, 931–936. Horvath, D.P., Anderson, J.V., Chao, W.S. and Foley, M.E. (2003) Knowing when to grow: signals regulating bud dormancy. Trends in Plant Science 8, 534–540. Howe, G.T., Aitken, S.N., Neale, D.B., Jermstad, K.D., Wheeler, N.C. and Chen, T.H.H. (2003) From genotype to phenotype: unravelling the complexities of cold adaptation in forest trees. Canadian Journal of Botany 81, 1247–1266. Ingvarsson, P.K., Garcia, M.V., Luquez, V., Hall, D. and Jansson, S. (2008) Nucleotide polymorphism and phenotypic associations within and around the phytochrome B2 locus in European aspen (Populus tremula, Salicaceae). Genetics (published online doi: 10.1534/genetics.107.082354). IPCC (2007) Climate Change 2007: Synthesis Report. http://www.ipcc.ch/pdf/assessment-report/ar4/syr/ar4_ syr.pdf (accessed January 2009). Jump, A.S. and Penuelas, J. (2005) Running to a stand still: adaptation and the response of plants to rapid climate change. Ecology Letters 8, 1010–1020. Junttila, O. (2007) Regulation of annual shoot growth cycle in northern tree species. In: Taulavuori, E. and Taulavuori, K. (eds) Physiology of Northern Plants Under Changing Environment. Research Signpost, Kerala, India, pp. 177–210. Lang, G.A. (1987) Dormancy: a new universal terminology. HortScience 22, 817–820. Mazzitelli, L., Hancock, R.D., Haupt, S., Walker, P.G., Pont, S.D.A., McNico, J., Cardle, L., Morris, J., Viola, R., Brennan, R., Hedley, P.E. and Taylor, M.A. (2007) Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.). Journal of Experimental Botany 58, 1035–1045. Menzel, A. and Fabian P. (1999) Growing season extended in Europe. Nature 397, 659. Mølmann, J.A., Asante, D.K.A., Jensen, J.B., Krane, M.N., Ernstsen, A., Junttila, O. and Olsen, J.E. (2005). Low night temperature and inhibition of gibberellin biosynthesis override phytochrome action and induce bud set and cold acclimation, but not dormancy in PHYA overexpressors and wild-type of hybrid aspen. Plant Cell & Environment 28, 1579–1588. Nitsch, J.P. (1957) Photoperiodism in woody plants. Proceedings of the American Society of Horticultural Science 70, 526–544. Reusch, T.B.H. and Wood, T.E. (2007) Molecular ecology of global change. Molecular Ecology 16, 3973–3992. Rohde, A. and Bhalerao, R.P. (2007) Plant dormancy in a perennial context. Trends in Plant Science 12, 217–223. Rohde, A., Howe, G.T., Olsen, J.E., Moritz, T., Van Montagu, M., Junttila, O. and Boerjan, W. (2000) Molecular aspects of bud dormancy in trees. In: Jain, S.M. and Minocha, S.C. (eds) Molecular Biology of Woody Plants, Vol. 1 (Forestry Sciences, Vol. 64). Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 89–134. Rohde, A., Ruttink, T., Hostyn, V., Sterck, L., Van Driessche. K. and Boerjan W. (2007) Dynamic gene expression changes during the induction and maintenance of dormancy in poplar apical buds. Journal of Experimental Botany 58, 4047–4060. Ruttink, T., Arend, M., Morreel, K., Storme, V., Rombauts, S., Bhalerao, R., Boerjan, W. and Rohde, A. (2007) A molecular timetable for apical bud formation and dormancy induction in poplar. The Plant Cell 19, 2370–2390. Savolainen, O., Bokma, F., Garcia-Gil, R., Komulainen, P. and Repo, T. (2004) Genetic variation in cessation of growth and frost hardiness and consequences for adaptation of Pinus sylvestris to climatic changes. Forest Ecology & Management 197, 79–89.

98

A. Rohde

Saxe, H., Cannell, M.G.R., Johnson, Ø., Ryan, M.G. and Vourlitis, G. (2001) Tree and forest functioning in response to global warming. New Phytologist 149, 369–400. Søgaard, G., Johnsen, Ø., Nilsen, J. and Junttila, O. (2008) Climatic control of bud burst in young seedlings of nine provenances of Norway spruce. Tree Physiology 28, 311–320. Stinchcombe, J.R., Weinig, C., Ungerer, M., Olsen, K.M., Mays, C., Halldorsdottir, S.S., Purugganan, M.D. and Schmitt, J. (2004) A latitudinal cline in flowering time in Arabidopsis thaliana modulated by the flowering time gene FRIGIDA. Proceedings of the National Academy of Sciences USA 101, 4712–4717. Sylven, N. (1940) Lang- och kortkagstyper av de svenska skogstraden (Long day and short day types of Swedish forest trees). Svensk Papperstidningen 43, 317–324, 332–342, 350–354. Tanino, K. (2004) Hormones and endodormancy induction in woody plants. Journal of Crop Improvement 10, 157–199. Taylor, G., Tallis, M.J., Giardina, C.P., Percy, K.E., Miglietta, F., Gupta, P.S., Gioli, B., Calfapietra, C., Gielen, B., Kubiske, M.E., Scarascia-Mugnozza, G.E., Kets, K., Long, S.P. and Karnoyski, D.F. (2008) Future atmospheric CO2 leads to delayed autumnal senescence. Global Change Biology 14, 264–275. Thompson, R. and Clark, R.M. (2008) Is spring starting earlier? The Holocene 18, 95–104. Veyrieras, J.-B., Goffinet, B. and Charcosset, A. (2007) MetaQTL: a package of new computational methods for the meta-analysis of QTL mapping experiments. BMC Bioinformatics 8, 49. Weiser, C.J. (1970) Cold resistance and injury in woody plants. Science 169, 1269–1278. Welling, A., Moritz, T., Palva, E.T. and Junttila, O. (2002) Independent activation of cold acclimation by low temperature and short photoperiod in hybrid aspen. Plant Physiology 129, 1633–1641. Zhang, X., Friedl, M.A., Schaaf, C.B. and Strahler, A.H. (2004) Climate controls on vegetation phenological patterns in northern mid- and high latitudes inferred from MODIS data. Global Change Biology 10, 1133–1145.

11

An Epigenetic Memory From Time of Embryo Development Affects Climatic Adaptation in Norway Spruce Ø. Johnsen, H. Kvaalen, I. Yakovlev, O.G. Dæhlen, C.G. Fossdal and T. Skrøppa

Introduction Conifer trees are long-lived plants that are able to acclimate from active growth to frost-tolerant winter dormancy and deacclimate back to active growth in a cyclic manner, synchronized with seasonal changes in temperature and day length. Some of the boreal species are remarkably tolerant to very low temperatures in the winter, yet easily injured by light frost incidences (−3 to −5°C) during active growth. Seasonal timing of growth, frost hardiness and dormancy influence or may even determine the geographic distribution of trees, and in consequence, phenology traits often display some kind of clinal relationships along latitudinal and altitudinal gradients (Aitken and Hannerz, 2001; Hänninen et al., 2001; Saxe et al., 2001; Howe et al., 2003; Olsen et al., 2004). Temperature, day length and light quality regulate transition between active growth, frost hardiness and dormancy in the autumn (e.g. Håbjørg, 1972; Heide, 1974; Junttila, 1980; Junttila and Kaurin, 1985; Clapham et al., 1998; Mølmann et al., 2005, 2006), and temperature increase in late winter and early spring is a major regulatory force affecting dehardening and bud burst timing in late spring (Saxe et al., 2001; Søgaard et al., 2008; Yakovlev et al., 2008). The transition between winter and summer hardiness proceeds with high speed (e.g. Beuker et al.,

1998), and conifers may be considered masters of adaptation (Rohde and Junttila, 2008), despite exhibiting a very long juvenile phase before flowers and seeds can be produced. Nevertheless, long generation intervals (>30 years) may make them less able to respond to rapid changes in temperature by evolutionary means (Rehfeldt et al., 1999, 2002). The anticipated change in global climate could then represent a significant challenge for rapid enough adaptation of the growth–dormancy cycle in trees. Despite this challenge, we have recently found that Norway spruce can adjust the adaptive performance by a rapid and likely epigenetic mechanism, through a kind of a long-term memory of temperature sum and (probably) photoperiod from the time of its embryo development. This memory can facilitate the conifer’s ability to cope with the anticipated, evolutionary speaking rapid change in temperature predicted from the steady increase in the emission of greenhouse gases. In the present chapter, we describe how we came across this unique phenomenon, how we deduced that the memory was epigenetic (without using molecular tools), how this phenomenon has changed our interpretation of clinal variation pattern in adaptive traits, and how we are currently working to understand the molecular basis and regulation of this epigenetic memory.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

99

100

Ø. Johnsen et al.

The Parental Seed Orchard Environment Seed production in Norway spruce stands is scarce in northern areas or near the timberline at high elevations in Norway. To increase seed productions for these areas, two seed orchards were established in the late 1960s: one with clones of northern origins (64–66°N), grafted in a southerly located seed orchard (Lyngdal, 58°N); and another with clones from high elevation areas in south-eastern Norway (600–900 m above sea level), located at a site with high spring and summer temperatures (Kaupanger, 20 m above sea level). Both orchards were placed outside the natural geographic range distribution of Norway spruce, quite far away from planted as well as natural Norway spruce stands. The orchards have yielded amounts of seeds with good storage and germination ability. However, when plants from seeds of these orchards were grown for commercial production in nurseries, we received reports that the seedlings displayed an unusual southern or low-land performance. Seedlings expressed a much later timing of growth cessation and bud set in the autumn than seedlings from seeds of commercial provenances produced in natural stands in the north and at high elevation. These unexpected and seemingly casual observations prompted us to look more carefully into the phenomenon, and our experimental studies started in 1979. Bjørnstad (1981) confirmed the validity of these unexpected observations in a study where he looked at the first year’s growth and bud set of progenies from controlled crosses in the Lyngdal seed orchard. Seedlings from seed produced in the orchard set bud three weeks later than progenies from the same mother clones derived from open pollination in the their native habitats. Bjørnstad suggested, among other hypotheses, that the seed orchard environment could directly modify bud phenology of the progeny. Johnsen (1989a) extended the study to see if frost hardiness in the autumn may be similarly affected, and found that the seed orchard progenies were more injured by frost during cold acclimation in the autumn. He also showed (Johnsen, 1989b) that progenies flushed later in the spring, terminated leader shoot exten-

sion later in the summer, had higher frequency of lammas shoots, were delayed in lignification in the annual ring in the autumn and were 15% taller at age 7 years than their half-sibs produced in the northern forest. The unexpected, altered progeny performance was thus found to be of a long-lasting character (Johnsen et al., 1989) and may endure for more than 17 years from seeds (Edvardsen et al., 1996; Skrøppa et al., 2007). Several hypotheses to explain the altered progeny performance were raised after these initial studies (Johnsen, 1989a). One of the many objections to the initial studies and hypotheses put forward was that the fathers were different in the two seed production environments (seed orchards and native forest). The pollen mixture used in crosses in the seed orchard were all from selected plus trees, and it could reasonably well be argued for that they were genetically different in phenology from the majority of father trees in the northern forest which delivered pollen to receptive female flowers there. We tested this hypothesis, but had to reject it because progenies from plus trees performed equal to progenies from randomly chosen trees in native northern stands when tested for bud set and autumn frost hardiness (Johnsen and Østreng, 1994). In the same study, we also rejected another hypothesis; that the increased seed weight due to the warm seed orchard environment could lead to delayed bud set and cold acclimation in the progeny. This rejection was later corroborated by Johnsen et al. (1995). Furthermore, Skrøppa (1988) had in fact shown earlier, using another experimental approach, that seed weight did not significantly affect first year’s bud set in Norway spruce.

Proper Experimental Material and Conditions We soon realized the need for the use of movable, potted grafts that could be induced to flower, and further, could be used to produce viable seeds in different environments to study this phenomenon under more controlled genetic and environmental conditions. Using such grafts, we could utilize cloned fathers and mothers (replicated as grafts) to repeat control

Epigenetic Memory and Embryo Development in Norway Spruce

crosses and produce full-sib families with identical genetic backgrounds across several environments. The full-sib progenies from such crosses could then be tested in common gardens to quantify and report carry-over effects from the reproductive environment to the progeny. During the late 1980s and early 1990s, numerous reports from flower induction experiments were published. In collaboration with Biri Nursery and Seed Improvement Centre, we managed to efficiently induce male and female cones and produce seeds from such potted grafts in a repeatable manner (Fig. 11.1; Johnsen et al., 1994a,b). We now had the genetic material and methods to proceed with a proper experimental design. First, we reproduced the unexpected observations we had discovered, but this time it was performed with properly defined genetic material (i.e. full-sib families; Johnsen et al., 1995). Next, we found that any differences in day length and tempera-

101

ture applied during microsporogenesis (pollen formation) did not affect progeny performance. However, the environmental conditions present during female flowering did affect performance, and the effect found could indeed account for all the adaptive difference we had reported earlier (Johnsen et al., 1996). We thus concluded that at some time during the reproduction period in the female flower, environmental signals could facilitate genetic selection at pre- or post-meiotic stages, and/or that some epigenetic influence at various stages in reproduction could later affect the seedlings and saplings in common garden tests. Because we had treated all the grafts equally up until the reproductive period was started, we could reject the hypothesis that the change in phenotypic performance, caused by the seed orchard environment, could be due to some earlier somatic changes in meristems that later gave rise to the reproductive cones.

Temperature Treatment at Various Reproductive Stages

A

B

C Fig. 11.1. Potted graft of Norway spruce can be induced to produce female (A) and male (B) cones, and control crosses (C) can be performed in controlled or semi-controlled environments. (Photo: Ola Gram Dæhlen.)

After achieving this level of understanding, we wanted to relate applied temperature, and later, the timing of temperature treatments, to the various reproductive stages in the female flower and further test progeny performance in common gardens. We knew in advance the approximate temperature sums that were required to reach various reproductive stages in the female flowers (Sarvas, 1968), but we needed to calibrate the data to our experimental situation. John Owens, University of Victoria, British Columbia, Canada, kindly helped us to generate appropriate data (Owens et al., 2001), and we then had the tool needed to separate temperature effects applied to preand post-meiotic stages in the female flower and relate those treatments to progeny performance in common gardens. In a rather extensive paper, reporting the results of five experiments conducted in a period from 1993 to 2004, we were able to deduce that climatic adaptation of Norway spruce is most likely affected by the temperature sum during zygotic embryogenesis and seed maturation. No other stages in the reproduction were shown to accumulate temperature differences that later could

102

Ø. Johnsen et al.

alter the progeny performance (Johnsen et al., 2005a). In a separate study, using eight phytotron rooms, seeds from three unrelated crosses were made in an environmental 2×2 factorial combination of long and short days and high and low temperatures. In the second growth season, the progenies’ adaptive performances were affected by both the photoperiod and temperature given while seeds were produced. Long days produced more hardy progenies even at high temperatures (Johnsen et al., 2005b). This shows that northern ecotypes maintain their short critical photoperiods and early hardiness development in the autumn partly because of a carry-over effect of the prevailing long photoperiods when seeds are produced in the north.

Pre- and Post-Meiotic Selection The reproductive process offers several possibilities for directional selection. However, the potential effects of gametophytic and sporophytic selection are rather limited due to the low number of pollen grains in each pollen chamber, and of embryos in a developing seed of spruce (Sarvas, 1968; Owens and Blake, 1985). We have found little evidence for temperature-driven selection during the reproductive stages prior to fertilization that could explain the observed change in phenotypic performance (Johnsen et al., 2005a). In a study where genetic markers were used to test if pre- or post-zygotic selection could be a possible causal factor (Besnard et al., 2008), two full-sib families of Norway spruce were produced in two contrasted maternal environments (warm and cold conditions). One family expressed large and the other small phenotypic differences in response to these crossing environments. Although four parental genetic maps covering 66 to 78% of the genome (using 190 to 200 loci) were constructed, no evidence of a locus under strong and repeatable selection could be found. Moreover, in some of our experimental materials, we found clear evidence for differences in embryo abortion (Owens et al., 2001), but this difference in abortion rate was not linked to the difference in progeny performance (Johnsen et al., 2005a). Thus, we concluded, based on the

accumulated evidence, that selection among gametes, among embryos within the developing seeds or among the seeds could hardly be the main explanation to the major phenotypic difference in progeny performance that was repeatedly found. Webber et al. (2005) argued for these possible driving forces but we came to the conclusion that even the highest possible selection intensity in these phases of reproduction was simply not high enough to account for the adaptive difference we reported (Skrøppa and Johnsen, 2000).

A Memory from Embryo Development The jury was still out on whether the phenomenon of embryo memory was due to selection or an epigenetic mechanism because we had yet to completely rule out the potential selection effects of embryo competition on progeny performance. This could not be easily done experimentally using an in vivo reproductive system. A new hypothesis, however, could be postulated using an in vitro system of somatic embryogenesis. Could embryos themselves, detached from their mothers, process temperature signals that further display phenotypic difference in bud set when the plants were later grown in common garden? If so, could the magnitude of such difference account for earlier observations that were similar to a provenance separation of 3–4° of latitude (Kohmann and Johnsen, 1994; Johnsen et al., 2005a)? We decided to test the hypothesis using in vitro propagated clones, to see if the memory operated within single genotypes. We excised mature embryos from seeds of a full-sib family which was produced in a cold (outdoor) and a warm (inside a glasshouse) environment, and embryonic clones from both reproductive environments were cultured at 18, 23 and 28°C during the proliferation and embryo maturation steps (Fig. 11.2). The regenerated plants not only remembered their temperature during zygotic embryogenesis, but also the temperature applied during somatic embryogenesis in vitro (Kvaalen and Johnsen, 2008; Fig. 11.3). The warmer the temperature applied during embryo formation, the later plants formed terminal buds. The total difference shown in Fig. 11.3 (combined effects of zygotic and

Epigenetic Memory and Embryo Development in Norway Spruce

A

B

C

D

103

A

B

50

50

40

40

30

30

0

Number of days to bud set from 1 July

Number of days to bud set from 1 July

Fig. 11.2. Excised zygotic embryos (A) are induced to form polyembryonal masses (B), and then embryos are matured (C), dried and thereafter germinated in vitro (D). (Photos: Harald Kvaalen and Lars S. Dalen.)

0 18

23

28

Temperature during somatic embryogenesis (°C)

647

1341

Heat sum during zygotic embryogenesis (degree-days)

Fig. 11.3. Effects of (A) temperature during somatic embryogenesis (SE) and (B) heat sum during zygotic embryogenesis ( , 23°C SE; , 28°C SE) on the timing of bud set in clones of Picea abies propagated through SE derived from cold and warm seed production environment for one full-sib family. Values are least square means with their standard errors shown by vertical bars. (Data and figure from Kvaalen and Johnsen, 2008; reproduced with kind permission from New Phytologist.)

Ø. Johnsen et al.

somatic temperature) were similar to those produced by a provenance separation of 4–6° of latitude. Because the effect on bud set resulted in a longer growth season, plants also became taller when embryos were formed at high temperatures. This indicates that a surprisingly major part of the variability between natural provenances with respect to bud phenology and frost hardiness can be directly attributed to an epigenetic memory of the local temperature (and probably photoperiod) when embryos develop. We feel confident to conclude that the effects of the prevailing temperature during sexual reproduction and seed maturation can entirely be explained by an epigenetic memory mechanism operating in the somatic cells within the embryo itself. This does not necessarily mean that biochemical changes in the female gametophyte, surrounding the embryo, do not influence the expression of the memory, but that the response to such signals resides in the embryo itself, and thus could be affected genetically by both paternal and maternal alleles (that means, potential allelic variants in both structural and regulatory parts of the genes involved).

Phenotypic Clinal Variation is Inflated These findings impact our interpretation of clinal variation in adaptive traits, especially when measured on young seedlings (e.g. Johnsen and Skrøppa, 2000; Garcia-Gil et al., 2003). We have made a simple figure to illustrate our point (Fig. 11.4). Ideally, genetic differences among populations, inferred from phenotypic difference, should be studied with seedlings and trees from seed collected from parents who have been grown at least for one generation in common gardens. Moreover, the seeds should be collected from the same seed year to avoid phenotypic difference caused by year-to-year variation in temperature when seed production takes place (Kohmann and Johnsen, 1994). In practice, however, studies of provenance variation in conifers have almost exclusively been made from seed produced from natural and/or planted forest stands from widespread geographical areas, from north to south and from high to low elevation within the natural distribution of the species, and without any regard to

seed years as well. The temperature during embryo formation and development is correlated with latitude, altitude and local conditions, and the memory from embryo development we have discovered may well inflate the phenotypic difference among the provenances, thus bringing the clinal relationship to appear steeper and more perfect (solid line with population means) than the true genetic difference can account for (dashed theoretical line). In our opinion, this phenomenon has misled plant biologists (ourselves included) to accredit too much to the efficiency of directional, natural selection among genotypes within a contemporary generation as being the causal factor shaping population differentiation in coniferous species. The population differentiation could be caused by more than allele frequency differences in structural genes. Nevertheless, environmentally induced parental effects have a genetic basis on their own (Shaw and Byers, 1998). They may be subjected to evolutionary change (Wade, 1998) and affect progeny fitness (Donohue and Schmitt, 1998; Lacey and Herr, 2000). Moreover, we have unpublished data indicating

Freezing injury (normal scores)

104

0.6 0.4 0.2 0 −0.2 −0.4 −0.6 59 60 61 62 63 64 65 66 67 Latitudinal origin (°N)

Fig. 11.4. The relationship between freezing injury least square means of provenances and their latitudinal origins, based on real data recalculated from Dæhlen et al. (1995). The theoretical dashed line has been drawn on the assumption that all the seed lots have been produced in the southern part of Norway, where the epigenetic memory reduces the hardiness level of the northern provenances in a southern direction, similar to the 3° of latitudinal origin of observed data (Johnsen et al., 2005a).

Epigenetic Memory and Embryo Development in Norway Spruce

that the memory is expressed differently among unrelated as well as related genotypes. Thus, we suggest that the ability of embryos to memorize local temperature is an adaptive trait in its own right (Kvaalen and Johnsen, 2008).

Search for Genes Involved in a Molecular Mechanism We need to understand the molecular basis of the epigenetic mechanism causing this adaptive difference between progenies, and have started to work with this challenging task, as explicitly suggested by Rohde and Junttila (2008). The epigenetic memory could be defined as adaptive phenotypic plasticity, and we want to find and characterize genes involved in its regulation. Recently, we have reported correlations between the transcription of some genes and the memory expression (Johnsen et al., 2005a), but better experimental material and a larger set of candidate genes to work with are warranted. The search strategy we have chosen to start with is based on subtractive library constructions (Yakovlev et al., 2006). Two subtractive cDNA libraries were made from seedlings grown for 6 days at 12 h photoperiod of a full-sib family expressing profoundly the epigenetic memory. Annotation revealed considerable difference in the transcriptomes. More than 50% of the

105

contigs were unknown, representing valuable sources of candidate genes for future research. We then used quantitative RT-PCR to verify the expression patters of 34 chosen candidate genes. A cold and a warm version of six full-sib families expressing differences in the epigenetic memory were used in this verification study. Among the candidate genes, only five showed constitutive differences in transcription that were closely correlated to the genetic variation in the epigenetic memory, three of them with unknown functions and two sequences with similarity to retrotransposons. Time will tell if these genes are somehow involved in reverse transcription. We have plans to include microRNAs in our studies, as well as to use microarrays made from spruce to screen for more candidates. We hope to weed out the majority of false positive genes and single out the most pertinent candidates for future research. The plan is to study DNA methylation of specific genes, as well as their interaction with histone methylation, acetylation and microRNAs. This is, however, a timedemanding ‘road to walk’, and not easy to conduct in Norway spruce. Our ultimate dream, however, is to use these genes to identify parental members in breeding populations of Norway spruce that give rise to progenies with high expression of adaptive phenotypic plasticity, fit to perform better under future climatic conditions.

References Aitken, S.N. and Hannerz, M. (2001) Gene ecology and gene resource management strategies for conifer cold hardiness. In: Bigras, F.J. and Colombo, S.J. (eds) Conifer Cold Hardiness. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 23–53. Besnard, G., Acheré, V., Jeandroz, S., Johnsen, Ø., Faivre Rampant, P., Baumann, R., Müller-Starck, G., Skrøppa, T. and Favre, J.-M. (2008) Does maternal environmental condition during reproductive development induce genotypic selection in Picea abies? Annals of Forest Science 65, 109–114. Beuker, E., Valtonen, E. and Repo, T. (1998) Seasonal variation in the frost hardiness of Scots pine and Norway spruce in old provenance experiments in Finland. Forest Ecology and Management 107, 87–98. Bjørnstad, Å. (1981) Photoperiodical after-effect of parent plant environment in Norway spruce (Picea abies (L.) Karst) seedlings. Meddelelser fra Norsk Institutt for Skogforskning No. 36. Norwegian Forest Research Institute, Ås, Norway, 30 pp. Clapham, D.H., Dormling, I., Ekberg, I., Eriksson, G., Qamaruddin, M. and Vince-Prue, D. (1998) Latitudinal cline of requirement for far-red light for the photoperiodic control of budset and extension growth in Picea abies (Norway spruce). Physiologia Plantarum 102, 71–78. Dæhlen, A.G., Johnsen, Ø. and Kohmann, K. (1995) Autumn frost hardiness in young seedlings of Norway spruce from Norwegian provenances and seed orchards. Research Paper of Skogforsk No. 1/95. Norwegian Forest Research Institute, Ås, Norway, 24 pp. (in Norwegian with English summary, figure and table legends).

106

Ø. Johnsen et al.

Donohue, K. and Schmitt, J. (1998) Maternal environmental effects in plants. Adaptive plasticity? In: Mousseau, T.A. and Fox, C.W. (eds) Maternal Effects as Adaptations. Oxford University Press, Oxford, UK, pp. 137–158. Edvardsen, Ø.M., Johnsen, Ø. and Dietrichson, J. (1996) Growth rhythm and frost hardiness in northern progeny trials with plants from Lyngdal seed orchard. Research Paper of Skogforsk No. 9/96. Norwegian Forest Research Institute, Ås, Norway, 9 pp. (in Norwegian with English summary, figure and table legends). Garcia-Gil, M.R., Mikkonen, M. and Savolainen, O. (2003) Nucleotide diversity at two phytochrome loci along a latitudinal cline in Pinus sylvestris. Molecular Ecology 12, 1195–1206. Håbjørg, A. (1972) Effects of photoperiod and temperature on growth and development of three latitudinal and three altitudinal populations of Betula pubescens Ehrh. Scientific Reports of The Agricultural University of Norway 51, 1–27. Hänninen, H., Beuker, E., Johnsen, Ø., Leinonen, I., Murray, M., Sheppard, L. and Skrøppa, T. (2001) Impacts of climate change on cold hardiness. In: Bigras, F.J. and Colombo, S.J. (eds) Conifer Cold Hardiness. Kluwer Academic Publishers Dordrecht, The Netherlands, pp. 305–333. Heide, O. M. (1974) Growth and dormancy in Norway spruce ecotypes (Picea abies) I. Interaction of photoperiod and temperature. Physiologia Plantarum 30, 1–12. Howe, G.T., Aitken, S.N., Neale, D.B., Jermstad, K.D., Wheeler, N.C. and Chen, T.H.H. (2003) From genotype to phenotype: unraveling the complexities of cold adaptation in forest trees. Canadian Journal of Botany 81, 1247–1266. Johnsen, Ø. (1989a) Phenotypic changes in progenies of northern clones of Picea abies (L.) Karst. grown in a southern seed orchard. I. Frost hardiness in a phytotron experiment. Scandinavian Journal of Forest Research 4, 317–330. Johnsen, Ø. (1989b) Phenotypic changes in progenies of northern clones of Picea abies (L.) Karst. grown in a southern seed orchard. II. Seasonal growth rhythm and height in field trials. Scandinavian Journal of Forest Research 4, 331–341. Johnsen, Ø. and Østreng, G. (1994) Effects of plus tree selection and seed orchard environment on progenies of Picea abies. Canadian Journal of Forest Research 24, 32–38. Johnsen, Ø. and Skrøppa, T. (2000) Provenances and families show different patterns of relationship between bud set and frost hardiness in Picea abies. Canadian Journal of Forest Research 30, 1858–1866. Johnsen, Ø., Dietrichson, J. and Skaret, G. (1989) Phenotypic changes in progenies of northern clones of Picea abies (L.) Karst. grown in a southern seed orchard. III. Climatic damage in a progeny trial. Scandinavian Journal of Forest Research 4, 343–350. Johnsen, Ø., Haug, G., Dæhlen, O.G., Grønstad, B.S. and Rognstad, A.T. (1994a) Effects of heat treatment, timing of heat treatment, and gibberellin A4/7 on flowering in potted Picea abies grafts. Scandinavian Journal of Forest Research 9, 333–340. Johnsen Ø, Dæhlen, O.G., Haug, G., Grønstad, B.S. and Rognstad, A.T. (1994b) Female cone abortion and full seed production in an indoor seed orchard with potted grafts of Picea abies grafts. Scandinavian Journal of Forest Research 9, 329–332. Johnsen, Ø., Skrøppa, T., Haug, G., Apeland, I. and Østreng, G. (1995) Sexual reproduction in a greenhouse and reduced autumn frost hardiness of Picea abies progenies. Tree Physiology 15, 551–555. Johnsen, Ø., Skrøppa, T., Junttila, O. and Dæhlen, O.G. (1996) Influence of the female flowering environment on autumn frost-hardiness of Picea abies progenies. Theoretical and Applied Genetics 92, 797–802. Johnsen, Ø., Fossdal, C.G., Nagy, N., Mølmann, J., Dæhlen, O.G. and Skrøppa, T. (2005a) Climatic adaptation in Picea abies progenies is affected by the temperature during zygotic embryogenesis and seed maturation. Plant, Cell & Environment 28, 1090–1102. Johnsen, Ø., Dæhlen, O.G., Østreng, G. and Skrøppa, T. (2005b) Daylength and temperature during seed production interactively affect adaptive performance of Picea abies progenies. New Phytologist 168, 589–596. Junttila, O. (1980) Effect of photoperiod and temperature on apical growth cessation in two ecotypes of Salix and Betula. Physiologia Plantarum 48, 347–352. Junttila, O. and Kaurin, Å. (1985) Climatic control of apical growth cessation in latitudinal ecotypes of Salix pentandra L. In: Kaurin, Å., Junttila, O. and Nilsen, J. (eds) Plant Production in the North. Norwegian University Press, Oslo, pp. 83–91. Kohmann, K. and Johnsen, Ø. (1994) The timing of bud-set in seedlings of Picea abies from seed crops of a cool versus a warm summer. Silvae Genetica 43, 328–332. Kvaalen, H. and Johnsen, Ø. (2008) Timing of bud set in Picea abies is regulated by a memory of temperature during zygotic and somatic embryogenesis. New Phytologist 177, 49–59.

Epigenetic Memory and Embryo Development in Norway Spruce

107

Lacey, E.P. and Herr, D. (2000) Parental effects in Plantago lanceolata L. III. Measuring parental temperature effects in the field. Evolution 54, 1207–1217. Mølmann, J.A., Asante, D.K., Jensen, J.B., Krane, M.N., Ernstsen, A., Junttila, O. and Olsen, J.E. (2005) Low night temperature and inhibition of gibberellin biosynthesis override phytochrome action and induce bud set and cold acclimation, but not dormancy in PHYA overexpressors and wild type of hybrid aspen. Plant, Cell & Environment 28, 1579–1588. Mølmann, J.A., Junttila, O., Johnsen, Ø. and Olsen, J.E. (2006) Effects of red, far-red and blue light in maintaining growth in latitudinal populations of Norway spruce (Picea abies). Plant, Cell & Environment 29, 166–172. Olsen, J.E., Jensen, J.B., Møllman, J.A., Ernstsen, A. and Junttila, O. (2004) Photoperiodic regulation of apical growth cessation in northern tree species: the role of phytochrome and gibberellin. Journal of Crop Improvement 10, 77–112. Owens, J.N. and Blake, M.D. (1985) Forest tree seed production. Information Report PI-X-53. Petawawa National Forestry Institute, Chalk River, Ontario, Canada, 161 pp. Owens. J.N., Johnsen, Ø., Dæhlen, O.G. and Skrøppa, T. (2001) Potential effects of temperature on early reproductive development and progeny performance in Picea abies (L.) Karst. Scandinavian Journal of Forest Research 16, 221–237. Rehfeldt, G.E., Ying, C.C., Spittlehouse, D.L. and Hamilton, D.A. Jr (1999) Genetic responses to climate in Pinus contorta: niche breadth, climate change, and reforestation. Ecological Monographs 69, 375–407. Rehfeldt, G.E., Tchebakova, N.M., Parfenova, Y.I., Wykoff, W.R., Kuzmina, N.A. and Milyutin, L.I. (2002) Intraspecific responses to climate in Pinus sylvestris. Global Change Biology 8, 912–929. Rohde, A. and Junttila, O. (2008) Remembrances of an embryo: long-term effects on phenology traits in spruce. New Phytologist 177, 2–5. Sarvas, R. (1968) Investigations on the flowering and seed crop of Picea abies. Communicationes Instituti Forestalis Fenniae 67.5, 1–84. Saxe, H., Cannell, M.G.R., Johnsen, Ø., Ryan, M.G. and Vourlitis, G. (2001) Tansley review no. 123. Tree and forest functioning in response to global warming. New Phytologist 149, 369–400. Shaw, R.G. and Byers, D.L. (1998) Genetics of maternal and paternal effects. In: Mousseau, T.A. and Fox, C.W. (eds) Maternal Effects as Adaptations. Oxford University Press, Oxford, UK, pp. 97–111. Skrøppa, T. (1988) The seed weight did not affect first year’s bud-set in Picea abies. Scandinavian Journal of Forest Research 3, 437–439. Skrøppa, T. and Johnsen, Ø. (2000) Patterns of adaptive genetic variation in forest tree species; the reproductive environment as an evolutionary force in Picea abies. In: Mátyás, C. (ed.) Forest Genetics and Sustainability. Kluwer Academic Publications, Dordrecht, The Netherlands, pp. 49–58. Skrøppa, T., Kohmann, K., Johnsen, Ø., Steffenrem, A. and Edvardsen, Ø.M. (2007) Field performance and early test results of offspring from two Norway spruce seed orchards containing clones transferred to warmer climates. Canadian Journal of Forest Research 37, 515–522. Søgaard, G., Johnsen, Ø., Nilsen, J. and Junttilla, O. (2008) Climatic control of bud burst in young seedlings of nine provenances of Norway spruce. Tree Physiology 28, 311–320. Wade, M.J. (1998) The evolutionary genetics of maternal effects. In: Mousseau, T.A. and Fox, C.W. (eds) Maternal Effects as Adaptations. Oxford University Press, Oxford, UK, pp. 5–21. Webber, J., Ott, P., Owens, J. and Binder, W. (2005) Elevated temperature during reproductive development affects cone traits and progeny performance in Picea glauca×engelmannii complex. Tree Physiology 25, 1219–1227. Yakovlev, I.A., Fossdal, C.G., Johnsen, Ø., Junttila, O. and Skrøppa, T. (2006) Analysis of gene expression during bud burst initiation in Norway spruce via ESTs from subtracted cDNA libraries. Tree Genetics and Genomes 2, 39–52. Yakovlev, I.A., Asante, D.K.A., Fossdal, C.G., Partanen, J., Junttila, O. and Johnsen, Ø. (2008) Dehydrins expression related to timing of bud burst in Norway spruce. Planta 228, 459–472.

12

The Influence of Temperature on Dormancy Induction and Plant Survival in Woody Plants L. Kalcsits, S. Silim and K. Tanino

Introduction The timing of growth cessation, dormancy, cold acclimation, subsequent deacclimation and the overall depth of cold hardiness are all critical components of winter survival for plants in temperate climates. Within the next 75 years, the annual mean global temperature is forecast to increase by 1.1 to 6.4°C (IPCC, 2007), with greater increases in temperature at northern latitudes. During the critical autumn period when growth cessation, dormancy development and cold acclimation in many deciduous trees occur, temperatures are forecast to increase by 3 to 5°C on the prairies (Wheaton, 2001). In spite of this elevated temperature, plants may be more prone to freezing injury due to increasing fluctuations in climate (Gu et al., 2008). Worldwide, lack of plant synchrony with the environment continues to be the primary cause of temperature stress injury that results in significant economic loss of domesticated crops. Growth, growth cessation and dormancy are sequential and interconnected processes in the life annual cycle of plants (Dormling, 1989; Heide, 2003; Horvath et al., 2003; Junttila et al., 2003; Tanino, 2004). Accumulating evidence indicates that the timing of initial cold acclimation is associated with growth cessation (Weiser, 1970; Fuchigami et al., 1971; Ruttink et al., 2007; Kalcsits, 2008) rather than dor108

mancy induction. While cold acclimation subsequently continues in concert with dormancy progression, dormancy may be more important for the maintenance and release of cold hardiness (Tanino et al., 1989). The timing of dormancy induction and environmental conditions jointly contribute to influence dormancy, maintenance and release (O. Junttila, Tromso, Norway, 2007, personal communication). The timing of bud set expressed high genetic variation and high heritability in Pinus sylvestris (Savolainen et al., 2004). Growth cessation may be a simple yet useful measurement in plant adaptation to climate change. Our work has focused on temperature–photoperiod interactions during growth cessation and dormancy induction. Timing of these measurable traits is important in short-season, northern temperate regions where late growth cessation and dormancy attainment would not permit attainment of full low-temperature tolerance (Smithberg and Weiser, 1968). A meta-study linked unfavourable autumn conditions and low midwinter temperatures to winter injury in Finnish apple orchards over a 71-year period (Lindén, 2001). The analysis suggested that the timing of hardiness development was as important as the absolute cold hardiness. In addition, Heide (2003) found that warm autumn temperatures resulted in an observed delay in spring bud break. Temperature, during the autumn period in which the induction of growth cessation and

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Temperature and Dormancy Induction in Woody Plants

dormancy occurs, is a critical regulating factor within the annual cycle in trees. Dormancy is induced by short photoperiod (Garner and Allard, 1924). This response is mediated through the phytochrome protein pigment which perceives the length of the photoperiod (Wareing, 1956; Williams et al., 1972). Short day (SD) photoperiod is widely accepted to be the key regulator of growth cessation and dormancy induction in deciduous woody plants (Kramer, 1936; Downs and Borthwick, 1956; Nitsch, 1957; Weiser, 1970; Allona et al., 2008). However, recent studies confirmed that temperature may be as important as photoperiod in dormancy induction for northern plant ecotypes (Howe et al., 1995, 2000; Heide, 2003; Juntilla et al., 2003; Tanino, 2004; Svendsen et al., 2007). Some crops such as apples and pears are insensitive to photoperiod but are sensitive to temperature (Heide and Pestrud, 2005). There is increasing evidence that growth cessation is the result of a combined effect of night length and air temperature (Hänninen and Kramer, 2007). Warm temperatures have been shown to induce earlier growth cessation and deep dormancy development (Fuchigami et al., 1971, 1982; Dormling, 1989; Heide, 2003; Junttila et al., 2003). Palonen (2006) reported earlier growth cessation and deeper dormancy under short photoperiod and constant 20°C warm temperatures than 4°C cool temperatures in raspberry (Rubus idaeas L.). Our recent results with hybrid poplar clones showed night temperature was more highly correlated with growth cessation and subsequent dormancy induction and cold hardiness development than day temperature (Kalcsits, 2008; Table 12.1). Growth cessation was affected most by temperature, which subsequently affected dormancy development and cold acclimation potential. Treatments where growth cessation occurred early were more dormant and had higher levels of cold hardiness. Dormancy and cold hardiness were more varied in ‘Walker’ poplar between temperature treatments (Table 12.1 and Fig. 12.1) under short photoperiod. Therefore, in certain years it would be susceptible to early freezing stress due to lack of environmental synchrony under cool night autumn temperatures. By contrast, the poplar clone ‘Okanese’ was relatively

109

Table 12.1. Days to growth cessation during dormancy induction in four hybrid poplar clones (‘Walker’, ‘Okanese’, ‘Katepwa’ and ‘Prairie Sky’) under short photoperiod and two temperature regimes (18.5°C/3.5°C and 18.5°C/13.5°C day/night temperature). (Modified from Kalcsits, 2008.) Clone ‘Katepwa’ ‘Prairie Sky’ ‘Walker’ ‘Okanese’

Day/night temperature (°C)

Days to growth cessation

18.5/3.5 18.5/13.5 18.5/3.5 18.5/13.5 18.5/3.5 18.5/13.5 18.5/3.5 18.5/13.5

50.9 29.1 42.4 27.4 57.5 28.2 30.6 27.4

insensitive to temperature and was induced into growth cessation and dormancy by both cool and warm night temperatures (Table 12.1 and Fig. 12.1). ‘Prairie Sky’ did not enter into dormancy under any inductive treatments, indicating that genetic variation also exists in dormancy development under different temperatures and short photoperiod (Kalcsits, 2008). Cool temperatures (<10°C) have been reported to override the effect of photoperiod on woody species and ecotypes of plants from northern populations. Low temperatures have been shown to induce dormancy in northern ecotypes of Cornus sericea L. (Svendsen et al., 2007), birch (Junttila, 1980), apple and pear (Heide and Pestrud, 2005) and Populus (Howe et al., 2000; Welling et al., 2002), even under long days (LD). Mölmann et al. (2005) reported that cool temperatures (5°C) induced bud set and cold acclimation in hybrid aspen (Populus tremula L.×tremuloides Michx.). However, endodormancy induction did not occur under the low-temperature treatments. Van der Veen (1951) observed bud set in Populus species exposed to 5°C and 9 h photoperiods. However, trees not exposed to an SD warm period did not attain endodormancy and resumed growth when placed under favourable growth conditions. Fuchigami et al. (1982) stated trees must undergo a warm period under SD before dormancy development can be induced. It may be possible that

110

L. Kalcsits et al.

45 40

Days to bud break

35 30 25 20 15 10 5 0 ‘Katepwa’

‘Prairie Sky’

‘Walker’

‘Okanese’

Fig. 12.1. Dormancy levels (days to bud break) of four hybrid poplar clones (‘Katepwa’, ‘Prairie Sky’, ‘Walker’ and ‘Okanese’) at 60 days of dormancy induction under four (day/night) temperature regimes ( , 18.5°C/13.5°C; , 13.5°C/8.5°C; , 23.5°C/8.5°C; , 18.5°C/3.5°C) and short photoperiod. (Modified from Kalcsits, 2008.)

dormancy is inhibited under cool temperatures and SD for some species or ecotypes (Junttila et al., 2003; Heide and Pestrud, 2005). Thermoperiodism, defined as the effect of day–night temperature differences on plant growth and development (Went, 1944), influences dormancy induction in woody plants. Junttila (1980) and Stevenson (1994) reported that diurnal temperature differences were important for dormancy induction in Salix/ Betula and C. sericea L., respectively. C. sericea ecotypes were subjected to either 22 h or 8 h photoperiods with 16 h of low night temperature regimes (5°C/5°C, 10°C/5°C and 20°C/5°C) beginning with the night period. Even under LD, cool night temperatures stimulated dormancy development in northern ecotypes but required a diurnal temperature difference since the treatment at 5°C/5°C was ineffective (Stevenson, 1994). By contrast, Palonen (2006) reported that dormancy development occurred earlier and was greater in raspberry (R. idaeus L.) under warm night temperature (20°C/20°C day/night temperature) compared with cool night temperatures (5°C). Collectively, the wide range of temperature effects on dormancy development emphasizes the importance of identifying responses

of woody plants to temperature change. What is consistent is the emerging recognition of temperature, in addition to photoperiod, as a key controlling factor in the timing of growth cessation and dormancy induction. The relative importance of temperature will profoundly increase with predicted climate change and increased variability in temperature. With expanding interest in agroforestry and Populus species, knowledge of how temperature may influence timing of growth cessation, dormancy (induction, maintenance, release) and subsequent cold hardiness will be particularly important for the distribution of cultivars. In the present chapter we present some of our hypotheses on the mechanism of temperature–photoperiod interactions during growth cessation and dormancy induction.

Phytochrome and Temperature In addition to its sensitivity to light, phytochrome has been clearly shown to be temperature-sensitive (see Hennig, 2006 for a review on phytochrome degradation and dark reversion). Both phytochrome Pfr destruction in maize coleoptiles (Butler and Lane, 1965) and

Temperature and Dormancy Induction in Woody Plants

dark reversion of Pfr to Pr in yeast (Schäfer and Schmidt, 1974; Hennig and Schäfer, 2001) were temperature-dependent. Therefore, responses which are regulated by the Pr:Pfr ratio may be affected by changes in environmental temperature. Böhlenius et al. (2006) reported that the poplar orthologue (PtFT1) of the FLOWERING TIME LOCUS (FT) gene in Arabidopsis was induced by the poplar CONSTANS (PtCO2) gene and controlled growth cessation and dormancy in Populus tremula×tremuloides ecotypes. PtFT1 expression inhibits growth cessation and dormancy development. Under LD conditions, PtCO2 expression peaks after dawn – depending upon its latitudinal adaptation – and induces PtFT1. Peak expression appears to be mediated by phytochrome B (PhyB) in the morning and partially by phytochrome A (PhyA) in the evening (Valverde et al., 2004). Under SD conditions, PtCO2 peaks at night and hence fails to induce PtFT1. During the night, SUPRESSOR OF PHYA (SPA family genes) controls expression of CO. Interestingly, Halliday et al. (2003) demonstrated that regulation of the FT gene in Arabidopsis also involved the interaction between PhyB and temperature. Temperature was found to alter flowering time in Arabidopsis, controlled by the Pr:Pfr ratio of PhyB. Since temperature affects expression of the FT gene in Arabidopsis, it is not unreasonable to suggest that temperature could also modify the expression of PtFT1 in woody plants.

111

both tropical (Frankie et al., 1974) and temperate tree species such as Betula pubescens (Rinne et al., 1994; Welling et al., 1997). An increase in abscisic acid (ABA), a stress-signalling hormone, was reported in buds during drought stress and cold stress. In temperate climates, periods of severe drought or frost periods may result in earlier growth cessation in woody plants (Rinne et al., 1994). Abiotic stresses can increase the rate of bud set to provide protection against unfavourable conditions in some species. Photoxidative stress is induced under conditions of low temperature in combination with light (Öquist and Huner, 2003; Ensminger et al., 2006). Previous studies which showed low-temperature induction of growth cessation may have induced responses through oxidative stress. Van der Veen (1951) observed rapid bud set of Populus plants placed in 5°C constant temperature. Junttila (1980), Stevenson (1994) and Mölmann et al. (2005) also induced growth cessation but not dormancy under LD/low temperature combinations. Multiple pathways likely exist that regulate bud set: a stress-induced pathway, which may not necessarily lead to true bud dormancy; and an SD-induced pathway, which classically results in bud dormancy development. Stress-induced growth cessation may be independent of photoperiod-induced growth cessation. However, under field conditions, the two processes may occur in concert to promote rapid cessation of growth and/or induction of dormancy in late summer or autumn, and therefore overlapping effects may be present at this time.

Abiotic Stress and Temperature Abiotic stress can also affect the timing and rate of growth cessation (Chen and Li, 1978; Stevenson, 1994). Evolution of angiosperms originated in the tropics where temperature and photoperiod were relatively stable, but an annual dry/wet seasonal cycle was present. Plants evolved to tolerate this cyclic precipitation by losing their leaves and entering a dormant state prior to the dry season (Frankie et al., 1974). In the tropics, dormancy is induced by moisture stress in combination with SD photoperiod. A decrease in water potential has been shown to initiate leaf abscission in

Sucrose and Temperature Day/night temperature alterations primarily induce changes in net carbon accumulation (see Öquist and Huner, 2003; Ensminger et al., 2006 for reviews). It is well established that simple sugars such as sucrose increase dramatically in cells in response to low autumn night temperatures (Levitt, 1980). The regulatory effect of sugars on photosynthetic activity and plant metabolism is widely recognized; however, the concept of sugars as central signalling molecules is relatively novel (Rolland et al., 2002).

112

L. Kalcsits et al.

There is a growing body of evidence demonstrating that sucrose acts as a signalling molecule in plant development, regulating the cell cycle, the phytohormones gibberellic acids (GAs) and ABA, and stress-associated genes (Riou-Khamlichi et al., 2000; Borisjuk et al., 2002; Eckardt, 2002; Gibson, 2004; Chao et al., 2006). Sucrose appears to regulate GA signal transduction which then regulates dormancy status of root buds in leafy spurge (Horvath et al., 2002; Chao et al., 2006). Furthermore, while ABA and glucose appear to have antagonistic effects, ABA and sucrose both promote storage reserve accumulation (Finkelstein and Gibson, 2002). ABA has long been associated with bud dormancy induction (Ramsay and Martin, 1970; Rinne et al., 1994; Rohde et al., 2002; see Tanino, 2004 for a review). ABA arrested cells in the G2 phase and buds remained dormant (Le Bris et al., 1999). Tanino et al. (1991) monitored [14C]sucrose accumulation and distribution during ABA treatment and showed that ABA induced [14C]sucrose cellular accumulation by 97% over the control in addition to significantly elevating freezing tolerance in bromegrass cell suspension cultures. Phytochrome action also mediates changes in ABA levels in Lemna gibba genes (Weatherwax et al., 1998). Dijkwel et al. (1997) indicated that sucrose controlled PhyA signalling in Arabidopsis. PhyA is strongly implicated in regulating bud dormancy induction in poplar (Olsen et al., 1997) and aspen (Eriksson, 2000). In hybrid poplar, the overexpression of PhyA prevented SD-induced cold acclimation (Olsen et al., 1997), but development of cold tolerance in response to low temperature was not disturbed under LD conditions (Welling et al., 2002). Kim et al. (2002) showed PhyB to be the primary photoreceptor responsible for the activation of cold-stress signalling in response to light in Arabidopsis. Furthermore, Short (1999) reported that the presence of sucrose and an overexpression of PhyB combined to inhibit PhyA function in Arabidopsis. Our results also indicate that the northern dogwood ecotypes have greater tolerance to low-temperature photoinhibition than the southern types (G. Gray, K.M. Cherry, J.N. Baerr, R.K. Stevenson, W. Hrycan and K.K. Tanino, unpublished results). Under steady-

state conditions, the northern ecotype showed increased capacity for electron transport and higher non-photochemical quenching compared with the southern Utah ecotype. We would assume that under low-temperature dormancy-inducing conditions, the northern ecotype would have more potential for CO2 fixation than the southern ecotype but this aspect still needs to be assessed.

Phytohormones and Temperature Phytohormones have long been associated with dormancy induction (see Tanino, 2004 for a review). While many reports indicate temperature-regulated ABA induction in other physiological responses, temperature-regulated hormone-mediated growth and dormancy induction has been largely associated with GA biosynthesis. Pinthus et al. (1989) found that endogenous GA1 concentration increased with increasing temperature in isogenic lines of Triticum. Additionally, a thermoperiodic effect was observed on the synthesis of GA in plants. Moe (1990) suggested the observed differences in stem elongation and growth under high day/ night alterations was a result of changes in the sensitivity and metabolism of GA. More specifically, daily temperature fluctuations affect the endogenous level of bioactive GA1 in the stems. A positive day–night temperature difference increases GA1, GA12, GA19 and GA20 concentrations in Begonia×hiemalis Fotsch (Myster et al., 1997). Positive day–night temperature differences resulted in less 2β-hydroxylation of bioactive GA1 to inactive GA8 than negative day–night temperature difference in Pisum sativum (Grindal et al., 1998). Since carbon fixation is strongly affected by temperature, sucrose as a signalling molecule may be mediating the phytohormone response. Endogenous concentration of GAs correlates with stem elongation and growth cessation (Jansen et al., 1986; Juntilla et al. 1991; Olsen et al., 1995; Mölmann et al., 2003) and bud break (Wurzberger and Farkash, 1976) in woody plants. Furthermore, GA concentrations dropped when willow trees were exposed to short photoperiod (Junttila and Jensen, 1988). Olsen et al. (1997) reported that exogenous GA applied to developed terminal buds

Temperature and Dormancy Induction in Woody Plants

of Salix pentandra initiated cell division at the apical meristem. This suggests that GA concentrations may have a regulatory effect on bud set and bud break in woody plants. GA is present in many different forms in plants. Chen (1994; cited in Tanino, 2004) showed that GA1 and GA8 were associated with bud break in Euphorbia longana while others (GA9 and GA32) increased during growth cessation and flowering initiation. Growth cessation and dormancy control ultimately revolve around control of meristematic activity (Rohde and Bhalerao, 2007) within woody plants. The cell cycle has an obvious impact on dormancy through regulation of meristem activity (see Horvath et al., 2003 for a review). The influence of phytohormones such as ABA on the cell cycle (Rohde et al., 1997; Le Bris et al., 1999) can be directly mediated by temperature through kinetic influences on metabolism.

Water Status and Temperature Physiological changes that regulate dormancy and cold hardiness in plants can also be affected by temperature. Temperature can impact the biophysical interaction between water, the catalyst in which many metabolic processes occur in plants, and other molecules that accumulate during dormancy induction. Binding and tissue dehydration may be two of the processes that regulate water status in the cell. For example, accumulation of hydrophilic molecules can bind water and restrict water movement within plant tissue (Faust et al., 1995). In addition to changes in biophysical interactions between water and hydrophilic molecules, restriction of movement both in and out of plant tissue may regulate the state of dormancy in vegetative buds. Ashworth (1982) identified that the lack of xylem differentiation prevented ice propagation into the bud primordia, forming a blockage that prevented injury to the dormant bud. Changes in plasmodesmata that regulate movement of water may also be responsible for the regulation of dormancy in woody plants (Van der Schoot, 1996). Erez et al. (1998) showed that peach shoots exposed to a long photoperiod and warm temperatures had more free water than buds exposed to a short photope-

113

riod and warm temperatures or a long photoperiod and cool temperatures during dormancy induction. Water is largely transported between cells through the plasmodesmata and aquaporin water channels. Intercellular communication is thought to be restricted during endodormancy by blockage of the plasmodesmata through differential Ca deposition (Jian et al., 1997) or by 1,3-b-glucan (Rinne et al., 2001). Blocking of the plasmodesmata would restrict access to water, essential for resumption of growth. Higher accumulation of 1,3-b-glucan has been reported in bean (Phaseolus vulgaris) under warmer temperatures (Abeles and Forrence, 1970). Therefore, more rapid accumulation of 1,3-b-glucan during dormancy induction (Rinne et al., 2001) could occur under warmer temperatures. Chilling increases the activity of 1,3b-glucanase at the plasmodesmata, providing some evidence that the breakdown of 1,3-bglucan is associated with low temperatures (Rinne et al., 2001). 1,3-b-Glucanase, the enzyme responsible for degradation of 1,3-bglucan, is present in plasmodesmata of endodormant woody plants (Rinne et al., 2001; Rinne and Van der Schoot, 2003). Recently, Yooyongwech et al. (2008) also reported alterations in water status and aquaporin gene expression, particularly in the basal portion of the bud, during peach dormancy induction. Aquaporins are widespread and increasingly accepted to be significant regulators of water transport (Sakurai et al., 2008). Opening of the plasmodesmata or changes in aquaporin gene expression could allow increases in cell– cell water movement within the plant. If warmer temperatures induce greater accumulation of 1,3-b-glucan and dormancy is regulated by this accumulation, the physiological basis by which temperature affects dormancy development could lie in the plasmodesmata and restriction of water and metabolite movement between plant tissues and cells.

Dehydrins and Temperature Dehydrins, a group of late embryogenesisabundant (LEA)-type proteins, were originally discovered to accumulate in tissues under increasing drought-like conditions in plants

114

L. Kalcsits et al.

(Close et al., 1993; Robertson and Chandler, 1994). These proteins are highly hydrophilic, heat-stable and contain at least one lysine-rich sequence comparable to EKKGIMDKIKEKLPG (Karlson et al., 2003). Accumulation of these proteins is induced by changes in temperature, water stress and photoperiod (Rowland and Arora, 1997). Salzman et al. (1996) studied protein induction in Vitis labruscana var. ‘Concord’ under short photoperiods with and without low temperature and 27-kDa and 47-kDa proteins accumulated under short photoperiods and warm temperature. The 47-kDa protein was later found to be a dehydrin-type protein. Wake and Fennell (1996) examined protein changes during dormancy induction of Vitis riparia and Vitis vinifera and their F1 hybrids. V. riparia entered dormancy earlier than V. vinifera. In both species and hybrids, proteins of 18–22 kDa and 16–19 kDa accumulated under SD exposure regardless of the dormancy status. However, in V. riparia, accumulation of a 17–20-kDa protein was attributed to endodormancy induction. The role of dehydrin proteins may have more impact on cold hardiness than dormancy but their role in stabilization of water cannot be discounted. Changes in water status may be regulated by temperature changes but the control of water movement between tissues may be closely related to endodormancy.

Conclusion It is apparent that temperature influences phenological and physiological changes related to

growth cessation, dormancy and cold hardiness. The impact of increasing temperatures due to global warming is largely unknown for woody plants introduced or adapted to a geographic location. The interaction between photoperiod and temperature is integral in assessing the impact of increasing temperatures on dormancy development. Changes in dormancy development may be regulated by changes of diverse factors from photosynthesis to water mobility within plant tissue. Physiological blockages at the base of buds and at the apical meristem may reduce water movement and associated susceptibility to deacclimation. Additionally, examining how temperature affects sensing of photoperiod through the phytochrome pathway may yield further insights into the physiological basis as to how temperature affects dormancy development. Identifying whether temperature affects the circadian rhythm-regulated expression of CO under SD and LD photoperiods may be useful in determining whether this hypothesis has validity. Comprehensive molecular characterization under temperature-induced growth cessation and dormancy, similar to the approach of Ruttink et al. (2007), is required before understanding can be gained at that level. Further work is required to identify the physiological basis of temperature-driven dormancy induction and related processes in woody plants. Further work is also needed to evaluate the interspecific, genetic, epigenetic (Johnsen et al., Chapter 11, this volume) and geographical variation in dormancy development in response to changes in temperature that may exist in a broad spectrum of woody plants.

References Abeles, F.B. and Forrence, F.E. (1970) Temporal and hormonal control of b-1,3-glucanase in Phaseolus vulgaris L. Plant Physiology 45, 395–400. Allona, I., Ramnos, A., Ibanez, C., Contreras, A., Casado, R. and Aragoncillo, C. (2008) Molecular control of dormancy establishment in trees. Spanish Journal of Agricultural Research 6, 201–210. Ashworth, E. (1982) Properties of peach flower buds which facilitate supercooling. Plant Physiology 70, 1475–1479. Böhlenius, H., Huang, T., Charbonnel-Campaa, L., Brunner, A.H., Jansson, S., Strauss, S.H. and Nilsson, O. (2006) CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312, 1040–1043. Borisjuk, L., Walenta, S., Rolletscheck, H., Mueller-Klieser, W., Wobus, U. and Weber, H. (2002) Spatial analysis of plant metabolism: sucrose imaging within Vicia faba cotyledons reveals specific developmental patterns. The Plant Journal 29, 521–530.

Temperature and Dormancy Induction in Woody Plants

115

Butler, W.L. and Lane, H.C. (1965) Dark transformation of phytochrome in vivo II. Plant Physiology 40, 13–17. Chao, W.S., Serpe, M.D., Anderson, J.V., Gesch, R.W. and Horvath, D.P. (2006) Sugars, hormones and environment affect the dormancy status in underground adventitious buds of leafy spurge (Euphorbia esula). Weed Science 54, 59–68. Chen, H.H. and Li, P.H. (1978) Interaction of low temperature, water stress, and short days in the induction of stem frost hardiness in red-osier dogwood. Plant Physiology 62, 833–835. Close, T.J., Fenton, R.D. and Moonan, F. (1993) A view of plant dehydrins using antibodies specific to the carboxy terminal peptide. Plant Molecular Biology 23, 279–286. Dijkwel, P.P., Huijser, C., Weisbeek, P.J., Chua, N.-H. and Smeekens, S.C.M. (1997) Sucrose control of phytochrome A signaling in Arabidopsis. The Plant Cell 9, 583–595. Dormling, I. (1989) The role of photoperiod and temperature in the induction and release of dormancy in Pinus sylvestris L. seedlings. Annals of Forest Science 46, 228–232. Downs, R.J. and Borthwick, H.A. (1956) Effects of photoperiod on growth of trees. Botanical Gazette 117, 310–326. Eckardt, NA (2002) Abscisic acid biosynthesis gene underscores the complexity of sugar, stress, and hormone interactions. The Plant Cell 14, 2645–2649. Ensminger, I., Busch, F. and Huner, N.P.A. (2006) Photostasis and cold acclimation: sensing low temperature through photosynthesis. Physiologia Plantarum 126, 28–44. Erez, A., Faust, M. and Line, M.J. (1998) Changes in water status in peach buds in induction, development and release from endodormancy. Scientia Horticultarae 73, 111–123. Eriksson, M.E. (2000) The role of phytochrome A and gibberellins in growth under long and short day conditions: studies in hybrid aspen. PhD thesis, Swedish University of Agriculture Sciences, Umeå, Sweden. Faust, M., Liu, D., Line, M.J. and Stutte, G.W. (1995) Conversion of bound water to free water in endodormant buds of apple is an incremental process. Acta Horticulturae 395, 113–117. Finkelstein, R.R. and Gibson, S.I. (2002) ABA and sugar interactions regulating development: cross-talk or voices in a crowd? Current Opinion in Plant Biology 5, 26–32. Frankie, G.W., Baker, H.G. and Opler, P.A. (1974) Comparative phenological studies of trees in tropical wet and dry forests in the lowlands of Costa Rica. Journal of Ecology 62, 881–919. Fuchigami, L.H., Weiser, C.J. and Evert, D.R. (1971) Induction of cold acclimation in Cornus stolinifera Michx. Plant Physiology 47, 98–103. Fuchigami, L.H., Weiser, C.J., Kobayashi, K., Timmis, R. and Gusta, L.V. (1982) A degree growth stage (°GS) model and cold acclimation in temperate woody plants. In: Li, P.H. and Sakai, A. (eds) Plant Cold Hardiness and Freezing Stress. Academic Press, New York, New York, pp. 93–116. Garner, W.W. and Allard, H.A. (1924) Further studies in photoperiodism, the response of the plant to relative length of day and night. Journal of Agricultural Research 23, 871–920. Gibson, S. (2004) Sugar and phytohormone response pathways: navigating a signalling network. Journal of Experimental Botany 55, 253–264. Grindal, G., Junttila, O., Reid, J.B. and Moe, R. (1998) The response to gibberelin in Pisum sativum grown under alternating day and night temperature. Journal of Plant Growth Regulation 17, 161–167. Gu, L., Hanson, P.J., MacPost, W., Kaiser, D.P., Yang, B., Nemani, R., Pallardy, S.G. and Meyers, T. (2008) The 2007 Eastern US spring freeze: increasing cold damage in a warming world? BioScience 58, 253–262. Halliday, K.J., Salter, M.G., Thingnaes, E. and Whitelam, G.C. (2003) Phytochrome control of flowering is temperature sensitive and correlates with expression of floral integrator FT. The Plant Journal 33, 875–885. Hänninen, H. and Kramer, K. (2007) A framework for modelling the annual cycle of trees in boreal and temperate regions. Silva Fennica 41, 167–205. Heide, O.M. (2003) High autumn temperature delays spring bud burst in boreal trees, counterbalancing the effect of climatic warning. Tree Physiology 23, 931–936. Heide, O.M. and Pestrud, A.K. (2005) Low temperature, but not photoperiod, controls growth cessation and dormancy induction and release in apple and pear. Tree Physiology 25, 109–114. Hennig, L. (2006) Phytochrome degradation and dark reversion. In: Schäfer, E. and Nagy, F. (eds) Photomorphogenesis in Plants and Bacteria, 3rd edn. Springer, Dordrecht, The Netherlands, pp. 131–153. Hennig, L. and Schäfer, E. (2001) Both subunits of the dimeric plant photoreceptor phytochrome require chromophore for stability of the far-red light absorbing form. Journal of Biological Chemistry 276, 7913–7918. Horvath, D.P., Chao, W.S. and Anderson, J.V. (2002) Molecular analysis of signals controlling dormancy and growth in underground adventitious buds of leafy spurge. Plant Physiology 128, 1439–1446.

116

L. Kalcsits et al.

Horvath, D.P., Anderson, J.V., Chao, W.S. and Foley, M.E. (2003) Knowing when to grow: signals regulating bud dormancy. Trends in Plant Science 8, 534–540. Howe, G.T., Hackett, W.P., Furnier, G.R. and Klevorn, R.E. (1995) Photoperiodic responses of a northern and southern ecotype of black cottonwood. Physiologica Plantarum 93, 695–708. Howe, G.T., Saruul, P., Davis, J. and Chen, T.H.H. (2000) Quantitative genetics of bud phenology, frost damage, and winter survival in an F2 family of hybrid poplars. Theoretical and Applied Genetics 101, 632–642. IPCC (2007) Climate Change 2007: Synthesis Report. http://www.ipcc.ch/pdf/assessment-report/ar4/syr/ar4_ syr.pdf (accessed January 2009). Jansen, E., Rivier, L., Junttila, O. and Crozier, A. (1986) Identification of abscisic acid from shoots of Salix pentandra. Physiologica Plantarum 66, 406–408. Jian, L.-C., Li, P.H., Sun, L.-H. and Chen, T.H.H. (1997) Alterations in ultrastructure and subcellular localization of Ca2+ in poplar apical bud cells during the induction of dormancy. Journal of Experimental Botany 48, 1195–1207. Junttila, O. (1980) Effect of photoperiod and temperature on apical growth cessation in two ecotypes of Salix and Betula. Physiologica Plantarum 48, 347–352. Junttila, O. and Jensen, E. (1988) Gibberellins and photoperiodic control of shoot elongation in Salix. Physiologica Plantarum 74, 371–375. Juntilla, O., Jensen, E. and Ernstsen, A. (1991) Effects of prohexadione (BX-112) and gibberellins on shoot elongation in Salix. Physiologica Plantarum 83, 17–21. Junttila, O., Nilsen, J. and Igeland, B. (2003) Effect of temperature on the induction of bud dormancy in ecotypes of Betula pubescens and Betula pentandra. Scandinavian Journal of Forestry Research 18, 208–217. Kalcsits, L. (2008) Exploring how temperature affects dormancy induction and cold acclimation initiation in hybrid poplar. MSc. thesis, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Karlson, D.T., Zeng, Y., Stirm, V.I., Joly, R.J. and Ashworth, E.N. (2003) Photoperiodic regulation of a 24-kD dehydrin-like protein in red-osier dogwood (Cornus sericea L.) in relation to freezing tolerance. Plant & Cell Physiology 44, 25–34. Kim, H.-J., Kim, Y.-K., Park, J.-Y. and Kim, J. (2002) Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. The Plant Journal 29, 693–704. Kramer, P.J. (1936) Effect of variation in length of day on growth and dormancy of trees. Plant Physiology 11, 127–137. Le Bris, M., Michaux-Ferrière, N., Jacob, Y., Poupet, A., Barthe, P., Guigonis, J.M. and Le Page Degivry, M.T. (1999) Regulation of bud dormancy by manipulation of ABA in isolated buds of Rosa hybrida cultured in vitro. Australian Journal of Plant Physiology 26, 273–281. Levitt, J. (1980) Responses of Plants to Environmental Stresses, Vol. I. Academic Press, New York, New York. Lindén, L. (2001) Re-analyzing historical records of winter injury in Finnish apple orchards. Canadian Journal of Plant Science 81, 479–485. Moe, R. (1990) Effect of day and night temperature alternations and of plant growth regulators on stem elongation and flowering of the long-day plant Campanula isophylla Moretti. Scientia Horticulturae 43, 291–305. Mölmann, J.A., Berhanu, A.T., Stormo, S.K., Ernstsen, A., Junttila, O. and Olsen, J.E. (2003) Metabolism of gibberellin A19 is under photoperiodic control in Populus, Salix and Betula, but not in daylength-insensitive Populus overexpressing phytochrome A. Physiologica Plantarum 119, 278–286. Mölmann, J.A., Asante, D.K.A., Jensen, J.B., Krane, M.N., Ernstsen, A., Junttila, O. and Olsen, J.E. (2005) Low night temperature and inhibition of gibberellin biosynthesis override phytochrome action and induce bud set and cold acclimation, but not dormancy in PHYA overexpressors and wild-type of hybrid aspen. Plant, Cell & Environment 28, 1579–1588. Myster, J., Junttila, O., Lindgaard, B. and Moe, R. (1997) Temperature alternations and the influence of gibberellins and indoleacetic acid on elongation growth and flowering of Begonia×hiemalis Fotsch. Plant Growth Regulation 21, 135–144. Nitsch, J.P. (1957) Photoperiodism in woody plants. Proceedings of the American Society for Horticultural Science 70, 526–544. Olsen, J.E., Jensen, E., Junttila, O. and Moritz, T. (1995) Photoperiodic control of endogenous gibberellins in roots and shoots of elongating Salix pentandra seedlings. Physiologica Plantarum 90, 378–381. Olsen, J.E., Junttila, O., Nilsen, J., Eriksson, M.E., Martinussen, I., Olsson, O., Sandberg, G. and Moritz, T. (1997) Ectopic expression of oat phytochrome A in hybrid aspen changes critical daylength for growth and prevents cold acclimation. The Plant Journal 12, 1339–1350.

Temperature and Dormancy Induction in Woody Plants

117

Öquist, G. and Huner, N.P.A. (2003) Photosynthesis of overwintering evergreen plants. Annual Review of Plant Biology 54, 329–355. Palonen, P. (2006) Vegetative growth, cold acclimation, and dormancy as affected by temperature and photoperiod in six red raspberry (Rubus idaeus L.) cultivars. European Journal of Horticultural Science 72, issue 4, 6 pp. Pinthus, M.J., Gale, M.D., Appleford, N.E.J. and Lenton, J.R. (1989) Effect of temperature on gibberelin (GA) responsiveness and on endogenous GA1 content of tall and dwarf wheat genotypes. Plant Physiology 90, 854–859. Ramsay, J. and Martin, G.C. (1970) Isolation and identification of a growth inhibitor in spur buds of apricot. Journal of the American Society for Horticultural Science 95, 569–574. Rinne, P.L.H. and Van der Schoot, C. (2003) Plasmodesmata at the crossroads between development, dormancy and defense. Canadian Journal of Botany 81, 1182–1197. Rinne, P., Saarelainen, A. and Junttila, O. (1994) Growth cessation and bud dormancy in relation to ABA level in seedlings and coppice shoots of Betula pubescens as affected by a short photoperiod, water stress and chilling. Physiologica Plantarum 90, 451–458. Rinne, P.L.H., Kaikuranta, P. and Van der Schoot, C. (2001) The shoot apical meristem restores its symplastic organization during chilling-induced release from dormancy. The Plant Journal 26, 249–264. Riou-Khamlichi, C., Menges, M., Healy, J.M. and Murray, J.A. (2000) Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Molecular and Cellular Biology 20, 4513–4521. Robertson, M. and Chandler, P.M. (1994) A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression. Plant Molecular Biology 26, 805–816. Rohde, A. and Bhalerao, R.P. (2007) Plant dormancy in the perennial context. Trends in Plant Science 12, 217–223. Rohde, A., Van Montagu, M., Inze, D. and Boerjan, W. (1997) Factors regulating the expression of cell cycle genes in individual buds of Populus. Planta 201, 43–52. Rohde, A., Prinsen, E., de Rycke, R., Engler, G., van Montagu, M. and Boerjan, W. (2002) PtI3 impinges on the growth and differentiation of embryonic leaves during bud set in poplar. The Plant Cell 14, 1885–1901. Rolland, F., Moore, B. and Sheen, J. (2002) Sugar sensing and signalling in plants. The Plant Cell 14, Suppl., 185–205. Rowland, L.J. and Arora, R. (1997) Proteins related to rest (endodormancy) in woody perennials. Plant Science 126, 119–144. Ruttink, T., Arend, M., Morreel, K., Storme, V., Rombauts, S., Bhalerao, R., Boerjan, W. and Rohde, A. (2007) A molecular timetable for apical bud formation and dormancy induction in poplar. The Plant Cell 19, 2370–2390. Sakurai, J., Ahamed, A., Murai, M., Maeshima, M. and Uemura, M. (2008) Tissue and cell-specific localization of rice aquaporins and their water transport activities. Plant & Cell Physiology 49, 30–39. Salzman, R.A., Bressan, R.A., Hasegawa, P.M., Ashworth, E.N. and Bordelon, B.P. (1996) Programmed accumulation of LEA-like proteins during desiccation and cold acclimation of overwintering grape buds. Plant, Cell & Environment 19, 713–720. Savolainen, O., Bokma, F., García-Gil, R., Komulainen, P. and Repo, T. (2004) Genetic variation in cessation of growth and frost hardiness and consequences for adaptation of Pinus sylvestris to climatic changes. Forest Ecology and Management 197, 79–89. Schäfer, E. and Schmidt, W. (1974) Temperature dependence of phytochrome dark reversions. Planta 116, 257–266. Short, T.W. (1999) Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose. Plant Physiology 119, 1497–1506. Smithberg, M.H. and Weiser, C.J. (1968) Patterns of variation among climatic races of red-osier dogwood. Ecology 49, 495–505. Stevenson, R.K. (1994) Dormancy and acclimation in dogwood clonal ecotypes. MSc. dissertation, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Svendsen, E., Wilen, R., Stevenson, R., Liu, R. and Tanino, K. (2007) A molecular marker associated with lowtemperature induction of dormancy in red osier dogwood (Cornus sericea). Tree Physiology 27, 385–397. Tanino, K. (2004) The role of hormones in endodormancy induction. Journal of Crop Improvement 10, 157–199.

118

L. Kalcsits et al.

Tanino, K.K., Fuchigami, L.H., Chen, T.H.H., Gusta, L.V. and Weiser, C.J. (1989) Dormancy-breaking agents on acclimation and deacclimation of dogwood. HortScience 24, 353–354. Tanino, K.K., Chen, T.H.H., Fuchigami, L.H. and Weiser, C.J. (1991) ABA-induced frost hardiness and cellular alterations. Journal of Plant Physiology 137, 619–624. Valverde, F., Mouradov, A., Soppe, W., Ravenscroft, D., Samach, A. and Coupland, G. (2004) Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303, 1003–1006. Van der Schoot, C.E. (1996) Dormancy and symplastic networking at the shoot apical meristem. In: Lang, G.A. (ed.) Plant Dormancy: Physiology, Biochemistry and Molecular Biology. CAB International, Wallingford, UK, pp. 59–81. Van der Veen, R. (1951) Influence of daylength on the dormancy of some species of the genus Populus. Physiologica Plantarum 4, 35–40. Wake, M.F. and Fennell, A. (2000) Morphological, physiological and dormancy responses of three Vitis genotypes to short photoperiod. Physiologica Plantarum 109, 203–210. Wareing, P.F. (1956) Photoperiodism in woody plants. Annual Review of Plant Physiology 7, 191–214. Weatherwax, S.C., Williams, S.A., Tingay, S. and Tobin, E.M. (1998) The phytochrome response of the Lemna gibba NPR1 gene is mediated primarily through changes in abscisic acid levels. Plant Physiology 116, 1299–1305. Weiser, C.J. (1970) Cold resistance and injury in woody plants. Science 169, 1269-1278. Welling, A., Kaikuranta, P. and Rinne, P. (1997) Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens: involvement of ABA and dehydrins. Physiologica Plantarum 100, 119–125. Welling, A., Moritz, T., Palva, E.T. and Junttila, O. (2002) Independent activation of cold acclimation by low temperature and short photoperiod in hybrid aspen. Plant Physiology 129, 1633–1641. Went, F.W. (1944) Plant growth under controlled conditions. II. Thermoperiodicity in growth and fruiting of tomato. American Journal of Botany 31, 135–150. Wheaton, E. (2001) Changing Climates: Exploring Possible Future Climates of the Canadian Prairie Provinces. Saskatchewan Research Council, Saskatoon, Saskatchewan, Canada. Williams, B.J., Pellett, N.E. and Klein, R.M. (1972) Phytochrome control of growth cessation and initiation of cold acclimation in selected woody plants. Plant Physiology 50, 262–265. Wurzburger, J. and Farkash, B. (1976) Endogenous gibberelin level during dormancy break of Diospyros virginiana and its possible effect on water content. Plant Science Letters 6, 1–4. Yooyongwech, S., Horigane, A.K., Yoshida, M., Yamaguchi, M., Sekozawa, Y., Sugaya, S. and Gemma, H. (2008) Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy. Physiologica Plantarum 124, 522–533.

13

Winter Hardiness and the CBF Genes in the Triticeae E.J. Stockinger

Introduction The temperate-climate cereal crops of the Triticeae – wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereal) – are cool-season annual plants generally classified as being of either a winter or a spring growth habit. Winter growth habit types are sown in the autumn, grow vegetatively for a short period, overwinter and then develop reproductively the following spring. If springsown they fail to properly develop reproductively. Their committed transition from a vegetative to a reproductive growth phase is gated by a vernalization requirement, an extended period at low temperatures (Takahashi and Yasuda, 1971). Yet vernalization is not an absolute because winter genotypes eventually flower, and certain genotypes can even bypass the vernalization requirement if grown under short day (SD) photoperiod (Dubcovsky et al., 2006). In contrast to winter types, spring growth habit types are inherently reproductively competent, do not have a vernalization requirement and can be sown in the spring (Takahashi and Yasuda, 1971). The choice of which growth habit form to grow is usually dictated by seasonal temperatures and water availability during critical growth phases. In certain temperate regions, such as the Great Plains of North America, winter wheats are grown because they are

much more efficient at using available water during the prolonged vegetative growth phase and as a result can yield 40% more than spring genotypes. In other cultivation systems, such as the US Upper Midwest, winter wheat is an essential component in the crop rotation cycle of maize–soyabean–wheat. Successful cultivation in these systems is critically dependent on the winter hardiness of the plant. Winter hardiness is a broad term used to describe the capacity of Triticeae cereal plants, and many other plants, to successfully overwinter the temperate-climate winter. It is a complex trait influenced by many environmental and genetic factors. Diverse environmental variables such as cultural practices, snow cover, growth conditions at and following planting, and the timing and duration of extremes in low temperatures all influence winter survival (Gusta et al., 1997). At the genetic level winter hardiness is primarily associated with vernalization requirements and photoperiod responsiveness (Hayes et al., 1997). An assumption also made is that there are genotypic differences in lowtemperature tolerance limits that can be separated from these other components. Winter hardiness is particularly interconnected with the growth habit form. Spring genotypes essentially lack winter hardiness. Comparisons between winter and spring genotypes show that the freezing tolerance levels attainable by spring genotypes of barley, wheat

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

119

120

E.J. Stockinger

and rye are −7, −9 and −12°C, respectively, whereas their winter genotype counterparts are able to attain temperatures of −17, −22 and −33°C (Wilen et al., 1996). Freezing tolerance levels are also tightly interconnected with the reproductive stage of the plant. Vernalized seedlings of these same winter genotypes are only able to survive freezing temperatures equivalent to that of their spring counterparts (Wilen et al., 1996). Moreover, as winter genotypes become increasingly vernalized they show a concomitant decrease in the levels of freezing tolerance (Fowler et al., 1996, 1999). Thus, a shift in the developmental program is accompanied by a change in the capacity of these plants to survive freezing temperatures. However, the vernalization requirement is not an absolute for winter hardiness. Nor is the length of the vernalization requirement directly correlated with the level of freezing tolerance. In barley, the ‘facultative’ agronomic class defines a group of genotypes capable of surviving the temperate-climate winter, but which also do not have an obligate vernalization requirement (von Zitzewitz et al., 2005). The prototypical winter-hardy North American barley is the facultative cultivar ‘Dicktoo’, which has no vernalization requirement (Hayes et al., 1997). In wheat, long vernalization requirements are frequently associated with cultivars grown in more maritime climates but as a group the maritime-climate cultivars tend to be about 6°C less freezing-tolerant than cultivars grown in the northern North American Great Plains (Gusta et al., 1997, 2001). What separates these two groups is that the northern Great Plains cultivars have a greater endurance for prolonged exposure to freezing temperatures and desiccating conditions (Gusta et al., 1997). Recent genetic analyses in wheat and barley indicate that two major loci in each of these two plants affect winter hardiness: Frost Resistance-1 (FR-1) and FR-2. FR-2 consists of a cluster of at least 12 genes encoding C-repeat binding factor (CBF) transcriptional factor proteins (Miller et al., 2006; Skinner et al., 2006; Stockinger et al., 2006; Francia et al., 2007; Knox et al., 2008). Homologues of the CBFs in Arabidopsis thaliana play a key role in effecting acquisition of freezing tolerance. In

Arabidopsis, increasing CBF levels through overexpression in transgenic plants increases freezing tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Jaglo et al., 2001) and decreasing CBF expression results in decreased freezing tolerance (Chinnusamy et al., 2003; Alonso-Blanco et al., 2005). The second locus, FR-1, maps coincident with VRN-1, one of two key loci effecting the vernalization requirement. One commonality that unites the winter-hardy Triticeae cereals is the presence of the winter vrn-1 allele at VRN-1. In these genotypes VRN-1 is not expressed unless vernalization or day length requirements have been met, or until an endogenous developmental program takes over (Danyluk et al., 2003; Trevaskis et al., 2003, 2006; Yan et al., 2003). Expression of VRN-1 then results in repression of the CBF genes at FR-2 (Stockinger et al., 2007). As winter hardiness is intimately interconnected with the vernalization requirement and FR-1 may in fact be the pleiotropic effect of VRN-1 (Stockinger et al., 2007), a discussion of the molecular genetics underlying the vernalization requirement is warranted.

Molecular Genetics of the Vernalization Requirement and the Barley Facultative Genotype The vernalization requirement in cereals is controlled primarily by two key loci, VRN-1 and VRN-2 (Takahashi and Yasuda, 1971; Dubcovsky et al., 1998). Nomenclature may also include notation to identify the specific Triticeae genome because chromosomes form a homoeologous series in which there is genetic colinearity, or synteny, between chromosomal regions of the different genomes. Thus when referring specifically to barley, VRN-H1 and VRN-H2 are used in which H identifies Hordeum, or when referring to the A genome of hexaploid wheat VRN-A1 and VRN-A2 are used. These Triticeae VRN genes are also completely dissimilar to Arabidopsis loci with the same name. To be consistent throughout the current chapter, we adopt an all upper-case italic form to identify the gene locus (e.g. VRN-1), upper-case first letter only and italics when referring to the dominant

Winter Hardiness and CBF Genes

allele (Vrn-1) and all lower case and italics for the recessive allele (e.g. vrn-1). An all uppercase italic form is also used to identify the transcript and an all upper-case non-italic form is used to identify the protein when describing these forms in context. VRN-2 is epistatic to VRN-1 (Takahashi and Yasuda, 1971). At the molecular level, VRN-2 effects repression of VRN-1 (Yan et al., 2004; Loukoianov et al., 2005). During vernalization expression of VRN-2 is down-regulated while that of VRN-1 is upregulated. Additional data suggest that VRN-1 might also act to repress VRN-2 (Loukoianov et al., 2005). Thus VRN-1 and VRN-2 appear to have an antagonistic relationship. The current model posits that at some point during vernalization VRN-1 levels reach a critical threshold required to effect the reproductive transition (Loukoianov et al., 2005). In barley, the recessive spring vrn-H2 allele is manifest as a complete deletion of the VRN-H2 gene (Yan et al., 2004). In einkorn wheat, Triticum monococcum ssp. monococcum, a point mutation occurs in the VRN-A2 coding sequence (Yan et al., 2004). The spring Vrn-1 allele differs from the winter vrn-1 allele such that it is non-responsive to the repressive effects of VRN-2. These differences occur in the form of deletions in either the VRN-1 promoter or the VRN-1 first intron (Yan et al., 2003; Fu et al., 2005). The coding regions of the winter vrn-1 and spring Vrn-1 alleles do not differ (Yan et al., 2003). In Vrn-1 spring genotypes, VRN-1 is expressed to increasingly higher levels during growth and development (Loukoianov et al., 2005; Trevaskis et al., 2006). Certain barley genotypes that lack VRN-H2 and possess the winter vrn-H1 allele are, by classical definition, spring genotypes because they do not have a vernalization requirement (Takahashi and Yasuda, 1971), but they are referred to as facultative because they are capable of surviving the temperate-climate winter (von Zitzewitz et al., 2005). ‘Dicktoo’ is one such cultivar and expression analyses in ‘Dicktoo’ indicate that there is at least a 10-week delay between detectable VRN-1 transcripts in plants grown under SD in comparison to plants grown under long day (LD) photoperiod (Danyluk et al., 2003).

121

Exactly what determines the length of vernalization requirement is not clear. Alleles at both VRN-1 and VRN-2 appear to be critical (Fu et al., 2005; Loukoianov et al., 2005; Szücs et al., 2007). But the time to flowering is determined in large part by the alleles at VRN-1. Hayes and colleagues found a significant correlation between the time to flowering and the size of the deletion in the VRN-1 intron; the larger the deletion, the shorter the time to flowering (Szücs et al., 2007). In bread wheat the situation is more complex because wheat is an allohexaploid comprised of three different diploid genomes: the A, B and D genomes. A dominant spring homoeoallele at any one of the three VRN-1 loci, VRN-A1, VRN-B1 or VRN-D1, will result in spring growth habit. Each homoeoallele also has a different ‘potency’ in effecting the length of time required to flower, with Vrn-A1 being the most potent and the Vrn-D allele being the least potent (Koemel et al., 2004). Notably, this potency also inversely correlates with freezing tolerance levels (Koemel et al., 2004). At some point transcripts are eventually produced from the winter vrn-1 allele. In the facultative barley cultivar ‘Dicktoo’ that lacks the VRN-H2 repressor, LD photoperiod is a key factor promoting induction of VRN-H1 transcription (Danyluk et al., 2003; Stockinger et al., 2007). In einkorn wheat, the winter vrn-Am1 allele is transcribed earlier in heterozygous Vrn-Am1/vrn-Am1 plants than in homozygous vrn-Am1/vrn-Am1 plants, indicating that expression of the Vrn-Am1 allele somehow promotes transcription of the recessive allele (Loukoianov et al., 2005). In hexaploid wheat the recessive vrn-B1 and vrn-D1 alleles are also transcribed earlier in Vrn-A1 plants than in vrn-A1 plants. The Vrn-B1 and Vrn-D1 spring alleles have a similar effect upon the other recessive homoeoalleles (Loukoianov et al., 2005). These data suggest that expression of Vrn-A1 initiates and establishes a positive feedback loop upon its own expression. Although plants that have the recessive vrn-1 allele will eventually flower in the absence of vernalization, the VRN-1 gene is absolutely essential for the reproductive transition as deletion of VRN-1 results in plants that remain in the vegetative stage indefinitely (Shitsukawa et al., 2007).

122

E.J. Stockinger

Genetics of Winter Hardiness Although winter hardiness and lowtemperature tolerance in the Triticeae cereals is complex and each of the seven chromosomes is implicated, homoeologous group 5 chromosomes are consistently identified as having the greatest effect (Thomashow, 1990). Early genetic studies indicated that there were two major chromosome 5 loci: one that directly controlled the capacity to cold harden and a second locus that sensed temperature, triggered vernalization and induced cold hardening (Roberts, 1990). The first mapped locus was named Frost Resistance-1 (FR-1) (Sutka and Snape, 1989). FR-1 plays an important role in affecting the freezing tolerance of vegetative tissues. FR-1 has been identified on the A, B and D chromosomes of hexaploid wheat (Sutka and Snape, 1989; Galiba et al., 1995; Snape et al., 1997; Tóth et al., 2003) and in barley (Hayes et al., 1993; Francia et al., 2004). In all these instances the FR-1 allele conferring greater freezing tolerance co-segregates with the recessive vrn-1 allele at VRN-1; genotypes possessing the spring Vrn-1 allele are less freezing-tolerant and have no vernalization requirement (Stockinger et al., 2006). The second major locus affecting winter hardiness is FR-2, and it maps 20–50 cM proximal of FR-2. FR-2 has been identified in multiple mapping populations including the barley ‘Nure’בTremois’ (NT) (Francia et al., 2004, 2007), the einkorn wheat DV92×G3116 (Vágújfalvi et al., 2003) and the hexaploid wheat ‘Norstar’× winter ‘Manitou’ (Båga et al., 2007). Nearly 45% of the phenotypic variance for freezing tolerance levels in these populations is explained by FR-2. The identification of FR-2 also utilized expression quantitative trait locus (e-QTL) analyses. Essentially, COR14b at either the RNA level (Vágújfalvi et al., 2000, 2003) or the protein level (Francia et al., 2004) is significantly higher in recombinants possessing the winter FR-2 allele. COR14b and many other highly expressed cold-regulated genes (e.g. DEHYDRINS, DHN; WHEAT COLD SPECIFIC, WCS) show a coordinated upregulation in response to low temperatures and the gene product abundances exhibit a strong

positive correlation with the individual genotype’s capacity to cold-acclimate and develop freezing tolerance (Houde et al., 1992a,b; Danyluk et al., 1994, 1998; Crosatti et al., 1996; Fowler et al., 1996; Limin et al., 1997; Sarhan et al., 1997; Grossi et al., 1998; NDong et al., 2002). As COR14b and many other Triticeae cereal genes whose expression are robustly induced by low temperature harbour the CRT/DRE (C-repeat/dehydrationresponsive element) motif in their upstream regions (Stockinger et al., 2007), the question was raised whether genes encoding CBF transcription factors might be the underlying molecular basis of COR14b expression level differences mapping to FR-2. Expressed sequence tag (EST) sequences encoding CBFs do indeed map to FR-2 (Vágújfalvi et al., 2003; Båga et al., 2007; Francia et al., 2007), and mapping of single EST sequences to FR-2 has been followed by discoveries that a very large CBF gene family resides at FR-2 (Loukoianov et al., 2005; Miller et al., 2006; Skinner et al., 2006; Stockinger et al., 2006).

The Arabidopsis CBFs In A. thaliana, the CBF/DREB1 (C-repeat binding factor/dehydration-responsive element binding 1) proteins play key roles in the pathway leading to cold acclimation and increased freezing tolerance. They are AP2/ERF domain containing, DNA-binding, transcriptional activators that bind to the CRT/DRE (Stockinger et al., 1997). The CRT/DRE, which has at its core the 5-bp motif CCGAC, was originally identified as the DNA regulatory element imparting abscisic acid-independent, low-temperature induction to the Arabidopsis COR genes (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994). The CRT/DRE is now known to effect cis-acting regulatory control over a much larger network of regulatory and structural genes referred to as the CBF regulon (Fowler and Thomashow, 2002; Vogel et al., 2005). The CBF regulon in turn functions to increase freezing tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Jaglo et al., 2001). The CBFs themselves are induced in response to low temperatures, initiating the regulatory cascade (Gilmour et al., 1998).

Winter Hardiness and CBF Genes

The CBFs are distinguished from the other 150 or so Arabidopsis AP2 domaincontaining proteins by signature sequences that flank the AP2 domain (Jaglo et al., 2001). In Arabidopsis, six protein-encoding genes harbour the CBF signature sequence, three of which form a cluster of head-to-tail, tandemly linked genes on chromosome 4 (Gilmour et al., 1998). Transcripts encoding these three CBFs accumulate within 15 min after plants are exposed to low temperatures (Gilmour et al., 1998). More recent studies indicate that expression is not the result of a cold shock; rather absolute temperature is monitored by an internal cold-sensing mechanism that yields greater CBF transcript output in a temperaturedependent manner in which lower temperatures effect greater CBF transcript output (Zarka et al., 2003). In contrast, the other CBF/DREB1 genes are dispersed across the genome and appear not be low-temperatureresponsive (Haake et al., 2002; Sakuma et al., 2002). The functional loss of just one of the three low-temperature-responsive CBFs significantly reduces the capacity of Arabidopsis to cold-acclimate and decreases the freezing tolerance of the plant (Chinnusamy et al., 2003; Alonso-Blanco et al., 2005).

Monocot CBF Discovery, Nomenclature and Phylogeny In the monocots, the CBF gene family is much larger than that of Arabidopsis. Rice harbours ten open-reading frames encoding CBFs (Skinner et al., 2005). Phylogenetic divisions of the monocot CBFs using barley nomenclature initially divided the genes into three clades, the HvCBF1, HvCBF3 and HvCBF4 subgroups (Skinner et al., 2005). The identification of each CBF gene is based on the chronological order in which it was isolated. Some were first identified as cDNA clones, while others were coding sequences (CDSs) residing on genomic clones. Nineteen different genes have been recovered from a single barley genotype (Skinner et al., 2005). Genes of the HvCBF1 clade are scattered across the barley genome, while the HvCBF3 and HvCBF4 clade genes, with the exception of the CBF8A, CBF8B and CBF8C pseudo-

123

genes, all co-localize to the Fr-H2 interval (Skinner et al., 2006). At about the same time, investigations in einkorn wheat carried out by Dubcovsky and colleagues revealed 11 CBF genes at the homoeologous FR-Am2 locus (Miller et al., 2006). Eight of the T. monococcum CBF genes residing at FR-Am2 had clearly identifiable barley orthologues, while three were new. Most recently, the groups of Sarhan and Danyluk at the University of Quebec, Montreal, Canada have added numerous CBF genes from hexaploid wheat to the picture (Badawi et al., 2007). Badawi and colleagues (2007) also carried out an extensive phylogenetic analysis showing that there are ten clearly discernable monophyletic CBF clades in the Poaceae (Badawi et al., 2007). To facilitate comparisons across the monocots, Badawi proposed inserting a roman numeral identifier corresponding to the original HvCBF subgroup, and a lower-case letter to designate the monophyletic clades to which the CBF belonged (Badawi et al., 2007). Using this nomenclature, barley CBF2A and CBF3 are thus identified as Hv-CBFIVa-2A and Hv-CBFIIIc-3, respectively. But since nomenclature of the CBF orthologues is consistent across the different Triticeae cereals – i.e. Hv-CBF2, Tm-CBF2 and Ta-CBF2 are orthologues, and Hv-CBF14, Tm-CBF14 and Ta-CBF14 are orthologues, etc. – the nomenclature proposed by Badawi and colleagues is used below only when collectively referring to the monophyletic clade; while HvCBF subgroup is used when making generalizations about HvCBF subgroups. As the HvCBF3 and HvCBF4 subgroup genes comprise FR-2, greater discussion is also placed upon these genes. The HvCBF3 and HvCBF4 subgroup CBFs at FR-2 are highly conserved in their NH3-terminal CBF signature sequences, and in the NH3-terminal half of the AP2 DNAbinding domain, which encompasses the three b-sheets (Skinner et al., 2005). Subgroupspecific differences arise in the region comprising the α-helix at the very COOH terminal portion of the AP2 domain, and in the COOHterminal CBF signature sequence (Skinner et al., 2005). For the most part, individual orthologues differ primarily in both the NH3terminal leader region of the protein, which

124

E.J. Stockinger

varies in length from approximately 20 to 70 residues, and in the acidic COOH terminal region, which also varies in length, from about 100 to 200 residues (Skinner et al., 2005). Orthologues from the different Triticeae cereals are easily identified by shared conservation in these flanking regions (Skinner et al., 2005). An additional feature of barley is that there are numerous highly identical duplicated genes, or paralogues. A single barley genotype harbours both CBF2A and CBF2B, and CBF10A and CBF10B (Skinner et al., 2005; Stockinger et al., 2006).

Genetic Analyses and Physical Relationships of the CBF Genes Encompassing FR-2 Both the barley and T. monococcum CBF intervals encompass approximately 0.8 cM and are about 30 cM proximal to VRN-1 on the long arm of homoeologous group 5 chromosomes (Vágújfalvi et al., 2003; Francia et al., 2004, 2007; Miller et al., 2006; Stockinger et al., 2007; Knox et al., 2008). In the ‘Dicktoo’בMorex’ barley mapping population Hv-CBF2A, Hv-CBF4B and Hv-CBF9 cosegregate as a single block, and Hv-CBF3, HvCBF6, Hv-CBF10A, Hv-CBF12, Hv-CBF13 and Hv-CBF14 co-segregate as a second block (Skinner et al., 2006). The larger NT barley F2 population resolves seven CBF genes, with Hv-CBF2B being the most proximal and HvCBF6 the most distal (Francia et al., 2007). Additional structural information and physical relationships are discerned from the genomic clones. Hv-CBF2A and Hv-CBF4B are present on single bacteriophage λ genomic clones from the cultivar ‘Dicktoo’, as are Hv-CBF3 and Hv-CBF13, and Hv-CBF10B

CBF17 BAC 60J11 103kb 37kb

CBF9 CBF4

CBF14 BAC 119P22

CBF2

17kb 12kb Fully sequenced BAC clones

To centromere

and Hv-CBF10A (Stockinger et al., 2006). Hv-CBF3, Hv-CBF10A and Hv-CBF6, in that arrangement, are also physically linked on the ‘Morex’ BAC clone 804E19 (Skinner et al., 2006). A similar structural picture of the locus comes from sequencing from T. monococcum BAC clones (Fig. 13.1). Several trends are apparent from these analyses. Genes of the HvCBF4 subgroup appear to be more proximal (to the centromere), genes of the HvCBF3 subgroup are more distal and CBF14 appears to be where the transition occurs. Genes within each monophyletic clade also tend to co-cluster. TmCBF17 is an oddity in that it is in the CBFIIId clade and is separated from the other CBFIIId clade genes by Tm-CBF14, and possibly also Tm-CBF2, Tm-CBF4 and Tm-CBF9. Each CBF is also separated from its neighbouring genes by large tracts of repetitive DNA.

Regulation of Cereal CBF Expression Expression analyses of the monocot CBFs indicate that many are expressed in response to cold, drought and salinity with similar kinetics as the Arabidopsis CBFs. Wheat and rye CBFs are induced by cold temperatures in nearly identical time frames as Arabidopsis CBF1 (Jaglo et al., 2001). In barley, it was noted that Hv-CBF2 is expressed to higher levels in the winter-hardy genotype ‘Dicktoo’ than in the non-winter-hardy spring genotype ‘Morex’, but that the opposite appears to be true for Hv-CBF4 (Skinner et al., 2005). Hv-CBF1 is induced by drought, while Hv-CBF7 is induced by high salinity (Skinner et al., 2005). In hexaploid wheat, Ta-CBF14, Ta-CBF15 and Ta-CBF16 are expressed at more than fourfold higher levels in the winterhardy genotypes than they are in the non-

CBF16

CBF15 BAC 21C6 48kb 190 kb 92kb

CBF13

BAC 284l15

CBF3 CBF10 BAC 511C10

CBF12 0.8 cM >650 kb

CBF gene and flanking sequence only

Fig. 13.1. Triticum monococcum FR-Am2 CBF gene cluster. (Knox et al., 2008 with kind permission of Springer Science and Business Media.)

Winter Hardiness and CBF Genes

winter-hardy genotypes (Vagujfalvi, 2005). In rice (Oryza sativa), Os-DREB1A is responsive to both cold and salinity (Dubouzet et al., 2003), and in maize (Zea mays), Zm-DREB1A is rapidly induced by drought (Qin et al., 2004). Thus the monocot CBF genes show similar differential induction by environmental conditions that impose a cellular dehydration stress upon the plant as the Arabidopsis CBFs. Of the 13 barley CBF genes at FR-H2, nine show induction in response to low temperatures (Stockinger et al., 2007). The four low-temperature non-responsive genes, Hv-CBF3, Hv-CBF10A, Hv-CBF10B and Hv-CBF13, are all CBFIIIc clade members. Expression analyses also indicate that the hexaploid wheat orthologues of these genes are also not expressed (Badawi et al., 2007). Exactly what developmental or environmental cues induce expression of the CBFIIIc clade genes is an open question. Across the different Triticeae their coding regions remain intact, and functional analyses indicate that they bind to CRT/DRE motifs and are capable of activating COR genes when overexpressed in Arabidopsis (Skinner et al., 2005). Time course studies in both barley and wheat indicate that transcript levels of the lowtemperature-responsive CBFs peak 4 to 12 h after plants are subjected to a temperature decrease (Badawi et al., 2007; Stockinger et al., 2007). Peak expression levels occur in this window regardless of whether plants are subjected to large temperature decreases, such as a cold shock, or whether the temperature decreases are more moderate and gradual (Stockinger et al., 2007). Transcripts are also detectable earlier in vrn-1 genotypes (Badawi et al., 2007; Stockinger et al., 2007). Transcript levels are decreased in abundance at 24 h and later in these low-temperature time courses, but they still remain above the levels detected in non-cold-treated plants growing at 18°C prior to the temperature decrease (Stockinger et al., 2007). CBF expression has also been detected in non-cold-treated plants (Xue, 2003; Kobayashi et al., 2005; Stockinger et al., 2007), which may be due to some as yet unidentified environmental factor. Recently VRN-1/FR-1 was shown to be a negative regulator of the CBF genes at FR-2 (Stockinger et al., 2007). Transcript levels of

125

all low-temperature-responsive CBF genes at FR-H2 are significantly higher in recombinants harbouring the vrn-H1 winter allele than in recombinants harbouring the Vrn-H1 spring allele (Stockinger et al., 2007). In vrn-H1 genotypes that require vernalization, CBF transcript levels are decreased in abundance relative to transcript levels detected in nonvernalized plants of similar leaf number; and this dampening in CBF levels is accompanied by robust expression of VRN-1 (Stockinger et al., 2007). CBF expression levels are also affected by photoperiod. Significantly higher CBF transcript levels accumulate under SD growth conditions than under LD conditions (Stockinger et al., 2007). However, the effect of photoperiod on transcript levels may be further compounded by a circadian clock. Growth chamber experiments in which wheat plants are grown under a 16 h photoperiod show a peak response to a low-temperature input between 8 and 14 h into the light period (Badawi et al., 2007). Although expression levels of the individual CBF genes differ, for the most part the overall trends are similar. Most of the HvCBF4 subgroup genes are expressed to higher levels than the HvCBF3 subgroup CBFs. Nearly identical temporal expression patterns are detected, and all of the low-temperature-responsive CBF genes at FR-2 are expressed to lower levels in Vrn-1 genotypes. These data support a model in which CBF levels are under regulatory control by other factors until they are subject to the repressive effects of VRN-1.

Identification of Candidate CBF Genes at FR-2 One driving question has been which CBF gene or genes at FR-2 is the critical gene. This question is being addressed using a combination of genetics and comparative genomic sequencing strategies. In barley the freezing tolerance phenotype attributed to FR-H2 is currently resolved to a 4.6 cM interval that encompasses the CBF cluster (Francia et al., 2007). Sequencing CBF-harbouring bacteriophage λ genomic inserts of the parental genotypes used for this mapping population, alongside two additional parental genotypes

126

E.J. Stockinger

from a second mapping population also segregating for winter hardiness, suggests that multiple CBF genes rather than a single individual gene underlies FR-2. Both winter-hardy genotypes ‘Dicktoo’ and ‘Nure’ share a segmental chromosomal duplication that encompasses the Hv-CBF2A and Hv-CBF4B genomic regions (Knox et al., unpublished data). Neither ‘Morex’ nor ‘Tremois’ possesses the segmental duplications (Knox et al., unpublished data). In addition, ‘Dicktoo’ and ‘Nure’ harbour the Hv-CBF2B gene. ‘Morex’ has only single copies of Hv-CBF2 and Hv-CBF4. ‘Tremois’ also harbours only single copies of Hv-CBF4 and Hv-CBF2. Fine mapping of the T. monococcum Fr-Am2 locus indicates that recombinants possessing the G3116 central cluster allele are better able to recover after freezing to −11°C than recombinants possessing the DV92 allele (Knox et al., 2008). A number of minor polymorphisms in the Tm-CBF14 and Tm-CBF15 gene sequences distinguish G3116 and DV92 alleles from one another, but the most noteworthy difference occurs in Tm-CBF12. The DV92 allele (freezing-sensitive genotype) has a 15-nucleotide deletion that results in the loss of five amino acid residues between the β1- and β2-sheets of the AP2 DNA-binding domain. Recombinant DV92 CBF12 protein expressed in Escherichia coli does not bind to CRT/DRE sequences whereas rG3116 CBF12 does (Knox et al., 2008). These data suggest that the loss of a single CBF gene reduces freezing tolerance in the Triticeae cereals just as it does in Arabidopsis.

discriminates the tropical from temperateclimate monocots is the expansion of the HvCBF3 and HvCBF4 subgroup genes coclustering on the Triticeae chromosome 5 homoeologs. The Triticeae cereals have at least 15 different CBFs in the combined HvCBF3 and HvCBF4 subgroups, while the rice genome harbours only two HvCBF3 subgroup genes, Os-DREB1A and Os-DREB1H, and a single HvCBF4 subgroup gene, Os-DREB1H (Skinner et al., 2005). The structural organization and the physical distances separating the barley and einkorn wheat CBF genes differ tremendously from that of the dicots Arabidopsis and tomato, and also the monocot rice. Both dicots and rice harbour three CBF genes tandemly linked head to tail in a 10-kb region (Gilmour et al., 1998; Zhang et al., 2004; Ohyanagi et al., 2006). In contrast, the CBF genes in barley and einkorn wheat are bordered by large tracks of repetitive DNA. Some of these repetitive DNA tracks are only 500–1000 bp distal to the coding sequences. In addition, numerous pseudogenes are interspersed in the CBF genomic regions (Miller et al., 2006; Stockinger et al., 2006). In the Triticeae cereals, differences between more winter-hardy and less winter-hardy genotypes are attributable to differences in CBF gene copy numbers and their functionality (Stockinger et al., 2007; Knox et al., 2008). A fundamental question thus raised is why the considerable expansion in number of the CBF genes forming the chromosome 5 cluster; if more is better why not simply increase the expression levels of a single CBF?

Conclusion

Acknowledgements

In conclusion, the monocot CBF gene family is much larger than that of the dicots. It is phylogenetically divided into three clades, genes of which are present in the tropical and temperate-climate monocots. A key difference that

This research was supported in part by a grant from the National Science Foundation Plant Genome Program (DBI 0110124) and The Ohio State University/Ohio Agricultural and Research Development Center.

References Alonso-Blanco, C., Gomez-Mena, C., Llorente, F., Koornneef, M., Salinas, J. and Martinez-Zapater, J.M. (2005) Genetic and molecular analyses of natural variation indicate CBF2 as a candidate gene for underlying a freezing tolerance quantitative trait locus in Arabidopsis. Plant Physiology 139, 1304–1312.

Winter Hardiness and CBF Genes

127

Badawi, M., Danyluk, J., Boucho, B., Houde, M. and Sarhan, F. (2007) The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs. Molecular Genetics and Genomics 277, 533–554. Båga, M., Chodaparambil, S.V., Limin, A.E., Pecar, M., Fowler, D.B. and Chibbar, R.N. (2007) Identification of quantitative trait loci and associated candidate genes for low-temperature tolerance in cold-hardy winter wheat. Functional & Integrative Genomics 7, 53–68. Baker, S.S., Wilhelm, K.S. and Thomashow, M.F. (1994) The 5′-region of Arabidopsis thaliana cor15a has cisacting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Molecular Biology 24, 701–713. Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B.H., Hong, X., Agarwal, M. and Zhu, J.K. (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes & Development 17, 1043–1054. Crosatti, C., Nevo, E., Stanca, A.M. and Cattivelli, L. (1996) Genetic analysis of the accumulation of COR14 proteins in wild (Hordeum spontaneum) and cultivated (Hordeum vulgare) barley. Theoretical and Applied Genetics 93, 975–981. Danyluk, J., Houde, M., Rassart, E. and Sarhan, F. (1994) Differential expression of a gene encoding an acidic dehydrin in chilling sensitive and freezing tolerant gramineae species. FEBS Letters 344, 20–24. Danyluk, J., Perron, A., Houde, M., Limin, A., Fowler, B., Benhamou, N. and Sarhan, F. (1998) Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. The Plant Cell 10, 623–638. Danyluk, J., Kane, N.A., Breton, G., Limin, A.E., Fowler, D.B. and Sarhan, F. (2003) TaVRT-1, a putative transcription factor associated with vegetative to reproductive transition in cereals. Plant Physiology 132, 1849–1860. Dubcovsky, J., Lijavetzky, D., Appendino, L. and Tranquilli, G. (1998) Comparative RFLP mapping of Triticum monococcum genes controlling vernalization requirement. Theoretical and Applied Genetics 97, 968–975. Dubcovsky, J., Loukoianov, A., Fu, D., Valarik, M., Sanchez, A. and Yan, L. (2006) Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2. Plant Molecular Biology 60, 469–480. Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G., Miura, S., Seki, M., Shinozaki, K. and YamaguchiShinozaki, K. (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. The Plant Journal 33, 751–763. Fowler, D.B., Chauvin, L.P., Limin, A.E. and Sarhan, F. (1996) The regulatory role of vernalization in the expression of low-temperature-induced genes in wheat and rye. Theoretical and Applied Genetics 93, 554–559. Fowler, D.B., Limin, A.E. and Ritchie, J.T. (1999) Low-temperature tolerance in cereals: model and genetic interpretation. Crop Science 39, 626–633. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14, 1675–1690. Francia, E., Rizza, F., Cattivelli, L., Stanca, A.M., Galiba, G., Toth, B., Hayes, P.M., Skinner, J.S. and Pecchioni, N. (2004) Two loci on chromosome 5H determine low-temperature tolerance in a ‘Nure’ (winter)בTremois’ (spring) barley map. Theoretical and Applied Genetics 108, 670–680. Francia, E., Barabaschi, D., Tondelli, A., Laido, G., Rizza, F., Stanca, A.M., Busconi, M., Fogher, C., Stockinger, E.J. and Pecchioni, N. (2007) Fine mapping of a HvCBF gene cluster at the frost resistance locus Fr-H2 in barley. Theoretical and Applied Genetics 115, 1083–1091. Fu, D., Szucs, P., Yan, L., Helguera, M., Skinner, J.S., von Zitzewitz, J., Hayes, P.M. and Dubcovsky, J. (2005) Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat. Molecular Genetics and Genomics 273, 54–65. Galiba, G., Quarrie, S.A., Sutka, J., Morounov, A. and Snape, J.W. (1995) RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat. Theoretical and Applied Genetics 90, 1174–1179. Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M. and Thomashow, M.F. (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. The Plant Journal 16, 433–442. Grossi, M., Giorni, E., Rizza, F., Stanca, A.M. and Cattivelli, L. (1998) Wild and cultivated barleys show differences in the expression pattern of a cold-regulated gene family under different light and temperature conditions. Plant Molecular Biology 38, 1061–1069.

128

E.J. Stockinger

Gusta, L.V., O’Connor, B.J. and MacHutcheon, M.G. (1997) The selection of superior winter-hardy genotypes using a prolonged freeze test. Canadian Journal of Plant Science 77, 15–21. Gusta, L.V., O’Connor, B.J., Gao, Y.P. and Jana, S. (2001) A re-evaluation of controlled freeze-tests and controlled environment hardening conditions to estimate the winter survival potential of hardy winter wheats. Canadian Journal of Plant Science 81, 241–246. Haake, V., Cook, D., Riechmann, J.L., Pineda, O., Thomashow, M.F. and Zhang, J.Z. (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiology 130, 639–648. Hayes, P.M., Blake, T., Chen, T.H.H., Tragoonrung, S., Chen, F., Pan, A. and Liu, B. (1993) Quantitative trait loci on barley (Hordeum vulgare L.) chromosome 7 associated with components of winterhardiness. Genome 36, 66–71. Hayes, P.M., Chen, F.Q., Corey, A., Pan, A., Chen, T.H.H., Baird, E., Powell, W., Thomas, W., Waugh, R., Bedo, Z., Karsai, I., Blake, T. and Oberthur, L. (1997) In: Li, P.H. and Chen, T.H.H. (eds) Fifth International Plant Cold Hardiness Seminar. Plenum Press, New York, New York, pp. 77–87. Houde, M., Danyluk, J., Laliberte, J.F., Rassart, E., Dhindsa, R.S. and Sarhan, F. (1992a) Cloning, characterization, and expression of a cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat. Plant Physiology 99, 1381–1387. Houde, M., Dhindsa, R.S. and Sarhan, F. (1992b) A molecular marker to select for freezing tolerance in Gramineae. Molecular & General Genetics 234, 43–48. Jaglo, K.R., Kleff, S., Amundsen, K.L., Zhang, X., Haake, V., Zhang, J.Z., Deits, T. and Thomashow, M.F. (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor coldresponse pathway are conserved in Brassica napus and other plant species. Plant Physiology 127, 910–917. Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O. and Thomashow, M.F. (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, 104–106. Knox, A.K., Li, C., Vágújfalvi, A., Galiba, G., Stockinger, E.J. and Dubcovsky, J. (2008) Identification of candidate CBF genes for the frost tolerance locus Fr-Am2 in Triticum monococcum. Plant Molecular Biology 67, 257–270. Kobayashi, F., Takumi, S., Kume, S., Ishibashi, M., Ohno, R., Murai, K. and Nakamura, C. (2005) Regulation by Vrn-1/Fr-1 chromosomal intervals of CBF-mediated Cor/Lea gene expression and freezing tolerance in common wheat. Journal of Experimental Botany 56, 887–895. Koemel, J.E. Jr, Guenzi, A.C., Anderson, J.A. and Smith, E.L. (2004) Cold hardiness of wheat near-isogenic lines differing in vernalization alleles. Theoretical and Applied Genetics 109, 839–846. Limin, A.E., Danyluk, J., Chauvin, L.P., Fowler, D.B. and Sarhan, F. (1997) Chromosome mapping of lowtemperature induced Wcs120 family genes and regulation of cold-tolerance expression in wheat. Molecular & General Genetics 253, 720–727. Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. The Plant Cell 10, 1391–1406. Loukoianov, A., Yan, L., Blechl, A., Sanchez, A. and Dubcovsky, J. (2005) Regulation of VRN-1 vernalization genes in normal and transgenic polyploid wheat. Plant Physiology 138, 2364–2373. Miller, A.K., Galiba, G. and Dubcovsky, J. (2006) A cluster of 11 CBF transcription factors is located at the frost tolerance locus Fr-Am2 in Triticum monococcum. Molecular Genetics and Genomics 275, 193–203. NDong, C., Danyluk, J., Wilson, K.E., Pocock, T., Huner, N.P. and Sarhan, F. (2002) Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses. Plant Physiology 129, 1368–1381. Ohyanagi, H., Tanaka, T., Sakai, H., Shigemoto, Y., Yamaguchi, K., Habara, T., Fujii, Y., Antonio, B.A., Nagamura, Y., Imanishi, T., Ikeo, K., Itoh, T., Gojobori, T. and Sasaki, T. (2006) The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Nucleic Acids Research 34, D741–D744. Qin, F., Sakuma, Y., Li, J., Liu, Q., Li, Y.Q., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2004) Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant and Cellular Physiology 45, 1042–1052. Roberts, D.W.A. (1990) Identification of loci on chromosome 5A of wheat involved in control of cold hardiness, vernalization, leaf length, rosette growth habit, and height of hardened plants. Genome 33, 247–259.

Winter Hardiness and CBF Genes

129

Sakuma, Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2002) DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydrationand cold-inducible gene expression. Biochemical and Biophysical Research Communications 290, 998–1009. Sarhan, F., Ouellet, F. and VazquezTello, A. (1997) The wheat wcs120 gene family. A useful model to understand the molecular genetics of freezing tolerance in cereals. Physiologia Plantarum 101, 439–445. Shitsukawa, N., Ikari, C., Shimada, S., Kitagawa, S., Sakamoto, K., Saito, H., Ryuto, H., Fukunishi, N., Abe, T., Takumi, S., Nasuda, S. and Murai, K. (2007) The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene. Genes & Genetic Systems 82, 167–170. Skinner, J.S., von Zitzewitz, J., Szucs, P., Marquez-Cedillo, L., Filichkin, T., Amundsen, K., Stockinger, E.J., Thomashow, M.F., Chen, T.H. and Hayes, P.M. (2005) Structural, functional, and phylogenetic characterization of a large CBF gene family in barley. Plant Molecular Biology 59, 533–551. Skinner, J.S., Szucs, P., von Zitzewitz, J., Marquez-Cedillo, L., Filichkin, T., Stockinger, E. J., Thomashow, M.F., Chen, T.H. and Hayes, P.M. (2006) Mapping of barley homologs to genes that regulate low temperature tolerance in Arabidopsis. Theoretical and Applied Genetics 112, 832–842. Snape, J.W., Semikhodskii, A., Fish, L., Sarma, R.N., Quarrie, S.A., Galiba, G. and Sutka, J. (1997) Mapping frost tolerance loci in wheat and comparative mapping with other cereals. Acta Agronomica Hungarica 45, 265–270. Stockinger, E.J., Gilmour, S.J. and Thomashow, M.F. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proceedings of the National Academy of Sciences USA 94, 1035–1040. Stockinger, E.J., Cheng, H. and Skinner, J.S. (2006) In: Chen, T.H.H., Uemura, M. and Fujikawa, S. (eds) Cold Hardiness in Plants: Molecular Genetics, Cell Biology and Physiology. CAB International, Wallingford, UK, pp. 53–63. Stockinger, E.J., Skinner, J.S., Gardner, K.G., Francia, E. and Pecchioni, N. (2007) Expression levels of barley Cbf genes at the Frost resistance-H2 locus are dependent upon alleles at Fr-H1 and Fr-H2. The Plant Journal 51, 308–321. Sutka, J. and Snape, J.W. (1989) Location of a gene for frost resistance on chromosome 5A of wheat. Euphytica 42, 41–44. Szücs, P., Skinner, J. S., Karsai, I., Cuesta-Marcos, A., Haggard, K.G., Corey, A.E., Chen, T.H. and Hayes, P.M. (2007) Validation of the VRN-H2/VRN-H1 epistatic model in barley reveals that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity. Molecular Genetics and Genomics 277, 249–261. Takahashi, R. and Yasuda, S. (1971) In: Nilan, R.A. (ed.) Barley Genetics II; Proceedings of the Second International Barley Genetics Symposium. Washington State University Press, Pullman, Washington, pp. 388–408. Thomashow, M.F. (1990) Molecular genetics of cold acclimation in higher plants. Advances in Genetics 28, 99–131. Tóth, B., Galiba, G., Feher, E., Sutka, J. and Snape, J.W. (2003) Mapping genes affecting flowering time and frost resistance on chromosome 5B of wheat. Theoretical and Applied Genetics 107, 509–514. Trevaskis, B., Bagnall, D.J., Ellis, M.H., Peacock, W.J. and Dennis, E.S. (2003) MADS box genes control vernalization-induced flowering in cereals. Proceedings of the National Academy of Sciences USA 100, 13099–13104. Trevaskis, B., Hemming, M.N., Peacock, W.J. and Dennis, E.S. (2006) HvVRN2 responds to daylength, whereas HvVRN1 is regulated by vernalization and developmental status. Plant Physiology 140, 1397–1405. Vágújfalvi, A., Crosatti, C., Galiba, G., Dubcovsky, J. and Cattivelli, L. (2000) Two loci on wheat chromosome 5A regulate the differential cold-dependent expression of the cor14b gene in frost-tolerant and frostsensitive genotypes. Molecular & General Genetics 263, 194–200. Vágújfalvi, A., Galiba, G., Cattivelli, L. and Dubcovsky, J. (2003) The cold-regulated transcriptional activator Cbf3 is linked to the frost-tolerance locus Fr-A2 on wheat chromosome 5A. Molecular Genetics and Genomics 269, 60–67. Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G. and Thomashow, M.F. (2005) Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. The Plant Journal 41, 195–211.

130

E.J. Stockinger

von Zitzewitz, J., Szücs, P., Dubcovsky, J., Yan, L., Francia, E., Pecchioni, N., Casas, A., Chen, T.H.H., Hayes, P.M. and Skinner, J.S. (2005) Molecular and structural characterization of barley vernalization genes. Plant Molecular Biology 59, 449–467. Wilen, R.W., Fu, P., Robertson, A.J. and Gusta, L.V. (1996) In: Li, P.H. and Chen, T.H.H. (eds) Fifth International Plant Cold Hardiness Seminar. Plenum Press, Corvallis, Oregon, pp. 191–201. Xue, G.P. (2003) The DNA-binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low-temperature responsive genes in barley is modulated by temperature. The Plant Journal 33, 373–83. Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. The Plant Cell 6, 251–264. Yan, L., Loukoianov, A., Tranquilli, G., Helguera, M., Fahima, T. and Dubcovsky, J. (2003) Positional cloning of the wheat vernalization gene VRN1. Proceedings of the National Academy of Sciences USA 100, 6263–6268. Yan, L., Loukoianov, A., Blechl, A., Tranquilli, G., Ramakrishna, W., SanMiguel, P., Bennetzen, J.L., Echenique, V. and Dubcovsky, J. (2004) The wheat VRN2 gene is a flowering repressor down-regulated by vernalization. Science 303, 1640–1644. Zarka, D.G., Vogel, J.T., Cook, D. and Thomashow, M.F. (2003) Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiology 133, 910–918. Zhang, X., Fowler, S.G., Cheng, H., Lou, Y., Rhee, S.Y., Stockinger, E.J. and Thomashow, M.F. (2004) Freezingsensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. The Plant Journal 39, 905–919.

14

Regulation of Stress-responsive Signalling Pathways by Eudicot CBF/DREB1 Genes A. Nassuth and M. Siddiqua

Introduction Many temperate plants acquire tolerance to freezing temperatures after exposure to low, non-freezing temperatures in a process called cold acclimation. During the cold acclimation period, several transcription factors called C-repeat binding factors or dehydrationresponsive element binding factors (CBF or DREB), depending on the research group, are synthesized that initiate downstream coldregulated (COR) gene expression and subsequent accumulation of cryoprotectants which results in the acquisition of freezing tolerance. The importance of this CBF signalling pathway for low-temperature tolerance in eudicots is supported by a number of observations. First, CBF genes have been found in all higher plants studied to date, including cold-tolerant (Arabidopsis, Brassica napus, poplar, Vitis riparia) and cold-sensitive (tomato, cherry) eudicot species. Second, overexpression of a CBF gene alone (e.g. an endogenous gene (Jaglo-Ottosen et al., 1998; Haake et al., 2002; Savitch et al., 2005), an Arabidopsis thaliana CBF (AtCBF ) gene expressed in other plant species (Jaglo et al., 2001; Kasuga et al., 2004; Benedict et al., 2006a) or the expression of CBFs from other species in Arabidopsis (Kitashiba et al., 2004) ) conferred increased freezing tolerance. Indeed, AtCBF transgenic Arabidopsis at 20–22°C display at least 12% of the cold-

induced transcriptional changes (Fowler and Thomashow, 2002; Hannah et al., 2005; Vogel et al., 2005) and a large portion of the biochemical changes associated with cold acclimation (Gilmour et al., 2000; Cook et al., 2004). Third, freezing tolerance in natural accessions appears, at least in part, to be mediated through the CBF pathway. A major Arabidopsis quantitative trait locus, explaining approximately 20% of the variation in acclimated freezing tolerance between Cape Verde Islands (Cvi) and Landsberg erecta (Ler) accessions, was associated with AtCBF2 (AlonsoBlanco et al., 2005), and the accumulation of AtCBF1 and AtCBF2 transcripts had significant positive correlations with cold-acclimated freezing tolerance in nine geographically diverse accessions (Hannah et al., 2006). Consistent with the important role for the CBF pathway in cold acclimation is the observation that impairment of the pathway correlates with a decrease in freezing tolerance. For example, CBF transcripts are induced later and are less abundant in the freeze-sensitive Citrus species Citrus paradisi compared with the freeze-tolerant species Poncirus trifoliata (Champ et al., 2007), or are maintained for a shorter period of time in freezing-sensitive varieties of B. napus (Gao et al., 2002) and Arabidopsis (Cook et al., 2004) or in a freezing-sensitive plant such as Lycopersicon esculentum (Jaglo et al., 2001; Zhang et al., 2004). In addition,

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

131

132

A. Nassuth and M. Siddiqua

the CBF regulon size is smaller (fewer COR genes are induced) in L. esculentum compared with A. thaliana (3 versus 30 of approximately 8000 genes examined; Zhang et al., 2004). Taken together, there is strong evidence that the CBF pathway plays a prominent role in acquired freezing tolerance. The present chapter focuses on a discussion of CBF genes and their encoded proteins in eudicots. The reader is referred to the contribution of Stockinger (this volume) for a discussion of CBF genes in monocots, and to several recent reviews for an extended discussion of cold-induced pathways and their crosstalk with drought and high salinity response pathways (Van Buskirk and Thomashow, 2006; Yamaguchi-Shinozaki and Shinozaki, 2006; Chinnusamy et al., 2007).

Eudicots Contain Several CBF Genes Eudicots contain a family of CBF genes (paralogues). At least six paralogues can be identified for Arabidopsis (Haake et al., 2002; Sakuma et al., 2002), Brassica (Zhao et al., 2006), Populus (Benedict et al., 2006a) and Vitis (see http://www.genoscope.cns.fr/externe/Gen omeBrowser/Vitis/ ), although Thlaspi arvense has replaced AtCBF1, AtCBF2 and AtCBF3 with a single homologous CBF (Zhou et al., 2007). It was suggested that the CBF genes might have redundant functional activities because their overexpression appeared to have similar effects on growth, development, proline and sugar composition, freezing tolerance and gene expression (Gilmour et al., 2004). However, recent data discussed in the following sections support the notion that many of the CBF paralogues likely have unique functions.

Eudicot CBF Paralogues Can Have Unique Induction Patterns Induction of CBF paralogues can be differently affected by the genotype and physiological state of the plant (Knight et al., 2004; Van Buskirk and Thomashow, 2006). At ambient temperatures, low levels of AtCBF1, AtCBF2 and AtCBF3 transcripts accumulate according to a circadian rhythm (Fowler et al., 2005).

Cold stress amplifies this rhythmic accumulation. Some CBF genes are also induced by other stresses such as drought and/or salt (e.g. CaDREBLP1: Hong and Kim, 2005; Vitis CBF1, CBF2 and CBF3: Xiao et al., 2006; GhDREB1L: Huang et al., 2007; BcCBF1 and BcCBF2: Zhang et al., 2006). AtCBF4 (DREB1D) is induced by drought but not by cold (Haake et al., 2002). The presence and quality of light also modulate the accumulation of CBF transcripts (AtCBF1, AtCBF2 and AtCBF3: Kim et al., 2002; Fowler et al., 2005; Eucalyptus Egu CBF1a and EguCBF1b: El Kayal et al., 2006; Brassica pekinensis: Zhang et al., 2006). Experiments with different light qualities and Arabidopsis mutants suggest that phytochromes B and D repress AtCBF1, AtCBF2 and AtCBF3 transcript induction and therefore a low ratio of red light to far red light (low R/ FR), which inactivates the phytochromes, induces CBF genes (Franklin and Whitelam, 2007). Induction by low R/FR light also amplifies the accumulation of CBF transcripts according to the circadian rhythm. Low R/FR light exists during autumn and induction of the CBF pathway at this time may prepare the plant for freezing temperatures during the following winter (Franklin and Whitelam, 2007). CBF gene induction was initially thought to occur rapidly (within 15 min) after the application of the inductive cue. Closer examination has revealed that transcript accumulation of different paralogues occurs at different times and is transient, likely as a result of a combination of sequential induction, a short half-life of the transcripts (Zarka et al., 2003) and negative regulation among CBFs. For example, it appears that cold first induces higher expression of the AtCBF1 and AtCBF3 genes, which temporarily escape the negative control by AtCBF2 until AtCBF2 gene expression is also induced (Novillo et al., 2004). The poplar PtCBF3 and PtCBF4 transcripts in leaves reach maximum levels before PtCBF1 and PtCBF2 (Benedict et al., 2006a). The up-regulation of PtCBF3 in AtCBF1 transgenic poplar stem tissues shows that regulation by CBF paralogues might also occur in poplar. In B. napus, BnDREBI-5 transcripts reached a maximum 1.5 h after the start of a cold stress, whereas BnDREBII-1 expression peaked only after 4.5 h (Zhao et al., 2006). A longer period of time was required for Vitis

Stress-responsive Signalling Pathways

Time (h)

0

0.5

1

2

4

8

12

133

1d

2d

5d

VrCBF1 VrCBF2 VrCBF3 VrCBF4 MDH Fig. 14.1. Northern blot showing the expression of Vitis riparia CBF1, CBF2, CBF3 and CBF4 and of MDH (endogenous control) at the indicated times after the start of cold treatment. (Based on Figures 6 and 6 in Xiao et al., 2006, 2008, with permission from Blackwell Publishing.)

CBF transcripts (Xiao et al., 2006). While Vitis CBF1 shows maximum accumulation in about 30 min after transfer to low temperatures, Vitis CBF2 transcripts required about 8 h and Vitis CBF3 transcripts levels increased from about 2 days until at least 5 days after transfer (Fig. 14.1). Cold enhancement of Vitis CBF4 transcripts started after about 1 h and continued for at least 2 days. Putative cold-inducible elements (named ICEr1 and ICEr2), light-inducible elements and rapid stress response elements are found in the upstream flanking regions of CBF genes in Arabidopsis (Zarka et al., 2003; Walley et al., 2007), Vitis (Siddiqua et al., 2005) and T. arvense (Zhou et al., 2007). So far, only a few transcription factors involved in the cold regulation of AtCBF genes have been identified. AtICE1 is a MYC-type bHLH transcription factor that can bind to MYC elements in the AtCBF3 promoter (Chinnusamy et al., 2003). AtICE1 is constitutively expressed but induces AtCBF3 expression only after cold stress, which stabilizes and activates AtICE1 by SUMO E3 ligase SIZ1-mediated sumoylation (Miura et al., 2007) and possibly by cold-activated MAP kinase-mediated phosphorylation (Chinnusamy et al., 2007). Sumoylation apparently reduces access for the ubiquination complex, which may include the ubiquitin E3 ligase HOS1 (Dong et al., 2006), and thereby reduces proteasome degradation of AtICE1. Sumolylation of AtICE1 also negatively affects AtMYB15 transcript abundance. AtMYB15, an R2R3-MYB family protein, is expressed at low levels in the absence of cold stress but upon cold treatment is upregulated and then represses gene expression of AtCBF1, AtCBF2 and AtCBF3, apparently

after binding to MYB elements in their promoters (Agarwal et al., 2006). Another negative regulator of AtCBF1, AtCBF2 and AtCBF3 expression, possibly indirectly, might be a coldinduced C2H2 zinc finger transcription factor called ZAT12 (Vogel et al., 2005; Benedict et al., 2006b). Chinnusamy et al. (2007), Franklin and Whitelam (2007) and Zhu et al. (2007) present further details of the proposed regulation of CBF expression.

Domains in Different CBF Proteins Can Regulate Activation of COR Gene Transcription Differently The CBF/DREB1 proteins are distinguished from other types of AP2/ERF family transcription factors by an AP2 DNA-binding domain that is flanked at the N-terminus side by a basic amino acid domain (PKKPAGRxKFxETRHP), which is a putative nuclear localization sequence (NLS), and at the C-terminus side by a DSAWRL sequence (Jaglo et al., 2001; Owens et al., 2002). Many CBF proteins also contain a LWSY-type motif at their ultimate C-terminal end (Fig. 14.2). The AP2 domain is responsible for binding to promoter elements. CBF/DREB1 proteins bind in vitro better to the C-repeat (CRT; TGGCCGACNT) or dehydration-responsive element (DRE; TACCGACNT) than to the similar GCC-box (AGCCGCC) promoter element. Changing Ala37 or Val14 and Glu19 of the AP2 domain to another amino acid eliminates binding to both DRE and GCC-box; but changing just Val14 eliminates binding to CRT/DRE

134

A. Nassuth and M. Siddiqua

VrCBF1 VrCBF4

1* ------NLS------- ---MDSDHEEFSASSSSSSSRTNSNPSDS-----LLPLQCIGHKRKAGRKKFRETRHPIYRGV MNTTSPPYSDPHPLVCNWDSLNLPDSDGGSEELMLASTHPKKRAGRKKFRETRHPVYRGV

VrCBF1 VrCBF4

----AP2----------4*----------------------------------DSAWRL RQRNGNKWVCEVREPLKKSRIWLGTFPTPEMAARAHDVAALALRGRFASLNFPDSAWRLP RRRNSGKWVCEVREPNKTSRIWLGTFPTAEMAARAHDVAALALRGRGACLNFADSAWRLH

VrCBF1 VrCBF4

4,1* 1* 1* RPKSSSAEDIQVAALEATKAFNPTAPSSSSLASALDNMSGVADSKKVLETSPNVESPKLK VPSSRDAKDIQKAAAEAAEAFRPMEND------------GVMQDERREES--EVRTP---

VrCBF1 VrCBF4

1* 1* 1* 1* HC2 HC3 1*/HC4 HC5/4* SQRMVLEVSPVDTKRSEKVGDGSTPVFMDEEAMFNMQGLINSMAEGLLLTPP-AMCKGFS ----------------------ENVFVMDEEDVFGMPGLLVNMAEGLLMPPPHSVADGYG

VrCBF1 VrCBF4

1* 1* LWSY WDDATDSHIDLSLWNDDGDD-MAADADMSLWSYSI

251aa 218aa

Fig. 14.2. Alignment of CBF1 and CBF4 from Vitis riparia (AY390370 and AY706986). Indicated are the domains common to all CBF proteins (top line), plus possible ubiquination (‘PEST’ domain, underlined) and phosphorylation sites (S/TXXE/D or S/TXK/R, in italics and identified in top line by 1* or 4* for respectively VrCBF1 or VrCBF4). See text for further details.

only (Sakuma et al., 2002; Liu et al., 2006). The binding preference of CBF proteins is also affected by the sequence context for a CRT/ DRE. For example, AtCBF3 (DREB1A) protein bound to A/GCCGACNT more efficiently than to A/GCCGACNA/G/C (Sakuma et al., 2002), and the B. napus BNCBF5 has a more stringent requirement for a particular CRT sequence than BNCBF17 (Gao et al., 2002). These binding preferences can have implications for regulon composition since different COR genes have slightly different CRT sequences (Suzuki et al., 2005). Indeed, the AtCBF1, AtCBF2 and AtCBF3 genes likely each have their own preferred set of downstream target genes. Downregulation of AtCBF3 but not AtCBF1 and AtCBF2 expression in the ice1 mutant substantially decreased the expression of the COR genes RD29A, COR15A and COR47 (Chinnusamy et al., 2003). Also, fewer COR gene transcripts accumulated in transgenic plants expressing BNCBF5 instead of BNCBF17 (Savitch et al., 2005). Little is known about the function of other domains, apart from the NLS. Recent experiments in our laboratory showed that nuclear localization is abolished upon deletion of the putative bipartite NLS from VrCBF1 or VrCBF4 (M. Siddiqua and A. Nassuth, unpublished results). No function has yet been

assigned to the other domains characteristic for CBF proteins, DSAWRL and LWSY, although transactivation experiments with AtCBF1-derived protein constructs showed that replacement of LWSY with LAAA increased the activation (Wang et al., 2005). It is thought the C-terminus is responsible for the transactivation of downstream gene expression, as was demonstrated for AtCBF1, CaDREBLP1 from Capsicum annuum and BnDREBI-5 from B. napus (Hong and Kim, 2005; Wang et al., 2005; Zhao et al., 2007). The C-terminus contains hydrophobic clusters (HCs) and flanking residues that favour the formation of loops, a structural pattern that is conserved in CBFs across plant species (Wang et al., 2005). HC2, HC3 and HC4 contribute 93% of AtCBF1 transactivation activity but likely require specific, mostly polar flanking amino acids to do so (Wang et al., 2005; Zhao et al., 2007). Amino acids in other CBF regions also contribute to the transactivation ability of B. napus CBFs (Zhao et al., 2007), possibly because they affect the efficiency of nuclear localization, DNA binding and/or interaction with the polymerase II complex, or protein stability or activity through the presence of post-translational modification sites. Putative ubiquination (PEST) domains, which could direct the protein for degradation by protea-

Stress-responsive Signalling Pathways

somes, and putative phosphorylation sites are present in some CBFs (A. Nassuth, unpublished results; see also Fig. 14.2). The presence of a CBF protein does not automatically mean that a downstream regulon is expressed. The protein might be inactive or less active than another CBF in its ability to induce COR expression, as is the case for Brassica CBFs (Savitch et al., 2005; Zhao et al., 2006). We observed higher levels of GUSPlus ( b-glucuronidase) reporter gene transactivation by VrCBF4 compared with VrCBF1 (Fig. 14.3) which might be due to differences in binding affinity to the TACCGACAT promoter elements.

Effect of CBF Gene Expression The CBF pathway differs between different tissues, and this might be attributed to tissuespecific expression of different CBF paralogues or to the activation of different regulons in the different tissues by the same CBF. Even though AtCBF1, AtCBF2 and AtCBF3 are coldinduced in both leaves and roots of Arabidopsis (Sakuma et al., 2002), 86% of the coldinduced genes are not shared between these tissues (Kreps et al., 2002). In the same vein,

(a)

overexpressed AtCBF1 activated different regulons in leaves and stems of poplar (Benedict et al., 2006a). Tissue-specific expression of CBF genes has been demonstrated in poplar where PtCBF1 and PtCBF3 are inducible in leaves, stems and dormant buds whereas PtCBF2 and PtCBF4 are inducible in leaves only (Sterky et al., 2004; Benedict et al., 2006a). Similarly, Vitis CBF4 was induced in young and mature leaves and buds, but Vitis CBF1, CBF2 and CBF3 only in young leaves and buds (Xiao et al., 2006, 2008). Expression of CBFs can increase not only tolerance to freezing but also to other osmotic stresses, such as drought and high salt (Kasuga et al., 1999; Haake et al., 2002). Some drought-tolerant Arabidopsis overexpressing VrCBF1 have a dwarf phenotype (M. Siddiqua and A. Nassuth, unpublished results; Fig. 14.4). Severe growth retardation has also been observed for Arabidopsis overexpressing At CBF3 or sweet cherry CIG-B and for tomato overexpressing AtCBF1, AtCBF3 or LeCBF1 (Kasuga et al., 1999; Hsieh et al., 2002; Kitashiba et al., 2004; Zhang et al., 2004). This might be related to a function for CBFs during development. Indeed, GUS reporter expression was observed in young CBF::GUS transgenic Arabidopsis seedlings, although it

(b)

(c) Gus activity (nm/mg/min) 14000

12000

10000

8000

6000

4000

2000

0

Reporter

Gus plus AtCBF1 VrCBF1 VrCBF4 MDH

Effector

135

(1) Non agroinfiltrated plants (2) min 35S::gusplus (3) 4CRTmin 35S::gusplus (4) 35S::AtCBF1

+

(5) 35S::AtCBF1

+4CRTmin 35S::gusplus

min 35S::gusplus

(6) 35S::VrCBF1

+

(7) 35S::VrCBF1

+4CRTmin 35S::gusplus

min 35S::gusplus

(8) 35S::VrCBF4+min 35S::gusplus (9) 35S::VrCBF4+4CRTmin 35S::gusplus

Fig. 14.3. Transactivation of CRT-containing promoter by AtCBF1, VrCBF1 or VrCBF4. (a) Combinations of reporter and effector plasmids used to co-infiltrate tobacco leaves. The GUSPlus reporter gene (CAMBIA) is driven by a minimal (min) 35S promoter with or without four CRT elements (TACCGACAT); the effector plasmid is driven by the full 35S promoter. (b) Representative semi-quantitative RT-PCR detection of GUSPlus, CBF and MDH (control) expression. (c) GUS activity. (Adapted from Xiao et al., 2008, with permission from Blackwell Publishing.)

136

A. Nassuth and M. Siddiqua

Co1-0

AtCBF1

VrCBF1

VrCBF4

Fig. 14.4. Comparison of VrCBF1 and VrCBF4 transgenic Arabidopsis lines with wild-type (Col-0) and AtCBF1 transgenic Arabidopsis. Shown are growth retardation in some 5-week-old plants (top), enhanced tolerance of seedlings to a 20 h treatment at −7 to −10°C (second row) and enhanced survival of 5-week-old plants after water deprivation (fourth row; watered controls in third row). Note correlation between the dwarf phenotype and enhanced drought tolerance. (For details see Siddiqua, 2007.)

has not been excluded that this is due to the dehydration stress caused by taking the seedlings out of their humid culture dish (Novillo et al., 2007; M. Siddiqua and A. Nassuth, unpublished results).

Conclusions The data presented in the current chapter show that CBF genes can be expressed to different levels at different times and tissues, and that the encoded CBF proteins can vary in their ability to bind to certain CRT or DRE sequences and activate downstream genes. This suggests that CBFs have unique regulons which result in different responses, such as increased tolerance to a particular stress or an effect on growth development. Use of constitutive promoters in experiments to study CBFs may not reveal unique functions as CBF expression is up-regulated in all tissues and at all times. In addition, transgenic CBF protein production is so high that subtle differences in

binding affinities to CRT and DRE sequences may be obscured. Experiments with (silencing) mutants might provide more information on CBF function (Novillo et al., 2007). The idea that CBF paralogues have unique functions has an extra dimension with woody perennial plants since they develop dormancy in response to shortened day length and subsequently deep winter hardiness in response to low temperatures (Welling and Palva, 2006). This, and the fact that many perennial woody plants are economically important, has driven a flurry of research activity into the CBF genes of woody plants, even though these plants are more difficult to work with. Information on CBF genes is now available for Prunus cerasus (Owens et al., 2002), Prunus avium (Kitashiba et al., 2004), Populus balsamifera (Benedict et al., 2006a), V. riparia and Vitis vinifera (Xiao et al., 2006, 2008), Eucalyptus gunni (El Kayal et al., 2006) and Eucalyptus globulus (Gamboa et al., 2007). Much of what has been learned from the herbaceous plants regarding the signalling and COR gene expression related to cold acclima-

Stress-responsive Signalling Pathways

tion appears to be similar and interchangeable in woody plants. Indeed, overexpression of birch CBFs (Welling and Palva, 2006) or grape CBFs (M. Siddiqua and A. Nassuth, unpublished data; see Fig. 14.4) can enhance the freezing tolerance of Arabidopsis and overexpression of AtCBF1 can enhance the freezing tolerance of birch (Welling and Palva, 2006).

137

Acknowledgements Work on stress tolerance in the Nassuth laboratory is supported by grants from the National Science and Research Council and from the Ontario Ministry of Agriculture and Food. We thank Raymond Lee for constructive comments on this manuscript.

References Agarwal, M., Hao, Y., Kapoor, A., Dong, C.-H., Fujii, H., Zheng, X. and Zhu, J.-K. (2006) A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. Journal of Biological Chemistry 281, 37636–37645. Alonso-Blanco, C., Gomez-Mena, C., Llorente, F., Koornneef, M., Salinas, J. and Martinez-Zapater, J.M. (2005) Genetic and molecular analyses of natural variation indicate CBF2 as a candidate gene for underlying a freezing tolerance quantitative trait locus in Arabidopsis. Plant Physiology 139, 1304–1312. Benedict, C., Skinner, J.S., Meng, R., Chang, Y., Bhalerao, R., Huner, N.P.A., Finn, C.E., Chen, T.H.H. and Hurry, V. (2006a) The CBF1-dependent low temperature signaling pathway, regulon and increase in freeze tolerance are conserved in Populus spp. Plant, Cell & Environment 29, 1259–1272. Benedict, C., Geisler, M., Trygg, J., Huner, N. and Hurry, V. (2006b) Consensus by democracy. Using metaanalyses of microarray and genomic data to model the cold acclimation signaling pathway in Arabidopsis. Plant Physiology 141, 1219–1232. Champ, K.I., Febres, V.J. and Moore, G.A. (2007) The role of CBF transcriptional activators in two Citrus species (Poncirus and Citrus) with contrasting levels of freezing tolerance. Physiologia Plantarum 129, 529–541. Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B., Hong, X., Agarwal, M. and Zhu, J.-K. (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes & Development 17, 1043–1054. Chinnusamy, V., Zhu, J. and Zhu, J.K. (2007) Cold stress regulation of gene expression in plants. Trends in Plant Science 12, 444–451. Cook, D., Fowler, S., Fiehn, O. and Thomashow, M.F. (2004) A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis. Proceedings of the National Academy of Sciences USA 101, 15243–15248. Dong, C.-H., Agarwal, M., Zhang, Y., Xie, Q. and Zhu, J.-K. (2006) The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proceedings of the National Academy of Sciences USA 103, 8281–8286. El Kayal, W., Navarro, M., Marque, G., Keller, G., Marque, C. and Teulieres, C. (2006) Expression profile of CBF-like transcriptional factor genes from Eucalyptus in response to cold. Journal of Experimental Botany 57, 2455–2469. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14, 1675–1690. Fowler, S.G., Cook, D. and Thomashow, M.F. (2005) Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiology 137, 961–968. Franklin, K.A. and Whitelam, G.C. (2007) Light-quality regulation of freezing tolerance in Arabidopsis thaliana. Nature Genetics 39, 1410–1413. Gamboa, M.C., Rasmussen-Poblete, S., Valenzuela, P.D.T. and Krauskopf, E. (2007) Isolation and characterization of a cDNA encoding a CBF transcription factor from E. globulus. Plant Physiology and Biochemistry 45, 1–5. Gao, M.-J., Allard, G., Byass, L., Flanagan, A.M. and Singh, J. (2002) Regulation and characterization of four CBF transcription factors from Brassica napus. Plant Molecular Biology 49, 459–471. Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D. and Thomashow, M.F. (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology 124, 1854–1865.

138

A. Nassuth and M. Siddiqua

Gilmour, S.J., Fowler, S.G. and Thomashow, M.F. (2004) Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Molecular Biology 54, 767–781. Haake V., Cook D., Riechmann, J.L., Pineda O., Thomashow, M.F. and Zhang J.Z. (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiology 130, 639–648. Hannah, M.A., Heyer, A.G. and Hincha, D.K. (2005) A global survey of gene regulation during cold acclimation in Arabidopsis thaliana. PLoS Genetics 1, 179–196. Hannah, M.A., Wiese, D., Freund, S., Fiehn, S., Heyer, A.G. and Hincha, D.K. (2006) Natural genetic variation of freezing tolerance in Arabidopsis. Plant Physiology 142, 98–112. Hong, J-P. and Kim, W.T. (2005) Isolation and functional characterization of the Ca-DREBLP1 gene encoding a dehydration-responsive element binding-factor-like protein 1 in hot pepper (Capsicum annuum L. cv. Pukang). Planta 220, 875–888. Hsieh, T., Lee, J., Yang, P., Chiu, L., Charng, Y., Wang, Y. and Chan, M. (2002) Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiology 129, 1086–1094. Huang, B., Jin, L. and Liu J. (2007) Molecular cloning and functional characterization of a DREB1/CBF-like gene (GhDREB1L) from cotton. Science in China, Series C: Life Sciences 50, 7–14. Jaglo, K.R., Kleff, S., Amundsen, K.L., Zhang, X., Haake, V., Zhang, J.Z., Deits, T. and Thomashow, M.F. (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiology 127, 910–917. Jaglo-Ottosen K.R., Gilmour S.J., Zarka D.G., Schabenberger O. & Thomashow M.F. (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, 104–106. Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnology 17, 287–291. Kasuga, M., Setsuko, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiology 45, 346–350. Kim, H.-J., Kim, Y.-K., Park, J.-Y. and Kim J. (2002) Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. The Plant Journal 29, 693–704. Kitashiba, H., Ishizaka, T., Isuzugawa, K., Nishimura, K. and Suzuki, T. (2004) Expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance. Journal of Plant Physiology 161, 1171–1176. Knight, H., Zarka, D.G., Okamoto, H., Thomashow, M.F. and Knight, M.R. (2004) Abscisic acid induces CBF genes transcription and subsequent induction of cold-regulated genes via the CRT promoter element. Plant Physiology 135, 1710–1717. Kreps, J.A., Wu, Y., Chang, H.S., Zhu, T., Wang, X. and Harpe, J.F. (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiology 130, 2129–2141. Liu, Y., Zhao, T.-J., Liu, J.-M., Liu, W.-Q., Liu, Q., Yan, Y.-B. and Zhou, H.-M. (2006) The conserved Ala37 in the ERF/AP2 domain is essential for binding with the DRE element and the GCC box. FEBS Letters 580, 1303–1308. Miura, K., Jin, J.B., Lee, J., Yoo, C.Y., Stirm, V., Miura, T., Ashworth, E.N., Bressan, R.A., Yun, D.-J. and Hasegawa, P.M. (2007) SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. The Plant Cell 19, 1403–1414. Novillo, F., Alonso, J.M., Ecker, J.R. and Salinas, J. (2004) CBF2/DREB1C is a negative regulator of CBF1/ DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proceedings of the National Academy of Sciences USA 101, 3985–3990. Novillo, F., Medina, J. and Salinas, J. (2007) Arabidopsis CBF1 and CBF3 have a different function than CBF2 in cold acclimation and define different gene classes in the CBF regulon. Proceedings of the National Academy of Sciences USA 104, 21002–21007. Owens, C.L., Thomashow, M.F., Hancock, J.F. and Iezzoni, A.F. (2002) CBF1 orthologs in sour cherry and strawberry and heterologous expression of CBF1 in strawberry. Journal of the American Society for Horticultural Science 127, 489–494. Sakuma, Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2002) DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communications 290, 998–1009.

Stress-responsive Signalling Pathways

139

Savitch, L.V., Allard, G., Seki, M., Robert, L.S., Tinker, N.A., Huner, N.P.A., Shinozaki, K. and Singh, J. (2005) The effect of overexpression of two Brassica CBF/DREB1-like transcription factors on photosynthetic capacity and freezing tolerance in Brassica napus. Plant Cell Physiology 46, 1525–1539. Siddiqua, M. (2007) Functional analysis of grape CBF genes and their promoters. PhD thesis, University of Guelph, Guelph, Ontario, Canada. Siddiqua, M., Xiao, H. and Nassuth A. (2005) In silico analysis of three CBF genes from both V. riparia and V. vinifera. In: Qiu, W. and Kovacs, L.G. (eds) Proceedings of the International Grape Genomics Symposium, St Louis, MO, USA. Missouri State University, Department of Fruit Science, Mountain Grove, Missouri, pp. 105–112. Sterky, F., Bhalerao, R.R., Unneberg, P., Segerman, B., Nilsson, P., Brunner, A.M., Charbonnel-Campaa, L., Lindvall, J.J., Tandre, K., Strauss, S.H., Sundberg, B., Gustafsson, P., Uhlén, M., Bhalerao, R.P., Nilsson, O., Sandberg, G., Karlsson, J., Lundeberg, J. and Jansson, S. (2004) A Populus EST resource for plant functional genomics. Proceedings of the National Academy of Sciences USA 101, 13951–13956. Suzuki, M., Ketterling, M.G. and McCarty, D.R. (2005) Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis. Plant Physiology 139, 437–447. Van Buskirk, H.A. and Thomashow, M.F. (2006) Arabidopsis transcription factors regulating cold acclimation. Physiologia Plantarum 126, 72–80. Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G. & Thomasow, M.F. (2005) Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. The Plant Journal 41, 195–211. Walley, J.W., Coughlan, S., Hudson, M.E., Covington, M.F., Kaspi, R., Banu, G., Harmer, S.L. and Dehesh, K. (2007) Mechanical stress induces biotic and abiotic stress responses via a novel cis-element. PLoS Genetics 3, e172. Wang, Z., Triezenberg, S.J., Thomashow, M.F. and Stockinger, E.J. (2005) Multiple hydrophobic motifs in Arabidopsis CBF1 COOH-terminus provide functional redundancy in trans-activation. Plant Molecular Biology 58, 543–559. Welling, A. and Palva, A.T. (2006) Molecular control of cold acclimation in trees. Physiologia Plantarum 127, 167–181. Xiao, H., Siddiqua, M., Braybrook, S. and Nassuth, A. (2006) Three grape CBF/DREB1 genes are regulated by low temperature, drought and abscisic acid. Plant, Cell & Environment 29, 1410–1421. Xiao, H., Tattersall, E., Siddiqua, M., Cramer, G.R. and Nassuth, A. (2008) CBF4 is a unique member of the CBF transcription factor family of Vitis vinifera and Vitis riparia. Plant, Cell & Environment 31, 1–10. Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology 57, 781–803. Zarka, D.G., Vogel, J.T., Cook, D. and Thomashow, M.F. (2003) Cold induction of Arabisopsis CBF gene involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiology 133, 910–918. Zhang, X., Fowler, S.G., Cheng, H., Lou, Y., Rhee, S.Y., Stockinger, E.J., and Thomashow, M.F. (2004) Freezingsensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. The Plant Journal 39, 905–919. Zhang, Y., Yang, T.-W., Zhang, L-J., Zhang, T.-G., Di, C.-X., Xu, S.-J. and An L.-Z. (2006) Isolation and expression analysis of two cold-inducible genes encoding putative CBF transcription factors from Chinese cabbage (Brassica pekinensis Rupr.). Journal of Integrative Plant Biology 48, 848–856. Zhao, T.J., Sun, S., Liu, Y., Liu, J.M., Liu, Q., Yan, Y.B. and Zhou, H.M. (2006) Regulating the droughtresponsive element (DRE)-mediated signaling pathway by synergistic functions of trans-active and transinactive DRE binding factors in Brassica napus. Journal Biological Chemistry 281, 10752–10759. Zhao, T.J., Liu, Y., Yan, Y.B., Feng, F., Liu, W.Q. and Zhou, H.M. (2007) Identification of the amino acids crucial for the activities of drought responsive element binding factors (DREBs) of Brassica napus. FEBS Letters 581, 3044–3050. Zhou, N., Robinson, S.J., Huebert, T., Bate, N.J. and Parkin, I.A.P. (2007) Comparative genome organization reveals a single copy of CBF in the freezing tolerant crucifer Thlaspi arvense. Plant Molecular Biology 65, 693–705. Zhu, J.H., Dong, C.H. and Zhu, J.-K. (2007) Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation. Current Opinion in Plant Biology 10, 290–295.

15

Evolution of Plant Cold Hardiness and its Manifestation along the Latitudinal Gradient in the Canadian Arctic J. Svoboda

Introduction The plant kingdom can be traced to the emergence of green algae some 800 million years (My) ago, which invaded the land about 450 My ago. They spread around the world and adapted to most diverse environments. From the autotrophic mega-eukaryotes, only the plants (Plantae) invaded the land. The transition from the aquatic to terrestrial environments was gradual and punctuated by the appearance of transient forms (e.g. rhyniophytes and giant lycophytes), long extinct. The terrestrification was never complete since even the most resilient xerophytes require water saturation at a certain season or stage of their lifespan. This fundamental land-based transition would not be possible without the development of supporting structures, a cuticle layer and division of labour. ‘Ecomorphic’ modifications included multi-cellularity and development of vascular and skeleton structures. Division of labour embodied the development of root (anchored in the moist soil) and shoot (stem, leaf and flower). It also involved alternation of generations of haploid gametophytes, still prevalent in bryophytes, and diploid sporophytes, predominant in vascular plants. Plants vary in size (from miniature micro-algae to towering redwoods) and in lifespan (from minutes to millennia). In terms of global biomass, they exceed a 100:1 ratio in compari140

son with animals, so that the visible biosphere is in essence a phytosphere. Plants occupy all available niches on the globe and are the working horses of every photo-autotrophic selfsustainable ecosystem. On the other hand, in the theatre of the biosphere, their role is that of supporting actors. This is, if we consider animals, with their mobility, neuro-sensuality and eventually intelligence, as superior life forms and thus the main actors. The plant special kingdom is ‘wedged between the rocks upon which it thrives and the animals which it supports’ (Svoboda, 1989).

Evolution of Plants Like the various phyla and subgroups of the animal kingdom, plants have evolved from their ‘lower’ forms. Disregarding the freshwater algae, the multicellular plants are summarily called embryophytes. As far as timing of the land invasion is concerned, plants ascended the land in tandem with, or ahead of the land animals they have sustained. In the late Ordovician (~450 My ago), the precursors of bryophytes and lycophytes became established on the land. Their simplest and oldest forms lived symbiotically with fungi, producing primitive proto-lichens (Tudge, 2000). Interestingly, these organisms may represent the earliest adaptation to cold hardiness

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Plant Cold Hardiness in the Canadian Arctic

and can still be found in the coldest Arctic and Antarctic soils. With no higher plants present due to overly harsh conditions, the ground is still beaming with life in a form of a dark, layered organic crust loaded with algae at the surface and fungi and bacteria beneath (personal observation; Elster et al., 1999). Clubmosses (lycophytes) and their tree-size forms thrived from the mid-Devonian (380 My ago), through the Carboniferous (oldest coal deposits!), to the end of the Permian (230 My ago). Ferns (pteridophytes) and their giant ancestors, with well-developed water- and sap-conductive tracheids forming a vascular system, also date back to the Carboniferous period. Seed plants, the latest established group, first appeared in the fossil record as ‘progymnosperms’ during the Devonian period. At present, the true ‘gymnosperms’ include the cycads, ginkgos and conifers which prevailed from 320– 280 My ago. Flowering plants (angiosperms) of the Cretaceous origin (130 My ago) are the youngest of the seed plant group. These evolutionarily most advanced forms dominate the tropical rainforest, the temperate deciduous forests and vast grasslands (Tudge, 2000). It should to be stressed that, over the millennia, the primeval evolutionary landscape had not been a smooth ‘tabula rasa’ open to life expansion and fostering diversification. Often to the contrary! During the last 500 My, six major and numerous minor spasms devastated the life on Earth. Plants, as their animal counterparts, had to endure great cataclysms due to asteroid, volcanic and other impacts, yet a few groups had always recovered from the repeated mass devastation (Wilson, 1992). However, the radical ‘shock and awe’ experiences – in which a large percentage of species, even of higher classes of organisms perished – opened the emptied ecological niches to the surviving groups. In reality, ‘the engine of (macro)evolution has been fuelled more by external environmental change than by internal competition’ (Leakey and Lewin, 1995), and could not successfully advance without a periodic mass devastation of the extant biota (Wilson, 1992; Svoboda, 2006). While evolution of animal species has become a favoured focus of phylogenetic studies, it was the plant world which first vigorously responded to the new opportunities of altered landscape

141

conditions and re-colonized the devastated earth. In fact, the plants’ post-cataclysmic recovery enabled and strongly guided the repopulation of the newly re-vegetated landscapes by the most befitting fauna. Evolution runs along two modes, which Eldredge and Gould (1972) and Gould (2002) described as ‘punctuated equilibria’. During our planetary history, the long periods of evolutionary inactivity, called ‘stasis’, were followed by relatively short periods of accelerated emergence of new species, often triggered by a cataclysmic or other detrimental impacts. This ‘STOP and GO’ pattern, or alternation of a micro- and macroevolution, shows the dynamism of evolution akin to a time-lapse film. The successional ‘GO’ phase, with the radically new, highly competitive species emergence, can be described as ‘climbing’ – to higher floors of complexity. The ‘stasis’ or ‘STOP’ phase, typified by micro-adjustment to newly established conditions, represents the species ‘spreading’. Consequently, after the initial burst of competitive colonization, the physical landscape becomes crowded with large populations of fewer winning species, and the intraspecific competition becomes more prevalent. The ‘STOP’ phase landscape forces the established occupiers to a more narrow specialization, mutualism, even symbiosis (Svoboda, 2006). As we explain below, the evolution of coldhardy plants had been occurring during the ‘STOP’ or ‘stasis’ phase.

Succession The ecological term ‘succession’ was primarily understood as the progressive landscape colonization by invading plant species, their gradual replacement in time, increase in the ground cover and build-up of the phytomass; in short, restoring newly functional ecosystems optimally to their desired climax conditions. The process also has broad application on a large timescale and a geographical scale and is fundamental in understanding the causes and mechanism of evolution. The primary succession starts in a landscape without any higher plants (e.g. on lava fields or colonization of a freshly deglaciated terrain); the secondary succession takes place in a terrain stripped of

142

J. Svoboda

vegetation to a greater or lesser degree, but with surviving individuals of various species and their seeds (e.g. regeneration of a forest after a fire or clear-cutting).

Evolution of Cold-hardened Plants, the Cryophytes The present arcto-alpine flora has common ancestors in the ‘normal’ temperate families and genera. Cryophytes (cryos=ice, freezing) are a product of the evolutionary process similar to other niche-specialized species. As their ancestors became exposed to gradually deteriorating climate conditions, they specialized to these conditions through adaptation and natural selection. From the green algae to the most advanced angiosperms, plants have diversified in their forms and survival strategies to fill all reachable niches over a great range of conditions. According to Dansereau’s (1971) ‘Law of inoptimum’, no living individual experiences optimal conditions anywhere anytime. Those organisms living in the most adverse habitats are called extremophiles (‘lovers’ of extremes: hot/cold, wet/dry, alkaline/acid, etc.), although, as shown below, they may not necessarily ‘love’ but rather only tolerate their extreme habitat conditions.

tion. Only some alpine grounds are frozen permanently. In the arctic tundra, especially far north above the Arctic Circle (66°30′N), the summer is a single nightless day (‘land of the midnight sun’). However, solar radiation arrives at a low angle, prone to higher reflection (albedo) from the snow and ground surface. Summer temperatures are low (<10°C) but rather steady, rarely dropping below freezing point. UV radiation is lower but spread over a 24 h period. The annual energy budget is on the negative side, resulting in a permanently frozen ground (permafrost) in the Asian and North American Arctic. The growing season is very short. Counting from snowmelt to maturing seeds, it lasts only 40–50 days. Moreover, in contrast with the alpine environment, snowmelt is late and arctic plants benefit from the solar radiation and associated heat input after the summer solstice, when the radiation levels are already declining. The ground is permanently frozen (permafrost) except for the surface ‘active layer’ (a few centimetres to >1 m deep), which warms up above the freezing temperatures after the snowmelt. Still, a steep temperature gradient, from the warm surface to the frost line beneath, is maintained during the entire season. This makes drawing soil water solution against the temperature gradient for the plant difficult and results in draught stress in shoots even in the moist soils.

Basic characteristics Cryophytes are plants covering cold barren terrains of high altitudes and latitudes, mostly in tundra landscapes. However, although the alpine and arctic tundra endure cold climates, there are significant differences between these two environments (Billings, 1973). In the alpine tundra the normal circadian rhythms are preserved. Diurnal temperatures fluctuate wildly, exposing the plants to ‘winter’ and ‘summer’ conditions practically every night and day. The alpine plants are also exposed to high UV radiation, sweeping winds and drought. The growing season is relatively long, depending on the slope, aspect and snow accumulation. The snowmelt is usually completed before the summer solstice, allowing the plants maximum benefit of the peak seasonal insola-

Ecological succession Following a method of a ‘Google maps’ search, but in ascending order from a local to the global scale, the ecological succession could be as ‘elementary’ (as in an abandoned field) or less obvious as during and after minor climate change (Little Ice Age), vast terrain recolonization (after the retreat of a continental ice sheet), ground uplift due to isostatic recovery (the Hudson Bay Lowland), terrain raise due to orogeny (Himalayan Plateau), migration of continents or, ultimately, the ‘succession’ after a major global upheaval (asteroid impact). Evidently, the timescale ranges from a few years to hundreds of million years and some of

Plant Cold Hardiness in the Canadian Arctic

the long-term processes have involved species replacement due to climate and other changes as well as the evolutionary progression. Sudden cataclysmic events had crushing impacts on the organisms of the particular era. Yet, despite their destructive strike, they speeded up natural selection and macroevolution. It is now clear that there were also silent disasters which descended upon the extant biota during their evolutionary history. They were no less destructive. In the review of the present chapter, we deal with three such majorimpact processes which resulted in large-scale successional changes in species composition and evolution, and are closely related to our topic of plant cold hardiness.

Migration of continents Over the geological history of our planet, the incoherent land masses cracked and split several times. The continents twisted and, floating on the plastic layer of the basaltic ocean floors like gigantic rafts, they travelled around the globe. In a slow motion, they carried their particular contingents of the biota with them (Hurley, 1968). The process of the continents splitting and migrating pre-dates our ability to fully retrace their path. Reconstructions of former positions of the land masses reach to the Cambrian era but at that time the Earth was already 4 billion years (By) old (Tudge, 1996). The super-continent, Pangaea, which included North and South America, Africa, the Indian subcontinent and Antarctica (still joined with Australia), was situated mostly in the southern hemisphere. The long globepositional ‘stasis’ was disrupted during the Jurassic period, ~200 My ago, when Pangaea began to disintegrate. A new landmass, Laurasia, containing North America and Eurasia, occupied the northern hemisphere, while the huge remnant of Pangaea, renamed Gondwana, lingered below the equator. However, the splitting, separation and northward migration of the continents (carried on floating ‘plates’, therefore ‘plate tectonics’) continued. The enormous Tethys Sea between Africa and Laurasia had closed, producing the Mediterranean Sea, and at the west, the Atlantic Ocean began to open.

143

How does all that relate to the evolution of cold-hardened plants? Let us follow the dramatic story a bit further. Our planet is a massive gyroscope spinning at the equator at approximately 1700 km/h, which maintains Earth’s enormous momentum and stability of its circular motion. Its N–S axis is slightly tilted, presently at 23.5° to the plane of the orbit, causing annual seasonality. During the Earth’s ‘thermal normal’, which existed for most of the Earth’s history, with the exception of the far-spaced Ice Ages, the overall global climate was much warmer than at present. It is assumed that even the polar regions were mild enough to support tree-size vegetation. Nevertheless, the poles were always colder and the higher latitudes more prone to seasonality than the equatorial girth. Thus, climatic zones always existed, although their poles versus equator differences were not as extreme as at present until about the late Tertiary. All biota, confined and conditioned within distinct ecosystems of the period, have been rafted through these climatic zones, exposed to pressure to adapt, evolve or perish. All kinds of plants and animals emerged during the time of migration to be extinct again when the continent entered another climatic zone. A most striking example is the plate of the Indian subcontinent, which separated from Antarctica during the Jurassic era and began to travel northward. It reached and crossed the equator, and finally collided with Eurasia 35 My ago, creating the Himalayas. Unfortunately, all traces of this ancient subcontinent’s flora and fauna, as they had evolved during their long journey, completely vanished under the ashes of a Deccan Volcano during the Eocene (Tudge, 1996). In late Eocene also Australia, the flattest, driest and most isolated continent (with the island of New Zealand), separated from Antarctica but did not make it too far from its polar motherland. Consequently, its flora remained distinctly different and less diverse. Especially the animals, although originally rich in forms, now extinct, did not evolve beyond primitive mammals (marsupials: koala, kangaroo), duck-billed platypus and ostriches. Antarctica, the only remnant of Gondwana, if not of the original Pangaea, with its rich Eocene (58–40 My ago) temperate

144

J. Svoboda

rainforests (ferns–conifers–dicots), was not granted time to evolve any further. With the deteriorating late Tertiary climate, its lush flora declined steadily. If any cold-hardened plants developed during the cooling periods of Miocene and Pliocene, they all perished under a huge ice sheet 5 My ago. At present, only sparse mossy tundra forms the vegetation along the continent’s deglaciated rims, complemented by two tiny flowering herbs found at the peninsula. The North American continent migrated in the north-west direction at a speed of about 2 latitudinal degrees per million years. This land mass was originally carrying the tropical vegetation and assemblages of animals, including large, water-adapted reptiles. At Axel Heiberg and Ellesmere Islands, Nunavut, localities presently at 80–83°N, hundreds of wellpreserved fossil tree stumps were found and the vast extent of the ancient forest stands was reconstructed (Francis and McMillan, 1987; Thurston et al., 1989). These early Tertiary, ~45 My old forests were populated with towering Metasequoia trees (still found in a small locality in China) and deciduous hardwoods (Basinger et al., 1994). This ancient Nemoral (open woodland) vegetation is sometimes called ‘Arcto-Tertiary’, although the northern fringes of the North American continent may have been 10–20 latitudinal degrees lower than now (Sjörs, 1963). In the late Miocene (15–20 My ago) the Nemoral vegetation still occupied the northern margins of the continent. Eventually, however, with the upper portions of the continent reaching the polar latitudes and the climate deteriorating at the same time, the thermophilic plant taxa became extinct. As earlier in Antarctica, also the Arctic entered the Ice Age ~2 My ago and many species, hardened and pre-adapted to colder climate, perished under the ice sheet. The present, Quaternary Period began.

Orogeny The northwards rafting of biota has been one of the most significant evolutionary engines during the last 200 My. Yet, under the prevalent warm climate of that era, plants could not develop cold hardiness, at least not in the low-

lying topography. Several authors attempted to explain the origin of arctic and alpine flora. Old botanists observed the relationship between the Scandinavian and Central European flora. They concluded that the alpine floras are remnants of the arctic vegetation retreated ahead of the advancing glaciation. However, the question of the true origin, from where the arctic flora came, was not addressed. Nobody knows how many mountain ranges have risen and eroded away during the history of the Earth. There is a possibility that at the end of Tertiary, the present polar landscapes were populated by alpine plants from the nearby mountain ranges from the Tertiary orogeny (Ellesmere and Axel Heiberg Islands) (Saville, 1972). If there were cohorts of plants adapted to this arcto-alpine environment, they disappeared during the Ice Age, with only traces possibly surviving on non-glaciated peaks. Yet, high mountains with their severe climate are the only possible explanation for the evolution of the cold-hardened plants. Some alpine plants have a very wide distribution, being present on both hemispheres. Many other species are much more local, bound to certain mountain ranges. Alpine plants of the tropical highlands are very different from their kin in the temperate zone (Löve and Löve, 1974). The present high-altitude regions can be grouped into three great mountain systems: (i) Eurasian–Australasian (Alps–Caucasus– Himalayas). The Himalayas are the most impressive recent creation on our planet. The associated Tibetan Plateau, the world’s largest upland with the mean elevation of almost 5000 m, is still rising at a rate 1.5 m per century (Velasco and Vessels, 2008); (ii) Western American (Rocky Mountains and the Andes with their extensive ‘altiplano’ plateau, >3000 m); and (iii) African (Atlas Mountains, Kilimanjaro). Löve and Löve (1974) maintain that the oldest alpine flora can be traced through the Tibetan range to a mountain chain north of Tethys Sea, before separation of the continents. These cosmopolitan plants can also be found in the Southern Rocky Mountains, and might even be related to the Antarctic elements which escaped from glaciation. This suggests that alpine flora, although diverse, yet having

Plant Cold Hardiness in the Canadian Arctic

common features, has evolved in the high altitudes. Some of its elements are older than the mountains which are presently harbouring them. They have evolved from the ordinary populations of lowland floras due to gradual changes in altitude owing to orogeny, and/or latitude owing to continental drift, over a long period of time. They are therefore a typical product of microevolution. True cold-hardened arctic and alpine species are extremophiles belonging to a specific subgroup of cryophytes. The cryophytes replaced the lush Nemoral vegetation even in the low-lying areas when the continents entered higher latitudes and the global climate deteriorated. The altitudinal treeline, and the permanent snow line above it, delineate the viable zone for alpine plants. In the tropics, the treeline is at ~4000 m level. With increasing latitude it descends, and in the highest Arctic/ Antarctic it drops to sea level. At these latitudes, the alpine and arctic tundra practically merge. As we find later, the descending alpine zone represents a corridor through which the alpine flora could reach the bare arctic landscape after the continental ice retreat.

Glaciations It goes against common sense to argue that the Ice Ages were not only unsupportive for the evolution of cold-hardened plants, but that they also strongly assisted in weakening and eradication of their populations around the world. This for the simple reason that the expanding continental ice sheet and surging mountain glaciers suffocated all higher structured life underneath the fallen snow which would not melt in summer and thus became perennial by changing to ice. During its geological history, planet Earth experienced several deep cooling periods known as Ice Ages. The oldest known was the Huronian Glaciation, 1.8 By ago, followed by the Eo-Cambrian (650 My), African (450 My), Permo-Carboniferous (280 My) and finally Quaternary, as recent as 2 My ago. Less prominent glacial advances may have occurred at other times as well. Although there are plausible theories interpreting climate and ice fluctuations within the glaciation period, the causes

145

of the widely separated great Ice Ages are still unknown. The Quaternary Glaciation was heralded by a progressive temperature decrease in the late Tertiary, during the ‘rainy’ epoch, the Pliocene. Thick sand and gravel deposits preceding the younger glacial drift deposits, found in North Athabasca River beds, suggest heavy precipitation at the end of Tertiary. During the subsequent Pleistocene epoch, rapid fluctuations of temperature resulted in repeated glaciations and deglaciations of both hemispheres, changes in sea level and migration of flora and fauna. As many as ten glacial advances and retreats have been identified over the last 1.8 My. The major glacial periods got their names after the present North American states, indicating how far south their ice reached and where they deposited terminal moraines. From the oldest known to the most recent ones, these were: Pre-Nebraskan, Nebraskan, Kansan, Illinoian and Wisconsinan glacials (~100,000 years each). Interglacials, the in-between warm periods, with their particular names, lasted longer (~200,000 years). We are still in the midst of the Ice Age but live in the early phase of an interglacial called the Holocene. The epoch started about 11,000 years ago, after the Wisconsinan ice sheet began to rapidly melt away. The present global temperature is some 5°C higher than that during the full-blown glacial, but also by about the same margin lower than was the ‘global normal’ before and up to the mid-Tertiary. Prior to the present Ice Age, cryophytes, which had evolved over many millions of years on high plateaus, on top of the mountains and their north-facing slopes, were prolific in their particular ranges and specialized habitats. When the climate began cooling down, mountains developed ice caps, even ice fields, which covered and killed the extant cold-hardened and often also all the vegetation of the particular range. As the ice expanded, the physical realm of the cryophytes shrunk, forming only a girdle around the mountains. This alpine zone became sandwiched between the permanent snowline above and the altitudinal tree line below. None the less, alpine environments with their extreme conditions, cold-adapted plants and other biota must have always

146

J. Svoboda

existed. They have been dispersed over all latitudes otherwise there would be no coldhardened plants at present. The Pleistocene glaciations spread over a significant portion of the affected continents. At the peak of expansion, 18,000 years ago, the ice covered almost a third of the global land surface compared with ~10% at present (Lange, 2005). The Wisconsinan ice sheet occupied 40.3 million km2, while the rapidly melting ice of the present day still covers 14.9 million km2, most of it being confined in the Antarctic.

Survival in (Marginal) Cold-region Environments Arctic plants are derivatives of their alpine cousins, and these, in turn, evolved from a ‘general’ non-specialized flora of a particular geographical region and era. In spite of their multisource origin, the cryophytes share common requirements for survival in their non-necessarily supportive, if not hostile environment. While the inter- and intraspecific competition for space and resources is the main struggle for plants in the mesic temperate and tropical ecosystems, the cryophytes simply strive to survive in their physically taxing niches, often each plant on its own.

Climate and microclimate Cold regions denote a cold climate. The climate (or macroclimate), in short, is defined by long-term weather patterns prevailing in a large region or a zone. Weather is described by short-term atmospheric conditions such as solar radiation, temperature, precipitation, humidity, wind and other physical parameters. However, plants, and organisms in general, do not recognize the climate. They respond to a climate near the ground, the microclimate, which, with the microtopography, soil moisture, nutrient availability, presence of other organisms, canopy shading, etc., determines the habitat’s microenvironment. The thickness of the microclimate layer varies. Generally, it diminishes with latitude (or altitude) and so does the height of the vegeta-

tion canopy. This, in turn, determines the nature, structure and composition of the plant community in a particular site or zone. Remarkably, during the July sunny noon, the nearground temperatures of the tropical forest and tundra vegetation are a surprisingly similar ~30°C. This suggests that even tundra plants require warm spells, to experience the ‘tropical’ milieu from which they have evolved, to initiate flowering and seed ripening. In polar regions the few centimetres of warm air near the ground make a difference between the vegetated and bare sites. Often such sheltered conditions exist only in a tiny cavity between rocks (Fig. 15.1). Curiously, while cold-hardened plants are not limited by light for photosynthesis, they are very much temperature (heat) limited. At the Alexandra Fiord upland, Ellesmere Island, the mean July temperature is ~5°C. Yet, when we tried to grow some of the local plants at 5°C in the growth chamber, they hardly produced new leaves and never flowered. In the tropics, the warm microclimate prevails for most of the year, in the temperate zone during the warmer part of the growing season and in the Arctic only for several days during the short summer.

Pre-selection and pre-adaptation The basic characteristic of evolution is its proverbial slowness. Sudden changes destroy the species, slow changes promote speciation. Pre-adaptation has become advantageous in the process of cold hardening. Only plants able to miniaturize, to squeeze within the shallow layer of the favourable microclimate, could occupy the cold-environment niche. In other words, as terrains have been slowly rising due to orogeny, only species producing smaller and smaller forms were predisposed to be preserved by natural selection. In time, they became cold-hardened and UV-tolerant alpine species. Only a few tree species could do it. Certain conifers changed slowly into prostrate shrubs and krummholz (Pinus mugo, Pinus banksiana). Plants dependent on the presence or protection of other species in a complex community (cf. ‘dominant’ versus ‘rare’ species) have died out.

Plant Cold Hardiness in the Canadian Arctic

147

Fig. 15.1. The tiny herb Epilobium arcticum in a shelter niche among the rocks.

Although the cryophytes belong to a number of families and genera, there is a preponderance of monocots, mainly grasses and sedges. These graminoids are short even in warmer regions and are therefore pre-adapted (predisposed) for harsher environment. According to Raunkier’s classification, cold-hardened dicots are cryptophytes with perennating buds under the ground surface or hemi-cryptophytes, protected by a litter layer. Less often they are chamaephytes, mostly dwarf shrubs with overwintering buds protected by snow against exposure, desiccation and abrasion by fast-moving snow crystals. Other pre-adaptations favoured by natural selection include wind- and selfpollination even in species with showy flowers (apparent relict of their pre-alpine origin with the abundance of insects), agamospermy (asexual production of seed without fertilization by

pollen; e.g. genus Antennaria), apomixis (vegetative propagation by bulbils, stolons, rhizomes and roots; e.g. Polygonum viviparum, Saxifraga cernua) and polyploidy (duplication of chromosome numbers, which increases species survival in extreme high-latitude conditions), all a result of natural selection.

Adaptation We mentioned that a speciation across the environmental spectrum symbolizes a microevolution, a lateral genetic shift in a degree. Alpine and arctic semi-deserts and deserts represent extreme environments where the main task of any living creature is survival (Svoboda, 1978). No major evolutionary

148

J. Svoboda

advancements are likely in such marginal conditions, especially since the genetic pool allowing for the generation of mutants is small due to smaller metapopulation sizes and the very long time (ranging in decades) required from seed germination to reproductive stage of the plant. Nevertheless, the necessity to survive has led to a development of adaptations, and to amazingly diverse survival strategies in order to grow, endure the hardships and to reproduce. Many authors became preoccupied with the way alpine and arctic plants have established in the cold-challenging environment, and with their long-term survival (Löve, 1963; Saville, 1972; Billings, 1974; Bell and Bliss, 1977; Körner and Larcher, 1988; Crawford, 2005; and others). Measured by human standards, the ten months frozen and two months cold Arctic was deemed extreme for man as well as for ‘beast’ (cf. Robert Frost’s poems and Jack London’s stories). The actual reality with respect to arctic biota, vascular plants included, is not far from this deep-rooted perception. Adaptations or not, marginal arctic and alpine zones, such as wind-swept ridges and plateaus, represent living conditions at the

very edge of survival almost for every creature (Svoboda and Henry, 1987).

Survival strategies Billings (1974) describes the adaptations of arctic and alpine plants in terms of their general morphology and their physiology. The more severe the milieu, the more likely the plants become dwarfed to fit within the thin layer of warmer air – the more favourable microclimate. Shrubs, if any, are found pressed to rock faces, in winter seeking protection in snow drifts and snow beds. Semi-erect and prostrate willows (Salix arctica) are often severely grazed right after the spring thaw and frost-heaved from the ground during their lifespan. Yet they show the most remarkable resilience to physical stresses and other privation (Fig. 15.2). Herbs, almost all perennials, develop extensive root systems (root/shoot ratio 5:1 and higher) to draw nutrients and store carbohydrates, yet there are exceptions. On frozen grounds with the shallowest active layer, some herbs produce spider web-like roots under ‘warm’ thin rocks, having almost no weighable biomass. At the

Fig. 15.2. Grad student Glenda Jones in the willow ‘forest’ (Salix arctica) in Sverdrup Pass, Ellesmere Island. Pressed to the ground, hundreds of years old plants are beaten by the elements and continuously grazed.

Plant Cold Hardiness in the Canadian Arctic

end of the seasonal life cycle, shoots of some species die back to ground level where their perennating buds are protected over the winter. Annual herbs are rare and even these often exist as perennial ecotypes. There are three main types of tundra perennials: graminoids, leafy dicots and cushion dicots. Grasses, sedges and rushes are omnipresent, owing their prevalence to their natural ability to miniaturize and adapt from aquatic (Carex aquatilis) to the driest habitats (Carex nardina). They flower late, ~20 days after the snowmelt, yet produce viable seed. The dicots’ common denominator is that during the growing season they produce a pre-formed shoot and flower primordia and overwintering buds. Flowering occurs early next spring and ripening of seeds takes place shortly after, so that the entire process is completed within 40–50 days, usually in early August. Thus these plants complete their life cycle in two, rather than in a single season. Longevity is a winning ticket (Svoboda, 1977; Fig. 15.3). The purple saxifrage (Saxifraga oppositifolia) blooms usually 3–5 days after its release from snow. Its pre-formed flower buds enlarge while still snow-covered because they absorb

149

solar radiation and form a tiny bubble greenhouse around each bud. The flowers open their petals just at the plant surfaces to keep the ovaries warm, yet during the seed ripening, the flower peduncle starts to elongate high enough for the wind to shake the fruit and disperse the seeds. Arctic avens (Dryas integrifolia) and other arctic/alpine species utilize the same reproductive and dispersal strategy. These species also display phenomenal phenotypic plasticity. In favourable (mesic and sheltered) habitats the purple saxifrage and arctic avens produce loose, creeping patches; in a less protected site they form compact clumps or cushions; and on an exposed ridge they grow as thin prostrate mats. Dryas cushions as semi-closed micro-ecosystems In extreme conditions, plants develop structures and defence mechanisms to be almost self-sustaining and self-contained in the hostile environment (cacti in deserts, tree islands in the Subarctic). In polar semi-deserts, Dryas cushions serve as an example of such an ingenious and efficient arrangement of features by

Fig. 15.3. An 800-year-old individual (>100 cm diameter) of arctic avens (Dryas integrifolia) lives only on its periphery. The core of the clump has died out and weathered away. A large stand of similar old clumps which escaped the Little Ice Age neoglaciation was found on a gravel terrace of Coats Island.

150

J. Svoboda

combining slow incremental growth with a prolific seed production. Arctic avens and the purple saxifrage are the most representative species of polar semidesert communities. They maintain a large above-ground biomass compacted into a shape of a cushion. At its surface, Dryas produces a layer of tiny green leaves which are photosynthetically active for two years. When they die, they remain attached and form a distinctly coloured layer similar to a tree ring. By counting these ‘rings’, the age of the clump can be established. This standing dead mass remains useful in several ways. It holds moisture and decays slowly. Its nutrients are released gradually within the cushion to be resorbed and recycled by the secondary roots developed within the cushion. The plant also catches fine dust particles which fill the cushion core providing minerals for the secondary roots. Mycorrhizal associations with Dryas roots (and other arctic dicots) infer their N-fixing function (Bledsoe et al., 1990; Kohn and Stasovski, 1990). Nematodes and tiny invertebrates cohabit in the cushions’ protected environment to a mutual benefit. Physiology Phenotypic and morphological difference among various ecotypes, ecoforms and perhaps even acclimated populations of cold-hardened plants result in physiological dissimilarities as well. These might be related to timing their life cycle, favouring self-pollination, apomixis over sexual reproduction, germination, dormancy, frost resistance, and many other physiological and eco-physiological characteristics, studied by a great number of researchers. As an example, at Alexandra Fiord (79°N), during the summer continuous daylight, we measured a 24 h transpiration activity of S. cernua. In spite of a full midnight sun, albeit with a lower light intensity, there was a 2 h ‘midnight’– a pause in transpiration due to stomata closure. We believe that was a residual property carried over from the species’ low-latitude alpine origin with a regular circadian rhythmicity still active. McNulty and Cummins (1987) found that dark respiration rate in S. cernua collected at the same locality and grown in chambers at a constant 10°C was higher than in plants

grown at 20°C. Crawford (2008b) reported distinct yet opposing metabolic strategies between the extreme forms of S. oppositifolia. As there are many environmental gradients, so there are gradients of evolutionary progression from induced acclimation, to temporary ecoforms, to more stable ecotypes and finally to a new species. Over a long timespan and several generations, some of the acquired physiological responses may ultimately serve the particular populations to cope better in their accustomed habitat. ‘Although the ecoforms are merely temporary, their production is continuous and may be one of the ways that the ancient autochthonous flora of the Arctic has survived’ (R.M. Crawford, St Andrews, Scotland, 2008, personal communication). However, unless such shifts become genetically fixed, they will remain a mere temporary phenomenon, prone to be reversed when the living conditions change.

Postglacial Plant Reinvasion During the Pleistocene, polar regions were subjected to long- and short-term climate oscillations resulting in vegetation and soil burials under the ice, circum-continental and longterm exposure of continental shelves due to sea level drop, damping of glacial drifts over the formerly vegetated lands, subsequent terrain erosion, sea and periglacial flooding, isostatic rebound, re-colonization, etc. All of these forces and factors had a considerable impact on the pre-Ice Age and interglacial flora: preserving it in some areas, moving it around as the situation changed, and, indeed, mostly decimating it during the great glaciations but also during minor neo-glaciations, as was the recent ‘Little Ice Age’ (Bergsma et al., 1984; Lévesque and Svoboda, 1999). The Pleistocene climatic upheavals which started the elimination, mixing and sorting of the biota have continued also in Holocene, in reality until the present. One must wonder that patterns of any floristic elements can be still recognized and that indigenous plant populations can be traced to their sources where they carried on in exile during the glaciations. The reconstruction of the dramatic saga has been possible largely due to the advancement of modern analytical techniques.

Plant Cold Hardiness in the Canadian Arctic

After the Pleistocene’s repeated ice advances and retreats, a smaller number of the cryophytes and proportionately even fewer animal species survived to return to the deglaciated terrain. Ice-free Alaska and Siberia together with fringe tundra zones adjacent to the glacial fronts, and for millennia exposed continental shelves during glaciations, had been the most plausible sources of the cryophytes’ reinvasion. Hultén (1972) described in detail radiation pathways of arctic plants from various continental, coastal, amphi-Atlantic, arctic-montane and boreal centres and refuges into their deglaciated realms. Some species may have survived within the glaciated landscape on nunataks, i.e. on rocky mountain tops protruding the ice field, giving a foundation for the ‘nunatak refugia hypothesis’ (Ives, 1974). However, at least at high latitudes, the potential pool of cryophytic species on these rock islands has been very limited. The presentday new-world nunataks still harbour remnants of the depaupered ice age flora. Only a small number of species at negligible frequency has persevered on them, although the present climate is kinder than was that during the fullstrength glacial. Of the world’s 235,000 species of flowering plants (Tudge, 2000), only about 1700 taxa populate the Nearctic realm of mostly Beringia, Alaska and Yukon Territory at present (Hultén, 1974). A mere 0.4% of the known vascular plant species are established in the Arctic (Billings, 1992). For the continental Northwest Territories of Canada, Porsild and Cody (1980) list 1100 species, mostly overlapping with those of Hultén (1972). However, both lists include many taxa of the species-rich tundra– taiga ecotone, of which about 500 comprise the flora of the Alaskan Arctic slope (Murray, 1995) and 350 occur in the Canadian Arctic Archipelago (Porsild, 1964). With increasing latitude, the number of vascular plants diminishes rapidly, dropping to less than 20 species on the vast regions of polar uplands at a dismally low total ground cover of 0.3% (Bliss et al., 1984). These uplands are a true polar desert. Most of the high arctic plant and animal life is concentrated in sheltered bays and lowlands, called thermal oases. At the Alexandra Fiord lowland, 79°N, Ellesmere Island, 92 vascular plants were identified (Ball and Hill, 1994).

151

By contrast, the largest northernmost oasis at Lake Hazen, 82°N, Ellesmere Island, sustains 117 vascular species (Soper and Powell, 1985). The non-vascular elements are represented by 750 bryophyte and 1200 lichen species (Murray, 1992).

The tardy journey to the promised land The Wisconsin glaciation ended some 11,000 years ago, when the continental ice ceased spreading and, in turn, started melting vigorously, first at its margins. The Holocene, the recent ameliorated era and likely a new interglacial, began. It took 8000 years for the continental ice masses to incompletely melt away (Greenland and eastern islands of the Arctic archipelago are still glaciated, and polar regions had been subjected to temporary neoglaciations during minor cold climate anomalies). At the south, huge periglacial lakes developed in front of the end moraines. The largest of them, Lake Agassiz (1100 km×400 km), was draining its waters for thousands of years. The forest–tundra plant communities adjacent to the glacial front entered the spatial void left by the shrinking continental ice. However, much of the vegetation, which followed the glacial retreat, drowned in these newly formed lakes. Nevertheless, the vegetation had steadily advanced. A plethora of temperate and boreal species invaded the icefree terrain; not all of them, however, succeeded in entering it. Walker (1995) describes the extant arctic plant diversity as a community of species which have passed through a series of ‘filters’. The ‘pores’ of the first filter have been so fine that only a tiny fraction of the existing vascular flora could pass through it. The second and third sets of filters are represented by the climatic, geological and habitat gradients, and biological interactions. In this imaginative scenario, these ‘filters’ further screened, and thus eliminated, many taxa which passed through the first filter. The melting of the continental ice sheet was triggered by a relatively sudden climate warming and also the climate in the melting zones became much more favourable for plant growth. A great number of species began marching north and would do well. However, a fierce interspecific competition made many species only temporarily successful. As the ice

152

J. Svoboda

receded into higher latitudes, a climatic gradient began emerging. Only those better adapted to the increasingly harsh conditions (shorter and cooler growing season, shallow active layer, etc.) would prevail migrating to higher latitudes. The less resilient species would remain behind, holding on their achieved positions. The Ice Age was a continental phenomenon and so was the phenomenon of plant recolonization of the immense ice-free landscape. The terrain to be reclaimed stretched over 40° of latitude from the present Wisconsin State to the top of Ellesmere Island, and acted as a large sheet of chromatographic paper. Cohorts of vascular species had travelled along it as far north as they could tolerate the increasingly hostile conditions and positioned themselves in the most plausible area or zone, out of reach of their less resilient competitors. For the sake of completeness, it has to be added that the deglaciated realm has been invaded also from the western and north-western side in a rather semicircular fashion; nevertheless, the pattern of re-colonization was very similar. Walker (1995) pointed to a remarkably high correlation between the regional summer climate and the number of species of the regional high arctic floras, ranging from more than 200 species at 9°C to less than 50 species at 3°C of the July mean temperature. At present, the North American tundra complexes represent a huge biome composed of various ecosystems characterized by similar vigour, competitive strength and tolerance of the environmental conditions (Bliss, 1997). In this biome distinct vegetation zones have reached a dynamic equilibrium with the present climate. However, the zones have shifted in the past and are bound to shift even more, as the climate gets warmer (Crawford, 2008a).

Competition versus stress tolerance Svoboda and Henry (1987) described the postglacial plant advancement and classified the process as a primary succession of continental dimensions. Three ongoing successional phases can be recognized. 1. Phase I: directional replacement succession, typical in mesic temperate environments and also manifested in the low-arctic contigu-

ous tundra. The invading plant cohorts meet little resistance here; however, due to fierce competition, only the winners have survived and established more or less permanent plant communities. 2. Phase II: directional non-replacement succession in near-marginal environments, where populations of invading species keep progressing slowly without much competitive interference. 3. Phase III: non-directional non-replacement succession, a virtually stagnant succession in marginal, extremely unfavourable environments. Plant propagules reach the area, of which some may germinate but most of the seedlings will sooner or later die. A handful of diminutive individuals may very slowly reach maturity, some even produce viable seed. Eventually, these few cold-hardened pioneers establish their presence in areas marginal for survival of any higher-structured life. Out of several thousands of the initially invading species, these final cold-hardened pioneers proved to be best pre-adapted. Their journey was the longest and they now grow far beyond the reach of their closest potential competitors. In other words, as the conditions along the latitudinal gradient worsened, the emphasis became less on species replacement and more on the establishment and survival of an individual. One more point: all the pioneer finalists of the long journey were the underdogs at the start line. They were the ‘rare species’ in the original plant community of the ‘big and tall’ dominants, composed of trees, shrubs, heath and graminoids. Presently, in the huge expanse of the Nearctic tundra, phase I predominates in the climatically least unfavourable zone, known as the Low Arctic. Phase II prevails in the Mid-Arctic, climatically more severe. Phase III is typical in the High Arctic polar desert with the most severe climate, being the extreme end of the latitudinal severity gradient. It should to be emphasized that the tundra plant communities, as we find them spread from the lowest to the highest latitudes (and altitudes in their alpine version), are still in the phase of colonization, ready to move forward when the conditions improve. In fact, this occurred during the warm hypsithermal period 6000 to 4000 years ago, at which time the treeline extended up to 350 km north of the

Plant Cold Hardiness in the Canadian Arctic

present forest–tundra boundary. This has also been happening after the termination of the more recent cold climatic episode, the Little Ice Age, and is gaining momentum during the present global warming (Svoboda et al., 1995; Crawford, 2008b). Time-lapse photography would show a busy activity of individual tundra plants and their entire cohorts, gaining ground and losing it again, as the year-to-year weather and decade-to-decade climate differ in completion of snowmelt, in precipitation patterns, in total degree-days, and other relevant parameters. As the Red Queen explained to Alice: ‘it takes all the running, to keep in the same place’.

Distribution of closely related species The arctic tundra vegetation is made of a relatively small number of species, yet these belong to an even disproportionately smaller number of genera. Moreover, certain genera are widely out of the expected range in terms of species they contain. Thus Porsild and Cody (1980) list 105 species of sedges (Carex spp.), 57 willows (Salix spp.), 29 buttercups (Ranunculus spp.), 27 blue grass (Poa spp.), 25 saxifrages (Saxifraga spp.), 23 cinquefoils (Potentilla spp.), 22 dandelions (Taraxacum spp.), 21 drabas (Draba spp.) and 20 oxytropis (Oxytropis spp.). Only a few of these species fully overlap in their ranges of distribution. For instance, Taraxacum lacerum, Taraxacum hyparcticum and Taraxacum phymatocarpum, and another set of closely related species, Salix planifolia, Salix arctophylla and Salix arctica, occupy separate zones in the Low, Mid and High Arctic (Svoboda and Henry, 1987). The topographical distribution of all these species reflects the source of their origin or, in the case of the above-named dandelions and willows, a fine-tuning of their cold hardiness along the latitudinal severity gradient. While, at the generic level, the dandelions and willows can be easily recognized by a non-taxonomist, each of the many saxifrage species looks different, as they arrived to the mountain tops and lastly to the arctic tundra domain via quite heterogeneous phylogenetic paths. Aside from their essential cladistic similarities (easily overlooked by a non-expert), they developed organs and use-

153

ful adaptations particular to each species. The spider plant (Saxifraga flagellaris) sends out long naked stolons, terminated by rooting rosettes; the most common purple saxifrage (S. oppositifolia) exists in dry habitats as a densely tufted (cushion-like) form and in wet snowbanks as a loosely trailing mat; the tiny nodding saxifrage (S. cernua) reproduces prolifically by clusters of white bulblets at its base and by clusters of small purple bulbils along the stem; the brook saxifrage (Saxifraga rivularis) specializes in wet places and reproduces frequently by thread-like rooting stolons; in contrast the prickly saxifrage (Saxifraga tricuspidata) is very successful in dry rocky places. Its succulent-like leaves are arranged on densely crowded shoots. Long flowering stems produce plenty of wind-dispersed seeds. In contrast to the homogeneous dandelions or willows which tend to occupy separate climatic zones, many of the heterogeneous saxifrages coexist in various habitats of the same, curiously, the most severe environment; both types being an amazing example of the various convergent cold-hardening pathways and, indeed, of the natural selection.

Small but prolific, or big yet rare? Why this dilemma? Plant vigour and species ground cover along the increasing severity and decreasing competition gradient reveal an opposite trend. In mesic habitats, at their southern rim of distribution, the wide-range species produce robust individuals but they are usually only a minor component of the plant community. Plant vigour continually diminishes because of increasing gradient severity. In contrast, due to decreasing competition, the species’ ground cover builds up, peaking at a certain midpoint of the distribution range, and then starts to decrease up to the point of extinction, as the physical environment becomes too hostile. In other words, as the less-adapted species keep dropping out of the picture and the competitive drive diminishes, the more stress-hardened species are able to cover more ground. The trade-off is a reduction of physical vigour for a higher presence (Svoboda and Henry, 1987).

154

J. Svoboda

Arctic avens (D. integrifolia) may serve as a classic example and model of arctic plants’ strategy of survival. This official flower of the Canadian Northwest Territories belongs to the most common species in the Nearctic tundra. As described above, at its southern margins, Dryas forms semi-erect shrubs, yet its ground cover is very low; at mid-range it becomes

prolific and produces substantial cushions; while at its northern fringes it becomes sporadic again forming thin, scattered mats. At the lower margin the strong competition with other shrubs and heath limit the species presence while at its northern margin, the intolerable severity of the physical environment becomes the ultimate limiting factor (Fig. 15.4).

Vigour/abundance

(a) s al ent stres

High m iron

v En

En vir on

Vigour

me

nta

ls

tre

ss

Distribution Low 40

50

60

Potential

70

80°

N

Actual Tolerance range

(b) High

A

Relative vigour

B C D E

Low Latitude/severity gradient (c)

A

Relative abundance

High

B C D E

Low Latitude/severity gradient Fig. 15.4. Effects of interspecific competition and environmental stress on (a) vigour and distribution of a single species, (b) relative vigour of a series of species and (c) relative abundance/standing crop of a series of species along the latitudinal stress gradient, as demonstrated on a Dryas integrifolia model. (Used with permission by Arctic and Alpine Research Journal.)

Plant Cold Hardiness in the Canadian Arctic

The Non-vascular Components The most ‘dwarfed’ cryophytes are mosses, lichens, algae, fungi and bacteria. These opportunistic organisms take advantage of the momentary changes of temperature and moisture, able to turn their metabolism on and off in minutes. They flourish far beyond the range of vascular plant distribution and some function even in subfreezing temperatures. They can also remain dormant for a very long time. Lichens grow at elevations ≥6000 m and their plaques and discs are known for their extreme longevity (>5000 years), allowing use for dating purposes (lichenometry). Various microbiota were found in deep ice cores (Miteva and Brenchley, 2005; Mosier et al., 2006) and in the permafrost (Steven et al., 2006). Algae flourish in the snow and glacial ice. As in the primeval times of algal invasion on to bare land, they still play a crucial role in re-colonization of the modern open terrain. In high latitudes, the resulting exposed landscape, the polar desert, is a climatic remnant of the past ice age. Here, cyanobacteria (blue-green algae) are first to operate. They fix N on the surface of the pre-washed, N-starving regosols, accompanied in tandem by the green algae. Together they produce the first organic matter and accumulate biomass, storing nutrients which, after their slow decomposition, are made available to fungi, mosses and soon vascular plants (Elster and Benson, 2004). Thus these photosynthetically active, cold-hardened microbes are essential in the process of initial primary succession following landscape deglaciation. Algal fertilization and ground preconditioning are highly efficient in the barren landscape revitalization and tundra ecosystem restoration.

Field studies In Sverdrup Pass (79°N), Ellesmere Island, we have conducted research on algal diversity, abundance and productivity in a glacial stream running from the melting front of the large Teardrop Glacier and merging about 500 m down with the fast rivulet in the pass’s valley (Elster and Svoboda, 1995, 1996). At the glacial front, the meltwater was only 0.5°C,

155

increasing gradually to a maximum of 10°C at the merger with the rivulet. We made the following observations. 1. The dissolved nutrient loading (nitrate- and ammonia-N, ortho- and total phosphate, Ca and Mg) was highest in the glacial front zone and diminished along the stream. 2. This wet and coldest zone was inhabited mainly by Cyanoprocaryota (blue-greens) clearly responsible for the highest reading in available N, mostly fixed by them. Further from the glacier, the blue-greens were largely replaced by the green algae. 3. As the water temperature increased with distance from the glacier, distinct taxonomical groups showed a proclivity for separation along the stream, proving that even algae are coldhardened to different degrees and thus temperature-sensitive. 4. The algal visible biomass (mostly dense filamentous curtains) peaked about 100 m from the glacial front and diminished virtually to zero before the stream merged with the rivulet about 400 m further on. The algae consumed practically all of the dissolved nutrients, thus impeding their own growth in the lower section. Here, the stream bottom showed the original substrate (clean sand and gravel) with no visible coating by algae. In contrast, the elevated stream banks consisted of a thick peat layer, rich with mosses and vascular plants. This was a classical demonstration of a gradual build-up of organic matter as a function of time and distance from the receding glacial front (Fig. 15.5). At the end of the growing season, the glaciers stop melting and the stream beds dry out and freeze. The algal biomass breaks and is blown into the valley, fertilizing the already established vegetation and facilitating further growth. A significant amount (kilograms) of dry algal biomass is being produced by a single stream every season, and thousands of such streams and seepages run down a great number of outlet glaciers descending into the 80 km long Sverdrup Pass from the surrounding ice fields at Ellesmere Island alone. Evidently, the quantity of organic matter produced and contributed by algae annually and summed up over years, even centuries, has been staggering. Presently, the ice margins of the 2000 km2 glaciated area in the circumpolar Arctic (1975 estimate) are melting, nourishing algal growth

156

J. Svoboda

Fig. 15.5. One of many glacial streams at Sverdrup Pass, Ellesmere Island. At the glacial front, the stream is loaded with algae. Their floating biomass diminishes along the stream, and the water and the gravelly stream bottom are free of visible algae before the stream merges with the valley rivulet.

in zones adjacent to their glacial fronts. As a consequence, also the neighbouring landscapes abound with life. The revitalized periglacial environment represents a net carbon sink, scrubbing CO2 from the atmosphere and depositing it as organic carbon upon the land. The cold-hardened meltwater algae are the primary facilitators of the initial primary succession after deglaciation.

Cold origin of life? A prolific presence of prokaryotes in ice cores and the permafrost points to their ability to metabolize in subfreezing temperatures, condi-

tions existing as well on cold astral bodies. These are the true extremophiles! Microbes which fit into micro-veins of liquid water within the ice can utilize nutrients and energy the chemo-autotrophic way. In a well-corroborated review paper, Price (2007) explains that: On the early Earth, and on icy planets, prebiotic molecules in veins in ice may have polymerized to RNA and polypeptides by virtue of the low water activity and high rate of encounter with each other in nearly one dimensional trajectories in the veins.

This is certainly a fascinating idea, contrasting with the more well-known theory of the hot underground and oceanic hydrothermal vents origin.

Plant Cold Hardiness in the Canadian Arctic

Horticultural Experiment Near 80°N In the early 1980s our research group was involved in a 7-year-long ecological study of the polar oasis at Alexandra Fiord lowland (79°N), Ellesmere Island (Svoboda and Freedman, 1994). The lowland’s mesoclimate seemed to be favourable enough to grow some lettuce and radishes in the local soil, under plastic mulch, for the camp consumption. The results were promising and the next year we arrived equipped with light, specially designed umbrella-shaped structures covered with Du Pont Fabrene® tear-resistant plastic. Twelve of them were reach-in (3 m diameter) and two were walk-in (6 m diameter) structures. We set them at a sunny site and for their circular shape, reflecting the 24 h path of the sun, we named them ‘igloos’. Two outlet glaciers nearby created a contrasting background. We called the experiment the Green Igloos Farm (Fig. 15.6). A parallel experiment, the Keewatin Gardens, a set-up of 40 A-frame rectangular greenhouses, was assembled at Rankin Inlet, (63°N), Northwest Territories, with the objective to grow native arctic plants as potential food crops. What was intended as a side project to our polar oasis study has developed into a seri-

157

ous research endeavour (four Masters theses) at two arctic localities in collaboration with Guelph University and supported by a significant grant (Romer et al., 1981; Cummins et al., 1988). At Alexandra Fiord, the local soil was worked out, fertilized, and used to fill black plastic grow-bags to avoid root contact with the permafrost. The bags were arranged in circles in the sun-heated greenhouses. A large variety of vegetables, potatoes and ornamental plants were grown with unanticipated success. Many were grown from seeds (radishes, beets, carrots, beans, etc.), others from seedlings (cabbages, lettuces, broccoli, tomatoes) or tubers (seven varieties of potatoes) (Fig. 15.7). In addition to the southern cultivars, two native herbs, arctic poppy (Papaver radicatum) and bulblet saxifrage (S. cernua), were extracted from the nearby tundra soon after the snowmelt and transplanted to one empty greenhouse. The very first season the transplants responded to the warm greenhouse environment by growing five times taller than the tundra controls and the next season they produced clusters from seeds and bulblets dropped the previous autumn (Figs 15.8 and 15.9).

Fig. 15.6. Green Igloos Farm at Alexandra Fiord, Ellesmere Island, with two outlet glaciers in the background.

158

J. Svoboda

Fig. 15.7. Assortment of vegetables, grown in black plastic grow-bags, in the walk-in ‘igloo’ greenhouse.

Fig. 15.8. Gigantic clumps of arctic poppy (Papaver radicatum – yellow flowers) and bulblet saxifrage (Saxifraga cernua – the reddish inflorescences), produced in an ‘igloo’ from seed dropped by single plants at the end of the previous growing season. NB: See Plate 3 for colour representation.

Plant Cold Hardiness in the Canadian Arctic

159

Fig. 15.9. Control plant collected in the open tundra (left of the scale bar), a single plant and a clump of bulblet saxifrage (Saxifraga cernua) grown in the ‘igloo’ greenhouse (right of the bar).

The horticultural experiment demonstrated that the southern produce plants respond well to a favourable greenhouse microclimate in spite of the continuous daylight. The formation of sizeable tubers under prolifically flowering potato plants surprised the Guelph experts who predicted that since potatoes originate in the equatorial zone with 12 h/12 h photoperiod, no tubers would be formed. The tundra transplant experiment showed clearly that their native environment was much below a desired optimum. Their immediate and vigorous growth confirmed that these plants, although tolerant to the stressful polar conditions, were still genotypically and phenotypically ready to take advantage of the ameliorated conditions which resembled the environment of their southern alpine origin. There is a fine yet critical distinction between plant adaptation (to feel home at the site) and plant tolerance (to be able to cope with the taxing situation).

Conclusion Over their evolutionary history, organisms have been adapting to most diverse environments. Plants, from the green algae to the most advanced angiosperms, have diversified in their forms and survival strategies to fill all reachable niches over a great range of conditions. Those living in the most contrasting habitats are called extremophiles (‘lovers’ of extremes: hot/cold, wet/dry, alkaline/acid, etc.), although, as manifested, some of them may not necessarily ‘love’ but rather only tolerate their habitat conditions. The cold-hardened arctic and alpine species, the cryophytes, belong to this category. They evolved in regions subjected to orogeny by being slowly carried up with the rising mountains to high altitudes and, in some cases, had been rafted to the polar regions by the northward migration of the continents. During the Interglacials and after the last Ice Age, the clean slate of the deglaciated

160

J. Svoboda

North American continental landmass acted as a large sheet of chromatographic paper along which the plant species travelled as far north as they were able to tolerate the increasingly hostile conditions. Some tundra species are more cold-hardened than others, although additional limiting factors are also involved. According to the degree of cold and stress tolerance, various species reached and established at different geographical positions and now form separate or overlapping ranges of their spatial distribution. All tundra species would prefer more favourable environments than the one they occupy most. In a less supporting environment, these plants are under stress. However, they recover fast if the conditions change for the better. At present, vegetation complexes of similar vigour, competitive strength and stress tolerance form distinct vegetation zones in the North American tundra biome. The vegetation of these zones is in a dynamic equilibrium with the extant climate. However, the zones have shifted in the past and are bound to shift again, as the climate ameliorates. The cold-hardened algae and bryophytes play a crucial role in the colonization of freshly deglaciated terrain by fixing N and

building the first biomass for the higher plants’ establishment. The horticultural experiment with southern cultivars at Alexandra Fiord confirmed that even warm-climate vegetables grow well in an artificially ameliorated space bubbles (‘igloos’) in a generally hostile climate. Similarly, the native tundra plants, transplanted into the same sun-heated greenhouse, grew much taller and produced several times more seeds and bulblets than plants in the nearby tundra.

Acknowledgements I am very grateful to Professor Karen Tanino for inviting me, an ageing arctic ecologist, to present an inaugural lecture to mostly the agrobiologists at the 8th International Plant Cold Hardiness Seminar, and for her involvement in editing of my manuscript. I am also thankful to Professor Robert M. Crawford, University of St Andrews, Scotland, for his constructive comments on the manuscript. Michael Svoboda provided vital assistance by resolving some defiant computer glitches and with preparing the figures for the publication.

References Ball, P. and Hill, N. (1994) Vascular plants at Alexandra Fiord. In: Svoboda, J. and Freedman, B. (eds) Ecology of a Polar Oasis Alexandra Fiord, Ellesmere Island, Canada. Captus University Publications, Toronto, Ontario, Canada, pp 255–256. Basinger, J.F., Greenwood, D.R. and Sweda, T. (1994) Early Tertiary vegetation of Arctic Canada and its relevance to paleoclimatic interpretation. In: Boulter, M.C. and Fisher, H.C. (eds) Cenozoic Plants and Climates of the Arctic. NATO ASI Series, Vol. 127. Springer Verlag, Berlin, pp. 175–198. Bell, K.L. and Bliss, L.C. (1977) Overwinter phenology of plants in a polar semi-desert. Arctic 30, 118–121. Bergsma, B.M., Svoboda, J. and Freedman, B. (1984) Entombed plant communities released by a retreating glacier at central Ellesmere Island, Canada. Arctic 37, 49–52. Billings, W.D. (1973) Arctic and alpine vegetations: similarities, differences, and susceptibility to disturbance. BioScience 23, 697–704. Billings, W.D. (1974) Arctic and alpine vegetation: plant adaptations to cold summer climates. In: Ives, J.D. and Barry, R.G. (eds) Arctic and Alpine Environments. Methuen, London, pp. 403–443. Billings, W.D. (1992) Phytogeographic and evolutionary potential of the arctic flora and vegetation in a changing climate. In: Chapin, F.S. III, Jefferies, R.L., Reynolds, J.F., Shaver, G.S. and Svoboda, J. (eds) Arctic Ecosystems in a Changing Climate: An Ecophysiological Perspective. Academic Press, San Diego, California, pp. 91–108. Bledsoe, C., Klein, P. and Bliss, L. (1990) A survey of mycorrhizal plants on Truelove Lowland, Devon Island, N.W.T., Canada. Canadian Journal of Botany 68, 1848–1856. Bliss, L.C. (1997) Arctic ecosystems of North America. In: Wielgolaski, F.E. (ed.) Polar and Alpine Tundra. Ecosystems of the World 3. Elsevier, Amsterdam, pp. 551–683. Bliss, L.C., Svoboda, J. and Bliss, D. (1984) Polar deserts, their plant cover and plant production in the Canadian High Arctic. Holarctic Ecology 7, 305–324.

1

2

Plate 1. Observed frost damage to heads of wheat following freezing. Left, following frost testing, left head froze, right 3 heads supercooled. Right, typical head damage observed in the field during head development. Plate 2. Infrared images taken during freezing and thawing of wheat plants at ear emergence.

3

4

Plate 3. Integrative molecular timetable of bud development in poplar. Autumnal bud development is a composite of bud formation (red), acclimation to dehydration and cold (blue), and dormancy (orange). Selected genes or processes that specifically belong to one of these aspects are highlighted accordingly. Simultaneous with bud development, elongation growth (green) gradually ceases in young derivatives that are displaced from the apex. Bud development is characterized by the sequential activation of light, ethylene, and ABA signal transduction pathways. The major transcriptional changes at the regulatory and target process levels are depicted at the time that the respective genes show their maximal change in expression. The two major phases of transcriptional and metabolic response are indicated by grey boxes. Below, tentative levels of cellular responses and/or the quantity of major metabolites are indicated with a graded scale. Arrows connect regulators and transcription factors to their putative downstream processes, without implying a genetic or direct molecular interaction. Because of its putative nature, the link between low sugar and ethylene signal transduction is shown with a dashed arrow. Genes shown in grey and within brackets are only found differentially expressed in ABI3-overexpressing poplars. Plate 4. Gigantic clumps of arctic poppy (Papaver radicatum - yellow flowers), and bulblet saxifrage (Saxifraga cernua - the reddish inflorescences), produced in an ʻIglooʼ from seed dropped by single plants at the end of the previous growing season.

Plant Cold Hardiness in the Canadian Arctic

161

Crawford, R.M.M. (2005) Long-term plant survival at high latitudes. Botanical Journal of Scotland 56, 1–23. Crawford, R.M.M. (2008a) Plants at the Margins – Ecological Limits and Climate Change. Cambridge University Press, Cambridge, UK. Crawford, R.M.M. (2008b) Cold climate plants in a warmer world. Plant Ecology and Diversity 1, 285–297. Cummins, W.R., Bergsma, B.M., Romer, M.J. and Svoboda, J. (1988) Food from the Northern Land: The Potential for Small-scale Food Production in Arctic Canada. Occasional Papers of the Prince of Wales Northern Heritage Centre No. 3. Government of the Northwest Territories, Yellowknife, Northwest Territories, Canada, pp. 93–110. Dansereau, P. (1971) Dimensions of environmental quality. Sarracenia 14, 1–109. Eldredge, N. and Gould, S.J. (1972) Punctuated equilibria: an alternative to phyletic gradualism. In: Schopf, T.J.M. (ed.) Models in Paleobiology. Freeman, Cooper & Company, San Francisco, California, pp. 82–115. Elster, J. and Benson, E.E. (2004) Life in the polar terrestrial environment – a focus on algae and cyanobacteria. In: Fuller, B., Lane, N. and Benson, E.E. (eds) Life In The Frozen State. Taylor and Francis, London, pp. 111–149. Elster, J. and Svoboda, J. (1995) In situ simulation and manipulation of a glacial stream ecosystem in the Canadian High Arctic. In: Jenkins, A., Ferrier, R.S. and Kirby, C. (eds) Ecosystem Manipulation Experiments: Scientific Approaches, Experimental Design and Relevant Results. Proceedings of a Symposium at Bowness-on-Windermere, Lake District, England, 16–21 October 1994. Ecosystem Research Report No. 20. Commission of the European Communities, Brussels, pp. 254–264. Elster, J. and Svoboda, J. (1996) Alagal diversity, seasonality and abundance in, and along glacial stream in Sverdrup Pass, 79°N, Central Ellesmere Island, Canada. Special Issue, Memoirs of the National Institute of Polar Research 51, 99–118. Elster, J., Lukešová, A., Svoboda, J., Kopecký, J. and Kanda, H. (1999) Diversity and abundance of soil algae in the polar desert, Sverdrup Pass, central Ellesmere Island. Polar Record 35, 231–254. Francis, J.E. and McMillan, N.J. (1987) Fossil forests in the far north. Geos 16, 6–9. Gould, S.J. (2002) The Structure of Evolutionary Theory. Harvard University Press, Cambridge, Massachusetts. Hultén, E. (1972) Outline of the History of Arctic and Boreal Biota during the Quaternary Period. Verlag von J. Cramer/Wheldon & Wesley, New York, New York. Hultén, E. (1974) Flora of Alaska and Neighboring Territories. Stanford University Press. Stanford, California. Hurley, P.M. (1968) The Confirmation of Continental Drift. Freeman, San Francisco, California. Ives, J.D. (1974) Biological refugia and the nunatak hypothesis. In: Ives, J.D. and Barry, R.G. (eds) Arctic and Alpine Environments. Methuen, London, pp. 605–636. Kohn, L.M. and Stasovski, E. (1990) The mycorrhizal status of plants at Alexandra Fiord, Ellesmere Island, Canada, a high arctic site. Mycologia 82, 23–35. Körner, C. and Larcher, W. (1988) Plant life in cold climates. In: Long, S.F. and Woodward, F.I. (eds) Plants and Temperature. Symposium of the Society for Experimental Biology, Vol. 42. The Company of Biologists Ltd, Cambridge, UK, pp. 25–57. Lange, M.A. (2005) Ice Ages. In: Nuttall, M. (ed.) Encyclopedia of the Arctic, Vol. 2. Routledge, New York, New York, pp. 905–907. Leakey, R. and Lewin, R. (1995) The Sixth Extinction. Patterns of Life and the Future of Humankind. Doubleday, New York, New York. Lévesque, E. and Svoboda, J. (1999) Vegetation re-establishment in polar ‘lichen-kill’ landscapes: a case study of the Little Ice Age impact. Polar Research 18, 221–228. Löve, D. (1963) Dispersal and survival of plants. In: Löve, A. and Löve, D. (eds) North Atlantic Biota and Their History. McMillan, New York, New York, pp. 189–205. Löve, A. and Löve, D. (1974) Origin and evolution of the arctic and alpine floras. In: Ives, J.D. and Barry, R.G. (eds) Arctic and Alpine Environments. Methuen, London, pp. 571–603. McNulty, A.K. and Cummins, W.R. (1987) The relationship between respiration and temperature in leaves of the arctic plant Saxifraga cernua. Plant, Cell & Environment 10, 319–325. Miteva, V.I. and Brenchley, J.E. (2005) Detection and isolation of ultrasmall microorganisms from a 120 000-year-old Greenland glacier ice core. Applied Environmental Microbiology 71, 7806–7818. Mosier, A.C., Murray, A.E. and Fritsen, C.H. (2006) Microbiota within the perennial ice cover of Lake Vida, Antarctica. FEMS Microbiological Ecology 59, 274–288. Murray, D.F. (1992) Vascular plant diversity in Alaskan arctic tundra. Northwest Environment 8, 29–52. Murray, D.F. (1995) Causes of arctic plant diversity: origin and evolution. In: Chapin, F.S. III and Körner, C. (eds) Arctic and Alpine Biodiversity. Patterns, Causes and Ecosystem Consequences. Springer Verlag, New York, New York. pp, 21–32.

162

J. Svoboda

Porsild, A.E. (1964) Illustrated Flora of the Canadian Arctic Archipelago. Bulletin No. 146, Biological Series No. 50. National Museum of Canada, Ottawa, Ontario, Canada. Porsild, A.E. and Cody, W.J. (1980) Vascular Plants of Continental Northwest Territories, Canada. National Museum of Natural Sciences, National Museums of Canada, Ottawa, Ontario, Canada. Price, P.B. (2007) Microbial life in glacial ice and implications for a cold origin of life. FEMS Microbiological Ecology 59, 217–231. Romer, M.J., Cummins, W.R. and Svoboda, J. (1981) Is there a potential for Canadian northern agriculture? (A justification for research on northern native plants as potential foodcrops). In: Freeman, M. (ed.) Proceedings of the First International Symposium on Renewable Resources and the Economy of the North, Banff, Alberta. Association of Canadian Universities for Northern Studies and Canada Man and the Biosphere (MAB) Program, Ottawa, Ontario, Canada, pp. 161–165. Saville, D.B.O. (1972) Arctic Adaptations in Plants. Monograph No. 6. Canada Department of Agriculture, Ottawa, Ontario, Canada. Sjörs, H. (1963) Amphi-Atlantic zonation, Nemoral to Arctic. In: Löve, A. and Löve, D. (eds) North Atlantic Biota and Their History. MacMillan, New York, New York, pp. 107–125. Soper, J.H. and Powell, J.M. (1985) Botanical Studies in the Lake Hazen Region, Northern Ellesmere Island, N.W.T. Canada. Publications in Natural Sciences No 5. National Museums of Canada, National Museum of Natural Sciences, Ottawa, Ontario, Canada. Steven, B., Briggs, G., McKay, C.P., Pollard, W.H., Geer, C.W. and Whyte, L.G. (2006) Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods. FEMS Microbiological Ecology 59, 513–523. Svoboda, J. (1977) Ecology and primary production of raised beach communities, Truelove Lowland. In: Bliss, L.C. (ed.) Truelove Lowland, Devon Island, Canada: A High Arctic Ecosystem. University of Alberta Press, Edmonton, Alberta, Canada, pp. 185–216. Svoboda, J. (1978) Plants of the High Arctic: how do they manage? In: Milligan, S. and Kupsch, W.O. (eds) Living Explorers of the Canadian Arctic. Outcrop, The Northern Publishers, Yellowknife, Northwest Territories, Canada, pp. 194–225. Svoboda, J. (1989) The reality of the phytosphere and (ultimate) values involved. Ultimate Reality and Meaning 12, 104–112. Svoboda, J. (2006) Life as an unfolding biocosmos. In: Seckbach, J. (ed.) Life as We Know It. Springer, Dordrecht, The Netherlands, pp. 431–444. Svoboda, J. and Freedman, B. (eds) (1994) Ecology of a Polar Oasis Alexandra Fiord, Ellesmere Island, Canada. Captus University Publications, Toronto, Ontario, Canada. Svoboda, J. and Henry, H.G.R. (1987) Succession in marginal arctic environments. Arctic and Alpine Research 19, 373–384. Svoboda, J., Henry, G. and Lévesque, E. (1995) Long- and short-term plant community dynamics of the Nearctic tundra. Presented at the BES Symposium: The Ecology of Arctic Environments, University of Aberdeen, UK, 27–30 March. Thurston, T., Raillard, M. and Svoboda, J. (1989) New fossil forest site at Irene Bay, Ellesmere Island, N.W.T. Canada. Musk-ox 37, 98–102. Tudge, C. (1996) The Time Before History. Scribner, New York, New York. Tudge, C. (2000) The Variety of Life. A Survey and Celebration of All Creatures That Have Ever Lived. Oxford University Press, Oxford, UK. Velasco, J. and Vessels, J. (2008) China (a map). Supplement to National Geographic 213(3), Special Issue. Walker, M.D. (1995) Patterns and causes of Arctic plant community diversity. In: Chapin, F.S. III and Körner, C. (eds) Arctic and Alpine Biodiversity. Patterns, Causes and Ecosystem Consequences. Springer Verlag, New York, New York, pp. 3–20. Wilson, E.O. (1992) The Diversity of Life. Harvard University Press, Cambridge, Massachusetts.

16

Ice Encasement Damage on Grass Crops and Alpine Plants in Iceland – Impact of Climate Change B.E. Gudleifsson

Introduction

Winter Damage in Iceland

Many kinds of stresses attack plants during winter (Griffith et al., 2001). The stresses cause strains in plants which subsequently can cause injury or death (Levitt, 1980). Frost stress is the most studied winter stress to plants and much knowledge on frost damage has been collected. Another type of winter stress, ice encasement, appears occasionally or frequently in some areas of the world. Ice encasement has especially damaged grasses and winter cereals or other perennial herbage plants in northern Scandinavia, Iceland, eastern Canada and northern Japan. The process of ice encasement damage in plants is not as thoroughly studied as the process of frost damage. Ice encasement damage is related to the absence of oxygen and the exact cause of death has mainly been studied on winter cereals in Canada (Andrews and Pomeroy, 1991). In the present chapter, information is presented on the impact of winter damage, especially ice encasement, on grass survival and agriculture in Iceland in the past, present and future. The impact of winter climate and the principal cause of plant death in ice encasement are explained and the impact of predicted climate change is discussed.

In Iceland, freezing damage is mainly harmful to trees because buds, the most sensitive organs to freezing, experience the ambient air temperature directly. The surviving organs of grasses and cereals, on the other hand, are located close to the soil surface where the temperature usually fluctuates around zero (Baadshaug, 1973) due to snow and old straw insulating the grass buds from extremely low air temperatures (Andrews and Pomeroy, 1977). The impact of different types of winter damage on hayfields and forest trees in Iceland has been evaluated by the author (Guðleifsson and Örvar, 2000). This survey confirms the dominance of frost damage of trees and ice damage of grasses (Table 16.1). Tolerance to these two stresses should be taken into account when species and cultivars are chosen for cultivation. This is particularly important in forestry as almost all plant species used in afforestation in Iceland are imported and therefore not adapted to the northern oceanic climate of the island. Also, the trees planted today will grow for 70–100 years and therefore will experience the climate of the future, which will surely be subject to climate change.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

163

164

B.E. Gudleifsson

Table 16.1. Relative impact of different types of winter damage in hayfields and forests in Iceland. Evaluation is based on information, studies and written records from 1950 to 2000. (From Guðleifsson and Örvar, 2000.) Stress Frost heaving Drought Frost Starvation

Strain

Drought Drought Freezing Energy shortage Flooding Suffocation Ice encasement Suffocation Snow moulds Rotting Ice-nucleating Freezing bacteria

Hayfields Forests (%) (%) 1 2 5 +

8 5 85 ?

+ 90 2 ?

? − ? 2

Impact of Ice Damage on Agriculture in Iceland The agriculture in Iceland is very single-tracked. Milk and meat production is based on cattle and sheep, which need to be fed indoors for 7–8 months a year. In earlier times, the hay production relied completely on uncultivated pastures and periodically years occurred when the yield was severely reduced because of climatic extremes. This had catastrophic consequences as the herd had to be decreased to match the food supply and, with fewer livestock, the human population was sometimes reduced as a consequence. Thus, from the 17th to the 19th century, annals describe between 25% and 37% of the years as ‘grassless years’ (Friðriksson, 1954). In the 20th century hay was mainly harvested from cultivated permanent hayfields. The figures on hay yield and hayfield area are rather unreliable regarding average hay yield. No figures are available on winter damage, but subjective evaluations could be found from descriptions of the agricultural situation each year. The years could only be classified as years with no damage, years with little damage and years with great damage. In the 20th century, there were seventeen years with great winter damage and twenty-one with little damage, i.e. 38% of the years. All these rather unreliable data have been used to produce Fig. 16.1, where the

annual yields for 1900–2006 for the whole country are presented. The years with great damage are marked (grey columns) and annual mean temperature in Stykkishólmur in western Iceland is inserted. In most cases, years with great damage have decreased mean yield and, during these years, the annual average temperature was fairly low.

Climate and Ice Damage The yield (Fig. 16.1) is not a very exact indication of the intensity of winter damage in hayfields in Iceland. This is partly because data were collected for purposes other than yield evaluation. There are many other factors besides winter climate and winter damage that influence hay yield, summer climate being one of these. The information on winter damage indicates that the distribution of such damage is highly localized, and mean yield for the whole country may therefore not be a very revealing figure. As an example, when damage is intense in northern Iceland it may be absent in the rest of the country and vice versa. Also it should be remembered that climatic factors other than temperature are involved in winter damage in the field. In spite of these weaknesses, the correlations between monthly temperature measurements and hay yield in 1900–2006 were calculated, indicating that March and April temperatures were the most closely related to hay yield (r=0.40, P<0.001). The temperatures in different seasons showed the following correlations to hay yield: Annual temperature (September–August) Summer temperature (June–August) Autumn temperature (September–November) Winter temperature (December–May)

0.49 (P<0.001) 0.28 (P<0.001) −0.15 (NS) 0.57 (P<0.001)

It is notable that winter temperatures have a greater impact on yield than summer temperatures. This is unusual, because for most crops in other countries the summer temperature in the growing season is far more important for growth than the winter temperature. Winter

Ice Encasement Damage

165

55

8 7

45 Hay yield (t /ha)

40

6

35 5

30 25

4

20 3

15 10

2

Average annual temperature (°C)

50

5 2000

1990

1980

1970

1960

1950

1940

1930

1920

1910

1 1900

0

Year Fig. 16.1. Annual hay yield in Iceland and mean annual temperature in Stykkishólmur, 1900–2006. Mean hay yield was 4.1 t/ha, average annual temperature was 3.7°C. Grey columns indicate years with great and extensive winter damage. The available statistical information on hay yield was collected by advisors in order to secure sufficient feed for the animals through the following winter. The hayfield area in the country was registered annually until 1988, but not thereafter; it was assumed that, from 1988, the hayfield area decreased by 2.6% annually related to the downward trend in agriculture in Iceland. (Adapted from Guðleifsson, 2004.)

temperatures can explain about 30% of the variations in hay yield in Iceland in the last century, mostly because of winter kill of grasses. The impact of low winter temperatures on hay yield has also been explained by delayed and decreased grass growth because of the cold soil and late start of growth (Bergthórsson et al., 1987). Survival of plants is much more likely to influence the yield drastically. To provide more reliable data on winter damage, it was evaluated in eight counties in northern Iceland in 1962–1969 (Guðleifsson, 1975). This should give more accurate information on winter damage than hay yield. These data on damage were related to the traditionally measured climatic parameters during winter period (October–May). The correlations between winter damage and climatic parameters were: Temperature Precipitation Snow depth

−0.34 (P<0.01) 0.47 (P<0.001) 0.60 (P<0.001)

These figures indicate that damage was most intensive at low temperatures, high precipitation

and high snow depth. These figures are surely related to better conditions for formation of ice cover on the ground as the snow gives material to ice formations in thaw periods. Multiple regression analysis of winter damage and traditionally measured climatic parameters gave a satisfactory relationship (R=0.60, P<0.001) but using calculated parameters such as the amount of snow, precipitation during winter thaws, intensity and number of winter thaws and duration of ice cover, the relationship increased (R=0.70, P<0.001). About 50% of the variation in winter damage is thus explained, confirming that ice encasement is the main cause of winter damage in Iceland (Guðleifsson, 1975).

Ice and Frost Tolerance of Herbage Species Hardening of plants in the autumn results in increased tolerance to all types of winter damage. However, freezing damage and ice damage are two quite different processes and

166

B.E. Gudleifsson

Table 16.2. Laboratory measurements of frost and ice tolerance of four naturally hardened plant species. Frost tolerance was tested by dropping temperature by 1°C/h and ice tolerance by keeping the plants ice encased at −2°C for different lengths of time.

Species

Cultivar

Land of origin

Alpine arctic cuckweed, Omalotheca supina (L.) Cand. Winter wheat, Triticum aestivum L. Red clover, Trifolium pratense L. Timothy grass, Phleum pratense L.

Wild type

Iceland

−19

7

Norstar Bjursele Korpa

Canada Sweden Iceland

−18 −15 −13

14 20 33

Metabolism Under Ice Plants respire during winter, using carbohydrate reserves as an energy source. Plants respire, even at subzero temperatures, and use the energy supply that was collected during hardening (Fig. 16.2). The respiration is slow at these low temperatures as limited oxygen is available, but it is finally used up and the plants gradually turn from aerobic to anaerobic respiration.

100 80 % O2

inheritance of freezing tolerance and ice tolerance are different (Gudleifsson and Larsen, 1993). Plant species therefore demonstrate very different levels of freezing and ice tolerance. This has been verified in laboratory experiments where tolerant species of three crops have been compared – winter cereals, legumes and grasses – as well as one alpine plant species growing in snow patches (Table 16.2). These results have established that grasses are relatively ice-tolerant but not frosttolerant, while this is reversed in winter cereals. The alpine plant holds the highest frost tolerance and lowest ice tolerance. In this laboratory test, the ice tolerance ranged from 7 to 33 days. Under natural conditions, plants usually survive much longer under ice cover, probably because the stress in the field is not as intensive as in an ice cube in a freezer. In the freezer plants are completely encased in compact ice, whereas in the field the ice and soil contain more or less air spaces. The rule of thumb for Icelandic farmers is that grasses tolerate up to 3 months of ice cover, legumes about 3–4 weeks, winter cereals 1–2 weeks and the natural ice tolerance of alpine plants is not known.

Frost tolerance, Ice tolerance, LT50 (°C) LD50 (days)

60 24°C

40

4°C

20 0

0

30

60 90 Minutes in ice

−2°C 120

150

Fig. 16.2. Respiration of timothy grass cells (cultivar Vega, 0.2 g fresh weight in 4 ml of water) acclimated for 4 days at 4°C. The signal was measured with a potentiometer recorder using an oxygen electrode and the results transformed into percentage O2 saturation.

During anaerobic respiration under ice, herbage grasses and cereals accumulate metabolites which they cannot release as they are incorporated into compact and more or less impermeable ice. Andrews and Pomeroy (1979) have studied the accumulation of metabolites in winter cereals under ice. Winter wheat accumulates mainly CO2, ethanol and lactate during ice encasement (Pomeroy and Andrews, 1978). The metabolism of ice-encased grasses is similar, but a number of other metabolites have been detected (Gudleifsson, 1994, 1997b). In three laboratory experiments where timothy plants were ice-encased for 9 and 38 days, ethanol, lactate and citrate were accumulated to highest concentrations whereas CO2 and malate were collected to fairly low concentrations (Fig. 16.3). In experiments where timothy plants were encased for different lengths of time, similar results were obtained except that the concentration of malate was higher than that of lactate (Fig. 16.4).

Ice Encasement Damage

167

60

60 Ethanol

50

50

40

40

30

Lactate

30

20

Citrate

20

10

CO2 Malate Butyrate Propionate

10

0

5

15

25 Days in ice

35

Survival (%)

Metabolite (mg/g dry weight)

Survival

0

45

Fig. 16.3. Accumulation of metabolites by timothy seedlings in three laboratory experiments where plants were encased in ice at −2°C for 9 and 38 days. Metabolites were measured by HPLC with an ion exclusion column. During melting CO2 was retained in meltwater by the addition of NaOH to pH 4.8–5.2 and analysed with a carbon dioxide electrode. (Adapted from Gudleifsson, 1994, 1997b.)

250

100 Survival

200

80

150

60 Malate

100

40

Citrate Lactate

50

Survival (%)

Metabolite (mg/g dry weight)

Ethanol

20

CO2 Propionate 0

0

10

20

30 Days in ice

40

50

0

Fig. 16.4. Survival and metabolite accumulation of timothy seedlings during ice encasement. Plants were encased for 0, 6, 20, 33 and 40 days. Metabolites were measured using HPLC and CO2 using a carbon dioxide electrode. (Adapted from Gudleifsson, 1994, 1997b.)

Comparisons of metabolite concentrations accumulated by winter cereals and grasses indicate that the more tolerant grasses accumulate metabolites to higher concentrations. Thus the higher tolerance of grasses is not related to slower metabolism but rather to higher tolerance of cell organelles. Plants of the alpine species creeping sibbaldia (Sibbaldia procumbens L.) were ice-encased

and the accumulation of metabolites (not CO2) analysed after 49 days of ice encasement. At that time the survival was only 2%. Ethanol, malate and citrate were not detected, whereas lactate and oxalate were accumulated to 70 and 80 mg/g dry weight respectively, much higher concentrations than in grasses. This indicates a lower tolerance and a quite different metabolism under ice in alpine plants than in grasses.

168

B.E. Gudleifsson

Causes of Plant Death in Ice Encasement Andrews and Pomeroy (1979, 1991) have established that membrane damage is the main cause of cell death in winter wheat under ice as a consequence of metabolite accumulation and that CO2 plays a more significant role in this damage than ethanol and lactate. The death caused by accumulation of metabolites happens during the ice encasement. It has also been proposed that plants killed by ice encasement are not killed until after the disappearance of the ice. This conclusion is supported by observations that plants escaping from lethal ice look green and healthy at first but wilt and die within a couple of days (Gudleifsson, 1986; Tanino and McKersie, 1985) and unlike with freezing stress, all tissues within the crown are killed during anaerobic stress (Tanino and McKersie, 1985). The transition from anoxia to air is a great shock, and reactive oxygen species (ROS) might develop which in turn can kill the cells (McKersie and Leshem, 1994). In this case, antioxidants should help in scavenging the ROS and could reduce the damage. Acclimated timothy cells were kept in ice at −2°C for 5 days. In one treatment, ascorbate, an antioxidant, was added to 5 mM concentration before icing. Survival was measured by fluorescein diacetate staining and results compared with survival in water. Survival in water was 100%, survival in ice 50%, and when ascorbate was added into the water before icing survival increased to 81%, indicating that ROS might be participating in killing plants after ice encasement. ROS are supposed to damage cell membranes (McKersie and Leshem, 1994) as do the accumulated metabolites. Thus toxicity of metabolites and formation of ROS during ice melting might both participate in cell membrane damage in ice encasement.

Ice Encasement and Phytotoxins When long-lasting ice cover is melting from hayfields in late winter or spring in Iceland, a strong sour odour has been detected, probably developing from metabolites evaporating from the plants (Guðleifsson, 1977). Meltwater from

long-lasting ice cover on hayfields was analysed in March 1993 (Gudleifsson, 1994) and the results are summarized in Table 16.3. The distinct odour is probably mainly related to metabolites such as butyrate and acetate, but the main anaerobic plant products are lactate, ethanol and malate. Propionate, tartarate and butyrate are collected later on in the ice cover period, probably partly as a result of microbe activity (Gudleifsson, 1997a). Several species of bacteria have been isolated from ice-encased plants under field and laboratory conditions (Gudleifsson, 1994, 1997a). Severely damaged hayfields in Iceland are usually recultivated and new grass plants are sown. If the damage is only in patches, direct drilling has been used to plant grasses into the patches. In many cases, the seedlings in recultivated fields do not establish successfully (Gudleifsson, 1986) and seedlings planted by direct drilling after ice encasement damage turn yellow and do not thrive (Guðleifsson, 1999). This might be explained by toxic substances accumulating in the soil as a consequence of the anaerobic respiration under ice. In a laboratory experiment (Brandsæter et al., 2005), orchardgrass (Dactylis glomerata L.) and colonial bentgrass (Agrostis capillaris L.) plants grown in soil were killed by snow moulds, freezing (0 to −20°C) or ice encasement (5–15 weeks). The soil water from these three treatments was tested for phytotoxins using a bioassay on filter paper and soil cores with radish plants (Raphanus sativus L.). Only ice encasement caused severe phytotoxicity, and out of 19 analysed metabolites, the metabolites in Table 16.4 dominated. Table 16.3. HPLC analysis of meltwater from hayfields in Iceland on 8 March 1993. Concentration (mg/l) Butyrate Acetate Lactate Tartarate Ethanol Oxalate Malonate Citrate Malate

365 161 118 82 54 38 38 21 14

Ice Encasement Damage

169

Table 16.4. Relative radish root growth and metabolites dominating in soil water after three different winter damage conditions tested in the laboratory. (From Brandsæter et al., 2005.)

Relative root growth (%) Butyrate Acetate Succinate Malate Ethanol Citrate Lactate Malonate

Snow moulds

Freezing

Ice encasement

104

93

76

Metabolite concentration (mg/l) 14 14 5 10 15 41 30 65 0 0 6 40 16 10 1 1

Component analysis indicated that butyrate was the major component in reducing root length of radish plants after ice encasement, while succinate, malate and citrate slightly reduced root length after freezing (Brandsæter et al., 2005). As can be seen from Figs 16.3 and 16.4, these three metabolites also accumulate under ice cover and will further add to the phytotoxic effects of ice encasement. As these phytotoxic substances are volatile, they are expected to evaporate from the soil in the first summer after winter kill.

Impact of Climate Change on Winter Damage in Iceland In eastern Canada, predicted climate change after 2040 may increase the risk of winter injury to perennial crops because of less favourable hardening conditions during the autumn and reduced protective snow cover during winter, which will increase exposure of plants to killing frosts, soil heaving and ice encasement (Bélanger et al., 2002). It is notable in Fig. 16.1 that the last seven years, 2000–2006, in Iceland have been free of winter damage and annual mean temperatures are fairly high, possibly as a result of the already coming climate change. The expected climate change in the Arctic includes increased temperature and precipitation, and the temperature increase is expected to be higher in winter than summer (ACIA, 2004). The Arctic

164 46 29 19 18 17 12 7

Climate Impact Assessment report presents scenarios to year 2100. If we look forward only to the year 2050, the summer and winter temperatures in Iceland might increase by 1.5°C and 3°C, respectively, and the winter precipitation might increase by 15%. These are drastic changes which surely will have a positive impact on the (until now) marginal Icelandic agriculture; the yield will increase, winter damage caused by ice encasement will decrease or disappear and new crops will be taken into use (Guðleifsson, 2004). On the other hand, the higher winter temperatures will result in increased probability of new winter stresses at higher altitudes and latitudes (Fig. 16.5). Alpine plants, which have been protected from frost and ice by stable snow cover during winter, will probably experience frost and ice stress for which they have not been selected (Guðleifsson, 2005). The danger of ice damage to alpine plants is supported by the relatively low ice tolerance of alpine arctic cuckweed presented in Table 16.2. The metabolism of these plant species might be different from the ice-tolerant grasses as exemplified by creeping sibbaldia in this text.

Conclusions Ice encasement often causes damage to herbage plants in some parts of the northern hemisphere. In the past, it has frequently decreased hayfield yield drastically in Iceland. Ice cover is

B.E. Gudleifsson

FROST ICE

ICE

FROST

Year 2000

FROST

ICE

FROST

ICE

FROST

ICE

FUNGI

FUNGI

170

Year 2050

Fig. 16.5. Present and future locations of winter stresses as a result of expected climate change in Iceland.

formed in thawing periods during winter. Grasses are rather ice-tolerant compared with winter cereals and alpine plants and can survive many weeks of ice encasement. Under the ice, plants turn to anaerobic respiration and accumulate metabolites, mainly ethanol, malate, lactate, citrate and CO2. Some of these metabolites are harmful to the plant cell, especially CO2, and can be accumulated to toxic levels. The ice-tolerant grasses accumulate metabolites to higher levels than winter cereals and seem to tolerate higher concentrations. In addition, when ROS are scavenged by antioxidants such as ascorbic acid, survival increases; thus ROS may be produced when plants return to ice after long-lasting and lethal ice cover.

Both accumulated metabolites and ROS seem to participate in ice damage and the cell membrane is probably the site of damage. Metabolites accumulating to high concentrations under ice, such as butyrate, seem to leak into the soil in spring and can have phytotoxic effects on new seedlings planted into the damaged areas. Expected climate change may decrease or eliminate the occurrence of ice damage in areas where it has dominated, but increase it in other areas where plants, possibly with low ice tolerance, are growing. Thus, cultivated crops in Iceland might become free of ice damage and alpine plants at higher altitudes, which are not ice-tolerant, might experience increased ice stress.

Ice Encasement Damage

171

References ACIA (2004) Impacts of a Warming Arctic – Synthesis Report of the Arctic Climate Impact Assessment. Cambridge University Press, Cambridge, UK, 140 p. Andrews, C.J. and Pomeroy, M.K. (1977) Changes in survival of winter cereals due to ice cover and other simulated winter conditions. Canadian Journal of Plant Science 57, 1141–1149. Andrews, C.J. and Pomeroy, M.K. (1979) Toxicity of anaerobic metabolites accumulating in winter wheat seedlings during ice encasement. Plant Physiology 64, 120–125. Andrews, C.J. and Pomeroy, M.K. (1991) Low temperature anaerobiosis in ice encasement damage to winter cereals. In: Jackson, M.B., Davies, D.D. and Lambers, H. (eds) Plant Life Under Oxygen Deprivation. SPB Academic Publishing BV, The Hague, The Netherlands, pp. 85–99. Baadshaug, O.H. (1973) Effects of soil type and soil compaction on the wintering of three grass species under different wintering conditions. Acta Agriculturae Scandinavica 23, 77–86. Bélanger, G., Rochette, P., Casonguay, Y., Bootsma, A., Mongrain, D. and Ryan, D.A.J. (2002) Climate change and winter survival of perennial forage crops in eastern Canada. Agronomy Journal 94, 1120–1130. Bergthórsson, P., Bjornsson, H., Dyrmundsson, Ó., Gudmundsson, B., Helgadottir, Á. and Jónmundsson, J. (1987) The effect of climatic variations on agriculture in Iceland. In: Parry, M.L., Carter, T.R. and Konjin, N.T. (eds) The Impact of Climatic Impacts on Agriculture. Vol. I. Assessment in Cool Temperate and Cold Regions. Reidel, Dordrecht, The Netherlands, pp. 383–509. Brandsæter, L.O., Haugland, E., Helgheim, M., Gudleifsson, B.E. and Tronsmo, A.M. (2005) Identification of phytotoxic substances in soil following winter injury of grasses as estimated by a bioassay. Canadian Journal of Plant Science 85, 115–123. Friðriksson, S. (1954) Rannsóknir á kali túna 1951–1952 (Winter Injury of Plants in Icelandic Hayfields). Department of Agriculture Reports, Series B, No. 7. University Research Institute, Reykjavik, 72 p. Griffith, M., Gudleifsson, B.E. and Fukuta, N. (2001) Abiotic stresses in overwintering crops. In: Iriki, N., Gaudet, D.A., Tronsmo, A.M., Matsumoto, N., Yoshida, M. and Nishimune, A. (eds) Low Temperature Plant–Microbe Interactions Under Snow. Hokkaido National Agricultural Experiment Station, Hokkaido, Japan, pp. 101–114. Gudleifsson, B.E. (1986) Ice encasement damages on grasses and winter cereals. In: Lantbruksväxternas Övervintring. NJF Seminar No. 84. Lantbrukets forskningscentral, Jockis, Finland, pp. 59–65. Gudleifsson, B.E. (1994) Metabolic accumulation during ice encasement of timothy grass (Phleum pratense L.). Proceedings of the Royal Society of Edinburgh 103B, 373–380. Gudleifsson, B.E. (1997a) Microbes active under snow and ice in hayfields in Iceland. In: Proceedings of the International Workshop on Plant–Microbe Interaction at Low Temperature under Snow. Hokkaido National Agricultural Experiment Station, JISTEC, Sapporo, Japan, pp. 109–118. Gudleifsson, B.E. (1997b) Survival and metabolite accumulation by seedlings and mature plants of timothy grass during ice encasement. Annals of Botany 79, Suppl. A, 93–96. Gudleifsson, B.E. (2005) Winter stresses to crops and native plants during climate change. NJF Seminar No. 380, Adaptation of Crops and Cropping Systems to Climate Change, Odense, Denmark. NJF Report 1, No. 3, 39. Gudleifsson, B.E. and Larsen, A. (1993) Ice encasement as a component of winter kill in herbage plants. In: Li, P.H. and Christersson, L. (eds) Advances in Plant Cold Hardiness. CRC Press, Boca Raton, Florida, pp. 229–249. Guðleifsson, B.E. (1975) Um kal og kalskemmdir IV. Samband veðurfars og kalskemmda (On winter damage. Relationhip between weather and damages). Ársrit Ræktunarfélags Norðurlands 72, 45–64. Guðleifsson, B.E. (1977) Svellkal og kallykt (Ice damage and ice damage odour). Ársrit Ræktunarfélags Norðurlands 74, 70–76. Guðleifsson, B.E. (1986) Tilraunir með endurræktun kalins lands á Norðurlandi (Experiments in northern Iceland on recultivation of winter damaged hayfields). Fjölrit BRT 13, 16 pp. Guðleifsson, B.E. (1999) Ísáning – sáð í gróinn svörð (Direct drilling into grass sward). Ráðunautafundur 1999, 90–99. Guðleifsson, B.E. (2004) Heyfengur og kalskemmdir í túnum á Íslandi á síðustu öld (Hay yield and winter damage in hayfields in Iceland on the last century). Freyr 100, issue 5, 29–32. Guðleifsson, B.E. (2005) Áhrif væntanlegra loftslagsbreytinga á landbúnað á Íslandi (Impact of expected climate change on agriculture in Iceland). Fræðaþing landbúnaðarins 2004, 17–25.

172

B.E. Gudleifsson

Guðleifsson, B.E. and Örvar, B. (2000) Kalskemmdir í túnum á síðustu öld – og framtíðarhorfur (Winter damage in hayfields in last century – and predictions for the future). Ráðunautafundur 2000, 323–329. Levitt, J. (1980) Responses of Plants to Environmental Stresses. Vol. I. Chilling, Freezing and High Temperature Stresses. Academic Press, New York, New York. McKersie, B.D. and Leshem, Y.Y. (1994) Stress and Stress Coping in Cultivated Plants. Kluwer Academic Publishers, Dordrecht, The Netherlands, 256 p. Pomeroy, M.K. and Andrews, C.J. (1978) Metabolic and ultrastructural changes in winter wheat during ice encasement under field conditions. Plant Physiology 61, 806–811. Tanino, K.K. and McKersie, B.D. (1985) Injury within the crown of winter wheat seedlings after freezing and icing stress. Canadian Journal of Botany 63, 432–436.

17

Impact of Simulated Acid Snow Stress on Leaves of Cold-acclimated Winter Wheat K. Arakawa, H. Inada and S. Fujikawa

Introduction Acid precipitation is a global environmental problem that causes damage to ecosystems directly or indirectly. Acid precipitation originates from air pollutants such as oxides of sulfur and nitrogen, which are produced by the consumption of fossil fuels (Holloway et al., 2002; Richter et al., 2005; Ohara et al., 2007). Acid precipitation means acidification of rain, fog, mist, snow, etc. to less than pH 5.6, which is the value of saturation of CO2 in water. It is known that acid precipitation of rain causes damage to historical buildings, ecosystems, human health and agriculture, especially in Europe, North America and Eastern Asia, in which industries are developing and populations are large (Evans, 1989; Likens and Bormann, 1995). In cold climate regions, acid precipitation appears as acid snow in winter. The pH of snow meltwater has become more acidic than that of pure water (pH 5.6), indicating the precipitation of acid snow during winter over the past decade (Fushimi et al., 2001; Noguchi et al., 2001). In Hokkaido, northern island of Japan, the area of acid precipitation has been spreading and the decrease in pH has been progressing, especially in the coastal area along the Sea of Japan (Noguchi et al., 2001; Fujita et al., 2003; Han et al., 2006). Localization of acid pollutants in snow changes drastically as the air temperature shifts.

Snowflakes with acid pollutants thaw as soon as they reach the ground surface and refreeze as the temperature gradually declines. When acid snow stays in the snow layer, snow and ice crystals with acid pollutants near the ground recrystallize due to heat transmission from the ground and change into a crust of frozen snow. In these processes, acid pollutants in the snow are preferentially rearranged on the surface areas of ice crystals. When snow and ice crystals with acid pollutants melt as the temperature rises, acid pollutants on the surfaces of the ice crystals are preferentially dissolved in meltwater in the beginning and thus the pH of the meltwater decreases significantly for a certain period of time. At the early and/or late stages of the snowmelt season, meltwater temporarily dissolves acid pollutants of the snow layer at twoto fivefold higher concentrations than the average concentrations in snow layers, and the acidic meltwater temporarily decreases the pH of water in the soil and the water of rivers and lakes, a phenomenon known as acid shock (Johannessen and Henriksen, 1978; Fushimi et al., 2001). Therefore, acid snow may change the growth conditions of wintering plants and other organisms living in the soil and water in winter and early spring and influence their survival and growth. Although results of studies on the impact of acid rain on plants have been reported, there have been no studies on the impact of acid snow stress on wintering plants.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

173

174

K. Arakawa et al.

In midwinter, wintering plants in cold climate regions with snowfall are subjected to longterm freezing under the snow layer. The duration and temperature of the freezing conditions are limiting factors of freezing injury of plants (Sakai and Larcher, 1987). As indicated by the results of freezing tests of many plants, lowering of freezing temperature promotes the injury of plant cells since irreversible ultrastructural changes of cell membranes, such as the plasma membrane, are caused by severe dehydration and deformation due to extracellular freezing of the cells (Steponkus, 1984; Fujikawa and Miura, 1986; Fujikawa et al., 1999). In wheat seedlings, survival rates of leaves decreased gradually as freezing temperature declined (Zhou et al., 1994; Inada et al., 2006). Long-term freezing also promotes injury. The survival rate of cold-acclimated (CA) wheat seedlings decreased gradually after exposure to a temperature of −6°C and declined to about 56% after 3 weeks (Pomeroy et al., 1985). Also, the survival rate of CA wheat leaf segments decreased gradually after exposure to a temperature of −4°C and declined to about 60% after 4 weeks (Inada et al., 2006). The survival rate of CA Arabidopsis leaves was markedly decreased after exposure to a temperature of −2 or −4°C and declined to less than 50% after 3 days or 1 day, respectively (Nagao et al., 2008). Long-term freezing at a high subzero temperature resulted in additional ultrastructural changes in plasma membranes of CA Arabidopsis leaves that are distinct from the ultrastructural changes in plasma membranes caused by interbilayer events during short-term freezing (Nagao et al., 2008). Since the additional membrane damage of Arabidopsis leaves caused by long-term freezing at a high subzero temperature was partially induced by salt treatment at a non-freezing temperature, this ultrastructural change in the plasma membrane may be caused by exposure of plant cells to the condensed electrolyte solution that remains unfrozen at a high subzero temperature. Therefore, wintering plants may be subjected to long-term freezing at a high subzero temperature and strong acid stress due to condensation of acid pollutants under the acid snow layer in winter. Additionally, light may be a potent stress factor, especially for wintering plants after

severe freeze–thawing at a low subzero temperature, although the protective mechanism of photosynthetic machinery is developed in the process of cold acclimation (Huner et al., 1998; Asada, 1999; Ensminger et al., 2006). Therefore, it is reasonable to assume that light irradiation after acid snow stress may be stressful for wintering plants. However, the impact of acid snow stress and subsequent light irradiation on wintering plants had not been studied before. We therefore studied this issue by carrying out simulation experiments on acid snow stress by means of freeze–thawing with sulfuric acid, a typical air pollutant. In the present chapter, we summarize our recent results regarding the impact of acid snow stress on winter wheat.

Cell Damage Caused by Simulated Acid Snow Stress First, we examined the impact of simulated acid snow (SAS) stress on the leaf tissue of CA wheat by the use of a freezing test, which was simply modified by the addition of sulfuric acid solution instead of water to the assay system, and amino acid leakage measurement for the estimation of survival rate. When leaf segments were frozen with sulfuric acid solution (pH 3.0 or 4.0) and cooled slowly to −8°C as SAS stress treatment, survival rate of the samples after thawing was hardly decreased (Inada et al., 2006). On the other hand, the survival rate of wheat leaf segments was significantly decreased by SAS stress with about 3 mM sulfuric acid (pH 2.0) compared with the survival rate of samples that were treated with water (pH 5.6) (Fig. 17.1A). Supercooling to −8 or −12°C with sulfuric acid (pH 2.0) without ice-seeding hardly decreased the survival rate of samples (Fig. 17.1A). Therefore, the existence of sulfuric acid during freeze–thawing may be a predisposing factor for cell survival. Additionally, SAS stress with hydrochloric acid or nitric acid (pH 2.0) instead of 3 mM sulfuric acid (pH 2.0) also decreased the survival rate of leaf tissues to levels similar to those with sulfuric acid, although freeze– thawing with 3 mM sodium sulfate or potassium sulfate instead of 3 mM sulfuric acid (pH

Simulated Acid Snow Stress

2.0) did not decrease the survival rates (Inada et al., 2006). Therefore, extracellular freezinginduced condensation of protons (acidification), rather than sulfate anion, might be the main cause of the decrease in survival rate of wheat leaf tissues. We also examined the impact of prolonged SAS stress on wheat leaf tissue. When leaf segments were subjected to long-term SAS stress treatment with sulfuric acid solution (pH 2.0, 3.0 or 4.0) at −4°C for 7 days, survival rate of the samples after thawing decreased gradually as a function of the duration of SAS stress treatment. Long-term SAS stress treatment (pH 3.0 or 4.0) at −4°C for 7 days decreased survival rates by about 10% compared with those of samples that were treated with water (pH 5.6) (Inada et al., 2006), and long-term SAS stress treatment (pH 2.0) at −4°C for 7 days decreased survival rates by about 30% (Fig. 17.1B). Lowering the temperature of long-term SAS stress treatment also decreased the survival rate of wheat leaf tissues (Inada et al., 2006). However, long-term supercooling with sulfuric acid (pH 2.0) at −4°C for 7 days without iceseeding hardly decreased the survival rate of samples (Fig. 17.1B).

It has also been suggested that the photosynthetic apparatus in mature leaves was damaged by SAS stress. After SAS stress treatment (pH 2.0) of CA wheat seedlings at −4°C for 1 week, chlorophyll degradation was detected spectrophotometrically in mature wheat leaves, but not in young leaves (Inada et al., 2007). In the chlorophyll fraction of SAS-stressed mature leaves, the major peak of chlorophyll was shifted slightly from 663 to 665 nm and the peak height was lowered, and some small peaks at 606, 535 and 507 nm that are distinct from the absorption spectrum of chlorophyll in mature leaves treated with water (pH 5.6) were detected (Fig. 17.2). This spectral change in the chlorophyll fraction caused by SAS stress is very similar to the changes caused by simulated acid rain (SAR) treatments (Shan, 1998), suggesting accumulation of the degraded intermediate of chlorophyll, pheophytin, in SAS-stressed mature leaves. Additionally, an SAR study suggested that chlorophylls were sensitive to low pH and degraded by SAR treatment of leaves of lichen (Rao and LeBlanc, 1965). Chlorophyll degradation in SAS-stressed mature leaves caused a decrease in photosynthetic activities, such as maximal quantum yield of photosystem

(B) 100

100

80

80

Survival (%)

Survival (%)

(A)

175

60 40 20 0

60 40 20

4

−4 −8 −12 Temperature (°C)

0

0

3 Period (days)

7

Fig. 17.1. Impact of simulated acid snow (SAS) stress on the leaves of cold-acclimated winter wheat. Leaf segments of cold-acclimated winter wheat were examined by equilibrium freezing or supercooling (A) and long-term freezing or supercooling (B) in the presence of sulfuric acid solution (SAS stress) or water (control). After sulfuric acid solution of pH 2.0 ( , ) or pure water of pH 5.6 ( , ) was added to leaf samples, samples were frozen by ice-seeding ( , ) or maintained in a supercooling state without ice-seeding ( , ) and cooled to the desired temperature at a rate of 2.4°C/h. In the long-term SAS test (B), samples were kept at –4°C for the desired period. Survival rates of the leaf samples after thawing were estimated by measuring amino acid leakage. Data are means, with standard deviations represented by vertical bars, from 18–20 samples. (Adapted from Inada et al., 2006.)

176

K. Arakawa et al.

(A)

(B) 0.5

Absorbance

0.4 0.3 0.2 0.1 0 500

540

580 620 660 Wavelength (nm)

700

500

540

580 620 660 Wavelength (nm)

700

Fig. 17.2. Absorption spectra of chlorophyll extracts of mature and young leaves of seedlings subjected to simulated acid snow (SAS) stress. After wheat seedlings had been subjected to long-term freezing at −4°C for 7 days in the presence of sulfuric acid solution of pH 2.0 (SAS stress) or pure water of pH 5.6 (control), chlorophyll extracts were prepared from mature (A) and young (B) leaves of SAS-stressed ( ) and control ( ), wheat seedlings. The chlorophyll extracts were analysed by UV spectrophotometry. (Adapted from Inada et al., 2007.)

II complex, PSII Fv / Fm, in mature wheat leaves (Table 17.1). On the other hand, such damage was not detected in SAS-stressed young leaves, suggesting that young leaves have higher tolerance to SAS stress than do mature leaves. It is also thought that mature wheat leaves were subjected to oxidative stress during SASstress treatments because the level of lipid peroxidation (Table 17.1) and hydrogen peroxide contents (data not shown) were increased by SAS stress in mature leaves but not in young leaves.

Possible Cause of Injury by Simulated Acid Snow As mentioned above, the predisposing effects by SAS stress may be caused mainly by freeze-induced condensation of sulfuric acid in the extracellular space during freeze–thawing cycles. When a solution is frozen at a subzero temperature, the concentrations of solutes in the residual unfrozen solution of a freezing solution will depend on the subzero temperature in the process of freezing point depres-

Table 17.1. Changes in levels of maximal quantum yield of photosystem II complex, PSII Fv /Fm, and membrane lipid peroxidation (measured as thiobarbituric acid-reactive substances, TBARS) of cold-acclimated wheat leaves by long-term simulated acid snow stress (prolonged freezing with sulfuric acid solution of pH 2.0 or water of pH 5.6 at −4°C for 7 days). (Data from Inada et al., 2007.)

Mature

Young

Treatment

PSII Fv/Fm

TBARS (µmol/g dry weight)

Untreateda pH 5.6 pH 2.0 Untreateda pH 5.6 pH 2.0

0.79±0.02 0.66±0.06 0.49±0.08 0.80±0.01 0.68±0.03 0.57±0.05

0.28±0.03 1.07±0.27 1.28±0.03 0.32±0.03 1.31±0.18 1.33±0.20

Data are mean±standard deviation from three to nine independent plants. a Data before freezing.

Simulated Acid Snow Stress

sion (Mazur, 1966). Therefore, sulfuric acid should be condensed to the same concentration in the residual unfrozen solutions at given subzero temperatures in all SAS stress treatments, regardless of the initial pH and volume of the solution for SAS stress. According to this theory, sulfuric acid should be condensed to about 358 mM in a residual unfrozen solution at −2°C. When samples are frozen to −2°C with 3 mM of sulfuric acid solution (pH 2.0), the samples would be condensed more than 100-fold in the residual unfrozen solution. Since strong acidification of the residual unfrozen solution and freezing-induced dehydration of the cells would be promoted as freezing temperature declines, SAS-stressed plants might be damaged at subzero temperatures. Additionally, volumes of the residual unfrozen solutions at a subzero temperature would be dependent on the initial pH (concentration) of the sulfuric acid solution. In the present context, consequently, the more acidic the initial pH of acid snow becomes, the greater the volume of residual unfrozen solution at a subzero temperature would be. Thus, wheat leaves might be locally subjected to strong acidification caused by freezing-induced condensation of acid pollutants at subzero temperatures during SAS stress. They may be damaged by SAS stress as the pH of the snow is lowered and the amount of acid snow (or amount of acid meltwater) is increased. Furthermore, oxidative stress that occurred during SAS stress may increase the damage of mature wheat leaves. As already stated, chlorophyll degradation was detected during SAS stress (Fig. 17.2). Since it has been reported that chlorophyll was sensitive to a low pH condition (Rao and LeBlanc, 1965), it is assumed that cytoplasmic pH might be lowered during SAS stress treatment. Some studies have suggested that exogenous acidification caused acidification of the cytoplasm in plant cells (Gout et al., 1992; Velikova et al., 1999). Therefore, SAS stress may result in intracellular acidification of the cells in mature wheat leaves. Intracellular acidification may cause the disturbance of cell metabolism and cell injury through structural and functional changes in macromolecules such as cell membranes and enzymes in plant cells.

177

Promotion of Simulated Acid Snow Damage by Light In the early spring, wintering plants commence regrowth with increasing air temperature and light period. However, damage may be promoted because reactive oxygen species (ROS) will easily be produced in damaged mature leaves under a light condition when the wintering plants are injured under a snow layer (Mittler, 2002). It has been reported that the induction of ROS under the condition of environmental stresses inhibited growth of plants because ROS caused oxidative damage to vital molecules such as the photosynthetic apparatus (Apel and Hirt, 2004). Recent studies have suggested that ROS produced under the condition of temperature or salt stress initially attack the protein synthesis machinery and inhibit the repair of damaged components of PSII such as D1 protein (Murata et al., 2007). The efficiency of chlorophyll usage in photosynthesis might be reduced in SASdamaged mature leaves because of degradation of chlorophyll to pheophytin (Fig. 17.2, Table 17.1). Since some intermediates of chlorophyll catabolism potentially generate radicals (Mach et al., 2001; Tanaka et al., 2003), SAS-induced chlorophyll degradation in plastids might promote oxidative damage by light irradiation in the early stage of the regrowth period. When CA wheat seedlings were regrown with light irradiation after SAS treatment, Fv/Fm values of mature leaves were decreased more greatly than those of young leaves, with accompanying increases in the level of lipid peroxidation in mature leaves under a continuous light condition of the regrowth period (Fig. 17.3). This means the promotion of injury by photooxidative stress in mature leaves that have been severely damaged by SAS treatment (Inada et al., 2007).

Simulated Acid Snow Stress Tolerance of Wheat Leaves As shown in the table and figures above, young leaves were more tolerant to SAS stress and subsequent light irradiation than were mature leaves. This means that CA young leaves have

178

K. Arakawa et al.

(A)

(B) TBARS (µmol/g dry weight)

5 0.8

Fv /Fm

0.6 0.4 0.2 0

0

12

24 36 Period (h)

48

4 3 2 1 0

0

12

24 36 Period (h)

48

Fig. 17.3. Changes in maximal quantum yield of photosystem II complex (Fv/Fm) (A) and level of membrane lipid peroxidation (B) of wheat leaves during the regrowth period after long-term simulated acid snow (SAS) stress. After SAS stress wheat seedlings had been regrown at 10°C under a continuous light condition (approximately 60 µmol/m2/s) for 48 h, Fv/Fm values and levels of membrane lipid peroxidation of mature and young leaves were measured. 䊉, 䉱, mature leaves; 䊊, 䉭, , young leaves. 䊉, 䊊, samples that had been freeze–thawed with water of pH 5.6 (control) before regrowth; 䉱, 䉭, samples that had been freeze–thawed with sulfuric acid solution of pH 2.0 (SAS stress) before regrowth. (A) Fv /Fm values of wheat leaves were measured at room temperature in the dark with a portable fluorometer (MINI-PAM; Walz, Germany) after adaptation of seedlings in the dark at 10°C for 20 min. Data are means, with standard deviations represented by vertical bars, from five to nine independent plants. (B) Levels of membrane lipid peroxidation of wheat leaves were measured by the thiobarbituric acid test and represented as production of thiobarbituric acidreactive substances (TBARS), which are typical indicators of oxidative damage. Data are means, with standard deviations represented by vertical bars, from three or four independent plants. (Adapted from Inada et al., 2007.)

higher levels of acid, freezing and/or oxidative stress tolerance than do mature leaves. The molecular mechanism of acid tolerance in plants remains unclear although many studies have been done on the harmful effects of acid rain on plant cells (Evans, 1989; Stoyanova, 1998; Velikova et al., 1999; Yu et al., 2002; Gabara et al., 2003; Wyrwicka and Skłodowska, 2006). However, the mechanism may be related to the ability of intracellular pH stasis including the proton pumping activities and capacity of buffering action by some solutes in the cytoplasm and the stabilization of macromolecules against strong acid (Träuble and Eibl, 1974; Pfanz and Heber, 1986; Cevc, 1987; Gout et al., 1992; Martz et al., 2006). In our preliminary experiment, tolerance to SAS stress of wheat leaves was significantly increased by cold acclimation. Freezing tolerance increased during cold acclimation, while it seemed that acid tolerance estimated from the tolerance to cooling at 4°C

with sulfuric acid (pH 2.0) also increased to some extent during cold acclimation (data not shown). Further studies on CA-induced physiological factors in wheat seedlings are needed to clarify the molecular mechanism of acid tolerance. In addition, the freezing tolerance (LT50) of CA young leaves was higher than that of mature leaves. Osmotic concentration and sugar content of CA young leaves were also higher than those of CA mature leaves (Table 17.2). In Arabidopsis, the level of freezing tolerance, osmotic pressure and proline and sugar contents during cold acclimation were dependent on leaf order (Takagi et al., 2003). Wheat leaves accumulate sugars, proline and glycine betaine as compatible solutes (Kamata and Uemura, 2004). Therefore, CA young leaves might accumulate higher levels of these compatible solutes than CA mature leaves in wheat. Since some of these solutes that were accumulated during cold acclimation might be involved

Simulated Acid Snow Stress

179

Table 17.2. Levels of freezing tolerance (LT50), osmotic concentration and content of soluble sugars in mature and young leaves of cold-acclimated wheat seedlings. (Data from Inada et al., 2007.)

Mature Young

LT50 (°C)

Osmotic concentration (mol/kg)

Soluble sugars (mmol/g dry weight)

−8.4±1.1 −12.1±1.8

0.75±0.03 0.88±0.03

0.57±0.02 0.74±0.05

Data are mean±standard deviation from three to nine independent plants.

in the development of freezing tolerance of cells due to freezing point depression, amelioration of cell dehydration, cryoprotective activity and reduction of oxidative damage (Smirnoff and Cumbes, 1989; Kishitani et al., 1994; Shen et al., 1997; Vágújfalvi et al., 1999; Wanner and Junttila, 1999; Takagi et al., 2003; Kamata and Uemura, 2004; Parvanova et al., 2004), accumulation of compatible solutes may promote the level of SAS stress tolerance of CA young leaves. As shown in Table 17.3, the level of superoxide dismutase (SOD) activity in CA young leaves was much higher than that in CA mature leaves after a long-term SAS stress (pH 2.0) at −4°C for 7 days. SOD activity in CA young leaves remained at almost the same level after the long-term SAS stress, but that in CA mature leaves was decreased by half. On the other hand, levels of ascorbate peroxidase and catalase activities were decreased slightly in both CA young and CA mature leaves by the long-term SAS stress. The level of membrane

lipid peroxidation in SAS-stressed young leaves remained at basal levels during the regrowth period under a continuous light condition, although that in CA mature leaves was increased significantly (Fig. 17.3B). The maximal quantum yield of PSII in CA young leaves was temporarily decreased slightly but gradually recovered (Fig. 17.3A). On the other hand, that in CA mature leaves was markedly decreased during the regrowth period under a light condition after SAS stress. Therefore, oxidative damage was enhanced by light irradiation during the regrowth period after a long-term SAS stress in CA mature leaves, but was reduced by antioxidative factors such as SOD activity in CA young leaves. It is also possible that other physiological factors such as cold-inducible stress proteins might contribute to the induction of SAS stress tolerance (Guy, 1990; Thomashow, 1999). Further study on molecular mechanisms of SAS stress tolerance, especially acid tolerance, is necessary in the future.

Table 17.3. Changes in levels of the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) of cold-acclimated wheat leaves by long-term simulated acid snow stress (prolonged freezing with sulfuric acid solution of pH 2.0 or water of pH 5.6 at −4°C for 7 days). (Data from Inada et al., 2007.)

Treatment Mature

Young

Untreateda pH 5.6 pH 2.0 Untreateda pH 5.6 pH 2.0

SOD (U/µg dry weight) 0.70±0.18 4.06±0.18 1.86±0.63 0.75±0.32 13.4±2.25 12.5±2.98

APX (µmol/min/mg dry weight)

CAT (µmol/min/mg dry weight)

0.031±0.006 0.041±0.012 0.028±0.014 0.025±0.006 0.065±0.013 0.045±0.005

3.71±0.84 4.45±0.34 4.00±0.79 1.51±0.16 5.33±0.19 3.54±0.71

Data are mean±standard deviation from three or four independent plants. a Data before freezing.

180

K. Arakawa et al.

Conclusion In the present chapter, we have demonstrated the impact of acid snow stress on wheat seedlings. Simulation experiments of acid snow stress suggest that acid snow stress is a potent stress factor since contamination of acid pollutants in snow might cause local but strong acidification due to freezing-induced condensation in the process of freeze–thawing in winter. The intensity of acid snow stress would be influenced by the severity of the winter conditions, such as duration and freezing temperature, and the severity of the acid snow conditions, such as acidity and amount of snowfall. In addition, damage caused by acid snow stress may be expanded by light irradiation in the early stage of the regrowth period. In wheat, mature leaves are less tolerant to SAS stress and subsequent photooxidative stress than are young leaves because of the lower levels of tolerance to freez-

ing stress and oxidative stress in mature leaves. It is also assumed that the acidification of snow in winter and subsequent environmental stimuli such as light, UV, ozone and fungal attacks may affect the survival or productivity of winter crops with less freezing tolerance and/or acid tolerance. Since consumption of fossil fuels will continue to cause acid precipitation, acid snow may become an environmental stress factor in cool climate regions of the world.

Acknowledgements We are grateful to Dr H. Saito and Dr Y. Sano (Research Faculty of Agriculture, Hokkaido University) for useful discussion and support. The study reported was partly supported by a Grant-in-Aid for Scientific Research (No. 16580270) from the Japanese Society for the Promotion of Science to K.A.

References Apel, K. and Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology 55, 373–399. Asada, K. (1999) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50, 601–639. Cevc, G. (1987) How membrane chain melting properties are regulated by the polar surface of the lipid bilayer. Biochemistry 26, 6305–6310. Ensminger, I., Busch, F. and Huner, N.P.A (2006) Photostasis and cold acclimation: sensing low temperature through photosynthesis. Physiologia Plantarum 126, 28–44. Evans, L.S. (1989) Effects of acidic precipitation on crops. In: Adriano, D.C. and Johnson, A.H. (eds) Acidic Precipitation. Springer Verlag, New York, New York, pp. 29–60. Fujikawa, S. and Miura, K. (1986) Plasma membrane ultrastructural changes caused by mechanical stress in the formation of extracellular ice as a primary cause of slow freezing injury in fruit-bodies of Basidiomycetes (Lyophylum ulmarium (Fr.) Kuhner). Cryobiology 23, 371–382. Fujikawa, S., Jitsuyama, Y. and Kuroda, K. (1999) Determination of the role of cold acclimation-induced diverse changes in plant cells from the viewpoint of avoidance of freezing injury. Journal of Plant Research 112, 237–244. Fujita, S., Takahashi, A. and Sakurai, T. (2003) The wet deposition of acid and some major ions over the Japanese Archipelago. Tellus B 55, 23–34. Fushimi, H., Kawamura, T., Iida, H., Ochiai, M., Nakajima, T. and Azuma, Y. (2001) Internal distribution of acid materials within snow crystals. In: Satake, K. (ed.) Acid Rain 2000, Vol. 3. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 1709–1714. Gabara, B., Skłodowska, M., Wyrwicka, A., Gliñska, S. and Gapiñska, M. (2003) Changes in the ultrastructure of chloroplasts and mitochondria and antioxidant enzyme activity in Lycopersicon esculentum Mill. leaves sprayed with acid rain. Plant Science 164, 507–516. Gout, E., Bligny, R. and Douce, R. (1992) Regulation of intracellular pH values in higher plant cells. Journal of Biological Chemistry 267, 13903–13909. Guy, C.L. (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 41, 187–223.

Simulated Acid Snow Stress

181

Han, Z., Ueda, H. and Sakurai, T. (2006) Model study on acidifying wet deposition in East Asia during wintertime. Atmospheric Environment 40, 2360–2373. Holloway, T., Levy, H. II and Carmichael, G. (2002) Transfer of reactive nitrogen in Asia: development and evaluation of a source–receptor model. Atmospheric Environment 36, 4251–4264. Huner, N.P.A., Öquist, G. and Sarhan, F. (1998) Energy balance and acclimation to light and cold. Trends in Plant Science 3, 224–230. Inada, H., Nagao, M., Fujikawa, S. and Arakawa, K. (2006) Influence of simulated acid snow stress on leaf tissue of wintering herbaceous plants. Plant & Cell Physiology 47, 504–512. Inada, H., Fujikawa, S., Saito, H. and Arakawa, K. (2007) Effects of light condition after simulated acid snow stress on leaves of winter wheat. Environmental Sciences 14, 53–71. Johannessen, M. and Henriksen, A. (1978) Chemistry of snow meltwater: changes in concentration during melting. Water Resources Research 14, 615–619. Kamata, T. and Uemura, M. (2004) Solute accumulation in wheat seedlings during cold acclimation: contribution to increased freezing tolerance. Cryo Letters 25, 311–322. Kishitani, S., Watanabe, K., Yasuda, S., Arakawa, K. and Takabe, T. (1994) Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants. Plant, Cell & Environment 17, 89–95. Likens, G. E. and Bormann, F. H. (1995) Chemistry. In: Biogeochemistry of a Forested Ecosystem, 2nd ed. Springer-Verlag, New York, New York, pp. 32–45. Mach, J.M., Castillo, A.R., Hoogstraten, R. and Greenberg, J.T. (2001) The Arabidopsis-accelerated cell death gene ACD2 encodes red chlorophyll catabolite reductase and suppresses the spread of disease symptoms. Proceedings of the National Academy of Sciences USA 98, 771–776. Martz, F., Sutinen, M.-L., Kiviniemi, S. and Palta, J.P. (2006) Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation. Tree Physiology 26, 783–790. Mazur, P. (1966) Physical and chemical basis of injury in single-celled microorganisms subjected to freezing and thawing. In: Meryman, H.T. (ed.) Cryobiology. Academic Press, New York, New York, pp. 213–315. Mittler, R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7, 405–410. Murata, N., Takahashi, S., Nishiyama, Y. and Allakhverdiev, S.I. (2007) Photoinhibition of photosystem II under environmental stress. Biochimica et Biophysica Acta 1767, 414–421. Nagao, M., Arakawa, K., Takezawa, D. and Fujikawa, S. (2008) Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells. Planta 227, 477–489. Noguchi, I., Katoh, T., Sakai, S., Iwata, R., Akiyama, M., Ohtsuka, H., Sakata, K., Aga, H. and Matsumoto, Y. (2001) Snowcover components in northern Japan. In: Satake, K. (ed.) Acid Rain 2000, Vol. 2. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 421–426. Ohara, T., Akimoto, H., Kurokawa, J., Horii, N., Yamaji, K., Yan, X. and Hayasaka, T. (2007) An Asian emission inventory of anthropogenic emission sources for the period 1980–2020. Atmospheric Chemistry and Physics Discussions 7, 6843–6902. Parvanova, D., Ivanov, S., Konstantinova, T., Karanov, E., Atanassov, A., Tsvetkov, T., Alexieva, V. and Djilianov, D. (2004) Transgenic tobacco plants accumulating osmolytes show reduced oxidative damage under freezing stress. Plant Physiology and Biochemistry 42, 57–63. Pfanz, H. and Heber, H. (1986) Buffer capacities of leaves, leaf cells, and leaf cell organelles in relation to fluxes of potentially acidic gases. Plant Physiology 81, 597–602. Pomeroy, M.K., Andrews, C.J., Stanley, K.P. and Gao, J.-Y. (1985) Physiological and metabolic responses of winter wheat to prolonged freezing stress. Plant Physiology 78, 207–210. Rao, D.N. and LeBlanc, B.F. (1965) Effects of sulfur dioxide on the lichen alga, with special reference to chlorophyll. The Bryologist 69, 69–75. Richter, A., Burrows, J.P., Nüß, H., Granier, C. and Niemeier, U. (2005) Increase in tropospheric nitrogen dioxide over China observed from space. Nature 437, 129–132. Sakai, A. and Larcher, W. (1987) Frost Survival of Plants: Responses and Adaptation to Freezing Stress. Springer-Verlag, Heidelberg, Germany. Shan, Y. (1998) Effects of simulated acid rain on Pinus densiflora: inhibition of net photosynthesis by the pheophytization of chlorophyll. Water, Air, and Soil Pollution 103, 121–127. Shen, B., Jenson, R.G. and Bohnert, H.J. (1997) Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts. Plant Physiology 113, 1177–1183. Smirnoff, N. and Cumbes, Q.J. (1989) Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28, 1057–1060.

182

K. Arakawa et al.

Steponkus, P.L. (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology 35, 543–584. Stoyanova, D. (1998) Effects of simulated acid rain on anatomy of primary leaves of Phaseolus vulgaris. Biologia Plantarum 40, 581–588. Takagi, T., Nakamura, M., Hayashi, H., Inatsugi, R., Yano, R. and Nishida, I. (2003) The leaf-order-dependent enhancement of freezing tolerance in cold-acclimated Arabidopsis rosettes is not correlated with the transcript levels of the cold-inducible transcription factors of CBF/DREB1. Plant & Cell Physiology 44, 922–931. Tanaka, R., Hirashima, M., Satoh, S. and Tanaka, A. (2003) The Arabidopsis-accelerated cell death gene ACD1 is involved in oxygenation of pheophorbide a: inhibition of the pheophorbide a oxygenase activity dose not lead to the ‘stay-green’ phenotype in Arabidopsis. Plant & Cell Physiology 44, 1266–1274. Thomashow, M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50, 571–599. Träuble, H. and Eibl, H. (1974) Electrostatic effects on lipid phase transitions: membrane structure and ionic environment. Proceedings of the National Academy of Sciences USA 71, 214–219. Vágújfalvi, A., Kerepesi, I., Galiba, G., Tischner, T. and Sutka, J. (1999) Frost hardiness depending on carbohydrate changes during cold acclimation in wheat. Plant Science 144, 85–92. Velikova, V., Tsonev, T. and Yordanov, I. (1999) Light and CO2 responses of photosynthesis and chlorophyll fluorescence characteristics in bean plants after simulated acid rain. Physiologia Plantarum 107, 77–83. Wanner, L.A. and Junttila, O. (1999) Cold-induced freezing tolerance in Arabidopsis. Plant Physiology 120, 391–399. Wyrwicka, A. and Skłodowska, M. (2006) Influence of repeated acid rain treatment on antioxidative enzyme activities and on lipid peroxidation in cucumber leaves. Environmental and Experimental Botany 56, 198–204. Yu, J.Q., Ye, S.F. and Huang, L.F. (2002) Effects of simulated acid precipitation on photosynthesis, chlorophyll fluorescence, and antioxidative enzymes in Cucumis sativus L. Photosynthetica 40, 331–335. Zhou, B.-L., Arakawa, K., Fujikawa, S. and Yoshida, S. (1994) Cold-induced alterations in plasma membrane proteins that are specifically related to the development of freezing tolerance in cold-hardy winter wheat. Plant & Cell Physiology 35, 175–182.

18

Elevated Atmospheric CO2 Concentrations Enhance Vulnerability to Frost Damage in a Warming World M.C. Ball and M.J. Hill

Introduction Climates are becoming progressively warmer in association with increasing atmospheric concentrations of greenhouse gases such as CO2. There is considerable uncertainty in future conditions, but scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) predict a doubling of atmospheric [CO2] and an increase in mean global temperature of 1.1 to 6.4°C by 2100 (IPCC, 2007). Indeed, increases in minimum temperatures have already been measured over the past 30 years, and further changes in annual average and regional seasonal temperature regimes are expected (IPCC, 2001). These changes will affect the occurrence, severity and distribution of frost, including extreme freezing events. The most responsive species to these changes are likely to occur in the cool to cold climates at high latitudes and high altitudes, where seasonal temperatures and the length of the frost-free period are important determinants of the growing season (Chen et al., 1995). Indeed, shifts in phenological patterns, such as earlier flowering (Fitter and Fitter, 2002), are occurring in temperate climates consistent with climate warming (Walther, 2003). However, there are also recent reports of severe frost damage in tundra (Molgaard and Christensen, 1997), temperate forests (Norby et al., 2003) and alpine vegetation (Inouye, 2008), and con-

cerns have been raised that severe frost damage is increasing despite climate warming (Gu et al., 2008).

Climate Warming Affects Phenology and Seasonal Variation in Acclimation Paradoxically, changes in the timing and duration of growing seasons due to climate warming can also increase the vulnerability of plants to freezing damage from early- or late-season frosts (Gu et al., 2008). The problem arises because seasonal change in acclimation to freezing or hot conditions occurs in response to combined effects of temporal variation in day length and temperature. Acclimation is the process by which tolerance to an environmental stress is increased and it is usually triggered by an environmental cue (Lee et al., 1995). Cold acclimation is activated by exposure to low temperatures and an accompanying reduction in photoperiod (Chen et al., 1995). Cold acclimation induces a state of minimum growth in plants and generates a level of freezing tolerance that enhances survival through the winter (Kalma et al., 1992; Li and Christersson, 1993; Chen et al., 1995). Although there are interspecific differences in the relative importance of different environmental cues, plants generally acclimate to freezing temperatures in autumn in response

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

183

184

M.C. Ball and M.J. Hill

to declining temperatures and day length, and deacclimate in spring as temperatures increase in association with increasing day length. Thus, warmer temperatures can cause plants to delay acclimation to freezing in autumn, and accelerate deacclimation in late winter or early spring, making them more vulnerable, respectively, to late autumn or early spring frosts. For example, the severe spring frost of 2007 in North America causedwidespread damage to a variety of frosttolerant vegetation types because it followed an unseasonally warm period in late winter that induced bud break, and hence also deacclimation (Gu et al., 2007).

Elevated CO2 Concentration Amplifies Freezing Stress However, prediction of plant responses to climate warming is complicated by the recent discovery that growth under elevated [CO2] can lower freeze tolerance, causing greater frost damage at warmer freezing temperatures even in highly freeze-tolerant species (Repo et al., 1996; Lutze et al., 1998; Barker et al., 2005; Loveys et al., 2006). Elevated [CO2] appears to affect freezing tolerance through at least two processes: the nucleation of ice and the acclimation state of the plant.

Elevated CO2 Concentration Increases Ice-nucleation Temperatures Elevated [CO2] increases the temperature at which ice nucleation occurs in plant tissues in a variety of frost-tolerant species (Lutze et al., 1998; Beerling et al., 2001; 2002; Royer et al., 2002). Woldendorp et al. (2008) evaluated published data to examine the relationship between the temperature at which ice nucleates in leaves and the [CO2] in which the plants were grown. The results showed that there was considerable variation between species in the temperatures at which ice nucleation occurred, but no significant differences between species in the effects of [CO2] on ice nucleation. Icenucleation temperatures increased rapidly with increasing [CO2] up to approximately 350 ppm,

and subsequently increased at a slower rate with further increase in [CO2]. The mechanism remains elusive, but a doubling of [CO2] from 350 to 700 ppm can be expected to increase the ice-nucleation temperature by as much as 1 to 2°C. The implication is that plants growing under present atmospheric conditions may be subject to greater freezing stress today than prior to the Industrial Revolution (Woldendorp et al., 2008). Further, if the ice-nucleation temperature were to increase by 2°C from say −5 to −3°C, then the incidence of frost damage will not necessarily decrease with climate warming by 2°C in areas where frosts persist (Lutze et al., 1998). An increase in the ice-nucleation temperature necessarily increases the extent of freezing damage at warmer freezing temperatures if species are sensitive to ice formation within tissues. However, many temperate evergreens, including both woody species such as Eucalyptus pauciflora (Ball et al., 2004) and herbaceous species (e.g. McCully et al., 2004), are seasonally tolerant of extracellular freezing within tissues. Whether or not a freezing event will injure these freeze-tolerant plant species depends on the acclimation state of the plant, which is also affected by elevated [CO2] (Loveys et al., 2006).

Elevated CO2 Concentration Affects Acclimation to Freezing Temperatures How might elevated [CO2] affect freezing tolerance? The answer is likely to involve a complex combination of biochemical and biochemical processes. Growth under elevated [CO2] is well known to affect the water status, nitrogen concentrations and carbohydrate contents of leaves (Drake et al., 1997), all factors that also influence freezing tolerance (Xin and Browse, 2000). However, there have been no reports linking any of these factors to the adverse effects of elevated [CO2] on freeze tolerance. An important clue was found serendipitously during a study of the effects of elevated [CO2] on growth of the snow gum (E. pauciflora Sieb. ex Spreng), one of the most freezetolerant of eucalypts (Sakai et al., 1981). Barker et al. (2005) grew snow gum seedlings

Elevated Atmospheric CO2 Concentrations

under field conditions in open-top chambers flushed with either ambient or elevated [CO2]. The chambers were partitioned with a vertical shade screen such that plants on the east or west side of the screen received direct sunlight only during the morning or afternoon, respectively. All plants received the same total irradiance and were subject to the same minimum temperatures. During early autumn, the plants experienced several light frosts of −2°C or warmer without measurable damage until a minimum temperature of −5.8°C occurred. This event caused greater frost damage in the plants receiving direct sunlight in the afternoon than in the morning, and ten times greater damage in plants grown under elevated than ambient [CO2]. Specifically, the loss in canopy leaf area under ambient [CO2] averaged 3 and 5% in plants grown with morning and afternoon sun, respectively, but averaged 37 and 67% in plants grown with morning and afternoon sun, respectively, under elevated [CO2] (Barker et al., 2005). These observations led Barker et al. (2005) to suggest that the greater frost damage observed in plants grown under elevated [CO2] might be related to the effects of elevated [CO2] on leaf temperatures. Specifically, the sustained reduction in stomatal conductance that is often seen under elevated CO2 (Morison, 1985; Drake et al., 1997) can reduce transpiration (Li et al., 2003), leading to increased leaf temperature due to reduced evaporative cooling (Siebke et al., 2002). This raised the possibility that warmer daytime leaf temperatures under elevated [CO2] could adversely affect the capacity to tolerate freezing leaf temperatures at night. If this were true, then greater frost damage would be expected on the west than on the east side of the screen, and the effect would be amplified under elevated [CO2], as observed (Barker et al., 2005). Loveys et al. (2006) tested this hypothesis in a subsequent field-based study of snow gum. They used open-top chambers and infra-red lamps for free air temperature increase (FATI) to subject snow gums to two experimental regimes: elevated/ambient [CO2] or elevated/ ambient daytime leaf temperatures. In the FATI treatment, the lamps were positioned to raise daytime leaf temperatures by as much as 3°C to simulate the average elevation of daytime

185

leaf temperatures (Barker et al., 2005) due to stomatal closure in snow gum seedlings grown under elevated [CO2] (Roden et al., 1999). The seedlings were planted in late summer and subject to the natural vagaries in weather including the decline in minimum ambient temperature as winter approached. This study was designed to determine whether there were similarities between effects of elevated CO2 and elevated daytime leaf temperature on the development of freeze tolerance. Loveys et al. (2006) showed that acclimation to freezing was delayed by at least 3 weeks in leaves of seedlings subject to either the artificial daytime warming or the elevated [CO2] treatment. Importantly, all plants achieved similar levels of freeze tolerance by the end of the study, showing that the treatments affected the timing but not the extent of acclimation. The similarity in effects of these treatments on acclimation to freezing temperatures was interpreted as indication of a common cause, namely higher daytime leaf temperatures. As both treatments only affected leaf temperature during daytime and had no effect on minimum leaf temperatures at night, the results showed that the diurnal range in leaf temperature, not just the temperature minima, affects acclimation to freezing temperatures. Loveys et al. (2006) concluded that the increase in leaf temperature due to stomatal closure under elevated [CO2] could contribute to the effects of elevated [CO2] on acclimation to freezing temperatures. However, Loveys et al. (2006) were cautious in their interpretation as the effects of elevated [CO2] on acclimation are unlikely to be due solely to indirect effects on daytime leaf temperature. They noted that how a plant senses the decreasing temperatures that induce freeze tolerance could be confounded by interactions between elevated [CO2], leaf temperature and the signalling pathways responsible for initiating freeze tolerance. Specifically, they suggested three mechanisms associated with development of freeze tolerance (Xin and Browse, 2000) that might be altered by growth under elevated [CO2]. First, elevated leaf temperature can affect membrane fluidity, which is thought to be one of the main triggers for the activity of calcium channels (Monroy and Dhindsa, 1995). Cytosolic calcium increases transiently when

186

M.C. Ball and M.J. Hill

a plant is exposed to cold temperatures (Monroy and Dhindsa, 1995; Sheen, 1996; Angeli et al., 2003). If elevated leaf temperatures were to reduce calcium influx to the cytosol, then calcium signalling and subsequent cold-regulated gene expression would be disrupted. Second, increasing concentrations of the hormone abscisic acid play a role in the development of freeze tolerance (Chen et al., 1983). Reduced stomatal conductance and changes in xylem sap pH caused by exposure to elevated [CO2] could influence the concentration of abscisic acid available to act as a signal for the perception of cold temperatures. Finally, changes in xylem sap pH, due to growth under elevated [CO2], can reduce nitrate assimilation resulting in a decline in protein synthesis (Rachmilevitch et al., 2004) which in turn could affect the rate of acclimation. Greater frost damage has been observed in plants growing under elevated than ambient [CO2] during both autumn (Barker et al., 2005) and spring (Lutze et al., 1998), raising questions about the effects of elevated [CO2] on deacclimation. Although the process of cold deacclimation has received less attention than acclimation (Rapacz, 2002), the rate of deacclimation to freezing temperatures is equally as important as acclimation for plant survival (Svenning et al., 1997). As elevated air temperatures can accelerate deacclimation in numerous plant species (Taulavuori et al., 1997; Rapacz, 2002), Loveys et al. (2006) predicted that higher leaf temperatures associated with lower stomatal conductance under elevated [CO2] could increase rates of deacclimation to freezing temperatures in spring. In this way, growth under elevated [CO2] could alter the timing of seasonal change in acclimation to freezing temperatures, causing acclimation to be delayed in autumn and accelerated in spring, thereby increasing vulnerability to earlyand late-season frosts. The effect of elevated [CO2] on plant freeze tolerance reported in the literature is variable. For example, growth under elevated [CO2] increased tolerance in Betula alleghaniensis (Wayne et al., 1998), decreased tolerance in Vaccinium myrtillus (Taulavuori et al., 1997) and had no effect on Picea abies (Wiemken et al., 1996). This lack of consistency is likely due to a range of factors including the timing of

the measurements themselves. For example, given the effects of elevated [CO2] on the timing of acclimation or deacclimation, measurements made when plants are fully acclimated are unlikely to show effects. Similarly, effects of elevated [CO2] on freeze tolerance are less likely to occur in species which respond more strongly to changes in day length than temperature. It follows that little effect would be expected in species with crassulacean acid metabolism because their stomata are closed during the day, and hence there would be no increase in leaf temperature under elevated [CO2]. Thus, interspecific differences in sensitivity to [CO2]dependent changes in freeze tolerance might reflect differences in functional attributes of the species, potentially enabling prediction of species that are most likely to be positively or negatively affected by rising atmospheric [CO2] in regions where frosts persist.

Combined Effects of Elevated CO2 Concentration and Climate Warming on Frost Damage Woldendorp et al. (2008) used a modelling approach to explore whether the increased susceptibility to frost damage under elevated [CO2] would be counteracted by climate warming. Their model focused on the incidence and severity of frost damage to E. pauciflora in a sub-alpine region of Australia for current and future conditions using the A2 IPCC elevated [CO2] and climate change scenario. Woldendorp et al. (2008) modified a model for predicting frost sensitivity of E. pauciflora seedlings (King and Ball, 1998) to include the effects of elevated [CO2] on acclimation and deacclimation to freezing temperatures. This was achieved by assuming that stomatal closure in response to increasing [CO2] would increase daytime leaf temperature; in effect, elevated [CO2] would amplify temporal effects of climate warming on acclimation states because the leaves would sense warmer conditions. However, the model did not take into account the increase in icenucleation temperature in the leaves of plants grown under elevated [CO2] (Lutze et al., 1998; Terry et al., 2000; Beerling et al., 2001; 2002), and hence may have underestimated

Elevated Atmospheric CO2 Concentrations

the [CO2]-dependent increase in frost sensitivity. Instead, the model assumed that plants froze if the temperature was below 0°C and that frost damage depended on the acclimation state relative to the temperature of the freezing event. Woldendorp et al. (2008) calibrated the model with field data and applied it to a study region in Victoria using climate scenario data from the Commonwealth and Scientific Research Organization’s Global Climate Model C-CAM for current (1975–2004) and future (2035–2064) 30-year climate sequences. The model predicted that a future climate would have a lower frequency of air temperatures below 0°C, but the occurrence of the coldest temperatures (i.e. those below −8°C) would be similar to the current climate. Both elevated [CO2] and climate warming affected the timing and rates of acclimation and deacclimation of snow gum to freezing temperatures, potentially reducing the length of time that plants are fully frost-tolerant and increasing the length of the growing season. Despite fewer days with freezing temperatures in the future, with consequently fewer damaging frosts with lower average levels of impact, the model predicted individual weather sequences that still resulted in widespread plant mortality due to severe frosts. Furthermore, delayed acclimation due to either warming or rising [CO2] combined with an early severe frost could lead to more frost damage and higher mortality than would occur in current conditions. Importantly, the model showed that the effects of elevated [CO2] on frost damage were greater in autumn, while cli-

187

mate warming induced more frost damage in spring. Woldendorp et al. (2008) concluded that frost damage will continue to be a management issue for plantation and forest management in regions where frosts persist.

Conclusion Recent studies have shown that plants can be more vulnerable to frost injury under elevated [CO2], with losses in leaf area (Lutze et al., 1998; Barker et al., 2005) or reduction in photosynthetic capacity (Terry et al., 2000) decreasing the capacity for growth under subsequent conditions (Barker et al., 2005). Indeed, a 2°C increase in temperature minima will not necessarily decrease the severity of frost damage to plants grown under elevated atmospheric [CO2] (Woldendorp et al., 2008) and, hence, the potential for elevated [CO2] to stimulate productivity (Poorter and Pérez-Soba, 2001) may be reduced under future climate scenarios (Lutze et al., 1998). This may have major implications for agriculture, forestry and vegetation dynamics (Lutze et al., 1998), especially in temperate regions where frosts persist (Beerling et al., 2002).

Acknowledgements The authors gratefully thank Larry Gusta, Karen Tanino and Michael Wisniewski, the organizers of the Eighth International Plant Cold Hardiness Seminar, for making this project possible.

References Angeli, S., Malho, R. and Altamura, M. (2003) Low-temperature sensing in olive tree: calcium signalling and cold temperature. Plant Science 165, 1303–1313. Ball, M.C., Canny, M.J., Huang, C.X. and Heady, R. (2004) Structural changes in acclimated and unacclimated leaves during freezing and thawing. Functional Plant Biology 31, 29–40. Barker, D.H., Loveys, B.R., Egerton, J.J.G., Gorton, H., Williams, W.E. and Ball, M.C. (2005) CO2 enrichment predisposes foliage of a eucalypt to freezing injury and reduces spring growth. Plant, Cell & Environment 28, 1506–1515. Beerling, D., Terry, A., Mitchell, P., Callaghan, T., Gwynn-Jones, D. and Lee, J. (2001) Time to chill: effects of simulated global change on leaf ice nucleation temperatures of subarctic vegetation. American Journal of Botany 4, 628–633. Beerling, D., Terry, A., Hopwood, C. and Osborne, C. (2002) Feeling the cold: atmospheric CO2 enrichment and the frost sensitivity of terrestrial plant foliage. Palaeogeography, Palaeoclimatology, Palaeoecology 182, 3–13.

188

M.C. Ball and M.J. Hill

Chen, H., Brenner, M. and Li, P. (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiology 71, 362–365. Chen, T., Burke, M. and Gusta, L. (1995) Freezing tolerance in plants: an overview. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 115–135. Drake, B.G., Gonzalez-Meler, M.A. and Long, S.F. (1997) More efficient plants: a consequence of rising atmospheric CO2. Annual Review of Plant Physiology and Plant Molecular Biology 48, 609–639. Fitter, A.H. and Fitter, R.S.R. (2002) Rapid changes in flowering time in British plants. Science 296, 1689–1691. Gu, L., Hanson, P.J., Post, W.M., Kaiser, D.P., Yang, B., Nemani, R., Pallardy, S.G. and Meyers, T. (2008) The 2007 eastern US spring freeze: increased cold damage in a warming world? Bioscience 58, 253–262. Inouye, D.W. (2008) Effects of climate change on phenology, frost damage, and floral abundance of montane flowers. Ecology 89, 353–362. IPCC (2001) Climate Change 2001: The Scientific Basis. Contribution of Working Group I to the Third Assessment Report of the Intergovernmental Panel on Climate Change (Houghton, J.T., Ding, Y., Griggs, D.J., Noguer, M., van der Linden, P.J., Dai, X., Maskell, K. and Johnson, C.A., eds). Cambridge University Press, Cambridge, UK. IPCC (2007) Climate Change 2007: Synthesis Report, Summary for Policymakers. IPCC Plenary XXVII, Valencia, Spain. http://www.ipcc.ch/pdf/assessment-report/ar4/syr/ar4_syr_spm.pdf (accessed January 2009). Kalma, J., Laughlin, G., Caprio, J. and Hamer, P. (1992) Advances in Bioclimatology 2: The Bioclimatology of Frost. Springer-Verlag, Berlin. King, D. and Ball, M.C. (1998) A model of frost impacts on seasonal photosynthesis of Eucalyptus pauciflora. Australian Journal of Plant Physiology 25, 27–37. Lee, R., Warren, G. and Gusta, L. (eds) (1995) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota. Li, J., Dugas, W., Hymus, G.J., Johnson, D., Hinkle, C. and Drake, B.G. (2003) Direct and indirect effects of elevated CO2 on transpiration from Quercus myrtifloia in a scrub-oak ecosystem. Global Change Biology 9, 96–105. Li, P. and Christersson, L. (1993) Advances in Plant Cold Hardiness. CRC Press, Boca Raton, Florida. Loveys, B.R., Egerton, J.J.G. and Ball, M.C. (2006) Higher daytime leaf temperatures contribute to lower freeze tolerance under elevated CO2. Plant, Cell & Environment 29, 1077–1086. Lutze, J.L., Roden, J.S., Holly, C., Wolfe, J., Egerton, J.J.G. and Ball, M.C. (1998) Elevated atmospheric CO2 promotes frost damage in evergreen tree seedlings. Plant, Cell & Environment 21, 631–635. McCully, M.E., Canny, M.J. and Huang, C.X. (2004) The management of extracellular ice by frosted, acclimated herbaceous petioles. Annals of Botany 94, 665–674. Molgaard, P. and Christensen, K. (1997) Response to experimental warming in a population of Papaver radicatum. Global Change Biology 3, 116–124. Monroy, A.F. and Dhindsa, R.S. (1995) Low-temperature signal-transduction – induction of cold acclimationspecific genes of alfalfa by calcium at 25°C. The Plant Cell 7, 321–331. Morison, J.I.L. (1985) Sensitivity of stomata and water-use efficiency to high CO2. Plant, Cell & Environment 8, 467–474. Norby, R.J., Hartz-Rubin, J.S. and Verbrugge, M.J. (2003) Phenological responses in maple to experimental atmospheric warming and CO2 enrichment. Global Change Biology 9, 1792–1801. Poorter, H. and Pérez-Soba, M. (2001) The growth response of plants to elevated under non-optimal environmental conditions. Oecologia 129, 1–20. Rachmilevitch, S., Cousins, A.B. and Bloom, A.J. (2004) Nitrate assimilation in plant shoots depends on photorespiration. Proceedings of the National Academy of Sciences USA 101, 11506–11510. Rapacz, M. (2002) Regulation of frost resistance during cold de-acclimation and re-acclimation in oilseed rape. A possible role of PSII redox state. Physiologia Plantarum 115, 236–243. Repo, T., Hänninen, H. and Kellomäki, S. (1996) The effects of long term elevation of air temperature and CO2 on the frost hardiness of Scots pine. Plant, Cell & Environment 19, 209–216. Roden, J.S., Egerton, J.J.G. and Ball, M.C. (1999) Effect of elevated CO2 on photosynthesis and growth of snow gum (Eucalyptus pauciflora) seedlings during winter and spring. Australian Journal of Plant Physiology 26, 37–46. Royer, D.L., Osbourne, C.P. and Beerling, D.J. (2002) High CO2 increases the freezing sensitivity of plants: implications for paleoclimatic reconstructions from fossil floras. Geology 30, 963–966.

Elevated Atmospheric CO2 Concentrations

189

Sakai, A., Paton, D.M. and Wardle, P. (1981) Freezing resistance of trees of the south temperate zone, especially subalpine species of Australasia. Ecology 62, 563–570. Sheen, J. (1996) Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274, 1900–1902. Siebke, K., Ghannoum, O., Conroy, J.P. and von Caemmerer, S. (2002) Elevated CO2 increases the leaf temperature of two glasshouse-grown C-4 grasses. Functional Plant Biology 29, 1377–1385. Svenning, M.M., Rosnes, K. and Junttila, O. (1997) Frost tolerance and biochemical changes during hardening and dehardening in contrasting white clover populations. Physiologia Plantarum 101, 31–37. Taulavuori, K., Laine, K., Taulavuori, E., Pakonen, T. and Saari, E. (1997) Accelerated dehardening in bilberry (Vaccinium myrtillus L.) induced by a small elevation in air temperature. Environmental Pollution 98, 91–95. Terry, A., Quick, P. and Beerling, D. (2000) Long-term growth of ginkgo with CO2 enrichment increases leaf ice nucleation temperatures and limits recovery of the photosynthetic system from freezing. Plant Physiology 124, 183–190. Walther, G.R. (2003) Plants in a warmer world. Perspectives in Plant Ecology Evolution and Systematics 6, 169–185. Wayne, P.M., Reekie, E.G. and Bazzaz, F.A. (1998) Elevated CO2 ameliorates birch response to high temperature and frost stress: implications for modeling climate-induced geographic range shifts. Oecologia 114, 335–342. Wiemken, V., Kossatz, L. and Ineichen, K. (1996) Frost hardiness of Norway spruce grown under elevated atmospheric CO2 and increased nitrogen fertilizing. Journal of Plant Physiology 149, 433–438. Woldendorp, G., Hill, M.J., Doran, R. and Ball, M.C. (2008) Frost in a future climate: modelling interactive effects of warmer temperatures and rising atmospheric [CO2] on the incidence and severity of frost damage in a temperate evergreen (Eucalyptus pauciflora). Global Change Biology 14, 294–308. Xin, Z. and Browse, J. (2000) Cold comfort farm: the acclimation of plants to freezing temperatures. Plant, Cell & Environment 23, 893–902.

19

The Occurrence of Winter-freeze Events in Fruit Crops Grown in the Okanagan Valley and the Potential Impact of Climate Change H.A. Quamme, A.J. Cannon, D. Neilsen, J.M. Caprio and W.G. Taylor

Abstract The main limitation to fruit production in the Okanagan Valley (British Columbia, Canada) is winter injury. Examination of historical records between 1916 and 2006 revealed 15 severe winter-kill events with two occurring in November, seven in December, four in January and two in February. An iterative c2 method applied to long-term production records verified that extreme low minimum temperatures had the strongest association with poor production of grape, apple, sweet cherry, pear, apricot and peach, and revealed the time when each crop was most at risk. Although all were subject to winter injury during most of the fourmonth period, variability occurred in the time when these crops were most susceptible. Grape, apple and sweet cherry were most susceptible in the early stages of acclimation during November to mid-January, whereas pear, peach and apricot were most susceptible during January and February. In western North America the trend in climatic warming has been in winter and spring with no or little change in autumn. The synoptic weather pattern associated with these winter-freeze events was the Arctic outflow. This was a relatively rare weather event but has had a great impact on production. A decrease in the frequency and temperature extremes of Arctic outflows appeared to be associated with the warming trends of the region. A warmer climate might not result in as great an extension of the northern range of these crops in this region as anticipated, and this may vary with crop.

Introduction Winter injury is a serious recurring problem in northern climates and is the main climatic limitation to growing many fruit crops in British Columbia (Boyer, 1977; Krueger, 1983). An iterative c2 technique was used to determine the association of daily weather occurrences with production of apple (Caprio and Quamme, 1999), grape (Caprio and Quamme, 2002), apricot, peach and sweet cherry (Caprio and Quamme, 2006) during a 78-year period in the Okanagan Valley. The greatest impact of weather on production in all of these crops was that of minimum temperature, which causes winter injury during late autumn and winter (November to February). 190

Two long-term temperature records exist in the Okanagan Valley, one taken at the Coldstream Ranch (CR) (1900–1996) on the northern edge of fruit production and the other at the Pacific Agri-Food Research Centre (PARC) in Summerland (1916 onwards) in the centre of fruit production, that allowed examination of the long-term trends in minimum temperature and hence of the impact of climate change on winter injury. Brewer and Taylor (2001) first reported a rise in minimum temperature at CR and other sites in southern British Columbia from 1970 to 2000 in winter. Taylor and Barton (2004) later reported that the average minimum temperature had risen at PARC and CR and that the trend was greatest in winter and spring. These changes reflect a

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Winter-freeze Events in Fruit Crops

larger pattern identified for Western Canada (Zhang et al., 2000). The same c2 analysis that was used to determine the weather associated with production allowed the comparison of minimum temperatures in a recent time period with an earlier period throughout autumn and winter on a daily basis. An increase in daily minimum temperature in the period 1974–1991 compared with 1938–1973 was observed at CR (Caprio and Quamme, 2002, Caprio et al., 2008) in mid-January and February and early March using this method. The present chapter describes a study in which the iterative c2 outputs of minimum temperature during autumn and winter associated with production from previous studies of apple (Caprio and Quamme, 1999), grape (Caprio and Quamme, 2002), apricot, peach and cherry (Caprio and Quamme, 2006) were compared with the iterative c2 outputs demonstrating change in minimum temperature in recent years (Caprio and Quamme, 2002; Caprio et al., 2008). These comparisons allow the determination of the time of year that crops are at greatest risk of winter injury and if the risk of winter injury coincides with the time of year when the climate has changed. The association of minimum temperature with pear production, which has not previously been reported, is also included in the comparison. Winter injury is usually associated with episodes of extremely low temperatures that are often termed ‘winter freezes’ and which affect all fruit crops. These winter freezes have been attributed to outflows of cold Arctic air masses that flood down the mountain valleys of British Columbia from the north-east (Krueger, 1983). The years when winter freezes occurred were determined from station reports at PARC beginning in 1916 and from annual reports of the British Columbia Department of Agriculture beginning in 1917 (H.A. Quamme, unpublished results). Synoptic climatology examines relationships between atmospheric circulation and surface weather conditions (Barry and Perry, 1973). Classification techniques can be used to group synoptic-scale atmospheric conditions into discrete map patterns, which can then be correlated to surface variables at single or multiple points (Yarnal, 1993). Regression tree or

191

recursive partitioning models use circulation and surface weather data to generate discrete map patterns by defining a set of rules which are applied to the atmospheric input variables and observed surface variables to create potential classes in the synoptic climatology. Synoptic weather typing was used successfully to identify map patterns associated with high surface level ozone concentrations in the Vancouver, British Columbia area (Cannon and Whitfield, 2002) and the occurrence of rare ice storms in south central Canada, both under historic and future climatic conditions (Cheng et al., 2004, 2007). In the present study, we determined the effects of climate change on rare, cold events which may limit the production of high-value perennial crops and used synoptic map typing as a technique to link the occurrence of surface temperatures associated with winter injury to atmospheric circulation during historic times.

Method and Materials Caprio and Quamme (1999) and Caprio et al. (2009) gave an in-depth discussion of the application the iterative c2 method to determine weather associated with production and gave examples of the interpretation of computer output. To summarize, annual production was first indexed by comparing each year with three previous years and three subsequent years. This was done to minimize the effects of variable tree populations and advancements in horticultural technology. The indexed production data was then ranked and separated into quartiles. In the present study, only the lowest quartile (poor years) and two mid-quartiles (normal years) were used. The production data come from the Okanagan Valley (180 km long) and the adjacent Similkameen Valley (40 km long). Data pertaining to the Okanagan Valley and the Similkameen Valley covered the 72-year period from 1920 to 1991, and were for all varieties combined in each crop (British Columbia Legislative Assembly Select Standing Committee, 1978; British Columbia Department of Agriculture, 1977– 1992). The minimum temperatures are those recorded at PARC (49°34'N, 119°39'W, 482 m above sea level).

192

H.A. Quamme et al.

Next, the c2 test was used to compare daily weather occurrence of the poor years with the normal years. Climate data were taken from two Environment Canada weather stations with long-term records: Summerland CDA/CS and Vernon Coldstream Ranch/ Vernon CS. The climatic variables of temperature, which were recorded in imperial units, were broken down into classes of 2°F and tallies were made of the frequency with which the minimum temperature measurements fell into each class. For a given 3-week period, the c2 test was used to test the difference in frequency of days between of the poor quartile and the two mid-quartiles for each of the classes. The c2 test was applied to the total number of days accumulating in each class in succession, ordered in this case from low to high, generating a c2 value for each class in a low–high scan. If the frequency distribution of the lowest quartile deviated from that of the combined midquartiles, the c2 values generated in the scan increased and then decreased. When c2 reached its maximum (or turning) point in the scan, the temperature at that point was referred to as the ‘cardinal’ value (CV). A c2 value of 7 was taken as the critical value for significance of temperature (P£0.01, df=1). For each week, the computer generated the maximum c2, the accumulated counts of daily weather occurrences and the associated CV of the climatic factor being scanned. Temperatures were converted to metric after the c2 scan. To determine if climate change had occurred during the study period, minimum temperature occurrences (November–February) for two selected periods, 36 early years (1937– 1973) and 18 later years (1974–1991), were compared using the same iterative c2 technique. Temperature data from CR (50°14 N, 119°12'W, 482 m above sea level) were used to study temporal changes because this weather station was removed from the influence of urbanization. The 3-week running averages of the weekly c2 values for the temperature–production associations and climate change were plotted together for all six crops. Examination of station and provincial horticultural reports indicated that reference was made to 15 major winter-freeze events in

the Okanagan Valley over 90 years (1916– 2006). These events were presumed to occur as a result of periods of extreme minimum temperatures, with two events occurring in November, seven in December, three in January and three in February (H.A. Quamme, unpublished results). Nine of these events fell within the period 1950 to 2006 during which it was possible to determine if these events were related to particular synoptic weather patterns. The synoptic map-types controlling surface weather conditions in the Okanagan Valley were determined via the synoptic classification scheme of Cannon and Whitfield (2002) that was modified to use the multivariate regression tree model described by De’Ath (2002). Predictors in the classification model were mean sea-level pressure (i.e. surface-level circulation), 85 kPa relative humidity (i.e. boundarylayer moisture), 85–50 kPa thickness (i.e. low to mid-troposphere) temperature and 50 kPa geo-potential height data (i.e. mid-troposphere circulation) from the US NCEP/NCAR model reanalysis (Kalnay et al., 1996). Daily mean maps from 1948 to 2006 were obtained for a region covering western North America and the North Pacific Ocean (30–70°N, 160– 110°W). Data were sub-sampled from the 2.5° by 2.5° resolution grid to a 5° by 7.5° grid to facilitate later use with coarser spatial resolution Global Climate Model (GCM) data. Concurrent daily weather conditions in the Okanagan Valley were represented by precipitation amounts, mean temperatures and diurnal temperature ranges at four surface observing stations (Okanagan Centre, Oliver STP, PARC and CR). To emphasize day-to-day weather events rather than seasonality, synoptic circulation data and surface weather data were expressed as anomalies from their respective seasonal cycles (Yarnal, 1993). The regression tree used to identify synoptic map-types operates by recursively splitting the synoptic-scale circulation data into days with similar surface weather, in which similarity is measured by the within-group sums of squares of the observed precipitation amounts, mean temperatures and diurnal temperature ranges. The overall structure of the model is that of a binary tree in which each of

Winter-freeze Events in Fruit Crops

the bottom branches corresponds to a different synoptic map-type (shown in Cannon and Whitfield, 2002). The optimum number of synoptic map-types is determined objectively based on cross-validation performance. Once the tree structure has been determined, new observed (or modelled) synoptic-scale circulation data can be entered into the model and assigned to map-types based on the bottom branches they reach in the tree. Trends in the monthly occurrence of synoptic patterns (November to February) and associated absolute minimum temperatures were analysed using the Mann–Kendall nonparametric test to determine statistical significance and Sen’s method was used to determine the magnitude of the trend (Salmi et al., 2002). In order to remove the effects of years where individual synoptic patterns did not occur, trend analyses were performed on running 5-year averaged data.

Results and Discussion Association of production with minimum temperature and climate change Significant associations of poor production with minimum temperature (c2 values) were present in all crops during autumn and winter, but the strength of the association varied with season and crop (Fig. 19.1). The c2 values became significant in the last week in October for grape (Fig. 19.1A), early November for sweet cherry, apple, pear and peach (Fig. 19.1B,C, D and F) and early December for apricot (Fig. 19.1E). For grape (Fig. 19.1A), the c2 values were greatest in November, dropped in late December and the beginning of January, and peaked again in late January. In sweet cherry (Fig. 19.1B) and apple (Fig. 19.1C), the c2 values increased to a maximum in November and plateaued until January, with those of apple declining before sweet cherry. The c2 values in apple increased slightly again in February and March. In pear (Fig. 19.1D), apricot (Fig. 19.1E) and peach (Fig. 19.1F), the c2 values reached a peak in late January and then declined.

193

In late autumn and winter the cause of poor production was winter injury in apple (Caprio and Quamme, 1999), grape (Caprio and Quamme, 2002), apricot, peach and sweet cherry (Caprio and Quamme, 2006). In short, the CV indicated for each of the crops in Fig. 19.1 was closely related to the killing temperature of certain tissues: xylem in apple and pear, the buds and xylem of grape, and the flower buds and xylem of apricot and peach. These tissues are critical for the production and survival of these crops, and were injured at their deep supercooling point which in turn coincided with the average annual minimum temperature isotherm at their northern limit of production (Quamme, 1976, 1986; Quamme et al., 1982). The small peaks of c2 values in sweet cherry (Fig. 19.1B), apricot (Fig. 19.1E) and peach (Fig. 19.1F) (Caprio and Quamme, 2006) that occur in March and April coincided with the time of flower development and were associated with spring frosts (radiation freezes). At flowering, an association of minimum temperature with apple (Caprio and Quamme, 1999) and pear production (data not shown) showed up as deficit days at £−3°C on the good production c2 plot for April. Flowering in grape occurs at a later date than was shown on the plot (Fig. 19.1A), at a time when frosts do not occur in the Okanagan Valley. The trend in climate change indicated by the c2 values on each part of Fig. 19.1 indicated that most of the recent warming has occurred beginning the first week in January to mid-March (deficit in days, CV£−24°C, 25 January; £−8°C, 8 March). During a threeweek period in late November an excess of days of minimum temperature (CV£−17°C, 29 November) occurred. Grape, sweet cherry and apple (Fig. 19.1A, B and C) were at greatest risk of winter injury in the Okanagan Valley in November, December and early January before the trend to warmer weather. Grape and apple remained at risk throughout February, although the risk had declined. Pear, apricot and peach (Fig. 19.1E, D and F) were at highest risk in January and February when the trend to warmer weather occurred. These crops would appear to be more favoured by the warming trend.

60

Excess days Deficit days

0

≤−11°C

≤−23°C

≤−14°C ≤−16°C

Poor production

≤−24°C

≤−9°C

≤−23°C

≤−13°C

20

≤−17°C

≤−17°C Climate trend

≤−23°C ≤−8°C

≤−24°C

Climate trend

≤−17°C ≤−24°C

≤−8°C

–20 60

Climate trend ≤−24°C

≤−8°C ≤−16°C

D

≤−8°C ≤−13°C

E

F

Excess days Deficit days

≤−22°C 40

0

Poor production

Poor production

Poor production

≤−11°C

≤−5°C

20

≤−17°C

≤−17°C

≤−24°C –20 40

Climate trend 45

50

Climate trend

≤−8°C 3

8

≤−5°C

≤−15°C

≤−17°C

13

4–10 8–11 13–12 18–1 22–2 27–3

40

45

50

≤−24°C

Climate trend

≤−8°C 3

8

13

4–10 8–11 13–12 18–1 22–2 27–3

40

45

50

≤−24°C ≤−8°C 3

8

13

4–10 8–11 13–12 18–1 22–2 27–3

Week number (upper label) and date (day-month, lower label)

Fig. 19.1. The association of daily minimum temperature with (A) grape, (B) sweet cherry, (C) apple, (D) pear, (E) apricot and (F) peach production for week 40 to week 18 (4 September–3 May) and its comparison with climate trends for 36 years (1937–1973) with a later 18 years (1974–1991) over the same period. For the association of minimum temperature with production, the c2 values compare the daily weather occurrences of poor years (extreme low quartile of years ranked by production) each with that of neutral years (combined mid-quartiles of years ranked by production). All comparisons are based on a 3-week running average. Cardinal values (the weather value at the maximum c 2) are indicated on the plots. c2=7 (indicated by the dotted lines) is the critical value for significance (P£0.01, df=1).

H.A. Quamme et al.

χ2 for minimum temperature (low–high scan)

Poor production

Poor production 40

C

B

194

A

≤−6°C

Winter-freeze Events in Fruit Crops

195

Table 19.1. Major synoptic map patterns identified for the Okanagan Basin, British Columbia, Canada. Upper air feature

Cold/warm

Season

4 5 7 8 11 12 14 16 17

Arctic outflow North-west flow Cyclonic Zonal/unstable Anticyclonic Moist south-west flow Idaho high Convective Stagnant

Pacific ridge Weak ridge to west Weak trough – Weak ridge Offshore trough Strong ridge coast Offshore trough Blocking high

Very cold Cool Cool – Warm Warm – Warm Very hot

Winter All All All Winter Winter Autumn–winter Summer Summer

100

A

0

80

−10

60

−20

40

−30

20

−40 10

0 4

5

7

8

11 12 14 100

B

0

80

−10

60

−20

40

−30

20

−40 10

0 4

5

7

8

11 12 14 100

C

0

80

−10

60

−20

40

−30

20

−40

0 4

5

Map-type frequency (%)

10

Map-type frequency (%)

Of the nine winter-freeze events identified from historical records during the period 1949–2000 (H.A. Quamme, unpublished results), all had low-temperature extremes that coincided with the Arctic outbreak synoptic classification (Table 19.1). This verifies previous conclusions of studies of winter injury on fruit production in British Columbia (Boyer, 1977; Krueger, 1983). To capture the three phases of climate change identified in Fig. 19.1, the late autumn and winter season was subdivided into three critical periods: (i) weeks 41–52 (Julian days 305–366); (ii) weeks 1–6 (Julian days 1–42); and (iii) weeks 7–13 (Julian days 43–91). Between 1949 and 2006 the frequency of Arctic outflows (map type 4) in these three periods was 4.6%, 6.9% and 4.4% respectively (Fig. 19.2). Overall, the lowest temperatures were associated with Arctic outflow as were the most extreme cold events. Trends in the occurrence of Arctic outflows indicated an increase in frequency during weeks 41–52, a decrease in frequency during weeks 1–6 and no change in frequency during weeks 7–13 (Table 19.2). A significant increase in daily minimum temperatures associated with Arctic outflows occurred during weeks 1–6 and weeks

Minimum temperature (°C)

Relationship of Arctic outbreaks to winterfreeze events and climate change

Minimum temperature (°C)

Thus, it appears that climate change may affect the survival of these crops differently. The production zones of pear, peach and apricot should increase relative to grape, sweet cherry and apple.

Map-type frequency (%)

Circulation pattern

Minimum temperature (°C)

Map number

7 8 11 12 14 Map type

Fig. 19.2. Box-and-whisker plots of extreme minimum temperatures associated with each synoptic map-type occurring in three critical periods: (A) weeks 1–6 (Julian days 1–42), (B) weeks 7–13 ( Julian days 43–91) and (C) weeks 41–52 ( Julian days 305–366), during the period 1948– 2007. Bars show the overall relative frequency of map-type occurrence over the same period.

196

H.A. Quamme et al.

Table 19.2. Trends in the frequency of Arctic outflow events during critical periods and associated absolute minimum temperatures for 1948 to 2006. Analyses are based on a running 5-year average (n=54).

Critical period Weeks 41–52, Julian days 305–366 Weeks 1–6, Julian days 1–42 Weeks 7–13, Julian days 43–91

Linear trend (days/year) 0.028 −0.056 0.029

Significance P£0.05 P≤0.001 NS

Linear trend (°C/year) −0.004 0.070 0.083

Significance NS P£0.001 P£0.05

NS, not significant.

7–13 and no change in weeks 41–52, confirming the trends observed in minimum temperature in Fig.19.1. Although warm weather during autumn and winter can negatively affect production by dehardening the trees or vines, the impact was not as great as that of low minimum temperature per se and depended on the occurrence of subsequent periods of low temperature (Caprio and Quamme, 1999, 2002, 2006) to cause injury. In warmer climates than in the Okanagan Valley where Arctic outbreaks do not occur as frequently, the survival of these crops is better. Our conclusion is that the production of these six crops in the Okanagan Valley is limited by the occurrence of Arctic outflows. This is a relatively small region but the average annual minimum isotherm delineates the northern range of these crops in other regions (Quamme, 1976; Quamme et al., 1982). Furthermore, northern range coincides with the average annual minimum isotherm in many other woody plants that show deep supercooling (George et al., 1974; Quamme, 1989) (93 species listed). It is our contention that the Arctic

outflow is the prime synoptic pattern affecting winter survival of fruit crops in many northern regions and that this same weather event restricts the northern range of many ornamental crops and other perennial species. We did not determine a change in the occurrence of Arctic outflows with future climate change in the present work, but Taylor and Barton (2004) downscaled GCMs by correlating the grid output with local surface temperatures and projected that the average minimum temperature will continue to decrease, especially in midwinter and spring. Future changes in synoptic weather types can be projected from GCMs (Cheng et al., 2007). The next phase in our study of synoptic weather typing will be to identify future changes in Arctic outflow events in British Columbia and determine if it can be applied to other regions of Canada. Analysis of the synoptic map patterns of the Arctic outbreaks should lead to a better understanding of how low temperature limits the zones of perennial crop production and how future climate change may impact these limits.

References Barry, R.G. and Perry, A.H. (1973) Synoptic Climatology: Methods and Applications. Methuen, London. Boyer, J.C. (1977) Human Response to Frost Hazard in the Orchard Industry, Okanagan Valley, British Columbia. Public Series No. 8. Department of Geography, University of Waterloo, Waterloo, Ontario, Canada. Brewer, R. and Taylor, B. (2001) Climate change in the Okanagan Basin. In: Cohen, S. and Kulkarni, T. (eds) Water Management and Climate Change in the Okanagan Basin. Environment Canada and the University of British Columbia, Vancouver, British Columbia, Canada, pp. 14–20. British Columbia Department of Agriculture (1977–1992) Annual Horticultural Statistics. Ministry of Agriculture, Fisheries and Food, Government of British Columbia, Victoria, British Columbia, Canada. British Columbia Legislative Assembly Select Standing Committee (1978) The British Columbia Tree Fruit Industry, Phase III, Research Report, November 1978. Government of British Columbia, Victoria, British Columbia, Canada.

Winter-freeze Events in Fruit Crops

197

Cannon, A.J. and Whitfield, P.H. (2002) Synoptic map classification using recursive partitioning and principle component analysis. Monthly Weather Review 130, 1187–1206. Caprio, J.M. and Quamme, H.A. (1999) Weather conditions associated with apple production in the Okanagan Valley of British Columbia. Canadian Journal of Plant Science 79, 129–137. Caprio, J.M. and Quamme, H.A. (2002) Weather conditions associated with grape production in the Okanagan Valley of British Columbia and potential impact of climate change. Canadian Journal of Plant Science 82, 755–763. Caprio, J.M. and Quamme, H.A. (2006) Influence of weather on apricot peach and sweet cherry production in the Okanagan Valley of British Columbia. Canadian Journal of Plant Science 86, 259–267. Caprio, J.M., Quamme, H.A., and Redmond, K.T. (2009) Procedure to determine recent climate change of extreme daily meteorological data at two locations in northwestern North America. Climatic Change 92, 65–82. Cheng, C.S., Auld, H., Li, J., Klaassen, J., Tugwood, B. and Li, Q. (2004) An automated synoptic typing procedure to predict freezing rain. An application to Ottawa, Ontario. Weather and Forecasting 19, 751–768. Cheng, C.S., Auld, H., Li, J., Klaassen, J. and Li, Q. (2007) Possible impacts of climate change on freezing rain in south-central Canada using down-scaled future climate scenarios. Natural Hazards and Earth System Sciences 7, 71–87. De’Ath, G. (2002) Multivariate regression trees: a new technique for modeling species environment relationships. Ecology 83, 1105–1117. George, M.F., Burke, M.J., Pellet, H.M. and Johnson, A.G. (1974) Low temperature exotherms and woody plant distribution. HortScience 9, 519–522. Kalnay, E., Kanamitsu, M., Kistler, R., Collins, W., Deaven, D., Gandin, L., Iredell, M., Saha, S., White, G., Woolen, J., Zhu, Y., Chelliah, M., Ebisuzaki, W., Higgins, W., Janowiak, J., Mo, K.C., Ropelewski, C., Wang, J., Leetma, A., Reynolds, R., Jenne, R. and Joseph, D. (1996) The NCEP/NCAR 40-year reanalysis project. Bulletin of the American Meteorological Society 77, 437–471. Krueger, R.R. (1983) The orchard industry’s response to low-temperature injury in the Okanagan Valley. Canadian Geographer 27, 315–327. Quamme, H.A. (1976) Relationship of the low temperature exotherm to apple and pear production in North America. Canadian Journal of Plant Science 56, 493–500. Quamme, H.A. (1986) Use of thermal analysis to measure freezing resistance of grape buds. Canadian Journal of Plant Science 66, 945–952. Quamme, H.A. (1989) Deep supercooling in buds of woody plants. In: Lee, R.E. Jr., Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 183–199. Quamme, H.A., Layne, R.E.C. and Ronald, W.G. (1982) Relationship of supercooling to cold hardiness and the northern distribution of several cultivated and native Prunus species and hybrids. Canadian Journal of Plant Science 62, 137–148. Salmi, T., Maata, A., Anttila, P., Ruoho-Airola, T. and Amnell, T. (2002) Detecting Trends of Annual Values of Atmospheric Pollutants by the Mann–Kendall Test and Sen’s Slope Estimates. Publications on Air Quality #31. Finnish Meteorological Institute, Helsinki; available at http://www.fmi.fi/kuvat/MAKESENS_MANUAL.pdf. Taylor, B. and Barton, M. (2004) Climate. In: Cohen, S., Neilsen, D. and Welbourne, R. (eds) Expanding the Dialogue on Climate Change in the Okanagan Basin, British Columbia. Environment Canada and the University of British Columbia, Vancouver, British Columbia, Canada, pp. 25–44. Yarnal, B. 1993. Synoptic Climatology in Environmental Analysis: a primer. Bellhaven Press, London. 195pp. Zhang, X., Vincent, L., Hogg, W.D. and Niitsoo, A. (2000) Temperature and precipitation trends in Canada during the 20th century. Atmosphere–Ocean 38, 395–429.

20

Cold Hardiness in Antarctic Vascular Plants

L.A. Bravo, L. Bascuñán-Godoy, E. Pérez-Torres and L.J. Corcuera

Introduction The Antarctic is the coldest region in the world. About 98% of its territory is covered by ice. Thus, only a small portion of the Antarctic continent is available for colonization by plants. Most of the ice and snow-free land is found along the Antarctic Peninsula, in the so-called Maritime Antarctic. Although some of the lowest temperatures in the world are registered on this continent, temperatures are much milder in the costal areas of the Antarctic Peninsula and associated islands. The mean summer temperature near the coast of the South Shetland Islands is about 2.8°C (Zúñiga et al., 1996). Although temperatures remain with little variation during one day, weekly variations are higher. For example, low temperature may range from −4 to 0°C and high temperature from 0 to 6°C (Edwards and Smith, 1988; Zúñiga et al., 1996). During winter, the whole continent is covered by ice or snow. Even when winter air temperatures reach −20°C, plants are protected by the snow cover (Winkler et al., 2000). The winter snow cover usually melts by the end of spring, when the growing season for vascular plants begins. During summer, there is rain and occasional snow. The growing season ends by the end of February. In the South Shetland Islands, where most of the Antarctic vegetation is found, precipitation

198

is about 400 mm/year, and about 100 mm/ year in Marguerite Bay (Redón, 1985). In the past 50 years, along the west coast of the Antarctic Peninsula, the mean annual temperature has risen by about 2.6°C and the mean summer air temperature by 1.5°C (Day et al., 1999; Xiong et al., 2000). More evidence of regional warming comes from the dramatic retreat of ice shelves along the west coast of the Peninsula over this period (Sympson, 2000). Glaciers have been retreating rapidly throughout the Antarctic Peninsula, receding by up to 150 m/year (Cook et al., 2005). This is generating more ice-free land, where future colonization by plants may take place. Irradiance and photoperiod are highly variable in the Antarctic. The highest radiation and photoperiod occur in summer and the lowest in winter (Kappen, 1993). On clear summer days, irradiance reaches up to 2000 µmol photons/m2/s (Schroeter et al., 1995). In summer, day length at the northern end of the Antarctic Peninsula is shorter than at the southern end. Plants in the northern part of the Peninsula receive 3–4 h of darkness in the early summer. Those within the Antarctic Circle may not experience darkness in that period. These combined conditions of high irradiance and near-zero temperatures are generally unfavourable for plants because they

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Cold Hardiness in Antarctic Vascular Plants

may cause photoinhibition of photosynthesis (Demmig-Adams and Adams, 1992). An additional stress for plants may be the excess proportion of UV-B due to the stratospheric ozone hole over the Antarctic (Day et al., 1999; Xiong and Day, 2001). The Antarctic vegetation is very poor in the number of flowering plants. Only two have been able to naturally colonize parts of the Maritime Antarctic (Fig. 20.1). These are the hair grass Deschampsia antarctica Desv. (Poaceae) and the pearlwort Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae) (Smith, 2003). These two species are found in the South Orkney Islands and in most of the Maritime Antarctic down to ~68°S, but do not extend into the continental Antarctic (Greene and Holtom, 1971). D. antarctica has a remarkably wide ecological amplitude and competitive tolerance. It has colonized habitats ranging from mineral soils to organic soils, nutrient-deficient to highly nutrient-enriched, and from dry to waterlogged areas. C. quitensis is less tolerant to extreme conditions, preferring sparsely vegetated, sheltered, moist, well-drained mineral soils. The Antarctic hair grass is much more abundant and widely distributed than the pearlwort (Smith, 2003). The populations of these vascular species, especially D. antarctica, are expanding in the Maritime Antarctic (Casaretto et al., 1994; Smith, 1994; Day et al., 1999). For this reason, it has been suggested to use them as indicators of global warming and UV-B increment (Smith, 1994; Rozema et al., 2001). What is so special about these two species that has enabled them to be the only successful flowering plants in the Antarctic? This question was fully analysed in inspired articles by Smith (2003) and Alberdi et al. (2002). To survive in the Antarctic, plants must be able to cope with severe physiological stresses, especially during the growing season (Antarctic summer), caused by low temperature and repeated freezing and thawing, desiccation, low availability of water, and high irradiance. The aim of the present chapter is to make an up-to-date analysis of the properties of these plants that allow them to survive in the harsh Antarctic conditions.

199

Global Warming and UV-B Radiation Increase and their Effects on Antarctic Vascular Plants Over the last centuries, a series of changes in general climatic condition of the planet have been observed. This phenomenon has been termed ‘global change’ (Walther, 2003). One of the most studied and well documented is the global increase in the average planet temperature. It has been estimated that over the past century the average temperature has increased by 1°C. Several models predict that temperature will increase by 1.5–4°C in the next 100 years. It has been established that some organisms appear to be more sensitive than others to this change (Walther et al., 2002; Nemani et al., 2003). Maritime Antarctica is one of the ‘hot spots’ that is responding dramatically to the temperature increase and associated effects. Low summer temperature, water availability and short length of the growing season are the major limiting factors for plant reproduction in the Antarctic (Convey, 1996; Convey and Smith, 2006). Besides, as most of continental Antarctica and the sub Antarctic Islands are glaciated, soil availability is another important factor that limits plant growth. Global warming is altering these factors dramatically (Karentz, 2003). Therefore, the ecological success of flowering plants in the Antarctic will increase. Longer growing seasons with higher temperatures, ice retreat and higher frequency of rains have determined an expansion of D. antarctica and C. quitensis (Smith, 1994; Gerighausen et al., 2003). Increases in the surface covered by flowering plants, number of populations and increase in the size of plants have been reported in the last 20 years. As mentioned, the expansion rate of Antarctic flowering plants has been proposed as a bioindicator of global warming (Smith, 1994). Even though most studies about Antarctic flowering plants deal with their adaptations to cope with low temperatures (see below), there is a little evidence, at least from D. antarctica, that plants exposed to heat shock express heat-shock proteins (HSP70) and tolerate exposure to high temperature, exhibiting high temperature LT50 similar to heat-tolerant plants (Reyes et al., 2003).

200

L.A. Bravo et al.

90°

80°

70°

60°

°

50

50°

45°20’

B

Islas Orkney del Sur

60°38’

0

100°

30 Km

A

°

60

40°

0’

°3

66 110°

°

70

30°

120°

130°

° 80

140°

0

725 Km

N

Fig. 20.1. The Maritime Antarctic. Deschampsia antarctica and Colobanthus quitensis are mostly found in the ice-free coastal areas of the Antarctic islands (insets A and B) as far south as Marguerite Bay (68°30′S, 68°30′W). These plants are also found along the Andes in South America.

Cold Hardiness in Antarctic Vascular Plants

Therefore these plants may have physiological mechanisms to adapt their metabolism to higher temperatures resulting in an increase of net photosynthesis, maintenance of dark respiration and a higher allocation to leaf area production (Xiong et al., 2000). Furthermore, low genetic variation (Holderegger et al., 2003) and low seed viability due to a lack of seed maturation during the short growing season observed in these species in the Antarctic (Convey, 1996) predict that vegetative growth has been the major propagating strategy and hence in plant colonization. The increase in temperature and longer growing seasons may have a strong influence on seed maturation and reproductive structures formation which may favour sexual reproduction and seed formation and viability. These changes will contribute to increase the rapid expansion experienced by flowering plant species to newly available areas released by ice melting. Therefore, there is a general perception that flowering plants have benefited from the temperature rise; however, there is a very important and poorly studied phenomenon that may have serious consequences for the biological success of these plants. It is how these flowering plants will respond to potential inputs of new flowering competitive species that may be able to colonize as environmental conditions get milder. Ozone depletion of the stratosphere has occurred with more intensity in the coldest regions. For this reason, UV-B increase has been observed with more intensity in polar areas, especially in the Antarctic (Karentz, 2003). UV-B damages nucleic acids, promotes oxidative stress and therefore affects proteins, membrane integrity and many cell functions. Long-term effects of UV-B exposure have not been properly evaluated. None the less, apparent ecological damage by increased UV-B in the Antarctic is far from the very negative effects initially thought. UV-B radiation has indeed increased in the Antarctic, but it is still well below the levels found at lower latitudes and high altitude. In spite of this, the two Antarctic vascular plants respond to UV-B treatments. Substantially high concentrations of UV-B-absorbing flavonoids and carotenoids have been found in these plants when they are exposed to ambient UV-B (Valladares et al., 1997; Montiel et al., 1999; Ruhland and Day,

201

2001). UV-B radiation does not affect photochemical yield and photosynthetic rate (Day et al., 1999; Xiong and Day, 2001), but reduces dry matter production and total leaf area and increases tillering and number of inflorescences (Day et al., 2001; Lud et al., 2001). In general, it appears that the two Antarctic vascular plants are well prepared to handle the present levels of radiation found in the Antarctic.

Freezing Resistance in Antarctic Vascular Plants These unique vascular species have contrasting mechanisms to cope with freezing. While C. quitensis avoids freezing by moderate supercooling, D. antarctica is able to tolerate ice formation in its tissues (Bravo et al., 2001). This has been studied by thermal analysis combined with the LT50 injury index. The first method allows one to determine the temperature at which ice begins to form (ice-nucleation temperature, NT). C. quitensis experiences tissue injury at higher temperatures than apoplastic freezing (LT50=−5.8°C > NT=−9.4°C) when ice nucleators are included in the LT50 assays to avoid supercooling (Bravo et al., 2001). However, LT50 decreases when plants are allowed to supercool, especially in coldacclimated plants, which reach LT50 of −14°C (Gianoli et al., 2004). D. antarctica is more freezing-tolerant than C. quitensis, always exhibiting lower LT50 than NT. Besides it has a bigger capacity to cold-acclimate than the pearlwort, exhibiting LT50 of −12°C in the non-acclimated and −27°C in the cold-acclimated state (Bravo et al., 2001; Alberdi et al., 2002). Judging by their LT50, Antarctic vascular plants are not the most freezing-tolerant plants in the world. D. antarctica has a similar LT50 to those exhibited by winter cereals, such as coldacclimated winter rye. C. quitensis is within the range of LT50 observed in other plants from cold environments in high elevations or high-latitude temperate zones (Table 20.1). Therefore, what really distinguishes Antarctic plants is that they must cope with low temperatures year-round. This imposes two major constraints: first, they must grow and reproduce under continuous low-temperature conditions. This implies that

202

L.A. Bravo et al.

Table 20.1. Freezing tolerance (range of LT50) in Antarctic plants compared with other plants from different ecosystems, high elevations, temperate forests, winter cereals and Arabidopsis thaliana mutants. Species/habitat

LT50, NA/CA (°C) Reference

Antarctic plants Colobanthus quitensis Deschampsia antarctica

−6.8/−14.4 −12/−26.6

Alpine/Andean herbs C. quitensis Phacelia secunda Poa alpine Hordeum comosum Nastanthus spathulatus Ranunculus glacialis

−6.8/−10.3 −12.8/−16.8* −10.0/−14.5* −11.5/−20.0* −13.2/−18.0* −7.9/−8.4*

Temperate-zone trees Nothofagus nitida Nothofagus dombeyi Lomatia ferruginea Embothrium coccineum Gevuina avellana

−6.78/−10.1 −7.0/−14.3 −5.3/−10.3 −5.8/−13.9 −6.4/−12.1

Reyes-Díaz et al. (2005) Reyes-Díaz et al. (2005) Reyes-Díaz et al.a (unpublished results) Reyes-Díaz et al.a (unpublished results) Reyes-Díaz et al.a (unpublished results)

Crop cereals Hordeum vulgare Secale cereale (winter) Triticum aestivum (winter)

−4.8/−11.3 −8.0/−30.0 −6.0/−16.0

Bravo et al. (1998) Griffith and McIntyre (1993) Cloutier and Siminovitch (1982)

Other plants A. thaliana cv. Columbia A. thaliana, ESK-1 A. thaliana, Frs-1

−6.5/−8.5 −6.5/−12.4 −4.5/−8.3

Reyes-Díaz et al. (2006) Reyes-Díaz et al. (2006) Reyes-Díaz et al. (2006)

Gianoli et al. (2004) Bravo et al. (2001) Gianoli et al. (2004) Sierra-Almeida et al. (2009) Taschler and Neuner (2004) Sierra-Almeida et al. (2009) Sierra-Almeida et al. (2009) Taschler and Neuner (2004)

NA, non-acclimated; CA, cold-acclimated; *summer/winter field acclimation. a M.M. Reyes-Díaz, M.R. Alberdi, F. Fuentes-Neira, L.A. Bravo and L.J. Corcuera (unpublished results).

their metabolism is adapted to work under these conditions. For instance, low temperature may seriously impair respiration and photosynthesis or cause photoinhibition if it is combined with even moderate irradiance in sensitive plants (Huner et al., 1998). Second, these plants have to be prepared to undergo freezing even in the growing season. Actually, they are covered by snow during winter, which protects them from exposure to extremely low air temperature that certainly occurs in the coldest season (Smith, 2003). Several biochemical and physiological mechanisms of freezing tolerance are present in these plants during the growing season. For example, apoplastic antifreeze activity associated with apoplastic soluble proteins has been found in D. antarctica even in non-acclimated plants, being the first plant described that

possesses constitutive expression of these proteins (Bravo and Griffith, 2005). It is likely that this peculiarity is due to the constant lowtemperature selection pressure in which this plant has evolved. None the less, these plants are able to respond to low-temperature exposure, inducing an increase in antifreeze activity (Bravo and Griffith, 2005) as well as other stress-induced proteins such as dehydrins (OlaveConcha et al., 2004) during cold acclimation. Dehydrins contain a 15 amino acid-repeat motif; since this motif is rich in lysine, it is usually called K segment (K, one-letter code for lysine). This motif is in the carboxy terminal or several times repeated within the polypeptide. The K segment resembles a class A2 amphipathic a-helical, lipid-binding domain found in other proteins such as apolipoproteins and

Cold Hardiness in Antarctic Vascular Plants

a-synuclein (Davidson et al., 1998). There is evidence that the K segment plays an essential role in cryoprotection or in preventing freezeinduced dehydration injury of cells and macromolecular structures (Lin and Thomashow, 1992; Houde et al., 1995; Honjoh et al., 2000; Bravo et al., 2003). Dehydrin expression has also been shown in D. antarctica subjected to drought, osmotic and salt stress, and by abscisic acid treatment (Olave-Concha et al., 2004). Series of other biochemical responses, including soluble sugars and proline accumulation (Bravo et al., 2001), gene expression (Gidekel et al., 2003) and increase of antioxidants and antioxidant enzymes (Pérez-Torres et al., 2004a,b), have been found in these Antarctic species. The magnitude of these responses is more intense in D. antarctica than in C. quitensis, which probably explains their differences in freezing tolerance or may be related with their different mechanisms to cope with freezing temperatures (avoidance versus tolerance). None the less, a common biochemical response to low temperature of both species is sucrose accumulation and low-temperature up-regulation of sucrose-phosphate synthase (SPS), the key enzyme of sucrose biosynthesis (Zúñiga-Feest et al., 2003; Bascuñán-Godoy et al., 2006). There is plenty of evidence that sucrose cryoprotects macromolecules, proteins and membranes under freezing and that it may also prevent macromolecular aggregation caused by freezing-induced cell dehydration (Strauss and Hauser, 1986; Santarius, 1992). It is important to remark that even though it is likely that the major limitation in the Antarctic summer is low temperature, plants are also exposed to other stressors such as low physiological water availability, marine salt spray, increased UV-B (Smith, 2003) and a long period of snow cover or even ice encasement, which may impose a hypoxic environment (Gudleifsson and Larsen, 1992; Griffith et al., 2001). For instance, the Antarctic grass seems to be well prepared to deal with drought. Anatomically, D. antarctica exhibits xerophytic characteristics (reduced leaf surface, small epidermal cells, thick leaves, high stomata density and number of cells per area, thick cuticle). Besides, the vascular bundles of D. antarctica are surrounded by two bundle sheaths (Vieira and Mantovani, 1995): an outer sheath, with parenchymatous

203

cells with very small chloroplasts (Romero et al., 1999), and an inner sheath with thick lignified walls called mestome. It has been suggested that mestome functions as endodermis, limiting apoplastic movement of water to the mesophyll (Evert et al., 1985). In C. quitensis, the bundle sheath lacks a mestome, although leaf thickness and mesophyll surface area values are typical of xeric plants (Mantovani and Vieira, 2000). A decrease of water supply to the mesophyll could be advantageous to D. antarctica since water control could take place in vascular bundles, in addition to the stomata, with a high control capacity to water loss (Vieira and Mantovani, 1995). Additionally, low transpiration avoids heat loss in cold environments, being an important adaptation for this habitat (Alberdi et al., 2002). The colonization, survival and expansion of C. quitensis populations in the Maritime Antarctic are evidences that extreme freezing tolerance is not needed to inhabit Antarctica. It seems that functional metabolism (photosynthesis, respiration, etc.) and maintenance of active sinks at low temperature, combined with efficient excess energy dissipation (Bravo et al., 2007; Pérez-Torres et al., 2007) and a moderate freezing avoidance, are enough to deal with the Antarctic summer. How the global warming scenario may affect freezing tolerance or even the strategies of these species to cope with freezing are interesting questions that should be addressed in the near future. For instance, what physiological mechanisms do these plants possess to deal with high temperature? Will constitutive freezing resistance mechanisms persist or will they disappear if warmer summers reduce selective pressures?

Photosynthesis in the Cold Photosynthesis is a complex multi-step process that involves transformation of solar energy to chemical energy. Absorption of photons is mostly independent of temperature, but metabolic steps associated with the use of this energy have a close relationship with temperature. Cold in the dark reduces metabolic rates. This usually affects growth; however, during the day, when plants are actively harvesting light, low temperatures may have significant

204

L.A. Bravo et al.

effects in terms of energy balance, because more energy is absorbed than can be used or dissipated (Huner et al., 1998). Excess energy determines an increased production of active oxygen species such as singlet oxygen, due to the reaction of 3Chl and oxygen; superoxide is formed by the reaction of electrons leaked from the photosynthetic electron transport chain and oxygen (Suzuki and Mittler, 2006). Active oxygen species may cause irreversible damage to cell structures, with severe consequences for the plant (Alscher et al., 1997). Damage caused by absorption of excess energy can lead to photoinhibition, which is defined as a reduction in photosynthetic efficiency or maximum photosynthetic rates by light (Niyogi, 1999). The extent of photoinhibition is the result of the capacity of the plant to regulate light absorption, its efficiency for energy dissipation, the degree of damage and the capacity of repair mechanisms. Therefore, plants are constantly challenged to maximize photosynthetic rates and minimize damage by production of active oxygen species despite daily fluctuations in temperature and light. Plants have photoprotective mechanisms that allow either modulation of energy absorption (change in leaf angle, self-shading chloroplasts) or dissipation of absorbed energy (non-photochemical quenching (NPQ) and photochemical quenching (qP) ) (Demmig-Adams and Adams, 1992; Niyogi, 1999). In particular, NPQ involves a series of events in which absorbed energy is safely released as heat. In the northern hemisphere, higher-latitude ecotypes of dogwood (Cornus sericea L.) expressed higher NPQ values compared with lower latitude ecotypes under light and low temperature conditions (K. Tanino, Saskatoon, Canada, 2008, personal communication). NPQ is dependent on transthylakoidal ∆pH and is partially the result of conformational changes in photosystem II complex (PSII) and synthesis of zeaxanthin from violaxanthin (Horton and Ruban, 2005; Niyogi et al., 2005; Szabó et al., 2005; Ruban et al., 2007). qP involves processes that optimize the electron flux outside the reaction centre such, as the water–water cycle, photorespiration, the Calvin cycle and cyclic electron flow around photosystem I complex (PSI), that allow modulation of the redox status of the photosynthetic electron

transport chain (Holaday et al., 1992; Kozaki and Takeba, 1996; Asada, 1999; Niyogi, 1999; Johnson, 2005). Both strategies are fundamental to maximize energy absorption and minimize damage. They are highly relevant in plants exposed to cold environments. In this regard, Antarctic vascular plants constitute a unique system to study adaptations to maintain photosynthesis in the cold, considering that they are naturally exposed during the day to a temperature range from 0 to 6°C in the Maritime Antarctic summer (Edwards and Smith, 1988; Alberdi et al., 2002). Thus, D. antarctica and C. quitensis usually cope with simultaneous episodes of high light and low temperature. These episodes are known to increase over-reduction of the photosynthetic electron transport chain, favour the generation of active oxygen species and cause photodamage in other plants (Alberdi et al., 2002). C. quitensis and D. antarctica have optimum photosynthetic temperatures of 13 and 19°C, respectively. These plants maintain approximately 30% of their maximum photosynthetic rate at 0°C and sustain high qP at low temperature (Edwards and Smith, 1988; Xiong et al., 1999). These traits are thought to partially contribute to a net carbon gain under conditions that are usually lethal to other plants. These traits are, therefore, fundamental for their survival in the Maritime Antarctic. Despite living in the same environment, Antarctic vascular plants have been shown to use different photoprotective strategies in terms of using oxygen as an electron sink through the water–water cycle (Pérez-Torres et al., 2007). In particular, the water–water cycle involves the process by which electrons leaked from the photosynthetic electron chain form superoxide. This superoxide is safely scavenged by the consecutive action of superoxide dismutase and antioxidant enzymes of the Halliwell–Asada pathway, such as ascorbate peroxidase and glutathione reductase (Asada, 2000). In particular, we propose that D. antarctica promotes the use of oxygen as an electron sink by donating electrons to oxygen at PSII and superoxide formed would be later scavenged by antioxidant enzymes (Fig. 20.2). This is supported by the close relationship between PSII electron transport rate and

Cold Hardiness in Antarctic Vascular Plants

Deschampsia antarctica NADP + H

205

Colobanthus quitensis

NADPH

NADP + H

Calvin–Benson cyle

Calvin–Benson cyle

Cyclic electron flow

Cyclic electron flow

PSII

PQ

O2

Cyt b6f

PSI

PSII

O2•

SOD

heat

PQ

Cyt b6f

PSI PC

PC –

NADPH

heat

heat

heat

H2O2 Halliwell–Asada pathway

H2O

Fig. 20.2. Differences in photoprotective mechanisms in Antarctic vascular plants at low temperature. PSII, photosystem II; PSI, photosystem I; Cyt b6f, cytochrome b6f; PQ, plastoquinone; PC, plastocyanine; SOD, superoxide dismutase.

SOD activity (U/g fresh weight)

2000 1500 1000 500 0

D .a D ntar .a c n tic C tarc a ( . q ti C c A C uite a ( ) .q n N u sis A) A. iten (C th sis A) A ali (N L. . th ana A) es ali (C c a A. ule na A) f L. ist ntu (NA an ulo m ) gu su (NA st m ) ifo (N liu A s ) (N A)

oxygen levels under non-photorespiratory conditions and high levels of antioxidant enzymes, especially superoxide dismutase, reported for this plant (Fig. 20.3) (Pérez-Torres et al., 2004b, 2007). On the other hand, in C. quitensis, electron transport rates in neither PSI nor PSII are correlated with oxygen levels under non-photorespiratory conditions, suggesting that oxygen is not used significantly as an electron sink in this plant (Fig. 20.3) (PérezTorres et al., 2007). In line with these results, it has been reported that C. quitensis has average levels of antioxidant enzymes, suggesting a low contribution of the water–water cycle to the modulation of redox state of the photosynthetic electron transport chain (Pérez-Torres et al., 2004a). The use of oxygen as an alternative electron sink is potentially dangerous considering that active oxygen species may cause massive damage to cell structures. However, high levels of antioxidants coupled with their strategic distribution throughout the cell could be determinant to making this a secure photoprotective alternative. Thermal dissipation of excess energy (NPQ) is considered a safe and fast alternative

Fig. 20.3. Superoxide dismutase (SOD) activity in plants. SOD activity for Colobanthus quitensis, Deschampsia antarctica and Arabidopsis thaliana were determined in non-acclimated conditions (NA) and after cold acclimation (CA) for 21 days at 4°C. SOD activity for Lycopersicon esculentum, Allium fistulosum and Lupinus angustifolius is taken from literature values (Un-Haing and Jung-O, 2000; Stajner et al., 1998; Yu and Rengel, 1999, respectively).

206

L.A. Bravo et al.

to balance energy in plants (Demmig-Adams and Adams, 1992; Szabó et al., 2005). This mechanism is especially safe because it occurs before photochemistry and therefore involves energy rather than electron transfer. Both Antarctic plants increase NPQ with light intensity at low temperature, indicating active dissipation of energy as heat under these conditions (Pérez-Torres et al., 2007). In particular, it has been reported that NPQ in C. quitensis is mainly associated with the fast relaxing component of NPQ, which also depends on transthylakoidal ∆pH and the xanthophyll cycle (Bravo et al., 2007). This indicates that thermal dissipation in C. quitensis is mostly due to dynamic and reversible processes, and emphasizes adaptability to a changing light environment in the cold. Cyclic electron flow around PSI may have an important contribution to form the transthylakoidal ∆pH in Antarctic vascular plants. This process involves recycling of electrons from reduced ferredoxin or NADPH to plastoquinone, with the consequent transport of protons to the thylakoid lumen and ATP formation (Johnson, 2005). The contribution of this process to ATP formation in normal conditions is minimal in C3 plants; however, it has been suggested to have an important role under conditions of high light and low temperature (Bendall and Manasse, 1995). Both Antarctic plants showed evidence of cyclic electron flow around PSI at low temperature when exposed to increasing light intensities (Pérez-Torres et al., 2007). Interestingly, variations in PSI electron transport rate were closely related to NPQ and suggest a contribution of cyclic electron flow around PSI to thermal dissipation of energy. Therefore, cycling of electrons at this level may have an important contribution in terms of modulation of redox state of the photosynthetic electron transport chain (photochemical quenching) and dissipation of excess energy as heat (non-photochemical quenching). Key enzymes of the Calvin cycle remain highly active at low temperature in Antarctic vascular plants (Pérez-Torres et al., 2006). A robust activity of enzymes involved in CO2 assimilation at low temperature suggests that CO2 remains the main electron sink in the cold, and supports observations of positive photosynthetic rates of these plants even at

0°C (Edwards and Smith, 1988). Active photosynthesis at low temperature has repercussions not only in terms of use of the absorbed energy but also in the survival of the plant, as some carbohydrates have important roles in protection of cell structures at freezing temperatures (Bravo et al., 2001). In summary, Antarctic vascular plants rely on efficient photoprotective mechanisms that allow harvesting light in the cold, minimizing energy imbalance and damage by active oxygen species. Additionally, the safe absorption of light energy at low temperature is coupled with a robust enzymatic system that allows them to sustain positive photosynthetic rates in the cold, thus contributing to the success of these plants in the Antarctic environment.

Carbohydrates in Antarctic Plants Antarctic plants present high levels of carbohydrates in leaves. These are mainly soluble sugars, which are the main product of photosynthesis (Zuñiga et al., 1996; PiotrowiczCieslak et al., 2005). Several of these sugars are often compatible solutes, which can act as osmoregulators, maintaining the hydration state of tissues (Morgan, 1984). One of the most common sugars in plants is sucrose. In addition to its osmoregulatory role, sucrose can act as a cryoprotector, reducing the damage produced by freezing and thawing, stabilizing membrane lipids and proteins (Santarius, 1992; Uemura and Steponkus, 2003). Both Antarctic plants store sucrose at low temperature (Bravo et al., 2001; Zúñiga-Feest et al., 2003). In addition to sucrose, these plants present polymers of (fructosyl)n-sucrose. C. quitensis stores raffinose family oligosaccharides (RFOs) and D. antarctica accumulates fructans during cold acclimation (Zuñiga et al., 1996; Bravo et al., 2001; Piotrowicz-Cieslak, et al., 2005). The role of these polymers during cold acclimation is not completely understood (Livingston, 1996; Zuther et al., 2004). Sucrose accumulation and distribution is affected by photoperiod and growth temperature (Zúñiga-Feest et al., 2003). Accumulation of sucrose at long photoperiod (21 h light/3 h dark) and low temperature is well related with an increase in the export of sugars to the crown

Cold Hardiness in Antarctic Vascular Plants

in D. antarctica and to the root system in C. quitensis. In D. antarctica grown at a 16 h light/8 h dark photoperiod, sucrose also increases in leaves, but there is a decrease of sugars in the non-photosynthetic organs. From these results, we can suppose that the increase of sucrose in the leaves of D. antarctica at long photoperiod is due to an increase in the synthesis of sucrose. Since sugars in the crown decrease when D. antarctica plants are grown in a 16 h light/8 h dark photoperiod, it is likely that there is conversion of other sugars into sucrose or maybe just degradation. Sucrose content in this photoperiod is very similar to that found in February in the field when days get shorter and temperature decreases (Zuñiga et al., 1996). Accompanying a sucrose increase in the field, there is an increase in the amount of fructans. Their degree of polymerization is lower at 16 h light/8 h dark photoperiod than at 21 h light/3 h dark (Bravo et al., 2001). It has been suggested that the increase in sucrose in leaves in response to cold acclimation helps plants to resist freezing temperatures. Additionally, the accumulated sugars could be a useful carbohydrate source in times of negative carbon balance (Bravo et al., 2001). The amount of sucrose reached by D. antarctica with cold acclimation is unusually high compared with other cold-resistant grasses (Table 20.2). C. quitensis, the other Antarctic vascular plant, reaches lower sucrose content than D. antarctica.

207

The high concentration of sucrose in D. antarctica has been related with a high activity of SPS (Zúñiga-Feest et al., 2003). Since the SPS-catalysed reaction is the limiting step of sucrose biosynthesis, SPS is considered the key enzyme of the pathway. SPS activity is regulated by environmental factors that could affect transcription, translation and post-translation modifications (Reimholz et al., 1997; Toroser and Huber, 1997). SPS also presents allosteric modulation by inorganic phosphorus (Pi), an inhibitor, and glucose-6-phosphate, an activator (Amir and Preiss, 1982). SPS from Antarctic plants is regulated by light and cold (ZúñigaFeest et al., 2003, 2005; Bascuñán-Godoy et al., 2006). Diurnal changes have been observed in SPS activity of C. quitensis and D. antarctica with a maximum at noon. The increase in SPS activity during the light period has been related with the activation of phosphatase type II (Zúñiga-Feest et al., 2005, Bascuñán-Godoy et al., 2006). Studies in spinach and tomato leaves have suggested that the diurnal changes in SPS activity are produced by dephosphorylation–phosphorylation of a serine residue by phosphatases and kinases, respectively (Jones and Ort, 1997). Cold acclimation induces an increase in activity of SPS of both Antarctic plants, without an increase in the protein levels (ZúñigaFeest et al., 2005. Bascuñán-Godoy et al., 2006). This increase in activity without changes in the amount of protein was studied

Table 20.2. Changes in the content of sucrose (mg/g dry weight) in leaves of Colobanthus quitensis and Deschampsia spp. subjected to cold acclimation (21 days at 4°C, photon flux density of 120 µmol/ m2s) at different photoperiods: medium (16 h light/8 h dark) and long (21 h light/3 h light).

Species C. quitensis Deschampsia antarctica Deschampsia caespitosa Deschampsia beringensis

NA (15°C) (mg/g dry weight)

CA (4°C) (mg/g dry weight)

Medium photoperiod

Medium photoperiod

Long photoperiod

4±1 16±4 17±1 18±1

13±1 60±15 21±1 6±2

14±2 54±7 9±3 36±1

Values are means±standard error of two separate experiments with three replicates each. NA, non-acclimated; CA, cold-acclimated

208

L.A. Bravo et al.

Co ld

in more detail in C. quitensis. The working model establishes that cold acclimation decreases the affinity for Pi, an SPS inhibitor, tenfold (Fig. 20.4). This could be especially important because Pi increases tenfold in the cytosol of cold-acclimated Arabidopsis (Hurry et al., 2000). In fact, the decrease of SPS affinity for Pi could induce an increase for the affinity of the activator glucose-6phosphate, that at the same concentration induces a higher affinity for SPS substrates (UDP-glucose and fructose-6-phosphate) in cold-acclimated than in non-acclimated plants. These changes induce an increase in enzyme activation and produce sucrose accumulation (Bascuñán-Godoy et al., 2006). SPS substrates and other cytosolic sugars are products of the condensation of triose phosphates (TP) obtained from the Calvin cycle. Optimal photosynthesis requires a balance between the cytosolic sucrose synthesis and rates of carbon fixation in the chloroplast. Excessive sucrose synthesis depletes phosphorylated Calvin cycle intermediates and inhibits regeneration of

c ac

ribulose-1,5-bisphosphate. Conversely, inadequate sucrose synthesis leads to accumulation of phosphorylated intermediates and depletion of Pi, resulting in inhibition of ATP synthesis, accumulation of glycerate-3-phosphate and inactivation of RuBisCO (Ensminger et al., 2006). One of the most important reactions in the Calvin cycle is CO2 fixation, which is catalysed by RuBisCO. RuBisCO activity from Antarctic plants is regulated by light and cold (PérezTorres et al., 2006). The degree of activation of this enzyme in both cold-acclimated Antarctic plants is close to 100%. RuBisCO activity in cold-acclimated plants of C. quitensis is maintained, while this activity in D. antarctica decreases. In spite of this, both cold-acclimated plants are able to keep a high rate of CO2 assimilation at low temperature suggesting there are other regulatory mechanisms that affect the Calvin cycle (Pérez-Torres et al., 2006). Fructose-1,6-bisphosphatase (FBPase) has a key regulatory role in the destination of photoassimilated carbon. Higher stromal

ation lim RuBiscO

Triose phosphate

CO2 [Pi] Calvin cycle

Triose phosphate [Pi]

Fructose-1,6-biphosphate Fructose-6-phosphate

Fructose-1,6-biphosphate

Ki

Glucose-6-phosphate

FBP Glucose-1-phosphate Fructose-6-phosphate

Chloroplast

(-)

(+)

UDP-Glucose

SPS Fructose-1,6-biphosphate + UDP-Glucose Cytoplasm

Sucrose

Fig. 20.4. Mechanistic working model of sucrose-phosphate synthase (SPS) regulation by low temperature in leaves of Colobanthus quitensis. RuBisCO and stromal fructose-1,6-biphosphatase (FBP) activities at low temperature keep the interchange of triose phosphates and inorganic phosphorus (Pi) between the chloroplast and cytoplasm. Sugars are formed by the condensation of triose phosphates in the cytoplasm. SPS, the key enzyme in sucrose synthesis, is activated (+) by glucose-6-phosphate and inhibited (−) by Pi. Cold acclimation modulates SPS activity of C. quitensis by decreasing the affinity of SPS for Pi (tenfold increase of the inhibition constant, Ki) and increasing the activation by glucose-6-phosphate resulting in a high level of SPS activity (>) and sucrose accumulation (>) in C. quitensis leaves. (=) indicates equal activity before and after cold acclimation.

Cold Hardiness in Antarctic Vascular Plants

FBPase levels may involve lower export of TP to the cytosol and consequently affect sucrose synthesis. However, with respect to non-acclimated plants, activity of FBPase from both cold-acclimated Antarctic plants is maintained at high light intensity and low temperature. Maintaining high activities of key enzymes in the Calvin cycle and a high rate of sucrose synthesis may be partially responsible for the positive CO2 assimilation rates observed in cold-acclimated plants (Pérez-Torres et al., 2006). These factors could also be responsible for the unusual rate of photosynthesis of Antarctic plants at 0°C (30% of its optimal photosynthesis) (Xiong et al., 1999). The modulation of Calvin cycle enzymes and sucrose synthesis enzymes by light and cold could be of crucial physiological importance, considering the short growing season of Antarctic plants. Regulation by light and cold seems to be a convenient way to ensure accumulation of sucrose and other soluble sugars that can improve survival of Antarctic plants by providing a source of readily available energy during the period of negative balance and protect them from damage caused by low temperature.

Challenges Ahead The maintenance of a positive carbon balance at low temperature and the high accumulation of sugars in Antarctic plants are still poorly understood. There are just a few studies on the responses of these plants to other environmental factors such as salt, water stress, high temperature, and their cross-talk. Little is known about the properties of the enzymes and genes

209

that participate in these processes. Likewise, the possible biotechnological applications of genes and enzymes of these plants that function under permanent cold conditions are just beginning to be explored. From a biological point of view, the basic question with respect to why only two vascular plants have colonized the Maritime Antarctic remains unanswered. The existence of these plants in this harsh environment provides us with a useful tool to study the effects of global warming on reproduction, growth and colonization of new areas as ice melts. Additionally, studies on how this plant biota responds to biotic stressors, such as the presence of new plant competitors and pathogens favoured by global warming, are needed to predict the future of these populations in the Antarctic.

Acknowledgements The authors are grateful for financial support from Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt) grants 1010899 and 1060910, Fundación Andes C-13680/5. L.B.-G. is grateful to Comisión Nacional de Investigación Científica y Tecnológica (Conicyt) for Fellowship D-21060801 and to Instituto Antártico Chileno (INACH) for doctoral thesis support, logistics and permits to collect plants at Especially Protected Areas in Antarctica. Without their support, this chapter would not be the same. We also thank Alejandra Zúñiga who provided data on sugar contents of different Deschampsia species. L.A.B. thanks Angélica Cañete for a careful review of the literature and citations.

References Alberdi, M., Bravo, L.A., Gutiérrez, A., Gidekel, M., Corcuera, L.J. (2002) Ecophysiology of Antarctic vascular plants. Physiologia Plantarum 115, 479–486. Alscher, R.G., Donahue, J.L. and Cramer, C.L. (1997) Reactive oxygen species and antioxidants: relationships in green cells. Physiologia Plantarum 100, 224–233. Amir, J. and Preiss, J. (1982) Kinetic characterization of spinach leaf sucrose phosphate. Plant Physiology 69, 1027–1030. Asada, K. (1999) The water–water cycle in the chloroplast: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50, 601–639. Asada, K. (2000) The water–water cycle as alternative photon and electron sinks. Philosophical Transactions of the Royal Society B: Biological Sciences 355, 1419–1431.

210

L.A. Bravo et al.

Bascuñán-Godoy, L., Uribe, E., Zúñiga-Feest, A., Corcuera, L.J. and Bravo, L.A. (2006) Low temperature regulates sucrose-phosphate synthase activity in Colobanthus quitensis (Kunth) Bartl. by decreasing its sensitivity to Pi and increased activation by glucose-6-phosphate. Polar Biology 29, 1011–1017. Bendall, D.S. and Manasse, R.S. (1995) Cyclic photophosphorylation and electron transport. Biochimica et Biophysica Acta 1229, 23–38. Bravo, L.A. and Griffith, M. (2005) Characterization of antifreeze activity in Antarctic plants. Journal of Experimental Botany 56, 1189–1196. Bravo, L.A., Zuñiga, G.E., Alberdi, M. and Corcuera, L.J. (1998) The role of ABA in freezing tolerance and cold acclimation in barley. Physiologia Plantarum 103, 17–23. Bravo, L.A., Ulloa, N., Zuñiga, G.E., Casanova A., Corcuera, L.J. and Alberdi, M. (2001) Cold resistance in antarctic angiosperms. Physiologia Plantarum 111, 55–65. Bravo, L.A., Gallardo, J., Navarrete, A., Olave, N., Martínez, J., Alberdi, M., Close, T.J. and Corcuera, L.J. (2003) Cryoprotective activity of a cold induced dehydrin purified from barley. Physiologia Plantarum 118, 262–269. Bravo, L.A., Saavedra-Mella, F.A., Vera, F., Guerra, A., Cavieres, L.A., Ivanov, A.G., Huner, N.P.A. and Corcuera, L.J. (2007) Effect of cold acclimation on the photosynthetic performance of two ecotypes of Colobanthus quitensis (Kunth) Bartl. Journal of Experimental Botany 58, 3581–3590. Casaretto, J.A., Corcuera, L.J., Serey, I. and Zuñiga, G.E. (1994) Size structure of tussocks of a population of Deschampsia antarctica Desv. in Robert Island, Maritime Antarctic. Serie Cientifica INACH 44, 61–66. Cloutier, Y. and Siminovitch, D. (1982) Augmentation of protoplasm in drought- and cold-hardened winter wheat. Canadian Journal of Botany 60, 674–680. Convey, P. (1996) Reproduction of Antarctic flowering plants. Antarctic Science 8, 127–134. Convey, P. and Smith, R.I.L. (2006) Responses of terrestrial Antarctic ecosystems to climate change. Plant Ecology 182, 1–10. Cook, A.J., Fox, A.J., Vaugham, D.G. and Ferrigno, J.G. (2005) Retreating glacier fronts on the Antarctic Peninsula over the past half century. Science 308, 541–544. Davidson, W.S., Jonas, A., Clayton, D.F. and George, J.M. (1998) Stabilization of a-synuclein secondary structure upon binding to synthetic membranes. Journal of Biological Chemistry 273, 9443–9449. Day, T.A., Ruhland, C.T., Grobe, C.W. and Xiong, F. (1999) Growth and reproduction of Antarctic vascular plants in response to warming and UV radiation reductions in the field. Oecologia 119, 24–35. Day, T.A., Ruhland, C.T. and Xiong, F.S. (2001) Influence of solar ultraviolet-B radiation on Antarctic terrestrial plants: results from a 4-year field study. Photochemistry and Photobiology B. Biology 62, 78–97. Demmig-Adams, B. and Adams, W.W. III (1992) Photoprotecction and other responses of plants to high light stress. Annual Review of Plant Physiology and Plant Molecular Biology 43, 599–626. Edwards, J.A. and Smith, R.I.L. (1988) Photosynthesis and respiration of Colobanthus quitensis and Deschampsia antarctica from the Maritime Antarctic. British Antarctic Survey Bulletin 81, 43–63. Ensminger, I., Busch, F. and Huner, N.P.A. (2006) Photostasis and cold acclimation: sensing low temperature through photosynthesis. Physiologia Plantarum 126, 28–44. Evert, R.F., Botha, C.E.J. and Mierzwa, R.J. (1985) Free space marker studies on the leaf of Zea mays L. Protoplasma 126, 62–73. Gerighausen, U., Braütigam, K., Mustafa, O. and Peter, H.-U. (2003) Expansion of vascular plants on an Antarctic Island: a consequence of climate change? In: Huiskes, A.H.L., Gieskes, W.W.C., Rozema, J., Schorno, R.M.L., van der Vies, S.M. and Wolff, W.J. (eds) Antarctic Biology in a Global Context. Backhuys Publishers, Leiden, The Netherlands, pp. 79–83. Gianoli, E., Insotroza, P., Zuñiga, A., Reyes, M., Cavieres, L.A., Bravo, L.A. and Corcuera, L.J. (2004) Ecotypic differentiation in morphology and cold resistance in populations of Colobanthus quitensis (Caryophyllceae) from the Andes and Antarctica. Arctic, Antarctic and Alpine Research 36, 470–475. Gidekel, M., Destefano-Beltrán, L., García, P., Mujica, L., Leal, P., Cuba M., Fuentes, L., Bravo, L.A., Corcuera, L.J., Alberdi, M. and Concha, I. (2003) Identification and characterization of three novel cold acclimation-responsive genes from the extremophile hair grass Deschampsia antarctica Desv. Extremophiles 7, 459–469. Greene, D.M. and Holtom, A. (1971) Studies in Colobanthus quitensis (Kunth) Bartl. and Deschampsia antarctica Desv. III. Distribution, habitats and performance in the Antarctic botanical zone. British Antarctic Survey Bulletin 26, 1–29. Griffith, M. and McIntyre, H.C.H. (1993) The interrelationship of growth and frost tolerance in winter rye. Physiologia Plantarum 87, 335–344.

Cold Hardiness in Antarctic Vascular Plants

211

Griffith, M., Gudleifsson, B.E. and Fukuta, N. (2001) Abiotic stress in overwintering crops. In: Iriki, O., Gaudet, D.A., Tronsmo, A.M., Matsumoto, N., Yoshida, M. and Nishimune, A. (eds) Low Temperature Plant–Microbe Interactions Under Snow. Hokkaido National Agricultural Experimental Station, Sapporo, Japan, pp. 101–114. Gudleifsson, B.E. and Larsen, A. (1992) Ice encasement as a component of winterkill of herbage plants. In: Li, P.H. and Christersson, L. (eds) Advances in Plant Cold Hardiness. CRC Press, Boca Raton, Florida, pp. 229–249. Holaday, A.S., Martindale, W., Alred, R., Brooks, A.L. and Leegood, R.C. (1992) Changes in activities of enzymes of carbon metabolism in leaves during exposure of plants to low temperature. Plant Physiology 98, 1105–1114. Holderegger, R., Stehlik, I., Smith, R.I.L. and Abbott, R.J. (2003) Populations of Antarctic hairgrass (Deschampsia antarctica) show low genetic diversity. Arctic, Antarctic and Alpine Research 35, 214–217. Honjoh, K., Matsumoto, H., Shimizu, H., Ooyama, K., Tanaka, K., Oda, Y., Takata, R., Joh, T., Suga, K., Miyamoto, T., Iio, M. and Hatano, S. (2000) Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27. Bioscience, Biotechnology and Biochemistry 64, 1656–1663. Horton, P. and Ruban, A. (2005) Molecular design of the photosystem II light-harvesting antenna: photosynthesis and photoprotection. Journal of Experimental Botany 56, 365–373. Houde, M., Daniel, C., Lachapelle, M., Allard, F., Laliberté, J. and Sarhan, F. (1995) Immunolocalization of freezing-tolerance associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. The Plant Journal 8, 583–593. Huner, N.P.A., Oquist, G. and Sarhan, F. (1998) Energy balance and acclimation to light and cold. Trends in Plant Science 3, 224–230. Hurry, V., Strand, A., Furbank, R. and Stitt, M. (2000) The role of inorganic phosphate in the development of freezing tolerance and the acclimatization of photosynthesis to low temperature is revealed by the pho mutants of Arabidopsis thaliana. The Plant Journal 24, 383–396. Johnson, G.N. (2005) Cyclic electron transport in C3 plants: fact or artefact? Journal of Experimental Botany 56, 407–416. Jones, T.L. and Ort, D.R. (1997) Circadian regulation of sucrose phosphate synthase activity in tomato by protein phosphatase activity. Plant Physiology 113, 1167–1175. Kappen, L. (1993) Plant activity under snow and ice, with particular reference to lichens. Arctic 46, 297–302. Karentz, D. (2003) Environmental change in Antarctica: ecological impacts and responses. In: Huiskes, A.H.L., Gieskes, W.W.C., Rozema, J., Schorno, R.M.L., van der Vies, S.M. and Wolff, W.J. (eds) Antarctic Biology in a Global Context. Backhuys Publishers, Leiden, The Netherlands, pp. 45–55. Kozaki, A. and Takeba, G. (1996) Photorespiration protects C3 plants from photooxidation. Nature 384, 557–560. Lin, C. and Thomashow, M.F. (1992) A cold-regulated Arabidopsis gene encodes a polypeptide having potent cryoprotective activity. Biochemical and Biophysical Research Communications 183, 1103–1108. Livingston, D. P. III (1996) The second phase of cold hardening: freezing tolerance and fructan isomer changes in winter cereal crowns. Crop Science 36, 1568–1573. Lud, D., Huiskes, A.H.I., Moerdijk, T. and Rozema, J. (2001) The effects of altered levels of UV-B radiation on an Antarctic grass and lichen. Plant Ecology 154, 87–99. Mantovani, A. and Vieira, R.C. (2000) Leaf micromorphology of Antarctic pearlwort Colobanthus quitensis (Kuntz) Bartl. Polar Biology 28, 531–538. Montiel, P.O., Smith, A. and Keiller, D. (1999) Photosynthetic responses of selected Antarctic plants to solar radiation in the southern Maritime Antarctic. Polar Research 18, 229–235. Morgan, J.M. (1984) Osmoregulation and water stress in higher plants. Annual Review of Plant Physiology 35, 299–319. Nemani, R.R., Keeling, C.D., Hashimoto, H., Jolly, W.M., Piper, S.C., Tucker, C.J., Myneni, R.B. and Running, S.W. (2003) Climate-driven increases in global terrestrial net primary production from 1982 to 1999. Science 300, 1560–1563. Niyogi, K.K. (1999) Photoprotection revisited: genetic and molecular approaches. Annual Review of Plant Physiology and Plant Molecular Biology 50, 333–359. Niyogi, K.K., Li, X.-P., Rosenberg, V. and Jung, H.-S. (2005) Is PsbS the site of non-photochemical quenching in photosynthesis? Journal of Experimental Botany 56, 375–382.

212

L.A. Bravo et al.

Olave-Concha, N., Ruiz-Lara, S., Muñoz, X., Bravo, L.A. and Corcuera, L.J. (2004) Accumulation of dehydrin transcrips and proteins in response to abiotic stress in Deschampsia antarctica. Antarctic Science 16, 175–184. Pérez-Torres, E., Dinamarca, J., Bravo, L.A. and Corcuera, L.J. (2004a) Responses of Colobanthus quitensis (Kunth) Bartl. to high light and low temperature. Polar Biology 27, 183–189. Pérez-Torres, E., García, A., Dinamarca, J., Alberdi, M., Gutiérrez, A., Gidekel, M., Ivanov, A.G., Hüner, N.P.A., Corcuera, L.J. and Bravo, L.A. (2004b) The role of photochemical quenching and antioxidants in photoprotection of Deschampsia antarctica Desv. Functional Plant Biology 31, 731–741. Pérez-Torres, E., Bascuñán-Godoy, L., Sierra, A., Bravo, L.A. and Corcuera, L.J. (2006) Robustness of activity of Calvin cycle enzymes after high light and low temperature conditions in Antarctic vascular plants. Polar Biology 29, 909–916. Pérez-Torres, E., Bravo, L.A., Corcuera, L.J. and Johnson, G.N. (2007) Is electron transport to oxygen an important mechanism in photoprotection? Contrasting responses from Antarctic vascular plants. Physiologia Plantarum 130, 185–194. Piotrowicz-Cieslak, A., Gielwanowska, I., Bochenek. A., Loro, P. and Gorecki, R.J. (2005) Carbohydrates in Colobanthus quitensis and Deschampsia antarctica. Acta Societatis Botanicorum Poloniae 74, 209–218. Redón, J. (1985) Líquenes Antárticos. Instituto Antártico Chileno (INACH), Santiago. Reimholz, R., Geiger, M., Deiting, U., Krause, K.P., Sonnewald, U. and Stitt, M. (1997) Potato plants contain multiple forms of sucrose phosphate synthase, that show differences in their tissue distribution, their response during development, and their response to low temperature. Plant, Cell & Environment 20, 291–305. Reyes, M.A., Corcuera, L.J. and Cardemil, L. (2003) Accumulation of HSP70 in Deschampsia antarctica Desv. leaves under thermal stress. Antarctic Science 15, 345–352. Reyes-Díaz, M., Alberdi, M., Piper, F., Bravo, L.A. and Corcuera, L.J. (2005) Low temperature responses of Nothofagus dombeyi and Nothofagus nitida, two evergreen species from south central Chile. Tree Physiology 25, 1389–1398. Reyes-Díaz, M., Ulloa, N., Zúñiga-Feest, A., Gutiérrez, A., Gidekel, M., Alberdi, M., Corcuera, L.J. and Bravo, L.A. (2006) Arabidopsis thaliana avoids freezing by supercooling. Journal of Experimental Botany 57, 3687–3696. Romero, M., Casanova, A., Iturra, G., Reyes, A., Montenegro, G. and Alberdi, M. (1999) Leaf anatomy of Deschampsia antarctica (Poaceae) from the Marime Antarctic and its plastic response to changes in growth conditions. Revista Chilena de Historia Natural 72, 411–425. Rozema, J., Noordijk, A.J., Broekman, R.A., van Beem, A., Meijkamp, B.M., de Bakker, N.V.J., van de Staaij, J.W.M., Stroetenga, M., Bohncke, S.J.P., Konert, M., Kars, P.H., Smith, R.I.L. and Conway, P. (2001) (Poly) phenolic compounds in pollen and spores of Antarctic plants as indicators of solar UV-B. Plant Ecology 154, 11–26. Ruban, A.V., Berera, R., Ilioaia, C., van Stokkum, I.H.M., Kennis, J.T.M., Pascal, A.A., van Amerongen, H., Robert, B., Horton, P. and van Grondelle, R. (2007) Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450, 575–578. Ruhland, C.T. and Day, T.A. (2001) Size and longevity of seed banks in Antarctica and the influence of UV-B on survivorship, growth and pigments concentrations of Colobanthus quitensis seedlings. Environmental and Experimental Botany 45, 143–154. Santarius, K.A. (1992) Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol. Physiologia Plantarum 84, 87–93. Schroeter, B., Olech, M., Kappen, L. and Heitland, W. (1995) Ecophysiological investigations of Usnea antarctica in the Maritime Antarctic. I Annual microclimatic conditions and potential primary production. Antarctic Science 7, 251–260. Sierra-Almeida, A., Cavieres, L.A. and Bravo, L.A. (2009) Freezing resistance varies within the growing season and with elevation in high-Andean species of central Chile. New Phytologist (doi: 10.1111/j.1469-8137. 2008.02756.x). Smith, R.I.L. (1994) Vascular plants as bioindicators of regional warming in the Antarctic. Oecologia 99, 322–328. Smith, R.I.L. (2003) The enigma of Colobanthus quitensis and Deschampsia antarctica in Antarctica. In: Huiskes, A.H.L., Gieskes, W.W.C., Rozema, J., Schorno, R.M.L., van der Vies, S.M. and Wolff, W.J. (eds) Antarctic Biology in a Global Context. Backhuys Publishers, Leiden, The Netherlands, pp. 234–239.

Cold Hardiness in Antarctic Vascular Plants

213

Stajner, D., Milic, N., Lazic, B. and Mimica-Dukié, N. (1998) Study on antioxidant enzymes in Allium cepa L. and Allium fistulosum L. Phytotherapy Research 12, s15–s17. Strauss, G. and Hauser, H. (1986) Stabilization of lipid bilayer by sucrose during freezing. Proceeding of the National Academy of Sciences USA 83, 2422–2426. Suzuki, N. and Mittler, R. (2006) Reactive oxygen species and temperature stresses: a delicate balance between signaling and destruction. Physiologia Plantarum 126, 45–51. Sympson, S. (2000) In Focus: Melting away. Scientific American 281, 14–15. Szabó, I., Bergantino, E. and Giacometti, G.M. (2005) Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation. EMBO Report 6, 629–634. Taschler, D. and Neuner, G. (2004) Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary. Plant, Cell & Environment 27, 737–746. Toroser, D. and Huber, S.C. (1997) Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves. Plant Physiology 114, 947–955. Uemura, M. and Steponkus, P.L. (2003) Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves. Plant, Cell & Environment 26, 1083–1096. Un-Haing, C. and Jung-O, P. (2000) Mercury-induced oxidative stress in tomato seedlings. Plant Science 156, 1–9. Valladares, F., Sancho, L.G., Chico, J.M. and Manrique, E. (1997) Diferencias en la utilización fotosintética de radiaciones lumínicas elevadas por líquenes y plantas vasculares en la Antártida marítima. Boletín de la Real Sociedad Española de Historia Natural Sección Biología 93, 119–125. Vieira, R. and Mantovani, A. (1995) Anatomía foliar de Deschampsia antarctica Desv. (Gramineae). Revista Brasileira de Botânica 18, 207–220. Walther, G.R. (2003) Plants in a warmer world. Perspectives in Plant Ecology, Evolution and Systematics 6, 169–185. Walther, G.R., Post, E., Convey, P., Menzel, A., Parmesan, C., Beebee, T.J.C., Fromentin, J.M., Hoegh-Guldberg, O. and Bairlein, F. (2002) Ecological responses to recent climate change. Nature 416, 389–395. Winkler, J.B., Kappen, L. and Schulz, F. (2000) Snow as an important ecological factor for the cryptogams in the Maritime Antarctic. In: Davison, W., Howard-Williams, C. and Broady, P. (eds) Antarctic Ecosystems: Models for Wider Ecological Understanding. Caxton Press, Christchurch, New Zealand, pp. 258–262. Xiong, F.S. and Day, T.A. (2001) Effect of solar ultraviolet-B radiation during spring time ozone depletion on photosynthesis and biomass production of antarctic vascular plants. Plant Physiology 125, 738–751. Xiong, F.S., Ruhland, T.C. and Day, T.A. (1999) Photosynthetic temperature response of the Antarctic vascular plants Colobanthus quitensis and Deschampsia antarctica. Physiologia Plantarum 106, 276–286. Xiong, F.S., Mueller, E.C. and Day, T. (2000) Photosynthetic and respiratory acclimation and growth response of Antarctic vascular plants to contrasting temperature regimes. American Journal of Botany 87, 700–710. Yu, Q. and Rengel, Z. (1999) Micronutrient deficiency influences plant growth and activities of superoxide dismutases in narrow-leafed lupines. Annals of Botany 83, 175–182. Zúñiga, G.E., Alberdi, M. and Corcuera, L.J. (1996) Non structural carbohydrates in Deschampsia antarctica Desv. from South Shetland Islands, Maritime Antarctic. Environmental and Experimental Botany 36, 396–399. Zúñiga-Feest, A., Inostroza, P., Vega, M., Bravo, L.A. and Corcuera, L.J. (2003) Sugars and enzyme activity in the grass Deschampsia antarctica. Antarctic Science 15, 483–491. Zúñiga-Feest, A., Ort, D., Gutierrez, A., Gidekel, M., Bravo, L.A. and Corcuera, L.J. (2005) Light regulation of sucrose-phosphate synthase activity in the freezing-tolerant grass Deschampsia antarctica. Photosynthesis Research 83, 75–86. Zuther, E., Buchel, K., Hundertmark, M., Stitt, M., Hincha, D.K. and Heyer, A.G. (2004) The role of raffinose in the cold acclimation response of Arabidopsis thaliana. FEBS Letters 576, 169–173.

21

Patterns of Freezing in Plants: the Influence of Species, Environment and Experiential Procedures L.V. Gusta, M.E. Wisniewski and R.G. Trischuk

Introduction Through the use of ‘omics’ in the past decade, research on cold hardiness has focused on identification and regulation of the genes, proteins, metabolites and hormones responsible for this trait. This effort has resulted in the identification of specific genes or transcription factors that appear to play a significant role; however, global genetic analyses using microarray technologies have also revealed the complexity of the cold acclimation process. The realization that hundreds of genes can be either up- or down-regulated underscores the need to develop a better understanding of how whole plants respond to stress in order to understand the potential impact of any specific gene or sets of genes on the plant’s response to freezing stress. Understanding the factors that determine how plants freeze, how rapid, where ice propagates and which plant tissue or organ is the most freezing-sensitive provides valuable insights into the protective role of specific proteins and metabolites, as well as the adaptive advantage of the structural composition of plant architecture. It is highly probable that many of the genes observed to be up-regulated or downregulated in response to a low-temperature treatment are not directly associated with freezing tolerance. For example, many of the genes may be associated with low-temperature 214

growth acclimation, cold shock, vernalization, dormancy and chilling. In addition, many studies on freezing tolerance do not consider repair, post-traumatic events, anatomical and histological changes as they relate to freezing injury. A further complication, contributed by researchers, is how the cold acclimation protocol is designed and carried out. Many of the studies reported are on cold-shock-induced changes rather than cold-acclimation-induced changes. When researchers transfer plants growing in pots from 20 to 4°C, a cold shock is induced rather than a process of cold acclimation. Small pots tend to cool very quickly thus causing a change in the hydraulic conductivity of the roots which then results in a decrease in the water potential of the aerial tissue. In contrast, in nature soil cools very slowly, often over days to weeks in instead of minutes in a growth chamber, and this causes a water stress on the aerial tissue. This aspect will be covered in a later section. As stated above, a more thorough understanding of the factors involved in freezing tolerance may assist in eliminating many genes not directly involved in freezing tolerance. There are at least seven different freezing patterns in plant tissues subjected to subzero temperatures. The first pattern occurs in succulent tender tissues (not exposed to acclimating temperatures) that have high water content. Injury or death occurs close to 0°C

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Patterns of Freezing in Plants

(−2 to −5°C) when water crystallizes due to the presence of an extrinsic (e.g. ice, bacterial) or intrinsic nucleator (of plant origin). Ice crystals grow explosively throughout the tissue causing mechanical damage. It is not known if ice first forms in the apoplast and then grows into the protoplast or if nucleation actually occurs in the protoplast. This is very typical for the freezing of tropical plants, or plants such as beans or tomatoes or temperate plants growing in a growth chamber at warm non-acclimating temperatures. For example, winter rye seedlings grown in a growth chamber at 23°C are killed at −4°C; however, they can tolerate −8°C in the field in early July in Saskatoon, Canada when air temperature ranges from 23 to 28°C. Temperate plants growing in the field are always exposed to some form of stress (e.g. water, wind, UV) that results in cross adaptation. In the first pattern of freezing, when nucleation occurs, the whole leaf freezes in a matter of minutes and has a water-soaked appearance upon thawing. Because of the high tissue water content and few restrictions to ice growth, non-equilibrium freezing occurs (Olien, 1965). Intracellular or symplastic ice formation in one cell can initiate freezing in surrounding cells, presumably through the plasmodesmata (Hudson and Brustkern, 1965). Invariably death results and its symptoms are readily apparent. In tissue where water is not confluent, isolated pockets of water migrate in the vapour phase to the growing ice crystal which creates a different pattern of stress. Exposure to −3°C and freezing of non-acclimated wheat crowns may not kill the apical meristem; however, the cells in the basal part of the crown are killed (Chen et al., 1983). The apical meristem cells are small, non-vacuolated and have a higher solute content than the basal cells, which are large with high water content. Therefore, freezing in the basal cells tends to occur in a non-equilibrium fashion resulting in death. The first pattern of freezing can be altered by a slight water stress. Non-acclimated winter wheat seedlings increased in freezing tolerance by 5°C following a brief water stress (Tyler et al., 1981). A reduction in water content would reduce the mechanical forces of the growing ice crystal but it would also result in elevated

215

levels of abscisic acid and/or induction of stress-related genes and/or inhibition of the cell cycle; all of which are associated with increased frost tolerance. While cold-induced genes may be induced by a water stress, the seedlings never attain their full hardiness potential as simple sugars are not produced to enhance freezing tolerance. The second pattern occurs in partially acclimated tissues or mature tissues (LT50 −3 to −10°C). Freezing may occur as an equilibrium process and injury is dependent on the subzero temperature. In contrast to the process described for the first pattern, in the second pattern ice first forms at a relatively few select sites of nucleation in the apoplast (Garten and Head, 1965). Within a few hours or days after acclimating plants at temperatures at less than 10°C, ice formation at subzero temperature occurs in apoplastic spaces that accommodate growing ice crystals. Generally, ice-nucleating substances are found in these locations of the tissue and have to be wet to be active (Gusta et al., 2004). It is tempting to speculate that the role of antifreeze proteins described by Griffith and Yaish (2004) is to ensure ice does not form between cells that cannot accommodate large masses of ice or that they modify the rate of ice growth. Migration of water in the vapour phase to the growing ice crystals results in a watersoaked appearance of the thawed leaves which may or may not be lethal depending upon the temperature and degree of acclimation (Burke et al., 1976; Olien, 1965). Under these conditions there is insufficient energy for ice to adhere to plasma membranes, which can produce a shearing force that results in laceration of the plasmalemma when the cell undergoes cytorrhysis. Generally ice forms along or in the cell walls (Olien, 1965; Reaney and Gusta, 1999). Water-soluble cell-wall carbohydrates of rye such as arabino-xylans interface with icelattice formation and control the growth of ice crystals (Shearman et al., 1973; Olien, 1965). Griffith and Yaish (2004) have also proposed that antifreeze proteins may act in a similar fashion. Using IR video thermography (IRVT), Gusta et al. (2004) demonstrated that sugars interacting with proteins also inhibit the rate of ice growth. Depending on the degree of supercooling and water content, both non-equilibrium and equilibrium freezing can occur.

216

L.V. Gusta et al.

Leaf maturity is also another important factor affecting the second pattern of freezing. Non-acclimated, juvenile leaves of Minum undulatum tend to exhibit non-equilibrium, intracellular freezing, whereas freezing in mature, non-acclimated leaves is similar to mature acclimated leaves in that freezing is initiated extracellularly in the apoplast (Hudson and Brustkern, 1965). In young non-acclimated leaves, intracellular protoplastic freezing is initiated at −4°C in leaf apices and propagates through the leaf in a wave pattern and the whole leaf is frozen at −8°C. In older leaves freezing is initiated at −8°C within the waterconducting xylem elements of the midribs. These authors observed that the wave of freezing then spreads to the base of the leaf and all the cells were frozen at −12°C. Pearce and Fuller (2001) and Gusta et al. (2004) also observed distinct freezing patterns between young tender leaves and cold-acclimated leaves using IRVT. The third freezing pattern occurs in fully cold-acclimated hardy species such as winter cereals and trees that do not supercool. Freezing is initiated in the apoplast and equilibrium freezing occurs, provided extensive supercooling does not occur and the rate of freezing is moderate (less than 5°C/h). The rate of freezing is dependent in part on the amount of heat released due to crystallization and its rate of dissipation; both of which are affected by supercooling. This latent heat may provide sufficient energy for adhesion of ice to bind to membranes resulting in their laceration due to the growth of ice (Olien, 1974). Some species can undergo extensive supercooling without experiencing injury, e.g. canola leaves; whereas winter wheat leaves are more sensitive (L.V. Gusta, unpublished results). Generally, the third pattern of freezing occurs in tissue of low moisture content (less than 75% water). Shearing of membranes due to ice crystal growth is avoided by a reduction in apoplastic water (Olien, 1965). The structure of ice crystals can be modified by sugars (Reaney and Gusta, 1999), antifreeze proteins (Griffith and Yaish, 2004) and long-chain branched fructans (Olien, 1965). Under conditions of equilibrium freezing, injury does not occur until the cells are severely contracted due to freeze-induced dehydration. Over 80% of the tissue water is converted to ice at

−10°C for plant species that do not deep supercool (Gusta et al., 1975). At −20°C, virtually all of the freezable water is in the form of ice, the symplast is in a gel form and water, tightly associated with hydrophilic sites, slowly migrates to locations with apoplastic ice. Injury in this case is primarily associated with freeze-induced desiccation, probably in part to reactive oxygen species (ROS) and the interaction of proteins with each other (Levitt, 1980). Depending on the subzero temperature, injury may not manifest itself immediately but may require days to months. For example, Gusta, et al. (1997) reported that fully cold-acclimated ‘Norstar’ winter wheat survived −24°C when cooled 2°C/h but was killed at −12°C when held at that temperature for 15 days. Fully acclimated spring wheat can tolerate −9°C when cooled at 2°C/h; however, spring wheat succumbs to −3°C when it is held at that temperature for 48 h. McKersie and Leshem (1994) proposed that freeze-induced desiccation results in an overproduction of oxygen free radicals which results in freezing injury. Therefore the LT50 method, where tissues are cooled at 2°C/h and removed at selected test temperatures, may not be appropriate for determining winter survival for plants subjected to long periods of subzero temperatures (Gusta et al., 1997). These findings also demonstrate that there are several forms of freezing injury depending on the freezing protocol utilized. As stated earlier, the histological and anatomical tissue structure of plants has a direct effect on the freezing process, resulting in different patterns of freezing and water redistribution. Although all cells may be genetically identical, differences in cell size, cell water content and rigidity of the cell wall have a dramatic effect on freezing tolerance. For example, the crown of winter cereal is a complex tissue consisting of the apical meristem that contains densely packed non-vacuolated cells. The vascular transition zone at the base of the crown has large, highly vacuolated cells, whereas the intermediate zone consists of smaller vacuolated cells. Originally Olien (1961, 1964) suggested that the basal region of the crown was the most sensitive to freezing injury. Subsequently, however, Chen et al. (1983) demonstrated by tetrazolium staining that the basal part of the crown in winter cereals was

Patterns of Freezing in Plants

more freezing-sensitive than the intermediate zone. This is somewhat expected as the cells in the basal part have a high water content that would freeze in a non-equilibrium fashion. Surprisingly, the small non-vacuolated cells in the apical meristem were less freezing-tolerant than cells in the transition zone. Limin and Fowler (1994) maintain that cell size is directly related to freezing tolerance of winter cereals; however, as demonstrated by both Chen et al. (1983) and Tanino and McKersie (1985), due to the complexity of the crown tissue, cell size is not a factor. Limin and Fowler (1994) based their conclusion on the length of the leaf guard cells rather than the cell size of the crown tissue, the latter tissue being critical for shoot and root regeneration in the spring. In addition, Chen et al. (1983) demonstrated that root initiation is impaired due to freezing injury and a lack of root morphogenesis. This is a classical example demonstrating the importance of identifying the primary site of injury using conventional techniques. The fourth pattern of freezing involves the ability of certain cells to supercool. Bigg (1953) originally reported that, in the absence of a heterogeneous ice nucleator, small volumes of pure water remain in a liquid or supercooled state to temperatures as low as −39°C. This was once considered to be the homogeneous nucleation temperature for water or the temperature at which water is converted to ice in the absence of a heterogeneous nucleator (Rasmussen and Mackenzie, 1972). Salt (1961), in his seminal review, reported the supercooling of water in insects to temperatures lower than −47°C. Several Alaskan gall-forming insects supercool to −60°C (Miller, 1982). Supercooling of water beyond the homogeneous nucleation temperature (−38.1°C) is enhanced by the accumulation of low-molecularmass polyols and sugars such as glycerol, sorbitol and trehalose, at multimolar concentrations (Lee et al., 1995). Recently Chen et al. (2006) demonstrated water confined in nano-pores can supercool to −93°C. These results suggest water does not have a defined freezing temperature as once suggested by Rasmussen and McKenzie (1972). As reviewed by Wisniewski et al. (2003), Weigand (1906) was probably one of the first to record deep supercooling in trees to tem-

217

peratures as low as −26°C. Supercooling occurs primarily in the xylem ray parenchyma of trees (Burke et al., 1976; Wisniewski, 1995) and in flower buds (Dereuddre, 1979; Ishikawa and Sakai, 1981). While it is quite remarkable that water in the absence of nucleators can supercool to −40°C, it is more puzzling why water does not move (or does so very slowly) from the symplast to the apoplast of the xylem ray parenchyma cells in the wood even though ice is present in adjacent cells. For example, trees growing in north central Canada reported to display deep supercooling, if analysed in the frozen state by differential thermal analysis (DTA), display much smaller or no low-temperature exotherm (LTE) compared with trees growing in areas that do not have long, extremely cold winters (Gusta et al., 1983b). However, if twigs of trees are held at 10°C for several minutes and then recooled at 5°C/h the LTE either increases in size or becomes destable. If twigs are held at −10°C for several days and then cooled at 5°C/h, the LTE diminishes in size and occurs at a lower temperature. The difference in the freezing pattern between trees from northern and southern provenances is quite fascinating. In the case of the northern trees, xylem ray parenchyma cells in midwinter are subjected to freeze-induced desiccation. This may explain why Wisniewski et al. (1996) detected dehydrins in peach trees that display deep supercooling. In the case of extraorgan freezing of flower buds, ice is segregated from the supercooled organ by a barrier created when tissues are cooled slowly (<5°C/h) (Ishikawa and Sakai, 1981). The barrier consists of an air space created from collapsed areas of pith tissue. It is the creation of this dry barrier that prevents ice from propagating into the flower buds. If ice does propagate through the barrier then non-equilibrium intracellular freezing often occurs (Ishikawa and Sakai, 1981). The fifth pattern of freezing exists if the cooling rate of the tissue is greater than 3°C/h (Ishikawa and Sakai, 1981; Gusta et al., 2006) and is not regarded as true supercooling. Hydrated lettuce seeds deep supercool when subjected to cooling rates greater than 5°C/h (Junttila and Stushnoff, 1977) but not at slower cooling rates (Ishikawa and Sakai, 1981). When seeds are cooled slowly the LTE

218

L.V. Gusta et al.

disappears and the temperature of the LTE does not correspond to the killing temperature. This is not considered to be true supercooling as these fast cooling rates do not occur in the soil under natural conditions. Studying the freezing of seeds by DTA and NMR spectroscopy, Gusta et al. (2006) reported that water associated with deep supercooling migrated to the ice which formed at −3 to −5°C if the seeds were held for several hours at −3 to −5°C. A sixth pattern of freezing has been demonstrated in non-acclimated cereals by Single (1964) and Marcellos and Single (1976). Single (1964) demonstrated that wheat ears prior to emergence remained supercooled at −5°C even though other parts of the plant were frozen. Ice propagated rapidly in the conducting vessels in the stem and leaves of wheat plants but was arrested at the stem and rachis nodes. Single and Olien (1967) concluded that ice formation in the stem of wheat results in a redistribution of water resulting in the production of dry areas which act as a barrier to ice growth, somewhat analogous to supercooling of florets tissue or extraorgan freezing. The major difference is in the extent of supercooling. The formation of ice in the flower buds of plants that display extraorgan freezing can result in supercooling to −35°C (Ishikawa and Sakai, 1981), whereas cereal ears may only supercool to temperatures as low as −15°C (Gusta et al., 2000). Utilizing IRVT, Pearce and Fuller (2001) observed that if freezing initiated in one part of the plant at −3°C, it did not spread to the rest of the plant for at least 75 min. In contrast, flower buds which display extraorgan freezing may remain supercooled for days. Carter et al. (2001) also noted that flowers on strigs of blackcurrant (Ribes nigrum) could supercool for extended periods of time (hours) despite other portions of the strig and main stem being frozen. Workmaster et al. (1999) also observed that ice did not propagate into cranberry fruit through peduncles that joined them to frozen uprights. These examples support the idea that barriers to ice propagation exist in plants which affect the overall pattern of plant freezing. They must represent a direct mechanism of adaptation to freezing but have been largely neglected in recent years (see Wisniewski et al., Chapter 1, this volume).

The seventh pattern of freezing involves vitrification or glass formation. Vitrification occurs at subzero temperatures when water is converted into a glass-like amorphous solid that does not have a crystalline structure. For a more detailed discussion on vitrification, the reader is referred to Chapter 22 by Strimbeck and Schaberg, this volume. During slow, equilibrium freezing plant cells undergo severe freeze-induced dehydration (Gusta et al., 1975) which may or may not be lethal depending on the desiccation tolerance of the cells. Reasons given for lethality are loss of a fluid lipid bilayer that enables lateral diffusion of membrane components and translocation processes (Crowe et al., 1984). Other factors that result in lethality are related as concentration of ions, protein denaturation and loss of semi-permeability properties (Koster, 1991). The formation of intracellular glass is thought to protect embryos in seeds from desiccation injury (Koster, 1991). Smallmolecular-weight sugars induce glass formation during cryopreservation (Crowe et al., 1984). Hirsh et al. (1985) were among the first to report intracellular glass formation in Populus during slow cooling (≤5°C/h) to −28°C. While there is considerable evidence demonstrating that sugars are involved in the stabilization of glass transitions, recent work has also demonstrated that late embryogenesis-abundant proteins may also be involved (Tolleter et al., 2007). Since the type or form of freezing injury in plants is not universal, it is not to be expected that the genes or proteins involved will be also. In other words, the events or type of stress which kills beans is not the same as in Arabidopsis, which is different from that in winter cereals, which is different from that in trees. Additionally, mechanisms of stress tolerance exist not only at the cell level but also at the tissue and organ level.

Manifestation of Injury – Field versus Controlled Environments Listed below are several examples of differences in results obtained when plants are frozen artificially compared with freezing under natural con-

Patterns of Freezing in Plants

ditions. The age of the tissue plays an important role in frost tolerance. Hudson and Brustkern (1965) reported that non-acclimated mature leaves are more freezing-tolerant than younger non-acclimated leaves. Schilling (2004) noted in Brassica that the cotyledons were more freezing-sensitive than the true leaves. At the seedling stage, the true leaves of canola are more sensitive to injury than the petiole, whereas at the flowering stage the stem region just below the crown is the most sensitive region. For the producer, the flowering and seed setting stage is the most critical period of production as it is too late to re-plant. Thus, if this is not recognized, attempts to improve frost tolerance by either conventional breeding or genetically engineering will fall short. Often artificial freeze tests only measure the freezing tolerance of excised leaves without considering the whole plant or the environmental conditions following a frost. Following an artificial freeze test, researchers often either thaw the plants at 4°C in the absence of light or allow recovery at 100 µmol/ cm2/s or less. In nature, most frosts occur on clear windless nights just prior to sunrise. Frosted plants are then exposed to light intensities of 600 to 2000 µmol/cm2/s, which result in photobleaching. The appearance of these plants stands in stark contrast to the dull green, water-soaked plants observed in artificial freeze tests that are thawed in the dark and maintained at low light intensities (100 µmol/cm2/s or less). Thus, the production of free radicals due to impaired photosynthesis under natural conditions is not taken into account. Electrolyte leakage is often employed to measure frost tolerance; however, in nature, plants are also injured by the photobleaching that occurs following a frost. This type of injury manifests itself after 3 or 4 days under natural conditions of full sunlight. Since the electrolyte leakage test is generally conducted the day after an artificial freeze, photobleaching will not be detected nor will its effect on survival be taken into account. Thus the electrolyte leakage test may underestimate injury and also give no indication of the potential for repair. The roots of winter cereals are killed when exposed to −5 to −9°C; however, the apical meristem tolerates −20 to −30°C in midwinter (Chen et al., 1983). In the absence of viable moisture in the spring, the seedlings

219

may die before establishing a root system. Following a controlled freeze, Chen et al. (1983) observed that root initiation was more sensitive to freezing than shoot regrowth. The majority of the crown cells, as determined by viability tests, were alive; however, the cells for root initiation were impaired. This was further verified using wheat cell callus cultures which were able to grow and divide following a controlled freeze test but were unable to produce roots for survival. Thus, in the field, the lack of root initiation rather than just the death of a proportion of cells is the main cause of winter kill. Radiation frosts, which generally occur on clear windless nights when the crop is actively growing, can result in either complete crop loss or a drastic reduction in crop quality. Under these conditions the crop rapidly loses heat to the night sky which is approximately −90°C. Exposed horizontal leaves tend to lose heat faster than erect leaves, whereas the leaves within the canopy are somewhat protected by the heat released from the soil. The relative humidity of the atmosphere increases to saturation and water condenses as dew on leaves that are colder than the ambient air temperature. Water on the leaves appears to activate ice nucleators as wet leaves do not supercool to the same extent as dry leaves (Wisniewski et al., 2002a; Gusta et al., 2004). Non-wetted, cold-hardened canola leaves readily supercool to temperatures as low as −13 to −14°C, while wetted leaves supercool to approximately −3 to −5°C (Gusta et al., 2004). Generally ice first forms in the leaves and spreads via the vascular bundles to the sheath and into the stem. Also, the addition of ice to dry leaves to initiate freezing may not be effective and the plants will remain in a supercooled state. The difference in nucleation temperature of wet versus dry leaves suggests two strategies may have evolved: avoidance and tolerance. Plants with a thick waxy cuticle or upright growth habit that can readily shed water may avoid or escape an episodic frost because extrinsic nucleators are not activated. In contrast, plants with larger leaves, e.g. bean, or those that have pubescent leaves, would tend to hold water which in turn would freeze at warm subzero temperatures if extrinsic nucleators are

220

L.V. Gusta et al.

present. Wisniewski et al. (2002b) demonstrated that tomato plants supercooled to −7°C or lower if their leaves were coated with a hydrophobic particle film that prevented wetting. If we are to select and produce superior frost-tolerant plants, these factors should be considered.

Natural versus Artificial Acclimation Lack of progress in improving the frost tolerance of plants may be exacerbated because plants cold-acclimated in a growth chamber may respond differently compared with plants subjected to natural acclimation (Wisniewski et al., 1997; Dhanaraj et al., 2007). There are significant differences between natural and artificial cold acclimation conditions. Field-grown plants are exposed to light intensities 4- to 12-fold higher than those in a growth chamber; the light spectrum varies from summer to autumn versus a constant spectrum in a growth chamber; and varying day/night temperatures produce mixed messages in contrast to constant temperatures in a growth chamber. Plants in the field are exposed to strong winds from different directions that affect secondary wall development. Due to the large soil mass, roots cool slowly in the autumn compared with plants in pots in a growth chamber. This was demonstrated in a growth chamber study when plants were transferred from 23 to 4°C (cold shock) versus slow cooling (2°C/h) from 23 to 4°C (cold stress) (R.G. Trischuk, unpublished results). After 24 h, the water potential was −0.3 MPa for the cold-stressed plants versus −0.7 MPa for the cold-shocked plants. There were obvious signs of wilting in the coldshocked plants. A proteomic study revealed there were at least 29 different proteins between the cold-shocked plants versus the cold-stressed plants. The low-temperatureinducible transcription factor C-repeat binding factor (CBF), which controls the induction of a suite or regulon of cold-regulated genes, increased dramatically in the cold-shocked plants compared with the cold-stressed plants. These results suggest that a portion of CBF gene induction may reflect a cold-shock response rather than a programmed process of cold acclimation. For a very thorough review of

CBF, the reader is referred to the chapters by Nassuth and Siddiqua and Stockinger in this volume (Chapters 14 and 13, respectively). In the autumn, winter annuals and perennials are gradually exposed to temperatures approaching 0°C and light intensities of over 1000 µmol/cm2/s. This is in stark contrast to plants grown in growth chambers, which are shifted from 23 to 4°C within minutes and are exposed to irradiance of 150 µmol/cm2/s or less. A sudden shift from warm temperatures to low temperatures at high irradiance in a growth chamber results in photoinhibition in many plants (Hurry and Huner, 1991), whereas in nature plants are gradually exposed to decreasing temperatures in the autumn. Coldacclimated winter cereals, both in the field and in a growth chamber, rapidly develop resistance to photoinhibition in contrast to spring wheat which does not and is susceptible to photoinhibition (Hurry and Huner, 1991). It is well established that adequate photosynthesis for the production of simple sugars and energy is a requirement for the full development of freezing tolerance and this could partially explain why spring cereals do not acclimate to the same level as winter cereals. In comparing the growth of cereals in the field in the autumn, winter rye has the greatest increase in dry matter production of all the cereals, indicating high rates of photosynthesis under high irradiance and low temperatures. Photoinhibition is linked to the formation of ROS, which are produced when excess light energy is not converted to chemical energy. ROS can oxidize lipids, proteins and enzymes, membranes and the whole cell (Foyer and Noctor, 2005). Plants have evolved several mechanisms including enzymatic and non-enzymatic antioxidants to prevent and respond to ROS (Giacomelli et al., 2006). Wisniewski et al. (1996) reported enhanced expression of a peach dehydrin in response to low temperatures in a growth chamber; however, three weeks of short days at 20°C had no impact on expression. In contrast, under natural conditions the expression of the dehydrin gene was first observed in August and increased during the autumn. These results suggest that the dehydrin gene under natural conditions may be controlled by the autumn light spectrum and/or day/night temperature cycling

Patterns of Freezing in Plants

rather than by short days as indicated in the field study. Gray and Heath (2005) observed in Arabidopsis that leaves developed solely at low temperatures have a different metabolome compared with leaves shifted to a low temperature. In comparison, no simple correlation was observed between nine Arabidopsis accessions varying in frost tolerance that were cold-acclimated in growth chambers. Several metabolic differences were observed in a metabolomic study of winter cereals acclimated in a growth chamber compared with naturally acclimated seedlings. Significant differences were detected in the production of simple sugars and flavonoid compounds, which are well known to correlate with freezing tolerance (Levitt, 1980). The largest differences occurred in ‘Puma’ winter rye where there were 42 different putative metabolites. Svec and Hodges (1972) compared metabolite changes in winter cereal seedlings cold-acclimated in either controlled or natural environments. Total soluble carbohydrates, reducing sugars, total soluble N and amino acids were two- to fourfold higher in naturally acclimated plants and total lipids were twofold lower compared with plants from the growth chamber. Recently, Dhanaraj et al. (2007) documented a large number of genes induced in growth chambers that were not induced under field conditions. Also, genes related to light stress that were induced in plants in the field were not induced in growth chambers. In the field in the autumn, plants receive mixed messages because one day the air temperature may be 2°C and the next few days the plants may be exposed to 20°C and then back to 2°C. Meanwhile, plants in a growth chamber are often exposed to constant day/night temperatures which are constantly driving the cold acclimation response. Under field conditions in the autumn, seedlings of a spring cultivar of Brassica napus cold-acclimated to −7°C compared with −18°C at 2°C in a growth chamber (Schilling, 2004). This is comparable to the freezing tolerance of the hardiest winter canola and suggests that the major genes for cold acclimation are present in spring canola but what is different is how the two types perceive the environmental signal(s) for acclimation.

221

Sugars, Proteins, Vernalization and Winter Hardiness It has been suggested that in order for winter cereals to cold-acclimate they must be in a vegetative state or they will not be capable of responding to environmental cues (Andrews, 1960). Therefore, if vernalization is either partially or fully saturated, winter cereals are unable to acquire their full freezing tolerance potential. Gusta et al. (1983a) reported that fully hardened winter cereals, prior to vernalization saturation, could partially reharden following exposure to dehardening conditions but once saturation was complete the seedlings acclimated similar to spring cereals. Fowler et al. (1999) proposed that vernalization in cereals acts as a master switch controlling the duration of frost tolerance and that expression of cold-acclimation-associated genes could only occur in the vegetative phase. These results were based on holding plants at 4°C for several months. Although it seems plausible that vernalization is a master switch for the cold acclimation process, several research studies suggest that it is not involved in the maintenance of freezing tolerance. ‘Norstar’ winter wheat and ‘Puma’ winter rye, the most winterhardy wheat and rye cereals, respectively, have a very short vernalization requirement. In fact, both ‘Norstar’ and ‘Puma’ have their vernalization requirement saturated prior to the onset of winter (Gusta et al., 1997), yet are the most winter-hardy wheat and rye cultivars grown in western Canada. A Danish cultivar, ‘Jokkienen’, which has similar LT50 to ‘Norstar’, requires more than twice the time for vernalization saturation than ‘Norstar’; however, it loses more freezing tolerance over the winter months than ‘Norstar’. In contrast to ‘Norstar’ winter wheat, the vernalization saturation time for ‘Jokkienen’ is only partially saturated before winter sets in and the ground freezes. Olien (1964) previously noted that cereal plants undergo chilling injury if held at temperatures just above 0°C for prolonged periods of time. Gusta et al. (1983a) demonstrated that fully hardened winter cereals could be held at −3°C for 6 months with little or no loss of freezing tolerance. However, at 4°C or temperatures lower than −6°C the crowns lost freezing tolerance; the lower the temperature the greater the loss in

222

L.V. Gusta et al.

freezing tolerance. Kobayashi et al. (2004) also concluded that vernalization was not the master switch that leads to the expression of COR genes and freezing tolerance. Markowski and Rapacz (1994) reported the lack of a strong relationship between vernalization requirement and freezing tolerance in winter rape lines. Schilling (2004) demonstrated that both a winter and a spring cultivar of Brassica napus were capable of cold-acclimating to an LT50 of approximately −16°C. Upon exposure to dehardening conditions, all freezing tolerance was lost and upon re-exposure to acclimating conditions these two cultivars reacclimation to −18°C despite the fact that vernalization saturation was complete in the winter cultivar. Trischuk (2003) also found that while a spring wheat, which has no vernalization requirement, can be cold-acclimated to −9°C, it will not reharden following dehardening and is killed at −3°C. Carbohydrate levels, particularly sucrose, have long been correlated with increased freezing tolerance (Levitt, 1980). Sugars are believed to play numerous roles in the development of freezing tolerance including use as an energy source, cryoprotectants, chaperones and gene regulators. In general, levels of carbohydrates increase as a result of low-temperature exposure, with maximal levels occurring in fully acclimated plants. During cold acclimation, changes in gene expression and protein accumulation occur (reviewed by Guy, 1990), enabling the plant tissues to tolerate the induced dehydration. Among the proteins most commonly found are dehydrins (Wilen et al., 1996) or dehydrin-like, e.g. Wheat Cold Specific (Houde et al., 1992) and Cold Responsive protein (Gilmour et al., 1992). These lowtemperature proteins are primarily involved in either binding of water or protecting proteins from desiccation-induced denaturation. Interestingly, winter wheat which cannot be reacclimation following deacclimation still accumulated the dehydrins WCS-120 and COR78 when subjected to reacclimation conditions (Trischuk, 2003). Wilen et al. (1996) also reported that dehydrins accumulated in fully vernalized winter cereals collected in the spring and subjected to hardening conditions. Both re-acclimation spring and winter canola accu-

mulated dehydrins and WSC120 but not COR78. Unlike cereals, the canola plants accumulated water-soluble carbohydrates to a level similar to that observed during the initial acclimation period. While it is well established that sugars are involved in cold acclimation, it is not known why they do not accumulate during reacclimation in cereals. In contrast to spring cereals, winter cereals have a greater ability to maintain high rates of CO2 assimilation and sucrose accumulation at cold acclimating temperatures (Lebate and Leegood, 1988; Savitch et al., 1997). Öquist et al. (1993) have also reported that the capacity of plants to photosynthesize at low temperatures is strongly correlated with the development of freezing tolerance. Therefore, if photosynthesis is restricted, sugar production is decreased and the development of freezing tolerance is limited. This suggests vernalization is somehow associated with the capacity of winter cereals to efficiently photosynthesize at low temperatures. ‘Norstar’ winter wheat seedlings collected from the field in early April had an LT50 of −13°C in contrast to −24°C in early November (L.V. Gusta, unpublished results). Although these seedlings would not reacclimate, seedlings incubated in sucrose and abscisic acid increased in freezing tolerance to −19°C. These results suggest that there is a strong interaction between low-temperature-associated proteins and sugars in determining levels of freezing tolerance. Vernalization may control the ability of leaves to effectively photosynthesize and store sugars that are either involved directly with freezing tolerance or regulate genes involved in freezing tolerance in winter cereals. In contrast to winter cereals, canola seedlings readily produce new leaves that can photosynthesize even though the plants are vernalized.

Conclusion New ‘omic’ tools and technologies have revealed a high level of complexity with regard to the response of plants to low temperatures. Care has to be taken, however, in how we approach the subject. As outlined here, freez-

Patterns of Freezing in Plants

ing injury manifests itself in several different ways and the strategies that have evolved in plants to survive low temperatures vary with the species as well as the location (Wisniewski et al., 2003). This is clearly evident when one closely examines both cold acclimation and

223

freezing injury in plants grown and/or tested in the field versus environmental chambers. Results obtained from controlled environment studies should always be validated by field studies in order to develop approaches to improve cold hardiness.

References Andrews, J.E. (1960) Cold hardening of sprouting wheat as affected by duration of hardening and hardening temperature. Canadian Journal of Plant Science 40, 94–103. Bigg, E.K. (1953) The supercooling of water. Proceedings of the Physical Society B 66, 688–694. Burke, M.J., Gusta, L.V., Quamme, H.A., Weiser, C.J. and Li, P.H. (1976) Freezing injury in plants. Annual Review of Plant Physiology 27, 507–528. Carter, J., Brennan, R. and Wisniewski, M. (2001) Patterns of ice formation and movement in black currant. HortScience 36, 1027–1032. Chen, S.-H., Mallamace, F., Mou, C.-Y., Brocdo, M., Corsavo, C., Faraone, A. and Liu, L. (2006) The violation of the Stokes–Einstein relation in supercooled water. Proceedings of the National Academy of Sciences USA 103, 12974–12978. Chen, T.H.H., Gusta, L.V. and Fowler, D.B. (1983) Freezing injury and root development in winter cereals. Plant Physiology 73, 773–777. Crowe, J.H., Crowe, L.M. and Chapman, D. (1984) Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223, 701–703. Dereuddre, J. (1979) Etude comparative du comportement des bourgeons d’arbres en vieralentie, pendant un refroidissement gradual des rameaux. Bulletin de la Societe Botanique de France Lettres Botaniques 126, 399–412. Dhanaraj, A.L., Alkharouf, N.W., Beard, H.S., Chouikha I.B., Mathews, B.F., Wei, H., Arora R. and Rowland, L.J. (2007) Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions. Planta 225, 735–751. Fowler, D.B., Limin, A.E. and Richie, J.T. (1999) Low temperature tolerance in cereals: model and genetic interpretation, interpretive paper. Crop Science 39, 626–633. Foyer, C.H. and Noctor, G. (2005) Oxidant and antioxidant signaling in plants: a re-evaluation of the concept of oxidative stress in a physiological contest. Plant, Cell & Environment 28, 1056–1071. Garten, V.A. and Head, R.B. (1965) A theoretical basis of ice nucleation by organic crystals. Nature 205, 160–162. Giacomelli, L., Rudella, A. and van Wijk, J.K. (2006) High light response of the thylakoid proteome in Arabidopsis wild type and ascorbate deficient mutant vtc2-2. A comparative proteomic study. Plant Physiology 141, 685–701. Gilmour, S.J., Artus, N.N. and Thomashow, M.F. (1992) cDNA sequence analysis and expression of two coldregulated genes of Arabidopsis thaliana. Plant Molecular Biology 18, 13–21. Gray, G.R. and Heath, D. (2005) The low temperature metabolome of Arabidopsis. In: Nikolau, B.J. and Wurtele, E.S. (eds) Concepts in Plant Metabolomics. Springer, The Netherlands, pp. 239–246. Griffith, M. and Yaish, M.W.F. (2004) Antifreeze proteins in overwintering plants: a tale of two activities. Trends in Plant Science 9, 399–405. Gusta, L.V., Burke, M.J. and Kapoor, A.C. (1975) Determination of unfrozen water in winter cereals at subfreezing temperatures. Plant Physiology 56, 707–709. Gusta, L.V., Chen, T.H.H. and Fowler, D.B. (1983a) Factors affecting the cold hardiness of winter wheat. In: Fowler, D.B., Gusta, L.V., Slinkard, A.E. and Holm, B.A. (eds) New Frontiers in Winter Wheat Production. Proceedings of the Western Canada Winter Wheat Conference. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, pp. 1–25. Gusta, L.V., Tyler, W.J. and Chen, T.H.H. (1983b) Deep undercooling in woody taxa growing north of the −40°C isotherm. Plant Physiology 72, 122–128. Gusta, L.V., O’Connor, B.J. and MacHutcheon, M.G. (1997) The selection of superior winter hardy genotypes using a prolonged freeze test. Canadian Journal of Plant Science 77, 15–21.

224

L.V. Gusta et al.

Gusta, L.V., Wisniewski, M., Nesbitt, N.T. and Gusta, M.L. (2000) Freezing injury in cereals: an overall view and new approaches on increasing frost tolerance of cereals. In: Proceedings of the 8th International Barley Genetics Symposium. University of Adelaide, Adelaide, South Australia, pp. 260–264. Gusta, L.V., Wisniewski, M., Nesbitt, N.T. and Gusta, M.L. (2004) The effect of water, sugars and proteins on the pattern of ice nucleation and propagation in acclimated and non-acclimated canola leaves. Plant Physiology 135, 1642–1653. Gusta, L.V., Gao, Y.-P. and Benning, N.T. (2006) Freezing and desiccation tolerance of imbibed canola seed. Physiologia Plantarum 127, 237–246. Guy, C.L. (1990) Cold acclimation and freezing tolerance: role of protein metabolism. Annual Review of Plant Physiology 41, 187–233. Hirsh, A.G., Williams, R.J. and Meryman, H.T. (1985) A novel method of natural cryoprotection. Intracellular glass formation in deeply frozen Populus. Plant Physiology 79, 41–56. Houde, M., Danyluk, J., Laliberte, J.-F., Rassart, E., Dhindse, R.S. and Sarhan, F. (1992) Cloning, characterization and expression of cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat. Plant Physiology 99, 1381–1387. Hudson, M.A. and Brustkern, P. (1965) Resistance of young and mature leaves of Minium undulatum (L) to frost. Planta (Berlin) 66, 135–155. Hurry, V.M. and Huner, N.P.A. (1991) Low growth temperature effects a differential inhibition of photosynthesis in spring and winter wheat. Plant Physiology 96, 491–497. Ishikawa, M. and Sakai, A. (1981) Freezing avoidance mechanisms by supercooling in some rhododendron flower buds with reference to water relations. Plant & Cell Physiology 22, 953–967. Junttila, O. and Stushnoff, C. (1977) Freezing avoidances by deep supercooling in hydrated lettuce seeds. Nature 269, 325–327. Kobayashi, F. Takumi, S., Nakata, M., Ohno, R., Nakamura, T., Nakamura, C. (2004) Comparative study of the expression profiles of the Cor/Lea gene family in two wheat cultivars with contrasting levels of freezing tolerance. Plant Physiology 120, 585–594. Koster, K.L. (1991) Glass formation and desiccation tolerance in seeds. Plant Physiology 96, 302–304. Lebate, C.A. and Leegood, R.C. (1988) Limitation of photosynthesis by changes in temperature, factors affecting the response of carbon dioxide assimilation to temperature in barley leaves. Planta 173, 519–527. Lee, R.E., Lee, M.R. and Strong-Gunderson, J.M. (1995) Biological control of insect pests using ice-nucleating microorganisms. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 257–269. Levitt, J. (1980) Responses of Plants to Environmental Stresses. Vol. 1. Chilling, Freezing and High Temperature Stresses, 2nd edn. Academic Press Inc., New York, New York. Limin, A.E. and Fowler, D.B. (1994) Relationship between guard cell length and cold hardiness in wheat. Canadian Journal of Plant Science 74, 59–62. Marcellos, H. and Single, W.V. (1976) Frost injury in wheat ears after ear emergence. Australian Journal of Plant Physiology 11, 7–15. Markowski, A. and Rapacz, M. (1994) Comparison of vernalization requirement and frost resistance of winter rape lines derived from a double haploid. Journal of Agricultural Crop Science 173, 184–192. McKersie, B.D. and Leshem, Y.Y. (1994) Stress and Stress Coping in Cultivated Plants. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 79–128. Miller, K. (1982) Cold hardiness strategies of some adult and immature insects overwintering in interior Alaska. Comparative Biochemistry and Physiology 73A, 595–604. Olien, C.R. (1961) A method of studying stresses occurring in plant tissue during freezing. Crop Science 1, 26–28. Olien, C.R. (1964) Freezing processes in the crown of ‘Hudson’ barley from winter injuries. Crop Science 16, 201–204. Olien, C.R. (1965) Interference of cereal polymers and related compounds with freezing. Cryobiology 2, 47–54. Olien, C.R. (1974) Energies of freezing and frost desiccation. Plant Physiology 53, 764–767. Öquist, G., Hurry, V.M. and Huner, N.P.A. (1993) Low temperature effects on photosynthesis and correlation with freezing tolerance in spring and winter cultivar of wheat and size. Plant Physiology 101, 245–254. Pearce, R.S. and Fuller, M.P. (2001) Freezing of barley studied by infrared video thermography. Plant Physiology 125, 227–240. Rasmussen, D.A. and Mackenzie, A.P. (1972) Effects of solute on ice-solution interfacial free energy: calculation from measured homogeneous nucleation temperature. In: Jellineck, H.H.G. (ed.) Water Structure at the Water–Polymer Interface. Plenum Press, New York, New York, pp. 126–145.

Patterns of Freezing in Plants

225

Reaney, M.J.T. and Gusta, L.V. (1999) Modeling sequential responses of plant cells to freezing and thawing. In: Margesin, R. and Schinner, F. (eds) Cold Adapted Organisms: Ecology, Physiology, Enzymology and Molecular Biology. Springer-Verlag, Berlin, pp. 119–136. Salt, R.W. (1961) Principles of insect cold hardiness. Annual Review of Entomology 6, 55–74. Savitch, L.V., Gray, G.R. and Huner, N.P.A. (1997). Feedback-limited photosynthesis and regulation of sucrose–starch accumulation during cold acclimation and low-temperature stress in a spring and winter wheat. Planta 201, 18–26. Schilling, B.S. (2004) Physiological and molecular responses of spring and winter canola (Brassica napus) during cold acclimation. PhD thesis, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, p. 163. Shearman, L.L., Olien, G.R., Smith, M.N. and Kindel, P.K. (1973) Production of freeze inhibitor polysaccharides of rye and barley in tissue culture. Crop Science 26, 189–191. Single, W.V. (1964) Studies on frost injury to wheat. II. Ice formation within the plant. Australian Journal of Agricultural Research 15, 869–875. Single, W.V. and Olien, C.R. (1967) Freezing process in wheat stems. Australian Journal of Biological Sciences 20, 1025–1028. Svec, L.V. and Hodges, H.F. (1972) A comparison of chemical changes in plants during cold hardening and natural environments. Canadian Journal of Plant Science 52, 165–175. Tanino, K.K. and McKersie, B.D. (1985) Injury within the crown of winter wheat seedlings after freezing and icing stress. Canadian Journal of Botany 63, 432–436. Tolleter, D., Jaquinod, M., Mangavel, C., Passirani, C., Sauinier, P., Manon, S., Teyssier, E., Payet, N., AvelangeMacherel, H-H. and Macherel, D. (2007) Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation. The Plant Cell 19, 1580–1589. Trischuk, R.G. (2003) Photoperiod and cultivar effects during acclimation, de-acclimation and re-acclimation on spring and winter wheat (Triticum aestivum). MSc. thesis, University of Saskatchewan. Tyler, N.J., Gusta, L.V. and Fowler, D.B. (1981) The effect of water stress on the cold hardiness of winter wheat. Canadian Journal of Botany 59, 1717–1721. Weigand, K.M. (1906) some studies regarding the biology of buds and twigs in winter. Botanical Gazette 103, 372–424. Wilen, R.W., Fu, P., Robertson, A.J. and Gusta, L.V. (1996) A comparison of the cold hardiness potential of spring cereals and vernalized and non vernalized winter cereals. In: Li, P.H. and Chen, T.H.H. (eds) Plant Cold Hardiness: Molecular Biology, Biochemistry and Physiology. Plenum Press, New York, New York, pp. 192–202. Wisniewski, M. (1995) Deep supercooling in woody plants and the role of cell wall structure. In: Lee, R.E. Jr, Warren, G.J. and Gusta, L.V. (eds) Biological Ice Nucleation and Its Applications. APS Press, St Paul, Minnesota, pp. 163–181. Wisniewski, M.E., Close, T.J., Artlip, T. and Arora, R. (1996) Seasonal patterns of dehydrins and 70 kDa heat shock proteins in bark tissues of eight species of woody plants. Physiologia Plantarum 96, 476–505. Wisniewski, M., Lindow, S.E. and Ashworth, E.N. (1997) Observations of ice nucleation and propagation in plants using infrared video thermography. Plant Physiology 113, 327–334. Wisniewski, M., Fuller, M., Glenn, D.M., Gusta, L., Duman, J. and Griffith, M. (2002a) Extrinsic ice nucleation in plants: what are the factors involved and can they be manipulated. In: Li, P.H. and Palva, E.T. (eds) Plant Cold Hardiness: Gene Regulation and Genetic Engineering. Kluwer Academic/Plenum Publishers, New York, New York, pp. 211–221. Wisniewski, M., Glenn, D.M. and Fuller, M.P. (2002b) Use of a hydrophobic particle film as a barrier to extrinsic ice nucleation in tomato plants. Journal of the American Society for Horticultural Science 127, 358–364. Wisniewski, M., Bassett, C. and Gusta, L.V. (2003) An overview of cold hardiness in woody plants: seeing the forest through the trees. HortScience 38, 952–959. Workmaster, B.A., Palta, J.P. and Wisniewski, M. (1999) Ice nucleation and propagation in cranberry uprights and frits using infrared thermography. Journal of the American Society for Horticultural Science 124, 619–625.

22

Going to Extremes: Low-temperature Tolerance and Acclimation in Temperate and Boreal Conifers G.R. Strimbeck and P.G. Schaberg

Introduction Despite global warming, temperatures in the continental interiors of Canada and Siberia can still fall below −60°C and can remain below −40°C for weeks at a time (Latysheva et al., 2007). These extreme temperatures occur not in barren tundra regions, but taiga forests dominated by species of spruce (Picea), fir (Abies), pine (Pinus) and larch (Larix). While other plant and animal species may receive some protection from snow cover, the above-ground parts of trees, including the foliage of evergreen trees, must survive the full brunt of the winter environment. Sakai (Sakai, 1960; Sakai and Weiser, 1973) first showed that the stems, buds and evergreen needles of various boreal conifers and other woody species can survive immersion in liquid nitrogen (LN2) at −196°C, provided they are first slowly cooled to an intermediate temperature, usually in the range from −15 to −40°C. For all practical purposes, these plants have achieved absolute lowtemperature (LT) tolerance, far beyond that of most crop species, fruit trees and model species that have (for good reasons) been the focus of the majority of studies of plant LT tolerance. Many temperate-zone conifer species, including Picea, Abies and Pinus species, reach their lower limit of LT tolerance in the −20 to −50°C range. A fundamental understanding of the cryoprotective mechanisms of extreme LT tol226

erance and the acclimation process in boreal species may be of value in improving the LT tolerance of less hardy species, in developing technologies for the cryopreservation of food, drugs, cells and whole organs, and in predicting the effects of global warming on boreal forest ecosystems.

The Ringve ‘Experiment’ The Ringve Botanical Garden in Trondheim, Norway, is owned and maintained by the Trondheim Natural History Museum, a branch of the Norwegian University of Science and Technology. It includes a small arboretum with 52 conifer and about 70 deciduous tree and shrub species from temperate and boreal regions around the northern hemisphere. These were planted shortly after the founding of the arboretum in 1975, with many accessions from other plantings in Norway, so that the ultimate seed source of many species and individual trees is, unfortunately, not known. Nevertheless, under the assumption that all genotypes can be traced to somewhere within the original range of each species, the collection presents an opportunity for comparative studies of LT tolerance and acclimation in conifers growing in a common environment, albeit one with a relatively mild winter climate. We hope that these comparative studies will

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Going to Extremes

help us to identify and isolate biochemical components and biophysical processes that contribute to extreme LT tolerance. The arboretum is located just a few hundred metres from Trondheimsfjord and, like most of coastal Norway, the local climate is moderated by the North Atlantic Drift, a poleward-flowing branch of the Gulf Stream. The local climate is classified as southern boreal and moderately oceanic in the Norwegian system (Moen, 1999) or more generally as temperate oceanic in global climate classifications (Trewartha and Horn, 1971). Despite a latitude of 63°25′N, in December to February mean temperatures are in the range of −2 to −3°C, with (increasingly) frequent periods of mild (temperature >0°C) weather throughout the winter, and minimum temperatures are about −20°C, although these have become less common in recent years. We compared midwinter LT responses of foliage from several species in each of the genera Abies, Picea and Pinus, and profiled LT acclimation and deacclimation in a boreal–temperate species pair in each genus. Our methods included controlled freezing to temperatures ranging from 0 to −80°C at relatively slow rates, quenching in LN2 after slow cooling to intermediate temperatures, and assessment of injury by the well-established relative electrolyte leakage (REL) method or an image analysis method for quantifying visible symptoms of LT stress and injury (Strimbeck et al., 2007). We also measured concentrations of mono-, diand oligosaccharides in many of our samples, and used differential scanning calorimetry (DSC) to look for evidence of glass transitions in conifer needle tissue. Here we review our results along with some of the pertinent literature to develop a composite picture of the patterns of LT tolerance and acclimation in conifers, with some discussion of possible mechanisms.

Midwinter Low-temperature Tolerance Much of the current understanding of LT tolerance in conifers dates to the exhaustive work of Akira Sakai, who identified the minimum survival temperature in 10°C classes for twig, bud and needle tissues of nearly 150 conifer spe-

227

cies, mainly temperate and boreal species and varieties from the northern hemisphere (Sakai and Okada, 1971; Sakai and Weiser, 1973; Sakai, 1983) but also including some species from warmer regions and the southern hemisphere. Conifers have also been approximately grouped into US Department of Agriculture hardiness zones based on their natural distributions (Bannister and Neuner, 2001). While these data give an excellent ‘big picture’ of the overall range of LT tolerance among conifers, it does not shed much light on differences in the LT responses of different species or groups, which might help differentiate mechanisms involved in extreme LT tolerance. Using material collected at the Ringve arboretum, we compared the midwinter LT responses of needles from eight conifer species in each of the genera Picea, Abies and Pinus (Strimbeck et al., 2007). We used a non-linear procedure to fit symmetrical, sigmoid curves to electrolyte leakage data for each species (Fig. 22.1), using four parameters to describe the shape of the curve: (i) Ymin, the baseline or uninjured conductivity; (ii) Ymax, the maximum conductivity produced by slow freezing (−0.1°C/ min); (iii) k, a measure of the steepness of the curve in the response region between Ymin and Ymax; and (iv) Tm, the midpoint of the curve. As in many studies involving deeply LT-tolerant species, relative tolerance cannot always be expressed as a simple LT50 if slow freezing never results in 100%, or in some cases even 50%, injury; however, Tm can be interpreted as an LT50 if Ymax corresponds to 100% lethal injury. The ratio of Tm to Ymax provides a composite index of relative LT tolerance that includes both a measure of response to LT stress (Tm) and a measure of maximum LT injury (Ymax). This index is especially useful in distinguishing the extremely tolerant boreal species, which have low but variable Ymax values. In general, species originating in temperate oceanic climates or warm temperate mountains with sporadic frost had relatively high Ymax and Tm values, with slow freezing producing lethal injury generally in the −20 to −40°C range (Table 22.1). Species from boreal regions generally had lower Tm and Ymax, indicating not only that lower temperatures are required to stress or injure the needles, but that injury is incomplete even at −80°C. Species from

228

G.R. Strimbeck and P.G. Schaberg

1.0 0.8 0.6 0.4

Relative electrolyte leakage

0.2 Abies alba

Picea sitchensis

Pinus nigra

Abies lasiocarpa

Picea engelmannii

Pinus contorta

0.8 0.6 0.4 0.2

0.8 0.6 0.4 0.2 Abies balsamea

0 0

−20

−40

−60

−80 0

Picea obovata −20 −40 −60 Temperature (°C)

−80 0

Pinus sylvestris −20

−40

−60

−80

Fig. 22.1. Examples of temperature–relative electrolyte leakage curves for oceanic or warm temperate (top), temperate continental and montane (middle) and boreal (bottom) conifer species. The different symbols and lines represent three different trees of each species.

temperate continental climates showed intermediate responses. The Tm/Ymax ratio was generally >−60 for temperate oceanic and warm temperate montane species, −60 to −100 for continental species, and <−100 for boreal species. Thus, the LT tolerance of various conifer species correlates reasonably well with the climate in their region of origin, especially when elevation is taken into account, even when they are grown under common and relatively mild climatic conditions. Exceptions to this pattern include species with boreal (Pinus banksiana, Abies sibirica) and continental distributions (Picea jezoensis, Picea

rubens) that are more LT-sensitive than expected, and this may be related to the ecological history of the species, seed source of trees in the collection, or specific responses to the local climate and growing conditions.

Quantification of Visible Symptoms One of the problems with using REL as a response variable is that it leaves some uncertainty as to how it correlates with specific symptoms of LT injury, such as the orangebrown necrotic coloration characteristic of

Table 22.1. Original range, climate region and low-temperature tolerance parameters for foliage of 24 conifer species collected at Ringve Botanical Garden, Trondheim, Norway, on 13 February 2006. Main range (continenta)

Abies alba Abies procera Abies veitchii Abies amabilis Abies sibirica Abies homolepis Abies lasiocarpa s.l.c Abies balsamea Picea jezoensis Picea sitchensis Picea rubens

Alps & Pyrenees (EU) Pacific north-west mountains (NA) Mountains, central Japan (AS) Pacific north-west coast (NA) Siberia (AS) Mountains, southern Japan (AS) Rocky Mountains (NA) Eastern Canada NA) Japan and adjacent mainland (AS) Pacific north-west coast (NA) Appalachian Mountains and north-east coast (NA) Rocky Mountains (NA) Balkans (EU) Northern Europe (EU) Canada (NA) Siberia (AS) Sierra Nevada (NA) Mountains in Mediterranean region (EU) Eastern Canada (NA) North-east US and south-east Canada (NA) Rocky Mountains & Pacific north-west coast (NA) Korea and Japan (AS) Northern Europe to Siberia (EU, AS) Alps and Carpathians (EU)

Picea engelmanni Picea omorika Picea abies Picea glauca Picea obovata Pinus jeffreyi Pinus nigra Pinus banksiana Pinus strobus Pinus contorta Pinus koraiensis Pinus sylvestris Pinus cembra

Elevation (m)

Regional climateb

Tm (°C)

Ymax

Tm/Ymax

300–1950 60–2700 1600–1900 1000–2300 ?−2400 700–2200 600–3700 0–1700 40–1000 0–1000 0–2000

Do, Dca Do (H) Dca (Cf) Do E (Dcb) Cf BSk (H) E, Dcb Dcb, Dca, E Do Dcb

−28.8 −32.4 −32.6 −37.7 −30.6 −33.6 −37.4 −36.7 −32.7 −35.7 −38.3

0.73 0.68 0.55 0.63 0.45 0.47 0.48 0.34 0.70 0.68 0.69

−39.7 −47.6 −59.6 −59.7 −69.3 −72.2 −83.0 −107.1 −46.5 −52.7 −56.1

1000–3000 400–1700 0–2200 0–2100

BSk (H) Dca E, Dcb E E Cs (H) Cs, Dca E, Dcb Dcb, Dca BSk (H), Do Dca E Dca

−44.0 −43.8 −47.9 −41.0 −37.0 −30.4 −26.8 −27.3 −36.6 −39.5 −35.2 −38.1 −42.9

0.54 0.53 0.43 0.31 0.26 0.68 0.60 0.55 0.49 0.52 0.46 0.35 0.37

−81.7 −83.4 −112.7 −136.1 −144.6 −45.0 −44.9 −50.5 −81.1 −78.0 −77.5 −111.5 −118.0

2000–3100 200–2000 0–800 0–1500 0–3500 600–1800 0–1000 1300–2400

Going to Extremes

Species

a

AS, Asia; EU, Europe; NA, North America. Climate in original range estimated from global map in Trewartha and Horn (1971). E, boreal; Dc, temperate continental; Do, temperate oceanic; Cf, subtropical humid; Cs, subtropical dry summer (Mediterranean); BS, steppe or semi-arid; (H), montane, follows climate of surrounding region. c Abies lasiocarpa s.s. includes only populations in coastal mountain ranges; Rocky Mountain populations have been segregated as Abies bifolia (Earle, 1997–2008). The provenance of the trees in the arboretum is unknown, so they might have come from either region. b

229

230

G.R. Strimbeck and P.G. Schaberg

dried out before necrosis was complete. In temperate species, chlorosis is also an intermediate stress symptom, appearing after exposure to temperatures 5 to 10°C warmer than those resulting in necrosis. In boreal species, LT treatment produced up to 30% chlorosis but little necrosis. Winter chlorosis in conifer foliage is thought to be a result of the destruction of chlorophyll by a combination of LT and bright light, and appears to be reversible as chlorotic needles can regain a normal green colour in the spring (Baronius et al., 1991). We also measured REL in sub-samples of the shoots used to evaluate visible symptoms, giving paired REL and colour measurements for these samples. While there was no straightforward correlation between relative conductivity and chlorosis or necrosis across all six species, necrosis occurred only in temperate species at REL values above 0.5 (Fig. 22.3),

LT-injured needles under field conditions (Friedland et al., 1984). Exposing whole shoots to LT stress, then placing them under bright light in cool conditions for about two weeks gives symptoms of injury very similar to those observed in the field. We scanned samples of needle sections after development of injury symptoms and used an image analysis procedure to quantify the relative amounts of necrotic (red-orange), chlorotic (yellow) and healthy green tissue in boreal–temperate pairs of Abies, Picea and Pinus species (Strimbeck et al., 2007). In agreement with the REL results, the temperate species developed up to 100% necrosis after LT stress at temperatures ranging from −40 to −60°C (Fig. 22.2). Some samples from the temperate species maintained up to 40% chlorosis even when severely injured; we think this is because these samples desiccated rapidly so that the tissue died and

100 necrosis 80 60

% of needle area

40 20 green

chlorosis

0 100 80 60 40 20 0 0

−20 −40 −60 Temperature (°C)

−80 0

−20 −40 −60 Pre-quench temperature (°C)

−80

Fig. 22.2. Visible low-temperature stress and injury symptoms in Abies alba (top), a species from warmtemperate region mountains, and Abies balsamea (bottom), a boreal species, after slow freezing (left) and slow freezing followed by quenching in liquid nitrogen (right). Samples were collected at Ringve Botanical Garden, Trondheim, Norway (63°25′N), 13 March 2006.

Going to Extremes

100

% necrosis

80 60 40 20 0

0

0.2 0.4 0.6 0.8 Relative electrolyte leakage

Fig. 22.3. Relative electrolyte leakage and necrosis in needles of boreal ( ) and temperate ( ) Abies, Picea and Pinus species after slow cooling to temperatures from 0 to −80°C.

giving a minimum value of REL associated with the development of necrosis, the main visible symptom of lethal injury. We now use an REL of 0.5 as a threshold value for determining if a sample is lethally stressed by LT treatment, and where Ymax>0.5 we refer to the corresponding Tm as LT50, an estimate of the temperature giving 50% cell death. As REL of foliage from most boreal and some temperate continental and montane species does not exceed this value at temperatures as low as −80°C, we conclude that they can survive these extreme low temperatures.

Liquid Nitrogen Quench Tolerance Sakai (1960) first showed that twig and bud tissues of boreal willows could survive quenching in LN2 at −196°C provided they are first slowly cooled to some intermediate temperature in the −15 to −30°C range. He later showed that tissues of boreal conifers can survive similar treatment, and suggested that the highest pre-quench temperature that a tissue can survive be used as an index of relative LT tolerance in extremely hardy species (Sakai and Okada, 1971; Sakai and Weiser, 1973; Sakai, 1983). To confirm and build on these results, we quenched whole shoots of six boreal and tem-

231

perate conifer species in LN2 after slow cooling to temperatures ranging from 0 to −80°C, and measured both REL and visible symptoms (Strimbeck et al., 2007). The temperate species were always lethally injured by LN2 quenching, with REL values in the same range as Ymax for the same species, and developed a mixture of chlorosis and necrosis comprising usually 90% or more of needle area (Fig. 22.2). In contrast, boreal species were lethally injured by LN2 quenching from temperatures between 0 and −20°C, but maintained REL values <0.5 and a mixture of chlorosis and green tissue with <5% necrosis when quenched from −30°C or lower. Our results confirm that the foliage of boreal conifers can survive LN2 quenching if it is first cooled to −30°C, and we can conclude that the cells undergo a transition during slow cooling between −20 and −30°C that confers the capacity to survive further slow cooling and LN2 quenching.

Acclimation Patterns The acclimation process has been studied in various conifer species in both natural and controlled environments (reviewed in Bigras et al., 2001). Acclimation in conifers follows many of the general patterns established for other species. Early acclimation is triggered by a critical night length, and reinforced by chilling temperatures. It involves desaturation of fatty acids and changes in membrane lipid composition (e.g. Senser, 1982; Martz et al., 2006), and accumulation of sucrose and oligosaccharides (e.g. Parker, 1959; Hinesley et al., 1992; Schaberg et al., 2000). We used our REL method to profile acclimation and deacclimation in boreal–temperate pairs of Abies, Picea and Pinus species growing in the Ringve Botanical Garden from August 2006 to April 2007 (Strimbeck et al., 2008). Figure 22.4 shows the results for the Siberian species Picea obovata and the temperate rainforest species Picea sitchensis. In August, all six species had Ymax values >0.75 and LT50 values around −10°C. Despite the relatively mild winter climate of Trondheim and unusually warm conditions in early winter, the three boreal species acclimated rapidly, becoming tolerant of temperatures below

G.R. Strimbeck and P.G. Schaberg

1.0 0.8 0.6 0.4 30.0 0.2

20.0

Relative electrolyte leakage

232

Temperature (°C)

10.0 0.0 −10.0 −20.0 −30.0 −40.0 −50.0 −60.0 15 Aug 04 Sep 25 Sep 08 Oct 23 Oct 05 Nov 20 Nov 04 Dec 02 Jan 22 Jan 12 Feb 05 Mar 26 Mar 23 Apr 2006 2007

Sample date Fig. 22.4. Ymax (•, ), relative electrolyte leakage after quenching in liquid nitrogen (LN2) from −30 °C (▲, ) and Tm (■, ) of foliage of Picea sitchensis ( , , ) and Picea obovata (•, ▲, ■) growing in the Ringve Botanical Garden during acclimation and deacclimation. Maximum and minimum (-----) and 5-day mean air temperature ( ) at Værnes International Airport (Trondheim) in the middle. Error bars are standard errors for parameters of non-linear curves fitted to three trees of each species (Ymax and Tm) or standard deviations for three trees (LN2 quenching). Some symbols are offset by +1 day for clarity.

−40°C by late October, with only one night where the temperature dropped a few tenths of a degree below freezing, probably not low enough to induce freezing in the plant. By late November, foliage from these species survived LN2 quenching from −30°C, although daytime temperatures remained above freezing with only sporadic night frost for most of the month. The temperate species acclimated only to around −15°C by late October, then more rapidly to temperatures in the −20 to −30°C range by late November, where they remained throughout the mild early winter period. They reached minimum LT50 ranging from about −28 to −36°C following a period of lower temperatures in late January and early February. Although temperatures were similar to those in November, all species deacclimated during March and April. These results suggest that acclimation in boreal species follows a rigid program that is relatively unaffected by environmental temper-

atures, while temperate species maintain some responsiveness to temperature throughout the winter. It is interesting to note, however, the parallel course of Tm values in both Picea species over the midwinter period (Fig. 22.4); the changes in Ymax are the dominant factor in the differences between the two species. Changes in temperature response curves during acclimation reveal different styles of acclimation in the boreal and temperate species (Fig. 22.5). In temperate species, acclimation can be described as ‘parallel slope retreat’, a shift of the sigmoid curve towards a low temperature while it maintains its steepness and amplitude. In these species, Tm can always be interpreted as LT50, and this parameter alone is more or less adequate to describe the changes in LT tolerance. Acclimation in boreal species can be described as ‘slope decline’, where there is both a shift of Tm towards lower temperatures and a decrease in maximum REL (Ymax) until it falls below the 0.5 REL

Going to Extremes

233

1.0

Relative electrolyte leakage

0.88

0.91

0.90

0.6

0.96

0.99

0.96 0.87

0.6 0.87

0.75 15 Aug 25 Sep 08 Oct 23 Oct 05 Nov Tm

0.4

0.2

0

0

−10

−20

−30

−40

−50

0.68

−10

−20

−30

−40

−50

−60

Temperature (°C) Fig. 22.5. Temperature–relative electrolyte leakage curves for temperate (Pinus jeffreyi, left) and boreal (Pinus sylvestris, right) Pinus species over five sample dates during acclimation. Curves fit to three trees of each species, values are model R2.

threshold we use to distinguish lethal LT stress. This also involves a decrease in slope characterized by a third parameter, k, but Tm and Ymax together give an adequate picture of the changes in LT tolerance in extremely hardy species. In both groups, Tm drops after periods of temperatures below 0°C and may increase slightly during prolonged mild periods, and thus seems somewhat responsive to environmental temperature. These parameters may represent different mechanisms of LT tolerance. Tm is the midpoint of some process that results in an increase in electrolyte leakage. One explanation for this would be a temperature-dependent phase change in cell membranes, with the transition temperature depending on membrane lipid composition, another factor known to change during LT acclimation. Fatty acid desaturation and changes in lipid composition that occur during acclimation can affect membrane phase change temperatures and stability during freezing stress (Steponkus, 1984; Uemura and Steponkus, 1999). Regardless of the actual mechanism, the transition represented by Tm is ultimately lethal in temperate species, but a controllable stress response in boreal species. Thus, the lowering of Ymax may represent a different process that prevents lethal injury.

Sugars and Low-temperature Tolerance Sugar accumulation is closely associated with LT tolerance in numerous plant species, including conifers, and in vitro studies show that sugars have cryoprotective effects on proteins and membranes (Arakawa and Timasheff, 1982; Caffrey et al., 1988; Leborgne et al., 1995; Santarius and Franks, 1998). While sucrose is typically the most abundant sugar in acclimated plants, raffinose is also common, especially in conifers (Little, 1970; Hinesley et al., 1992; Schaberg et al., 1999), and smaller amounts of stachyose may also appear during acclimation. We measured dry weight concentrations of sucrose, raffinose, stachyose, glucose, fructose and xylose in foliar samples drawn from the same bulk samples we used in our LT tolerance testing in both the midwinter 24 species comparison and the seasonal profile of six boreal and temperate species. In the midwinter study, we found significant correlations between Tm and sucrose (R2=0.35, P=0.042, n=12) and raffinose (R2=0.46, P=0.015, n=12) in a subgroup of relatively LT-sensitive temperate and continental species (defined by Ymax>0.5), but no significant relationship in the remaining continental and boreal species, where Tm is an

234

G.R. Strimbeck and P.G. Schaberg

incomplete descriptor of LT tolerance (Strimbeck et al., 2007). Thus, the relationships between LT tolerance and sugars found within species also hold across species, despite likely variation in the proportion of dry weight invested in cuticle and cell walls in different genera and species, which may affect the sugar/dry weight ratio. Studies of seasonal variation in sugar content in conifers have shown both sustained winter increases in montane populations of Picea rubens (Schaberg et al., 2000) and fluctuations that correlated with environmental

°C

30 20 10 0 −10 −20

temperature in paired cold- and warmtemperate Pinus and Cupressaceae species (Hinesley et al., 1992). In the latter study, raffinose was again singled out as closely associated with LT tolerance. In our seasonal study (Strimbeck et al., 2008), total sugar concentrations, driven principally by sucrose, fluctuated dynamically throughout the measurement period, with increases generally following periods of freezing temperatures and gradual decreases during prolonged periods of mild weather (Fig. 22.6). Decreases in sucrose may be due to some combination of respiratory

Abies alba 80 60

−60 −50 −40 −30 −20 −10

Abies balsamea

−200 −150 −100 −50 0

40

0 Picea sitchensis 80 60

−60 −50 −40 −30 −20 −10

Picea obovata

−200 −150 −100 −50

Tm/Ymax

Sugar concentration (mg/g)

20

0

40 20 0 Pinus jeffreyi 80 60

−60 −50 −40 −30 −20 −10

Pinus sylvestris

−200 −150 −100 −50 0

40 20 0 Aug Sep Oct Nov Dec Jan Feb Mar Apr May Aug Sep Oct Nov Dec Jan Feb Mar Apr May 2006 2007 2006 2007

Fig. 22.6. Top panels: daily maximum and minimum temperature (-----) and 5-day running mean ( ) at Værnes International Airport (Trondheim). Other panels: Tm/Ymax (•) and concentrations of sucrose ( ), raffinose ( ) and stachyose ×5 (D). Tm and Ymax from non-linear curves fit to relative electrolyte leakage data for three trees of each species, sugar concentrations are mean of three trees. X-axis tick marks on the first day of each month.

Going to Extremes

depletion, phloem export or perhaps conversion to starch, while increases may depend on the reverse processes. Raffinose and, in some species, stachyose first appeared in significant quantities early in the acclimati on process, increased monotonically through the winter, and decreased rapidly during deacclimation. Glucose and fructose were also found in variable quantities throughout the winter, with glucose increasing temporarily in all species during deacclimation (data not shown). There were no clear patterns in total sugar fluctuations unique to the boreal and temperate species groups. As in previous analyses, simple correlations between LT tolerance parameters and sugars suggest a key role for raffinose in LT tolerance in both the boreal and temperate groups (Table 22.2). Correlations between raffinose and both Tm and Ymax are negative, indicating that higher sugar concentrations are associated with lower midpoint temperatures and lower maximum REL values in both groups. Over all dates, raffinose concentrations are significantly higher in boreal than temperate species as indicated by a significant main effect of group in analysis of variance (Strimbeck et al., 2008). Stachyose also correlates significantly and negatively with Ymax in the boreal group and Tm in both groups. Overall stachyose concentrations are higher in the boreal than the temperate Picea species, but higher in Pinus jeffreyi than Pinus sylvestris, so there is no clear association of this sugar with extreme LT

235

tolerance. Despite its association with LT tolerance in our and others’ previous studies, sucrose correlates weakly with LT tolerance variables in the seasonal study. Sugar concentrations, notably stachyose and raffinose, are intercorrelated, including significant negative correlations between glucose and raffinose and stachyose in the boreal group, making multiple regression models of LT tolerance parameters on sugar concentrations unstable.

Cytoplasmic Vitrification Intracellular vitrification has long been implicated as a general mechanism of extreme LT and desiccation tolerance in plant tissues (Burke, 1985; Hirsh, 1987). According to this model, cytoplasm goes through a glass transition as a result of dehydration (in desiccationtolerant organisms) or a combination of LT and dehydration in frozen tissues. Dehydration concentrates sugars and other cellular components, and both processes result in increased cytoplasmic viscosity and ultimately in a complete loss of rotational and translational molecular mobility, so that all molecules are more or less locked in place in an amorphous glassy matrix. This will slow further dehydration by several orders of magnitude, and prevent deleterious interactions between proteins, membranes and other cell components. Effectively, the glassy state is a kind of molecular suspended animation.

Table 22.2. R values of correlations among low-temperature tolerance parameters and sugar concentrations for the boreal and temperate species, based on measurements of data for nine trees in each group on 13 sample dates. Tm and Ymax for some trees in the boreal species group could not be determined on some dates due to poor curve fits. Temperate species (n=117) Tm Ymax Stachyose Raffinose Sucrose Glucose Fructose

Boreal species (n=106 for Tm and Ymax, n=117 between sugars) Tm

Ymax

0.704*** 0.283** −0.263** −0.151 −0.687*** −0.414*** 0.008 0.081 0.105 −0.165 −0.091 0.122

*P<0.05, **P<0.01, ***P<0.001.

Stachyose

Raffinose

Sucrose

Glucose Fructose

−0.272** −0.377***

−0.677*** −0.819*** 0.576***

0.092 0.187* −0.195* −0.094

0.153 0.039 0.342*** −0.022 −0.373*** −0.047 −0.315*** 0.119 0.457*** −0.017 −0.042 0.276**

0.339*** −0.354*** −0.151 0.213*

−0.053 0.020 −0.101

0.214* −0.161

236

G.R. Strimbeck and P.G. Schaberg

Our LN2 quenching results confirm that the foliage of boreal conifer species can survive temperatures as low as −196°C, and show that some transition occurs as the tissue is slowly cooled to about −30°C that allows the tissue to survive lower temperatures without further loss of membrane integrity or development of postfreezing necrosis. Although there may be other interpretations, these observations are consistent with the hypothesis that the concentrated cytoplasm in the freeze-dehydrated cells undergoes a glass transition. The well-characterized vitrification behaviour of sucrose and oligosaccharides provides a link between sugar accumulation and cytoplasmic vitrification. Under slow freezing, maximally freeze-concentrated sucrose solutions vitrify at about −41°C, without any apparent eutectic crystallization of the concentrated sucrose (Goff and Sahagian, 1996; Goff et al., 2003). Anhydrous raffinose and stachyose have glass transition temperatures as much as 40°C higher than sucrose, and can be expected to elevate glass transition temperatures in solutions as well (Buitink et al., 2000). The relatively high concentrations of these sugars in extremely LT-tolerant tissues suggests that vitrification is possible, if not probable, as tissues cool and cells are dehydrated by extracellular ice. Other cytoplasmic components, notably stress-associated proteins such as dehydrins (Buitink and Leprince, 2004), are also potential contributors to cytoplasmic vitrification. While vitrification has been convincingly demonstrated in dried orthodox seeds that remain viable for long periods at about 5% water content (Koster, 1991; Buitink and Leprince, 2004), the evidence for vitrification in frozen tissues is less persuasive (Hirsh, 1987) because it relies on rather subjective interpretation of DSC scans (Wolanczyk, 1989). A glass is functionally defined as a fluid with a viscosity >1012 Pa s (Franks, 1985), but there are no known methods for directly measuring viscosity in the extremely dehydrated protoplasts, surrounded by cell walls and buried in extracellular ice, that would be found in frozen conifer needles or other plant tissues at −30°C. DSC is the most commonly used method for studying glass transitions, and relies on identifying a step change in heat capacity as molecules lose rotational and

translational mobility in the glass transition. This is relatively clear in single materials such as plastics or in simple two-component systems such as aqueous sucrose solutions, but has proved much more difficult to detect in frozen tissue samples. More recently, modulated temperature DSC (MT-DSC) has been employed to distinguish changes in heat capacity from other thermal events that take place during a thermal scan, giving a much clearer picture of glass transitions (e.g. Goff and Sahagian, 1996). We employed MT-DSC to search for glass transitions in conifer needles and other extremely LT-tolerant plant tissues. We have detected glass transitions in a few samples from boreal species (Fig. 22.7) and they occur in the expected temperature range, but they are weak and not fully repeatable, so at best our direct evidence for LT-induced vitrification is equivocal. Because MT-DSC can only give the heat capacity of the whole sample and the freezedehydrated cells comprise only a small fraction of the total mass, we think that any change in cytoplasmic heat capacity may be masked by the stronger signal from extracellular ice and other components. Furthermore, while glass transitions in single materials or two-component systems occur over a range of a few degrees, in complex mixtures the shift in heat capacity would occur over a broader temperature range as different classes of molecules lose mobility. Other methods, such as electron spin resonance and NMR spectroscopies, which rely on detecting a relative change in molecular mobility, may suffer from similar problems when applied to frozen plant tissues. Cooling and freeze-concentration of cytoplasm may result in a more continuous change in viscosity and heat capacity, with the cytoplasm increasingly out of thermodynamic equilibrium with extracellular ice at progressively lower temperatures (Wolfe et al., 2002).

Conclusion LT tolerance in the foliage of conifers from north temperate and boreal regions varies according to the climate in the region of origin. Species from warm temperate mountains die at midpoint temperatures of −25 to −35°C,

Going to Extremes

237

−0.02 0.0

Non-reversing heat flow (W/g)

Heat flow (W/g)

−0.2

−0.2

−0.4

−0.6

−0.8 −0.6 −1.0 −50

−40

−30

−20 Temperature (°C)

−10

0

−0.06

−0.08

−0.4

−22.78°C(I)

−0.04

10

−0.10

Reversing heat flow (W/g)

0.0

−0.12

−0.14

Fig. 22.7. Modulated-temperature differential scanning calorimetry scan of four 5 mm Picea abies needle sections, sampled 3 March 2004, cooled slowly to −60°C, then scanned during warming at 0.2°C/min with temperature modulation of ±0.318°C in 60 s (Q1000 MT-DSC; TA Instruments, New Castle, Delaware, USA). Shown are total (——), non-reversing (- - -) and reversing (– – –) heat flows. Reversing heat flow corresponds to heat capacity, and the step change shown in the inset is a glass transition, measured using the inflection point.

while boreal species can survive slow freezing to −80°C or lower and quenching in LN2 after slow freezing to −30°C. Injury due to LT stress is expressed as electrolyte leakage of LT-stressed cells and as chlorosis and necrosis developed after exposure to light. Acclimation in temperate species involves a shift of the temperature response curve towards lower temperature, while in boreal species there is both a temperature shift and a decrease in the maximum

amount of injury produced by LT stress. Foliar concentrations of the oligosaccharides raffinose and stachyose correlate strongly with LT tolerance, indicating an important cryoprotective role for these sugars. During slow cooling from −20 to −30°C, boreal conifers undergo a transition, possibly cytoplasmic vitrification involving oligosaccharides, sucrose or other cytoplasmic components, that allows them to survive extreme LT stress.

References Arakawa, T. and Timasheff, S.N. (1982) Stabilization of protein structure by sugars. Biochemistry 21, 6536–6544. Bannister, P. and Neuner, G. (2001) Frost resistance and distribution of conifers. In: Bigras, F.J. and Colombo, S.J. (eds) Conifer Cold Hardiness. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 3–21. Baronius, G., Fiedler, H.J. and Montag, H.G. (1991) Comparative investigations by means of Munsell-color charts and the Cielab color system on the winter chlorosis of Pinus sylvestris L. in the pollution area of the Dueben Heath. Forstwissenschaftliches Centralblatt 110, 263–277.

238

G.R. Strimbeck and P.G. Schaberg

Bigras, F.J., Ryyppö, A., Lindström, A. and Stattin, E. (2001) Cold acclimation and deacclimation of shoots and roots of conifer seedlings. In: Bigras, F.J. and Colombo, S.J. (eds) Conifer Cold Hardiness. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 57–88. Buitink, J. and Leprince, O. (2004) Glass formation in plant anhydrobiotes: survival in the dry state. Cryobiology 48, 215–228. Buitink, J., Van Den Dries, I.J., Hoekstra, F.A., Alberda, M. and Hemminga, M.A. (2000) High critical temperature above Tg may contribute to the stability of biological systems. Biophysical Journal 79, 1119–1128. Burke, M.J. (1985) The glassy state and survival of anhydrous biological systems. In: Leopold, A.C. (ed.) Membranes, Metabolism, and Dry Organisms. Comstock Publishing Associates, Ithaca, New York, pp. 358–363. Caffrey, M., Fonseca, V. and Leopold, A. (1988) Lipid–sugar interactions. Relevance to anhydrous biology. Plant Physiology 86, 754–758. Earle, C.J. (1997–2008) The Gymnosperm Database. http://www.conifers.org/index.html (accessed June 2008). Franks, F. (1985) Biophysics and Biochemistry at Low Temperatures. Cambridge University Press, Cambridge, UK. Friedland, A.J., Gregory, R.A., Karenlampi, L. and Johnson, A.H. (1984) Winter damage to foliage as a factor in red spruce decline. Canadian Journal of Forest Research 14, 963–965. Goff, H.D. and Sahagian, M.E. (1996) Glass transitions in aqueous carbohydrate solutions and their relevance to frozen food stability. Thermochimica Acta 280, 449–464. Goff, H.D., Verespej, E. and Jermann, D. (2003) Glass transitions in frozen sucrose solutions are influenced by solute inclusions within ice crystals. Thermochimica Acta 399, 43–55. Hinesley, L., Pharr, D., Snelling, L. and Funderburk, S. (1992) Foliar raffinose and sucrose in four conifer species: relationship to seasonal temperature. Journal of the American Society for Horticultural Science 117, 852–855. Hirsh, A. (1987) Vitrification in plants as a natural form of cryoprotection. Cryobiology 24, 214–228. Koster, K. (1991) Glass formation and desiccation tolerance in seeds. Plant Physiology 96, 302–304. Latysheva, I.V., Belousova, E.P., Ivanova, A.S. and Pokemkin, V.L. (2007) Circulation conditions of the abnormally cold winter of 2005/06 over Siberia. Russian Meteorology and Hydrology 32, 572–575. Leborgne, N., Teulieres, C., Travert, S., Rols, M.P., Teissie, J. and Boudet, A.M. (1995) Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance. European Journal of Biochemistry 229, 710–717. Little, C.H.A. (1970) Seasonal changes in carbohydrate and moisture content in needles of balsam fir (Abies balsamea). Canadian Journal of Botany 48, 2021–2028. Martz, F., Sutinen, M.L., Kivineemi, S. and Palta, J.P. (2006) Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation. Tree Physiology 26, 783–790. Moen, A. (1999) National Atlas of Norway: Vegetation. Norwegian Mapping Authority, Hønefoss, Norway. Parker, J. (1959) Seasonal variations in sugars of conifers with some observations on cold resistance. Forest Science 5, 56–63. Sakai, A. (1960) Survival of the twigs of woody plants at −196°C. Nature 185, 393–394. Sakai, A. (1983) Comparative-study on freezing resistance of conifers with special reference to cold adaptation and its evolutive aspects. Canadian Journal of Botany–Revue Canadienne De Botanique 61, 2323–2332. Sakai, A. and Okada, S. (1971) Freezing resistance of conifers. Silvae Gentica 20, 91–97. Sakai, A. and Weiser, C.J. (1973) Freezing resistance of trees in North America with reference to tree regions. Ecology 54, 118–126. Santarius, K.A. and Franks, F. (1998) Cryopreservation of lactate dehydrogenase: interactions among various cryoprotectants. Cryo-Letters 19, 37–48. Schaberg, P.G., Strimbeck, G.R., Hawley, G.J., Dehayes, D.H., Shane, J.B., Murakami, P.F., Perkins, T.D., Donnely, J.R. and Wong, B.L. (1999) Cold tolerance and photosystem function in a montane red spruce population: physiological relationships with foliar carbohydrates. Journal of Sustainable Forestry 10, 173–180. Schaberg, P.G., Snyder, M.C., Shane, J.B. and Donnelly, J.R. (2000) Seasonal patterns of carbohydrate reserves in red spruce seedlings. Tree Physiology 20, 549–555. Senser, M. (1982) Frost resistance in spruce (Picea abies (L.) Karst). III. Seasonal changes in the phospho- and galactolipids of spruce needles. Zeitschrift fur Pflanzenphysiologie 105, 229–239.

Going to Extremes

239

Steponkus, P.L. (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology 35, 543–584. Strimbeck, G.R., Kjellsen, T.D., Schaberg, P.G. and Murakami, P.F. (2007) Cold in the common garden: comparative low-temperature tolerance of boreal and temperate conifer foliage. Trees – Structure and Function 21, 557–567. Strimbeck, G.R., Kjellsen, T.D., Schaberg, P.G. and Murakami, P.F. (2008) Dynamics of low-temperature acclimation in temperate and boreal conifer foliage in a mild winter climate. Tree Physiology 28, 1365–1374. Trewartha, G.T. and Horn, L.H. (1971) An Introduction to Climate. MGraw Hill International, London. Uemura, M. and Steponkus, P.L. (1999) Cold acclimation in plants: relationship between the lipid composition and the cryostability of the plasma membrane. Journal of Plant Research 112, 245–254. Wolanczyk, J.P. (1989) Differential scanning calorimetry analysis of glass transitions. Cryo-Letters 10, 73–76. Wolfe, J., Bryant, G. and Koster, K.L. (2002) What is ‘unfreezable water’, how unfreezable is it and how much is there? Cryo-Letters 23, 157–166.

23

The Rapid Cold-hardening Response in Insects: Ecological Significance and Physiological Mechanisms M.A. Elnitsky and R.E. Lee, Jr

Introduction Historically, investigations of insect cold hardiness have focused on seasonal adaptations acquired over a period of weeks to months, which promote low temperature and winter survival (Zachariassen, 1985; Duman et al., 1991; Lee, 1991; Storey and Storey, 1996). However, in addition to increased winter cold hardiness, many species of insects also possess the ability to quickly, on the order of minutes to hours, increase their cold tolerance at other times of the year. It has now been more than 20 years since the first report of this rapid cold hardening (RCH) response in insects (Chen et al., 1987; Lee et al., 1987). The RCH response is characterized by increased protection against cold shock (i.e. non-freezing) injury following a brief acclimation to moderately low temperature. Lee et al. (1987) originally hypothesized that RCH allows insects to ‘instantaneously’ enhance their cold tolerance by tracking changes in environmental temperature. Since then, strong support for this hypothesis has come from investigations documenting RCH under ecologically relevant cooling regimes and diurnal thermoperiods, and a significant body of literature has demonstrated the broader ecological implications – improved courtship behaviour, mating success and fecundity – of the RCH response. Therefore, the significance of RCH is not

240

restricted to the increased survival of low temperatures, but appears to function more generally to enhance organismal performance within a thermally variable environment. For many years the mechanisms underlying RCH remained poorly understood and only recently we have begun to gain a better understanding of the physiological mechanisms of the response. However, our knowledge remains limited as to the molecular and cellular events involved in the signalling pathway for induction of the cold-hardening response. The purpose of the present chapter is to review briefly our understanding of RCH in insects, especially as it relates to the ecological implications and physiological mechanisms of the response.

Ecological Significance of Rapid Cold Hardening Rapid cold hardening increases survival of low temperature Rapid cold hardening, as its name implies, contrasts with the slow, seasonal attainment of increased cold tolerance associated with preparation for winter. Within minutes to hours, the RCH response can dramatically increase an

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Rapid Cold-hardening Response in Insects

insect’s tolerance of cold shock. For example, flesh flies, Sarcophaga crassipalpis, reared at 25°C cannot survive direct exposure to −10°C for 2 h (Lee et al., 1987). However, if this lowtemperature treatment is preceded by RCH at 0°C for 2 h flies readily survive; even as little as 10 min at 0°C allows ∼50% of flies to survive subsequent exposure to −10°C. Similarly, in adult fruit flies, Drosophila melanogaster, chilling at 5°C for 30 min dramatically increases (from 0 to 50%) survival of subsequent exposure to −5°C for 2 h (Czajka and Lee, 1990). In nature, this response may be especially important to rapidly enhance the cold tolerance of insects during the early spring and late autumn months that are often accompanied by dramatic cold snaps. Most investigations have induced the RCH response by exposing insects to temperatures between 0 and 10°C for a few hours. However, the acquired cold tolerance is lost rapidly (within ∼2 h) if flies are returned to their rearing temperature of 25°C (Chen et al., 1991). Similar response profiles have been reported in most other insects in which RCH has been examined. RCH appears to be an extremely widespread response among freeze-intolerant insects, having been documented in nearly 30 species, including members of the orders Coleoptera (beetles), Diptera (flies), Hemiptera (bugs and aphids), Lepidoptera (butterflies and moths), Orthoptera (grasshoppers and crickets) and Thysanoptera (thrips). Within a species, the response can occur in multiple developmental stages, including eggs, larvae, pupae and adults (Czajka and Lee, 1990; Wang and Kang, 2005), and among both non-diapausing and diapausing individuals (Chen et al., 1987). Recently, we demonstrated that the RCH response not only confers increased tolerance of cold shock, but can also increase freeze tolerance, as we observed in the Antarctic midge, Belgica antarctica (Lee et al., 2006b).

Rapid cold hardening during ecologically relevant thermocycles Most early investigations of RCH induced the response by direct transfer of insects from their

241

laboratory rearing temperature to the cold hardening temperature (e.g. direct transfer from 23 to 0°C for 2 h). While such direct transfers clearly increase subsequent cold tolerance and are useful for exploring the physiological basis of the protection generated, the ecological relevance of such protocols for induction of RCH is questionable. In nature, insect populations rarely, if ever, experience such dramatic fluctuations and extremes of low temperature. A growing body of literature now indicates that RCH is also induced under ecologically relevant cooling regimes and natural diurnal thermocycles (Coulson and Bale, 1990; Kelty and Lee, 1999, 2001; Koveos, 2001; Powell and Bale, 2004, 2006; Kelty, 2007). For example, an RCH response is induced in D. melanogaster during cooling from 23 to 0°C at natural rates (0.05 or 0.1°C/min) (Kelty and Lee, 1999). Further, a significant decrease in the lower lethal temperature is observed when flies are cooled from 23°C to only 11°C, a temperature commonly experienced in nature. Kelty and Lee (2001) demonstrated that the cold tolerance of D. melanogaster increased during the cooling phase of an ecologically relevant, diurnal thermocycle and further increased with the number of thermoperiods to which flies were exposed. More recently, RCH has been documented in flies maintained in a field setting and exposed to ambient thermocycles (Koveos, 2001; Kelty, 2007). Such studies clearly demonstrate an ecological relevance for the RCH response and support the notion that RCH allows insects to ‘instantaneously’ enhance their cold tolerance within a thermally variable environment. However, most insects will never benefit from the reduction of the lower lethal temperature associated with RCH, as they are unlikely to experience temperatures low enough to cause mortality as a result of direct chilling injury. In this case, for the RCH response to have ecological relevance, it must prevent more subtle deleterious effects of chilling at temperatures that are likely to be encountered within an insect’s natural environment. Evidence for this may be seen in the effect of RCH on the critical thermal minimum (CTmin), the temperature at which an insect can no longer maintain activity

M.A. Elnitsky and R.E. Lee, Jr

and enters a cold-induced torpor or chill coma. In D. melanogaster, RCH during slow cooling (at 0.1°C/min) lowers the CTmin by >2.5°C (to 3.9°C) (Kelty and Lee, 1999). Powell and Bale (2006) reported a similar lowering of the CTmin (by ∼2.5°C) in the grain aphid, Sitobian avenae, during RCH. Further, the CTmin of D. melanogaster decreased during the cooling phase of a natural diurnal thermocycle (Kelty and Lee, 2001), seemingly ‘tracking’ changes in ambient temperature. Such reductions of CTmin may be explained by the preservation of the resting membrane potential in muscle fibres and nervous tissue, neural conduction velocities and neuromuscular coordination at lower temperatures following RCH (Kelty et al., 1996). Even a relatively modest 1–2°C reduction of the CTmin following RCH would allow insects to expand the thermal range over which activity, feeding and reproduction may be maintained, thereby optimizing performance at temperatures lower than otherwise possible.

Rapid cold hardening preserves courtship behaviour, reproductive success and longevity RCH appears to not only improve survival of low temperatures, but also ameliorates the sublethal effects associated with cold-shock injury (Rinehart et al., 2000; Powell and Bale, 2004; Shreve et al., 2004; but see Coulson and Bale, 1992). For example, in S. crassipalpis cold shock at −10°C for 1 h negatively affects adult longevity, egg production and fertilization success (Rinehart et al., 2000). However, RCH for 2 h at 0°C eliminates these negative effects following subsequent low-temperature exposure. Similar benefits of RCH on development, longevity and reproductive success in the aphid, S. avenae, were observed by Powell and Bale (2004). In adult D. melanogaster, a reduction in temperature from 23 to 16°C resulted in only half (11/22) of male– female pairs engaging in courtship behaviours and no pairs were observed mating (Shreve et al., 2004). However, if flies were allowed to acclimate for 2 h at 16°C, nearly all pairs (17/20) displayed courtship behaviours and

more than half (11/20) mated successfully. Further, the hardening response observed by Shreve et al. (2004) after 2 h at 16°C is the highest reported induction temperature for an RCH response. These results provide yet further evidence that RCH not only allows rapid enhancement of an insect’s cold tolerance, but represents a constant fine-tuning of behavioural and physiological processes to match environmental conditions. Consequently, the term ‘rapid cold hardening’, with its emphasis on low-temperature tolerance, is in some regards a misnomer, as it is too restrictive to encompass the wide range of induction temperatures and organismal benefits from this swift acclimatory response (Fig. 23.1).

Induction and Physiological Mechanisms of the Rapid Cold-hardening Response While a relatively clear picture has emerged for the ecological significance of the RCH response, understanding the underlying physiological mechanisms has proved a far greater challenge. This is especially true in light of documented species-specific differences in the biochemical/physiological responses that occur

Ambient temperature

242

Time of day Fig. 23.1. Theoretical representation of the rapid cold-hardening response. Rapid cold hardening allows insects (– – –) to track changes in ambient temperature (——), thereby optimizing their behavioural and physiological performance. Such fine-tuning may be crucial for important fitness traits, such as survival, activity, feeding and reproduction, within a thermally variable environment.

Rapid Cold-hardening Response in Insects

during RCH. More recent investigations have, however, begun to provide deeper insight into the mechanistic basis for the response.

243

and mechanistic role played by cryoprotective compounds in RCH is needed.

Heat-shock proteins and gene expression The role of cryoprotectants? Early investigations as to the mechanisms underlying the RCH response reported a correlation between the accumulation of cryoprotective compounds and the onset of increased cold hardiness (Chen et al., 1987; Lee et al., 1987). For example, cold-hardening pharate adults of S. crassipalpis at 0°C for 2 h results in a threefold increase in glycerol levels to ∼80 mM (Lee et al., 1987). While modest in comparison to the seasonal accumulations in many overwintering insects, such increases in cryoprotectants likely play a non-colligative role in stabilizing proteins and membrane structures at low temperatures (Carpenter and Crowe, 1988; Crowe et al., 1988). However, later investigations found no evidence for the accumulation of glycerol or other potential cryoprotectants during RCH in D. melanogaster (Kelty and Lee, 1999, 2001). Together these results suggest that, at least in some insect species, the accumulation of cryoprotective compounds alone cannot account for the increase in cold hardiness that occurs during RCH. More recent studies have taken a metabolomics approach to simultaneously monitor the relative concentrations of a large number of metabolites in insects exposed to low temperature and/or RCH. In S. crassipalpis, metabolomic analysis confirmed previous reports of the accumulation of glycerol during RCH, but also revealed increased concentrations of a variety of other known cryoprotectants including sorbitol, glucose and alanine (Michaud and Denlinger, 2007). Similarly, using metabolic profiling, Overgaard et al. (2007) reported increased concentrations of glucose and trehalose (∼1.5- to 2-fold) in adults of D. melanogaster during an ecologically relevant bout of cooling that induced RCH. This result differs from previous reports that found no evidence of cryoprotectant accumulation in D. melanogaster during RCH (Kelty and Lee, 1999, 2001). Clearly, further study concerning the accumulation

The synthesis of heat-shock proteins (i.e. stress proteins) and other molecular chaperones is a well-documented rapid response to hightemperature exposure in both plants and animals, including insects (Feder and Hofmann, 1999). However, the response of heat-shock proteins in insects exposed to low temperature is less well studied (Denlinger and Lee, 1998). Stress-protein synthesis in response to cold shock has been documented in D. melanogaster (Burton et al., 1988; Sejerkilde et al., 2003), S. crassipalpis (Joplin et al., 1990) and several other insect species, but appears to occur only during recovery from and not during the low-temperature exposure. For example, when Drosophila triauraria were subjected to 0°C for 24 h, Hsp70 mRNA was not detected immediately after the exposure but accumulated within 30 min upon return to 23°C (Goto and Kimura, 1998). Similarly, exposure of D. melanogaster to 0°C for 2 h, a temperature treatment known to induce a significant RCH response, fails to result in the accumulation of Hsp70 transcripts (Sinclair et al., 2007) or protein (Nielsen et al., 2005; Overgaard et al., 2005). This is consistent with the lack of Hsp70 induction during RCH of D. melanogaster using an ecologically relevant, diurnal thermocycle (Kelty and Lee, 2001). Further, Yi et al. (2007) found that RCH did not induce a significant increase in the expression of Hsp110, Hsp70 and Hsp27, or the heat-shock factor, Hsf-1. Together, these data suggest that increased expression of heat-shock proteins plays little to no significant role during RCH in insects, but are likely important during recovery from chilling injury. This suggestion is further supported by the finding that D. melanogaster undergo RCH even when protein synthesis is inhibited by treatment with cycloheximide (Misener et al., 2001). A recent investigation using microarray analysis revealed a change in the expression of 37 genes from D. melanogaster chilled at 0°C for 2 h followed by a 30 min recovery at 25°C

244

M.A. Elnitsky and R.E. Lee, Jr

(Qin et al., 2005). Of the 31 transcripts upregulated during cold hardening, nearly a third appeared to encode membrane proteins. These findings were perhaps not surprising, as cellular membranes are believed to be the primary site for cold-shock damage (Drobnis et al., 1993; Hazel, 1995) and reorganization of the membrane appears to be involved in the RCH response (see below). A number of other transcripts encoding stress proteins (Hsp83, Hsp26, Hsp23) were also up-regulated and likely function during recovery following chilling and/or in blocking cold-induced apoptosis (Yi et al., 2007). To date, studies investigating the role of heat-shock proteins in the RCH response have focused solely on single low-temperature exposures and the subsequent recovery from these events. However, in nature, most insects are exposed to thermocycles on a daily basis. Protection generated during the exposure or recovery from one thermocycle, such as the synthesis of heat-shock proteins, may confer significant protection during subsequent exposure to low or high temperature. This idea is supported by the finding that the cold tolerance of D. melanogaster increases with the number of thermocycles to which they are exposed (Kelty and Lee, 2001). Therefore, we believe the role of heat-shock proteins during RCH requires further examination under more ecologically relevant conditions of multiple diurnal thermocycles.

In vitro rapid cold hardening Recent evidence suggests that organismal survival is matched closely by increases in the cold tolerance of tissues during RCH. Yi and Lee (2004) demonstrated that both in vivo and in vitro RCH of tissues from S. crassipalpis protects cells against cold-shock injury. The survival of isolated tissues (fat body, gut, salivary gland, Malpighian tubules) cold-hardened at 0°C for 2 h significantly increased cell survival (by ∼25%) relative to tissues directly exposed to −8°C. We have since confirmed this result in the freeze-tolerant Antarctic midge, B. antarctica; RCH hardening of isolated tissues at −5°C, whether frozen or supercooled, for 1 h enhanced cell survival (N.M. Teets, unpublished

results). These results are significant as they demonstrate that neuroendocrine mediation from the intact organism is not required to elicit the RCH response; however, it is possible that the nervous and endocrine systems may enhance the cold-hardening effect (Yoder et al., 2006). Additionally, these results suggest a ‘cold-sensing’ role for the individual cells, as has been demonstrated in plants (see below), in the induction of the cold-hardening response. The fact that individual cells respond directly to low temperature without mediation from higher levels of organization helps to explain the defining characteristic of the RCH response: the swiftness of its induction.

Changes in membrane fluidity and phospholipid composition Cellular membranes are believed to be the primary sites of cold-shock damage as a result of lipid phase transitions (Drobnis et al., 1993; Hazel, 1995). As temperatures decrease, there is a concomitant decrease in membrane fluidity culminating in a transition from the liquidcrystalline to gel state at which time the membrane loses the ability to function as a selective barrier (Cossins, 1983). However, cells may counter this effect by adjusting the composition of membranes, including the phospholipid head groups and fatty acid chains, and the cholesterol content, to maintain membrane fluidity and the liquid-crystalline state at lower temperatures (Hazel, 1995). This response, termed homeoviscous adaptation (Sinensky, 1974), is well documented in microorganisms, plants and ectothermic animals during acclimation to low temperatures (Los and Murata, 2004). Recent investigations into the RCH response revealed an increase in the molar percentage of unsaturated phospholipid fatty acids, predominantly linoleic and oleic acid, within the cell membrane at the expense of saturated fatty acids (Overgaard et al., 2005; Michaud and Denlinger, 2006). Additionally, Overgaard et al. (2006) documented a similar increase in the degree of membrane unsaturation during an ecologically relevant cooling regime that induces an RCH response in D. melanogaster. These changes in membrane composition

Rapid Cold-hardening Response in Insects

would be expected to maintain membrane fluidity at low temperatures and prevent/reduce lipid phase transitions and resulting cellular damage. The significance of these results is further supported by the observation that membrane fluidity increases during RCH (2 h at 0°C) in fat body cells from Sarcophaga bullata (Lee et al., 2006a). Taken together, these results suggest that membrane modifications play a vital role in the RCH response protecting against cold-shock injury in insects.

Rapid cold hardening blocks cold-induced apoptosis Evidence from mammalian cells suggests chilling injury is linked to cold-induced apoptosis (Murakami et al., 1997). Cold shock may induce apoptosis by causing the release of cytochrome-c into the cytoplasm, likely as result of mitochondrial membrane damage, and activation of caspase-3 which initiates the proteolytic cascade ultimately leading to cell death (Cryns and Yuan, 1998). Yi et al. (2007) found that a cold shock treatment at −7°C for 2 h induced apoptosis in adult D. melanogaster. However, RCH treatment (2 h at 5°C) prior to the low-temperature exposure significantly reduced (by 38%) apoptotic cell death relative to the cold-shocked group. Expression of the anti-apoptosis protein, Bcl-2, perhaps along with heat-shock proteins, may be involved in blocking apoptosis following the RCH treatment (Yi et al., 2007). Further research as to the role of apoptosis in coldshock injury in insects and the pathway by which RCH prevents such programmed cell death is warranted.

Rapid cold hardening induction pathways While we are now beginning to gain insight into the physiological mechanisms involved in RCH, the molecular and cellular pathways for induction of the response remain largely unexplored. How is the signal of low environmental temperature ‘sensed’ and conveyed to induce modification of membrane composition and the synthesis of cryoprotectants ultimately resulting

245

in increased cold tolerance? The early stages of cold acclimation in plants suggest the plasma membrane functions as the primary cold sensor. In lucerne, Medicago sativa, cold acclimation is associated with a transient influx of Ca2+ and activation of Ca-dependent (CDPKs) and mitogen-activated protein kinases (MAPKs) (Sangwan et al., 2002). Such influx of Ca2+ appears to be mediated by low-temperatureinduced rigidification of the plasma membrane and cytoskeletal rearrangement resulting in the opening of Ca2+ channels (Orvar et al., 2000). Similarly to cold acclimation in plants, we recently determined that Ca2+ is required to elicit the RCH response in isolated tissues of B. antarctica (N.M. Teets, unpublished results). Further, p38 MAPK is activated in S. crassipalipis within 10 min of exposure to 0°C (Fujiwara and Denlinger, 2007), a temperature well-known to induce RCH in this species. These results suggest that a similar signalling pathway to that observed in plants is operating in the induction of the RCH response in insects.

Conclusion For some time now, the RCH response has been known to dramatically increase an insect’s protection against cold-shock injury. However, only recently have the broader ecological implications of the response become appreciated, including the reduction of the CTmax thereby extending the range of temperatures over which an insect may remain active, induction of RCH under natural thermal cycles and field conditions, and the preservation of reproductive behaviours and fecundity. RCH therefore appears to be part of a more general hardening response that allows insects to optimize behavioural and physiological performance within a thermally variable environment. Despite the apparent generality of the response among insects, the underlying physiological mechanisms remain poorly understood. While changes in the phospholipid fatty acid composition and the maintenance of membrane fluidity appear to be a component of RCH, the mechanistic role of other physiological changes associated with the response, such as the accumulation of cryoprotective compounds

246

M.A. Elnitsky and R.E. Lee, Jr

and the expression of heat-shock proteins, remains inconclusive. Further clarification as to the underlying mechanisms of the response, as well as investigation of the induction pathways of RCH and its role in blocking coldinduced apoptosis, will likely serve as focal points for future research.

Acknowledgements We thank Ben Philip and Yuta Kawarasaki for their comments on drafts of this chapter. Support from the National Science Foundation, grants #OPP-0337656 and #IOB-0416720, is gratefully acknowledged.

References Burton, V., Mitchell, H.K., Young, P. and Petersen, N.S. (1988) Heat shock protection against cold stress of Drosophila melanogaster. Molecular and Cellular Biology 8, 3550–3552. Carpenter, J.F. and Crowe, J.H. (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25, 244–255. Chen, C.P., Denlinger, D.L. and Lee, R.E. (1987) Cold-shock injury and rapid cold hardening in the flesh fly Sarcophaga crassipalpis. Physiological Zoology 60, 297–304. Chen, C.-P., Lee, R.E. and Denlinger, D.L. (1991) Cold shock and heat shock: a comparison of the protection generated by brief pretreatment at less severe temperatures. Physiological Entomology 16, 19–26. Cossins, A.R. (1983) The Adaptation of Membrane Structure and Function to Changes in Temperature. Cambridge University Press, Cambridge, UK. Coulson, S.J. and Bale, J.S. (1990) Characterization and limitations of the rapid cold-hardening response in the house fly Musca domestica (Diptera, Muscidae). Journal of Insect Physiology 36, 207–211. Coulson, S.J. and Bale, J.S. (1992) Effect of rapid cold hardening on reproduction and survival of offspring in the house fly Musca domestica. Journal of Insect Physiology 38, 421–424. Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Wistrom, C.A., Spargo, B.J. and Anchordoguy, T.J. (1988) Interactions of sugars with membranes. Biochimica et Biophysica Acta 947, 367–384. Cryns, V. and Yuan, J. (1998) Proteases to die for. Genes and Development 12, 1551–1570. Czajka, M.C. and Lee, R.E. (1990) A rapid cold-hardening response protecting against cold shock injury in Drosophila melanogaster. Journal of Experimental Biology 148, 245–254. Denlinger, D.L. and Lee, R.E. (1998) Physiology of cold sensitivity. In: Hallman, G.J. and Denlinger, D.L. (eds) Temperature Sensitivity in Insects and Application in Integrated Pest Management. Westview Press, Boulder, Colorado, pp. 55–95. Drobnis, E.Z., Crowe, L.M., Berger, T., Anchordoguy, T.J., Overstreet, J.W. and Crowe, J.H. (1993) Cold shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model. Journal of Experimental Biology 265, 432–437. Duman, J.G., Wu, D.W., Xu, L., Tursman, D. and Olsen, T.M. (1991) Adaptations of insects to subzero temperatures. Quarterly Review of Biology 66, 378–410. Feder, M.E. and Hofmann, G.E. (1999) Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annual Review of Physiology 61, 243–282. Fujiwara, Y. and Denlinger, D.L. (2007) p38 MAPK is a likely component of the signal transduction pathway triggering rapid cold hardening in the flesh fly Sarcophaga crassipalpis. Journal of Experimental Biology 210, 3295–3300. Goto, S.G. and Kimura, M.T. (1998) Heat- and cold-shock responses and temperature adaptations in subtropical and temperate species of Drosophila. Journal of Insect Physiology 44, 1233–1239. Hazel, J.R. (1995) Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annual Review of Physiology 57, 19–42. Joplin, K.H., Yocum, G.D. and Denlinger, D.L. (1990) Cold shock elicits expression of heat shock proteins in the flesh fly, Sarcophaga crassipalpis. Journal of Insect Physiology 36, 825–834. Kelty, J. (2007) Rapid cold-hardening of Drosophila melanogaster in a field setting. Physiological Entomology 32, 343–350. Kelty, J.D. and Lee, R.E. (1999) Induction of rapid cold hardening by cooling at ecologically relevant rates in Drosophila melanogaster. Journal of Insect Physiology 45, 719–726. Kelty, J.D. and Lee, R.E. (2001) Rapid cold-hardening of Drosophila melanogaster (Diptera: Drosophilidae) during ecologically based thermoperiodic cycles. Journal of Experimental Biology 204, 1659–1666.

Rapid Cold-hardening Response in Insects

247

Kelty, J.D., Killian, K.A. and Lee, R.E. (1996) Cold shock and rapid cold-hardening of pharate adult flesh flies (Sarcophaga crassipalpis): effects on behaviour and neuromuscular function following eclosion. Physiological Entomology 21, 283–288. Koveos, D.S. (2001) Rapid cold hardening in the olive fruit fly Bactrocera oleae under laboratory and field conditions. Entomologia Experimentalis et Applicata 101, 257–263. Lee, R.E. (1991) Principles of insect low temperature tolerance. In: Lee, R.E. and Denlinger, D.L. (eds) Insects at Low Temperature. Chapman and Hall, New York, New York, pp. 17–43. Lee, R.E., Chen, C.P. and Denlinger, D.L. (1987) A rapid cold-hardening process in insects. Science 238, 1415–1417. Lee, R.E., Damodaran, K., Yi, S.-X. and Lorigan, G.A. (2006a) Rapid cold-hardening increases membrane fluidity and cold tolerance of insect cells. Cryobiology 52, 459–463. Lee, R.E., Elnitsky, M.A., Rinehart, J.P., Hayward, S.A.L., Sandro, L.H. and Denlinger, D.L. (2006b) Rapid coldhardening increases the freezing tolerance of the Antarctic midge Belgica antarctica. Journal of Experimental Biology 209, 399–406. Los, D.A. and Murata, N. (2004) Membrane fluidity and its roles in perception of environmental signals. Biochimica et Biophysica Acta 1666, 142–157. Michaud, M.R. and Denlinger, D.L. (2006) Oleic acid is elevated in cell membranes during rapid coldhardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis. Journal of Insect Physiology 52, 1073–1082. Michaud, M.R. and Denlinger, D.L. (2007) Shifts in carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (Sarcophaga crassipalpis): a metabolomic comparison. Journal of Comparative Physiology B 177, 753–763. Misener, S.R., Chen, C.P. and Walker, V.K. (2001) Cold tolerance and proline metabolic gene expression in Drosophila melanogaster. Journal of Insect Physiology 47, 393–400. Murakami, K., Kondo, T., Sato, S., Li, Y. and Chen, P.H. (1997) Occurrence of apoptosis following cold injuryinduced brain edema in mice. Neuroscience 81, 231–237. Nielsen, M.M., Overgaard, J., Sorensen, J.G., Holmstrup, M., Justesen, J. and Loeschcke, V. (2005) Role of HSF activation for resistance to heat, cold and high-temperature knock-down. Journal of Insect Physiology 51, 1320–1329. Orvar, B.L., Sangwan, V., Omann, F. and Dhindsa, R.S. (2000) Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity. The Plant Journal 23, 785–794. Overgaard, J., Sorensen, J.G., Petersen, S.O., Loeschcke, V. and Holmstrup, M. (2005) Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster. Journal of Insect Physiology 51, 1173–1182. Overgaard, J., Sorensen, J.G., Petersen, S.O., Loeschcke, V. and Holmstrup, M. (2006) Reorganization of membrane lipids during fast and slow cold hardening in Drosophila melanogaster. Physiological Entomology 31, 328–335. Overgaard, J., Malmendal, A., Sorensen, J.G., Bundy, J.G., Loeschcke, V., Nielson, N.C. and Holmstrup, M. (2007) Metabolic profiling of rapid cold hardening and cold shock in Drosophila melanogaster. Journal of Insect Physiology 53, 1218–1232. Powell, S.J. and Bale, J.S. (2004) Cold shock injury and ecological costs of rapid cold hardening in the grain aphid Sitobion avenae (Hemiptera: Aphididae). Journal of Insect Physiology 50, 277–284. Powell, S.J. and Bale, J.S. (2006) Effect of long-term and rapid cold hardening on the cold torpor temperature of an aphid. Physiological Entomology 31, 348–352. Qin, W., Neal, S.J., Robertson, R.M., Westwood, J.T. and Waker, V.K. (2005) Cold hardening and transcriptional change in Drosophila melanogaster. Insect Molecular Biology 14, 607–613. Rinehart, J.P., Yocum, G.D. and Denlinger, D.L. (2000) Thermotolerance and rapid cold hardening ameliorate the negative effects of brief exposures to high or low temperatures on fecundity in the flesh fly, Sarcophaga crassipalpis. Physiological Entomology 25, 330–336. Sangwan, V., Orvar, B.L., Beyerly, J., Hirt, H. and Dhindsa, R.S. (2002) Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways. The Plant Journal 31, 629–638. Sejerkilde, M., Sorensen, J.G. and Loeschcke, V. (2003) Effects of cold- and heat hardening on thermal resistance in Drosophila melanogaster. Journal of Insect Physiology 49, 719–726. Shreve, S.M., Kelty, J.D. and Lee, R.E. (2004) Preservation of reproductive behaviors during modest cooling: rapid cold-hardening fine-tunes organismal response. Journal of Experimental Biology 207, 1797–1802.

248

M.A. Elnitsky and R.E. Lee, Jr

Sinclair, B.J., Gibbs, A.G. and Roberts, S.P. (2007) Gene transcription during exposure to, and recovery from, cold and desiccation stress in Drosophila melanogaster. Insect Molecular Biology 16, 435–443. Sinensky, M. (1974) Homeoviscous adaptation – a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proceedings of the National Academy of Sciences USA 71, 522–525. Storey, K.B. and Storey, J.M. (1996) Natural freezing survival in animals. Annual Review of Ecology and Systematics 27, 365–386. Wang, H.S. and Kang, L. (2005) Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs. Cryobiology 51, 220–229. Yi, S.-X. and Lee, R.E. (2004) In vivo and in vitro rapid cold-hardening protects cells from cold-shock injury in the flesh fly. Journal of Comparative Physiology B 174, 611–615. Yi, S.-X., Moore, C.W. and Lee, R.E. (2007) Rapid cold-hardening protects Drosophila melanogaster from cold-induced apoptosis. Apoptosis 12, 1183–1193. Yoder, J.A., Benoit, J.B., Denlinger, D.L. and Rivers, D.B. (2006) Stress-induced accumulation of glycerol in the flesh fly, Sarcophaga bullata: evidence indicating anti-desiccant and cryoprotectant functions of this polyol and a role for the brain in coordinating the response. Journal of Insect Physiology 52, 202–214. Zachariassen, K.E. (1985) Physiology of cold tolerance in insects. Physiological Reviews 65, 799–832.

24

Conifer Cold Hardiness, Climate Change and the Likely Effects of Warmer Temperatures on Photosynthesis I. Ensminger, N.P.A. Hüner and F. Busch

Abstract During winter and early spring, evergreen boreal conifers are severely stressed because light energy cannot be used when photosynthesis is pre-empted by low ambient temperatures. As a result of global warming, the growing season for boreal forest trees will increase, thereby altering the carbon sink of conifer forests. Some of these forests might also be negatively affected by increased land surface air temperatures, due to e.g. the disruption of regulatory processes and hence an incomplete exploitation of the increased length of the growing season. To isolate the effect of temperature from the cellular to the ecosystem level, we studied the dynamics of photosynthesis under boreal climate conditions in the field as well as in controlled experiments in phytotrons. While photosynthesis of many species responds positively to temperature, recent findings in pine suggest that photoperiod control of autumn dormancy appears to negate any potential for an increased carbon gain associated with higher temperatures during the autumn season. By contrast, the onset of CO2 assimilation in spring is clearly triggered by increasing air temperatures; however, low soil temperatures and intermittent frost can decrease the rate of the recovery of photosynthesis. We conclude that adaptation of photosynthesis to varying temperatures revolves around the trade-off between utilizing the full growing season and minimizing damage (frost, oxidative stress) through proper timing of hardening in autumn and dehardening in spring.

Introduction Boreal forests represent the largest biome of the northern hemisphere and cover 17% of the world’s and about 30% of Canada’s surface. Most importantly, these forests represent a net sink for the removal of atmospheric CO2 (Ciais et al., 1995). Evergreen conifers are the dominant trees of these forests. These forests are characterized by a strong seasonality, with peaks of maximum activity of e.g. photosynthesis and respiration during the short boreal summer and almost complete cessation of any obvious activity during extreme winter climatic conditions (Fig. 24.1). Despite the rather short growing season with a net CO2 uptake, these forests represent an integral part in the global climate system. Boreal conifers act as modula-

tors of climatic change while at the same time being affected by climate change. Over the past decades the northern latitudes have experienced a substantial increase in surface air temperature, especially in the winter, with increases of up to 4°C in the boreal forest (ACIA, 2005). This trend is expected to continue and an increase of 1.4–5.8°C in the global average surface air temperature is projected by the end of the century (IPCC, 2007). Most of this warming will occur as a greater than average increase in temperature during the night, in the winter and at high latitudes (Balling et al., 1998; Serreze et al., 2000; IPCC, 2007). In addition to the temperature increase observed over the last decades in high latitudes, an increase of up to 11°C by 2100 is possible (IPCC, 2007). Due to these changes

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

249

250

I. Ensminger et al.

(a)

PAR (mol/m2/day)

60

40

20

0 (b) Gross primary production

Flux (mol C/m2/day)

0.4 Ecosystem respiration

0.2 0.0 −0.2 −0.4

Net ecosystem exchange

Temperature (°C)

(c)

Soil temperature 1999

25 0

2000 −25 Air temperature

−50

Precipitation (mm)

(d)

30 20 10 0

J

S

N

J

M

M

J

S

N

J

M

M

J

S

N

Fig. 24.1. The seasonal pattern of (a) daily photosynthetically active radiation (PAR) integral; (b) gross primary productivity, ecosystem respiration and net ecosystem exchange rate; (c) daily average soil and air temperature; and (d) precipitation from 7 June 1998 to 31 December 2000 for the Zotino field site, central Siberia. During the growing season net uptake of carbon from the atmosphere and accumulation by the ecosystem is indicated by negative values of the net ecosystem exchange rate. Occurrence of the first daytime frosts causes ecosystem photosynthesis (gross primary production) to decline to values close to zero. During winter the net ecosystem exchange rate is slightly positive, indicating respiratory release of CO2. During the observation period, the magnitude of CO2 released during winter was offset by the much greater amount of carbon gained during summer. (Adapted from Lloyd et al., 2002.)

Conifer Cold Hardiness

in temperature, the length of the growing season is expected to increase by 20–30 days by 2080 (ACIA, 2005). It has been suggested that an increase in temperature and length of the growing season will stimulate the productivity and carbon sequestration of the northern hemisphere forests (Saxe et al., 2001). Nevertheless, factors determining the timing and rate of the spring photosynthetic recovery in these conifers are not well understood in terms of both underlying physiology and associated environmental triggers (Ensminger et al., 2004; Slot et al., 2005). In order to assess and mitigate effects of climate change on these forests, we need to be able to understand the mechanisms underlying the phenology of evergreen forests.

Why and How Photosynthetic Processes Constantly Acclimate Photosynthesis constantly adjusts its light-use efficiency to optimize the use of carbon and nitrogen resources and to minimize the damaging effects of excess absorbed energy under changing environmental conditions (Huner et al., 1998, 2003; Falkowski and Chen, 2003). Thus, for optimum plant performance, the amount of energy absorbed by the photosystems and the amount of energy utilized by metabolic sinks has to be balanced by a mechanism called photostasis (Öquist and Huner, 2003; Ensminger et al., 2006). Solar energy trapped by the photosystems can be used for photochemical processes which drive the photosynthetic electron transport. This process generates NADPH and a trans-thylakoid pH gradient. This pH gradient, in turn, is used to produce ATP through the process of photophosphorylation. Solar energy that has been converted into NADPH and ATP is then used to fix CO2 in the Calvin cycle to generate carbohydrates to fuel plant growth and general metabolism. Changes in environmental conditions have large effects on the energetic balance of photosynthesis. Changes in the source of energy, such as an increase in solar radiation, or changes in the metabolic sink capacity, e.g. by a change in temperature, can result in an energetic imbalance. Any imbalance between the

251

source of energy and the metabolic sink created e.g. by high light can be exacerbated by low temperatures. This is because the rates of the enzymatic reactions involved in the C, N and S reduction are more negatively affected than the photophysical and photochemical processes involved in light absorption and energy transfer (Huner et al., 1998; Ensminger et al., 2006). As a consequence, the rates of photochemical processes must be adjusted to meet the demand of the decreased sink capacity to avoid excess energy resulting in the formation of reactive oxygen species and photooxidative damage. A rapid adjustment of the energetic balance is facilitated by a process named nonphotochemical quenching (NPQ). Whenever solar energy absorbed by the photosynthetic apparatus exceeds the photochemical capacity, the excess light energy can be dissipated harmlessly by NPQ via heat. Hereby the transthylakoid pH gradient is an important regulatory element, since it affects NPQ through the xanthophyll cycle (Krause and Weis, 1991; Horton et al., 1999). Under conditions of decreased metabolic sink activity, feedback inhibition of the electron transport causes increases in NPQ (Fig. 24.2c). In order to achieve photostasis, the photosynthetic apparatus must be acclimated via a combination of various mechanisms described below (for a detailed review see Ensminger et al., 2006).

Decreasing the capacity for energy transfer from LHCII to PSII A decrease in the energy transfer from lightharvesting complex II (LHCII) to the photosystem II complex (PSII) by dissipation of excess energy through the xanthophyll cycle is a very rapid mechanism to adjust an energetic imbalance (Demmig-Adams and Adams, 1996; Demmig-Adams et al., 1996; Gilmore, 1997). The main photoprotective role of the xanthophyll cycle is achieved by an increased thermal dissipation of excess energy, which decreases the photochemical and Chl-a fluorescence efficiencies of PSII (Gilmore and Yamamoto, 1993; Gilmore et al., 1998). This photoprotective mechanism is facilitated by

252

I. Ensminger et al.

the light-dependent conversion of violaxanthin to antheraxanthin and the photoprotective zeaxanthin to decrease the energy transfer to Chl-a in the PSII reaction centre (RC) (Horton et al., 1999; Niyogi et al., 2005). Sustained zeaxanthin-dependent NPQ is an important protective mechanism that enables evergreen trees to maintain their leaves during winter, while the photosynthetic capacity is constitutively down-regulated. In this sustained NPQ mechanism, the component of the fast relaxation of the xanthophyll cycle-dependent NPQ is replaced by PSII core rearrangements. This mechanism is further accompanied by a decreased rate of re-conversion of zeaxanthin to violaxanthin (Demmig-Adams and Adams, 2006). In overwintering evergreens and other plants, an improved photoprotection has also been shown to occur through reorganization of LHCII into aggregates (Horton et al., 1991; Ruban et al., 1993; Ottander et al., 1995; Gilmore and Ball, 2000; Krol et al., 2002; Busch et al. 2007). It has been suggested that an uncoupling of these aggregates from the core protects PSII from photodamage (Busch et al., 2008).

Reaction centre quenching Zeaxanthin-independent RC quenching is another quenching mechanism complementing the antenna quenching via the xanthophyll cycle and the protein PsbS (Ivanov et al., 2001, 2002; Lee et al., 2001; Sane et al., 2003; Huner et al., 2006). Originally proposed by Briantais et al. (1979) and Krause and Weis (1991), RC quenching in pine is at a maximum in cold-acclimated needles during the winter months and decreases to a minimum during photosynthetically active periods in late spring (Ivanov et al., 2002; Sveshnikov et al., 2006).

Adjusting the physical size of the antenna or the abundance of photosynthetic components State transitions represent a further rapid mechanism to balance the energy distribution

between PSII and the photosystem I complex (PSI). When PSII is preferentially excited relative to PSI, the redox sensor plastoquinone (PQ) becomes reduced. This activates a thylakoid protein kinase which in turn phosphorylates LHCII. As a consequence of charge repulsion, a population of peripheral LHCII migrates and associates with PSI via the PSI-H subunit (Lunde et al., 2000). Through this mechanism, the light-harvesting chlorophyll is redistributed to PSI at the expense of PSII. This results in a balanced excitation of PSII relative to PSI to ensure optimal quantum efficiency for photosynthetic electron transport. In contrast to these rapid responses, longterm acclimation is achieved through regulation of photosynthesis genes coding e.g. for PSII or PSI. A decrease of the total amount of LHCII polypeptides results in a smaller total size of the antenna which protects PSII from excessive excitation by decreasing the amount of absorbed solar light. The stoichiometry of the photosynthetic RC of PSII versus the RC of PSI is controlled by the redox state of the PQ pool, which regulates the transcription of the psbA and psaAB genes coding for the RCs of PSII and PSI, respectively (Pfannschmidt et al., 1999). Under high excitation pressure, the transcription of psaAB is enhanced compared to psbA (Pfannschmidt et al., 1999). In Pinus banksiana this has been shown to result in a decrease of the ratio of PSII RCs to PSI RCs (Busch et al., 2008).

Acclimation of electron sink capacity Regulation of photosynthesis under low temperatures underscores the importance of both source and sink as control points to maintain photostasis and reveals differences between cold-hardy trees and cold-hardy herbaceous plants. In conifers, dormancy results in downregulation of photosynthesis and decreased assimilates due to growth cessation and decreased sink capacity. By contrast, herbs such as winter wheat continue to grow and maintain their need for photoassimilates even after cold acclimation (Savitch et al., 2002; Leonardos et al., 2003).

Conifer Cold Hardiness

Spring Recovery of Photosynthesis

253

warmer temperatures and hence there is no decrease in the amount of PSII RC protein.

Temperature and light control the onset of photosynthesis in spring The protective mechanisms mentioned above allow evergreen conifers to retain much of their photosynthetic apparatus during winter despite being exposed to adverse environmental conditions. As a consequence, once conditions become more favourable, photosynthesis has the ability to resume quickly through rapid reassembly and restructuring of the photosynthetic apparatus. Air temperature, soil temperature and light are the environmental triggers controlling the recovery of photosynthetic activity in spring (Ensminger et al. 2004; Porcar-Castell et al., 2008a,b). Interestingly, in Scots pine, a detectable photosynthetic uptake of CO2 is preceded by an increase of photochemical processes and a decrease in non-photochemical processes (Ensminger et al., 2004). For example, photosynthesis at the canopy level, as measured by an eddycovariance system above the canopy of a Siberian boreal Scots pine forest, increased in early spring but was preceded by increases in photochemical efficiency of PSII, as obtained from direct chlorophyll fluorescence measurements on the needle level (Fig. 24.2a and b). As photochemical efficiency of PSII increased, NPQ processes decreased, along with a decrease in the de-epoxidation state of xanthophyll pigments (Fig. 24.2c). To a large extent air temperature controls the recovery of thylakoid protein composition. Various proteins of photosynthesis closely followed air temperature in a Siberian Scots pine forest (Ensminger et al., 2004). The RCs of PSII and PSI showed a clear response to changes in environmental conditions (Fig. 24.2d). Low levels of D1, which is the RC protein of PSII, and light-harvesting complex proteins of both PSII and PSI were observed in spring when excitation pressure was highest due to a high photon flux density in combination with low temperature. At high air temperatures, high light intensities exert much smaller excitation pressure compared with low temperatures. This is due to the increased electron sink capacity in the Calvin cycle under

Spring warming and its effects on the dehardening in pine As described above, warm spring air temperatures rapidly release cold-induced inhibition of photosynthesis, resulting in an earlier onset of photosynthesis in boreal forest trees. However, the rate of the recovery of photosynthesis from winter stress is modulated by additional factors such as soil temperature and intermittent frosts (Ensminger et al., 2004, 2008). Under experimental spring conditions, the recovery of the quantum yield of PSII (Fv/Fm) did not indicate any differences under low soil temperature conditions compared with the control (Fig. 24.3a). By contrast, the recovery rates of net CO2 uptake were significantly slower in plants from cold or frozen soil compared with controls (Fig. 24.3b). In addition, under low soil temperatures a large fraction of absorbed light was not used photochemically, but was dissipated thermally via xanthophyll cycle pigments (Fig. 24.3c). This probably reflects restrictions in water uptake and root activity (Bergh and Linder, 1999; Jarvis and Linder, 2000). As a result, stomatal and non-stomatal effects impaired the ability of CO2 assimilation to act as an electron sink, providing a negative feedback to the recovery of electron transport (Ensminger et al., 2008). Intermittent frost events decreased photosynthetic capacity and increased thermal energy dissipation. In early spring, proteins of the photosynthetic apparatus such as D1, LHCII, CP29 and LHCI-730 were highly sensitive to intermittent frost, which reversed the spring reorganization of the thylakoid membrane proteins (Fig. 24.2d; Ensminger et al., 2004). Thus, both low soil temperature and intermittent frost decrease the rate of the photosynthetic spring recovery process. Earlier spring warming under future climatic conditions would facilitate the dehardening of conifers; however, spring frosts increase the risk of delayed recovery of photosynthesis. Low soil temperature effects on spring recovery of photosynthesis could become an issue in

254

NEEdaily mean (µmol/m2/s)

(a)

(b)

I. Ensminger et al.

1.0 0.5 0.0 −0.5 −1.0 −1.5 −2.0 −2.5 −3.0 −3.5 −4.0

CO2 uptake by the forest

1.0

FV /Fm

0.8 0.6 0.4 0.2 0.0 5.0

1.2

4.5

1.0

4.0 0.8

NPQ

3.5 3.0

0.6

2.5

0.4

2.0 0.2

1.5 1.0 0.5 Apr

(d)

0.0 12 Apr

19 Apr

26 Apr 03 May 10 May 17 May 24 May 31 May

9 Apr 18 Apr 20 Apr 25 Apr 2 May 4 May 12 May 14 May 21 May 29 May

Early spring PSII

Core

D1 (psbA)

Late spring KD 32

LHCIIb (major complexes) LHCII (Ihcb1)

27-28 LHCIIa (minor complexes)

CP29 (Ihcb4)

29

PSI LHCI-730 (Ihca1)

20

PsbS

22

Elip

16

DEPS (mol/mol)

(c)

Fig. 24.2. (a) Time course of the net ecosystem CO2 exchange rate (NEE) during spring 2001 for a boreal Pinus sylvestris stand in Zotino, central Siberia. Positive values indicate respiratory CO2 release, negative values indicate net photosynthetic CO2 uptake. (b) Optimum quantum yield of photosystem II (PSII) (Fv/Fm; –•–) and effective quantum yield of PSII (∆F/Fm′, –◊–) at 1300 mmol photons/m2/s. (c) Nonphotochemical quenching (NPQ; –•–) and de-epoxidation status (DEPS; – –䊐– –) of the xanthophyll cycle pigments [(0.5A+Z)/(V+A+Z), where A=antheraxanthin, Z=zeaxanthin and V=violaxanthin]. (d) Immunoblots reflecting changes in the amount of key proteins of the photosynthetic apparatus during recovery of photosynthesis in the spring. Data points in (b) and (c) represent the average of n = 1–3 (± SD) measurements. (Adapted from Ensminger et al., 2004.)

Conifer Cold Hardiness

PSII (Fv /Fm)

(a) 0.9

0.6

0.3

0

Asat (µmol CO2/m2/s)

(b)

0

5

10

15

20

15 12 9

255

natural forests. Globally a 10% reduction of snow cover has been observed over the past 40 years and this trend is supposed to continue (Houghton et al., 2001). Snow cover provides insulation of the soil and thus keeps the soil warmer during spring than uncovered bare soil (Decker et al., 2003). In snow-removal experiments the daily averages of soil temperature in snow-covered soil remained consistently above 0°C, whereas snow-free soil temperatures dropped below −3°C. This suggests that in the northern hemisphere the likely effects of increased land surface temperature for an earlier onset of photosynthesis in spring might be negated by generally lower soil temperatures in areas where a decrease in snow cover is predicted.

6 3 0

Down-regulation of Photosynthesis During Autumn 0

5

10

15

20

(c) 0.8

Photoperiod and temperature as triggers of growth cessation and low-temperature acclimation

DEPS

0.6 0.4 0.2 0.0

0 5 10 15 20 Days of experimental spring conditions

Fig. 24.3. Recovery of photosynthesis in 1-year-old Pinus sylvestris seedlings under different experimental spring conditions at different air and soil temperatures. (a) Optimum quantum yield of photosystem II (PSII) (Fv/Fm); (b) light-saturated CO2 assimilation (Asat); (c) de-epoxidation status (DEPS) of the xanthophyll cycle pigments [(0.5A+Z)/ (V+A+Z), where A=antheraxanthin, Z=zeaxanthin and V=violaxanthin]. ■, 15°C air temperature/15°C soil temperature (15/15); ▲, 15°C air temperature/+1°C soil temperature (15/+1); °, 15°C air temperature/−2°C soil temperature (15/−2); ——, 15/15 modelled; – – –, 15/+1 modelled; - - -, 15/−2 modelled. Each data point represents the average of n = 6 (± SD) measurements. (Adapted from Ensminger et al., 2008.)

In the boreal forest photosynthetic activity exhibits a seasonal pattern with the highest rates of CO2 assimilation occurring in August, as shown for Pinus sylvestris in a central Siberian forest (Fig. 24.1). Already in September photosynthesis can decline considerably. This is indicated by a decrease in the light-saturated rate via a stage involving a decrease in canopy quantum yield to a very slowly reversible, light-independent decrease of photosynthetic capacity after the first daytime frosts on sunny days (Lloyd et al., 2002) (Fig. 24.4a). Ensminger et al. (2004) have shown that this initial decrease is concomitant with a decrease in the amount of the PSII RC protein D1 and CP29 of the LHCII, reflecting a downregulation intimately linked with the coldhardening process (Fig. 24.4b). In contrast, the major light-harvesting complexes Lhcb1 were retained or even increased. Such rearrangements in the thylakoid protein composition were paralleled by a shift in the distribution of xanthophyll cycle pigments from antheraxanthin to the photoprotective zeaxanthin. This

256

I. Ensminger et al.

(a) 14

(b)

18 Aug 21 Sep 14 Dec 14 Feb

Sum Autu mer mn

Net CO2 assimilation (µmol/m2/s)

12 PSII

10

3 Mar 9 Apr 25 Apr 4 May 29 May

Early spring

Winter

Late spring

Core

kDa

D1 (psbA)

8

32 LHCIIb (main complexes)

LHCII (Ihcb1)

6

27–28 LHCIIa (minor complexes)

CP29 (Ihcb4)

4 2

29

PSI LHCI-730 (Ihca1)

20

PsbS

22

0 −2 0

1000 1250 250 500 750 Incoming photosynthetically active radiation 2 (µmol quanta/m /s)

CO2 exchange (µmol/m2/s)

(c) 3.0

(d)

Protein

Treatment LD/HT SD/HT

LD/LT

SD/LT

PsbA

1.00 ±0.07

0.68 ±0.05

0.46 ±0.06

0.55 ±0.05

PsaA/B

1.00 ±0.04

0.75 ±0.03

0.44 ±0.05

0.56 ±0.06

RbcL

1.00 ±0.03

0.86 ±0.02

0.75 ±0.03

0.81 ±0.03

Lhcb1

1.00 ±0.09

1.02 ±0.04

1.35 ±0.02

1.26 ±0.07

Lhcb2

1.00 ±0.06

1.14 ±0.06

1.12 ±0.06

1.02 ±0.04

Lhcb5

1.00 ±0.07

0.71 ±0.07

1.02 ±0.05

0.89 ±0.03

LD/HT SD/HT LD/LT SD/LT

2.5 2.0 1.5 1.0 0.5 0.0 −0.5 −1.0 −1.5

LD/HT

(e) PSII

k1

SD/HT

stroma k2 PQ/Cyt b6f

LD/LT

SD/LT

16

Elip

k6 k4

PSI

k5

CO2 assimilation

k3 PTOX

Fig. 24.4. (a) Canopy net CO2 assimilation rate in response to incoming photosynthetically active radiation of a Pinus sylvestris stand in Zotino, central Siberia, in 1999 (–•–, 27/28 August; ▼, 12/13 September; –▲–, 20/21 September; –*–, 5/6 October). (b) Immunoblots reflecting changes in the amount of key proteins of the photosynthetic apparatus during the seasons in P. sylvestris. (c) The effect of photoperiod and temperature on needle-level CO2 assimilation of Pinus banksiana seedlings, measured at 1000 ( ) and 350 ( ) mmol photons/m2/s; dark respiration was also measured after 20 min of dark acclimation ( ). All measurements were performed at growth temperature. LD/HT, 16 h photoperiod/22°C; SD/HT, 8 h photoperiod/22°C; LD/LT, 16 h photoperiod/7°C; SD/LT, 8 h photoperiod/7°C. Each bar represents the average of n = 7–8 (± SE) biological replicates. (d) The effect of photoperiod and temperature on the expression levels of key proteins of the photosynthetic apparatus in needles of Pinus banksiana. All values are relative to the amount of protein in the LD/HT plants. (e) Possible points of regulation for photosynthetic electron transport in SD/HT plants. k1 to k6 indicate rate constants for the flux of electrons. k4 is highlighted by a dashed line, as this is the probable site of limitation in photosynthetic electron transport between photosystem II (PSII) and photosystem I (PSI). PQ, plastoquinone; Cyt b6f, cytochrome b6f; PTOX, plastic terminal oxidase. (Adapted from Lloyd et al., 2002; Ensminger et al., 2004; Busch et al., 2007, 2008; www. plantphysiolo.org, copyright American Society of Plant Biologists.)

Conifer Cold Hardiness

combination enables a sustained NPQ through the mechanisms of decreased energy transfer from LHCII to PSII as explained above. These autumn processes are triggered to a large extent by photoperiod and temperature. Weiser (1970) suggested that growth cessation, the development of dormancy and a basal level of freezing tolerance are triggered by short photoperiod. Freezing tolerance is furthermore potentiated by low temperature, preparing the vegetative tissue and reproductive organs to survive boreal winter conditions (Weiser, 1970; Christersson, 1978; Bigras et al., 2001; Li et al., 2002; Beck et al., 2004; Puhakainen et al., 2004).

Autumn warming and its effects on photosynthesis in pine In a set of factorial experiments using Pinus banksiana, Busch et al. (2007, 2008) separated the effects of day length and temperature to investigate how warm autumn air temperature affects photosynthetic carbon gain, electron transport and the dissipation of excess energy. To distinguish between the effects of day length and temperature, controlled environments simulated the summer-to-autumn transition. Normal summer conditions (16 h photoperiod/22°C; LD/HT) and normal autumn conditions (8 h photoperiod/7°C; SD/LT) were compared with simulated warm autumn air temperatures (8 h photoperiod/22°C; SD/HT). In order to separate the effects of photoperiod and temperature, another treatment with summer photoperiod and autumn temperatures (16 h photoperiod/ 7°C; LD/LT) was tested. Under simulated warmer autumn air temperatures (SD/HT) a considerable decrease in CO2 assimilation was observed compared with summer conditions (LD/HT, Fig. 24.4c). CO2 assimilation under warm autumn conditions was also slightly lower than under normal autumn conditions (SD/LT). Most interestingly, respiration under warm autumn conditions increased twofold compared with normal autumn conditions (Busch et al., 2007). This finding clearly shows that an extended growing season due to warmer air temperatures does not necessarily increase photosynthetic carbon uptake. Recently, similar results were

257

obtained by simulated and eddy-covariancederived estimates of the carbon uptake by northern ecosystems (Piao et al., 2008). This is the first field evidence for the mechanistic physiological analysis done by Busch et al. (2007, 2008) under controlled environmental conditions. Changes in carbon uptake patterns during warm autumn conditions are tightly correlated with changes observed in the arrangement and the composition of thylakoid proteins (Fig. 24.4d). This observation, as well as an impairment of electron transport between PSII and PSI under warm autumn conditions (SD/HT), are the reasons for a decreased carbon uptake, created by an insufficient supply of reducing equivalent provided by the photosynthetic apparatus (Fig. 24.4e) (Busch et al., 2008). In addition, plants grown under warmer autumn air temperatures exhibited a shift in quenching of excess energy from a zeaxanthin-dependent mechanism to a photoprotection facilitated by zeaxanthin-independent aggregation of LHCII (Busch et al., 2007, 2008). Gene expression analysis using microarrays for pine exposed to the above-mentioned autumn conditions revealed that among 10,000 genes represented on the chip, 989 showed a significant (P<0.001) up- or down-regulation in response to day length and/or temperature (I. Ensminger, F. Busch, L.A. Tarca, S. Caron, J. MacKay, N.P.A. Hüner, unpublished results). While the majority of genes are exclusively upor down-regulated under normal autumn conditions (263 up- and 174 down-regulated in SD/ LT), there is also a large number of genes exclusively responding to warm autumn conditions (82 up- and 37 down-regulated in SD/HT). However, the overall number of 729 differentially expressed genes under normal SD/LT autumn conditions compared with 234 genes under SD/HT autumn conditions indicates that a single factor alone, either short photoperiod or low temperature, is unlikely to induce the complete range of changes required to achieve full acclimation. Thus, warmer autumn air temperature has the potential to disrupt a coordinated acclimation to winter conditions. Given these results, it is not surprising that the frost tolerance of trees grown at warm autumn air temperature lags behind that of trees grown at normal autumn air temperatures (Ensminger et al., 2005) (Fig. 24.5a). Four hours of frost

258

I. Ensminger et al.

(b) 1.0

1.0

0.8

0.8

PSII (Fv /Fm)

PSII (Fv /Fm)

(a)

0.6 0.4 0.2

0.6 0.4 0.2

0

0 –5°C

–10°C

–15°C

–25°C

–5°C

–10°C

–15°C

–25°C

Fig. 24.5. The effect of photoperiod and temperature on freezing tolerance of Scots pine photosynthesis after 21 days under artificial autumn conditions from Fig. 24.4c (LD/HT: 16h photoperiod/22°C; SD/HT: 8h/22°C; LD/LT: 16h/7°C; SD/LT: 8h/7°C). The degree of frost tolerance is indicated by the quantum yield of photosynthesis obtained (a) 4 hours or (b) 14 days after exposure to either -5 °C, -10 °C, -15 °C or -25 °C for 6 h. Each bar represents the mean of 3–5 (± SD) measurements. (Adapted from Ensminger et al., 2005.)

treatment at −10°C decreased the optimum quantum yield of PSII (Fv/Fm) in SD/HT trees by about 30% compared with the value obtained by properly cold-hardened pine seedlings under normal autumn conditions (SD/LT). Exposure to −15°C almost entirely inhibited any activity of PSII as was indicated by almost indiscernible values of Fv/Fm. Most interestingly, this effect was not reversible, as Fv/Fm did not recover but decreased even further over the next 14 days, when the seedlings were exposed to recovery conditions at 20°C (Fig. 24.5b).

Conclusions Climate change reveals itself as a temperature increase along with changes in precipitation pattern and the frequency of extreme events such as drought and heat (IPCC, 2007). Future growth and productivity of boreal forests will depend on the adaptability, vulnerability or resistance of trees to climatic change (Aitken et al., 2008). While photosynthesis of many species has a positive response to temperature, the response of boreal forests at the edges of the growing season, including regulatory and acclimatory changes, clearly requires further understanding. Increased autumn air temperatures are of particular interest, as they potentially affect the regulation of the seasonal development in conifer trees by an inadequate phasing of two environmental stimuli. These

findings are consistent with large-scale simulations combined with eddy-covariance data (Piao et al., 2008). There is evidence for an earlier date when net ecosystem CO2 exchange of northern forests crosses the zero line from a negative (photosynthetic uptake of CO2) to a positive value (respiratory loss of CO2) with increasing autumn temperatures. This indicates that in warmer years boreal forests might become a net carbon source earlier in the year. The decrease of CO2 uptake in autumn offsets the increase of CO2 uptake in spring if autumn warming occurs at a faster pace than warming in spring (Piao et al., 2008). It is therefore necessary to verify recent observations through controlled manipulative experiments in the field and to prove that they hold true in mature trees under field conditions.

Acknowledgements The work of many colleagues has contributed to this review. We are especially grateful to Gunnar Öquist, Alexander G. Ivanov, Marianna Krol, Douglas A. Campbell, Jon Lloyd, Olga Shibistova, Lilian Schmid, Dmitry Sveshnikov, John MacKay, Sebastien Caron and Laurentiu A. Tarca. Part of this work was supported by the Natural Science and Engineering Research Council of Canada. I.E. was supported by a Marie-Curie fellowship of the EU (PhysConFor, contract no. MOIF-CT-2004-002476).

Conifer Cold Hardiness

259

References ACIA (2005) Arctic Climate Impact Assessment – Scientific Report. Cambridge University Press, Canbridge, UK. Aitken, S.N., Yeaman, S., Holliday, J.A., Wang, T.L. and Curtis-McLane, S. (2008) Adaptation, migration or extirpation: climate change outcomes for tree populations. Evolutionary Applications 1, 95–111. Balling, R.C., Michaels, P.J. and Knappenberger, P.C. (1998) Analysis of winter and summer warming rates in gridded temperature time series. Climate Research 9, 175–181. Beck, E.H., Heim, R. and Hansen, J. (2004) Plant resistance to cold stress: mechanisms and environmental signals triggering frost hardening and dehardening. Journal of Biosciences 29, 449–459. Bergh, J. and Linder, S. (1999) Effects of soil warming during spring on photosynthetic recovery in boreal Norway spruce stands. Global Change Biology 5, 245–253. Bigras, F.J., Ryyppö, A., Lindström, A. and Stattin, E. (2001) Cold acclimation and deacclimation of shoots and roots of conifer seedlings. In: Bigras, F.J. and Colombo, S.J. (eds) Conifer Cold Hardiness. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 57–88. Briantais, J.M., Vernotte, C., Picaud, M. and Krause, G.H. (1979) Quantitative study of the slow decline of chlorophyll a-fluorescence in isolated-chloroplasts. Biochimica et Biophysica Acta 548, 128–138. Busch, F., Hüner, N.P.A. and Ensminger, I. (2007) Increased air temperature during simulated autumn conditions does not increase photosynthetic carbon gain but affects the dissipation of excess energy in seedlings of the evergreen Conifer Jack Pine. Plant Physiology 143, 1242–1251. Busch, F., Hüner, N.P.A. and Ensminger, I. (2008) Increased air temperature during simulated autumn conditions impairs photosynthetic electron transport between photosystem II and photosystem I. Plant Physiology 147, 402–414. Ciais, P., Tans, P.P., Trolier, M., White, J.W.C. and Francey, R.J. (1995) A large northern-hemisphere terrestrial CO2 sink indicated by the C-13/C-12 ratio of atmospheric CO2. Science 269, 1098–1102. Christersson, L. (1978) The influence of photoperiod and temperature on the development of frost hardiness in seedlings of Pinus silvestris and Picea abies. Physiologia Plantarum 44, 288–294. Decker, K.L.M., Wang, D., Waite, C. and Scherbatskoy, T. (2003) Snow removal and ambient air temperature effects on forest soil temperatures in northern Vermont. Soil Science Society of America Journal 67, 1234–1242. Demmig-Adams, B. and Adams, W.W. (1996) The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends in Plant Science 1, 21–26. Demmig-Adams, B. and Adams, W.W. (2006) Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation. New Phytologist 172, 11–21. Demmig-Adams, B., Adams, W.W., Barker, D.H., Logan, B.A., Bowling, D.R. and Verhoeven, A.S. (1996) Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiologia Plantarum 98, 253–264. Ensminger, I., Sveshnikov, D., Campbell, D.A., Funk, C., Jansson, S., Lloyd, J., Shibistova, O. and Öquist, G. (2004) Intermittent low temperatures constrain spring recovery of photosynthesis in boreal Scots pine forests. Global Change Biology 10, 995–1008. Ensminger, I., Schmidt, L., Tittmann, S. and Lloyd, J. (2005) Will photosynthetic gain of boreal evergreen conifers increase in response to a potentially longer growing season? In: van der Est, A. and Bruce, D. (eds) Photosynthesis: Fundamental Aspects to Global Perspectives volume 2, Allen Press Inc., Lawrence, USA, pp. 976–978. Ensminger, I., Busch, F. and Hüner, N.P.A. (2006) Photostasis and cold acclimation: sensing low temperature through photosynthesis. Physiologia Plantarum 126, 28–44. Ensminger, I., Schmidt, L. and Lloyd, J. (2008) Soil temperature and intermittent frost modulate the rate of recovery of photosynthesis in Scots pine under simulated spring conditions. New Phytologist 177, 428–442. Falkowski, P.G. and Chen, Y.-B. (2003) Photoacclimation of light harvesting systems in eukaryotic algae. In: Green, B.R. and Parson, W.W. (eds) Advances in Photosynthesis and Respiration. Vol. 13. Light Harvesting Antennas in Photosynthesis. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 423–447. Gilmore, A.M. (1997) Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves. Physiologia Plantarum 99, 197–209. Gilmore, A.M. and Ball, M.C. (2000) Protection and storage of chlorophyll in overwintering evergreens. Proceedings of the National Academy of Sciences USA 97, 11098–11101.

260

I. Ensminger et al.

Gilmore, A.M. and Yamamoto, H.Y. (1993) Linear-models relating xanthophylls and lumen acidity to nonphotochemical fluorescence quenching – evidence that antheraxanthin explains zeaxanthinindependent quenching. Photosynthesis Research 35, 67–78. Gilmore, A.M., Shinkarev, V.P., Hazlett, T.L. and Govindjee (1998) Quantitative analysis of the effects of intrathylakoid pH and xanathophyll cycle pigments on chlorophyll a fluorescence lifetime distributions and intensity in thylakoids. Biochemistry 37, 13582–13593. Horton, P., Ruban, A.V., Rees, D., Pascal, A.A., Noctor, G. and Young, A.J. (1991) Control of the light-harvesting function of chloroplast membranes by aggregation of the LHCII chlorophyll protein complex. FEBS Letters 292, 1–4. Horton, P., Ruban, A.V. and Young, A.J. (1999) Regulation of the structure and function of the light harvesting complexes of photosystem II by the xanthophyll cycle. In: Frank, H.A., Young, A.J., Britton, G. and Cogdell, R.J. (eds) Advances in Photosynthesis and Respiration. Vol. 8. The Photochemistry of Carotenoids. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 271–291. Houghton, J.T., Ding, Y., Griggs, D.J., Noguer, M., van der Linden, P.J. and Xiaosu, D. (2001) Climate Change 2001: The Scientific Basis. Contribution of Working Group I: The Third Assessment Report of the Intergovernmental Panel on Climate Change (IPCC). Cambridge University Press, Cambridge, UK. Huner, N.P.A., Öquist, G. and Sarhan, F. (1998) Energy balance and acclimation to light and cold. Trends in Plant Science 3, 224–230. Huner, N.P.A., Öquist, G. and Melis, A. (2003) Photostasis in plants, green algae and cyanobacteria: the role of light harvesting antenna complexes. In: Green, B.R. and Parson, W.W. (eds) Advances in Photosynthesis and Respiration. Vol. 13. Light Harvesting Antennas in Photosynthesis. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 401–421. Huner, N.P.A., Ivanov, A.G., Sane, P.V., Pocock, T., Krol, M., Balseris, A., Rosso, D., Savitch, L.V., Hurry, V.M. and Öquist, G. (2006) Photoprotection of photosystem II: reaction center quenching versus antenna quenching. In: Demmig-Adams, B., Adams, W.W. and Mattoo, A.K. (eds) Advances in Photosynthesis and Respiration. Vol. 21. Photoprotection, Photoinhibition, Gene Regulation, and Environment. Springer, Dordrecht, The Netherlands, pp. 155–173. IPCC (2007) Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, UK. Ivanov, A.G., Sane, P.V., Zeinalov, Y., Malmberg, G., Gardeström, P., Huner, N.P.A. and Öquist, G. (2001) Photosynthetic electron transport adjustments in overwintering Scots pine (Pinus sylvestris L.). Planta 213, 575–585. Ivanov, A.G., Sane, P.V., Zeinalov, Y., Simidjiev, I., Huner, N.P.A. and Öquist, G. (2002) Seasonal responses of photosynthetic electron transport in Scots pine (Pinus sylvestris L.) studied by thermoluminescence. Planta 215, 457–465. Jarvis, P. and Linder, S. (2000) Botany – constraints to growth of boreal forests. Nature 405, 904–905. Krause, G.H. and Weis, E. (1991) Chlorophyll fluorescence and photosynthesis – the basics. Annual Review of Plant Physiology and Plant Molecular Biology 42, 313–349. Krol, M., Hurry, V., Maxwell, D.P., Malek, L., Ivanov, A.G. and Huner, N.P.A. (2002) Low growth temperature inhibition of photosynthesis in cotyledons of jack pine seedlings (Pinus banksiana) is due to impaired chloroplast development. Canadian Journal of Botany–Revue Canadienne de Botanique 80, 1042–1051. Lee, H.Y., Hong, Y.N. and Chow, W.S. (2001) Photoinactivation of photosystem II complexes and photoprotection by non-functional neighbours in Capsicum annuum L. leaves. Planta 212, 332–342. Leonardos, E.D., Savitch, L.V., Huner, N.P.A, Oquist, G. and Grodzinski, B. (2003) Daily photosynthetic and C-export patterns in winter wheat leaves during cold stress and acclimation. Physiologia Plantarum 117, 521–531. Li, C.Y., Puhakainen, T., Welling, A., Vihera-Aarnio, A., Ernstsen, A., Junttila, O., Heino, P. and Pavla, E.T. (2002) Cold acclimation in silver birch (Betula pendula). Development of freezing tolerance in different tissues and climatic ecotypes. Physiologia Plantarum 116, 478–488. Lloyd, J., Shibistova, O., Zolotoukhine, D., Kolle, O., Arneth, A., Wirth, C., Styles, J.M., Tchebakova, N.M. and Schulze, E.D. (2002) Seasonal and annual variations in the photosynthetic productivity and carbon balance of a central Siberian pine forest. Tellus Series B – Chemical and Physical Meteorology 54, 590–610. Lunde, C., Jensen, P.E., Haldrup, A., Knoetzel, J. and Scheller, H.V. (2000) The PSI-H subunit of photosystem I is essential for state transitions in plant photosynthesis. Nature 408, 613–615.

Conifer Cold Hardiness

261

Niyogi, K.K., Li, X.P., Rosenberg, V. and Jung, H.S. (2005) Is PsbS the site of non-photochemical quenching in photosynthesis? Journal of Experimental Botany 56, 375–382. Öquist, G. and Huner, N.P.A. (2003) Photosynthesis of overwintering evergreen plants. Annual Review of Plant Biology 54, 329–355. Ottander, C., Campbell, D. and Öquist, G. (1995) Seasonal changes in photosystem II organization and pigment composition in Pinus sylvestris. Planta 197, 176–183. Pfannschmidt, T., Nilsson, A. and Allen, J.F. (1999) Photosynthetic control of chloroplast gene expression. Nature 397, 625–628. Piao, S.L., Ciais, P., Friedlingstein. P., Peylin, P., Reichstein, M., Luyssaert, S., Margolis, H., Fang, J.Y., Barr, A., Chen, A.P., Grelle, A., Hollinger, D.Y., Laurila, T., Lindroth, A., Richardson, A.D. and Vesala, T. (2008) Net carbon dioxide losses of northern ecosystems in response to autumn warming. Nature 451, 49–53. Porcar-Castell, A., Juurola, E., Nikinmaa, E., Berninger, F., Ensminger, I. and Hari, P. (2008a) Seasonal acclimation of photosystem II in Pinus sylvestris. I. Estimating the rate constants of sustained thermal energy dissipation and photochemistry. Tree Physiology 28, 1475–1482. Porcar-Castell, A., Juurola, E., Ensminger, I., Berninger, F., Nikinmaa, E. and Hari, P. (2008b) Seasonal acclimation of photosystem II in Pinus sylvestris. II. Using the rate constants of sustained thermal energy dissipation and photochemistry to study the effect of the light environment. Tree Physiology 28, 1483–1491. Puhakainen, T., Li, C.Y., Boije-Malm, M., Kangasjarvi, J., Heino, P. and Palva, E.T. (2004) Short-day potentiation of low temperature-induced gene expression of a C-repeat-binding factor-controlled gene during cold acclimation in silver birch. Plant Physiology 136, 4299–4307. Ruban, A.V., Young, A.J. and Horton, P. (1993) Induction of nonphotochemical energy-dissipation and absorbency changes in leaves – evidence for changes in the state of the light-harvesting system of photosystem II in vivo. Plant Physiology 102, 741–750. Sane, P.V., Ivanov, A.G., Hurry, V., Huner, N.P.A. and Öquist, G. (2003) Changes in the redox potential of primary and secondary electron-accepting quinones in photosystem II confer increased resistance to photoinhibition in low-temperature-acclimated Arabidopsis. Plant Physiology 132, 2144–2151. Savitch, L.V., Leonardos, E.D., Krol, M., Jansson, S., Grodzinski, B., Huner, N.P.A. and Öquist, G. (2002) Two different strategies for light utilization in photosynthesis in relation to growth and cold acclimation. Plant, Cell & Environment 25, 761–771. Saxe, H., Cannell, M.G.R., Johnsen, B., Ryan, M.G. and Vourlitis, G. (2001) Tree and forest functioning in response to global warming. New Phytologist 149, 369–399. Serreze, M.C., Walsh, J.E., Chapin, F.S., Osterkamp, T., Dyurgerov, M., Romanovsky, V., Oechel, W.C., Morison, J., Zhang, T. and Barry, R.G. (2000) Observational evidence of recent change in the northern highlatitude environment. Climatic Change 46, 159–207. Slot, M., Wirth, C., Schumacher, J., Mohren, G.M.J., Shibistova, O., Lloyd, J. and Ensminger, I. (2005) Regeneration patterns in boreal Scots pine glades linked to cold-induced photoinhibition. Tree Physiology 25, 1139–1150. Sveshnikov, D., Ensminger, I., Ivanov, A.G., Campbell, D., Lloyd, J., Funk, C., Hüner, N.P.A. and Öquist, G. (2006) Excitation energy partitioning and quenching during cold acclimation in Scots pine. Tree Physiology 26, 325–336. Weiser, C.J. (1970) Cold resistance and injury in woody plants: knowledge of hardy plant adaptations to freezing stress may help us to reduce winter damage. Science 169, 1269–1278.

25

Chemical Genetics Identifies New Chilling Stress Determinants in Arabidopsis J. Einset

Introduction Cold stress limits plant survival and productivity in agriculture and in natural systems worldwide. Although the effects of freezing temperatures have often been of primary interest for plant researchers, chilling temperatures in the range from 0 to 15°C can also be of importance. For example, chilling temperatures during spring can hinder establishment of vegetable seedlings or have damaging effects on already established plants. In Nordic areas especially, it is common that summer growing seasons can have periods of cold weather that cause significant effects on flowering, fruit set and general plant vigour. In the autumn, early chilling episodes can prevent effective winter hardening, resulting in winter damage and reduced crop yields during subsequent growing seasons. Finally, crop losses caused by postharvest chilling, in the field or in storage, can also be significant. Knowledge gaps in relation to fundamental questions about chilling stress are substantial. The following questions can be asked. How important is chilling stress as a factor limiting plant distribution and success? How do plants sense chilling temperatures? How is the perception of chilling temperatures translated into signal transduction, leading to the activation of genes that enable plants to cope with chilling? When are the critical times of the year in relation to sensitivity to chilling stress: spring, autumn or 262

winter? What is the molecular mechanism(s) of chilling injury? What are the mechanisms of tolerance? Opinions vary with respect to the best experimental approaches to answer these questions. On the one hand, it is often said that research should focus on our most important agricultural and landscape plants, since breakthroughs with these plants would be expected to result in rapid payoffs practically. On the other hand, other researchers emphasize the idea that plant research should be focused on Arabidopsis as a model system, keeping in mind the advantages of technologies based on knockout mutants, transformation and gene expression profiling. Arabidopsis was introduced as a model system for plant research about 25 years ago and approximately 50% of all plant researchers in the world today study this plant. The extent to which discoveries with Arabidopsis will be transferable to other plant species remains to be determined. The basis for the research reported in the present chapter were investigations pioneered by Professor Norio Murata during the last 10 years, demonstrating that glycine betaine (GB) can confer tolerance to several types of stress at low concentrations, including chilling stress, either after application to plants or in transgenics engineered to overproduce GB. GB is a widely distributed natural product in plants (Rhodes and Hanson, 1993; Ingram and Bartels, 1996) that has been demonstrated to improve

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

Chemical Genetics in Arabidopsis

stress tolerance in agriculturally important crops (Allard et al., 1998; Chen et al., 2000; Rahman et al., 2002). Examples of stress-tolerant transgenics engineered to produce GB (Sakamoto and Murata, 2000, 2001; Chen and Murata, 2002; Park et al., 2004) include rice, tobacco, tomato, Brassica napus, Brassica juncos and Diospyrus kaki. GB production has also been shown to improve stress tolerance in bacteria and algae (Nomura et al., 1995; Kempf and Bremer, 1998). GB levels in transgenic plants are often quite low, and the extreme examples are transgenic tobacco plants with improved salt and chilling tolerance associated with a GB concentration of 0.035 µmol/g fresh weight (Holmström et al., 2000). Given this fact and the demonstration that GB application can affect gene expression (Allard et al., 1998), we hypothesized that GB might confer stress tolerance, at least in part, via effects on gene expression rather than by its known effects on osmotic pressure or protein stability. If this could be shown, then a new approach for identifying chilling stress determinants in plants would be established, using GB in a chemical genetic screen. The rationale of this approach would be that at least some of the genes up- or down-regulated by GB play a role in chilling sensitivity and/or tolerance to chilling stress. In other words, by identifying GB-regulated genes, the potential exists to identify new chilling stress determinants. Because the best system for conducting these types of experiments was with Arabidopsis, we began conducting global gene expression studies with microarrays followed by confirmation on Northern blots to identify GB-regulated genes. Several up-regulated genes were detected in leaves (Einset, 2006) and roots (Einset et al., 2007a). That GB’s effect depends on gene expression was first proved by direct functional evidence using a knockout mutant for RabA4c GTPase (At5g47960). Although Arabidopsis is usually defined as chilling-resistant because it shows no obvious signs of chilling injury, chilling does have an effect because chilled plants show inhibited root growth upon transfer back to normal temperatures. Remarkably, when wild-type plants were pre-treated with GB, root growth rates after chilling were comparable to nonchilled plants. By contrast, the RabA4c GTPase

263

knockout showed no GB response in the chilling test, proving the requirement for a functional RabA4c GTPase gene for GB’s effect (Einset et al., 2007a). The chemical genetics idea was validated! The chapter begins by giving an update on studies using GB-based chemical genetics. After demonstrating the overall approach with results from a putative basic leucine zipper (bZIP) transcription factor, evidence is presented further implicating membrane trafficking processes and reactive oxygen species (ROS) in chilling stress. Finally, a model for ROS signalling in chilling stress is presented along with suggestions about important areas for future research.

Materials and Methods Plant materials, growth conditions, chilling and chemical treatments Arabidopsis thaliana plants were grown in 8.5 cm diameter plastic Petri plates containing MS medium, 30 g sucrose/l and 8 g agar/l under controlled environment conditions at 24°C, 16 h photoperiod and light intensity of 80 µM/m2/s. Seed for making homozygous knockouts of the bZIP gene (knockout SALK_ 004683) were obtained through SIGnAL (http://signal.salk.edu/tdna_protocols.html). In experiments testing recovery from chilling, plants at the rosette stage of growth, treated for 24 h either with GB or with water as controls, were transferred to 4°C and 30 µM/m2/s continuous light for 2 days, after which time they were transferred back to 24°C, 16 h photoperiod and 80 µM/m2/s light intensity and incubated vertically. Daily root growth increments were measured with a ruler. All data were subjected to ANOVA in groups. Significant differences between treatments were evaluated at the 95% confidence interval. For GB treatments, approximately 3-week-old plants at the rosette stage were sprayed on both leaves and roots with an aqueous solution containing 100 mM GB only while control plants were sprayed with water. For brefeldin A (BFA) experiments, plants were treated with a solution containing 10 µM BFA immediately before transfer to chilling conditions.

264

J. Einset

Detection of superoxide For detecting superoxide, plant material was vacuum-infiltrated with 0.1 mg nitroblue tetrazolium (NBT)/ml in 25 mM HEPES buffer, pH 7.6. Samples were incubated at room temperature in the dark for 0.5 h. In control treatments to test the validity of the method, 10 mM MnCl2 and 10 units of superoxide dismutase/ml were added to the buffer, in addition to NBT. These controls gave no formazan product.

Microscopy Microscopy was performed using a Leitz Laborlux K microscope with a Leica DC300F colour camera. The Leica DC program was used to import images of NBT-stained roots into Adobe Photoshop Elements 2.0. Images were then analysed for blue pixel intensities, representing formazan staining, using publicdomain ImageJ (http://rsb.info.nih.gov/ij/). At least eight roots from each chilled treatment with or without either GB or BFA (10 µM solution) pre-treatments were analysed relative to controls, with at least three independent experiments giving similar results. Data were normalized for chilled wild-type Columbia roots and evaluated using ANOVA between groups. Mean differences at the 95% confidence level were considered as significant.

Results Pursuing the goal of identifying genes regulated by GB treatments, we began gene expression profiling using DNA microarrays five years ago. Our first experiments were done with Incyte Genomics Arabidopsis GEM1 chip with 6000+ different genes. Next, we began collaboration with Atle Bones’ group at the Norwegian University of Science and Technology in Trondheim. Over the years, the Trondheim Group has developed their own unique oligonucleotide sets for genes that have been implicated in stress responses. One array (1.1K chip) comprises eight copies each of 1100 genes, while the other custom array has eight copies each of 2000 genes (2K chip).

Lately, we have used arrays printed with the 2K gene set plus Operon Biotechnologies GmbH’s 70-mer oligonucleotide set for over 26,000 Arabidopsis genes. In conducting experiments, tissue samples from control plants and GB-treated plants were used for RNA isolation and then this RNA was used as template to make cDNA that was hybridized to the microarrays. After quantification of signal intensities, the data were normalized to correct for differences in probe labelling, background levels, inconsistency in replicates on the same array and non-linearity of intensity distributions. Next, we identified genes showing significant and consistent upregulation by GB in at least three independent experiments. Finally, to confirm up-regulation using an alternative method, we conducted gene expression analyses using Northern blots. So far, we have obtained Northern blot confirmation for significant up-regulation of the genes encoding the following in roots and leaves: membrane trafficking RabA4c GTPase, a bZIP transcription factor (At3g62420), mitochondrial catalase 2 (At4g35090), cell wall peroxidase ATP3a (At5g64100) and a glutathione S-transferase (At1g02930). In roots only, we have confirmed up-regulation of genes encoding tonoplast aquaporin (At5g47450) and the NADPH-dependent ferric reductase FRO2 (At1g01580) localized to the plasma membrane. The fact that the level of mRNA for a particular gene increases upon GB pre-treatment is not sufficient by itself to show that GB’s effect on chilling tolerance requires this up-regulation of mRNA levels, even if the particular gene involved has already been implicated in relation to stress in other systems. What is needed is a direct demonstration of the role of specific genes in relation to stress tolerance and the effect of GB. This has already been accomplished using mutants for the membrane trafficking RabA4c GTPase (Einset et al., 2007a) and FRO2 (Einset et al., 2008). Figure 25.1 presents a similar demonstration for the putative bZIP transcription factor, showing the effect of GB pre-treatments on chilling recovery by wild-type plants and knockout mutants for the putative bZIP transcription gene. Twenty-four hours after treatment with GB, plants were transferred to

Chemical Genetics in Arabidopsis

A

B

C

265

D

Fig. 25.1. Effect of glycine betaine (GB) on recovery from chilling stress by wild-type and bZIP-knockout mutant Arabidopsis. Plants approximately 2 weeks old were sprayed with 100 mM GB, then incubated 24 h at 24°C, 16 h photoperiod and 40 µM/m2/s light intensity before being transferred to chilling conditions at 4°C and 30 µM/m2/s light intensity for 48 h. Photographs show plants four days after transfer back to normal growing conditions at 24°C, 16 h photoperiod and 40 µM/m2/s light intensity and incubated vertically. All photographs are presented with the same magnification and the vertical bar in (D) corresponds to 1 cm. Treatments were: (A) Columbia without GB pre-treatment; (B) Columbia with GB pre-treatment; (C) bZIP knockout without GB pre-treatment; (D) bZIP knockout with GB pre-treatment.

chilling conditions at 4°C and 20 µM/m2/s light intensity. Two days later plants were transferred back to normal growing conditions and recovery was monitored as root growth on Petri plates incubated vertically. As Fig. 25.1 shows, wild-type Columbia res-

ponded markedly to GB pre-treatments, as demonstrated by the large difference in shoot and root growth plus versus minus GB, while bZIP knockouts showed little, if any, response. This proves that bZIP is required for GB’s effect on chilling recovery. Figure 25.2 (b)

(a)

7 Growth rate (mm)

Growth rate (mm)

7 6 5 4 3 2

5 4 3 2 1

1 0

6

Day 1 Day 2 Day 3 Day 4 Day 5

0

Day 1 Day 2 Day 3 Day 4 Day 5

Fig. 25.2. Root growth rates during recovery from chilling by (a) wild-type Columbia and (b) bZIP-knockout Arabidopsis plants, pre-treated with glycine betaine (GB) (–䊐–) or with no GB pre-treatment (–◆–). Daily growth increments, measured as mm growth per day, were determined for randomly selected roots of plants grown on Petri plates incubated vertically after transfer from chilling conditions to normal growth conditions at 24°C, 16 h photoperiod and 40 µM/m2/s light intensity. Each point is mean value based on pooled results from five independent experiments with at least ten independent measurements per data point; standard errors are represented by vertical bars.

266

J. Einset

presents root growth rates during recovery and also shows that the response to GB by wild-type plants can be seen by measuring root growth as early as one day after plants were transferred back to normal growth conditions after the 2 days of chilling treatment. Five days later, all treatments had similar root growth rates, indicating that the bZIP knockouts are not simply less robust in their growth under normal conditions. So far, three genes have been identified with this approach: those encoding a membrane trafficking RabA4c GTPase, a NADPHdependent ferric reductase (FRO2) and a bZIP transcription factor. The fact that both the RabA4c GTPase and the plasma membrane NADPH-dependent ferric reductase FRO2 are both membrane proteins in epidermal cells of Arabidopsis roots has focused our attention on the possibility that membrane trafficking processes might be central to chilling sensitivity. For example, does the increase in ROS associated with chilling require membrane trafficking? An experiment to answer this question is shown in Fig. 25.3. In this case, roots of wild-type Columbia were pre-treated with a solution containing 10 µM BFA, an

Relative ROS Staining

100 80 60 40 20 0 Chilled

Chilled + GB Chilled + BFA

Fig. 25.3. Levels of reactive oxygen species (ROS), measured as blue pixel intensities using ImageJ with nitroblue tetrazolium-stained roots, in chilled Arabidopsis plants of wild-type Columbia not pre-treated with glycine betaine (GB), pre-treated with GB or pre-treated with brefeldin A (BFA). Data are means based on pooled results from three independent experiments with standard errors represented by vertical bars.

inhibitor of Golgi to plasma membrane vesicle trafficking, or with 100 mM GB prior to placing plants in the cold. Two days later, plants were removed from chilling and superoxide was visualized in roots using NBT staining and ROS staining was analysed in photographs using ImageJ. As Fig. 25.3 shows, ROS accumulation was markedly decreased in both GBand BFA-treated roots compared with control roots, indicating that inhibition of membrane trafficking can cause an inhibition of ROS accumulation during chilling. ROS staining of GB- versus BFA-treated roots was not significantly different.

Discussion Before the introduction of Arabidopsis as a model system, experiments to identify processes and genes involved in chilling tolerance were based on hypotheses generated via understanding of fundamental processes. In the 1970s, for example, Lyons and Raison proposed an explanation for the molecular mechanism determining chilling sensitivity based on membrane phase transitions occurring at low temperatures and resulting in destructive events such as membrane damage, ion leakage, impaired photosynthesis and respiration, as well as the production of toxic compounds (Lyons and Raison, 1970; Lyons, 1973). This focus on membrane phase transitions led to several studies examining the role of unsaturated membrane lipids on chilling sensitivity (Murata, 1983). Among other findings, a correlation was reported between the unsaturation level of membrane lipids and chilling sensitivity in tests of several herbaceous species (Nishida and Murata, 1996). In addition, it was shown that manipulation of the unsaturation in chloroplast membrane lipids in transgenic plants and blue-green algae had effects on chilling sensitivity (Murata et al., 1992). On the other hand, it has also been shown that many chilling-resistant plants do not have high levels of unsaturated membrane lipids. In addition, examples are found in Arabidopsis, for example, where mutants having large changes in chloroplast membrane lipids had no change in chilling sensitiv-

Chemical Genetics in Arabidopsis

ity (Wu and Browse, 1995). The conclusion, therefore, has been that membrane lipid composition and membrane phase transitions are not the only factors determining chilling sensitivity. With the beginning of the Arabidopsis era during the 1980s, new hypothesis-generating approaches became possible based on: (i) rapid screening for mutants that are less tolerant (Warren et al., 1996; Thorlby et al., 2004) or more tolerant (Xin and Browse, 1998) to cold temperatures; (ii) gene expression studies to identify genes affected by cold temperatures (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997; Thomashow, 2001; Viswanathan and Zhu, 2002); and (iii) chemical genetics approaches such as the one described in this chapter. At the same time that we were conducting experiments to identify genes required for GB’s effect on chilling tolerance, a close relationship was found between ROS accumulation and chilling stress. To summarize several independent lines of evidence in this regard, we found that: (i) chilling results in increases in ROS levels (superoxide and hydrogen peroxide) in both roots and leaves; (ii) during recovery from chilling under normal growth conditions, ROS disappears in parallel with growth recovery; (iii) GB pre-treatments prevent ROS accumulation during chilling, as well as the root growth inhibition seen after transfer of plants to normal growth temperatures; (iv) GB pre-treatments up-regulate several genes known to be involved in ROS scavenging; (v) mutants that are unresponsive to GB accumulate ROS with or without GB pre-treatments; (vi) GB does not prevent chilling effects in these mutants; and, finally, (vii) treatment of plants with hydrogen peroxide (0.05%) alone without chilling results in the same kinds of inhibition of root growth and elevated ROS levels as seen in chilled plants. Possibly the most remarkable finding cited above was that plants pre-treated with GB do not show reduced growth rates after chilling. This observation makes it seem as though chilled plants can avoid the effects of chilling stress as long as they can avoid ROS accumulation. To stimulate further mechanistic studies in relation to the role of ROS in chilling stress,

267

we present a model involving ROS signalling as the molecular mechanism determining chilling sensitivity in Fig. 25.4. According to this model, chilling stress begins with perception of low temperature by an unknown receptor, followed by activation of membrane NADPH oxidase (NOX), causing an increase in ROS levels which signals altered gene expression, resulting in inhibited growth under normal growing conditions in Arabidopsis and in more severe effects in chilling-sensitive plants. Although the actual mechanism of ROS production in the cold is not known, NOX activity is an interesting possibility inasmuch as NOX has been demonstrated in Arabidopsis to be involved in ROS production during root hair development by root epidermal cells (Gapper and Dolan, 2006) as well as being implicated in pathogen responses (Torres and Dangl, 2005) and stomatal control (Kwak et al., 2006). Considerable knowledge gaps exist with regard to plant NOX enzymes. In neutrophils activation of NOX involves membrane trafficking as well as other proteins such as membrane trafficking Rop GTPases (Bedar and Krause, 2007). If similar processes are occurring in Arabidopsis roots, this could

Chilling

GB FRO

Rop NOX

RabA4c bZIP ROS

‘Chilling stress’ gene expression

ROS scavenging

Fig. 25.4. Schematic representation of a model for chilling stress as reactive oxygen species (ROS) signalling and gene expression. A competing pathway based on glycine betaine (GB) action via gene expression, membrane trafficking and extracellular ferric ion reduction blocks ROS accumulation and alleviates chilling stress. bZIP, basic leucine zipper transcription factor; FRO, NADPH-dependent ferric reductase; NOX, NADPH oxidase; RabA4c, RabA4c GTPases; Rop, Rop GTPases.

268

J. Einset

explain our observation that BFA blocks ROS accumulation during chilling. In fact, a recent paper by Jones et al. (2007) suggests that enhanced Rop GTPase activity can heighten ROS production regulating root hair development in Arabidopsis. Based on all of these lines of evidence, we suggest that Rop GTPases may affect ROS production during chilling via NOX activation. As the model suggests, the working hypothesis is that GB upregulates membrane trafficking associated with RabA4c GTPases and that this process competes with Rop GTPase membrane trafficking associated with NOX activation. Another feature of the model is the involvement of Fe via markedly elevated levels of NADPH-dependent ferric reductase occurring during chilling in plants pre-treated with GB (Einset et al., 2007b). If one makes the assumption that NOX in plants is similar to neutrophil NOX (Bedar and Krause, 2007), then superoxide would initially be produced outside the plasma membrane. By increasing plasma membrane-localized ferric reductase activities, then GB increases transfer of reductant potential from cytoplasmic NADPH to the cell wall of root epidermal cells, effectively stopping ROS build-up in roots, both extra- and intracellularly, along with attendant ROS accumulation associated with chilling stress.

Conclusion In conclusion, results from chemical genetic studies on chilling stress seem to be focusing attention on ROS as well as associated processes such as membrane trafficking and Fe metabolism. Major unanswered questions involve the mechanism of ROS production and the factors that regulate it. Further questions revolve around downstream components of the ROS signalling system. In animal systems, ROS levels comparable to those we see in chilled Arabidopsis roots activate protein kinases (Burgoyne et al., 2007), initiating signalling cascades. The possibility exists that ROS might be regulatory in Arabidopsis in relation to mitogen-activated protein (MAP) kinase signalling, for example. If so, then one would have to ask whether it is ROS that is dominant in determining the system’s dynamics or other components such as the MAP kinases. Finally, by analogy to ROS signalling during root hair formation, the involvement of Ca2+ channels is likely. All in all, only the results of future experiments will be able to resolve the issue of whether chemical genetic studies with Arabidopsis can give us a fundamentally better understanding of cold tolerance mechanisms with the view to producing improved plant types for cold stress.

References Allard, F., Houde, M., Krol, M., Ivanov, A., Huner, N.P.A., and Sarhan, F. (1998) Betaine improves freezing tolerance in wheat. Plant & Cell Physiology 39, 1194–1202. Bedar, K. and Krause, K.-H. (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 87, 245–313. Burgoyne, J.R., Madhani, M., Cuello, F., Charles, R.L., Brennan, J.P., Schröder, E., Browning, D.D. and Eaton, P. (2007) Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science 317, 1393–1397. Chen, W.P., Li, P.H. and Chen, T.H.H. (2000) Glycinebetaine increases chilling tolerance and reduces chilling-induced lipid peroxidation in Zea mays L. Plant, Cell & Environment 23, 609–618. Chen, T.H.H. and Murata, N. (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Current Opinion in Plant Biology 5, 250–257. Einset, J. (2006) An extracellular mechanism of light protection in plants identified using a chemical genetic screen. Acta Horticulturae 711, 339–344. Einset, J., Nielsen, E, Connolly, E.L., Bones, A., Sparstad, T., Winge, P. and Zhu, J.-K. (2007a) Membrane trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis. Physiologia Plantarum 130, 511–518.

Chemical Genetics in Arabidopsis

269

Einset, J., Winge, P. and Bones, A. (2007b) ROS signaling pathways in chilling stress. Plant Signaling & Behaviour 2, 365–366. Einset, J., Winge, P., Bones, A. and Connolly, E.L. (2008) The FRO2 ferric reductase is required for glycine betaine’s effect on chilling tolerance in Arabidopsis roots. Physiologia Plantarum 134, 334–341. Gapper, C. and Dolan, L. (2006) Control of plant development by reactive oxygen species Plant Physiology 141, 341–343. Holmström, K., Somersalo, S., Mandal, A., Palva, T.E. and Welin, B. (2000) Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. Journal of Experimental Botany 51, 177–185. Ingram, J. and Bartels, D. (1996) Molecular basis of dehydration tolerance in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 377–403. Jones, M.A., Raymond, M.J., Yang, Z. and Smirnoff, N. (2007) NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase. Journal of Experimental Botany 58, 1261–1270. Kempf, B. and Bremer, E. (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Archives of Microbiology 5, 319–330. Kwak, J.M., Nguyen, V. and Schroeder, J.I. (2006) The role of reactive oxygen species in hormonal responses. Plant Physiology 141, 323–329. Lyons, J.M. (1973) Chilling injury in plants. Annual Review of Plant Physiology 24, 445–466. Lyons, J.M. and Raison, J.K. (1970) Oxidative activity of mitochondria isolated from plant tissues sensitive and resistant to chilling injury. Plant Physiology 45, 386–389. Murata, N. (1983) Molecular species composition of phosphatidylglycerols from chilling-sensitive and chilling-resistant plants. Plant & Cell Physiology 25, 1241–1245. Murata, N., Ishibaki-Nishizawa, O., Higashi, S., Hayashi, H., Tasaka, Y. and Nishida, I. (1992) Genetically engineered alteration in the chilling sensitivity of plants. Nature 356, 710–713. Nishida, I. and Murata, N. (1996) Chilling sensitivity in plants and cyanobacteria: the crucial contribution of membrane lipids. Annual Review Plant Physiology 47, 541–568. Nomura, M., Ishitani, M., Takabe, T., Rai, A.K. and Takabe, T. (1995) Synechococcos sp. PCC7942 transformed with Escherichia coli bet genes produces glycine betaine from choline and acquires resistance to salt stress. Plant Physiology 107, 703–708. Park, E.-J., Jeknic, Z., Sakamoto, A., DeNoma, J., Yuwansiri, R., Murata, N. and Chen, T.H.H. (2004) Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. The Plant Journal 40, 474–487. Rahman, S., Miyake, H. and Takeoka, Y. (2002) Effects of exogenous glycinebetaine on growth and ultrastructure of salt-stressed rice seedlings (Oryza sativa L.). Plant Production Science 5, 33–44. Rhodes, D. and Hanson, A.D. (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 44, 357–384. Sakamoto, A. and Murata, N. (2000) Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance. Journal of Experimental Botany 51, 81–88. Sakamoto, A. and Murata, N. (2001) The use of bacterial choline oxidase, a glycinebetaine-synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiology 125, 180–188. Stockinger E.J., Gilmour, S.J. and Thomashow, M.F. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domaincontaining transcription activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proceedings of the National Academy of Sciences USA 94, 1035–1040. Thomashow, M.F. (2001) So what’s new in the field of plant cold acclimation? Lots! Plant Physiology 125, 89–93. Thorlby, G., Fourrier, N. and Warren, G. (2004) The SENSITIVE TO FREEZING2 gene, required for freezing tolerance in Arabidopsis thaliana, encodes a β-glucosidase. The Plant Cell 16, 2192–2203. Torres, M.A. and Dangl, J.L. (2005) Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Current Opinion in Plant Biology 8, 397–403. Viswanathan, C. and Zhu, J.-K. (2002) Molecular genetic analysis of cold-regulated gene transcription. Philosophical Transactions of the Royal Society, London B 357, 877–886.

270

J. Einset

Warren, G., McKown, R., Martin, A.L. and Teutonico, V. (1996) Isolation of mutations affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Plant Physiology 111, 1011–1019. Wu, J.R. and Browse, J. (1995) Elevated levels of high-melting-point phosphatidylglycerols do not induce chilling sensitivity in an Arabidopsis mutant. The Plant Cell 7, 17–27. Xin, Z. and Browse, J. (1998) eskimo1 mutants of are constitutively freezing-tolerant. Proceedings of the National Academy of Sciences USA 95, 7799–7804. Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. The Plant Cell 6, 251–264.

26

Analysis of the Ascorbate Antioxidant Pathway in Overwintering Populations of Lucerne (Medicago sativa L.) of Contrasting Freezing Tolerance A. Bertrand, Y. Castonguay, S. Laberge, J. Cloutier and R. Michaud

Introduction The perennial growth habit of lucerne combined with its capacity to symbiotically fix N contributes to long-term sustainability of agriculture because it is a multi-harvest crop improving soil properties with benefits to successive crops. Furthermore, because of its symbiotic N fixation with rhizobia, lucerne reduces the reliance on fossil fuels required for N fertilizer production (Vance, 2001). However, lack of winter hardiness constitutes a major limitation to lucerne persistence under harsh winter conditions and there is a need to improve winter survival of cultivars of high agronomic value grown in northern climates (Volenec et al., 2002). We recently applied a recurrent selection protocol entirely performed under environmentally controlled conditions to develop lucerne populations selectively improved for their tolerance to freezing (TF populations). Several cycles of recurrent phenotypic selection have been performed in various genetic backgrounds and new populations have been produced using elite genotypes (Castonguay et al., 2006). Even though we measured significant increases in freezing tolerance in advanced cycles of selection, the molecular bases of the improved freezing tolerance are still largely unknown. Multiple mechanisms

are induced during the acquisition of freezing tolerance of lucerne including the synthesis of low-molecular-weight cryoprotective molecules such as proline (Paquin and Pelletier, 1981), sucrose and oligosaccharides of the raffinose family (Castonguay et al., 1995) as well as the induction of the expression of cold-regulated (COR) genes homologous to glycine-rich protein (msaCIA), putative nuclear protein (msaCIB), bimodular protein (msaCIC), pathogenesis-related protein PR10 (msaCID), glyceraldehyde-3P-dehydrogenase (msaCIE), galactinol synthase (msaCIF) and dehydrin (msaCIG) (Castonguay et al., 1997; Bertrand et al., 2007). It is however unclear whether these cold-induced changes are a consequence of low-temperature exposure or a cause of enhanced freezing tolerance. For instance, Wanner and Junttila (1999) observed that proline accumulation lagged behind the acquisition of freezing tolerance by one day in Arabidopsis and concluded that this accumulation is a stress response to cold rather than an adaptive mechanism. In that respect, TF populations obtained after successive cycles of selection for improved freezing tolerance could allow us to assess the relationship between cold-induced changes, such as the antioxidant metabolism, and the ultimate level of freezing tolerance of lucerne.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

271

272

A. Bertrand et al.

Freezing Stress and Antioxidant Pathways At the cellular level, it has been demonstrated that plasma membranes and endo-membranes are the primary site of freezing injury (Arora and Palta, 1991). Therefore, the maintenance of their integrity in the frozen state is an intricate component of the cold acclimation process and of the determination of freezing tolerance. Membrane damage under freezing stress can be mediated by various factors including destabilization of the phospholipid bilayer and embedded proteins in desiccated tissues, and an increased level of active oxygen species (AOS). When plants are exposed to freezing stress, different metabolic pathways could become uncoupled, leading to the transfer of electrons with high energy state to molecular oxygen to form AOS including oxygen ions, free radicals and peroxides, which initiate lipid peroxidation and damage to proteins and DNA (Mittler et al., 2004). Increased levels of AOS in plants exposed to environmental stress leading to cumulative damage to cell structures is known as oxidative stress. Recent studies have shown that, in addition to their potential to cause oxidative damage to cells during environmental stress, AOS could be involved as signal transduction molecules mediating responses to biotic and abiotic stresses (Pei et al., 2000; Torres and Dangl, 2005). How plants cope with this dual role of AOS as both toxic by-products of aerobic metabolism and key regulators of growth, development and defence pathways is unknown, but it is clear that the steady-state levels of AOS in cells needs to be tightly regulated (Mittler et al., 2004). To control levels of AOS, plants have evolved several enzymatic and non-enzymatic antioxidant mechanisms. These systems have the capacity to scavenge and detoxify AOS by their transformation into non-reactive molecules. The ascorbate–glutathione (A-G) cycle is a key H2O2-detoxifying system, located in the cytosol, peroxisome, mitochondria and chloroplast of plant cells. In the A-G cycle, superoxide radicals are eliminated by superoxide dismutase (SOD) in a reaction that yields H2O2. H2O2 is converted to oxygen and water by catalase (CAT) or to water alone through the

oxidation of ascorbate (ASC) by ascorbate peroxidase (APX). ASC can be regenerated by two mechanisms: the enzymatic reduction of monodehydroascorbate (MDHA) or the reaction of dehydroascorbate (DHA) with glutathione (GHS) to produce ASC and oxidized glutathione (GSSG) in a reaction catalysed by dehydroascorbate reductase (DHAR). Levels of AOS can also be reduced by the non-enzymatic antioxidants vitamin E and carotenoids (Bray et al., 2000).

Freezing Tolerance and Antioxidant Species Previous research has shown a close relationship between AOS scavenging and frost tolerance. For instance, Kendall and McKersie (1989) reported that acclimation to cold stress enhanced the concentration of antioxidant molecules in winter wheat and that freeze–thaw injury at the membrane level is exacerbated by a treatment with superoxide radicals. Zhou and Zhao (2004) showed that the level of freezing tolerance of perennial roots of alpine grasses was correlated with the activities of key antioxidant enzymes and concluded that antioxidative metabolism plays an important role in limiting the production of free radicals to protect membrane integrity. In lucerne, McKersie et al. (1999) reported that transgenic plants expressing Mn-SOD had increased vigour after freezing stress and increased winter survival under field conditions. Zhang et al. (2006) observed higher SOD and APX activities in acclimated plants of a cold-tolerant cultivar, as compared with those of a cold-sensitive cultivar of bermuda grass. Recent results obtained during freezing acclimation of Populus cuttings showed a close correlation between freezing tolerance of cuttings and levels of the enzymes of the A-G cycle including APX, DHAR, monodehydroascorbate reductase (MDHAR) and glutathione reductase (GHSR) (Lei et al., 2007). Cumulatively, these evidences from the literature allow us to conclude that the antioxidant metabolism in general, and more specifically the A-G cycle, plays an important role in the enhancement of freezing tolerance during cold acclimation.

Ascorbate Antioxidant Pathway

LT50 (°C)

−36 −32 −28 −24 −20 −16

ATF0

ATF5

ATF6

Fig. 26.1. Lethal freezing temperature for 50% of plants (LT50) of the original lucerne cultivar Apica (ATF0) and of populations obtained after five and six cycles of recurrent selection for improved freezing tolerance in the same genetic background (ATF5 and ATF6, respectively). The evaluation of the LT50 was done in January 2004 on lucerne plants acclimated under natural conditions in an unheated greenhouse. Error bars represent the lower confidence limits (5% probability level) of LT50 estimated by the PROBIT procedure of SAS software.

(a) mg/g dry weight

Several cycles of recurrent selection performed within the cultivar Apica (Michaud et al., 1983) have resulted in a progressive increase in the level of freezing tolerance from −27 to −31°C between the initial cultivar ATF0 and the populations obtained after five (ATF5) or six (ATF6) selection cycles (Fig. 26.1). As part of our ongoing effort to unravel the molecular and genetic bases of the improvement in freezing tolerance in TF populations, we monitored the level of key metabolites of the A-G cycle in plants of the original cultivar ATF0 and in the ATF7 populations obtained after seven cycles of recurrent selection. Our results indicated that the concentration of ASC in perennial crowns was significantly lower in the more freezing-tolerant ATF7 than in those of the initial background (ATF0) when plants were non-acclimated in October (Fig. 26.2a). ASC concentration decreased rapidly in both populations during the first stage of cold acclimation in November and its level was similar in both until the end of January. Although ASC concentration increased in ATF0 in February, it was significantly lower in ATF7. The seasonal pattern of DHA concentration, from which ASC is regenerated, was similar to that

of ASC, although no significant differences occurred between the two populations (Fig. 26.2b). Our observation of a marked decline in ASC concentration during the cold acclimation process is in agreement with reports of lower activity of antioxidant enzymes in perennial roots of alpine grasses overwintering under natural conditions by Zhou and Zhao (2004). They observed a decrease in the activity of antioxidant enzymes during the second stage of cold acclimation, from November to April, when the temperature was constantly below 0°C, and concluded that antioxidant metabolism was sufficient to protect overwintering roots against oxidative damage. In non-photosynthetic tissues, respiration is the primary source of AOS. In our experiment, plants were frozen from

5 4 3 2 1 0

Oct

Nov Dec Jan Month

Feb

Mar

Oct

Nov Dec Jan Month

Feb

Mar

(b) mg/g dry weight

Ascorbate–Glutathione Cycle in Lucerne Populations with Contrasting Levels of Freezing Tolerance

273

1.0 0.8 0.6 0.4 0.2 0.0

Fig. 26.2. Concentrations of ascorbate (a) and dehydroascorbate (b) in overwintering taproots of the original lucerne cultivar Apica (ATF0; —■—) and of a population obtained after seven cycles of recurrent selection for improved freezing tolerance in the same genetic background (ATF7; - -•- -). On October 20, plants were transferred to an unheated greenhouse for overwintering under natural temperature variations (Castonguay et al., 1995). From December until the end of February, the soil remained frozen with an average temperature of −1.1°C. Ascorbate and dehydroascorbate were extracted and analysed by HPLC according to Graham and Annette (1992). Values are means of five samples, with their standard errors represented by vertical bars.

A. Bertrand et al.

Photostimulated light (PSL ⫻ 105)

(a) 20 15 10 5 0

(b) Photostimulated light (PSL ⫻ 105)

December to March (average soil temperature of −1.1°C) and it could be hypothesised that low levels of stress-induced AOS were generated under reduced metabolic activity, including low respiration rate, at sub-freezing temperature. Since the major source of AOS under freezing stress is the uncoupling of metabolic processes, it is reasonable to speculate that less uncoupling occurs in the freezingtolerant population ATF7 than in the coldsensitive ATF0. Lower levels of ASC in freezing-tolerant ATF7 could be indicative of a lower rate of production of AOS or that a more efficient scavenging system is present in ATF7 than in ATF0. To address this question, we measured transcript levels of three genes encoding key enzymes of the A-G cycle: APX, MDHAR and DHAR (Fig. 26.3a, b and c). APX is a key H2O2-detoxifying enzyme (Murgia et al., 2004) which uses ASC as an electron donor to reduce H2O2 to water with the concomitant generation of MDHA. MDHA is recycled back to ASC through the action of DHAR and MDHAR. It is noteworthy that transcripts of genes encoding these enzymes accumulated to significantly lower levels in the more freezingtolerant ATF7 than in the less-freezing tolerant ATF0. This observation is consistent with our observation of lower levels of ascorbate in ATF7 than in ATF0 and indirectly suggests a lower level of production of H2O2 in ATF7; or, alternatively, that the signal for the induction of stress response may be lower in the tolerant population (Torres and Dangl, 2005). Lower level of expression of genes of the A-G cycle is not a part of a more general response of the ATF7 genome to low-temperature acclimation. This effect appears to be specific to the AOS metabolism since the level of expression of the cold-regulated (COR) gene msaCIA, typically induced in cold-acclimated lucerne, was significantly higher in freezing-tolerant ATF7 than in ATF0 throughout the overwintering period (Fig. 26.4). Similar inductions of other COR genes were observed (data not shown). This could be an indication that, in overwintering lucerne, the induction of the A-G cycle is a consequence of freezing damage to the perennial organs while the activation of COR genes is associated with both the

30 25 20 15 10 5 0

(c) Photostimulated light (PSL ⫻ 105)

274

20 15 10 5 0

Oct

Nov

Dec Jan Month

Feb

Mar

Fig. 26.3. Expression levels of genes encoding key enzymes of the ascorbate–glutathione pathway: ascorbate peroxidase (a), monodehydroascorbate reductase (b) and dehydroascorbate reductase (c), in overwintering taproots of the original lucerne cultivar Apica (ATF0; —■—) and of a population obtained after seven cycles of recurrent selection for improved freezing tolerance in the same genetic background (ATF7; - -•- -). Expression levels were assessed by dot blot analyses. Relative gene expression for each of the enzymes was assessed using a Phosphoimager (Typhoon 9400; Amersham Biosciences). Values are means of five samples, with their standard errors represented by vertical bars.

acquisition of and the level of freezing tolerance achieved by the plants.

Bulk Segregant Analysis of Key Antioxidant Genes in Lucerne of Contrasting Freezing Tolerance Populations of lucerne selected for improved tolerance to freezing (TF populations) are

Ascorbate Antioxidant Pathway

Photostimulated light (PSL ⫻ 106)

25 20 15 10 5 0

Oct

Nov

Dec Jan Month

Feb

Mar

Fig. 26.4. Expression levels of a lucerne coldregulated (COR) gene homologous to glycine-rich protein (msaCIA) in overwintering taproots of the original lucerne cultivar Apica (ATF0; —■—) and of a population obtained after seven cycles of recurrent selection for improved freezing tolerance in the same genetic background (ATF7; - -•- -). Values are means of five samples, with their standard errors represented by vertical bars.

particularly useful to identify genes conferring superior freezing tolerance (Castonguay et al., 2006). Using the bulk segregant approach (BSA) described by Michelmore et al. (1991), candidate genes potentially associated with phenotypic variation can be tested for differences in allele frequency between populations derived from a given genetic background and selectively improved for that trait of interest. In the present study, we pooled DNA from about 40 cold-acclimated genotypes of the cultivar Apica and from its derived ATF populations and hybridized the digested DNA with candidate genes coding for enzymes of the antioxidant metabolism and associated with cold tolerance. Homologues of genes coding for enzymes that have been related to tolerance to low temperature, including Cu/Zn-SOD (McKersie et al., 1999), CAT (Karpinski et al., 2002), MDHAR and DHAR (Baek and Skinner, 2003), were selected from an expressed sequence tag (EST) bank (Dr S. Laberge, Agriculture and Agri-Food Canada). Comparison of restriction fragment length polymorphism patterns resulting from hybridization with EST homologues to the four candidate genes involved in antioxidant metabolism revealed an intensification and/or disappearance of specific DNA fragments in response to selection (Fig. 26.5). For Cu/Zn-SOD, we observed the intensification of one band (A)

275

while three others declined (B, C and D) in response to the cycles of selection. For CAT, MDHAR and DHAR, we observed the intensification of at least one band in populations obtained after five or six cycles of selection for improved freezing tolerance. The likelihood that these polymorphisms are genetically linked to a quantitative trait locus controlling freezing tolerance is high, considering that selection was performed within a single genetic background and was solely targeted towards the improvement of this trait. Based on this information, it seems likely that recurrent selection for freezing tolerance has changed the frequency of alleles encoding AOS-detoxifying enzymes that could contribute to more effective AOS scavenging in TF populations exposed to low temperature.

Conclusion Our results show that the lucerne populations selected for improved freezing tolerance have lower levels of metabolites as well as lower levels of expression of genes coding for enzymes of the A-G cycle in perennial overwintering taproots. Contrary to what is generally reported in the literature, the improvement of freezing tolerance in lucerne appears to be reversely correlated with the activity of the antioxidant metabolism. Since the major source of AOS under freezing stress is the uncoupling of metabolic processes, we could speculate that less uncoupling occurs in a more freezing-tolerant population (ATF7) than in the original cultivar (ATF0) and that a lower level of antioxidant is needed to control AOS. Another explanation is that, in overwintering lucerne, the induction of the A-G cycle is a consequence of freezing damages to the perennial organs and is not associated with the acquisition or the level of freezing tolerance achieved by the plants. On the other hand, the BSA approach showed that recurrent selection for freezing tolerance has changed the frequency of alleles encoding AOS-detoxifying enzymes. These selected allelic forms might be more effective in AOS scavenging in response to low-temperature stress.

276

ATF0 ATF2 ATF4 ATF5 ATF6

ATF0 ATF2 ATF4 ATF5 ATF6

ATF0 ATF2 ATF4 ATF5 ATF6

ATF0 ATF2 ATF4 ATF5 ATF6 A

A A

B

A. Bertrand et al.

C A

D

B

(a)

(b)

(c)

(d)

Fig. 26.5. Southern blot hybridization of pooled genomic DNA extracts from about 40 genotypes each of the cultivar Apica (ATF0) and the populations (ATF2, 4, 5 and 6) derived from this cultivar through recurrent cycles of selection for superior freezing tolerance. Total genomic DNA was extracted using the CTAB procedure (Rogers and Bendich, 1988) and 10 mg were digested with DraI, separated by electrophoresis on 0.8% agarose gels, and transferred on to nylon membranes. Homologues of genes involved in antioxidant metabolism: Cu/Zn-superoxide dismutase (a), catalase (b), monodehydroascorbate reductase (c) and dehydroascorbate reductase (d), isolated from cold-acclimated lucerne, were used for hybridization. Relative gene expression for each of the enzymes was assessed using a Phosphoimager (Typhoon 9400; Amersham Biosciences).

Ascorbate Antioxidant Pathway

277

References Arora, R. and Palta, J.P. (1991) A loss in plasma membrane ATPase activity and its recovery coincides with incipient freeze–thaw injury and post-thaw recovery in onion bulb scale tissue. Plant Physiology 95, 845–852. Baek, K.-H. and Skinner, D.Z. (2003) Alteration of antioxidant enzyme gene expression during cold acclimation of near-isogenic wheat lines. Plant Science 165, 1221–1227. Bertrand, A., Prévost, D., Bigras, F.J. and Castonguay, Y. (2007) Elevated atmospheric CO2 and strain of rhizobia alter cold tolerance and expression of COR genes in alfalfa (Medicago sativa L.). Annals of Botany 99, 275–284. Bray, E.A., Bailey-Serre, J. and Weretilnyk, E. (2000) Responses to abiotic stresses. In: Buchanan, B.B., Gruissem, W. and Jones, R.L. (eds) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, Maryland, pp. 1158–1203. Castonguay, Y., Nadeau, P., Lechasseur, P. and Chouinard, L. (1995) Differential accumulation of carbohydrates in alfalfa cultivars of contrasting winterhardiness. Crop Science 35, 509–516. Castonguay, Y., Nadeau, P., Laberge, S. and Vézina, L.-P. (1997) Temperature and drought stress. In: McKersie, B.D. and Brown, D.C.W. (eds) Biotechnology and the Improvement of Forage Legumes. CAB International, Wallingford, UK, pp. 175–202. Castonguay, Y., Cloutier, J., Laberge, S., Bertrand, A. and Michaud, R. (2006) A bulk segregant approach to identify genetic polymorphisms associated with cold tolerance in alfalfa. In: Chen, T.H.H., Uemura, M. and Fujikawa, S. (eds) Cold Hardiness in Plants: Molecular Genetics, Cell Biology and Physiology. CAB International, Wallingford, UK, pp. 88–102. Graham, W.D. and Annette, A. (1992) Determination of ascorbic and dehydroascorbic acid in potatoes (Solanum tuberosum) and strawberries using ion-exclusion chromatography. Journal of Chromatography 594, 187–194. Karpinski, S., Wingsle, G., Karpinska, B. and Hallgren, J. (2002) Low-temperature stress and antioxidant defense mechanisms in higher plants. In: Inze, D. and Van Montagu, M. (eds) Oxidative Stress in Plants. Taylor & Francis, London, pp. 69–104. Kendall, E.J. and McKersie, B.B. (1989) Free radical and freezing injury to cell membranes of winter wheat. Physiologia Plantarum 76, 86–94. Lei, L., Lin, S., Zheng, H., Lei, Y., Zhang, Q. and Zhang, Z. (2007) The role of antioxidant system in freezing acclimation-induced freezing resistance of Populus suaveolens cuttings. Forestry Studies in China 9, 107–113. McKersie, B.D., Bowley, S.R. and Jones, K.S. (1999) Winter survival of alfalfa overexpressing superoxide dismutase. Plant Physiology 119, 839–847. Michaud, R., Richard, C., Willemot, C. and Gasser, H. (1983) Apica alfalfa. Canadian Journal of Plant Science 63, 547–549. Michelmore, R.W., Paran, I. and Kesseli, R.V. (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences USA 88, 9828–9832. Mittler, R., Vanderauwera, S., Gollery, M. and Van Breusegem, F. (2004) Reactive oxygen gene network of plants. Trends in Plant Science 9, 490–498. Murgia, I., Tarantino, D., Vannini, C., Bracale, M., Carravieri, S. and Soave, C. (2004) Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increase resistance to paraquat-induced photo-oxidative stress and to nitric oxide-induced cell death. The Plant Journal 38, 940–953. Paquin, R. and Pelletier, G. (1981) Acclimatation naturelle de la luzerne (Medicago media Pers.) au froid. I. Variations de la teneur en proline libre de feuilles et des collets. Physiologie Végétale 19, 103–117. Pei, Z.-M., Murata, Y., Benning, G., Thomine, S., Klüsener, B., Allen, G.J., Grill, E. and Schroeder, J.I. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406, 731–734. Rogers, S.O. and Bendich, A.J. (1988) Extractions of DNA from plant tissues. In: Gelvin, S.B. and Schilperoort, S.A. (eds) Plant Molecular Biology Manual. Kluwer Academic Publishers, Boston, Massachusetts, pp. 1–8. Torres, M.A. and Dangl, J.L. (2005) Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Current Opinion in Plant Biology 8, 397–403. Vance, C.P. (2001) Symbiotic nitrogen fixation and phosphorus acquisition. Plant nutrition in a world of declining renewable resources. Plant Physiology 127, 390–397.

278

A. Bertrand et al.

Volenec, J.J., Cunningham, S.M., Haagenson, D.M., Berg, W.K., Joern, B.C. and Wierama, D.M. (2002) Physiological genetics of alfalfa improvement: past failures, future prospects. Field Crops Research 75, 97–110. Wanner, L.A. and Junttila, O. (1999) Cold-induced freezing tolerance in Arabidopsis. Plant Physiology 120, 391–400. Zhang, X., Ervin, E.H. and LaBranche, A.J. (2006) Metabolic defense responses of seeded bermudagrass during acclimation to freezing stress. Crop Science 46, 2598–2605. Zhou, R. and Zhao, H. (2004) Seasonal pattern of antioxidant enzyme system in the roots of perennial forage grasses grown in alpine habitat related to freezing tolerance. Physiologia Plantarum 121, 399–408.

27

Identification of Proteins from Potato Leaves Submitted to Chilling Temperature J. Renaut, S. Planchon, M. Oufir, J.-F. Hausman, L. Hoffmann and D. Evers

Introduction Abiotic stresses limit agricultural production in many areas of the world. One of the success keys to reach sustainability in agriculture would be to access crops adapted to changing climatic conditions. Indeed, limited tolerance to abiotic stresses, like temperature extremes or drought for example, restricts the cropping alternatives available to farmers (Viner et al., 2006). Many authors describe the importance of environmental stresses including cold exposure and their impact on growth, productivity and plant development (Viner et al., 2006; Beck et al., 2007). Two types of cold stress are known: (i) chilling stress at temperatures above zero; and (ii) freezing stress at subzero temperatures. Chilling stress induces some metabolic changes at the plant level: it reflects the plant’s ability to tolerate temperatures above zero without damage. On the other hand, at subzero temperatures, the tissue water freezes outside the cell (Palva and Heino, 1998) and withdrawal of water from the cells leads to dehydration of the cytoplasm. This stress is thus characterized by a freeze-induced dehydration that can be compared to a droughtstress response (Uemura et al., 2006). Plants can evolve and adapt to freezing stress by increasing their tolerance through a process called cold acclimation. It can be achieved by

an exposure to low non-freezing temperatures over a period of time (Guy et al., 1985). At the biochemical level, some metabolites are known to accumulate in cells upon cold exposure (Kaplan et al., 2004). They include soluble sugars, amino acids, organic acids, polyamines and lipids (Guy et al., 2008). These metabolites can function as osmolytes by reducing cellular dehydration, as compatible solutes by stabilizing membranes, as chelating agents by neutralizing toxic molecules and as energy sources (Guy et al., 2008). On the molecular level, changes in the transcriptome and the proteome include differential expression of dehydrins and antifreeze proteins, chaperones and detoxification enzymes on one hand and, on the other, changes in biosynthetic enzymes producing the previously described lowmolecular-weight compounds (Fowler and Thomashow, 2002; Bassett et al., 2005; Taylor et al., 2005; Amme et al., 2006; Renaut et al., 2006). Moreover, regulatory proteins such as transcription factors, protein kinases and phospholipases are known to be differentially regulated upon cold stress (Chapman, 1998; Knight and Knight, 2001). Part of this knowledge results from model studies on Arabidopsis thaliana and tobacco, but also from cold response studies in poplar (Renaut et al., 2005). However, few results are available on the response of important crop species to cold stress.

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

279

280

J. Renaut et al.

Potato is an important crop worldwide. It is grown mainly in temperate areas and in the Andean regions from where it originates. The commonly grown potato, Solanum tuberosum L., possesses little tolerance to cold stress while other South American species are more tolerant to this factor (Gopal and Khurana, 2006). However, many of these species are not very productive, and it would be interesting to combine the productivity traits with the cold tolerance traits to breed for plants that can produce well under harsher climates. To achieve this, better knowledge of the physiological, biochemical and molecular processes underlying the cold-stress response and cold acclimation is of primary importance. Here, we present results on the cold response in two S. tuberosum genotypes, called ‘PS3’ and ‘Désirée’.

Materials and Methods Plant materials and stress treatments Two potato genotypes were used for cold treatment: (i) a dihaploid S. tuberosum, hereafter called ‘PS3’; and (ii) a tetraploid S. tuberosum, cv. ‘Désirée’. In vitro-cultured plants were acclimated and grown ex vitro for 2 months in a mixture of soil and sand (3:1) before coldstress treatment. Cold treatment of acclimated potato plants was performed in growth chambers at 7°C/2°C (day/night) and control conditions at 21°C/18°C (day/night) under a light intensity of 150 µmol/ m2/s (Sylvania Grolux fluorescent lamps; Osram GmbH, Munich, Germany). Their morphological and biochemical reactions to cold are partly described in Evers et al. (2007). Cold-stressed and control plant leaves were sampled after 0, 1, 3 and 8 days. Samples harvested on days 3 and 8 were used for proteomic analysis. Potato leaves (first and second) were collected and stored at −80°C until analysis.

after treatment periods of 0, 7, 14 and 21 days at 4°C by measuring changes in electrical conductivity of the leaves and by estimating the LT50 corresponding to the temperature inducing 50% of injured cells. Eight leaves were used for each freezing tolerance assay (0, −3, −6, −9, −12, −15 and −18°C). Samples were cooled at a rate of 2°C/h in a temperature-controlled bath (model F12-MP; Julabo Labortechnik, Seelbach, Germany) initially maintained at 4°C. One drop of deionized water was added in each tube to promote ice nucleation. Tubes were removed and allowed to reach room temperature before the addition of 40 ml of double-distilled water. Tubes were shaken for 2 h at room temperature and conductivity was measured with a Cyberscan CON400 (Eutech Instrument, Nijkerk, The Netherlands). After autoclaving (1.21 bar, 120°C, 20 min) and cooling, conductivity was measured again. The percentage of ion leakage and injury was determined following Arora et al. (1996). Percentage of electrolyte leakage was estimated as a relative conductivity, i.e. the ratio between conductivity after freezing and total conductivity after killing of the tissue by autoclaving, when remaining electrolytes are released. In order to take into account the electrolyte leakage of the control leaves, which was slightly different from zero, the following expression was used to determine the percentage of injury: Percentage injury = [percentage L(t) − percentage L(c) / 100 − percentage L(c)] × 100, where percentage L(t) and percentage L(c) are the measurements of percentage electrolyte leakage for the respective freezing treatment temperatures and the unfrozen control, respectively (Arora et al., 1996). LT50 was defined as the temperature at which 50% of freezing injury was observed. A response curve for freezing injury versus temperature was modelled for each treatment. A non-linear regression was applied to the set of points. The validation of the results was performed with ANOVA followed by a t test (GraphPad Instat version 3.05; GraphPad Software, Inc., La Jolla, CA, USA).

Measurements of cold acclimation Carbohydrate measurement Freezing tolerance (FT) was determined as described by Renaut et al. (2004), according to Lim et al. (1999). Briefly, FT was determined

Carbohydrates and polyols were measured on approximately 100 mg of fresh leaf material

Identification of Proteins from Potato Leaves

according to Evers et al. (2007) with some modifications.

Two-step real-time RT-PCR Real-time RT-PCR was performed as previously published (Nicot et al., 2005). Primer sequences are presented in Table 27.1. Elongation factor 1-α (ef1-α) was used as a reference gene and the comparative Ct method was used to calculate relative gene expression. This method uses the arithmetic formula 2−∆∆Ct to calculate relative quantification using a reference and a calibrator gene.

Protein extraction, labelling and separation by electrophoresis Protein extraction and separation were carried out as described previously (Renaut et al., 2008). In short, approximately 300 mg of fresh leaf material was crushed in liquid nitrogen and proteins were extracted by acetone precipitation. Then, proteins were resolubilized and quantified prior to their labelling with CyDyes™ (GE Healthcare, Little Chalfont, UK). After labelling, proteins were separated on Immobiline DryStrips (pH 4–7, 24 cm; GE Healthcare) by isoelectrofocusing in an IPGphor IEF unit (GE Healthcare). The second dimension (SDS-PAGE) was performed on 12.5% w/v acrylamide containing 0.1% N,N'methylenebisacrylamide at 15°C in Ettan Dalt II tank (GE Healthcare). After capture of the images on a Typhoon laser scanner, spots were detected on the gels and matched between gels. A statistical analysis of spot abundances was performed using the Decyder

281

software version 6.5.14 (GE Healthcare). Differentially expressed spots were excised from the gels, digested with trypsin and spotted on MALDI targets as described previously (Renaut et al., 2008). Spectra were captured on an Applied Biosystems 4800 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) and used for protein identification. Proteins were identified by searching against a potato expressed sequence tags (EST) database downloaded from the National Center for Biotechnology Information (NCBI) database using an in-house MASCOT server (Matrix Science, London; www.matrixscience.com). All searches were carried out using a mass window of 150 ppm for the precursor and 0.75 Da for the fragments. The search parameters allowed for carboxyamidomethylation of cysteine, oxidation of methionine as well as tryptophan to kynurenine, and double oxidation of tryptophan to N-formylkynurenine. Identifications were validated manually with at least two identified peptides with a score above homology.

Results Measurements of cold acclimation The ability of potato clones to cold-acclimate was evaluated during 3 weeks of exposure at 4°C (Fig. 27.1). The cold acclimation ability was determined by an estimation of the FT of potato plants exposed to low temperatures. This FT was assessed by measuring the lethal temperature LT50 on eight detached potato leaves for each temperature assay (0, −3, −6, −9, −12, −15 and −18°C). Measurements of the LT50 indicate that ‘Désirée’ was not able to

Table 27.1. Forward and reverse primer sequences of the genes tested in real-time RT-PCR. Gene identity

Forward primer sequence 5′–3′

Reverse primer sequence 5′–3′

Sucrose synthase Glucose-6Pdehydrogenase Pyruvate decarboxylase Galactinol synthase Raffinose synthase

ATGGAACAAGTTGAGCCTGAAAAT TCACTATTTTTGGCTATGCCCG

TTCCGCAAGCTGGTAAGTCTCT CCACAATTTTCCCTCTTATCAATCC

GGGTTCCTTCTATCACCACCATAA CTCCGTATTTGGGAGTTTGTGGA AACTTCTGCCGGTGAGCAAA

TTCAGTGCCATGGAAGTTGAGAC CCATCTGGCAAGTCAAACAAGTG GGTCACCCTGTCCCAAACTTC

282

J. Renaut et al.

−6 −7 a LT50 (°C)

a

a

−8 a −9

b

−10 c −11 −12

0

2

4

6 8 Days at 4°C

10

12

14

16

Fig. 27.1. Variation of the lethal temperature for detached leaves harvested from potato, cultivars ‘PS3’ (•) and ‘Désirée’ (°), after exposure at 4°C for 0, 7, 14 and 21 days. a,b,cValues with unlike lower-case letters were significantly different, P<0.001.

acclimate as no significant difference could be observed for ‘Désirée’ between the LT50 values measured each week. In contrast to ‘Désirée’, the values of LT50 determined for ‘PS3’ decreased during the first two weeks. Therefore, the behaviour of ‘PS3’ suggests that it is able to cold-acclimate. Based on these observations, the different results obtained on ‘Désirée’ and ‘PS3’ are discussed further below as a response to cold and an acclimation process, respectively.

Two-dimensional gel analysis Three replicates of each condition (i.e. control and cold-exposed plants of the two cultivars) were used for statistical analysis. A first analysis of gels resulted in the selection of 57 protein spots from ‘Désirée’ with variation in their abundance, and 150 spots in ‘PS3’. More precisely, 31 and 26 spots in ‘Désirée’ and 92 and 58 spots in ‘PS3’ were differentially expressed (respectively up- and down-regulated). In total, given that some spots were common to the two accessions, 177 spots were picked. Analysis of the gels by principal components revealed: (i) a limited variability between gels of the same condition; and (ii) more simi-

larities between the six gels from ‘Désirée’ samples than between the controls of both clones (Fig. 27.2, right panel). From this loading plot it can be seen that more differences between control and cold-exposed leaves are expected in the accession ‘PS3’ than in ‘Désirée’. On this loading plot, the first axis (PC1) corresponds to the separation between cultivars and explains more than 53% of the variability between the gels; while the second axis (PC2) is related to the separation between control and lowtemperature conditions and explains more than 38% of the variability. From the score plot (Fig. 27.2, left panel) showing the distribution of the 118 identified proteins, it can be observed that spots in cold-treated samples (open circles) are also clustered together. Among these proteins, 118 could be identified by MS and can be seen in Fig. 27.3. Considering results from the cultivar ‘Désirée’, the abundance of enzymes or proteins involved in mechanisms such as photosynthesis (e.g. OEE protein 1 and 2, chlorophyll a/b-binding proteins), carbohydrate metabolism (e.g. malic enzymes, phosphoglycerate mutase) and protease inhibitors mainly decreased upon lowtemperature exposure. On the other hand, proteins involved in defence mechanisms (e.g. chaperones, superoxide dismutase, catalase),

0.60 0.55 0.50 0.45 0.40 0.35

PC2

0.30

Cold-treated samples

0.25

Identification of Proteins from Potato Leaves

PC2

3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 −0.2 −0.4 −0.6 −0.8 −1.0 −1.2 −1.4 −1.6 −1.8 −2.0 −2.2

PS3

0.20 0.15 0.10 0.05 0 −0.05 Control samples

−0.10 −0.15 Désirée −3

−2

−1

0

1 PC1

2

3

4

5

−0.1

0

0.1

0.3 0.2 PC1

0.4

0.5

0.6

Fig. 27.2. Principal components analysis based on the abundances of 118 identified proteins differentially expressed (score plot, left panel) and on the variability between gels (loading plot, right panel) of potato cultivars ‘Désirée’ and ‘PS3’. °, Protein spots (left panel) and gels (right panel) corresponding to cold-treated samples; •, protein spots (left panel) and gels (right panel) corresponding to control samples.

283

284

J. Renaut et al.

Fig. 27.3. Example of an image provided by a two-dimensional difference gel electrophoresis (2D-DiGE) experiment: 2D-DiGE gel (labelled with Cy5) of a ‘Désirée’ sample kept under control conditions for 7 days. Identified proteins are presented with their number. Different symbols surrounding the protein spots correspond to the behaviour of the proteins: closed circles, proteins whose abundance increases exclusively in ‘PS3’; closed squares, proteins whose abundance increases in ‘Désirée’ and ‘PS3’; closed triangles, proteins whose abundance increases exclusively in ‘Désirée’; closed stars, proteins whose abundance decreases in ‘PS3’ and increases in ‘Désirée’; dotted circles, proteins whose abundance decreases exclusively in ‘PS3’; dotted squares, proteins whose abundance decreases in ‘Désirée’ and ‘PS3’; dotted triangles, proteins whose abundance decreases exclusively in ‘Désirée’; dotted star, proteins whose abundance increases in ‘PS3’ and decreases in ‘Désirée’.

energy/carbohydrate metabolism (e.g. carbonic anhydrase, aldolase, ATP synthases, inositol-3-phosphate synthase) and methionine synthesis (methionine synthase) were more abundant in the leaves of cold-treated plants. In ‘PS3’ subjected to cold treatment at 4°C, 59 proteins with a higher abundance and 28 less abundant proteins were identified. Among the first category were an important number of (isoforms of) enzymes involved in carbohydrate metabolism, mainly glycolysis and the pentose phosphate pathway (e.g. glyceral-

dehyde-3-phosphate dehydrogenases, phosphoglucomutases, transketolases, aldolases), but also defence proteins (e.g. chaperones) and storage proteins (e.g. germins, patatin). Five isoforms of methionine synthase and some proteins from the photosynthesis pathway were also more abundant in cold-treated leaves. Among the proteins down-regulated upon cold exposure, a majority of those identified were linked with photosynthesis. All of these results are summarized in Table 27.2. An interesting observation is that,

Table 27.2. Summary results of the analysis of two-dimensional gels. Spot, the number of the spot on the master gel; Ratio, the abundance ratio of the proteins in the cold-treated sample to that in the control sample in D (‘Désirée’) and P (‘PS3’), respectively; t Test, P value of the t test of the corresponding ratio; Name, name of the protein as given in the NCBInr database (http://www.ncbi.nlm.nih.gov/); GI accession, accession number in the NCBInr database; Species, species in which the protein has been described. Spot

Ratio D

t Test

Carbohydrate metabolism 1.04 884 9.10×10−1 1.59 918 6.70×10−4

t Test

−1.27 −1.85 −3.10

4.70×10−1 4.80×10−2 4.70×10−5

1.76 −2.12 −2.54 −1.79 −1.17 −1.46 −1.04 1.06 −1.47 3.97 1.76 1.16 1.31 3.25 3.15 3.03 2.98 3.43 1.53 −2.54

1.50×10−5 5.70×10−4 6.70×10−4

5.02 1.92

GI accession

Species

7.20×10−6 3.20×10−4 7.10×10−2 6.20×10−3 7.20×10−4 5.20×10−3 3.90×10−2 1.10×10−3 6.00×10−3 6.10×10−2 6.70×10−4

ascorbate peroxidase superoxide dismutase [Fe] superoxide dismutase [Fe] catalase SOUL haem-binding family protein thioredoxin peroxidase thioredoxin peroxidase Hsp90-2-like 60 kDa chaperonin alpha subunit, chloroplast chaperonin-60 beta subunit chaperonin-60 beta subunit chaperonin-60 beta subunit chaperonin-60 beta subunit HSP70; chloroplast HSP70 chaperonin 21 precursor chaperonin 21 precursor chaperonin 21 precursor heat shock cognate 70 kDa protein; putative chaperonin-60 beta subunit CPN60A; ATP binding/protein binding chaperonin-60 beta subunit CPN60A; ATP binding/protein binding CPN-60 beta thioredoxin peroxidase

GI:21039134 GI:33413303 GI:33413303 GI:40950550 GI:15220033 GI:21912927 GI:3328221 GI:81074298 GI:15226314 GI:1762130 GI:1762130 GI:1762130 GI:1762130 GI:124245039 GI:7331143 GI:7331143 GI:7331143 GI:108864707 GI:1762130 GI:15226314 GI:1762130 GI:15226314 GI:110349923 GI:3328221

Solanum lycopersicum S. lycopersicum S. lycopersicum Solanum tuberosum Arabidopsis thaliana Nicotiana tabacum Secale cereale S. tuberosum A. thaliana S. tuberosum S. tuberosum S. tuberosum S. tuberosum Cucumis sativus S. lycopersicum S. lycopersicum S. lycopersicum Oryza sativum S. tuberosum A. thaliana S. tuberosum A. thaliana Solanum commersonii S. cereale

9.20×10−5 1.20×10−1

plastidic aldolase NPALDP1 plastidic aldolase

GI:4827251 GI:4827251

Nicotiana paniculata N. paniculata Continued

2.80×10−1 7.60×10−2 8.80×10−1

285

Name

Identification of Proteins from Potato Leaves

Antioxidant and defence system 1075 −1.50 5.90×10−4 1350 1.52 6.60×10−5 1351 1.52 3.50×10−5 1.86 326 2.60×10−4 1238 −1.16 1.80×10−2 1310 1.11 5.00×10−1 1352 −1.42 1.70×10−2 1.18 113 5.20×10−1 2.27 295 5.60×10−4 2.17 331 3.30×10−3 1.77 343 8.60×10−3 2.15 344 4.00×10−4 1.66 351 1.10×10−2 1.30 173 5.00×10−3 1246 2.13 5.20×10−3 1267 2.14 2.30×10−4 1272 1.70 1.00×10−4 1.10 208 8.60×10−1 2.27 341 1.30×10−4 2.83 299 1.50×10−5 2.29 342 1.00×10−5 2.61 301 4.70×10−6 2.13 338 4.70×10−5 1352 −1.42 1.70×10−2

Ratio P

286

Table 27.2. Continued Ratio D

t Test

Ratio P

t Test

894 896 273 298 700

−1.11 1.64 −1.58 −1.01 1.38

6.50×10−1 3.10×10−2 2.90×10−2 9.20×10−1 1.40×10−2

3.65 1.63 1.72 1.97 3.30

4.40×10−2 1.30×10−2 4.50×10−2 2.30×10−2 4.80×10−4

777 754 786 146 280 290 697

1.10 1.08 1.01 −1.71 −1.82 −2.14 1.16

6.90×10−1 1.90×10−2 9.50×10−1 4.90×10−2 1.30×10−4 1.60×10−3 2.50×10−2

6.94 5.23 5.36 1.88 2.02 1.70 −1.92

224 893 232 398

−1.12 1.55 −1.21 1.59

3.90×10−2 1.00×10−3 1.60×10−3 3.40×10−2

483 286 198 180 194 199 182

−1.11 −2.22 1.22 1.05 1.12 1.20 −1.79

2.00×10−1 1.40×10−2 7.20×10−3 3.40×10−1 3.90×10−1 8.50×10−2 1.00×10−1

Amino acid metabolism 1.14 390 1.56 391 1.42 92 1.54 112 −1.05 55

2.20×10−1 4.20×10−2 3.20×10−5 1.70×10−1 7.80×10−1

Name

GI accession

Species

GI:4827251 GI:4827251 GI:4582924 GI:4582924 GI:82400156

N. paniculata N. paniculata S. tuberosum S. tuberosum S. tuberosum

1.60×10−4 8.40×10−4 4.80×10−4 1.30×10−1 4.90×10−3 3.20×10−1 1.80×10−2

plastidic aldolase NPALDP1 plastidic aldolase phosphoglycerate mutase phosphoglycerate mutase phosphoglycerate kinase precursor-like protein glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase formate–tetrahydrofolate ligase cytosolic NADP-malic enzyme cytosolic NADP-malic enzyme hydroxypyruvate reductase

GI:22094840 GI:22094840 GI:22094840 GI:2507455 GI:2150029 GI:2150029 GI:118723307

1.76 3.64 3.03 1.04

1.70×10−4 1.10×10−3 1.70×10−3 8.60×10−1

phosphoglucomutase; cytoplasmi inositol-3-phosphate synthase phosphoglucomutase; cytoplasmic RuBisCO large subunit

GI:12585316 GI:14548096 GI:12585316 GI:126216288

2.48

1.80×10−3

5.17 4.19 4.17 12.03 1.68

5.70×10−6 8.10×10−5 1.10×10−3 7.20×10−6 6.50×10−4

RuBisCO large subunit succinate dehydrogenase 1-1 transketolase; chloroplast precursor (TK) transketolase 1 transketolase; chloroplast precursor (TK) transketolase; chloroplast precursor (TK) LEXYL1

GI:21634023 GI:15240075 GI:2501358 GI:3559814 GI:2501358 GI:2501358 GI:37359706

S. tuberosum S. tuberosum S. tuberosum Spinacia oleracea S. lycopersicum S. lycopersicum Solenostemon scutellarioides S. tuberosum N. tabacum S. tuberosum Neoalsomitra capricornica Calystegia sepium A. thaliana Rhizobium sp. NGR235 Capsicum annum Rhizobium sp. NGR234 Rhizobium sp. NGR236 S. lycopersicum

−1.78 1.57 1.58

4.30×10−2 1.70×10−1 8.00×10−2

8.65

8.60×10−5

threonine dehydratase biosynthetic threonine dehydratase biosynthetic methionine synthase methionine synthase methionine synthase

GI:401179 GI:401179 GI:8439545 GI:8439545 GI:8439545

S. tuberosum S. tuberosum S. tuberosum S, tuberosum S. tuberosum

J. Renaut et al.

Spot

1.26 1.33 1.45 1.53

2.80×10−2 9.00×10−3 2.40×10−3 4.70×10−3

8.06 3.38 2.70 1.46

3.50×10−5 1.10×10−3 8.60×10−3 2.20×10−2

470 680

−1.18 1.20

2.00×10−2 1.40×10−1

4.35 3.77

4.40×10−3 5.40×10−4

Energy and photosynthesis 1.62 360 4.20×10−1 1.00 522 9.50×10−1 1.56 851 1.80×10−3 1.69 839 1.10×10−4 1.60 387 4.60×10−4 577 −1.70 4.50×10−2 1020 −1.63 3.70×10−3

−2.53 −1.55 4.34 2.04 1.14 −1.53 −2.16

1.40×10−3 1.00×10−2 3.90×10−4 3.60×10−1 1.80×10−1 1.40×10−1 5.30×10−3

1.07 1.02 −1.62 −1.53 −1.28 −1.37 −1.75

3.80×10−1 8.50×10−1 1.00×10−2 1.40×10−2 4.50×10−3 1.70×10−1 4.30×10−3

−2.79 −4.27 −2.57 −3.08 −3.40 −3.31 −2.40

4.00×10−4 1.60×10−3 1.10×10−3 7.90×10−3 6.10×10−5 7.80×10−4 1.20×10−4

1175 982 1182 1456

−1.42 −1.66 −1.53 2.27

1.80×10−3 5.60×10−3 9.00×10−3 1.30×10−4

-5.22 −2.66 −1.54 −1.79

4.40×10−5 6.50×10−4 9.30×10−3 5.20×10−4

1346

−1.57

9.00×10−4

1.85

9.20×10−5

1034

−1.42

3.10×10−2

1.76

2.60×10−4

1394

−1.63

2.10×10−2

2.72

1.60×10−5

1298

−1.13

2.50×10−3

3.61

2.90×10−2

GI:8439545 GI:8439545 GI:8439545 GI:15238686

S. tuberosum S. tuberosum S. tuberosum A. thaliana

GI:15223186 GI:18418270

A. thaliana A. thaliana

ATP synthase CF1 alpha chain ATP synthase beta subunit ATP synthase gamma chain, chl precursor ATP synthase gamma chain, chl precursor ATP synthase CF1 alpha chain chlorophyll a-b binding protein 3C-like oxygen-evolving enhancer protein 1; chl precursor chlorophyll a; b binding protein type I chlorophyll a; b binding protein type ferredoxin–NADP reductase, chl precursor chlorophyll a/b-binding protein Cab-3C ferredoxin–NADP reductase; chl precursor chlorophyll a/b binding protein oxygen-evolving enhancer protein 2, chl precursor chlorophyll a/b binding protein ferredoxin–NADP reductase; chl precursor chlorophyll a/b-binding protein Cab-3C. photosystem I reaction centre subunit II; chl precursor oxygen-evolving enhancer protein 2, chl precursor oxygen-evolving enhancer protein 1; chl precursor photosystem I reaction centre subunit II; chl precursor chlorophyll a/b-binding protein type III precursor

GI:89280620 GI:6688527 GI:231610 GI:231610 GI:82754614 GI:81074613 GI:131385

S. lycopersicum S. lycopersicum N. tabacum N. tabacum S. tuberosum S. tuberosum S. tuberosum

GI:511153 GI:511153 GI:3913651 GI:170404 GI:3913651 GI:693914 GI:11134035

S. tuberosum S. tuberosum N. tabacum S. lycopersicum N. tabacum S. tuberosum S. tuberosum

GI:693914 GI:3913651 GI:170404 GI:131166

S. tuberosum N. tabacum S. lycopersicum S. lycopersicum

GI:11134035

S. tuberosum

GI:131385

S. tuberosum

GI:131166

S. lycopersicum

GI:82080

S. lycopersicum Continued

287

1262 1100 966 1171 967 1176 1334

methionine synthase methionine synthase methionine synthase cobalamin-independent methionine synthase isozyme alanine transaminase transaminase

Identification of Proteins from Potato Leaves

72 81 88 98

288

Table 27.2. Continued Spot

Ratio D

t Test

732 599

1.27 1.43

1.60×10−2 2.30×10−2

6.06 3.59

1.10×10−1 1.20×10−5

RuBisCO activase; chl precursor sulfate adenylyltransferase

GI:10720247 GI:531495

Solanum pennellii S. tuberosum

Storage proteins 1.08 901 1353 −1.42 1371 −1.45 1.63 678 1356 −1.08 1337 −1.14

5.00×10−1 4.90×10−2 4.40×10−3 2.90×10−5 3.00×10−1

−2.46 −2.73 −3.68 2.84 3.53 4.38

5.00×10−5 7.10×10−3 8.90×10−3 2.60×10−4 5.70×10−3 2.10×10−3

hypothetical protein OsI_000986 germin-like protein germin-like protein patatin precursor 24K germin like protein 24K germin like protein

GI:125525025 GI:123965222 GI:123965222 GI:73426677 GI:31711507 GI:31711507

O. sativa Capsicum chinense C. chinense S. tuberosum N. tabacum N. tabacum

Protein turnover 105 −1.50 1071 −1.95 1091 −2.06 1149 1.76 1109 −1.68 1095 −2.10 1160 1.85 1.58 89

6.50×10−3 5.80×10−5 3.70×10−4 4.20×10−2 4.70×10−5 9.00×10−3 1.30×10−3 3.00×10−2

−1.50 −1.14 −2.29 -2.34 3.61 4.65 1.48

6.30×10−1 8.50×10−3 2.70×10−3

GI:3687301 GI:2144585 GI:6538776 GI:6538776 GI:6538776 GI:6538776 GI:6538776 GI:399213

S. lycopersicum Synsepalum dulcificum Nicotiana glutinosa N. glutinosa N. glutinosa N. glutinosa N. glutinosa S. lycopersicum

1.52

3.60×10−2

2.18

1.10×10−1

subtilisin-like protease miraculin precursor putative proteinase inhibitor putative proteinase inhibitor putative proteinase inhibitor putative proteinase inhibitor putative proteinase inhibitor ATP-dependent Clp protease ATP-binding subunit clpA homolog CD4B, chl precursor ATP-dependent Clp protease ATP-binding subunit clpA homolog CD4B, chl precursor

GI:399213

S. lycopersicum

Miscellaneous 1141 1.56 1235 1.70 1240 1.76 1275 1.48 1087 −2.02 1090 −2.00 1.16 99 1.09 100 1.21 769

9.50×10−4 3.70×10−5 1.50×10−5 5.10×10−3 2.80×10−2 1.60×10−3 2.20×10−1 8.50×10−1 3.80×10−3

1.46 3.38 15.19 14.94 −1.43 1.44 2.32 5.64 3.24

8.30×10−1 1.30×10−4 1.40×10−5 4.00×10−3 3.20×10−1 1.40×10−1 9.00×10−4 6.30×10−5 6.40×10−4

carbonic anhydrase carbonic anhydrase carbonic anhydrase ferritin single-stranded DNA binding protein precursor single-stranded DNA binding protein precursor translation elongation factor EF-G; chl translation elongation factor EF-G; chl putative RNA binding protein

GI:56562177 GI:56562177 GI:56562177 GI:50787937 GI:17432522 GI:17432523 GI:2119927 GI:2119927 GI:3850621

S. lycopersicum S. lycopersicum S. lycopersicum Conyza canadensis S. tuberosum S. tuberosum Glycine max G. max A. thaliana

t Test

1.60×10−3 6.90×10−2

Name

GI accession

Species

J. Renaut et al.

90

Ratio P

Identification of Proteins from Potato Leaves

in ‘Désirée’, the most important variation in abundance does not exceed a 2.83-fold change, whereas in ‘PS3’, some alterations reach more than 15-fold changes.

289

thase were more expressed in ‘Désirée’ as compared with ‘PS3’, whereas for the other two (encoding glucose-6P-dehydrogenase and pyruvate decarboxylase), the relative abundance was higher in ‘PS3’ as compared with ‘Désirée’.

Carbohydrate analysis Some carbohydrates were measured by HPLC in leaves from control and cold-treated ‘PS3’ and ‘Désirée’ during the 14 days of treatment. Here we present only the results after 8 days (Table 27.3). We observed a significant (P<0.05) accumulation of glucose, sucrose, raffinose and galactinol in cold-treated leaves. Moreover, contents of these carbohydrates were higher in cold-treated ‘PS3’ leaves than in cold-treated ‘Désirée’ leaves. Real-time RT-PCR results After 8 days of cold exposure, genes encoding galactinol synthase, raffinose synthase, sucrose synthase, glucose-6P-dehydrogenase and pyruvate decarboxylase were more expressed in cold-treated leaves as compared with control leaves, for both ‘PS3’ and ‘Désirée’ (Table 27.4). When comparing the two cultivars, one can observe that the genes encoding galactinol synthase, raffinose synthase and sucrose syn-

Discussion In this experiment, we used two cultivars of potato to compare their response to a shortterm chilling exposure. The results of the freezing tolerance experiment at the leaf level suggest that the dihaploid ‘PS3’ is able to coldacclimate whereas the tetraploid genotype ‘Désirée’ does not show any increase in its freezing tolerance, even after 3 weeks of treatment. Although the method used here to estimate this freezing tolerance was quite far from natural conditions, it is a fast method, relying on a number of important measures and providing good information about the plant’s behaviour (Lim et al., 1999) although the temperatures used may be far from reality. The use of quantitative and large-scale techniques such as two-dimensional difference gel electrophoresis (2D-DiGE) provides an important amount of data to be integrated into a general picture, concomitantly with studies based on metabolites and gene expression.

Table 27.3. Carbohydrate contents (per gram fresh weight, FW) in leaves of control and cold-treated potato plants, cultivars ‘PS3’ and ‘Désirée’, after 8 days. Results are shown as means and standard deviations (SD) for six independent assays. Control Cultivar Galactose (nmol/g FW) Glucose (µmol/g FW) Sucrose (µmol/g FW) Raffinose (nmol/g FW) Galactinol (nmol/g FW)

‘PS3’ ‘Désirée’ ‘PS3’ ‘Désirée’ ‘PS3’ ‘Désirée’ ‘PS3’ ‘Désirée’ ‘PS3’ ‘Désirée’

Mean 11.70 6.77 1.08 0.79 1.18 4.05 34.45 17.27 132.60 52.25

Cold treatment SD

Mean

4.55 2.17 0.16 0.28 0.80 0.36 9.44 5.54 77.67 16.24

n.d.** n.d.** 14.24** 10.06** 13.21** 9.46** 466.32** 228.41** 620.58** 287.66**

n.d., not detected. Mean values were significantly different from those of controls: *P<0.05, **P<0.01.

SD

1.91 6.61 3.30 3.36 208.89 49.76 128.74 117.58

290

J. Renaut et al.

Table 27.4. Relative expression of some genes linked to carbohydrate metabolism measured by real-time RT-PCR and expressed as the 2−∆∆Ct value in leaves of control and cold-treated potato plants, cultivars ‘PS3’ and ‘Désirée’, after 8 days. Results are shown as means and standard deviations (SD) for three independent measurements. Control Cultivar Galactinol synthase (2

−∆∆Ct

)

‘PS3’ ‘Désirée’ Raffinose synthase (2−∆∆Ct) ‘PS3’ ‘Désirée’ Sucrose synthase (2−∆∆Ct) ‘PS3’ ‘Désirée’ Glucose-6P-dehydrogenase (2−∆∆Ct) ‘PS3’ ‘Désirée’ Pyruvate decarboxylase (2−∆∆Ct) ‘PS3’ ‘Désirée’

A first bidimensional analysis was carried out on ‘PS3’ and ‘Désirée’ samples harvested after 3 days of cold exposure (results not shown). In ‘PS3’, after 3 days of cold exposure, seven proteins were more abundant (two transketolases [134, 135], two chaperonin 60 subunit A [212, 216], carbonic anhydrase [1151], 24K germin-like protein [1258] and putative RNA binding protein [730]) whereas two proteins displayed a slight decrease in their abundance (unknown [693] and a PSI reaction centre subunit II [1313]). In ‘Désirée’, no changes were observed after 3 days. After 8 days of cold exposure, it is clear that an important number of proteins had been quantitatively and qualitatively modified. In ‘PS3’, a large portion of the proteins that were more abundant after cold exposure are linked with carbohydrate metabolism (e.g. in glycolysis, gluconeogenesis or the pentose phosphate pathway). This could be linked to the increase in different carbohydrates that we measured (Table 27.3). Indeed, increases in glucose, sucrose, raffinose and galactinol contents have been observed in the cold-exposed plants. However, in cold-treated ‘Désirée’ plants, only a small set of the same enzymes showed a similar trend, and others were even less abundant. Furthermore, at the gene level (Table 27.4), galactinol, raffinose and sucrose synthases were up-regulated by cold, and they showed a more intense variation in ‘Désirée’

Cold treatment

Mean

SD

Mean

SD

4.49 0.64 1.25 13.56 1.32 3.18 0.79 1.18 1.40 0.92

0.08 0.04 0.01 0.70 0.22 0.27 0.13 0.06 0.09 0.04

8.92 32.15 2.29 66.10 9.96 119.98 4.31 1.48 9.10 1.84

0.39 1.04 0.25 1.78 0.86 4.32 0.30 0.18 0.90 0.16

than in ‘PS3’. The expression of two other genes analysed (glucose-6P-dehydrogenase and pyruvate decarboxylase) was also up-regulated by chilling exposure, but for these two, the variations were more intense in ‘PS3’ than in ‘Désirée’. One could try to explain the variation of carbohydrate contents by the combined effect of gene expression and protein abundances as observed here. It is also necessary to draw attention to the different genes that showed variation in their expression and that were not found in the proteomic data set. It should be kept in mind that several technical aspects could explain this phenomenon. Indeed, if we consider the pI range used for the experiment, some of the enzymes mentioned above are out of this range; moreover, their mass is quite important and high-molecular-weight proteins do not penetrate easily in the second dimension gel due to their size. Furthermore, due to their mass or pI, some of these enzymes could be hidden by the huge spots represented by RuBisCO on the gel. The timeframe is also important. Indeed, the analyses were done on samples harvested on the same day, and there is a delay between gene expression, the turnover of proteins and the production of metabolites. In addition, it should not be forgotten that changes in gene expression are not always correlated with the corresponding protein (Gygi et al.,

Identification of Proteins from Potato Leaves

1999). Finally, the treated plants were exposed to cold and this physical factor definitely has an effect on biological processes, explaining, at least partly, the apparent discrepancy between metabolites, proteins and gene transcripts. Another interesting point was the variation in abundance of different isoforms of methionine synthases. This enzyme is involved in the synthesis of methionine and, through the activated methyl cycle, is involved in the provision of single carbons in plants (Hanson et al., 2000). It is also the starting point of glycine betaine, methylated polyols and polyamine and ethylene biosyntheses via the formation of S-adenosyl-L-methionine and its decarboxylated form (Eckermann et al., 2000). This increase in methionine synthase has already been mentioned at the mRNA level in response to several abiotic constraints and at the protein level when barley was exposed to salt stress (Narita et al., 2004). In both genotypes, multiple isoforms of chaperone were also more abundant in leaves of potatoes kept at 4°C. These chaperones were chaperonin 60 α- and β-subunits [295, 299, 301, 331, 338, 341, 342, 343, 344, 351], HSPs 70 [173, 208] and chaperonin 21 [1246, 1267, 1272]. Increases in these different chaperones are commonly known for a wide range of stresses (Miernyk, 1999; Sung et al., 2001; Lopez-Matas et al., 2004; Taylor et al., 2005). Another common feature to the two genotypes is the identification of 17 proteins involved

291

in photosynthesis and showing a general trend to diminish in abundance. These proteins included several isoforms of chlorophyll a/bbinding proteins [577, 1100, 1171, 1175, 1176, 1182, 1262, 1298], ferredoxin–NADP reductase [966, 967, 982], oxygen-evolving enhancer protein 1 and oxygen-evolving enhancer protein 2 [1020, 1034, 1334, 1346] and photosystem I (PSI) reaction centre subunit II [1394, 1456].

Conclusion In conclusion, it appears from this study that ‘PS3’ plants can be cold-acclimated. This process induces many modifications in protein expression, mainly in relation to primary metabolism and methionine synthesis. This is sustained by carbohydrate level variations and gene transcript adjustments. In the cultivar ‘Désirée’, fewer changes could be observed at the protein level, indicating a weaker metabolic plasticity and explaining, at least partially, its very faint acclimation to cold.

Acknowledgements The authors gratefully acknowledge L. Solinhac for his technical assistance. They thank the International Potato Center (Lima, Peru) for the potato clones and the Ministry of Finance of Luxembourg for financial support.

References Amme, S., Matros, A., Schelsier, B. and Mock, H.-P. (2006) Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology. Journal of Experimental Botany 57, 1537–1546. Arora, R., Wisniewski, M. and Rowland, L.J. (1996) Cold acclimation and alterations in dehydrin-like and bark storage proteins in the leaves of sibling deciduous and evergreen peach. Journal of the American Society for Horticultural Science 121, 915–919. Bassett, C.L., Wisniewski, M.E., Artlip, T.S., Norelli, J.L., Renaut, J. and Farrel, R.E. Jr (2005) Global analysis of genes regulated by low temperature and photoperiod in peach bark. Journal of the American Society for Horticultural Science 131, 551–563. Beck, E., Fettig, S., Knake, C., Hartig, K. and Bhattarai, T. (2007) Specific and unspecific responses of plants to cold and drought stress. Journal of Bioscience 32, 501–510. Chapman, K.D. (1998) Phospholipase activity during plant growth and development and in response to environmental stress. Trends in Plant Science 3, 419–426. Eckermann, C., Eichel, J. and Schroder, J. (2000) Plant methionine synthase: new insights into properties and expression. Biological Chemistry 381, 695–703.

292

J. Renaut et al.

Evers, D., Bonnechère, S., Hoffmann, L. and Hausman, J.-F. (2007) Physiological aspects of abiotic stress response in potato. Belgian Journal of Botany 140, 236–245. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14, 1675–1685. Gopal, J. and Khurana, S.M. (2006) Handbook of Potato Production, Improvement, and Postharvest Management. Haworth Press, Binghamton, New York. Guy, C.L., Niemi, K.J. and Brambl, R. (1985) Altered gene expression during cold acclimation of spinach. Proceedings of the National Academy of Sciences USA 82, 3673–3677. Guy, C., Kaplan, F., Kopka, J., Selbig, J. and Hincha, D.K. (2008) Metabolomics of temperature stress. Physiologia Plantarum 132, 220–235. Gygi, S.P., Rochon, Y., Franza, B.R. and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Molecular Cell Biology 19, 1720–1730. Hanson, A.D., Gage, D.A. and Shachar-Hill, Y. (2000) Plant one-carbon metabolism and its engineering. Trends in Plant Science 5, 206–213. Kaplan, F., Kopka, J., Haskell, D.W., Zhao, W., Schiller, K.C., Gatzke, N., Sung, D.Y. and Guy, C.L. (2004) Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiology 136, 4159–4168. Knight, H. and Knight, M.R. (2001) Abiotic stress signalling pathways: specificity and cross-talk. Trends in Plant Science 6, 262–267. Lim, C.-C., Krebs, S.L. and Arora, R. (1999) A 25-kDa dehydrin associated with genotype and age-dependent leaf freezing-tolerance in Rhododendron: a genetic marker for cold hardiness? Theoretical and Applied Genetics 99, 912–920. Lopez-Matas, M.A., Nunez, P., Soto, A., Allona, I., Casado, R., Collada, C., Guevara, M.A., Aragoncillo, C. and Gomez, L. (2004) Protein cryoprotective activity of a cytosolic small heat shock protein that accumulates constitutively in chestnut stems and is up-regulated by low and high temperatures. Plant Physiology 134, 1708–1717. Miernyk, J.A. (1999) Protein folding in the plant cell. Plant Physiology 121, 695–703. Narita, Y., Taguchi, H., Nakamura, T., Ueda, A., Shi, W. and Takabe, T. (2004) Characterization of the saltinducible methionine synthase from barley leaves. Plant Science 167, 1009–1016. Nicot, N., Hausman, J.F., Hoffmann, L. and Evers, D. (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany 56, 2907–2914. Palva, E.T. and Heino, P. (1998) Molecular mechanism of plant cold acclimation and freezing tolerance.. In: Li, P.H. and Chen, T.H.H. (eds.) Plant Cold Hardiness. Molecular Biology, Biochemistry and Physiology. Plenum Press, New York, New York, pp. 3–14. Renaut, J., Lutts, S., Hoffmann, L., and Hausman, J.-F. (2004) Responses of poplar to chilling temperatures: proteomic and physiological aspects. Plant Biology 6, 81–90. Renaut, J., Hoffmann, L. and Hausman, J.F. (2005) Biochemical and physiological mechanisms related to cold acclimation and enhanced freezing tolerance in poplar plantlets. Physiologia Plantarum 125, 82–94. Renaut, J., Hausman, J.F. and Wisniewski, M.E. (2006) Proteomics and low-temperature studies: bridging the gap between gene expression and metabolism. Physiologia Plantarum 126, 97–109. Renaut, J., Hausman, J.-F., Bassett, C.L., Artlip, T., Witters, E. and Wisniewski, M. (2008) Quantitative proteomic analysis of short photoperiod and low temperature responses in bark tissues of peach (Prunus persica L. Batsch). Tree Genetics and Genomes 4, 589–600. Sung, D.Y., Vierling, E. and Guy, C.L. (2001) Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiology 126, 789–800. Taylor, N.L., Heazlewood, J.L., Day, D.A. and Millar, A.H. (2005) Differential impact of environmental stresses on the pea mitochondrial proteome. Molecular Cellular Proteomics 4, 1122–1133. Uemura, M., Tominaga, Y., Nakagawara, C., Shigematsu, S., Minami, A. and Kawamura, Y. (2006) Responses of the plasma membrane to low temperatures. Physiologia Plantarum 126, 81–89. Viner, D., Morison, J.I.L. and Wallace, C. (2006) Recent and future climate change and its implications for plant growth. In: Morison, J. and Morecroft, M. (eds) Plant Growth and Climate Change. Blackwell Publishing, Oxford, UK, pp. 1–16.

28

Genomics of Cold Hardiness in Forest Trees J. Holliday

Introduction Seasonal cold acclimation in coniferous and broadleaf trees of the temperate and boreal regions is a remarkable physiological transition. The same dormant trees that in some cases survive submersion in liquid nitrogen (−196°C) once acclimated, can be killed during active growth by ambient temperatures only slightly below freezing (Sakai, 1960; Weiser, 1970). Whereas maximum cold hardiness is of primary interest in many crop plants, susceptibility to frost damage in forest trees in their native environment usually occurs during acclimation or after deacclimation. Cold hardiness in forest trees is therefore often thought of as a set of component traits which include not only cold hardiness per se, but also timing of growth cessation and bud set, initiation of cold acclimation and timing of bud flush (Howe et al., 2003). Genecological studies in widely distributed species such as European aspen (Populus tremula), Sitka spruce (Picea sitchensis) and Scots pine (Pinus sylvestris) have revealed steep genetic clines along environmental gradients for cold hardiness-related traits associated with phase I of cold acclimation (Cannell and Sheppard, 1982; Aitken and Adams, 1996; Hurme et al., 1997; St Clair, 2006; Hall et al., 2007; Mimura and Aitken, 2007). Whereas there is generally little difference in cold hardiness during the active growing season, signifi-

cant variation both within and among populations reveals itself during the autumn. In the southern portion of the ranges, moderate temperatures enable extended growing seasons, which may be accentuated by greater interspecific competition for light resources. In contrast, the cold winter temperatures of the north necessitate a short growing season. The resources are now available to unravel the genomic basis for adaptive variation in cold hardiness-related traits in a comprehensive and rigorous way. Genomics has both revolutionized our understanding of the molecular basis of biological processes and expanded the realm of questions about those processes that can be addressed. Until recently, the resources necessary for large-scale functional and evolutionary genomics projects in plants were available only for model species. However, non-model plants provide more favourable biological frameworks for answering certain questions, and are generally of more ecological and/or economic importance. The decreasing cost and increasing efficiency of establishing genomics platforms (i.e. sequencing, expression profiling, high-throughput genotyping) has enabled the extension of these tools to non-model species. Investment in the development of genomic resources for forest trees has been substantial in recent years, resulting in the development of large expressed sequence tag (EST) databases,

©CAB International 2009. Plant Cold Hardiness: From the Laboratory to the Field (eds L. Gusta, M. Wisniewski and K. Tanino)

293

294

J. Holliday

dense genetic maps, gene expression microarrays and, in the case of black cottonwood (Populus trichocarpa), a genome sequence (Ralph et al., 2006a,b; Tuskan et al., 2006). Although economically important traits such as growth and wood quality have received substantial interest, the importance of forest health has led to large research projects in the area of plant–herbivore interactions and adaptation to local climate (Ralph et al., 2006a,b; Holliday et al., 2008). Though generally well adapted to their local environment at present, the changing climate will force forest trees to adapt, migrate or be extirpated (Aitken, 1999; Aitken et al., 2008). Conservation of existing variation in the genes involved in adaptation to local climate, particularly those involved in the growth and dormancy cycle and cold hardiness, is therefore crucial to maintain the adaptive capacity of our forests in the face of a changing climate (Aitken, 1999; Aitken et al., 2008). This goal can be facilitated by a thorough appraisal of the genomic architecture of local adaptation to climate, including the number of genes involved, their respective effect sizes, linkages among them and epistatic interactions.

Linking Gene Expression with the Molecular Physiology of Cold Hardiness Moving beyond model systems Many of the genes involved in cold tolerance in angiosperms have been identified, particularly in Arabidopsis thaliana (Thomashow, 1999; Yamaguchi-Shinozaki and Shinozaki, 2006). Studies of Arabidopsis mutants have provided our most detailed understanding of the mode of action and effects of a few of these genes, while expression profiling has provided the global picture of response genes that protect angiosperms from sudden cold temperatures (Seki et al., 2001, 2002; Fowler and Thomashow 2002; Lee, 2005). These data have increased our understanding of the molecular basis of cold hardiness substantially, but it is difficult and in some cases impossible to directly extrapolate them to forest trees. This is in part because summer annuals do not naturally have

the same requirements for cold hardiness as overwintering plants. The environmental cues regulating acclimation also vary between annuals and perennials, the former responding primarily to chilling (<5°C) temperatures and the latter largely to long nights followed by freezing temperatures (Weiser, 1970). Studies of overwintering angiosperms (e.g. Secale cereale) are more useful, but still limited by their annual nature, by the environmental cue (i.e. low temperature) regulating acclimation and by a lack of similar morphologies (e.g. buds, vascular cambium). In addition, angiosperms are evolutionarily distant from the conifers, a taxon of particular interest owing to its ecological and economic importance. Functional genomic and expression profiling studies of cold acclimation in forest trees are therefore needed as a first step in identifying candidate genes for this important adaptive process.

The global picture of gene expression during acclimation: microarray studies With only a handful of mechanistic studies available, selection of candidate genes for population genomic dissection of cold hardiness in forest trees relies on data from expression profiling across the cold acclimation period. Gene expression microarrays for aspen (Populus), spruce (Picea) and pine (Pinus) have provided the first near-global pictures of the autumn transcriptome in trees (Table 28.1), and the results are intriguing. Far from a period of cellular ‘quiescence’, cold acclimation involves extensive remodelling of the transcriptome. Some of the genes that are up-regulated appear to be involved in cold hardiness, though it is difficult to disentangle gene expression related to the concomitant process of dormancy induction. In Sitka spruce for example, similar numbers of genes were up-regulated between late summer and early winter as were down-regulated (Holliday et al., 2008). Most of the changes were early in the autumn, suggesting that they were regulated by the transition to short days. In contrast, few changes were noted between late autumn and early winter, and sampling 4 days before and after the first significant subfreezing temperatures did not reveal the coordi-

Genomics of Cold Hardiness in Forest Trees

295

Table 28.1. Summary of microarray studies in forest trees spanning the period of cold acclimation. Species

Type

Description

Populus tremula

Binary

Pinus sylvestris

Time course, transplant

P. tremula

Time course

P. tremula×Populus alba

Time course

Picea sitchensis

Time course, provenance variation

Comparison of active and dormant cambial meristem Apical buds sampled from a single provenance grown in multiple environments Monthly sampling of cambial meristem between April and December (no sampling in November) Apical buds from three lines (WT, ABI3-overexpression, ABI3-RNAi) sampled weekly following transition to short days Needle tissue from three phenotypically divergent populations sampled from a common garden

nated transcriptional event that was expected given the well-characterized increase in cold hardiness for many tree species following the first frost (i.e. phase II of cold acclimation; Weiser, 1970).

Cold hardiness candidate genes in trees Dehydrins Dehydrins (also known as late embryogenesisabundant, LEA) were among the first cold hardiness-related proteins identified in trees. Interest in them stems from their importance in the cold hardiness in annual plants and, from a practical perspective, their abundance in cold-hardy tissue (and hence their ease of isolation). LEA-like transcripts have been identified in several tree species, including peach (Prunus persica) (Wisniewski et al., 1996, 1999), Scots pine (Joosen et al., 2006), white spruce (Picea glauca) (Richard et al., 2000; Liu et al., 2004) and Sitka spruce (Holliday et al., 2008). Although they are clearly important to the cold acclimation process, the protective mechanism is unclear. In angiosperms, dehydrins have been found localized in the cytoplasm, nucleus and adjacent to cellular membranes (Close,

Array elements 13,824 1,500

Reference Schrader et al. (2004) Joosen et al. (2006)

13,824

Druart et al. (2007)

24,735

Ruttink et al. (2007)

21,840

Holliday et al. (2008)

1997; Koag et al., 2003). It has been suggested that they may function as solubilizing agents for cellular macromolecules (Close, 1997), though this is not proved. Interestingly, a study of a nematode LEA-like protein suggests structural alterations upon dehydration leading to a coiled coil-like structure similar to that of intermediate filaments (Goyal et al., 2003; Wise and Tunnacliffe, 2004). This suggests a possible additional role for dehydrins in increasing the mechanical strength of cells that have become dehydrated during cold acclimation (Wise and Tunnacliffe, 2004). Antifreeze proteins Antifreeze proteins (AFPs) are a class of secreted proteins with homology to pathogenesis-related (PR) proteins (chitinases, β-1,3-glucanases and thaumatin-like proteins) (Yu and Griffith, 1997). AFPs prevent the propagation of ice by binding irreversibly to the ice surface through multiple hydrophilic domains (Kuiper et al., 2001). Structural analysis of an insect AFP also suggests an important role for hydrophobic interactions in ice binding (Leinala et al., 2002). Extensive studies have been carried out in winter rye (S. cereale), and although ice-binding domains in some species have been identified

296

J. Holliday

(e.g. Kuiper et al., 2001) there is no consensus domain that would enable identification of novel AFPs from a database search (Jia and Davies, 2002; Griffith and Yaish, 2004). Functional or gene expression studies are therefore needed to establish candidate antifreeze genes in forest trees. The presence of gene products with antifreeze activity has been indirectly demonstrated in several tree species by a thermal hysteresis assay that measures differences in the freezing and melting points of water in cold-acclimated tissue. These include the woody angiosperms Populus deltoides (eastern cottonwood) and Quercus alba (white oak), as well as the gymnosperm Ginkgo biloba (ginkgo) (Duman and Olsen, 1993). In Norway spruce (Picea abies), a candidate antifreeze protein, af70, is expressed in response to cold treatment (Sabala et al., 1997), and an apoplastic Douglas-fir (Pseudotsuga menziesii) chitinase with antifreeze activity has also been isolated (Zamani et al., 2003). Expression profiling during cold acclimation in Sitka spruce showed that numerous PR transcripts are up-regulated, including chitinases, b-1,3-glucanases and thaumatin-like proteins (Holliday et al., 2008). Analysis of apoplastic fluid extracted from cold-acclimated needles reveals accumulation of proteins with sizes similar to these PR transcripts, and work is ongoing to further characterize the extracelluar spruce proteome through peptide sequencing and antifreeze assays (J.A. Holliday, unpublished results). Light signalling Phytochromes and their interactions with circadian oscillators form the basis for photoperiodism in plants (Thomas and Vince-Prue, 1997) and are thought to be the primary regulators of night length-mediated bud set in perennials (Howe et al., 1996; Horvath et al., 2003). Indeed, overexpression of an oat PHYA gene in hybrid aspen trees (P. tremula×Populus tremuloides) blocked growth cessation and cold acclimation under short days (Olsen et al., 1997). Conifer phytochrome genes similar to PHYA and PHYB have been isolated from Norway spruce and Scots pine, respectively (Clapham et al., 1999). In Sitka spruce, a PHYA-like gene is transiently up-regulated during cold acclimation, whereas a PHYB-like

gene is consistently down-regulated through the same period (Holliday et al., 2008). In Arabidopsis, PHYA controls expression of the flowering-time gene FLOWERING LOCUS T (FT) through regulation of CONSTANS (CO) (Yanovsky and Kay, 2002), and a crucial role for FT in growth cessation was recently demonstrated in hybrid aspen (Bohlenius et al., 2006). Whether functional orthologues to FT exist in conifers is currently unknown, but FT-like transcripts have been identified in EST databases. One of these transcripts was shown to be up-regulated strongly following transfer of Norway spruce plants to short days (Gyllenstrand et al., 2007), whereas a Sitka spruce FT-like gene showed the opposite pattern (although these studies are not directly comparable as the latter was conducted under field conditions) (Holliday et al., 2008). Given the similarity of angiosperm FT gene family members, it is difficult to assess function of putative gymnosperm FT-like genes. However, it seems likely that if they do exist and function in a similar fashion, conifer FT orthologues would exhibit similar expression patterns to those seen in aspen (i.e. down-regulation following transition to short days). Transcription factors Whereas the C-repeat binding factors (CBFs) and their upstream regulator, ICE1, are crucial for transient cold tolerance in annuals, their role in seasonal cold acclimation in forest trees is unclear (Stockinger et al., 1997; Chinnusamy et al., 2003; Van Buskirk and Thomashow, 2006). Gene expression microarray studies in both angiosperm trees and conifers across the growth cessation, cold acclimation and dormancy induction period show conflicting results in this area (Schrader et al., 2004; Joosen et al., 2006; Druart et al., 2007; Ruttink et al., 2007; Holliday et al., 2008). Whereas Schrader et al. (2004) found a CBF1-like gene in P. tremula up-regulated 6.7-fold in a contrast between active and dormant cambium, Ruttink et al. (2007) found no differential expression of either ICE1 or CBF genes in P. tremula×Populus alba buds up to 6 weeks following the transition to short days. In the latter, many canonical downstream targets of CBF were induced. It is possible that the coarse

Genomics of Cold Hardiness in Forest Trees

nature of the sampling contributed to variation in results for CBF expression, as CBF appears to be functional in angiosperm trees. P. trichocarpa transformed with an Arabidopsis CBF1 gene increased constitutive freezing tolerance (Benedict et al., 2006), and overexpression of a sweet cherry (Prunus avium) CBF in Arabidopsis conferred increased freezing tolerance (Kitashiba et al., 2004). The situation in conifers is less clear. Although phylogenetic analysis of AP2 transcription factors in spruce suggests the existence of a CBF/ DREB clade (S. Ralph, unpublished results), microarray studies during cold acclimation in Scots pine and Sitka spruce did not find conifer CBF-like genes to be induced (Joosen et al., 2006; Holliday et al., 2008). No heterologous expression studies have been carried out to date with conifer CBF-like transcripts. Metabolic remodelling Cellular membranes are the primary sites of freezing injury. Plasma membranes from nonacclimated plants suffer expansion-induced lysis and lipid phase transitions; however, membranes from cold-acclimated plants resist this damage (Steponkus, 1984; Webb and Steponkus, 1993; Webb et al., 1993). The hydrophilic nature of dehydrins may function in part to stabilize membranes, and changes in the composition of membranes themselves likely also play a role in their ability to resist rupture (Lynch and Steponkus, 1987; Uemura and Steponkus, 1994; Uemura et al., 1995; Close, 1997). Specifically, higher lipid to protein ratios in several conifer species increases membrane fluidity. In P. abies, membrane lipid content of frost-hardy needles is almost twice that of frost-sensitive needles (Senser and Beck, 1982). The degree of membrane phospholipid saturation also varies. Unfortunately, the difficulty in analysing the lipid fraction in metabolite screens has left the global picture of lipid remodelling during cold acclimation obscure. However, inferences from gene expression studies suggest some of the important players in this process. For example, in Arabidopsis, overexpression of the lipid transfer protein EARLI1 leads to reduced electrolyte leakage during freezing damage (Bubier and Schlappi, 2004), and a homologue to

297

EARLI1 was up-regulated 34-fold in Sitka spruce needles during the autumn (Holliday et al., 2008). In addition, 17-fold induction of a squalene synthase transcript in the same study suggests a role for changes in membrane sterol content. Although membrane remodelling likely plays a role in preventing rupture upon extracellular freezing, soluble carbohydrates have a dual role in protection of membranes in that they directly stabilize membranes (Bryant et al., 2001) and are thought to reduce osmotic potential across those membranes. A positive correlation between sugar content and hardiness has been observed in Scots pine, lodgepole pine (Pinus contorta), Norway spruce and red spruce (Picea rubens) (Aronson et al., 1976; DeHayes, 1992; Ogren et al., 1997). In particular, disaccharides such as sucrose and raffinose increase during cold acclimation (Wang and Zwiazek, 1999; Druart et al., 2007; Ruttink et al., 2007). Conversely, starch concentration has been shown to be at a minimum in winter in red pine (Pinus resinosa), Scots pine and red spruce (Alscher et al., 1989; Hansen et al., 1996), and in aspen, starch breakdown appears to be regulated by short days (Druart et al., 2007). There is good agreement among expression profiling studies that sugar metabolism genes are up-regulated during the autumn. In particular, galactinol synthase, raffinose synthase and sucrose synthase were induced in both Sitka spruce and P. tremula×P. alba (Ruttink et al., 2007; Holliday et al., 2008).

Population Genomics of Cold Hardiness Alternatives to functional studies Although functional genomics enables a more detailed and in some ways more satisfying account of the role of particular genes in plant cold hardiness, the recalcitrance of many forest trees species to transformation, long generation times, complex genomes, and lack of genome sequences for all but one, make modern population genomic approaches attractive alternatives to understanding the genomic architecture of cold hardiness (Neale and

298

J. Holliday

Savolainen, 2004; Gonzalez-Martinez et al., 2006b). Additionally, from an industrial standpoint, the goals of molecular biology in forest trees are quite different from those in crop species. The latter are often genetically depauperate and the most expedient route to genetic improvement consists in homologous or heterologous overexpression. In contrast, an explicit goal of forest tree improvement is the maintenance of genetic diversity, and deployment of genetically modified (GM) trees with a uniform genetic background is therefore problematic (in addition, moratoriums on planting of GM trees are in place in many locales). As such, improving or modifying traits such as cold hardiness is best achieved through the exploitation of standing genetic variation. Understanding the functional significance of particular genes, while interesting from an academic point of view, is of little interest to tree breeders if there is no variation in their nucleotide sequence to exploit. Hence, population genomics in forest trees facilitates an understanding of the genomic basis for local adaptation, i.e. elucidating the genetic variants that contribute to variation in the phenotype, which then guides breeding strategies for traits related to local adaptation, as well as conservation genetics to maintain the plasticity and adaptability of natural populations. Conservation genetics typically proceeds from the premise that maintenance of genetic diversity at neutral loci indirectly maintains diversity in phenotypic traits. However, in many cases neutral loci are unlikely to be a sufficient proxy for genes involved in adaptation. In forest trees, Fst and Qst values are often wildly divergent (Howe et al., 2003), and in general genetic diversity measured by traditional neutral marker studies is not a good predictor of trait variation measured in common garden experiments (Reed and Frankham, 2001, 2003). A high level of genetic differentiation for cold hardiness-related traits exists both within and among populations for many tree species, and although much is known about the ecological and quantitative genetics of these traits, few of the specific genes controlling them have been uncovered. At the molecular level, there are several ways in which adaptive divergence in cold hardiness traits could be manifested. These include dif-

ferences in gene expression timing, extent and/or constitution, and nucleotide differences that correspond to differences in protein function. In addition, a significant role for epigenetics in facilitating local adaptation has also been demonstrated in Norway spruce (Johnsen et al., 2005a,b; see Johnsen et al., Chapter 11, this volume). Where genetic variation can directly explain trait variation, quantitative trait loci (QTL) mapping and, more recently, association mapping are the methods of choice to determine the respective contributions of individual loci.

QTL mapping Cold hardiness has long been understood to be a quantitative trait, and its genetic dissection became possible in the 1990s through the combination of genetic maps and breeding populations to map cold hardiness QTL. QTL mapping takes advantage of the extensive linkage disequilibrium (LD) within known pedigrees to identify marker–trait associations (Lander and Botstein, 1989). While useful for determining the number of QTL and their relative effect, a major disadvantage of QTL mapping is resolution: the few meioses present in the mapping population means that QTL can only be mapped to a genomic region (with a genome sequence these regions can be interrogated further). Also, marker– trait associations are population-specific, and cannot be transferred between mapping populations or to natural stands (Howe et al., 2003). Nevertheless, these data provide the first glimpse of the complexity of this trait. Results of QTL studies have been summarized elsewhere (Howe et al., 2003; Savolainen et al., 2007). In general, the results point to many genes of small effect (percentage of variation explained, PVE, up to 15%) governing variation in cold hardiness (Howe et al., 2003). For example, a series of QTL studies of bud phenology and cold hardiness in Douglas fir, which is widely distributed in western North America, identified 11 QTL for autumn cold hardiness and nine for spring cold hardiness, with PVE ranging from 2.0 from 7.5% (Jermstad et al., 2001).

Genomics of Cold Hardiness in Forest Trees

Association mapping While QTL mapping has substantially increased our understanding of the genomics of cold hardiness, association mapping is the current method of choice in this area. Ever expanding sequence resources in the form of large EST databases provide the foundation for the extension of this technique to non-model species such as trees (Neale and Savolainen, 2004). Association mapping is similar in many respects to QTL mapping, as both of these methods attempt to track the co-segregation of marker alleles and phenotypic variants. In contrast to QTL mapping, association mapping employs large unstructured populations with minimal LD and highly abundant markers (single-nucleotide polymorphisms, SNPs) to identify genetic variants that associate with phenotypic variation (Neale and Savolainen, 2004). A marker may associate with the phenotype because it is the actual functional variant or due to tight linkage with the causative locus. Candidate gene-based association studies involve first selecting a set of genes that may contribute to the trait of interest. Annotations from angiosperms and gene expression studies are the principal lines of evidence to guide the choice of candidates, and where a QTL map exists, co-localization of candidates with QTL can provide additional support. Although sequence similarity to cold hardiness-related genes in Arabidopsis or other model angiosperms may be sufficient to choose candidate genes in angiosperm trees, in the absence of corroborating evidence for function (e.g. gene expression), this is unlikely to be successful in gymnosperms. Not only have gymnosperms diverged from angiosperms for close to 400 million years (Kenrick and Crane, 1997), but the evolution of their genomes has followed a very different trajectory from that of angiosperms. Whereas the difference in genome size and gene family size between poplar and Arabidopsis, for example, can be largely explained by whole genome duplication (Tuskan et al., 2006), the genomes of Pinus and Picea species appear to have expanded to their current massive sizes in large part through tandem gene duplications and expansion of multigene families (Kinlaw and Neale, 1997; Ahuja and Neale, 2005). As

299

such, BLASTX searches between Arabidopsis and gymnosperms typically return many possible gymnosperm orthologues with no way to differentiate them. This problem is confounded by the redundancy present in EST collections, which hold by far the most sequence information for conifers. In spite of the potential complexities in identifying candidate genes, forest trees in general and conifers in particular are well suited to the genetic dissection of cold hardiness using candidate gene-based association studies. One of the major challenges in designing an effective association study is population structure. In crop plants in particular, breeding history in domesticated lines and limited gene flow in wild populations has created complex stratification that could lead to spurious association (Flint-Garcia et al., 2003). Similar issues have led to false association in other systems, most notably (or infamously) in man (Knowler et al., 1988). Fortunately, most temperate and boreal tree species do not suffer from these problems to a large extent. Population structure at selectively neutral sites is generally low and gene flow via pollen is efficient (Hamrick and Godt, 1996). In species where disjunct peripheral populations exist, population structure can be elevated (Mimura, 2006), but this can be accounted for by either excluding these populations or explicitly modelling population structure and/or kinship in the analysis (Yu et al., 2006). Association mapping of cold hardiness in forest trees has recently begun to yield results, but these data are yet to be published. Among the projects underway is the ‘Douglasfir genome project’, which has re-sequenced >100 cold hardiness-related candidate genes in Douglas fir (D.B. Neale, unpublished results), as well as ‘Treenomix’, which to date has successfully re-sequenced 200 cold hardiness-related genes, which will be tested for associations in a panel of 500 Sitka spruce genotypes (J.A. Holliday, unpublished results). Preliminary data from the Sitka spruce re-sequencing effort reveal a number of genes that appear to be under diversifying selection and suggest that the majority have been subject to purifying selection (J.A. Holliday, unpublished results). Genes in the former category may be of most interest in this species due to the linear variation in

300

J. Holliday

climate conditions along its range (the west coast of North America). That many genes are being constrained by purifying selection is not surprising, and appears to be the norm in most forest trees (Krutovsky and Neale, 2005; Gonzalez-Martinez et al., 2006a; Pavy et al., 2006). However, it is an important point worth considering when selecting candidate genes. The most obvious choices may also be so important that nucleotide variation is not tolerated.

now be achieved for non-model tree species with a modest input of research funds within the reach of most individual research programmes (Margulies et al., 2005). Recent advances in genotyping platforms have enabled assays of thousands of SNPs simultaneously, making true population genomics possible (Fan et al., 2003). The goal of understanding the genomic complexity of adaptation of woody perennials to life in a cold climate has long been contemplated; we are now on the verge of a coherent description of this important adaptive trait.

Perspectives The pace of genomics research today is stunning. Driven largely by the human biomedical community, rapid advances in sequencing and genotyping technologies have put goals within reach that were unimaginable only a few years ago. Acquiring a large amount of expressed sequence data, which previously required a coordinated and well-funded approach, can

Acknowledgements I would like to thank Sally Aitken for guidance and thoughtful discussions related to this work. Funding has been provided by Genome British Columbia, Genome Canada, and the Natural Science and Engineering Research Council of Canada.

References Ahuja, M.R. and Neale, D.B. (2005) Evolution of genome size in conifers. Silvae Genetica 54, 126–137. Aitken, S.N. (1999) Conserving adaptive variation in forest ecosystems. Journal of Sustainable Forestry 8, 1–10. Aitken, S.N. and Adams, W.T. (1996) Genetics of fall and winter cold hardiness of coastal Douglas-fir in Oregon. Canadian Journal of Forest Research–Revue Canadienne de Recherche Forestiere 26, 1828–1837. Aitken, S.N., Yeaman, S., Holliday, J.A., Wang, T. and Curtis-McLane, S. (2008) Adaptation, migration or extirpation: climate change outcomes for tree populations. Evolutionary Applications 1, 95–111. Alscher, R.G., Amundson, R.G., Cumming, J.R., Fellows, S., Fincher, J., Rubin, G., Vanleuken, P. and Weinstein, L.H. (1989) Seasonal changes in the pigments, carbohydrates and growth of red spruce as affected by ozone. New Phytologist 113, 211–223. Aronson, A., Ingestad, T. and Loof, L.G. (1976) Carbohydrate metabolism and frost hardiness in pine and spruce seedlings grown at different photoperiods and thermoperiods. Physiologia Plantarum 36, 127–132. Benedict, C., Skinner, J.S., Meng, R., Chang, Y.J., Bhalerao, R., Huner, N.P.A., Finn, C.E., Chen, T.H.H. and Hurry, V. (2006) The CBF1-dependent low temperature signalling pathway, regulon and increase in freeze tolerance are conserved in Populus spp. Plant, Cell & Environment 29, 1259–1272. Bohlenius, H., Huang, T., Charbonnel-Campaa, L., Brunner, A.M., Jansson, S., Strauss, S.H. and Nilsson, O. (2006) CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312, 1040–1043. Bryant, G., Koster, K.L. and Wolfe, J. (2001) Membrane behaviour in seeds and other systems at low water content: the various effects of solutes. Seed Science Research 11, 17–25. Bubier, J. and Schlappi, M. (2004) Cold induction of EARLI1, a putative Arabidopsis lipid transfer protein, is light and calcium dependent. Plant, Cell & Environment 27, 929–936. Cannell, M.G.R. and Sheppard, L.J. (1982) Seasonal changes in the frost hardiness of provenances of Picea sitchensis in Scotland. Forestry 55, 137–153.

Genomics of Cold Hardiness in Forest Trees

301

Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B.H., Hong, X.H., Agarwal, M. and Zhu, J.K. (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes & Development 17, 1043–1054. Clapham, D.H., Kolukisaoglu, H.U., Larsson, C.T., Qamaruddin, M., Ekberg, I., Wiegmann-Eirund, C., Schneider-Poetsch, H.A. and von Arnold, S. (1999) Phytochrome types in Picea and Pinus. Expression patterns of PHYA-related types. Plant Molecular Biology 40, 669–678. Close, T.J. (1997) Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiologia Plantarum 100, 291–296. DeHayes, A.L. (1992) Winter injury and developmental cold tolerance in red spruce. In: Eagar, C. and Adams, M.B. (eds) Ecology and Decline of Red Spruce in the Eastern United States. Ecological Studies. SpringerVerlag, Berlin, pp. 295–337. Druart, N., Johansson, A., Baba, K., Schrader, J., Sjodin, A., Bhalerao, R.R., Resman, L., Trygg, J., Moritz, T. and Bhalerao, R.P. (2007) Environmental and hormonal regulation of the activity–dormancy cycle in the cambial meristem involves stage-specific modulation of transcriptional and metabolic networks. The Plant Journal 50, 557–573. Duman, J.G. and Olsen, T.M. (1993) Thermal hysteresis protein activity in bacteria, fungi, and phylogenetically diverse plants. Cryobiology 30, 322–328. Fan, J.B., Oliphant, A., Shen, R., Kermani, B.G., Garcia, F., Gunderson, K.L., Hansen, M., Steemers, F., Butler, S.L., Deloukas, P., Galver, L., Hunt, S., McBride, C., Bibikova, M., Rubano, T., Chen, J., Wickham, E., Doucet, D., Chang, W., Campbell, D., Zhang, B., Kruglyak, S., Bentley, D., Haas, J., Rigault, P., Zhou, L., Stuelpnagel, J. and Chee, M.S. (2003) Highly parallel SNP genotyping. Cold Spring Harbor Symposia on Quantitative Biology 68, 69–78. Flint-Garcia, S.A., Thornsberry, J.M. and Buckler, E.S. (2003) Structure of linkage disequilibrium in plants. Annual Review of Plant Biology 54, 357–374. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14, 1675–1690. Gonzalez-Martinez, S.C., Ersoz, E., Brown, G.R., Wheeler, N.C. and Neale, D.B. (2006a) DNA sequence variation and selection of tag single-nucleotide polymorphisms at candidate genes for drought-stress response in Pinus taeda L. Genetics 172, 1915–1926. Gonzalez-Martinez, S.C., Krutovsky, K.V. and Neale, D.B. (2006b) Forest tree population genomics and adaptive evolution. New Phytologist 170, 227–238. Goyal, K., Tisi, L., Basran, A., Browne, J., Burnell, A., Zurdo, J. and Tunnacliffe, A. (2003) Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein. Journal of Biological Chemistry 278, 12977–12984. Griffith, M. and Yaish, M.W. (2004) Antifreeze proteins in overwintering plants: a tale of two activities. Trends in Plant Science 9, 399–405. Gyllenstrand, N., Clapham, D., Kallman, T. and Lagercrantz, U. (2007) A Norway spruce FLOWERING LOCUS T homolog is implicated in control of growth rhythm in conifers. Plant Physiology 144, 248–257. Hall, D., Luquez, V., Garcia, V.M., St Onge, K.R., Jansson, S. and Ingvarsson, P.K. (2007) Adaptive population differentiation in phenology across a latitudinal gradient in European aspen (Populus tremula, L.): a comparison of neutral markers, candidate genes and phenotypic traits. Evolution 61, 2849–2860. Hamrick, J.L. and Godt, M.J.W. (1996) Effects of life history traits on genetic diversity in plant species. Philosophical Transactions of the Royal Society of London Series B – Biological Sciences 351, 1291–1298. Hansen, J., Vogg, G. and Beck, E. (1996) Assimilation, allocation and utilization of carbon by 3-year-old Scots pine (Pinus sylvestris, L.) trees during winter and early spring. Trees –Structure and Function 11, 83–90. Holliday, J.A., Ralph, S.G., White, R., Bohlmann, J. and Aitken, S.N. (2008) Global monitoring of autumn gene expression within and among phenotypically divergent populations of Sitka spruce (Picea sitchensis). New Phytologist 178, 103–122. Horvath, D.P., Anderson, J.V., Chao, W.S. and Foley, M.E. (2003) Knowing when to grow: signals regulating bud dormancy. Trends in Plant Science 8, 534–540. Howe, G.T., Gardner, G., Hackett, W.P. and Furnier, G.R. (1996) Phytochrome control of short-day-induced bud set in black cottonwood. Physiologia Plantarum 97, 95–103. Howe, G.T., Aitken, S.N., Neale, D.B., Jermstad, K.D., Wheeler, N.C. and Chen, T.H.H. (2003) From genotype to phenotype: unraveling the complexities of cold adaptation in forest trees. Canadian Journal of Botany– Revue Canadienne de Botanique 81, 1247–1266.

302

J. Holliday

Hurme, P., Repo, T., Savolainen, O. and Paakkonen, T. (1997) Climatic adaptation of bud set and frost hardiness in Scots pine (Pinus sylvestris). Canadian Journal of Forest Research–Revue Canadienne de Recherche Forestiere 27, 716–723. Jermstad, K.D., Bassoni, D.L., Wheeler, N.C., Anekonda, T.S., Aitken, S.N., Adams, W.T. and Neale, D.B. (2001) Mapping of quantitative trait loci controlling adaptive traits in coastal Douglas-fir. II. Spring and fall cold-hardiness. Theoretical and Applied Genetics 102, 1152–1158. Jia, Z.C. and Davies, P.L. (2002) Antifreeze proteins: an unusual receptor–ligand interaction. Trends in Biochemical Sciences 27, 101–106. Johnsen, O., Daehlen, O.G., Ostreng, G. and Skroppa, T. (2005a) Daylength and temperature during seed production interactively affect adaptive performance of Picea abies progenies. New Phytologist 168, 589–596. Johnsen, O., Fossdal, C.G., Nagy, N., Molmann, J., Daehlen, O.G. and Skroppa, T. (2005b) Climatic adaptation in Picea abies progenies is affected by the temperature during zygotic embryogenesis and seed maturation. Plant, Cell & Environment 28, 1090–1102. Joosen, R.V.L., Lammers, M., Balk, P.A., Bronnum, P., Konings, M., Perks, M., Stattin, E., Van Wordragen, M.F. and van der Geest, A.H.M. (2006) Correlating gene expression to physiological parameters and environmental conditions during cold acclimation of Pinus sylvestris, identification of molecular markers using cDNA microarrays. Tree Physiology 26, 1297–1313. Kenrick, P. and Crane, P.R. (1997) The origin and early evolution of plants on land. Nature 389, 33–39. Kinlaw, C.S. and Neale, D.B. (1997) Complex gene families in pine genomes. Trends in Plant Science 2, 356–359. Kitashiba, H., Ishizaka, T., Isuzugawa, K., Nishimura, K. and Suzuki, T. (2004) Expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance. Journal of Plant Physiology 161, 1171–1176. Knowler, W.C., Williams, R.C., Pettitt, D.J. and Steinberg, A.G. (1988) Gm3-5,13,14 and type-2 diabetes-mellitus – an association in American-Indians with genetic admixture. American Journal of Human Genetics 43, 520–526. Koag, M.C., Fenton, R.D., Wilkens, S. and Close, T.J. (2003) The binding of maize DHN1 to lipid vesicles. Gain of structure and lipid specificity. Plant Physiology 131, 309–316. Krutovsky, K.V. and Neale, D.B. (2005) Nucleotide diversity and linkage disequilibrium in cold-hardiness- and wood quality-related candidate genes in Douglas fir. Genetics 171, 2029–2041. Kuiper, M.J., Davies, P.L. and Walker, V.K. (2001) A theoretical model of a plant antifreeze protein from Lolium perenne. Biophysical Journal 81, 3560–3565. Lander, E.S. and Botstein, D. (1989) Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121, 185–199. Lee, B.H., Henderson, D.A. and Zhu, J.K. (2005) The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. The Plant Cell 17, 3155–3175. Leinala, E.K., Davies, P.L. and Jia, Z.C. (2002) Crystal structure of b-helical antifreeze protein points to a general ice binding model. Structure 10, 619–627. Liu, J.J., Ekramoddoullah, A.K.M., Taylor, D., Piggott, N., Lane, S. and Hawkins, B. (2004) Characterization of Picg5 novel proteins associated with seasonal cold acclimation of white spruce (Picea glauca). Trees – Structure and Function 18, 649–657. Lynch, D.V. and Steponkus, P.L. (1987) Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale). Plant Physiology 83, 761–767. Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J., Braverman, M.S., Chen, Y.J., Chen, Z.T., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V., Godwin, B.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C., Alenquer, M.L.I., Jarvie, T.P., Jirage, K.B., Kim, J.B., Knight, J.R., Lanza, J.R., Leamon, J.H., Lefkowitz, S.M., Lei, M., Li, J., Lohman, K.L., Lu, H., Makhijani, V.B., McDade, K.E., McKenna, M.P., Myers, E.W., Nickerson, E., Nobile, J.R., Plant, R., Puc, B.P., Ronan, M.T., Roth, G.T., Sarkis, G.J., Simons, J.F., Simpson, J.W., Srinivasan, M., Tartaro, K.R., Tomasz, A., Vogt, K.A., Volkmer, G.A., Wang, S.H., Wang, Y., Weiner, M.P., Yu, P.G., Begley, R.F. and Rothberg, J.M. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380. Mimura, M. (2006) Adaptation and gene flow in Sitka spruce. PhD thesis, University of British Columbia, Vancouver, British Columbia, Canada. Mimura, M. and Aitken, S.N. (2007) Adaptive gradients and isolation-by-distance with postglacial migration in Picea sitchensis. Heredity 99, 224–232. Neale, D.B. and Savolainen, O. (2004) Association genetics of complex traits in conifers. Trends in Plant Science 9, 325–330.

Genomics of Cold Hardiness in Forest Trees

303

Ogren, E., Nilsson, T. and Sundblad, L.G. (1997) Relationship between respiratory depletion of sugars and loss of cold hardiness in coniferous seedlings over-wintering at raised temperatures: indications of different sensitivities of spruce and pine. Plant, Cell & Environment 20, 247–253. Olsen, J.E., Junttila, O., Nilsen, J., Eriksson, M.E., Martinussen, I., Olsson, O., Sandberg, G. and Moritz, T. (1997) Ectopic expression of oat phytochrome A in hybrid aspen changes critical daylength for growth and prevents cold acclimatization. The Plant Journal 12, 1339–1350. Pavy, N., Parsons, L.S., Paule, C., MacKay, J. and Bousquet, J. (2006) Automated SNP detection from a large collection of white spruce expressed sequences: contributing factors and approaches for the categorization of SNPs. BMC Genomics 7, 174. Ralph, S., Oddy, C., Cooper, D., Yueh, H., Jancsik, S., Kolosova, N., Philippe, R.N., Aeschliman, D., White, R., Huber, D., Ritland, C.E., Benoit, F., Rigby, T., Nantel, A., Butterfield, Y.S.N., Kirkpatrick, R., Chun, E., Liu, J., Palmquist, D., Wynhoven, B., Stott, J., Yang, G., Barber, S., Holt, R.A., Siddiqui, A., Jones, S.J.M., Marra, M.A., Ellis, B.E., Douglas, C.J., Ritland, K. and Bohlmann, J. (2006a) Genomics of hybrid poplar (Populus trichocarpa×deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags and a cDNA microarray for the study of insect-induced defences in poplar. Molecular Ecology 15, 1275–1297. Ralph, S.G., Yueh, H., Friedmann, M., Aeschliman, D., Zeznik, J.A., Nelson, C.C., Butterfield, Y.S.N., Kirkpatrick, R., Liu, J., Jones, S.J.M., Marra, M.A., Douglas, C.J., Ritland, K. and Bohlmann, J. (2006b) Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome. Plant, Cell & Environment 29, 1545–1570. Reed, D.H. and Frankham, R. (2001) How closely correlated are molecular and quantitative measures of genetic variation? A meta-analysis. Evolution 55, 1095–1103. Reed, D.H. and Frankham, R. (2003) Correlation between fitness and genetic diversity. Conservation Biology 17, 230–237. Richard, S., Morency, M.J., Drevet, C., Jouanin, L. and Seguin, A. (2000) Isolation and characterization of a dehydrin gene from white spruce induced upon wounding, drought and cold stresses. Plant Molecular Biology 43, 1–10. Ruttink, T., Arend, M., Morreel, K., Storme, V., Rombauts, S., Fromm, J., Bhalerao, R.P., Boerjan, W. and Rohde, A. (2007) A molecular timetable for apical bud formation and dormancy induction in poplar. The Plant Cell 19, 2370–2390. Sabala, I., Franzen, H. and von Arnold, S. (1997) A spruce gene, af70, constitutively expressed in somatic embryos and induced by ABA and low temperature in seedlings. Physiologia Plantarum 99, 316–322. Sakai, A. (1960) Survival of the twig of woody plants at −196°C. Nature 185, 393–394. Savolainen, O., Pyhajarvi, T. and Knurr, T. (2007) Gene flow and local adaptation in tees. Annual Review of Ecology Evolution and Systematics 38, 595–619. Schrader, J., Moyle, R., Bhalerao, R., Hertzberg, M., Lundeberg, J., Nilsson, P. and Bhalerao, R.P. (2004) Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome. The Plant Journal 40, 173–187. Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P., Hayashizaki, Y. and Shinozaki, K. (2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. The Plant Cell 13, 61–72. Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., Kamiya, A., Nakajima, M., Enju, A., Sakurai, T., Satou, M., Akiyama, K., Taji, T., Yamaguchi-Shinozaki, K., Carninci, P., Kawai, J., Hayashizaki, Y. and Shinozaki. K. (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal 31, 279–292. Senser, M. and Beck, E. (1982) Frost resistance in spruce (Picea abies): the lipid composition of frost resistant and frost sensitive spruce chloroplasts. Zeitschrift fur Pflanzenphysiologie 105, 241–253. St Clair, J.B. (2006) Genetic variation in fall cold hardiness in coastal Douglas-fir in western Oregon and Washington. Canadian Journal of Botany–Revue Canadienne de Botanique 84, 1110–1121. Steponkus, P.L. (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology and Plant Molecular Biology 35, 543–584. Stockinger, E.J., Gilmour, S.J. and Thomashow, M.F. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proceedings of the National Academy of Sciences USA 94, 1035–1040.

304

J. Holliday

Thomas, B. and Vince-Prue, D. (1997) Photoperiodism in Plants. Academic Press Limited, London. Thomashow, M. F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50, 571–599. Tuskan, G.A., DiFazio, S., Jansson, S., Bohlmann, J., Grigoriev, I., Hellsten, U., Putnam, N., Ralph, S., Rombauts, S., Salamov, A., Schein, J., Sterck, L., Aerts, A., Bhalerao, R.R., Bhalerao, R.P., Blaudez, D., Boerjan, W., Brun, A., Brunner, A., Busov, V., Campbell, M., Carlson, J., Chalot, M., Chapman, J., Chen, G.L., Cooper, D., Coutinho, P.M., Couturier, J., Covert, S., Cronk, Q., Cunningham, R., Davis, J., Degroeve, S., Dejardin, A., Depamphilis, C., Detter, J., Dirks, B., Dubchak, I., Duplessis, S., Ehlting, J., Ellis, B., Gendler, K., Goodstein, D., Gribskov, M., Grimwood, J., Groover, A., Gunter, L., Hamberger, B., Heinze, B., Helariutta, Y., Henrissat, B., Holligan, D., Holt, R., Huang, W., Islam-Faridi, N., Jones, S., Jones-Rhoades, M., Jorgensen, R., Joshi, C., Kangasjarvi, J., Karlsson, J., Kelleher, C., Kirkpatrick, R., Kirst, M., Kohler, A., Kalluri, U., Larimer, F., Leebens-Mack, J., Leple, J.C., Locascio, P., Lou, Y., Lucas, S., Martin, F., Montanini, B., Napoli, C., Nelson, D.R., Nelson, C., Nieminen, K., Nilsson, O., Pereda, V., Peter, G., Philippe, R., Pilate, G., Poliakov, A., Razumovskaya, J., Richardson, P., Rinaldi, C., Ritland, K., Rouze, P., Ryaboy, D., Schmutz, J., Schrader, J., Segerman, B., Shin, H., Siddiqui, A., Sterky, F., Terry, A., Tsai, C.J., Uberbacher, E., Unneberg, P., Vahala, J., Wall, K., Wessler, S., Yang, G., Yin, T., Douglas, C., Marra, M., Sandberg, G., de Peer, Y.V. and Rokhsar, D. (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 1596–1604. Uemura, M. and Steponkus, P.L. (1994) A contrast of the plasma membrane lipid composition of oat and rye leaves in relation to freezing tolerance. Plant Physiology 104, 479–496. Uemura, M., Joseph, R.A. and Steponkus, P.L. (1995) Cold acclimation of Arabidopsis thaliana – effect on plasma membrane lipid composition and freeze induced lesions. Plant Physiology 109, 15–30. Van Buskirk, H.A. and Thomashow, M.F. (2006) Arabidopsis transcription factors regulating cold acclimation. Physiologia Plantarum 126, 72–80. Wang, Y.F. and Zwiazek, J.J. (1999) Spring changes in water relations, gas exchange, and carbohydrates of white spruce (Picea glauca) seedlings. Canadian Journal of Forest Research–Revue Canadienne de Recherche Forestiere 29, 332–338. Webb, M.S. and Steponkus, P.L. (1993) Freeze-induced membrane ultrastructural alterations in rye (Secale cereale) leaves. Plant Physiology 101, 955–963. Webb, M.S., Hui, S.W. and Steponkus, P.L. (1993) Dehydration-induced lamellar-to-hexagonal-II phase transitions in DOPE–DOPC mixtures. Biochimica et Biophysica Acta 1145, 93–104. Weiser, C.J. (1970) Cold resistance and injury in woody plants. Science 169, 1269–1278. Wise, M.J. and Tunnacliffe, A. (2004) POPP the question: what do LEA proteins do? Trends in Plant Science 9, 13–17. Wisniewski, M., Close, T.J., Artlip, T. and Arora, R. (1996) Seasonal patterns of dehydrins and 70-kDa heatshock proteins in bark tissues of eight species of woody plants. Physiologia Plantarum 96, 496–505. Wisniewski, M., Webb, R., Balsamo, R., Close, T.J., Yu, X.M. and Griffith, M. (1999) Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiologia Plantarum 105, 600–608. Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology 57, 781–803. Yanovsky, M.J. and Kay, S.A. (2002) Molecular basis of seasonal time measurement in Arabidopsis. Nature 419, 308–312. Yu, J.M., Pressoir, G., Briggs, W.H., Bi, I.V., Yamasaki, M., Doebley, J.F., McMullen, M.D., Gaut, B.S., Nielsen, D.M., Holland, J.B., Kresovich, S. and Buckler, E.S. (2006) A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nature Genetics 38, 203–208. Yu, X.M. and Griffith, M. (1997) Characterization of winter rye antifreeze proteins in their native forms. Plant Physiology 114, 602–602. Zamani, A., Sturrock, R., Ekramoddoullah, A.K.M., Wiseman, S.B. and Griffith, M. (2003) Endochitinase activity in the apoplastic fluid of Phellinus weirii-infected Douglas-fir and its association with over wintering and antifreeze activity. Forest Pathology 33, 299–316.

Index

Note: page numbers in italics refer to figures and tables abscisic acid (ABA) 32 bud development signal 93 bud dormancy induction 112 cold stress 111 drought stress 111 mutants 47 phytochrome effects 112 regulation by sucrose 112 acetate 168, 169 acid precipitation 173 acid shock 173 acid snow stress 173–180 cause of injury 176–177 cell damage 174–176 light damage 174, 177 tolerance of wheat 177–179 acid tolerance, wheat leaves 177–179 actin filaments 68 adaptation of cryophytes 147–148 adaptive traits, clinal variation 104–105 agamospermy, cryophytes 147 AGL15 48, 49 alanine aminotransferase (AAT) gene 81, 82 alcohol dehydrogenase (ADH) activity in Arabidopsis shoots 84 NADH+-dependent activity 84, 86 alcohol dehydrogenase (ADH) gene 80 expression during cold shock 81, 82, 86 algae, Arctic 155–156 Alpine plants adaptation 148 cold-hardened 146 Alpine tundra 142

Alpine zone 145–146 amplified fragment length polymorphism (AFLP) 93 Antarctic 143–144 climate change 198 irradiance variation 198–199 photoperiod variation 198–199, 206–207 vegetation 199 Antarctic vascular plants Calvin cycle enzymes 206 carbohydrates 206–209 climate change effects 199, 201 cold hardiness 198–209 freeze resistance 201–203 photoprotective strategies 204–205 photosynthesis 203–206 rate 209 sucrose 206–208 UV-B radiation effects 199, 201 antifreeze activities 26 antifreeze glycoproteins 35 antifreeze proteins 6, 26, 35 cold acclimation 279 cold hardiness candidate gene in trees 295–296 role 215 xylem parenchyma cell deep supercooling 34–35 antifreeze sugars 6 anti-ice nucleators 6 antifreeze proteins/glycoproteins 35 deep supercooling in woody plants 7–8, 36–37 secondary metabolites 35–37 305

306

Index

anti-nucleating activity 23–25 antioxidant pathways freezing stress 271, 272 freezing tolerance 271, 272–275, 276 antioxidant species, lucerne 271, 272–275, 276 apomixis, cryophytes 147 apoplasts antifreeze proteins 26 freeing patterns 216 freezing patterns 215 ice nucleation sites 215 pathogenesis-related proteins 26 apoptosis, cold-induced 245 aquaporin water channels 113 aquaporins 67–68, 77 water transport regulation 113 Arabidopsis chilling stress determinants 262–268 cold-shock responses 80–88 C-repeat binding factor 122–123 freezing tolerance 271 FT gene 111 proline accumulation 271 VrCBF1 and VrCBF4 transgenic 135, 136 Arabidopsis cold-shock proteins see AtCSP(s) Arctic see Canadian Arctic Arctic outbreaks, winter-freeze event relationship 195–196 Arctic plants adaptation 148 environmental stress 154 non-vascular 155–156 phenotypic plasticity 149 survival in cold-region environments 146–150 transplant experiment 157, 158, 159 Arctic tundra distribution of closely related species 153 energy budget 142 plant communities 152–153 solar radiation 142 ascorbate antioxidant pathway 271–275, 276 ascorbate peroxidase cold-acclimated wheat leaves 179 lucerne 272, 274 ascorbate–glutathione (A–G) cycle 272, 273–274 lucerne 273–274, 275 aspen, microarray studies 294, 295 association mapping, cold hardiness 299–300 AtCSP(s) 45, 46–49, 55–60 DNA polymorphic variation of promoter region 48–49 expression developmental-related 47–48 response to cold 57 stress-related 46–47 transcriptional regulation 48 AtCSP2 56–60

developmental control 58–59 expression regulation 58–59 response to cold 57 GUS fusion gene 58–59 nucleic acid-binding activity 57 RNA chaperone activity 57–58 subcellular localization 59–60 AtCSP4 48–49, 57 Australia 143 autumn warming, conifers 257–258 avens, Arctic 149–150, 154

bacteria 155 bark, ice-nucleating activities 25 Bcl-2 anti-apoptosis protein 245 betaine/proline transporter 76 biochemical regulatory mechanisms for plant tissue freezing behaviours 23–26 antifreeze activities 26 anti-nucleating activities 23–25 ice-nucleating activity 25–26 supercooling stabilizing factors 23–25 boreal forests 249 cold hardiness of trees 293–300 brefeldin A 263, 266 bud(s) deep supercooling 7–8, 19, 20–22 ice-nucleating activities 25–26 bud acclimation to dehydration/cold 93 bud break 73 gibberellic acid regulatory effect 113 bud development components 93 molecular timetable 94 stages 95 transcription factors 93 bud dormancy 93 abscisic acid in induction 112 chilling 93 transcriptome data 93 bud flush 91 bud formation 93 climate change 92 bud scales, ice-nucleating activities 25–26 bud set 91–96 genetic approaches 95–96 gibberellic acid regulatory effect 113 metabolomic approach 92–93, 94 quantitative trait loci 95–96 regulatory pathways 111 temperature 109 timing 108 transcriptomic approach 92–93, 94 butyrate 168, 169 bZIP-like transcription factor 77

Index

calcium influx, cold acclimation 245 calcium-dependent protein kinases (CDPKs) 245 Calvin cycle carbon dioxide fixation 208 electron sink capacity 253 enzymes 208, 209 Antarctic vascular plants 206 Canadian Arctic algae 155–156 cold hardiness latitudinal gradient 140–160 distribution of closely related species 153 horticultural experiment 157, 158, 159 non-vascular plants 155–156 postglacial plant reinvasion 150–154 survival in cold-region environments 146–150 carbohydrates Antarctic vascular plants 206–209 freezing tolerance 222 plasma membrane protection 297 potato leaf measurement 280–281, 289, 290–291 see also soluble sugars; sucrose carbon, gain in summer 250 carbon dioxide accumulation cereals 222 under ice 166 ice encasement causing plant death 168 elevated concentrations 183–187 cold acclimation effects 184–186 fixation by Calvin cycle 208 freezing stress effects 184 release in winter 250 temperature effects on assimilation 249 catalase 272, 275, 276 CBF genes 119–126 cold-inducible elements 133 encompassing FR-2 124 eudicot 132–133 expression 120, 135–136 regulation in cereals 124–125 genetic analyses 124 identification of candidate at FR-2 125–126 light-inducible elements 133 monocots 123–124 paralogues 132–133 rapid stress response elements 133 CBF proteins COR gene transcription activation regulation 133–135 C-terminus 134 nuclear localization sequence 133, 134 PEST domains 134–135 CBF/DREB transcription factors 47, 122–123 CBF/DREB1 genes 131–137 CBF/DREB1 proteins 133–135 CCHC zinc fingers 45

307

Cdc20 homologue 75 cell cycle, dormancy/phytohormone effects 113 cereals apical meristem 219 carbon dioxide accumulation 222 CBF genes 119–126 expression regulation 124–125 cell size 217 cold acclimation 221–222 crown 216–217 freezing tolerance 217 metabolomics 221 roots 217, 219 sucrose accumulation 222 vernalization 120, 221, 222 winter hardiness 119–126 genetics 122 growth habit form 119–120 see also wheat chalcone synthase 73 chamaephytes 147 chaperones, cold acclimation 279, 291 chilling bud dormancy 93 injury in wheat 17 chilling stress determinants in Arabidopsis 262–268 ferric reductase 268 glycine betaine 264–268 membrane lipid composition 267 reactive oxygen species 266, 267–268 root growth rate 265–266 see also cold stress; freezing stress chi-squared technique, iterative 190–193, 194 chlorophyll, simulated acid snow stress damage 175, 176, 177 chlorosis, conifers 230 citrate 169 Citrus 76–77 genes upregulated in response to low temperature 77 protein profiles 76 transcript profiles 76–77 climate 146 ice damage 164–165 synoptic climatology 191 climate change 108 Antarctic Peninsula 198 Antarctic vascular plant effects 199, 201 autumn warming 257–258 colonization of Arctic tundra plant communities 153 conifers 249–258 continental ice sheet melting 151–152 elevated carbon dioxide concentration 183–187 freezing injury 108

308

climate change (continued) fruit tree production impact 190–196 growing season 249, 251 growth cessation 108 Iceland 163–170 phenology changes 91, 183–184 photosynthesis 249–258 tree growth cessation 92 winter damage in Iceland 169 winter-freeze event impact on fruit trees 190–196 climatic adaptation, Norway spruce 99–105 climatic zones 143 clubmosses 141 cold acclimation 26, 279 antifreeze proteins 279 artificial 220–221 ascorbate–glutathione cycle enzymes 272 calcium influx 245 carbon dioxide concentration effects 184–186 cereals 221–222 chaperones 279, 291 cold tolerance 56 conifers 226–237 patterns 231–233 dehydrins 202–203, 220–221, 279 DRM fraction function 66–68 protein composition 64–66 fatty acid desaturation 231 freezing pattern 216–217 gene expression 294–295 growth cessation 108 membrane component reconstitution 68 membrane microdomain protein composition 64–66 function 66–68 membrane microdomains 62–68 metabolite accumulation 279 natural 220–221 phases 92 photoperiod 255, 256, 257 plasma membrane lipid composition 231 plasma membrane proteins 63–68 potato 280, 281–282 seasonal variation 183–184 signal transduction 68 sucrose-phosphate synthase 207–208 supercooling capability of xylem parenchyma cells 30, 32 temperature 109, 255, 256, 257 response curves 232–233 trees 73 winter wheat 173–180 woody perennials 72–73

Index

cold deacclimation conifers 231–232 supercooling capability of xylem parenchyma cells 30, 32 cold exposure, potato leaf proteins 279–291 cold hardening buds 92 see also rapid cold-hardening response cold hardiness adaptation 147–148 Antarctic vascular plants 198–209 association mapping 299–300 candidate genes in trees 295–297 conifers 249–258 environmental stress |154 evolution 140–160 forest tree genomics 293–300 gene expression linkage with molecular physiology 294–297 physiology of plants 150 plasma membrane 297 population genomics 297–300 postglacial plant reinvasion 150–151 pre-adaptation 146–147 QTL mapping 298, 299 survival in cold-region environments 146–150 strategies 148–150, 154 cold shock 214 ADH gene expression 82, 86 ethanol levels 82, 84 ethanolic fermentation 80–88 gene knockout studies 86 hypoxia 86–87 insect tolerance 240–241 oxygen deficit 86–87 see also cold-shock domain (CSD) proteins cold stress abscisic acid levels 111 CBF gene induction 132 phospholipases 279 protein kinases 279 transcription factors 279 see also chilling stress; freezing stress cold tolerance 56 CBF gene expression 135 conifers 226–237 midwinter low-temperature 227–228 parameters 229 sugars 233–235 cytoskeleton 68 exogenous ethanol application 84–85 herbage species 165–166 plasma membranes 62–69 sugars 233–235 vitrification 235–236, 237 see also freezing tolerance; frost tolerance cold-induced genes 32–33

Index

cold-region environments survival in 146–150 see also Antarctic; Arctic entries; Canadian Arctic cold-responsive genes, fruit trees 72–78 Citrus 76–77 Malus 73–74 Prunus 74–75 Vaccinium 75–76 cold-responsive plasma membrane proteins 63 cold-shock domain (CSD) proteins 43–51 AtCSPs 45, 46–49 developmental-related expression 47–48 DNA polymorphic variation of promoter region 48–49 stress-related expression 46–47 transcriptional regulation 48 eukaryotic 44, 55–56 evolution 45 FRSL motif 45–46 gene expression analyses 46–49 localization 49–50 novel 56 plant 44–51 post-translational modification 45–46 RNA secondary structure 43 sumoylation sites 46 tissue-specific expression patterns 50 cold-shock proteins (CSPs) 43, 44–51 E. coli 55 evolution 45 sumoylation sites 46 Colobanthus quitensis (pearlwort) 199, 200, 201–208 colonization, Arctic tundra plant communities 152–153 competition, post-glacial plant reinvasion 152–153, 154 conifers acclimation 226–237 autumn warming 257–258 carbon sink 249 chlorosis 230 climate change 249–258 cold acclimation gene expression 294–295 cold deacclimation 231–232 cold hardiness 249–258 cold tolerance 226–237 midwinter low-temperature 227–228 parameters 229 cytoplasmic vitrification 235–236, 237 frost tolerance 257–258 gene response to day length/temperature 257 liquid nitrogen quench tolerance 230, 231 low temperature injury 228, 230–231 microarray studies 294, 295 necrosis 230–231

309

photosynthesis 249–258 spring warming/temperature 253, 254, 255 vitrification 235–236, 237 see also Norway spruce continental migration 143–144 cooling rate, freezing pattern 217–218 copper/zinc–superoxide dismutase (Cu/Zn–SOD) 275, 276 cortical parenchyma cells (CPCs) 29 extracellular freezing 33 soluble sugars 34 courtship behaviour, rapid cold-hardening response 242 C-repeat binding factor (CBF) transcriptional factor 120, 122–123, 220 cold hardiness candidate gene in trees 296–297 see also CBF genes; CBF proteins critical thermal minimum (CTmin), rapid cold-hardening response 241–242 cryophytes adaptation 147–148 distribution of closely related species 153 ecological succession 142–143 environmental stress |154 evolution 142–146 monocots 147 non-vascular 155–156 physiology 150 post-glacial plant reinvasion 150–154 pre-adaptation 146–147 survival in cold-region environments 146–150 strategies 148–150, 154 cryoprotectants, rapid cold-hardening response 243, 245 cryptophytes 147 crystallization, heat release 216 CspA 43 CspB 43 CspG 43 CspI 43 cushion dicots 149 cyclins, B-type 75 cytoplasmic P-bodies 49–50 cytorrhysis 31, 215 cytoskeleton, cold tolerance 68

day length critical 95 see also photoperiod deep supercooling 1–2 buds 19, 20–22 differential thermal analysis 217 extra-organ freezing in shoot/flower primordia 21–22 ice nucleation inhibition 24, 25

310

deep supercooling (continued) temperature limit 31 trees 217 woody plant buds/xylem 7–8 xylem 19, 22, 29–37 freezing patterns 217 xylem parenchyma cells maximum temperature limit 30 dehydration, freeze-induced 218 dehydrins 6, 77 cold acclimation 202–203, 220–221, 279 cold hardiness candidate gene in trees 295 freezing tolerance 222 K segment 202–203 Malus 73 Prunus 74 temperature effect 113–114 water status changes 114 xylem parenchyma cells 35 dehydroascorbate (DHA) 273, 274 dehydroascorbate reductase (DHAR) 272, 274, 275, 276 Deschampsia antarctica (hair grass) 199, 200, 201–207 dicots cold-hardened 147 cushion 149 leafy 149 tundra perennial 149 differential scanning calorimetry (DSC) 227 modulated temperature 236, 237 differential thermal analysis (DTA) 7, 19 anti-nucleating activity 24–25 deep supercooling 217 extra-organ freezing 20, 21 NMR imaging 23 dormancy cell cycle 113 growth cessation 109 induction 109, 110 inhibition 110 levels 92 photoperiod 110 plasmodesmata function 113 processes 92 temperature influence on induction 108–114 see also growth cessation drought stress abscisic acid levels 111 CBF gene expression 135 Dryas cushions 149–150 Dryas integrifolia (Arctic avens) 149–150, 154

early light-inducible protein (ELIP) 76, 77 ecological succession 141–143

Index

electrolyte leakage frost tolerance 219 see also relative electrolyte leakage (REL) embryo development, epigenetic memory 99–105 genes involved 105 temperature 102, 103, 104, 105 embryophytes 140 endodormancy 72–73 Prunus 75 energy absorption modulation 204 charge in cold shock induction of ethanolic fermentation 87 dissipation of excess 203, 204, 205–206 environmental stress, Arctic species 154 environments, extreme 147–148 Escherichia coli, cold-shock proteins 55 ethanol fermentation during cold shock 80–88 pathway 83 fluidizing agent for membranes 84–85 levels in cold shock 82, 84 protoplast effects in freeze–thaw stress 85, 86 seasonal pattern of accumulation in trees 87–88 ethylene, bud development signal 93 eudicots CBF genes 132–133 stress-responsive signalling pathway regulation by CBF/DREB1 genes 131–137 evolution of plants 140–142 cold-hardened 142–146 continental migration 143–144 ecological succession 141–142 glaciations 145–146 orogeny 144–145 periodic mass devastation 141 punctuated equilibria 141 silent disasters 143–146 STOP and GO pattern 141 expansion-induced lysis (EIL) 86, 87 extracellular freezing 20, 21, 29 extreme environments 147–148 extremophiles 156

fatty acids, desaturation 231 ferns 141 ferric reductase, membrane-localized 268 flavonol glucosides 36–37 flowering plants 141 forests boreal 249, 293–300 temperate 293–300 FR-2 124, 125–126 freeze resistance, Antarctic vascular plants 201–203

Index

freezing cooling rate 217–218 extra-organ in shoot/flower primordia 20–22 initiation site 2–3, 4, 5 patterns 214–223 plant structure 216–217 plant tissue behaviours 19–27 biochemical regulatory mechanisms 23–26 slow equilibrium 218 supercooling 217 vitrification 218, 235–236 see also extracellular freezing; winter-freeze events freezing injury 174 cereal root initiation impairment 217, 219 climate fluctuations 108 cold-induced apoptosis 245 conifers 228, 230–231 manifestation 218–220 see also frost damage freezing stress antioxidant pathways 272 elevated carbon dioxide concentration 184 see also chilling stress; cold stress freezing tolerance 214 Antarctic vascular plants 202 antioxidant pathways 271, 272–275, 276 Arabidopsis 271 carbohydrate levels 222 cereals 217 cold-acclimated wheat seedlings 179 lucerne 271 genes 274–275, 276 potato 280 see also cold tolerance; frost tolerance frost 219 frost damage elevated carbon dioxide concentrations 183–187 combined effect with climate warming 186–187 photobleaching 219 trees 163, 164 wheat 12–17 see also freezing injury Frost Resistance (FR) genes 120, 122 CBF genes 124, 125–126 frost tolerance conifers 257–258 electrolyte leakage 219 herbage species 165–166 tissue age 219 see also cold tolerance; freezing tolerance fructans 206, 207 fructose, cold tolerance in conifers 233, 235 fructose-1,6-bisphosphatase (FBPase) 208–209

311

fruit trees climate change effects on production 190–196 cold-responsive gene expression 72–78 Malus 73–74 production association with minimum temperature 193, 194, 195 temperature sensitivity 109 winter-freeze events 190–196 FT gene 111 fungi 155

galactinol plasma membrane protection 297 potato content 289, 290 galactose, potato content 289 genomics cold hardiness in forest trees 293–300 population 297–300 gibberellic acids 112–113 glaciations 145–146 post-glacial plant reinvasion 150–154 glass formation, intracellular 218, 235–236, 237 glass transition 236, 237 1,3-beta-glucan 113 glucocerebrosides, plasma membrane 62 glucose cold tolerance in conifers 233, 235 potato content 289, 290 glucose-6P-dehydrogenase 289, 290 beta-glucouronidase (GUS) fusion gene 58–59, 135 glutathione see ascorbate–glutathione (A–G) cycle glycine betaine 262–268 reactive oxygen species decrease 264, 266, 267–268 glycolytic fermentation pathway 83 Gondwana 143 graminoids ice damage to grasses 163, 164 tundra perennial 149 see also cereals grasses, ice damage 163, 164 Green Igloos Farm (greenhouse experiment, Canadian Arctic) 157, 158, 159 ground cover snow 255 species 153 growth cessation abiotic stress 111 cold acclimation 108 dormancy 109 gibberellic acids 112–113 night length/temperature 109 photoperiod 255, 256, 257 plant adaptation to climate change 108

312

Index

growth cessation (continued) stress-induced 111 temperature 109, 255, 256, 257 see also dormancy growth cycle, seasonal in poplar 91–96 GUS reporter gene 58–59, 135

hair grass 199, 200, 201–207 Halliwell–Asada pathway 204 hay production damaged fields 168 Iceland 164, 165 winter damage 164–165 heat-shock proteins (HSPs) 32 rapid cold-hardening response 243–244, 245 hemi-cryptophytes 147 herbs, Arctic 148–149 heterogeneous nucleators 2 high-resolution IR thermography 1, 3, 4 high-temperature exotherm (HTE) 23, 24, 25 hinokitiol 35 homeoviscous adaptation 81, 87 homogeneous nucleation temperature 2 Hsp70 73, 77 hydrogen peroxide, simulated acid snow stress damage 176 hypoxia, cold-shock induced 86–87 hysteresis proteins (THPs) 6

ice, apoplastic 216 Ice Ages 144, 145 postglacial plant recolonization 150–152 ice crystals mechanical damage 215 structure 216 ice damage agriculture in Iceland 164 climate 164–165 grasses 163, 164 ice encasement damage 163–170 phytotoxins 168–169 plant death 168 ice nucleation 1–8 activity 25–26 carbon dioxide concentration effect on temperature 184 factors affecting 3–6 freezing patterns 215 hydrophobic barriers to ice propagation 4 inhibition 24, 25 initiation site 2–3, 4, 5 moisture factors 3–4, 219–220 plant structure factors 4–6 sites 215 soluble sugar effects on temperature 34

ice propagation 1–8 barriers 218 hydrophobic 4 direction 5–6 plant structure factors 4–6 prevention 22 rate 5–6 ice tolerance, herbage species 165–166 Iceland climate change 163–170 hay production 164, 165 ice damage climate 164–165 impact on agriculture 164 winter damage 163, 164 climate change impact 169 winter temperatures 164–165 ice-nucleating activity (INA) 25–26 ice-nucleating agents 3–4, 6 ice-nucleation-active (INA) bacteria 1, 3, 4, 6 deep supercooling in xylem parenchyma cells 36 ice nucleation inhibition 24, 25 ICEr1 and ICEr2 133 insects, rapid cold-hardening response 240–246 interglacial periods 145 isolated water droplet theory 30–31 iterative chi-squared technique 190–193, 194

kaempferol-3-O-beta-glucoside 37 kaempferol-7-O-beta-glucoside 37 krummholz 146

lactate dehydrogenase gene 81, 82 late embryogenesis-abundant (LEA) proteins 32, 33, 77 Prunus 74 temperature effects 113–114 see also dehydrins latent heat 216 Laurasia 143 leaf maturity and freezing pattern 216 moisture and ice nucleation temperature 3–4, 219–220 lichens 155 light acid snow stress 174, 177 Antarctic 198–199 bud development signal 93 CBF gene induction 132 photosynthesis onset in spring 253, 254 stress for wintering plants 174 see also photoperiod

Index

light signalling, cold hardiness candidate gene in trees 296 light-harvesting complex II (LHCII) 251–252 proteins 255, 256, 257 linkage disequilibrium 298 lipid peroxidation, cold-acclimated wheat leaves 179 liquid nitrogen quench tolerance, conifers 230, 231 longevity of insects, rapid cold-hardening response 242 loss of osmotic responsiveness (LOR) 86, 87 low-temperature exotherm (LTE) 24, 25, 217 lucerne antioxidant genes 274–275 antioxidant species 271, 272–275, 276 ascorbate antioxidant pathway 271–275, 276 ascorbate–glutathione cycle 273–274, 275 freezing tolerance 271

macroevolution 143 malate 168, 169 Malus trees genes upregulated in response to low temperature 77 protein profiles 73 temperature sensitivity 109 transcript profiles 73–74 metabolite concentrations ice encasement causing plant death 168 plant metabolism under ice 166–167 winter cereals/grasses 167 metabolomics bud set 92–93, 94 cereals 221 rapid cold-hardening response 243 methionine synthase 291 8-methoxykaempferol-3-O-beta-glucoside 37 microclimate 146 microevolution 145 microRNA (miRNA) processing 49–50 microtubules 68 mitogen-activated dependent protein kinases (MAPKs) 245 modulated temperature differential scanning calorimetry (MT-DSC) 236, 237 monocots CBF genes 123–124 cryophytes 147 monodehydroascorbate reductase (MDHAR) 272, 274, 275, 276 mosses, cryophyte 155 mountain formation 144–145 Myb family transcription factor 75 mycorrhizal associations, Dryas cushions 150

313

NADH/NADPH oxidation 86 NADPH 251 NADPH oxidase (NOX) 267–268 necrosis, conifers 230–231 Nemoral vegetation 144, 145 night length cold acclimation phase 92 growth cessation 109 sensing mechanism 91 see also photoperiod NMR micro-imaging 3 advantages 23 anti-nucleating activity 24–25 freezing behaviours in plant tissues 19–23 interpretation problems 22–23 technical problems 22–23 technique 20, 23 woody plant tissues 20–22 non-photochemical quenching (NPQ) 204, 206, 251 autumn downregulation of photosynthesis 257 zeaxanthin-dependent 252 North American continent 144 Norway spruce climatic adaptation 99–105 embryo abortion 102 environmental conditions during female flowering 101 genes in molecular mechanism for progeny adaptive differences 105 memory from embryo development 102, 103, 104, 105 orchard seed production 100 phenotypic clinal variation 104–105 pre-/post-meiotic selection 102 seed production 100 somatic embryogenesis 102, 103, 104 temperature treatment at reproductive stages 101–102 zygotic embryogenesis 102, 103, 104 nuclear localization sequence (NLS) of CBF proteins 133, 134 nunatak refugia hypothesis 151

orogeny 144–145 osmotic concentration cold-acclimated wheat seedlings 178, 179 intracellular of xylem parenchyma cells 34 oxidative stress, simulated acid snow stress damage 176 oxygen deficit in cold shock 86–87

pathogenesis-related (PR) proteins 26 PCA60 see dehydrins pearlwort 199, 200, 201–208 periglacial lakes 151

314

Index

permafrost 142 phenology 91 climate change 183–184 phenotypic plasticity, Arctic plants 149 pheophytin 177 phosphatidylcholine 62–63 phosphatidylethanolamine 63 phosphoenolpyruvate carboxylase (PEPCase) 76 phospholipases, cold stress 279 phospholipids, plasma membrane 62–63 phosphorus, inorganic (Pi) 207, 208 photobleaching 219 photoinhibition, Antarctic vascular plants 202, 204 photoperiod Antarctic 198–199, 207 autumn 257 CBF gene expression 125 cold acclimation 255, 256, 257 critical day length 95 dormancy 110 growth cessation 255, 256, 257 see also night length photosynthesis acclimation of processes 251–252 Antarctic vascular plants 203–206 rate 209 climate change effects 249–258 in the cold 203–206 conifers 249–258 downregulation in autumn 255, 256, 257–258 electron sink capacity acclimation 252 electron transport 251 light effects on onset in spring 253, 254 metabolic sink 251 protein abundance with cold exposure 291 spring recovery 253, 254, 255 temperature effects on onset in spring 253, 254 photosynthetic apparatus, simulated acid snow stress damage 175–176, 177 photosynthetic electron transport chain 204–205 photosystem I complex (PSI) 204, 205, 206, 252 reaction centre 252, 253, 254 photosystem II complex (PSII) 204–205, 251–252 reaction centre 253, 254, 255, 256, 257 quenching 252 photoxidative stress induction 111 phytochrome abscisic acid effects 112 cold hardiness candidate gene in trees 296 temperature sensitivity 110–111 phytohormones, temperature effects 112–113 phytotoxins 168–169 plant evolution see evolution of plants plant metabolism under ice 166–167, 168 plasma membrane

aquaporins 67–68 carbohydrates in protection 297 cold hardiness function 297 cold sensor function 245 composition 62–63, 297 detergent-resistant membrane fractions 62, 63–68 cold acclimation 66–68 protein composition 64–66, 67 ethanol as fluidizing agent 84–85 fluidity change cold acclimation 297 rapid cold-hardening response 244–245 glucocerebrosides 62 lipid composition 231 chilling sensitivity 267 low temperature tolerance 233 phospholipids 62–63 composition change in rapid coldhardening response 244–245 rapid cold-hardening response 244–245 remodelling 297 rupture prevention 297 sterols 62, 63 plasma membrane microdomains 63 functions 67–68 preparation 63–64 protein composition 64–66 protein function in cold acclimation 66–68 plasma membrane proteins, cold acclimation 63–68 plasmalemma, laceration 215 plasmodesmata 113 plastoquinone 252 Pleistocene glaciations 146 postglacial plant reinvasion 150–151 pollination, cryophytes 147 polypeptides low temperature response 75 Vaccinium 75 polyploidy, cryophytes 147 poplar, seasonal growth cycle 91–96 Populus (aspen), microarray studies 294, 295 post-glacial plant reinvasion 150–154 potato carbohydrate content 280–281, 289, 290–291 cold acclimation 280, 281–282 cold stress tolerance 280 freezing tolerance 280 methionine synthase 291 proteins cold-exposed leaves 279–291 extraction 281, 282, 283, 284, 285–288, 289 PpCSPs 49–50 PpDhn1 74 pre-adaptation/pre-selection 146–147

Index

proline 271 prostrate mats 149 prostrate shrubs 146, 148 protein kinases, cold stress 279 protein phosphatase 2A regulatory subunit (PP2A) 32 protoplasts cryoinjury 86, 87 ethanol effects in freeze–thaw stress 85, 86 Prunus 74–75 genes upregulated in response to low temperature 77 protein profiles 74–75 transcript profiles 75 PtF1 gene expression 111 pyridine nucleotide redox poise 87 pyruvate decarboxylase (PDC) gene 80, 289, 290 expression in cold-shock response 81, 82 pyruvate kinase (PK) gene 81, 82

quantitative trait loci (QTL) bud set 95–96 mapping for cold hardiness 298, 299 Quaternary Glaciation 145 quercetin-3-O-beta-glucoside 37

raffinose cold tolerance in conifers 233, 234, 235 deep supercooling in xylem parenchyma cells 34 glass transition temperature 236 plasma membrane protection 297 potato content 289, 290 raffinose family oligosaccharides (RFOs) 206 rapid cold-hardening response cold-induced apoptosis 245 courtship behaviour 242 critical thermal minimum 241–242 cryoprotectants 243, 245 ecological significance 240–242 gene expression 243–244 heat-shock proteins 243–244, 245 induction 242–245 pathways 245 insects 240–246 longevity 242 membrane fluidity/phospholipid changes 244–245 metabolomics 243 physiological mechanisms 242–245 reproductive success 242 survival of low temperature 240–241 thermocycles ecologically relevant 241–242 protection generation 244 in vitro 244

315

reaction centre quenching 252 reactive oxygen species (ROS) 168 acid snow stress 174 Arabidopsis 264, 266, 267–268 chilling stress 266, 267–268 freezing patterns 216 glycine betaine treatment 264, 266, 267–268 plant damage 204 relative electrolyte leakage (REL) conifer cold tolerance/acclimation 227, 228, 230–233 necrosis signs 230–231 temperature curve 228 reproductive success, rapid cold-hardening response 242 respiration under ice 166 RING (Really Interesting New Gene) finger protein 75, 77 Rop GTPases 268 RuBisCO 208–209 Citrus 76

Salix distribution 153 Saxifraga distribution 153 Saxifraga oppositifolia (purple saxifrage) 149, 150 seed plants 141 self-pollination in cryophytes 147 shrubs, Arctic 146, 148 Sibbaldia procumbens (creeping sibbaldia) 167 signal transduction, cold acclimation 68 silent disasters 143–146 snow cover 255 solar radiation, Alpine tundra 142 soluble sugars cold-acclimated wheat seedlings 178, 179 intracellular 33–34 species ground cover 153 stachyose cold tolerance in conifers 233, 234, 235 glass transition temperature 236 sterols, plasma membrane 62, 63 stress and ripening (ASR) proteins 32 stress tolerance, post-glacial plant reinvasion 152–153 stress-associated genes 112 stress-responsive signalling pathway regulation by CBF/DREB1 genes 131–137 succession, primary 152 succinate 169 sucrose accumulation in cereals 222 Antarctic vascular plants 206–208 cold tolerance in conifers 233–235 cryoprotection 203

316

sucrose (continued) deep supercooling in xylem parenchyma cells 34 freezing tolerance 222 glass transition temperature 236 level change with temperature 111–112 plasma membrane protection 297 potato content 289, 290 signalling molecule function 112 sucrose-phosphate synthase 203 cold acclimation 207–208 sugars antifreeze 6 cold tolerance 233–235 conifers 233–235 cytoplasmic vitrification 236 freezing tolerance 222 ice crystal structure modification 216 plasma membrane protection 297 protein interactions 215 see also soluble sugars sulfuric acid, simulated acid snow stress 174–177 SUMO (small ubiquitin-like modifier) 46 supercooling 217 freezing pattern 216 leaf moisture 219 stabilizing factors 23–25 wheat 14–15, 16, 17, 218 see also deep supercooling supercooling related xylem (SXL) genes 32 superoxide 264, 268 superoxide dismutase Antarctic vascular plants 204, 205 ascorbate–glutathione cycle 272 cold-acclimated wheat leaves 179 copper/zinc 275, 276 survival of woody plants, temperature influence 108–114 SXL (supercooling related xylem) genes 32 synoptic climatology 191

tachyose, deep supercooling in xylem parenchyma cells 34 Taraxacum distribution 153 temperate forests 293–300 temperature Alpine tundra 142 autumn 257 bud set 109 CBF gene expression 125 cold acclimation 109, 255, 256, 257 drop 92 response curves 232–233 conifers in spring 253, 254, 255 dehydrin effects 113–114 dormancy induction influence 108–114 drop in cold acclimation phase 92

Index

gibberellic acid effects 112–113 growth cessation 109, 255, 256, 257 ice nucleation effects of carbon dioxide concentration 184 night and growth cessation 109 photosynthesis onset in spring 253, 254 phytohormone effects 112–113 plant survival 108–114 rapid cold-hardening in insect survival 240–241 relative electrolyte leakage curve 228 soil impact of snow cover 255 spring 253, 254, 255 sucrose level change 111–112 water status 113, 114 winter in Iceland 164–165 thermal hysteresis proteins (THPs) 6 thermoperiodism 110 thylakoid protein composition 253, 255 transcription factors bud development 93 cold hardiness candidate gene in trees 296–297 cold stress 279 trans-thylakoid pH gradient 251 trees association mapping of cold hardiness 299–300 bud break 73 bud flush 91 bud set 91–96 cold acclimation 73 cold hardiness genomics 293–300 QTL mapping 298 deep supercooling 217 ethanol accumulation seasonal pattern 87–88 frost damage 163, 164 growth cessation in winter 72–73 see also conifers; forests; fruit trees; woody plants; xylem triose phosphates 208 tubulins 68

ultraviolet light B (UV-B) radiation, Antarctic vascular plant effects 199, 201

Vaccinium 75–76 genes upregulated in response to low temperature 77 protein profiles 75 transcript profiles 75–76 vernalization 120 cereals 221, 222 requirement 120–121 length 121

Index

violaxanthin 252 vitrification cold tolerance 235–236, 237 freezing pattern 218 VRN-1 gene 120–122, 125

water status, temperature effects 113, 114 water stress, freezing pattern alteration 215 water–water cycle 204–205 WCSP1 protein 44–45, 56 localization 59 weather 146 wheat chill injury 17 cold-acclimated 173–180 freezing pattern 14, 15 frost damage 14–15, 16 yield losses 12 frost resistance assessment 17 frost testing 13–14 ice formation in stem 218 ice nucleation 15, 17 leaf segment/tissue survival with acid snow stress 174–175 low-temperature damage 12–17 supercooling 14–15, 16, 17, 218 tolerance to freezing damage 13, 17 yield losses to frost damage 12 wind-pollination, cryophytes 147 winter hardiness of cereals 119–126 genetics 122 growth habit form 119–120 winter-freeze events Arctic outbreak relationship 195–196 fruit tree impact 190–196 iterative chi-squared technique 190–193, 194

317

woody plants anti-ice nucleators 7–8, 36–37 cold-responsive gene expression in fruit trees 72–78 deep supercooling 7–8 freezing behaviours 19 NMR micro-imaging 20–22 temperature influence on dormancy induction/ plant survival 108–114 see also xylem WXL (winter accumulated xylem) genes 32 WXL5 gene 37

xanthophyll cycle pigments 252, 255 xylem, deep supercooling 7–8, 19, 22, 29–37 freezing patterns 217 xylem parenchyma cells (XPCs) deep supercooling 29–37 anti-ice nucleation substances 35–37 gene expression in supercooling 31–33 intracellular substances 33–37 isolated water droplet theory 30–31 maximum temperature limit 30 protein effects 34–35 secondary metabolites 35–37 soluble sugars 33–34 stabilizing mechanisms 31 extracellular ice penetration barrier 31 intracellular osmotic concentration 34 protoplasts 30–31

Y-box proteins 44, 55–56

zeaxanthin 252, 255