Advances in Botanical Research, Volume 49

Advances in

BOTANICAL RESEARCH Series Editors JEAN-CLAUDE KADER

Laboratoire Physiologie Cellulaire et Mole´culaire des Plantes, CNRS, Universite´ de Paris, Paris, France

MICHEL DELSENY

Laboratoire Ge´nome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 32, Jamestown Road, London NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands First edition 2009 Copyright ß 2009, Elsevier Ltd. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@ elsevier.com. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made ISBN: 978-0-12-374735-8 ISSN: 0065-2296 For information on all Academic Press publications visit our Web site at elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6 5 4 3 2 1

CONTRIBUTORS TO VOLUME 49

GABRIELA AGUILETA Ecologie, Syste´matique et Evolution, CNRS, Universite´ Paris‐Sud, F‐91405 Orsay cedex, France TATIANA GIRAUD Ecologie, Syste´matique et Evolution, CNRS, Universite´ Paris‐Sud, F‐91405 Orsay cedex, France MICHAEL E. HOOD Department of Biology, McGuire Life Sciences Building, Amherst College, Amherst, MA, USA VAUGHAN HURRY Umea˚ Plant Science Centre, Department of Plant Physiology, Umea˚ University, Umea˚, Sweden JOHN Z. KISS Department of Botany, Miami University, Oxford, Ohio, USA MARIA LIA MOLAS Facultad de Agronomı´a, Universidad Nacional de La Pampa, Santa Rosa (LP), Argentina GUISLAINE REFRE´GIER Ecologie, Syste´matique et Evolution, CNRS, Universite´ Paris‐Sud, F‐91405 Orsay cedex, France ERIC RUELLAND Universite´ Pierre et Marie Curie, UMR7180, CNRS, 3 Rue Galile´e, Ivry‐sur‐Seine, France MARIE‐NOELLE VAULTIER Umea˚ Plant Science Centre, Department of Plant Physiology, Umea˚ University, Umea˚, Sweden ALAIN ZACHOWSKI Universite´ Pierre et Marie Curie, UMR7180, CNRS, 3 Rue Galile´e, Ivry‐sur‐Seine, France

CONTENTS OF VOLUMES 35–48 Series Editor (Volumes 35–44) J.A. CALLOW School of Biosciences, University of Birmingham, Birmingham, United Kingdom

Contents of Volume 35 Recent Advances in the Cell Biology of Chlorophyll Catabolism H. THOMAS, H. OUGHAM and S. HORTENSTEINER The Microspore: A Haploid Multipurpose Cell A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS The Seed Oleosins: Structure Properties and Biological Role J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY Compartmentation of Proteins in the Protein Storage Vacuole: A Compound Organelle in Plant Cells L. JIANG and J. ROGERS Intraspecific Variation in Seaweeds: The Application of New Tools and Approaches C. MAGGS and R. WATTIER Glucosinolates and Their Degradation Products R. F. MITHEN

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Contents of Volume 36 PLANT VIRUS VECTOR INTERACTIONS Edited by R. Plumb Aphids: Non-Persistent Transmission T. P. PIRONE and K. L. PERRY Persistent Transmission of Luteoviruses by Aphids B. REAVY and M. A. MAYO Fungi M. J. ADAMS Whitefly Transmission of Plant Viruses J. K. BROWN and H. CZOSNEK Beetles R. C. GERGERICH Thrips as Vectors of Tospoviruses D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL, A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN Virus Transmission by Leafhoppers, Planthoppers and Treehoppers (Auchenorrhyncha, Homoptera) E. AMMAR and L. R. NAULT Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB

CONTENTS OF VOLUMES 35–48

Contents of Volume 37 ANTHOCYANINS IN LEAVES Edited by K. S. Gould and D. W. Lee Anthocyanins in Leaves and Other Vegetative Organs: An Introduction D. W. LEE and K. S. GOULD Le Rouge et le Noir: Are Anthocyanins Plant Melanins? G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD Anthocyanins in Leaves: History, Phylogeny and Development D. W. LEE The Final Steps in Anthocyanin Formation: A Story of Modification and Sequestration C. S. WINEFIELD Molecular Genetics and Control of Anthocyanin Expression B. WINKEL-SHIRLEY Differential Expression and Functional Significance of Anthocyanins in Relation to Phasic Development in Hedera helix L. W. P. HACKETT Do Anthocyanins Function as Osmoregulators in Leaf Tissues? L. CHALKER-SCOTT The Role of Anthocyanins for Photosynthesis of Alaskan Arctic Evergreens During Snowmelt S. F. OBERBAUER and G. STARR Anthocyanins in Autumn Leaf Senescence D. W. LEE A Unified Explanation for Anthocyanins in Leaves? K. S. GOULD, S. O. NEILL and T. C. VOGELMANN

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Contents of Volume 38 An Epidemiological Framework for Disease Management C. A. GILLIGAN Golgi-independent Trafficking of Macromolecules to the Plant Vacuole D. C. BASSHAM Phosphoenolpyruvate Carboxykinase: Structure, Function and Regulation R. P. WALKER and Z.-H. CHEN Developmental Genetics of the Angiosperm Leaf C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE and R. A. MARTIENSSEN A Model for the Evolution and Genesis of the Pseudotetraploid Arabidopsis thaliana Genome Y. HENRY, A. CHAMPION, I. GY, A. PICAUD, A. LECHARNY and M. KREIS

Contents of Volume 39 Cumulative Subject Index Volumes 1–38

Contents of Volume 40 Starch Synthesis in Cereal Grains K. TOMLINSON and K. DENYER The Hyperaccumulation of Metals by Plants M. R. MACNAIR Plant Chromatin — Learning from Similarities and Differences J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI, P. ZIELENKIEWICZ and A. JERZMANOWSKI

CONTENTS OF VOLUMES 35–48

The Interface Between the Cell Cycle and Programmed Cell Death in Higher Plants: From Division unto Death D. FRANCIS The Importance of Extracellular Carbohydrate Production by Marine Epipelic Diatoms G. J. C. UNDERWOOD and D. M. PATERSON Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins A. K. CHARNLEY

Contents of Volume 41 Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection JAMES E. COOPER Investigating and Manipulating Lignin Biosynthesis in the Postgenomic Era CLAIRE HALPIN Application of Thermal Imaging and Infrared Sensing in Plant Physiology and Ecophysiology HAMLYN G. JONES Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and Transposable Elements CELIA HANSEN and J. S. HESLOP-HARRISON

Role of Plasmodesmata Regulation in Plant Development ARNAUD COMPLAINVILLE and MARTIN CRESPI

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Contents of Volume 42 Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON and IAN CUMMINS The Impact of Molecular Data in Fungal Systematics P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction HILARY J. ROGERS Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol ALYSON K. TOBIN and CAROLINE G. BOWSHER

Contents of Volume 43 Defensive and Sensory Chemical Ecology of Brown Algae CHARLES D. AMSLER and VICTORIA A. FAIRHEAD Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose Nonfermenting-1-Related Protein Kinase-1 and General Control Nonderepressible-2-Related Protein Kinase NIGEL G. HALFORD Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides AARON MAXWELL and JOHN K. SCOTT Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism MATTHEW J. DRING Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus ANNA AMTMANN, JOHN P. HAMMOND, PATRICK ARMENGAUD and PHILIP J. WHITE

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Contents of Volume 44 Angiosperm Floral Evolution: Morphological Developmental Framework PETER K. ENDRESS Recent Developments Regarding the Evolutionary Origin of Flowers MICHAEL W. FROHLICH Duplication, Diversification, and Comparative Genetics of Angiosperm MADS-Box Genes VIVIAN F. IRISH Beyond the ABC-Model: Regulation of Floral Homeotic Genes LAURA M. ZAHN, BAOMIN FENG and HONG MA Missing Links: DNA-Binding and Target Gene Specificity of Floral Homeotic Proteins RAINER MELZER, KERSTIN KAUFMANN ¨ NTER THEIßEN and GU Genetics of Floral Development in Petunia ANNEKE RIJPKEMA, TOM GERATS and MICHIEL VANDENBUSSCHE Flower Development: The Antirrhinum Perspective BRENDAN DAVIES, MARIA CARTOLANO and ZSUZSANNA SCHWARZ-SOMMER Floral Developmental Genetics of Gerbera (Asteraceae) TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI, SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM, ¨ NEN, SUVI BROHOLM, ROOSA LAITINEN, ¨ LLA EIJA PO PAULA ELOMAA and VICTOR A. ALBERT Gene Duplication and Floral Developmental Genetics of Basal Eudicots ELENA M. KRAMER and ELIZABETH A. ZIMMER

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Genetics of Grass Flower Development CLINTON J. WHIPPLE and ROBERT J. SCHMIDT Developmental Gene Evolution and the Origin of Grass Inflorescence Diversity SIMON T. MALCOMBER, JILL C. PRESTON, RENATA REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG Expression of Floral Regulators in Basal Angiosperms and the Origin and Evolution of ABC-Function PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM, ANDRE CHANDERBALI and MATYAS BUZGO The Molecular Evolutionary Ecology of Plant Development: Flowering Time in Arabidopsis thaliana KATHLEEN ENGELMANN and MICHAEL PURUGGANAN A Genomics Approach to the Study of Ancient Polyploidy and Floral Developmental Genetics JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE, ZHENGUI ZHENG, DAVID OPPENHEIMER and CLAUDE DEPAMPHILIS Series Editors (Volume 45– ) JEAN-CLAUDE KADER Laboratoire Physiologie Cellulaire et Mole´culaire des Plantes, CNRS, Universite´ de Paris, Paris, France MICHEL DELSENY Laboratoire Ge´nome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

Contents of Volume 45 RAPESEED BREEDING History, Origin and Evolution S. K. GUPTA and ADITYA PRATAP

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Breeding Methods B. RAI, S. K. GUPTA and ADITYA PRATAP The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA Development and Practical Use of DNA Markers KATARZYNA MIKOLAJCZYK Self-Incompatibility RYO FUJIMOTO and TAKESHI NISHIO Fingerprinting of Oilseed Rape Cultivars ´ ˇ URN and JANA ZˇALUDOVA VLADISLAV C Haploid and Doubled Haploid Technology L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG, Y. HE and W. J. ZHOU Breeding for Apetalous Rape: Inheritance and Yield Physiology LIXI JIANG Breeding Herbicide-Tolerant Oilseed Rape Cultivars PETER B. E. MCVETTY and CARLA D. ZELMER Breeding for Blackleg Resistance: The Biology and Epidemiology W. G. DILANTHA FERNANDO, YU CHEN and KAVEH GHANBARNIA Development of Alloplasmic Rape MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA Honeybees and Rapeseed: A Pollinator–Plant Interaction D. P. ABROL

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Genetic Variation and Metabolism of Glucosinolates NATALIA BELLOSTAS, ANNE DORTHE SØRENSEN, JENS CHRISTIAN SØRENSEN and HILMER SØRENSEN Mutagenesis: Generation and Evaluation of Induced Mutations SANJAY J. JAMBHULKAR Rapeseed Biotechnology VINITHA CARDOZA and C. NEAL STEWART, JR. Oilseed Rape: Co-existence and Gene Flow from Wild Species RIKKE BAGGER JØRGENSEN Evaluation, Maintenance, and Conservation of Germplasm RANBIR SINGH and S. K. SHARMA Oil Technology ¨ US BERTRAND MATTHA

Contents of Volume 46 INCORPORATING ADVANCES IN PLANT PATHOLOGY Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features Influencing Root Growth and Development ´ NICA CREUS, MARI´A CELESTE MOLINA-FAVERO, CECILIA MO LUCIANA LANTERI, NATALIA CORREA-ARAGUNDE, MARI´A CRISTINA LOMBARDO, CARLOS ALBERTO BARASSI and LORENZO LAMATTINA How the Environment Regulates Root Architecture in Dicots ´ RIE LEFEBVRE, PHILIPPE MARIANA JOVANOVIC, VALE LAPORTE, SILVINA GONZALEZ-RIZZO, CHRISTINE LELANDAIS-BRIE`RE, FLORIAN FRUGIER, CAROLINE HARTMANN and MARTIN CRESPI

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Aquaporins in Plants: From Molecular Structure to Integrated Functions OLIVIER POSTAIRE, LIONEL VERDOUCQ and CHRISTOPHE MAUREL Iron Dynamics in Plants JEAN-FRANC ¸ OIS BRIAT Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions VIVIENNE GIANINAZZI-PEARSON, NATHALIE SE´JALON-DELMAS, ANDREA GENRE, SYLVAIN JEANDROZ and PAOLA BONFANTE Dynamic Defense of Marine Macroalgae Against Pathogens: From Early Activated to Gene-Regulated Responses AUDREY COSSE, CATHERINE LEBLANC and PHILIPPE POTIN

Contents of Volume 47 INCORPORATING ADVANCES IN PLANT PATHOLOGY The Plant Nucleolus ´ EZ-VA ´ SQUEZ AND FRANCISCO JAVIER MEDINA JULIO SA Expansins in Plant Development DONGSU CHOI, JEONG HOE KIM AND YI LEE Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis WEN-CHIEH TSAI, YU-YUN HSIAO, ZHAO-JUN PAN, CHIACHI HSU, YA-PING YANG, WEN-HUEI CHEN AND HONG-HWA CHEN

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Molecular Physiology of Development and Quality of Citrus ´ S, JOSE´ M. FRANCISCO R. TADEO, MANUEL CERCO COLMENERO-FLORES, DOMINGO J. IGLESIAS, MIGUEL A. NARANJO, GABINO RI´OS, ESTHER CARRERA, OMAR RUIZ-RIVERO, IGNACIO LLISO, RAPHAE¨ L MORILLON, PATRICK OLLITRAULT AND MANUEL TALON Bamboo Taxonomy and Diversity in the Era of Molecular Markers MALAY DAS, SAMIK BHATTACHARYA, PARAMJIT SINGH, TARCISO S. FILGUEIRAS AND AMITA PAL

Contents of Volume 48 Molecular Mechanisms Underlying Vascular Development JAE-HOON JUNG, SANG-GYU KIM, PIL JOON SEO AND CHUNG-MO PARK Clock Control Over Plant Gene Expression ANTOINE BAUDRY AND STEVE KAY Plant Lectins ELS J. M. VAN DAMME, NAUSICAA LANNOO AND WILLY J. PEUMANS Late Embryogenesis Abundant Proteins MING-DER SHIH, FOLKERT A. HOEKSTRA AND YUE-IE C. HSING

Phototropism and Gravitropism in Plants

MARIA LIA MOLAS* AND JOHN Z. KISS{

*Facultad de Agronomı´a, Universidad Nacional de La Pampa, Santa Rosa (LP), Argentina { Department of Botany, Miami University, Oxford, Ohio, USA

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Phototropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Sensing the Direction of Light................................................ B. Intracellular Signaling of Light Stimulus ................................... C. Response to Phototropic Light Stimulus ................................... III. Gravitropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Sensing the Direction of Gravity............................................. B. Intracellular Signaling of Gravity and the Response ..................... IV. Interaction Between Phototropism and Gravitropism . . . . . . . . . . . . . . . . . . . . . . A. Effect of Light on Gravitropism ............................................. B. Effect of Gravity on Phototropism .......................................... V. Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 2 7 8 10 11 13 19 20 23 24 25 25

ABSTRACT Light and gravity are two of the most important environmental parameters aVecting plant growth and development. To maximize available light and nutrients, plants orient their stems toward the direction of illumination and away from the gravity vector, and, conversely, orient their roots away from light and toward gravity. In plant organs, there may be a competition between gravity (gravitropism) and light (phototropism). At the same time, the tropistic signaling and/or responses induced by both stimuli may be independent processes that coexist, or they may modulate each other. Our limited knowledge of the interaction of these tropistic Advances in Botanical Research, Vol. 49 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(08)00601-0

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stimuli is due to the ubiquitous nature of gravity on earth, which makes it diYcult to study a ‘‘pure’’ eVect of light without the interference of gravity. However, during the past decade, significant advances have been made in our understanding of gravity and light signal transduction pathways by using mutants and novel techniques for genome‐wide analysis. Thus, both unique and common elements in the signaling and response pathways of both tropisms have been identified.

I. INTRODUCTION An incredible diversity of sensory systems has evolved to perceive and transduce specific incoming environmental signals. For plants, light and gravity are two of the most important environmental parameters aVecting growth and development. Light provides the ultimate source of biological energy while gravity, a constant stimulus on the Earth surface, provides critical spatial information about the surroundings, which is vital for organ orientation (Iino, 2006; Liscum and Stowe‐Evans, 2000). While stem‐like organs orient their growth toward light (i.e. positive phototropism) and away from gravity (i.e. negative gravitropism), roots, in general, bend away from light (i.e. negative phototropism) in the direction of gravity (i.e. positive gravitropism). Both stimuli, light and gravity, operate together in nature, interacting and aVecting each other, thus resulting in adaptive growth movements of the plant. Hence, the knowledge of the mechanisms governing the responses to and gravity, as well as specific interaction between these tropisms, is of major interest in plant biology. In the last few years, significant advancements in our knowledge of the basic mechanisms of tropisms have been made. Mutants that are impaired in tropisms have played especially important roles, and, more recently, high throughput techniques investigating transcript and metabolite profiling (e.g. microarrays, protein arrays) provide important information about the participants in the process of sensing, transducing, and responding to light and gravity (Ghassemian et al., 2006; Jiao et al., 2007; Khanna et al., 2006; Kimbrough et al., 2004; Quail, 2007; Tepperman et al., 2006). In this article, we summarize the current knowledge in phototropism, gravitropism, and the interaction between these two key tropisms.

II. PHOTOTROPISM A. SENSING THE DIRECTION OF LIGHT

Phototropism, a response induced by UV/blue light and regulated by red light, provides researchers with an optimal system to study light perception and signaling in plants (Hangarter, 2006; Iino, 2006). Photobiological studies

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using shoots of dark‐grown seedlings from a variety of plant species revealed a high degree of complexity in the bending response toward blue light. Phototropism can be divided into two phases depending on the fluence and time requirement (Iino, 2006). First, positive curvature is generally described as the bending of shoots toward unilateral blue light delivered in short pulses at very low fluences. Second, positive curvature occurs with prolonged irradiation in a time‐dependent fashion. In the coleoptile of etiolated grass seedlings, illumination of seedlings with red light causes subsequent changes in the sensitivity of the tissue to phototropically active blue light, and this red‐light‐induced change is known to be controlled by phytochrome and the process is termed ‘‘enhancement’’ (Briggs and Olney, 2001). Enhancement of phototropism is most easily illustrated by the eVect of an exposure to red light on the subsequent phototropic curvature to blue light. Even though red light does not typically induce phototropism, maximum first‐positive curvature to blue light is increased when the seedlings are exposed to red light up to 2 h prior to irradiation with unilateral blue light. Moreover, the enhancement of phototropic curvature increases as the fluence of red light is increased (Janoudi et al., 1997), and this response to red light is controlled by the phytochrome family of photoreceptors (Parks et al., 1996; Whippo and Hangarter, 2003). Although fluence‐response measurements have provided important photobiological information about the sensory mechanism mediating phototropism (Franklin et al., 2005; Iino 2006), a greater understanding of the photodetection process involved has come from biochemical and molecular genetic approaches. In the next paragraphs, we will focus on the role of specific photoreceptors in the process of phototropism in lower and flowering plants. Phototropins are ubiquitous in higher plants and have been identified in several plant species (reviewed in Christie, 2007; Kimura and Kagawa, 2006). In higher plants (Fig. 1), sensing of the light direction is primarily regulated by blue‐light photoreceptors, phototropins (i.e. phototropin 1 and phototropin 2; PHOT1 and PHOT2, formerly known as nonphototropic hypocotyl 1 and nonphototropic hypocotyl 1‐like—nph1 and npl1) and cryptochromes (i.e. cryptochrome 1 and cryptochome 2; CRY1 and CRY2). nph1 mutants lack a phototropic bending of the hypocotyl under low‐fluence light (<1 mol m
2 s
1), while the bending of this mutant is normal under high‐ fluence light (1–100 mol m
2 s
1; Liscum and Briggs, 1995; Sakai et al., 2000). Hence, a second photoreceptor is acting under high‐fluence rate in Arabidopsis. Studies with npl1 mutant showed a normal response under low‐ and high fluence of blue light; however, the nph1 npl1 double mutant was found to exhibit impaired hypocotyl phototropism in response to both

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Blue light

PHOT1-PHOT2

CRY1-CRY2

Red light

PHYA-PHYB

NPH3 RPT2 PKS1, PKS2, PKS4 HY5

Signal transduction and response

EXPA1, EXPA8 SKS1 GH3.5, GH3.6 SAUR bHLH134 HAT2 NPH4/ARF7 AXR1 PIS1 Ethylene Auxin Brassinosteroids

Phototropism

Fig. 1. Some of the components involved in phototropism of Arabidopsis. Abbreviations are for elements described in the text.

low‐ and high‐fluence rates of blue light (Sakai et al., 2001). Thus, in the absence of NPH1, NPL1 mediates hypocotyl phototropism in response to only high‐fluence rates of unilateral blue light. Therefore, there is an overlap in function between NPH1 and NPLl photoreceptors that occurs only at these higher fluence rates. These findings also indicate that NPH1 and NPLl function in a fluence rate‐dependent manner to regulate phototropism. NPL1 appears to function at high‐fluence rates of blue light, whereas NPH1 appears to exhibit a broad fluence rate‐dependence, mediating the phototropic response at both low‐ and high‐fluence rates (Sakai et al., 2001). Phototropins have been identified in several plant species, such as the fern Adiantum capillus‐veneris (Kagawa et al., 2004), the moss Physcomitrella patens (Kasahara et al., 2004), the filamentous green alga Mougoetia scalaris

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(Suetsugu et al., 2005), and the biflagellate unicellular green alga Chlamydomonas reinhardtii (Huang et al., 2002). However, phototropins are not the major players controlling phototropic responses in lower organisms but are important in these processes in flowering plants. Cryptochromes, the other blue‐light sensing type of receptors, are evolutionarily conserved, and this family of photoreceptors has members in the animal kingdom as well as in many diVerent lower and higher plant species (reviewed in Briggs 2007; Li and Yang, 2007). Cryptochromes regulate the circadian rhythms of animals, and in plants regulate photomorphogenesis, stomatal opening, and phototropism (reviewed in Essen and Klar, 2006). Cryptochromes have two homologues in Arabidopsis, CRY1, and CRY 2. Originally designated as HY4 (long hypocotyl 4; the gene encoding for CRY1), an Arabidopsis mutant deficient in CRY1 was isolated based on its long hypocotyl phenotype in blue light (review in Li and Yang, 2007). Cryptochromes control the phototropic response to blue light (Ahmad and Cashmore, 1996; Christie, 2007). Despite the fact that cryptochromes are not required for low light induction of phototropism (Lasce`ve et al., 1999) or for the induction of phototropism under high illumination (Sakai et al., 2000), cryptochromes may have indirect eVect on phototropism because first‐ positive phototropism is attenuated in the cry mutant plants (Whippo and Hangarter, 2003). Cryptochromes and phototropins act in concert to enhance phototropism under low fluence of blue light, and this eVect may be the result of coordinating the balance between stimulation and inhibition of growth of the hypocotyl (Whippo and Hangarter, 2004). The phototropic curvature of a plant organ can be induced by red light as well as blue light (Fig. 1). In lower plants, such as mosses and ferns, the detection of the direction of light is primarily a role of phytochrome (Esch et al., 1999; Kawai et al., 2003). In some ferns, including A. capillus‐veneris, phototropism is controlled by red light as well as blue light, a feature that provides an enhanced light sensitivity to increase fitness in low‐light environments (Wada and Kadota, 1989). In A. capillus‐veneris, the red light eVect on the tropistic response is mediated by phytochrome 3 (i.e. PHY3), a chimerical protein with features of both phytochrome and the blue‐light receptor phototropin in Arabidopsis (Nozue et al., 1998). It has been established that, under red light, PHY3 mediates negative phototropism in rhizoid cells, contrasting with its role in regulating positive phototropism in protonemal cells (Tsuboi et al., 2006). The occurrence of a chimeric red light and blue light photoreceptor is observed in other lower plant groups as well. For instance, like Adiantum PHY3, the green alga Mougeotia scalaris have two genes (i.e. NEOCHROME 1 and NEOCHROME 2) that show a bilin‐binding domain and

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red/far‐red reversibility typical to phytochromes (Suetsugu and Wada, 2007). Additionally, chimeric phytochrome genes have been identified in the mosses Ceratodon purpureus and Phycomitrella patens. Thus, these nonflowering plants appear to evolve a sophisticated light sensing system to utilize red as well as blue light for regulation of photoresponses as an adaptation to low light environments (reviewed in Suetsugu and Wada, 2007). In the flowering plant Arabidopsis, red light induces a positive phototropic response in roots (Kiss et al., 2003; Ruppel et al., 2001), and this response is mediated by phytochrome A and B (PHYA and PHYB; Kiss et al., 2003), but red light alone cannot induce positive hypocotyl phototropism in Arabidopsis (Liscum and Briggs, 1996). Therefore, the phytochromes (a small gene family with five members: PHYA‐E) are probably not the principal photoreceptors inducing hypocotyl phototropism (Liscum and Stowe‐Evans, 2000). Instead, phytochrome activity probably modulates hypocotyl phototropism by interfering with the signaling phase of phototropism involving PHOT1 and PHOT2 (Sakai et al., 2001). Alternatively, the phytochromes may promote phototropism through their interaction with the cryptochrome (CRY1 and CRY2) family of blue light photoreceptors (Ahmad et al.,1998), which also aVect phototropism (Whippo and Hangarter, 2003). Seedlings of several flowering plants, when pretreated with red light, have increased blue‐light‐induced phototropic bending (i.e. enhancement; Janoudi et al., 1997). The red light dependence of this response implicates phytochrome in the enhanced response, and in Arabidopsis, this is mediated by PHYA and PHYB (Hangarter, 1997; Parks et al., 1996). The mechanism underlying phytochrome‐mediated phototropism enhancement is not clear and several hypotheses have been proposed. One speculation is that red light enhances phototropic response by attenuating the gravitropic response (Lariguet and Fankhauser 2004; Parks et al., 1996). Another idea is that PHYA signaling activates an auxin response factor system thereby amplifying the auxin diVerential gradient (Stowe‐Evans et al., 2001). Srinivas et al. (2004) proposed the existence of negative regulators of the phototropic signal transduction pathway that can be down regulated by phytochromes. At the moment, we cannot exclude any of these hypotheses. In addition, phytochromes modulate blue‐light‐mediated phototropism in the absence of a red light pretreatment (Whippo and Hangarter, 2004). It was found that phytochromes A, B, and D have conditionally overlapping functions in the promotion of blue‐light‐induced phototropism. Under very low blue light intensities (0.01  mol m
2s
1), PHYA activity is necessary for the progression of a normal phototropic response, whereas above 10 mol m
2s
1, PHYB and PHYD have functional redundancy with PHYA to promote phototropism. PHYA also contributes to attenuation

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of phototropism under high‐fluence rates of unilateral blue light, which was previously shown to be dependent on the phototropins and cryptochromes. Therefore, it appears that phytochromes are required to develop a robust phototropic response under low‐fluence rates, but at high irradiances, PHYA suppresses phototropism. B. INTRACELLULAR SIGNALING OF LIGHT STIMULUS

Despite the fact that the photoreceptors involved in phototropism are becoming increasingly well‐characterized, the downstream signal transduction pathways are just beginning to be elucidated and characterized (Fig. 1). Arabidopsis mutants provide us with valuable information about the signaling mechanisms involved in phototropism. The isolation of the nonphototropic hypocotyl mutant 3 (nph3; Liscum and Briggs, 1995, 1996) led to the identification of a PHOT1‐interacting protein that is essential for lateral auxin redistribution (Haga et al., 2005) and phototropism (Haga et al., 2005; Motchoulski and Liscum, 1999). NPH3 is a novel protein containing several protein interaction motifs and has been shown to interact with PHOT1 in vitro (Motchoulski and Liscum, 1999) and in vivo (Lariguet et al., 2006). Like the phototropins, NPH3 is hydrophilic in nature but is associated with the plasma membrane (Motchoulski and Liscum, 1999). Although its biochemical function is unknown, NPH3 most likely serves as a scaVold to assemble components of a phototropin receptor complex. NPH3 is a member of a gene family consisting of 31 members in Arabidopsis (Celaya and Liscum, 2005) and at least 24 in rice (Kimura and Kagawa, 2006). NPH3 appears to be phosphorylated in the dark and becomes dephosphorylated upon exposure to blue light (Motchoulski and Liscum, 1999). PHOT1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction (Pedmale and Liscum, 2007). The abrogation of both blue light‐dependent dephosphorylation of NPH3 and development of phototropic curvature by protein phosphatase inhibitors further suggests that this post‐translational modification represents a crucial event in PHOT1‐dependent phototropism (Pedmale and Liscum, 2007). A protein closely related to NPH3, designated root phototropism 2 (RPT2), has also been shown to bind PHOT1 and is required for both phototropism and stomatal opening by blue light (Inada et al., 2004; Sakai et al., 2000, 2001). RPT2 protein belongs to a family that includes NPH3 and plays a role in the second‐positive phototropism (Sakai et al., 2000). It has been suggested that NPH3 and RPT2 function as scaVold or adaptor proteins to bring together the early components of the phototropic signal cascade

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(Liscum and Stowe‐Evans, 2000). All three proteins (i.e. NPH1, NPH3, and RPT2) are required for normal phototropic responses (Liscum and Stowe‐ Evans, 2000). Recent photobiological studies identified a group of proteins that appear to mediate hypocotyl (Lariguet et al., 2006) and root phototropism (Boccalandro et al., 2008; Molas and Kiss, 2008). Phytochrome kinase susbtrate proteins (PKS 1–4) belong to a small gene family in Arabidopsis, and PKS1, PKS2 and PKS4 are required for blue‐light‐induced phototropism (Lariguet et al., 2006). More specifically, PKS proteins appear to act as positive regulators of PHOT1 signal transduction in blue light and the participation of PKS1 is PHYA‐dependent (Lariguet et al., 2006). PKS1 physically interacts with PHOT1 and NPH3 in vitro and in vivo (Lariguet et al., 2006), and additionally, it can be phosphorylated by PHYA in vitro and by PHYB in vivo (Fankhauser et al., 1999). Given the capability of interaction of PKS1 with blue and red light photoreceptors, PKS1 may represent a link between these two photoreceptor families that have long been known to cooperate during the early steps of phototropism (Lariguet et al., 2006). In roots, PKS1 mediates blue (Boccalandro et al., 2008) and red‐light‐ induced phototropism (Molas and Kiss, 2008). In the blue‐light‐induced phototropism, PKS1 promotes the negative phototropic curvature of the root, and this response is mediated by PHOT1, and it is PHYA dependent (Boccalandro et al., 2008). In the red‐light‐based phototropism, which is mediated by PHYA and PHYB (Kiss et al., 2003), PKS1 promotes a negative curvature to red light (Molas and Kiss, 2008). These findings indicate that PKS1 positively regulates negative phototropism in roots by unilateral blue and red light, and this family plays a significant role in the control of organ orientation (Boccalandro et al., 2008).

C. RESPONSE TO PHOTOTROPIC LIGHT STIMULUS

Both phototropism and gravitropism involve diVerential growth mechanisms. For example, in stem‐like organs, there is concomitant decreased growth on the irradiated side and stimulated growth on the shaded side (Baskin, 1986). The light‐induced transduction cascade leads to the diVerential growth most likely caused by lateral redistribution of the plant hormone auxin (Went and Thimann, 1937). At present, little is known about how phototropin activation by blue light leads to an accumulation of auxin on the shaded side of the stem. Unilateral irradiation has been shown to induce a gradient of PHOT1 autophosphorylation across oat coleoptiles, with the

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highest level of phosphorylation occurring on the irradiated side (Salomon et al., 1997a,b). A phosphorylation gradient model has therefore been proposed to account for the complex fluence‐response curve for phototropism (Salomon et al., 1997a), but the question still remains as to how a gradient in phototropin phosphorylation across the stem can produce a lateral gradient in auxin. Genetic analyses have shown that auxin responsiveness is necessary for phototropism. Auxin regulated transcription factors such as nonphototropic hypocotyl 4 (NPH4, Stowe‐Evans et al., 1998) and Massugu 2 (MSG2, Tatematsu et al., 2004) are required for normal phototropism and gravitropism, highlighting the need for auxin‐regulated gene expression. Activities of NPH4 and MSG2 are likely to be regulated by the recently identified auxin receptor transport inhibitor response 1 (TIR1), a subunit of the Skp1‐Cullin‐ F‐box (SCF)–TIR1 complex, which targets proteins for degradation in the presence of auxin (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). A study by Liscum and coworkers (Esmon et al., 2006) investigated the transcript profiling of phototropically stimulated hypocotyls of Brassica oleracea, a close relative of Arabidopsis that shares approximately 85% of the genome (Ayele et al., 2005; Yang et al., 2005). Because of the larger size of Brassica in comparison to Arabidopsis, it was possible to isolate the target tissue (opposing flanks from the most tropistically responsive region of the hypocotyl without central vasculature) by hand sectioning. Eight tropic stimulus‐induced (TSI) genes exhibited higher expression in the elongating (i.e. shaded) versus nonelongating flanks (i.e. illuminated) of phototropically stimulated B. oleracea seedlings. Among those genes, At1g69530_EXPA1, At2g40610_EXPA8, At4g25240_SKS1, At4g27260_GH3.5, At4g34760_SAUR50, At5g15160_bHLH134, At5g47370_HAT2, and At5g54510_ GH3.6_DFL1 showed such a pattern of diVerential expression in B. oleracea. Concomitantly, the analysis of gene expression performed in nph4 mutant and wild type (WT) of Arabidopsis seedlings demonstrated that the auxin‐dependent accumulation of TSI genes requires NPH4 in Arabidopsis. EXPA1 and EXPA8, two members of the ‐expansin family, showed diVerential transcript accumulation in the distal hypocotyl flank before noticeable tropic curvature. Members of the ‐expansin family mediate cell wall extension (Cosgrove, 2000), a prerequisite for cell elongation processes such as those occurring in the stem flank opposite to the incident light. Hence, it would appear that an increase in auxin concentration is capable of stimulating both the local accumulation of EXPA1 and EXPA8 mRNAs. GH3.5 and GH3.6 encode IAA‐amino synthetases and thus could be involved in feedback mechanism that reduce responsiveness over time by converting free active IAA to inactive amino‐IAA (Ljung et al., 2002).

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Thus, a photomorphogenic and phototropic interplay, via hormonal regulation, has been proposed. Additionally to auxin action, brassinosteroids may modulate phototropism by regulating the gene expression of PHOT1 (Nemhauser and Chory, 2004), Aux/IAA genes (Mussig et al., 2002; Nakamura et al., 2003; Nemhauser and Chory, 2004), as well as ARF‐ mediated transcription (Nemhauser and Chory, 2004). Whippo and Hangarter (2005) reported that elongated, a photomorphogenesis mutant, enhances high‐light phototropism. This mutation represents a unique allele of BAK1/SERK3, a receptor kinase implicated in brassinosteroid perception. In the same way, HYPOCOTYL HYPERSENSITIVE 5 (HY5), typically associated with photomorphogenesis, enhances phototropism under very low light conditions. HY5 activates the transcription of Aux/ IAA genes (Cluis et al., 2004), and it has been proposed that HY5 activity might control phototropism by modifying the levels of Aux/IAA proteins (Whippo and Hangarter, 2005).

III. GRAVITROPISM Gravitropism is the directed growth of plant organs in response to gravity. In general, stems grow upwards and roots grow downwards. However, plant organs often maintain a specific orientation in the gravity field that is not directly parallel to the gravity vector. The angle at which a plant organ is oriented with respect to gravity is defined as gravitropic set‐point angle (GSA). The GSA varies with the species, with respect to organ, and can also be modified by the plant in response to environmental cues such as light. Because gravity aVects plants in such complex ways, plants have developed sophisticated mechanisms to sense and respond to this ubiquitous stimulus. Gravitropism can be separated into three temporal events (Fig. 2): sensing the gravity signal (perception), transduction of the signal, and the diVerential growth of organs (reviewed in Palmieri and Kiss, 2006; Perrin et al., 2005; Terao and Tasaka, 2004). Although a substantial amount of work has been carried out on diVerent organisms, the main focus of this review is on the current state of knowledge of the molecular mechanisms that govern the response to gravity in flowering plants. The sites of perception and response to gravity in hypocotyls and roots of flowering plants are spatially separated (Weise et al., 2000). This physical separation between the primary sites of gravity perception (endodermal cells and root cap columella, respectively) and curvature responses (cortical tissue and elongation zone, respectively) provides a suitable model in the analysis of gravitropism.

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Gravity

Sensing

Signal transduction and response

PGM EAL1 Protoplasmic pressure SGR ARG others pH ARG AGR AUX1 RHG CLG1 RCN1 AXR4/RGR DWF AUX/IAA Flavonoids Reactive oxygen species NPH4/ARF7 AXR1 PIS1 Ethylene Auxin Brassinosteroids

Gravitropism

Fig. 2. Some of the components involved in gravitropism in roots and hypocotyls of Arabidopsis. Abbreviations are for elements described in the text.

A. SENSING THE DIRECTION OF GRAVITY

There are several mechanisms by which organisms can sense gravity. In the protonemata of the moss Ceratodon (Kuznetsov et al., 1999), gravity perception is located in the apical cells containing starch‐filled plastids (amyloplast) that function as statoliths (dense, movable intracellular particles). In the alga Chara, the apex of the rhizoid has membrane‐bound vesicles that seem to be involved in sensing gravity (Kiss, 1997). In the sporangiophores of the fungus Phycomyces, gravity sensing appears to be through three sensory mechanisms: wall strain (bending stress), the sedimentation of protein crystals, and the floating of lipid globules (Grolig et al., 2000, 2006).

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In flowering plants, there are two major hypotheses of how gravity is sensed: the starch statolith hypothesis and the protoplast pressure hypothesis (reviewed in Blancaflor and Masson, 2003; Palmieri and Kiss, 2006). The starch statolith hypothesis proposes that the force due to gravity is perceived by the sedimentation of dense, starch‐filled amyloplasts that function as statoliths within the statocytes (gravity‐perceiving cells) in the root cap and the endodermal starch sheath cells in shoots. This model is supported by the observation that genetic or physiological depletion of starch in these cells results in altered gravitropism. For example, a starchless mutant (pgm, phosphoglucomutase) in Arabidopsis shows reduced sensitivity to gravity compared to the WT, suggesting a strong correlation between gravity sensing and total mass of plastids (Fig. 2). Another starch‐deficient mutant, eal1 (endodermal amyloplast less 1) has no amyloplasts in the endodermal cells of hypocotyls and also exhibits reduced gravitropic response. Mutants that lack an endodermis (sgr1–shoot gravitropism/scr—scarecrow) and sgr7/shr (shoot‐root) have no gravtitropic response in stems or hypocotyls, although roots respond normally. However, the way in which amyloplast sedimentation is sensed remains unsolved. One possibility is that sedimenting statoliths contact the receptors embedded in sensitive membranes on the side of the statocyte, hence triggering gravity signaling within the cell (Kiss, 2000). In vertically growing plants, these receptors could be associated with the plasma membrane or with the cortical endoplasmic reticulum (ER), a specialized nodular ER found only in columella cells of the root cap (Yoder et al., 2001). This also occurs with PIN3 (named for the pin‐like shape of the inflorescence stems in the mutant) activity and/or recycling between plasma membrane and endosome, where this mechanism of regulation may take place (Fleming, 2006). However, this mechanism of perception might not be the exclusive operative system to sense gravity. For instance, starch‐deficient mutants lacking amyloplast sedimentation still display some gravitropic response (Kiss et al., 1997). Also, it is possible to trigger a productive signal transduction response by exposing seedlings to a succession of very short gravitropic stimuli that are insuYcient to promote amyloplast sedimentation (reviewed in Perbal et al., 2002). Alternatively, the protoplast pressure hypothesis suggests that the mass of the entire protoplasm causes tension and compression at the top and bottom of the plasma membrane, respectively (reviewed in Palmieri and Kiss, 2006). According to this model, these forces may relate the gravity signal to membrane‐bound stretch receptors located in the plasma membrane. Support for the gravitational pressure hypothesis is primarily based on studies of gravity‐dependent cytoplasmic streaming with the algae Nitellopsis and Chara, which have no sedimenting amyloplasts, but still sense and respond

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to gravity. For example, vertically oriented internodal cells of Characean algae exhibit a gravity‐dependent polarity of the cytoplasmic streaming. However, the data supporting this model are not conflicting with the other model, and the preponderant view support that multiple mechanisms may act in the perception of gravity (Palmieri and Kiss, 2006). The possibility of more than one pathway acting in concert to perceive gravity in plants is well accepted (Barlow, 1995; Guan et al., 2003; LaMotte and Pickard, 2004). However, a prospective candidate for a receptor to the gravity‐induced forces on sensitive intracellular structures does not exist at the present time. Activation of mechanosensitive ion channels has been suggested as the first step in gravity signal transduction. However, the molecular identity of these channels remains unknown. Transient receptor channels (TRC), that mediate mechanoperception in several animal cell types and yeast (Barritt and Rychov, 2005) has been recently revealed in the Arabidopsis genome annotation bank (Perrin et al., 2005). Several proteins sharing a functional domain with bacterial mechanosensitive channels were identified, and some of these genes are highly expressed in the root tip. For instance, one of them was found to be regulated by gravistimulation (Kimbrough et al., 2004). Hence, these channels may function as mechanoreceptors in the root tip. Another cellular component that may be involved in sensing and/or transducing gravitropism is the cytoskeleton (Balusˇka and Hasenstein, 1997; Nick et al., 1990). Mutations in the ARG gene (altered response to gravity) disrupt the gravity response. ARG1 encodes a DNA j‐like protein that may interact with the cytoskeleton (Sedbrook et al., 1999). The amyloplasts are enmeshed in microfilaments, and, upon sedimentation, these cytoskeletal elements may transmit the signal to receptors (Balusˇka and Hasenstein, 1997). Mutation in ARG1 (or its paralog protein ARL2) dramatically attenuates the lateral redistribution of auxin in gravistimulated roots (Boonsirichai et al., 2003) and eliminates the contribution of the auxin eZux carrier PIN3 to the root gravitropic response (Harrison and Masson, 2008). Therefore, ARG1 is needed for PIN3 localization and/or function and in consequence, for a full response to gravity stimulation (Harrison and Masson, 2008). B. INTRACELLULAR SIGNALING OF GRAVITY AND THE RESPONSE

After gravity stimulus is sensed, a series of events cause the activation of auxin‐dependent transduction pathways (Fig. 2). A number of signaling molecules and second messengers have been implicated in transducing the gravity signal into a diVerential growth response: ionic gradients (Monshausen et al., 1996), pH (Fasano et al., 2001; Scott and Allen, 1999),

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Ca2þ (Plieth and Trewavas, 2002), and inositol 1,4,5‐trisphosphate (InsP3; Perera et al., 2001). Changes in transcript abundance also occurs after gravistimulation in root tips of Arabidopsis whole seedlings (Moseyko et al., 2002) and root tips (Kimbrough et al., 2004). In addition, sequestration of specific transcripts in polysomes and rapid changes in protein distribution has been shown within the first 30 min of gravistimulation (Clore et al.,2008; Heilmann et al., 2001). Hence, transcriptional and translational regulations are also part of the early signaling events. Nevertheless, to date, the relevance of these signaling events and their possible interactions is not well understood. A number of inorganic ions have been proposed to play significant roles in gravity signal transduction such as cytosolic and apoplastic pH changes within seconds of gravity stimulation. Alkalinization of the statocytes in the root cap seems necessary for full responsiveness. This process may be accompanied by a hyper polarization of the plasma membrane and could be the result of the gravity‐activated vacuolar type proton ATPases (Fasano et al., 2001; Scott and Allen, 1999). Recently, two plasma membrane proteins that regulate intracellular cation homeostasis have been linked to cell growth and gravitropic response (Christopher et al., 2007). AtCNGC5 and AtCNGC10 belong to a class of cyclic nucleotide‐gated ion channels (CNGCs) that facilitate the movement of cations across cellular membranes. It has been proposed that AtCNGC10 traYcks from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity‐sensing columella cells, combined with the fact that antisense phenotypes decreased gravitropic and cell enlargement responses (Borsics et al., 2007), suggest roles of AtCNGC10 in modulating cation balance required for root gravitropism, cell division, and growth (Christopher et al., 2007). Calcium has long been suggested to play a role in gravity signal transduction. Since cytoplasmic calcium (Ca2þ) is a ubiquitous intracellular second messenger, its possible involvement in gravity response has been controversial (Blancaflor and Masson, 2003). Novel techniques of Ca2þ imaging revealed changes in the free cytoplasmic calcium concentration ([Ca2þ]c) induced by gravity in Arabidopsis seedlings that express the luminous Ca2þ reporting protein aquaporin (Plieth and Trewavas, 2002). Changes in cytosolic calcium have recently been confirmed to occur in hypocotyls and petioles of Arabidopsis (Toyota et al., 2008). Gravistimulated seedlings showed a transient biphasic peak in the cytoplasmic free [Ca2þ]c. The first peak seems to be produced by the speed of rotation of the device used to sense the changes in calcium concentration. The second peak of [Ca2þ]c

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appeared to be induced by changes in the gravity vector. This increase of [Ca2þ]c concentration in the second peak seems to occur via mechanosensitive [Ca2þ] ‐permeable channel (MSCC) in the plasma membrane and the Ca2þ release from intracellular stores. Actin filaments may be partially involved in the activation of [Ca2þ]‐permeable channels, since actin‐ disrupting drugs such as cytochalasin B and latrunculin B attenuated the second peak of calcium, suggesting that the second [Ca2þ]c increase via MSCCs is a gravity response in the hypocotyl and petiole of Arabidopsis seedlings (Toyota et al., 2008). This study confirmed the previous observation (Plieth and Trewavas, 2002) that using refined technologies of [Ca2þ]c imaging revealed changes in the [Ca2þ]c induced by gravistimulation in Arabidopsis seedlings expressing the luminous [Ca2þ] reporting protein, aquaporin. Another line of evidence that supports the involvement of Ca2þ as a second messenger is the participation of inositol‐3 phosphate (IP3) in the gravitropic signaling pathway (Perera et al., 2001). In oat (Avena sativa) pulvinus system IP3, which is known to trigger the release of Ca2þ from intracellular stores, increased threefold over vertical controls within 15 s of gravistimulation. Interestingly, gravitropic bending and the sustained increases in IP3 were inhibited by a phospholipase C antagonist, suggesting that phospholipase C‐mediated hydrolysis of phosphatidylinositol 4,5‐ bisphosphate into IP3 may regulate diVerential growth in cereal pulvini (Perera et al., 2001). Similar results were found in Arabidopsis, corn and tobacco, suggesting a widespread role of this system in mediating gravity transduction (Perera et al., 2006). Other elements involved in the gravitropic transduction pathway include reactive oxygen species (ROS; Joo et al., 2001). Gravistimulation or unilateral application of auxin to vertical roots resulted in a transient increase in ROS concentration in the convex endodermis. Moreover, unilateral application of hydrogen peroxide to the elongation zone of vertically oriented roots induced curvature, whereas inactivation of ROS by antioxidants inhibited root gravitropism (Joo et al., 2001). Along with these results, there is increasing evidence that Ca2þ regulates hydrogen peroxide homeostasis in plants (Neill et al., 2002). Conversely, free oxygen radicals were also shown to activate Kþ‐ and Ca2þ‐permeable channels and to promote a large Ca2þ influx in the elongation zone of Arabidopsis roots (Demidchik et al., 2003). Hence, it seems plausible that a cross talk between ROS and Ca2þ might contribute to regulate the auxin‐induced diVerential growth responsible for gravicurvature in roots (Blancaflor and Masson, 2003). In fact, an analysis of the transcript profiling of gravi‐stimulated seedlings showed that genes involved in oxidative burst were diVerentially regulated by gravity (Moseyko et al., 2002).

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ROS are not only involved in root gravitropism, but also in aerial parts (Clore et al., 2008). Studies in Zea mays showed that ROS levels increase rapidly in gravistimulated pulvini. This increase in ROS levels occurs in or near the amyloplasts immediately following gravistimulation and spread throughout the bundle sheath cells by 30 min poststimulation. The authors speculate that ROS may have a role in determining the direction of bending the pulvini and may also be a link between the amylolpast sedimentation and subsequent changes in the gene expression that facilitate directional growth. In an attempt to understand the signaling‐response network triggered by gravity, analysis of the gene profiling in seedlings and roots of Arabidopsis were investigated (Kimbrough et al., 2004; Moseyko et al., 2002). In whole seedlings, expression changes were detected in many genes whose products are already suspected players in gravitropism, including calcium‐binding and calmodulin‐like proteins, proton exchanging proteins, expansins, and auxin‐ induced proteins. Surprisingly, changes in expression levels of the auxin carriers AUX1 and PIN2 were not evident, and it was suggested that these might be regulated post‐translationally. However, some new genes were implicated in gravitropism, including ethylene‐responsive elements, oxidative burst proteins, and heat shock proteins (Moseyko et al., 2002). A similar genetic profiling study was performed in Arabidopsis root tips (Kimbrough et al., 2004). Results identified several genes that were diVerentially expressed specifically in response to gravity and, because the experiment was performed as a time course, temporal resolution of gene expression was possible. During the first hour of gravistimulation by reorientation, a group of 65 genes whose transcript abundance increases encoded proteins that belong to a variety of functional groups, including transcription factors, transporters, wall‐ modifying enzymes, and proteins involved in other stress responses. Five genes were identified that are specifically and transiently upregulated within 2 min following gravistimulation: a pentacyclic triterpene synthase, expressed protein At2g16005, a cys‐protease, S‐adenosyl‐L‐Met:carboxymethyltransferase, and a major latex related protein. The specific roles these proteins play in gravitropism are yet to be determined. Subsequent analyses using transgenic IP3‐depleted plants indicate that gravity‐induced expression of these five genes is IP3‐dependent, whereas that of other gravity‐ responsive genes, including most of those previously shown to be hormone‐ responsive, is not. Therefore, at least two signaling pathways may regulate gene expression in the root tip upon gravistimulation (Kimbrough et al., 2004; Salinas‐Mondragon et al., 2005). Most of the gravity‐specific changes in transcript abundance occurred between 5 and 15 min following gravistimulation. Several transcription factors of known function were identified, including: HFR1 (phytochrome A activator),

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17

AtHB‐12 (homeobox transcription factor) and KNAT1 (fate determination of shoot meristem cells). At 30 min following gravistimulation, NO APICAL MERISTEM transcription factors were upregulated. Although these transcription factors aVect diVerent stages of shoot and floral development, until now they had not been implicated in physiological processes of the root. Thus, this study points to new avenues of research that may further our understanding of gravitropism as well as the role of plastids in gravity perception and gravitropic signal transduction (Palmieri and Kiss, 2006). Polar auxin transport has been demonstrated to be necessary for tropic responses, providing a strong support to the Cholodny–Went hypothesis, which suggests that the diVerential growth of the gravitropic response occurs by the lateral redistribution of auxin (reviewed in Fleming, 2006; Fuchs et al., 2006). However, other phytohormones have been postulated to participate in gravitropism (Aloni et al., 2004; Steed et al., 2004). Studies using cytokinin‐ sensitive ARR5‐GUS reporter construct demonstrates diVerential reporter expression on opposite flanks of gravistimulated root caps, with strong activation in the lower lateral cap cells (Aloni et al., 2004). This response precedes the gravity‐induced asymmetric activation of the auxin‐sensitive DR5‐GUS reporter, and application of exogenous cytokinin on one side of vertical roots promotes a curvature in the direction of cytokinin application (Aloni et al., 2004). Hence, a lateral cytokinin gradient might be generated across the root cap upon gravistimulation, and transmitted to the distal elongation zone where it might contribute to the initial curvature phase of the response (Aloni et al., 2004). Ethylene participates in gravitropism of both plant stems (Lu et al., 2001) and roots (Guisinger and Kiss, 1999). In dark‐grown seedlings, ethylene reduces the hypocotyl gravitropic response (Kiss et al., 1999), whereas in the light‐grown seedlings, a prolonged ethylene treatment promotes the hypocotyl gravitropism (Golan et al., 1996). Studies with the egy1 mutant (ethylene‐dependent gravitropism‐deficient and yellow green 1; Chen et al., 2005) suggest that ethylene is a positive regulator of gravitropism in shoots (Guo et al., 2008). The EGY1 gene encodes a plastid targeted metalloprotease that is actively expressed in hypocotyl tissue and targets to endodermal and cortex plastid. EGY1 appears to control the size and number of plastids in endodermal cells (Guo et al., 2008), which are responsible for sensing the gravity stimuli. Hence, it is proposed that ethylene may enhance the sensitivities of endodermal cells to gravity signal triggered by sedimenting amyloplasts. In addition, the ethylene insensitive mutants etr1 and 2 (ethylene responses 1 and 2), ers2 (ethylene response sensor 2), and ein4 (ethylene‐ insensitive root 4) did not curve after gravistimulation supporting the idea that ethylene positively aVects gravitropism in hypocotyls (Guo et al., 2008).

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Ethylene may have a diVerent role in roots than in shoots (Buer and Muday, 2004; Buer et al., 2006). ACC (1‐aminocyclopropane carboxylic acid) is a precursor of ethylene synthesis in planta. When ACC is applied exogenously to WT plants, it reduces root curvature and growth; however, when ACC is applied to ethylene‐insensitive mutants, they exhibit the WT root gravity responses (Buer et al., 2006). Additionally, ACC also stimulated the synthesis of flavonoids in root tips of WT plants, but not in the ein2 and etr1 mutants. Flavonoid synthesis in epidermal tissues of the Arabidopsis root tip is induced after gravity stimulation (Buer and Muday, 2004); however, ACC prevented a transient peak in flavonoid synthesis in response to gravity (Buer et al., 2006). Thus, these authors hypothesize that elevated ethylene levels negatively regulate root gravitropism and that ACC inhibition of gravity response occurs through altering flavonoid synthesis. Brassinosteroids, in concert with auxin, have been proposed to regulate gravitropism (Kim et al., 2007). Exogenously applied brassinolide (BL) at high concentrations increased gravitropic curvature while it inhibited primary root growth. In addition, expression of a gene encoding an indole acetic acid (IAA) biosynthetic enzyme (i.e. CYP79B2) was suppressed in blue light‐ hypersensitive plants and enhanced in insensitive or deficient plants. The authors suggest that blue light interacts negatively with IAA in the regulation of plant gravitropic responses, and this regulation, in part, is achieved by modulating the biosynthesis of the counterpart hormone (Kim et al., 2007). Gravity signaling appears to be dissimilar in the aerial organs and the roots. These diVerences became apparent in mutants that have normal response in one organ and lack the response in the other. Such is the case of the above mentioned hormone ethylene‐insensitive mutants. In addition, the axr1 (auxin resistant 1) mutant does not show gravitropic response in roots, but it has a limited response in hypocotyls (Watahiki et al., 1999). Furthermore, agr1 (abnormal gravitropic response)/eir1 (ethylene‐insensitive root)/pin2/wav6 mutants (hereafter referred as agr) have disruptions in the gravitropism in roots but not in hypocotyls or inflorescence stems (Utsuno et al., 1998). The AGR gene appears to encode one of the multiple auxin eZux carriers and it is only expressed in roots (Utsuno et al., 1998). Thus, agr mutants have decreased sensitivity to ethylene, so therefore in roots, ethylene seems to target AGR (Rosen et al., 1999). AGR activity also appears to be dependent on a component of an enzyme involved in activating proteins that degrade repressors of auxin responses (i.e. AXR1, Sieberer et al., 2000). It has been postulated that AXR1 causes the degradation of AGR that is essential for establishing an auxin gradient involved in gravitropism. Other mutants have been studied to decipher the gravitropic signaling pathway. The rhg (root hypocotyl gravitropism) mutants lack gravitropic

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responses in roots and hypocotyls, but stems respond normally (Fukaki et al., 1997). Hence, the RHG gene product would be an organ‐specific factor in the signaling of gravity. Another root gravitropic mutant is clg1, which has slightly altered sensitivity to auxins and increased insensitivity to ethylene, supporting the involvement of these plant hormones in gravitropism (Ferrari et al., 2000). This mutant also has a slow gravity response and increased right‐ hand slanting. In addition, a new mutant of Arabidopsis, rha1 (named for its characteristic of showing reduced or inverted root slanting or handedness on agar plates) shows minimal right‐handed slanting, reduced gravitropic response, notable resistance to ethylene and 2,4‐D, but little resistance to IAA and NAA. Rha1 is the first gravitropic mutant described that is disrupted in a heat shock protein (Fortunati et al., 2008). Rha belongs to a group of mutants disturbed in slanting, gravitropism, and auxin physiology. When these Arabidopsis mutant seedlings are grown on an agar plate, their primary roots show characteristic spiraling movements, apparent as waves, coils and torsions, together with a slanting toward the right‐hand side. All these movements are believed to be the result of three diVerent processes acting on the roots: circumnutation, positive gravitropism, and negative thigmotropism (review in Migliaccio and Piconese, 2001). Another mutant that partially impairs the gravitropic response in rice is lazy1 (Li et al., 2007; Yoshihara and Iino 2007). While lazy1 phenotype has a prostrate growth habit of the aerial parts, the primary roots show normal gravitropism. LAZY1 is temporally and spatially expressed, and plays a negative role in polar auxin transport (Li et al., 2007). Loss‐of‐ function of lazy1 greatly enhances polar auxin transport and thus alters the endogenous IAA distribution in shoots, leading to reduced gravitropism (Li et al., 2007).

IV. INTERACTION BETWEEN PHOTOTROPISM AND GRAVITROPISM Phototropism and gravitropism act together in nature to confer adaptive growth movements to plants. Light influences gravitropic responses, and concomitantly, gravity aVects light responses (Hangarter, 1997). Integration of gravity and light signals make it possible for shoots to adjust their architecture to optimize photosynthetic activity and root growth within the soil to nourish and anchor the plant (Hangarter, 1997). In this section, we focus on the current knowledge of the interaction of these two tropic stimuli.

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M. L. MOLAS AND J. Z. KISS A. EFFECT OF LIGHT ON GRAVITROPISM

Light can either stimulate or inhibit the gravity signal/eVect (Fig. 3). In flowering plants, light enhances the gravitropic response in some species such as Pisum sativum (Britz and Galston, 1982), Sesamun indicum (Woitzik and Mohr, 1988), and Oryza sativa (Yoshihara and Iino, 2007) and reduces the gravitropic response in other species such as Licopersicum esculentum (Behringer and Lomax, 1999), Zea mays (Lu et al., 1996), and Arabidopsis thaliana (Poppe et al., 1996). In Arabidopsis, when seedlings are grown in darkness, the hypocotyls orient upward and roots orient downward; however, when seedlings are grown in red or far red light, their shoot growth becomes random. This behavior involves a very low fluence‐response induced by far‐red and blue light and mediated by PHYA (Poppe et al., 1996) and a low fluence‐response induced by red light and mediated by PHYB (Robson and Smith, 1996),

Gravity

pH ARG AGR AUX1 RHG CLG1 RCN1 AXR4/RGR DWF AUX/IAA Flavonoids Reactive oxygen species

B light

R/FR light

CRY1-CRY2

PHYA-PHYB

HFR1 SHY2-1 FIN2 GIL 1 FHY1 FHL

NPH4/ARF7 AXR1 PIS1 Ethylene Auxin Brassinosteroids

Gravitropism

Fig. 3. Some of the components involved in the gravity and light eVects on gravitropism in Arabidopsis. Abbreviations are for elements described in the text.

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CRY1, and CRY 2 (Ohgishi et al., 2004). Under red/far‐red illumination, it seems that gravity is inhibited by phytochromes in a way that hypocotyls cannot sustain upward growth. Under white or blue light, due to a strong inhibition of gravitropism, the hypocotyl orientation appears to be entirely determined by phototropism (Lariguet and Fankhauser, 2004). From an ecological perspective, the interplay between these two driving forces can be interpreted as an ability to undertake strong inhibition of gravitropism and strong enhancement of phototropism in the presence of light (Iino, 2006). Because of its rosette habit, Arabidopsis may not need to sustain an upward orientation of hypocotyls; therefore, it may redirect growth to optimize phototropism in environment depleted in light. Little is known about how these interactions occur at molecular level. In Arabidopsis, the disruption of gravitropism by far‐red is lacking in phyA, hfr1 (long hypocotyls in far‐red, Fairchild et al., 2000), fin2 (far‐red insensitive 2, Soh et al., 1998), shy2 (suppressor of hy2; Halliday and Fankhauser, 2003), and gil1 (gravitropic in the light; Allen et al., 2006) mutants. In addition, studies of the light eVect on gravity signal using pks1 mutant (phytochrome kinase substrate 1) and fhy/fhl double mutant (far‐red‐elongated hypocotyl 1/fhy1‐like) shown that PKS1, FHY1, and FHL proteins modulate the inhibition of hypocotyl gravitropism (Boccalandro et al., 2008, Ro¨sler et al., 2007). The FIN2 protein seems to act downstream to PHYA in the far‐red light signaling pathway (Soh et al., 1998). SHY2 can physically interact with PHYA and expression of SHY2 gene is regulated by light (Halliday and Fankhauser, 2003). As well, HFR1expression is light regulated and HFR1 protein has homology to PIF3 (phytochrome interacting factor, Fairchild et al., 2000). PIF3 is a transcription factor that activates several light‐dependent genes (Martinez‐Garcia et al., 2000). HFR1 binds to PIF3 and, as a complex, preferentially binds the activated forms of PHYA or PHYB (Fairchild et al., 2000). Hence, it is plausible that far‐red light, via PHYA activation, induces the activation of genes in the gravitropic signaling pathway through the formation of the complex PIF3‐HFR1 (Fairchild et al., 2000). In addition, SHY2 and HFR1 are localized in the nucleus, supporting the hypothesis that nuclear events of phytochrome signaling are involved in the light‐regulated inhibition of gravitropism (Lariguet and Fankhauser, 2005). GIL1 is required for light‐dependent randomization of hypocotyl growth orientation (Allen et al., 2006). In gil1 mutants, hyopcotyls continue to grow upwards after exposure of seedlings to red or far‐red light. In fact, the gil1 mutant displays no other obvious phenotype, suggesting that GIL1 plays a role in a much defined subset of phytochrome‐mediated responses (Allen et al., 2006).

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PKS1 is a light inducible gene and PKS1 (a cytosolic protein) can physically interact with PHYA and PHYB, PHOT1 and NPH3 (Fankhauser et al., 1999; Lariguet et al., 2006). In Arabidopsis, PKS1 regulates shoot and root phototropism (Boccalandro et al., 2008; Lariguet et al., 2006; Molas and Kiss, 2008) and root gravitropism (Boccalandro et al., 2008). The role of PKS1 in phototropism has been discussed in a previous section (IIB). In root gravitropism, PKS1 appears to negatively regulate the gravitropic response under blue light, in contrast with the positive eVect on root phototropism (Boccalandro et al., 2008). It is known that some of the features of the signaling process and response to gravity and light are shared, especially those related to auxin redistribution and diVerential growth (Correll and Kiss, 2002). Since PKS1 is involved in both tropistic responses, it may be a common intermediary in these processes. The dual eVect of PKS1 in root phototropism and gravitropism suggest that PKS1 may regulate these processes upstream of their convergence (Boccalandro et al., 2008). FHY1 and FHL proteins are required for PHYA translocation into the nucleus. In the fhl/fhy1 double mutant, nuclear accumulation of phyA:GFP is undetectable, and, consequently, it aggregates in the cytoplasm in a pathological manner (Rosler et al., 2007). Whereas fhl/fhy1 showed a phyA phenotype in respect to PHYA controlled responses (i.e. hypocotyl elongation, and cotyledon opening), no phenotype with respect to the PHYA dependent abrogation of negative gravitropism in blue light and red light enhanced phototropism (Rosler et al., 2007). Thus, most specific PHYA responses, such as inhibition of hypocotyl elongation in FR, can be attributed to nuclear PHYA; however, the abrogation of gravitropism and enhancement of phototropism in the fhl/fhy1 demonstrate clear cytoplasmic functions of PHYA (Rosler et al., 2007). The recently proposed role of PKS1, FHY1 and FHL in phytochrome‐dependent gravitropism points toward a cytosolic route of light‐dependent signaling, along with the demonstrated nuclear pathway. In lower plants, phytochromes also mediate a red light repression of gravitropism as it is the case of the moss Ceratodon (Lamparter et al., 1998). In the protonemata of this moss, this negative regulation of gravitropism is not controlled at the level of the gravity sensing, since the amyloplast sedimentation is not aVected. Thus, it has been suggested that phytochrome acts downstream of sensory mechanism in the signaling pathway (Kern and Sack, 1999). This repression of gravitropism is fluence dependent, since it occurs at high, but not at low fluence (Kern and Sack, 1999). Blue light can also regulate gravitropism in the Ceratodon. In the moss protonemata, blue light reverses the gravity response from negative to positive (Lamparter et al., 1998). This reversal gravitropic response in blue light has not been reported in other species.

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In the fungus Phycomyces blakesleemus,the presence of light can influence the response to gravity (Grolig et al., 2000). Sporangiophores grown in bilateral illumination have an enhanced gravitropic response compared with those grown in darkness. The growth orientation of the sporangiophores appears to be governed by a feedback loop where gravitropism and phototropism influence each other (Grolig et al., 2000). The light control of gravitropic response represents a prime example of crosstalk between environmental signaling systems. In Arabidopsis, it has been hypothesized that such inhibition of gravitropism by light is to allow for phototropic stimuli to determine the orientation in growing hypocotyls (Robson and Smith, 1996). Therefore, in low and patchy light environments, hypocotyl growth orientation would be expected to be determined only by phototropic stimuli in a way that maximize the probabilities of successful seedling emergence. While this agravitropic response has been observed in several species that have small‐size seeds (i.e. Godetia grandiflora, Lobelia erinus, Digitalis purpurea, and Arabidopsis; Allen et al., 2006), this response is absent in other Brassica species that have larger seeds (i.e. B. oleracea, B. rapa, and Synapses alba; Allen et al., 2006). However, this response is not widespread in other plant species with large seeds. Finally, interactions between light and gravity stimuli are very common in nature. Organisms have developed a set of strategies for a better adaptation to specific conditions, and these responses likely confer ecological adaptations to maximize survival in a competitive habitat. B. EFFECT OF GRAVITY ON PHOTOTROPISM

The eVects of gravity on phototropism are most obvious in plants with reduced gravitropic responses. In Arabidopsis, the response to unilateral light is enhanced in mutants that are impaired in gravitropic response (Okada and Shimura, 1992; Ruppel et al., 2001). For instance, in roots of the gravity impaired pgm mutants, a positive response to unilateral red light is observed; however, this response is undetectable in the WT (Ruppel et al., 2001). Hence, this is a case where gravity masks a phototropic response. In plant organs, there exists a competition between gravity and light and it is diYcult to establish whether these tropistic responses are independent processes that coexist or they interplay modulating each other. In Arabidopsis mutants showing reduced gravitropic response (i.e. sgr1/scr, sgr2, sgr4, sgr7/shr, and arg1), the phototropic response is normal (Firn et al., 2000). However, when a phototropic‐deficient mutant was studied in its response to gravity, hypocotyls of seedlings grown in blue light orient randomly since blue light simultaneously suppresses gravitropism (Lariguet and

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Fankhauser, 2005). While light‐controlled inhibition of gravitropism seems to be a better understood process, the gravity control of phototropism is more obscure. Because of the ubiquitous nature of gravity on Earth, the study of phototropism without the interference of gravity is complex (Kraft et al., 2000). One instrument that has been used to mitigate the eVects of gravity is termed a clinostat. Some clinostats rotate specimens around an axis while others work in three dimensions (e.g. random positioning machine). Studies with the fungus Phycomyces determined that sporangiophores are negatively gravitropic and positively phototropic (Grolig et al., 2000). When grown on a rotating clinostat, the sporangiophores display an increase in phototropic bending, with a greater final angle of curvature compared with those grown at 1 g (Grolig et al., 2000). Microgravity is the most eVective way to eliminate gravitational eVects on phototropism. Studies in the moss Ceratodon conducted in microgravity conditions of spacecraft in low Earth orbit demonstrated a fluence dependency in the interaction between phototropism and gravitropism, which was not detectable on Earth (Kern and Sack, 1999). While high fluences of red light have no eVect on the enhancement of phototropism in the protonemata of the moss, at low‐fluence rates the phototropic curvature is enhanced (Kern and Sack, 1999).

V. CONCLUSIONS AND FUTURE PROSPECTS Our comprehension of the molecular pathways of phototropic and gravitropic responses has improved considerably in recent years. Although we have some understanding of how plants sense gravity and light, much less is known of how these signals are transduced and how they result into tropistic responses. At present, these pathways becoming better resolved due to the development of new tools that permit the exploration of physiological, genetic, and biochemical foundation of such processes. Genomic and proteomic approaches with model plants are now widely used and are playing a significant role unraveling gravity‐ and light‐regulated networks. Furthermore, recent genomic analysis has started to address how light and gravity influence transcription at the whole genomic scale. Protein microarrays, extensively used to study protein functions in bacteria and yeast, are starting to being used in plants. Thus, these techniques open a complete new avenue to better understand the transcriptional networks in plants; in particular, in characterizing the networks that regulate light and gravity responses. Recently, it has also been

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proposed that photomorphogenic and phototropic responses interact via hormonal regulation. Whether this crosstalk is actually occurring can be tested by these new methods involving genome‐wide analysis. Recent studies have confirmed the high level of organ specificity of two model plants such as Arabidopsis (dicots) and rice (monocots). The organ specificity in response to gravity and light has long been established, and genome wide analysis of particular organs oVers the possibility of explaining such divergence. In the same way, a remarkable opportunity is opening to better understand how diVerent plant species sense and respond to light and gravity to adapt to diVerent environments. The continuing advances in bio‐imaging have made possible the study of intracellular events in real‐time in living cells. For instance, the involvement of calcium as a secondary messenger in gravitropism has been postulated for a long time. However, not until recently did we have direct evidence supporting its involvement in this process due to refined technologies of calcium imaging. We also expect that advances in the comprehension of the complex interaction between phototropism and gravitropism will be made by using microgravity as an experimental tool (Kiss et al., 2007). Thus, in the near future, the combination of new and refined technologies will help to unravel the signal cascade of light, gravity, and complex interactions between phototropism and gravitropism.

ACKNOWLEDGMENTS We thank Dr. Prem Kumar for his critical review of the manuscript.

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Cold Signalling and Cold Acclimation in Plants

ERIC RUELLAND,* MARIE‐NOELLE VAULTIER,{ ALAIN ZACHOWSKI* AND VAUGHAN HURRY{

*Universite´ Pierre et Marie Curie, UMR7180, CNRS, 3 Rue Galile´e, Ivry‐sur‐Seine, France { Umea˚ Plant Science Centre, Department of Plant Physiology, Umea˚ University, Umea˚, Sweden

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Physiology of Cold Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Effect of Low Temperatures .................................................. B. Responses to Low Temperatures............................................. C. Light and Hormones in Cold Response..................................... III. The CBF Pathway is Essential in Triggering Response to Cold Exposure .. . A. Identification of the CRT/DRE Element ................................... B. Identification of CRT/DRE Binding Factors: CBF....................... C. CBF Expression................................................................. D. Role of CBF in Freezing Tolerance ......................................... E. Regulation of CBF Expression ............................................... F. Others Regulators .............................................................. IV. Transduction of Cold Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Calcium .......................................................................... B. Protein Kinases/Protein Phosphatases ...................................... C. Phospholipases and Phospholipids .......................................... V. Summary and Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 49 Copyright 2009, Elsevier Ltd. All rights reserved.

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0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(08)00602-2

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ABSTRACT Exposure to low temperatures is one of the most important plant abiotic stress factors. In this review we describe the damages that chilling and/or freezing temperatures can cause to plant cells. Confronted to these damages, some plants are able to adapt through mechanisms based on protein synthesis, membrane composition changes, and activation of active oxygen scavenging systems. These adaptive mechanisms rely in part on gene induction. The best understood genetic pathway leading to gene induction upon a temperature downshift is based on C‐repeat‐binding factors (CBF) activating promoters through the C‐repeat (CRT) cis‐element. Such activation of transcription factors suggests that cold, as a signal, has been transduced into the cells. Calcium entry is a major signalling event occurring immediately after a temperature downshift. The increase in cytosolic calcium will activate many enzymes, such as phospholipases and calcium dependent‐protein kinases. A MAP‐kinase module has been shown to be involved in the cold response. Ultimately, the activation of those signalling pathways leads to changes to the transcriptome. In this review we have focused on the genetic and signalling pathways activated early after cold exposure. Much of the data cited is from the model plant Arabidopsis but when possible evidence from other plants is presented.

I. INTRODUCTION During growth and development plants are subjected to numerous biotic and abiotic stresses, and in Boyer’s classic study (Boyer, 1982) he demonstrated that the average yields of our existing crop genotypes were reaching only about 20% of their genetic potential. While some of the yield reductions could be attributed to biotic factors such as disease, insects, and weeds, the major loss in yield resulted from abiotic stresses, chiefly drought and sub‐ optimal temperatures. Many of our existing staple crop species (e.g., corn (Zea mays), rice (Oryza sativa), and potatoes (Solanum tuberosum)) originate from tropical and subtropical regions and are susceptible to damage when temperatures fall below 15 8C (McKersie and Leshem, 1994). The stage in the growth cycle most vulnerable to stress is the reproductive phase, which includes the formation of reproductive organs, flowering, fruiting, and seed development. Besides, these species also do not tolerate chilling temperatures and can suVer irreversible damage when the temperature goes down below 10 8C (Kodama et al., 1995). Other species, such as Pelargonium, are able to tolerate chilling but not freezing temperatures and still others from temperate regions, such as spinach (Spinacia oleracea), winter wheat (Triticum aestivum), canola (Brassica napus) are able to survive freezing temperatures. During the course of evolution many plant species have developed an array of mechanisms that enable them to minimize the negative eVects of cold stress. The ability to cold acclimate, i.e. to increase tolerance to severe cold (freezing) stress as a result of prior exposure to moderately sub‐optimal

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(chilling) temperatures, is a multigenic trait and research over the last 20 years, primarily using Arabidopsis thaliana as a model species, has identified a large number of genetic and biochemical changes that take place during cold acclimation (e.g. Chinnusamy et al., 2007, Nakashima and Yamaguchi‐ Shinozaki, 2006, Thomashow, 1999). In temperate latitudes, cold‐hardiness (or cold acclimation) is established in the autumn, when the temperatures are low but positive and the photoperiod decreases, and it reaches a maximum in winter (Fig. 1). While current research has identified primary and secondary regulators responsible for modulating cold acclimation, the proteins and processes involved in sensing temperature change and initiating acclimation remain to be identified. Furthermore, the modulation of cellular metabolism and the regulation of transcriptome and proteome composition that is the result of acclimation is made even more complex by the presence of three diVerent genomes within the cell (nucleus, chloroplast, and mitochondria), whose expressional activity must be coordinated depending on the status of the cell as a whole and of the individual organelles. Thus, proper cellular function can only be maintained in a fluctuating environment through the activity of a complex network of signalling pathways that respond to the metabolic status of the diVerent compartments and regulate the expression of the diVerent genomes in a coordinated fashion. Adding to this complexity, these regulators of cold acclimation have been identified in plants exposed to abrupt short‐term changes in temperature, not from plants exposed to

Temperature (⬚C) −2

−4

−6

−8

−10

Non Acclimated -acclimated

Wild-type

No treatment

Fig. 1. Cold acclimation of Arabidopsis. Plants either acclimated (8 days at 4 8C) or not were exposed at diVerent freezing temperatures.The plants placed in a temperature chamber with the following freezing temperature regimen: from 4 8C to
2 8C in 30 min, then hold at
2 8C for 1 h; then an identical timing sequence (30 min to reach the next temperature, hold there for 1 h) for successive 2 8C decreases until
10 8C was reached. Photos were taken 7 days later. While all no acclimated plants died at
6 8C, some cold acclimated plants could survive at
8 8C. From Zhu et al., 2005.

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prolonged cold such as experienced by herbaceous winter crops overwintering under snow or woody perennials overwintering above the snow cover. Closing these critical gaps in our understanding is the key goal of the research in this field. In this review, we will detail the eVects that low temperatures have on plants, and the diVerent responses triggered by cold treatment. The involvement of light and hormones on the response will be discussed. We will also describe the molecular web established in response to cold stress and necessary for adaptation to cold. Finally, the diVerent signalling pathways triggered by cold stress will be described. This review is focussed on the cold response in relation to low temperature tolerance. In addition to their negative eVects on plant cell metabolism detailed below, low temperatures can, in some species, induce developmental responses such as flowering (vernalization) or seed germination (stratification). These developmental eVects of cold are beyond the scope of this review. Readers interested in vernalization are invited to read Alexandre and Hennig (2008) and Schmitz and Amasino (2007) while cold stratification is discussed in Finch‐Savage and Leubner‐Metzger (2006).

II. PHYSIOLOGY OF COLD STRESS A. EFFECT OF LOW TEMPERATURES

1. General eVects of cold To understand the eVect of cold on plants, one has to distinguish between low but positive cold temperatures (chilling temperatures) and negative temperatures (freezing temperatures). Lowering temperatures will thermodynamically reduce the kinetics of metabolic reactions. Exposure to low temperatures will shift the thermodynamic equilibrium such that there will be an increased likelihood that non‐polar side chains of proteins will become exposed to the aqueous medium of the cell. This will directly aVect the stability and the solubility of many globular proteins (Siddiqui and Caviocchioli, 2006). This leads to a disturbance in the stability of proteins, or protein complexes and also to a disturbance of the metabolic regulations. Lower temperatures induce rigidification of membranes, leading to a disturbance of all membrane processes (e.g. opening of ion channels, membrane associated electron transfer reactions, etc.). Chilling is also associated with the accumulation of reactive oxygen species (ROS). The activities of the scavenging enzymes will be lowered by low temperatures, and the scavenging systems will then not be able to counterbalance the ROS formation that is always associated with mitochondrial and chloroplastic electron transfer reactions. Moreover, the chloroplast electron

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transfer chain will be over‐reduced during chilling, which will lead to increased ROS formation. The accumulation of ROS will have deleterious eVects, especially on membranes. This will result in ion leakage. In addition, the low temperatures will favour the formation of secondary structures in RNA, thus aVecting gene and protein expression (Fig. 2). Freezing temperatures have even more damaging eVects (Fig. 2). Under natural conditions, plants freeze slowly and extracellular freezing occurs. Due to the diVerence in chemical potential created by a growing ice crystal, cellular water migrates to this extracellular ice, causing cell dehydration and shrinkage (Dowgert and Steponkus, 1984). The degree of dehydration produced by such extracellular freezing depends upon the temperature, and its severity increases with lower temperatures. Ultimately, ice can penetrate the symplast (Gusta et al., 2004), causing a deterioration of the intracellular structures and death of tissues. 2. Detailed eVects of cold (chilling) on photosynthesis Photosynthesis transforms light energy into ATP and redox equivalents (NADPH). The primary metabolic sink for these forms of stored chemical energy is the Calvin cycle, where CO2 is fused to ribulose‐1,5‐bisphosphate to generate glycerate‐3‐phosphate, which is then reduced into triose‐phosphate to the expense of ATP and NADPH. Most of the triose‐phosphate remain in

Protein conformation change Protein complex destabilization

Photoinhibition Photosynthesis impairment

Lowering of enzymatics kinetics Chilling ROS accumulation Membrane rigidification Cytoskeleton depolymerization

Membrane leakage ARN secondary structure stabilization

Freezing

Ice nucleation and spreading in the apoplast

Cell dehydration Water efflux to apoplast Ice spreading into the cell

Osmotic contraction Membrane disruption

Fig. 2.

Cell death

Chilling and freezing eVects on plant cells.

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the chloroplasts to regenerate ribulose‐1,5‐bisphosphate, but a fraction of the triose‐phosphate pool is exported to the cytosol, where it is either used to support cellular metabolism or converted to sucrose for export. Sucrose synthesis releases inorganic phosphate that is then transported back to the chloroplast, in exchange for the exported triose‐phosphate, to support the continuing formation of ATP (Fig. 3). For photosynthesis to be optimal, the balance between light absorbed by the primary photochemical reactions in photosystem II (PSII) and photosystem I (PSI), the transformation of this energy into NADP and ATP, and its utilization in metabolism must constantly be fine‐tuned in response to fluctuations in the environment. This balance between the energy harvested by the photochemical reactions and its utilization by metabolic sinks is called photostasis (Ensminger et al., 2006; Huner et al., 1993). Low temperatures aVect diVerent aspects of photosynthesis. For example, low temperatures inhibit sucrose synthesis in the cytosol, leading to the

Lumen

Stroma

Light

H2O

CO2

PSII

H+

Sucrose-P Glu1-P

RU-1,5-P2

O2 H+

Sucrose UDP-Glu

b6/f

3-PGA

ATP

1,3-diPGA Light

Glu6-P

Fru6-P

Fru6-P NADPH

PSI

NADP+

+

Fru1,6-P2

H+ Fru1,6-P2

NADPH

G3P DHAP

G3P

DHAP ATP H+

CFo CF1 ADP + Pi

Pi

Pi Cytosol

Fig. 3. Simplified representation of phostosynthesis. PSI: photosystem I; PSII: photosystem II; b6/f: cytochrome b6/f complex; Fru1,6‐P2: fructose‐1,6‐bisphosphate; Fru6‐P: fructose‐6‐phosphate; Ru‐1,5‐P2: rubilose‐1,5‐bisphosphate; 3‐PGA: 3‐phosphoglycerate; 1,3‐diPGA: 1,3‐diphosphoglycerate; G3P: glyceraldehyde‐3‐phosphate; DHAP: Dihydroxyacetone phosphate; Glu6‐P: Glucose‐6‐phosphate; Glu‐1‐P: Glucose‐1‐phosphate; UDP‐Glu: UDP‐Glucose.

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accumulation of phosphorylated intermediates. This results in the depletion of the available inorganic phosphate and in the decreased cycling of inorganic phosphate between the cytosol and the chloroplast (Furbank et al., 1987; Hurry et al., 1993, 2000). This, in turn, impedes synthesis of the ATP necessary for the regeneration of ribulose‐1,5‐bisphosphate to support CO2 fixation. In addition, low temperatures can inhibit thylakoid electron transport by increasing membrane viscosity and restricting the diVusion of plastoquinone (GriYth et al., 1984). In contrast, light energy trapping by the antenna of PSI and PSII and the use of this energy to drive charge‐separation within the reaction centre cores are largely temperature independent. Therefore an imbalance can be created by an exposure to low temperatures because the chlorophyll antenna complexes trap more energy that can be processed biochemically (Ensminger et al., 2006; Huner et al., 1993). Under these conditions, thylakoid membranes become over‐energized. One of the consequences of this over-energized state is photodamage, caused primarily by the increased formation of ROS. The accumulation of excitation energy in the light‐ harvesting chlorophyll antennae of the photosystems favours the production of triplet excited chlorophyll molecules that can interact with O2 to generate reactive singlet oxygen (1O2). Superoxide can be produced during water oxidation. Over‐reduction of the photosynthetic electron carrier chain will also favour the direct reduction of O2 by PSI, leading to the formation of damaging ROS, such as superoxide (O2
), hydrogen peroxide (H2O2), and the hydroxyl radical ( OH) (Fig. 4). ROS synthesis by the photosynthetic electron transport system is, under warm growth conditions, counter‐balanced by an active ROS scavenging system, including Cu/Zn‐superoxide dismutase and ascorbate peroxidase. However, at low temperatures the eYciency of these enzymatic systems is compromised and, coupled to the increase in accumulation of reducing power at the acceptor side of PSI, this leads to photoinhibition and lipid peroxidation. Superoxide is the major cause of photodamage to PSI in the cold. This reactive species of oxygen attacks the iron–sulphur centres, FA, FB and Fx (Sonoike et al., 1996; Tjus et al., 1998). PSI photoinhibition in low light at chilling temperatures occurs not only in cold sensitive plants (cucumber, potato; Sonoike et al., 1996), but also in cold resistant plants such as barley (Tjus et al., 1998), winter rye (Ivanov et al., 1998), and Arabidopsis (Zhang and Scheller, 2004). One of the diVerences between cold sensitive and cold tolerant species is suggested to originate in diVering sensitivities of the ROS scavenging enzymes toward low temperatures, and in diVerences in how to cope with dissipation of excess excitation light (Tjus et al., 2001).The active oxygen produced at PSI thus contributes to photoinhibition of PSI. However when not scavenged, the rather long‐lived superoxide anions created at PSI can be



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O2 NADP+ O2

Stroma

Fd QA

QB

NADPH

FX,FA,FB

Phe e−

FNR

PQ

Cytb6 Fe/S Cytf

P680*

A1 e− P700*

[Mn]4 Lumen

PC O2

1O

2

3O

PC

H2O

2

O2

−

Fig. 4. ROS formation in chloroplasts. P680, Photosystem II primary electron donor; P700, Photosystem I primary electron donor; Mn4, Mn4 cluster; QA, quinone QA; QB, quinone QB; PQ, plastoquinone pool; Cytb6, cytochrome b6; Cytf, Cytochrome f; Fx, FA, FB, iron–sulphur centers of PSI; PC, plastocyanin; Fe/S, Fe/S centers; Fd, ferredoxin; FNR, ferredoxin NADP reductase.

converted into hydrogen peroxide through auto‐disproportionation or residual superoxide dismutase activity. These hydrogen peroxide and superoxide molecules can diVuse and inactivate photosystem II (PSII). This was shown in spinach thylakoids illuminated with far red light at 4 8C, i.e. a light exciting specifically PSI but not PSII. This treatment not only inactivated PSI but also led to a 90% inactivation of PSII. In addition, the use of oxygen scavengers suggests that superoxide is the major cause of photodamage to PSI whereas hydrogen peroxide and superoxide are the most damaging forms for PSII (Tjus et al., 2001). In Arabidopsis plants exposed to a low light chilling treatment, PSII functionality (measured as Fv/Fm) was shown to progressively decline to 19% after 24 h at 4 8C. In contrast, no loss of PSII activity took place in the darkness even after 24 h at 4 8C. PSI activity (measured as NADPþ photoreduction) was shown to decline by 32% after a similar low light chilling treatment for only 8 h, while no obvious changes occurred in the darkness at 4 8C. The low light chilling treatment had thus a much more pronounced eVect on PSI activity compared with PSII in Arabidopsis plants (Zhang and Scheller, 2004). The major site of photoinhibition in PSII is the inactivation of the D1 protein (Aro et al., 1993; Greenberg et al., 1987). Inactivated proteins must

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be replaced by newly synthesized D1 to restore PSII activity. The actual extent of PSII photoinhibition in vivo depends on the balance between inactivation of D1 and the recovery process, which involves insertion of new D1 molecules into the thylakoid and their incorporation into the PSII complex (van Wijk et al., 1997). Recovery from PSII photoinhibition is strongly temperature dependent, i.e. low temperature will decrease the rate of repair (Gombos et al., 1994). In addition to these photoinhibitory eVects on the two photosystems, the formation of ROS will lead to peroxidation of thylakoid membranes. Because of their unique lipid composition, characterized by a high proportion of galactolipids containing highly unsaturated fatty acids (18:3), chloroplast membranes are very sensitive targets for photodestruction by active forms of oxygen (Havaux and Niyogi, 1999). Moreover, the formation of ROS will expend a substantial portion of the chloroplast reductant reserves, thereby leading to a lowering of the redox poise. This will have a negative eVect on the reduction of thioredoxin f and its capacity to activate its target enzymes by reduction of disulfide groups (Ruelland and Miginiac‐Maslow, 1999). It has been shown that the light dependent‐reductive activation of sedoheptulose‐1,7‐bisphosphatase and fructose‐1,6‐bisphosphatase, two enzymes of the Calvin cycle, was inhibited following illumination at low temperature in tomato (Bru¨ggemann et al., 1994; Hutchison et al., 2000; Sassenrath et al., 1991). This could be explained by the redox midpoint potential of the sulfhydryl groups of these enzymes: if the cold and light treatments cause a lowering of the redox poise of thioredoxin f to its midpoint potential, sedoheptulose‐1,7‐bisphosphatase and fructose‐1,6‐bisphosphatase would be only 10% activated (Hutchison et al., 2000). In addition, recent proteomics studies with rice (Yan et al., 2006) and Arabidopsis (Goulas et al., 2006) have provided evidence for the cold‐induced degradation of photosynthetic proteins such as RcbA, proteins of the photosystem II oxygen evolving complex, sedoheptulose‐bisphosphatase and the ATP synthase a and b chains. Degradation of these proteins and protein complexes could be linked to the action of ROS in the cold. In chilling-sensitive cucumber, the large subunit of ribulose‐1,5‐bisphosphate carboxylase (Rubisco) is site‐specifically cleaved by a hydroxyl radical ( OH) generated in the illuminated chloroplast lysates, or in intact leaf, (Nakano et al., 2006). The eVects of low temperatures on photosynthesis are summarized in Fig. 5.



3. Detailed eVects of cold (Freezing) on membranes The ROS produced during chilling exposure also lead to lipid peroxidation, resulting in ion leakage. However, freezing temperatures induce more dramatic damages. Several types of freeze‐induced membrane lesions have been

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Chilling Lowering of enzyme activities

ROS scavenging enzymatic systems are exceeded

Over reduction of thylakoid membranes

ROS accumulation Photoinhibition of PSI and PSII

Breakage of stromal proteins (Rubisco)

Redox poise is more oxidized

Thioredoxin-dependent enzymes are less activated

Fig. 5. Overview of the eVects of chilling on photosynthesis. At light the chloroplast is a major site of ROS production. Chilling will thermodynamically reduce enzymatic activities such as Calvin cycle enzymes and ROS scavenging activities. At light, chilling provokes a photostasis imbalance resulting in an over reduction of electron transport chain in thylakoid membranes and ROS generation in PSII and PSI. As a consequence, ROS will accumulate, leading to degradation of D1 proteins in PSII, iron sulfur centers in PSI, and stromal enzymes such as Rubisco. The changes in the redox poise imposed by ROS accumulation will lead to lower activation of thioredoxin‐dependent Calvin cycle enzymes such as fructose1,6‐bisphosphatase and sedoheptulose1,7‐bisphosphatase, and to enhanced inhibition of photosynthesis.

described, and all are linked to freezing initiating ice nucleation in the apoplast. The formation of extra‐cellular ice will induce cellular water to move to the apoplast in response to the change in water potential, thus causing cellular dehydration and osmotic contraction of the cell. In contrast, thawing events will be accompanied by osmotic expansion (Fig. 6). a. Expansion‐induced lysis. Expansion‐induced lysis will predominate for species for which freezing and subsequent osmotic contraction are associated with the formation of endocytotic vesicles. This will result in a reduction in the surface area of the plasma membrane. During thawing, osmotic expansion is accompanied by surface area expansion. However, when too large areas are reduced during the freezing step, these reductions are irreversible. As a consequence, during thawing the protoplasts lyse before regaining their initial size because the material deleted into cytoplasmic vesicles is not readily available for reincorporation into the plasma membrane during subsequent expansion (Fig. 6A; Dowgert and Steponkus, 1984).

COLD SIGNALLING AND COLD ACCLIMATION IN PLANTS

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A

Freeze dehydration

Thawing

Contraction

Endocytotic vesicles

Expansion leads to lysis Cell is dead

B

Freeze dehydration

Thawing

Contraction Endocytotic vesicle and membrane apposition : HII phase formation

No expansion Cell is dead

C

Freeze dehydration

Thawing

Contraction Exocytic extrusions Expansion D

Freeze dehydration

Thawing

Contraction Exocytic extrusions and membrane apposition : fracture jump lesion

No expansion Cell is dead

Fig. 6. Consequences of osmotic contraction on membrane and cell viability. (A) In non‐acclimated protoplasts of rye, freeze‐induced osmotic contraction is accompanied by endocytotic vesicles. During thawing, osmotic expansion leads to lysis because the deleted membrane material is not available for reincorporation into the plasma membrane. (B) At lower freezing temperatures, freeze‐induced osmotic contraction is also accompanied by membrane apposition resulting in hexagonal II (HII) phase formation leading to irreversible loss of membrane integrity. Thawing is not accompanied by expansion. In both cases, freezing leads to cell death. (C) In non‐ acclimated Arabidopsis protoplasts or acclimated Rye protoplasts, freeze‐induced osmotic contraction is accompanied by exocytotic extrusions. During thawing, plasma membrane regains its initial shape and osmotic expansion does not lead to lysis. (D) At lower freezing temperatures, freeze‐induced osmotic contraction is also accompanied by membrane apposition resulting in fracture jump lesions leading to irreversible loss of membrane integrity. Thawing is not accompanied by expansion. The cell is dead. Drawings are adapted from Uemura et al., 2006.

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The incidence of expansion‐induced lysis depends on the species and the temperatures. In non‐acclimated protoplasts, the occurrence of expansion‐ induced lysis is quite low in A. thaliana (<10%) in comparison to that which occurs in rye (>40%) and oat (30%) over the range of
2 to
4 8C (Uemura et al., 1995). In rye and oat, expansion‐induced lysis no longer occurs once leaves are cold‐acclimated because the plasma membranes no longer undergo endocytotic reduction but instead produce exocytotic extrusions. Such extrusions are reversible during thawing and during expansion the extrusions retract into the plane of the plasma membrane and the protoplasts remain intact (Fig. 6C). The occurrence of expansion‐induced lysis is low in Arabidopsis because during freezing‐induced osmotic contraction non‐acclimated protoplasts form exocytotic extrusions rather than endocytotic vesicles. b. Freeze-induced formation of hexagonal II phase and fracture-jump lesions. At lower freezing temperatures, another consequence of freeze‐induced contraction is the fact that the plasma membrane is brought into close apposition with various endomembranes, and particularly with the chloroplast envelope. When lipid bilayers are in close apposition, they may form inverted micellar intermediates that can be converted into either the hexagonal II phase (a non‐lamellar phase that is a 3‐dimensional (3‐D) array of inverted cylindrical micelles with water in the central core of each cylinder) or interlamellar attachments (Fig. 7). Whether the inverted micellar intermediates are converted into hexagonal II phase or interlamellar attachments are thought to be dependent on the intrinsic curvature of the constituent monolayers: monolayers with a high intrinsic curvature form the hexagonal II phase whereas those with a low curvature form interlamellar attachments (Siegel, 1987; Uemura et al., 1995). Both will lead to loss of osmotic responsiveness: for the cells for which osmotic contraction has led to hexagonal II phase or interlamellar attachments, thawing is no longer accompanied by osmotic expansion. The threshold temperature for freeze‐induced formation of the hexagonal II phase is
3 8C in oat and
6 8C in rye, with a high incidence (in 50% of the protoplasts) occurring in oat at
5 8C and at
10 8C in rye (Fig. 6B). In Arabidopsis, after acclimation, freeze‐induced formation of hexagonal II phase is precluded, and injury is associated with fracture‐ jump lesions (Fig. 6D; Uemura et al., 1995). Fracture‐jump lesions result from the formation of interlamellar attachments. In freeze‐fracture electron micrograph, they are characterized by localized deviations of the plasma membrane fracture plane to closely appressed lamellae. We have just seen the different damages caused by chilling and/or freezing temperatures to plant cells. Plant responses to a temperature downshift are attempts to minimize or to prevent the deleterious eVects, either physical

COLD SIGNALLING AND COLD ACCLIMATION IN PLANTS

Freeze-induced osmotic contraction

47

Plasma membrane Membrane of chloroplast envelope

Inverted Micellar Intermediates

Interlamellar Attachments

Hexagonal II phase

Fig. 7. Destabilization in membrane structures triggered by freeze‐induced osmotic contraction. Osmotic contraction leads to membrane apposition between plasma membrane and membrane from chloroplast envelope, most likely outer membrane. The resulting inverted micellar intermediates can be converted into interlamellar attachments or hexagonal II phase.

(e.g., membrane rigidification, lowering of enzymatic kinetics), chemical (e.g., ROS production) or biochemical (e.g., metabolic disequilibrium), that are the consequences of the temperature downshift. We will now present those diVerent responses.

B. RESPONSES TO LOW TEMPERATURES

1. Stress‐related proteins a. COR/LEA and dehydrins. One of the best documented responses of plants to cold treatments is the accumulation of hydrophilic proteins predicted to form an amphipathic ‐helix. Many of the genes encoding those proteins were first characterized as being responsive to cold, drought and/or ABA. Most of these proteins have therefore been named COR (cold responsive), LTI (low temperature induced), RAB (responsive to abscisic acid), KIN (cold induced) or ERD (early responsive to dehydration).

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These proteins include dehydrins, which define the group II of late embryogenesis abundant (LEA) proteins (Bies‐Ethe`ve et al., 2008). The amino acid sequence of dehydrins is characterized by three highly conserved domains: (i) the K‐segment (EKKGIMDKIKEKLPG), which exists as one or more copies and that is the only conserved repeat found in all dehydrins; (ii) the Y‐segment (DEYGNP), usually found in 1–3 copies in the most N‐terminal part; and (iii) the S‐segment, which is a phosphorylable Ser‐rich tract (Puhakainen et al., 2004). In wheat, the accumulation of a dehydrin (WCOR410) is correlated with the capacity to develop freezing tolerance: during the whole period of cold acclimation, the freezing‐tolerant winter cultivar ‘‘Fredrick’’ accumulated more of the WCOR410 proteins compared with the less freezing‐tolerant spring cultivar ‘‘Glenlea’’ (Danyluk et al., 1998). However, the overexpression of single dehydrins does not necessarily lead to enhanced freezing tolerance. For example, the overexpression of RAB18, a cold induced dehydrin, had no eVect on freezing tolerance in Arabidopsis (La˚ng, 1993). Nevertheless, the overexpression of multiple dehydrins can enhance the freezing tolerance. For example, when either RAB18 and COR47 (two dehydrins) or LTI29 and LTI30 (two dehydrins) were ectopically expressed as pairs in transgenic Arabidopsis, this resulted in plants with reduced ion leakage during freezing test when compared to the wild‐type plants (Puhakainen et al., 2004). This increased resistance to freeze‐induced damage was observed with non‐cold acclimated plants and the eVect was even more pronounced when plants were exposed to an acclimation period. In addition, the eVect was more pronounced in plants overexpressing LTI29 and LTI30 than in plants overexpressing RAB18 and COR47. The fact that an eVect is seen in plants overexpressing two dehydrins, but not one, may be an indication that diVerent dehydrins have diVerent cellular functions and that to acquire freezing tolerance these diVerent functions are necessary. However, because freezing tolerance is further enhanced when the overexpressing plants have been subjected to a cold treatment might also indicate that, to fully play their role, dehydrins might be activated by a cold‐induced mechanism such as protein phosphorylation. The way dehydrins can interfere with freezing is still not clear. In wheat, WCOR410 accumulates in the vicinity of the plasma membrane (Danyluk et al., 1998). In Arabidopsis, LTI29 was detected in cytosol but also in the plastid envelope, in the plasma membrane and tonoplast (Puhakainen et al., 2004). However, membrane localization of LTI29 became predominant after cold exposure. In contrast, LTI30 was specifically localized to membranes (Puhakainen et al., 2004). These localizations could indicate a role for dehydrins in preventing membrane destabilization during dehydration.

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Indeed, the presence of amphipathic helices in dehydrins suggests that the proteins could act as an interface between membranes and the cytosol. A maize dehydrin, DHN1, can bind lipid vesicles containing acidic phospholipids (Koag et al., 2003). In addition to this postulated function in protecting membranes, dehydrins have been proposed to possess cryoprotective (Bravo et al., 2003) or antifreeze (Wisniewski et al., 1999) activity. They could improve enzyme activity under conditions of low cellular water (Rinne et al., 1999). A dehydrin from Citrus unshiu Marcov., CuCOR19, can act as a hydroxyl radical scavenger (Hara et al., 2004). In vitro, CuCOR19 prevented peroxidation of Soybean liposomes more eYciently than serum albumin, glutathione, proline, or glycine betaine. When overexpressed in Nicotiana tabaccum, it led to plants with enhanced freezing tolerance, the freezing tolerance being correlated to the level of CuCOR19 accumulation (Hara et al., 2003). In Arabidopsis, ERD14 can bind calcium. The phosphorylated form of ERD14 binds significantly more calcium than the non‐phosphorylated protein. ERD14 can be phosphorylated by protein extracts from cold treated plants, suggesting that the phosphorylated status of this protein could be modulated through cold‐regulated kinases or phosphatases. Such calcium binding dehydrins could be calcium buVers or act as calcium dependent chaperones (Alsheikh et al., 2003). Dehydrins are only one subgroup of hydrophilic proteins that accumulate in response to cold. In A. thaliana, COR15A is a hydrophilic protein but it is not a canonical dehydrin as defined above. It is located to the chloroplast stroma. Its expression is highly induced in response to cold. Ectopic expression of COR15A enhances the in vivo freezing tolerance of chloroplasts in non‐acclimated plants by almost 2 8C (Artus et al., 1996). The eVect of COR15A could be due to a cryoprotective eVects on stroma proteins. Recombinant COR15A exhibits in vitro cryoprotection of a freeze‐labile enzyme, l‐lactate dehydrogenase. COR15A is capable of associating with potential stroma substrates in vivo, such as the small and large subunits of Rubisco (Nakayama et al., 2007). It has also been suggested that COR15A could enhance freezing tolerance through stabilizing membranes. The transition to hexagonal II phase (see Section II.A.3.) is decreased in plants overexpressing COR15A subjected to freezing (Steponkus et al., 1998). Because COR15A is located in the stroma, the way it could have an eVect on those transitions is not clear. COR15A might defer freeze‐induced formation of the hexagonal II phase to lower temperatures (higher dehydration) by altering the intrinsic curvature of the inner membrane of the chloroplast envelope. As seen in Section II.A.3., chloroplast envelopes are involved in the formation of hexagonal II phase and high curvature of membranes favours the formation of such phases.

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b. Antifreeze proteins. In order to prevent ice nucleation and ice crystal coalescence, overwintering plants, like fish and insects, can secrete thermal hysteresis or antifreeze proteins (AFPs). AFPs are secreted into the apoplast, where they bind irreversibly to the surface of ice and are incorporated into the ice crystal lattice. Plants have no constitutive AFP activity, it is induced by cold. In winter rye (Secale cereale L. cv ‘‘Musketeer’’), AFPs are accumulated in response to cold in the apoplast of leaves and crowns (Antikainen et al., 1996). The level of accumulation of transcripts for an AFP, TaIR1, correlated with the freezing tolerance of diVerent wheat cultivars (Tremblay et al., 2005). In the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by about 1 8C. In vitro studies showed that apoplastic proteins extracted from cold‐ acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution‐saturated filter paper. The role of AFPs thus appear to be to directly interact with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice (GriYth et al., 2005). There is no consensus sequence or single structure identified as an ice‐ binding domain. The AFP from perennial ryegrass (Lolium perenne) is predicted to fold into ‐roll with two ice‐binding domains located on opposite sides of the protein (GriYth and Yaish, 2004). About 74% of carrot AFP is composed of a Leu‐rich repeat (Zhang et al., 2004a) and two AFPs from wheat contain 2 or 8 Leu‐rich repeats (Tremblay et al., 2005). In peach, a protein with homology to dehydrins has antifreeze activity (Wisniewski et al., 1999). In addition, Lipid Transfer Protein 1 from winter rye was shown to have antifreeze activity (Doxey et al., 2006). However, most plant AFPs are pathogenesis‐related (PR) proteins, including chitinases, ‐1,3‐glucanases, thaumatin‐like proteins. In winter rye, AFPs have antifungal activities and it is notable that PR proteins that accumulate in warm temperature in response to elicitors such as salicylic acid (SA) have no antifreeze activity. What features distinguish PR proteins with or without antifreeze activity is not known (GriYth and Yaish, 2004). c. Cold shock proteins and RNA binding proteins. One of the major problems that living organisms have to deal with when exposed to low temperatures is the formation and stabilization of RNA secondary structure. Indeed what have been named cold shock proteins (CSP) in bacteria are RNA chaperones that unwind RNA secondary structures. This function is critical for eYcient translation of mRNA at low temperatures. In Escherichia coli, CspA, the most prominent of the nine E. coli CSPs, can account for as much

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as 10% of total protein during cold stress (Jiang et al., 1997). The 3‐D structure of CspA forms a five stranded ‐barrel structure that contains two consensus RNA binding domains. This ‐barrel structure defines the cold shock domain (CSD). A CSD‐containing protein, WCSP1, was isolated from wheat. WCSP1 contains a three‐domain structure consisting of an N‐terminal CSD and an internal glycine‐rich region, which is interspersed with three C‐terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. WCSP1 mRNA and protein levels increase during cold acclimation (Karlson et al., 2002). In vitro melting assays demonstrated that WCSP1 could unwind dsDNA (Nakaminami et al., 2006). In A. thaliana, 4 genes encoding putative proteins with a N‐terminal CSD have been detected in silico. These proteins have glycine‐rich regions. AtCSP2 (At4g38680), At4g36020 and At2g17870 are induced by cold while AtGRP2b (At2g21060) is repressed (Karlson and Imai, 2003). AtCSP2 binds to RNA and unwinds the nucleic acid duplex (Sasaki et al., 2007). Other RNA‐binding proteins have been shown to be important for cold acclimation. In A. thaliana, transcript levels of AtGRP2 (At4g13850) increase markedly during cold stress. Root growth at 11 8C of plants overexpressing GRP2 plants was higher than that of wild‐type. After a freezing treatment at
5 8C for 2 h, while most of the wild‐type and grp2 plants died, about 70–80% of plants overexpressing GRP2 subjected to the same freezing treatment survived. GRP2 is localized in mitochondria in Arabidopsis, and 22 proteins putatively modulated by GRP2 were investigated by comparing mitochondrial protein fractions of Arabidopsis plants, either wild‐type or GRP2‐overexpressing, subjected to 4 8C for 3 days (Kim et al., 2007). Another glycine‐rich RNA‐binding protein, designated at RZ‐1a (At3g26420), has been studied in A. thaliana in relation to cold stress. The transcript level of atRZ‐1a markedly increased with cold stress. Germination and seedling growth of the loss‐of‐function mutants were retarded remarkably compared with those of the wild‐type under cold stress. In contrast, plants that overexpressed atRZ‐1a displayed earlier germination and better seedling growth than the wild‐type under cold stress (Kim et al., 2006). 2. Membrane composition changes a. Bulk composition of membranes. It is well documented that cold exposure induces important changes in membrane composition. For example, in chloroplasts of winter rye (Uemura and Steponkus, 1997) and Arabidopsis (Hendrickson et al., 2006; Ivanov et al., 2006) cold acclimation results in a decrease of monogalactosyldiacylglycerol and an increase of digalactosyldiacylglycerol, both in the inner or outer membranes. Cold exposure also leads to a decrease in plastidic phosphatidylcholine but only in outer membranes

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(Uemura and Steponkus, 1997). Similarly, in coVee, cold acclimation leads to an increase in digalactosyldiacylglycerol (Campos et al., 2003). Concerning plasma membranes, cold acclimation of A. thaliana results in an increase of phospholipids and to a decrease of cerebrosides and free sterols (Uemura et al., 1995). In potato, cold acclimation of the freezing tolerant Solanum commersonii was correlated with an increase of phospholipids in the plasma membrane, primarily due to an increase in phosphatidylethanolamine (Palta et al., 1993). Such an increase in the ratio phosphatidylethanolamine to phosphatidylcholine has also been observed in Spring oat ‘‘Ogle’’ and winter Rye ‘‘Puma’’, but not in Arabidopsis (Uemura and Steponkus, 1994; Uemura et al., 1995). The changes in the composition of plasma membranes and chloroplast envelopes have been proposed to have a role in preventing freeze‐induced membrane damage (see Section II.A.3). Of the lipids that comprise the chloroplast envelope, monogalactosyldiacylglycerol has a high propensity to form hexagonal II phase, while digalactosyldiacylglycerol and phosphatidylcholine have a low propensity to form hexagonal II phase, and would stabilize the bilayer lamellar configuration (Uemura and Steponkus, 1997). After cold acclimation, freeze induced hexagonal II phase formation does not occur and the fracture‐jump lesion is the predominant lesion observed in many plants. As mentioned above, both hexagonal II phase and the fracture‐jump lesions are associated with the formation of inverted micellar intermediates between the plasma membranes and the chloroplast envelope. It is the intrinsic curvature of the monolayers that will determine whether the inverted micellar intermediates are converted into interlamellar attachments (leading to fracture-jump lesions) or hexagonal II phase. In the plasma membrane of rye, an increase in the proportion of phospholipids and a decrease in the proportions of cerebrosides and free sterols during cold acclimation will result in an increase of water retained at the membrane surface together with a decrease of the intrinsic curvature of the monolayers. In the chloroplast envelope of rye, a decrease in the proportion of monogalactosyldiacylglycerol and an increase in the proportion of digalactosyldiacylglycerol would have the same consequences (increase of the hydration of envelope membranes and decrease of the intrinsic curvature). Consequently, after cold acclimation, a lower temperature must be imposed to remove water from the membrane surface and to bring the chloroplast envelope into close apposition with the plasma membrane. In a temperature range, loss of osmotic response is therefore precluded. But at lower temperatures, inverted micellar intermediates are nevertheless formed; they will lead to interlamellar attachments because of the intrinsic curvature of the monolayers. This leads to fracture‐jump lesions (Fig. 7; Uemura and Steponkus, 1997).

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b. Phospholipid desaturation. In Arabidopsis, the proportion of the diVerent phospholipids diVers little during cold acclimation. However, there is a marked diVerence in the composition of phospholipids. After one week at 2 8C, the proportion of di‐unsaturated species such as 18:1/18:3, 18:2/18:2, and 18:2/18:3 increased in both phosphatidylcholine (from 36.9 to 42.8 mol %) and phosphatidylethanolamine (from 28.6 to 34.9 mol%), and the proportion of mono‐unsaturated species such as 18:0/18:3 and 16:0/18:3 decreased from 60.8 to 54.8 mol% in phosphatidylcholine and from 67.6 to 61.4 mol% in phosphatidylethanolamine (Uemura et al., 1995). This increase in unsaturated species is a general effect of cold and was observed in plasma membranes of mulberry (Morus bombycis Koidz.), in tonoplast of the CAM plant Kalanchoe¨ daigrmontiana, and in mitochondria membranes of Arabidopsis or Soybean (Behzadipour et al., 1998; Davy de Virville et al., 2002; Matos et al., 2007; Yoshida et al., 1984). The level of unsaturation of lipids has a role in the response to cold. The fad2 mutants of Arabidopsis are deficient in the activity of microsomal 18:1 desaturase. These plants contain reduced levels of polyunsaturated fatty acids: phosphatidylcholine from the fad2.2 mutant contained 2% 18:2 fatty acid and 14% 18:3 fatty acid compared with wild‐type levels of 33% and 40%, respectively. Their growth characteristics at 22 8C were very similar to wild‐type. However, after transfer to 6 8C, rosette leaves of the mutants gradually died, and the plants died (Miquel et al., 1993). During cold treatment, the freezing tolerant, cold‐acclimating S. commersonii showed an increase in 16:0 to 18:2 ratio in polar lipids extracted from plasma membrane that was not observed in the freezing sensitive, non cold‐acclimating S. tuberosum (Palta et al., 1993). In rapeseed, the 18:3 species in reticulum lipids appeared much more rapidly in a cold tolerant cultivar than in a cold sensitive one (Tasseva et al., 2004). One of the goal of desaturation increase might be to counterpart cold‐ induced membrane rigidification (see Section II.A.1.) because unsaturated lipids yield to a more fluid membrane. In mulberry and B. napus, the fluidity of the membrane, as determined by fluorescence depolarization technique, increased with cold hardiness (Tasseva et al., 2004; Yoshida et al., 1984). However, as seen by Tasseva et al. (2004), this increase in membrane fluidity is not suYcient to compensate the rigidification imposed by cold. The fluidity of reticulum membranes extracted from B. napus plants treated at 4 8C and assayed at 4 8C is quite lower than the fluidity of membranes extracted from plants grown at 22 8C and assayed at 22 8C. No homeoviscous adaptation is attained (Tasseva et al., 2004). Another role of unsaturation of lipids during cold treatment is to prevent the formation of expansion‐induced lysis during freezing/thawing cycle (see Section II.A.3.): an increase in the proportion of unsaturated species of

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phosphatidylcholine causes decrease in the incidence of expansion‐induced lysis (Uemura and Steponkus, 1989). Finally, a role of the polyunsaturated fatty acids would be for chloroplast maintenance. The steady‐state level of transcripts for FAD8, a chloroplast omega‐3 desaturase, is strongly increased in Arabidopsis plants grown at low temperature (Gibson et al., 1994). Chilling is thought to induce liquid crystalline phase to gel phase transition in chloroplast membranes. The induction of trienoic acid is proposed to prevent the formation of this gel phase in chloroplast membranes (Roughan et al., 1985). Trienoic fatty acids of thylakoid membrane lipids are also required for low temperature recovery from photoinhibition in Arabidopsis. Lowered thylakoid unsaturation reduces the rate at which damaged D1 protein can be replaced (Vijayan and Browse, 2002). The roles of membrane composition changes in the response to cold are summarized in Fig. 8. 3. Sugars a. Sugars are accumulated during cold acclimation. The role of sugar accumulation in the development of freezing tolerance in plants is well documented (e.g. Levitt, 1980; Siminovitch, 1981). Many studies have shown that the content of soluble sugars in Arabidopsis leaves increases dramatically during cold acclimation (Kaplan et al., 2004; Klotke et al., 2004; Rohde et al., 2004; Strand et al., 1997, 1999, 2003; Takagi et al., 2003) and studies of transgenic Arabidopsis have demonstrated a causal link between the cold‐induced Response Lipid desaturation

Adaptative effects Cold-induced membrane rigidification is partly counterbalanced

Prevention of Expansion-induced lysis Chilling temperatures Chloroplast membrane gel phase transition is prevented

Increase of phospholids vs. cerebrosides Decrease of monogalactosyldiacylglycerol

Prevention of membrane apposition

vs. digalactosyldiacylglycerol Prevention of HII phase formation

Fig. 8. Low temperatures induce changes in lipid membrane composition. The roles of these changes are indicated.

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modulation of sucrose metabolism and cold tolerance (Strand et al., 2003). Data from winter rye (Koster and Lynch, 1992), winter wheat (Hurry et al. 1995; Kamata and Uemura, 2004; Sagisaka et al., 1991) and winter B. napus (Gusta et al., 2004; Hurry et al., 1995) have all been shown to strongly increase total soluble sugars during cold acclimation. Most of this increase can be attributed to the accumulation of sucrose or raYnose (Koster and Lynch, 1992). Furthermore, the heterosis of leaf freezing tolerance generated by crossing diVerent Arabidopsis ecotypes has been shown to be positively correlated with leaf sugar content (Rohde et al., 2004). In diVerent studies, Gray and Heath (2005) and Kaplan et al. (2007) used metabolite profiling analysis on Arabidopsis during a cold acclimation time course to show the dynamics of metabolic change during cold acclimation. Kaplan et al. (2007) showed that, during cold exposure, the time point when galactinol level reached 50% of its maximum value (T0.5[MAX]) was 8–9 h. Galactinol can combine with sucrose, leading to raYnose whose T0.5[MAX] ¼ 17–18 h. Galactinol maximum level was attained after 30 h of 4 8C exposure and stays constant during the rest of the experiment (till 96 h). In contrast, raYnose is still increasing in abundance at 96 h (Kaplan et al., 2007). In support of this evidence for raYnose playing a role in cold acclimation, of diVerent Arabidopsis ecotypes exposed to 4 8C for 14 days, ‘‘Cape Verde Islands‐1’’ plants accumulated less galactinol, fructose, glucose and raYnose than ‘‘Wassilewskija‐2’’ plants, a more freezing tolerant cultivar (Cook et al., 2004). Similarly, in wheat, cold acclimation induced a marked up‐regulation of transcripts for galactinol synthase, which was detected earlier in the spring cultivar ‘‘Quantum’’ than in the winter cultivar ‘‘CDC Clair’’. However, after 2 h of cold exposure, levels began to decrease in the spring cultivar but the winter cultivar showed sustained induction (Monroy et al., 2007). Furthermore when the rice gene OsUGE‐1, which encodes a UDP‐glucose 4‐epimerase, was ectopically expressed in Arabidopsis, transgenic plants showed a higher level of raYnose and increased freezing tolerance relative to wild‐type plants, suggesting that elevated levels of raYnose resulted in enhanced freezing tolerance (Liu et al., 2007). However, in contrast to these findings, transgenic lines of A. thaliana, ‘‘Columbia‐0’’ and ‘‘Cape Verde Islands,’’ that constitutively expressed a galactinol synthase gene from cucumber have been shown to accumulate up to 20 times more raYnose than the respective wild‐type under non‐acclimated conditions and up to 2.3 times more after 14 days at 4 8C without showing increases in the freezing tolerance of non‐acclimated leaves nor the ability to cold acclimate (Zuther et al., 2004). Moreover, a mutant carrying a knockout of the endogenous raYnose synthase gene, which resulted in raYnose not being detectable in any tissues, showed no change in freezing tolerance of non‐acclimated leaves, nor altered

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ability to cold acclimate (Zuther et al., 2004). This genetic data indicates that while raYnose accumulation correlates well with increased freezing tolerance, raYnose accumulation is not an absolute requirement for Arabidopsis to acquire increased freeze tolerance. Arabidopsis subjected to 14 days at 4 8C also show increases in trehalose (Cook et al., 2004). Similarly in rice, OsTPP1, which encodes a trehalose‐6‐ phosphate phosphatase shows low transcript levels under ambient conditions but is highly induced within 1–2 h of chilling stress (12 8C) in both root and shoot tissues of seedlings. This induction disappeared after 6 h of the chilling stress. Transient expression of OsTPP1 was also induced under severe chilling stress (4 8C). Both trehalose‐6‐phosphate phosphatase activity and trehalose levels were transiently increased after chilling (12 8C) stress, confirming that transient activation of trehalose biosynthesis is involved in early chilling stress response in rice (Pramanik and Imai, 2005). A construction of a chimeric translational fusion of yeast trehalose‐6‐phosphate synthase and trehalose‐6‐phosphate phosphatase was overexpressed in A. thaliana and these transgenic plants accumulated trehalose and displayed a significant increase in freezing tolerance (Miranda et al., 2007). b. Starch degradation is involved in cold‐induced sugar accumulation. In Arabidopsis, Kaplan et al. (2007) demonstrated that the first carbohydrates that increased after cold exposure were maltose and maltotriose, direct breakdown products of starch degradation by ‐amylase activity: their T0.5[MAX] (the time point where it reached 50% of its maximum or minimum value) were 10 min and 40 min, respectively. These increases for starch breakdown products were followed by glucose‐6‐phosphate and fructose‐6‐ phosphate (T0.5[MAX] are 40 min and 60 min, respectively). Hexose phosphates are immediate precursors for sucrose biosynthesis and sucrose increased over 1–4 h of cold acclimation with an estimated T0.5[MAX] of about 1 h. This increase was followed by increases in its breakdown products glucose and fructose with T0.5[MAX] values of about 1 h. Therefore, during early cold exposure of Arabidopsis, the dynamic data make a compelling argument for a sequence of starch degradation leading to glucose‐6‐ phosphate and fructose‐6‐phosphate, then to sucrose and then to fructose and glucose (Fig. 9). The fact that starch degradation is partly involved in cold‐induced sugar accumulation has been demonstrated through mutant analysis. An Arabidopsis gene encoding a ‐amylase (BMY8; At4g17090) is induced in response to cold (Kaplan and Guy, 2005; Lundmark et al., 2006). A bmy8 RNAi line exhibited a dramatic decrease in maltose, glucose, fructose and sucrose accumulation (Kaplan and Guy, 2005). Yano et al. (2005) further

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Starch GDW Amylase Maltose TPS

DPE

UDP-glucose

GP Glucose-1-P

Glucose-6-P

UDP-glucose SPS

UDP TPP Trehalose

Trehalose-6-P

MIPS

Fructose-6-P myo inositol-1-P UDP

UDP-glucose

IP

myo inositol Sucrose-6-P UDP-galactose

GS SPP Sucrose Invertase

UDP

RS myo inositol

Glucose + fructose

Galactinol

Raffinose SS Stachyose

Fig. 9. Starch metabolism. DPE, disproportionating enzyme; GP, glucan phosphorylase; SPS, sucrose phosphate synthase; SPP, sucrose phosphate monophosphatase; TPS, trehalose phosphate synthase; TPP, trehalose phosphate phosphatase; MIPS, myo‐inositol phosphate synthase; IP, myo‐inositol phosphate monophosphatase; GS, galactinol synthase; RS, raYnose synthase; SS, stachyose synthase.

documented the role of starch degradation in cold. In Arabidopsis, SEX1 (STARCH EXCESS 1), a gene encoding a starch related ‐glucan/water dikinase (GWD), is induced in response to cold. This enzyme phosphorylates the C‐3 and C‐6 positions of ‐glucans. This starch phosphorylation promotes starch degradation, possibly through the increase in hydrophilicity of water insoluble glucans, thereby ensuring better accessibility to starch degrading enzymes (Hejazi et al., 2008). A T‐DNA tagged Arabidopsis mutant, sex1–7, possesses 20% of GWD activity present in wild‐type plants. In this mutant, starch not only did not diminish in the first 24 h of exposure

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at 2 8C as seen in the wild‐type plants, but increased. Fructose and glucose contents still increased in sex1–7 plants, but at a much lower rate than that seen in wild‐type plants (Yano et al., 2005). Clearly, SEX1‐dependent starch degradation is at play in the first hours of cold exposure. c. Mode of action of sugars. A widespread response to low temperatures is the accumulation of osmolytes. DiVerent mechanisms have been proposed to explain how osmolytes can contribute to the increased freezing tolerance achieved during cold acclimation. One important aspect in which solutes might play a major role is in osmotic adjustment of the cell. The accumulation of carbohydrates and other solutes changes the osmotic potential of the cell and consequently diminishes the diVerence in water potential between the ice formed in the apoplastic space and the solution within the cell. As a result, the rate at which water is withdrawn from the cell will be reduced. A doubling of the internal solute concentration, which is not uncommon, will decrease the extent of cellular dehydration by 50% at any subzero temperature (Steponkus, 1984). Another role of sugar accumulation would be in avoiding ice nucleation in Arabidopsis, total soluble sugar accumulation during cold acclimation being correlated lower ice nucleation temperatures (temperatures at which ice crystals initiate) (Reyes‐Diaz et al., 2006). Similarly, it has been shown that at
3 8C cell sap extracted from non‐acclimated winter B. napus (cv ‘‘Express’’) leaves froze 3‐fold faster than extracts from acclimated leaves. After dialysis, sap extracts of both non‐acclimated and acclimated leaves froze at similar rate. When 0.5 M sucrose was added to these dialyzed samples, the freezing rate was similar to that of cell saps from acclimated leaves (Gusta et al., 2004). In addition, the ice nucleation temperatures of dialyzed cell extracts were lowered in presence of sucrose, illustrating a role of sugars in avoiding ice nucleation and ice growth during freezing in both monocot (Olien, 1974) and dicot (Gusta et al., 2004) crop species. Another role for solutes during cold acclimation is to protect the plasma membrane during freeze‐thaw cycles. All cells contain water that does not readily freeze. The amount of non‐freezable water is not large, varying within 20–40% of the dry weight. For example the phospholipid phosphatidylcholine contains about 0.25 g H2O g
1 lipid and this water is in some way associated with the polar head group (Crowe et al., 1990). The water associated with the membranes is required to create the hydrophilic environment necessary to stabilise the lipids in a bilayer. During freeze‐induced dehydration, non‐reducing sugars can replace the lost water. Arabidopsis sensitive to freezing 4 (sfr4) mutants exhibited a reduced accumulation of glucose and fructose, and impaired cold acclimation (McKown et al., 1996). When grown

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under non-acclimating conditions, sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts were injured (LT50) of
4.5 8C. After cold acclimation, the LT50 was decreased to only
5.6 8C for sfr4 protoplasts, compared with
9.1 8C for wild‐type protoplasts. Although expansion‐induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to loss of osmotic responsiveness. After incubation of seedlings in sucrose solution in the dark at 2 8C, the freezing tolerance (LT50
9 8C) and the incidence of loss of osmotic responsiveness were now similar for wild‐type and sfr4 isolated protoplasts. Sucrose treatment is expected to increase in vivo sugars such as sucrose, glucose or fructose. Indeed, incubation of Arabidopsis with glucose or fructose also decreased the incidence of loss of osmotic responsiveness. Therefore the freezing sensitivity of cold‐ acclimated sfr4 is due to its continued susceptibility to loss of osmotic responsiveness and this susceptibility is associated with its low sugar content (Uemura et al., 2003). Increased content in soluble sugars reduces the apposition of membranes, thus preventing fracture‐jump lesions, and loss of osmotic responsiveness. Sugars can also have a role in protecting integral membrane protein complexes. The Arabidopsis Bmy8 RNAi lines with reduced maltose content exhibited diminished PSII photochemical eYciency compared with wild‐type plants during freezing stress (Kaplan and Guy, 2005). This is in agreement with in vitro assays that showed that 14 mM maltose could preserve 24% of the electron transport chain activity of pea thylakoids that were frozen at
15 8C for 20 h. Trehalose and glucose also protected this thylakoid function but at a lesser extent (Kaplan and Guy, 2004). The stability of proteins is aVected by low temperature and enzyme inactivation may result from dissociation or aggregation (Guy, 1990). More than 40 years ago, Shikama and Yamazaki (1961) showed that the addition of solutes to catalase could increase the recovery of activity following thawing. They also reported that the cryoprotective property was not limited to a single class of compounds but was instead associated with diverse compounds, including diVerent sugars, polyalcohols, and salts. The fact that relatively high concentrations of solutes are usually needed to achieve cryoprotection argues against any specificity involved in protein stabilisation (Carpenter and Crowe, 1988). However, the exact mechanism by which solutes protect proteins during freezing is still unclear. Another mechanism for the protective eVects of solutes during freezing is the ability to form a glass instead of ice in freezing conditions. Glass is a liquid with the viscosity of a solid, and aqueous solutions of many diVerent solutes can form a glass. Plants utilise glass formation, or vitrification, as a cryoprotective strategy and the accumulation of solutes increases the

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glass‐forming tendency of the cytoplasm (Steponkus et al., 1992). Glass formation confers protection only at very low temperatures. Very cold resistant plants such as poplar form intracellular glass as a protection against intracellular ice formation at temperatures below
20 8C (Hirsh, 1987). Vitrification is thought to play an important role in the cold hardiness of woody species, such as poplar and birch, which are frequently exposed to severe winter temperatures. Oligosaccharides such as raYnose and stacchyose are thought to be major components of the glass‐forming solutions but sugar‐binding proteins are also believed to be important (Hirsh, 1987). Sugars could also have some ROS scavenging properties. The antioxidative properties of fructose and its phosphorylated forms were evaluated in vitro. Fructose‐6‐phosphate and fructose‐bisphosphate exhibited eight and four times higher superoxide scavenging capacity than fructose, and fructose also exhibited a very high eYciency for scavenging hydroxyl radical (Bogdanovic´ et al., 2008). Arabidopsis plants overexpressing galactinol synthase have high intracellular levels of galactinol and raYnose. The photoinhibition of PSII induced by high light and chilling was less important in those overexpressing plants than in wild type plants (Nishizawa et al., 2008). This could be explained by an increased scavenging capacity of the overexpressing plants. In the overexpressing plants, the chilling and cold temperature treatments resulted in ROS formation less important than in wild tye plants. This could be correlated with the fact that in vitro galactinol and raYnose have hydroxyl scavenging capacity (Nishizawa et al., 2008). 4. Compatible osmoticum other than sugars In addition to soluble sugars, compatible solutes are a heterogeneous group of molecules comprising amino acids (Ala, Gly, Pro, Ser), polyamines, and betaines. Compatible solutes are organic molecules of low molecular weight that are produced in response to many stresses such as desiccation, osmotic stress, or low temperature stress. Physiologically, compatible solutes have no adverse metabolic eVects even at very high concentrations. However, their role in stress tolerance is not always clearly established. As already mentioned for sugars, they may act by increasing the osmotic potential and prevent excessive cell dehydration, counterbalancing the osmotic eVect of ice formation in apoplastic space. They may stabilize proteins, assist refolding of proteins and stabilize membranes (Korn et al., 2008). During freezing, cell dehydration leads to cell volume reduction but the cell wall resists this reduction, and consequently the movement of water is prevented, which in turn results in the development of negative pressures that could lead to cavitation (Rajashekar and Burke, 1996). The accumulation of solutes during cold hardening may minimize cavitation by increasing tensile strength of

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cellular water (Rajashekar and Lafta, 1996). We will go into more detail on proline, glycine betaine, and polyamine in response to cold. a. Proline. It is well documented that proline robustly accumulates under cold stress. For example, in Arabidopsis a more than 2‐fold increase in proline occurs after 4 h of exposure to 4 8C, and this is followed by a continuous and dramatic increase up to 130‐fold the control level after 96 h (Kaplan et al., 2007). This accumulation of proline is sustained even after new leaves develop in the cold (Hurry et al., 2000). In S. cereale L. cv ‘‘Puma’’, the proline content did not change during the first three weeks of cold acclimation but then increased from 27 to 580 g g
1 fresh weight during the next three weeks (Koster and Lynch, 1992). In wheat, when proline is assayed in diVerent cultivars, proline increased in all cultivars after 1 week of cold acclimation but a prolonged cold acclimation resulted in diVerent profiles. No further increase occurred in cultivar ‘‘Haruyutaka’’, the least freezing tolerant cultivar assayed, while the increase continued in ‘‘Norstar’’ and ‘‘Chihokukomugi’’, more freezing tolerant cultivars (Kamata and Uemura, 2004). In Arabidopsis, the eskimo1 mutation resulted in a 5.5 8C improvement in freezing tolerance in the absence of cold acclimation. This mutant is characterized, amongst others changes, by a high accumulation of proline (Xin and Browse, 1998). All these studies suggest a role for proline in the acquisition of freezing tolerance. Supporting this conclusion, antisense transgenic Arabidopsis plants with reduced AtProDH expression, which encodes proline dehydrogenase catalyzing proline degradation, accumulated proline at higher levels than wild‐type plants. These antisense transgenics were more tolerant to freezing than wild‐type plants, showing again a positive correlation between proline accumulation and freezing tolerance in plants (Nanjo et al., 1999a). However, when the proline content and the freezing tolerance have been measured in diVerent Arabidopsis accessions and in the crosses between these accessions, no significant correlations between heterosis eVects in freezing tolerance and the content of proline could be established (Korn et al., 2008). This indicates that diVerences in proline accumulation play no major role in the establishment of heterosis eVects in Arabidopsis freezing tolerance. Thus, even though proline accumulates in response to cold exposure, its content is not the limiting factor explaining the diVerences in freezing tolerance capacity in a population of Arabidopsis cultivars. The mode of action of proline in freezing tolerance is not clearly elucidated. Proline is thought to be a compatible solute and it has been reported to play roles in protecting enzymes from denaturation (studies in Salsola soda; Nikolopoulos and Manetas, 1991), stabilizing the machinery of protein synthesis (studies in Eleucine coracana; Kadpel and Rao, 1985), regulating

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the cytosolic acidity (Venekamp, 1989), increasing water‐binding capacity (Schobert and Tschesche, 1978), and acting as a reservoir of carbon and nitrogen source (studies in Glycine max; Fukutaku and Yamada, 1984). Proline accumulation may also induce the expression of many genes, whose promoters contain the proline‐responsive element ACTCAT defined in Arabidopsis (Satoh et al., 2002). b. Glycine betaine. Glycine betaine is a quaternary ammonium compound which is synthesized in chloroplasts by a two step‐oxidation of choline, catalyzed by a choline monooxygenase (choline ! betaine aldehyde) and a betaine aldehyde dehydrogenase (betaine aldehyde ! betaine). Not all plants can accumulate glycine betaine in response to various abiotic stresses. For instance, spinach and wheat are natural accumulators of glycine betaine, while Arabidopsis, tomato, potato, and rice are not (Wyn Jones and Storey, 1981). In S. cereale L. cv ‘‘Puma’’, the amount of glycine betaine increased from 2900 to 1300 g g
1 fresh weight during the 4‐week acclimation period (Koster and Lynch, 1992). In wheat, in three cultivars assayed, a noticeable increase of glycine betaine was detected after the second week of cold acclimation. The amount of glycine betaine accumulation correlated with the freezing resistance of the cultivars (Kamata and Uemura, 2004). The role of glycine betaine in the response to cold has been studied by facilitating glycine betaine accumulation in plants that do not normally accumulate it. Decrease in PSII activity during chilling was less pronounced in plants treated with glycine betaine. When chilled plants were returned to 25 8C, plants treated with glycine betaine showed higher growth rates (Park et al., 2006). Glycine betaine also enhances freezing tolerance. When codA gene is expressed in Arabidopsis, freezing tolerance estimated via the LT50 of ion leakage is increased in non‐cold acclimated plants but it does not attain the freezing tolerance of cold acclimated plants. Possible roles for glycine betaine include stabilization of the transcriptional and translational machineries. Glycine betaine stabilizes protein complexes and membranes in vitro (Papageorgiou and Murata, 1995). A similar role might exist in response to low temperatures. In addition, glycine betaine may indirectly induce H2O2 mediated signalling pathways. H2O2 is a secondary messenger in plants and although toxic levels lead to programmed cell death, lower non toxic amounts modify gene expression and enhance plant stress responses. Low levels of H2O2 can stimulate protection against such oxidative stress by inducing the expression of antioxidant enzymes such as catalase, resulting in enhanced tolerance to chilling (see Section II.B.6.; Prasad et al., 1994). Both exogenous application of glycine betaine or H2O2 improves chilling tolerance in wild‐type tomato (Park et al., 2004). Glycine betaine can help form H2O2 (Sulpice et al., 2002) and it is likely that the induced

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chilling tolerance conferred by exogenously applied glycine betaine results from the induction of H2O2‐mediated antioxidant mechanisms. c. Polyamines. Polyamines are organic compounds having two or more primary amino groups. The polyamine biosynthetic pathway begins with the diamine putrescine that can be converted into spermidine and spermine through the consecutive activity of two distinct aminopropyl transferases, spermidine synthase, and spermine synthase. Both enzymes use decarboxylated S‐adenosyl‐l‐methionine (SAM) as an aminopropyl donor. In rice, a spermidine synthase gene, OsSPDS2, is up regulated in response to long term chilling in roots (Imai et al., 2004). Transcripts for SAM synthase are up‐regulated in Thlaspi arvense submitted to cold (Sharma et al., 2007), and rice in response to cold (Cui et al., 2005; Yan et al., 2006). In Arabidopsis submitted for 14 days at 4 8C, an increase in putrescine, ornithine, and citrulline, precursors of polyamines, is also observed (Cook et al., 2004). In rice exposed to 5 8C, transcripts of OsSAMDC increased for up to 72 h in the cold‐resistant ‘‘Yukihikari’’ genotype, but did not accumulate in the susceptible indica cultivar ‘‘TKM9’’ (Pillai and Akiytama, 2004). However, and this is a general remark, the fact that a gene is activated and/or a protein accumulates in response to low temperatures does not necessarily means that it is biologically relevant and significant. Yet experimental data suggest a role for polyamines in chilling tolerance linked to protection of photosynthetic functions. Polyamines are associated with the thylakoid membranes, especially the light harvesting complex II (LHCII) and the PSII (Kotzabasis et al., 1993). In Phaseolus vulgaris, a 52 h exposure at 6 8C resulted in a decrease of LHCII associated‐putrescine and an increase of LHCII associated‐spermine. A decrease of the putrescine/spermine ratio associated with LHCII has been reported to determine a re‐organization of LHCII, through an impact of polyamines on autoproteolytic degradation of LHCII (studies with green alga Scenedesmus obliquus Navakoudis et al., 2007). In addition, polyamines have been shown to have a role in alleviating oxidative stress. In cold treated plants, inhibition of polyamine synthesis led to increased oxidative damage, such as electrolyte leakage (studies with tomato; Kim et al., 2002a) or lower photochemical eYciency of PSII (studies with spinach; He et al., 2002). Conversely, chilling‐induced damage due to oxidative stress such as visible yellowing was attenuated in transgenic tobacco (N. tabacum L.) overexpressing S‐adenosylmethionine decarboxylase and accumulating higher levels of soluble total polyamines (Wi et al., 2006). Interestingly, in tobacco plants, transcripts for antioxidant enzymes such ascorbate peroxidase, superoxide dismutase, and glutathione S‐transferase (see Section II.B.6) were induced more significantly during stress treatment

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than in control plants (Wi et al., 2006). Whether this increase in antioxidant enzyme transcript is a direct or indirect consequence of polyamine accumulation remains an open question. It has been hypothesized that polyamines could play a role as signalling molecules, possibly through the promotion of DNA binding activity of transcription factors (Arabidopsis; Kasukabe et al., 2004). Another possible role of spermidine might be through direct inhibition of the chill‐induced activation of NADPH oxidases in microsomes as is seen in cucumber (Shen et al., 2000). 5. Photosynthesis and photosynthesis‐related pigments a. Adaptation of photosynthesis to cold. Plants have evolved diverse strategies to cope with low temperatures. For evergreen conifers such as Pinus sylvestris, cold acclimation induces cessation of primary growth and a resultant decrease in the sink demand for photoassimilates. The cessation of growth decreases the capacity for energy utilization and this, in turn, results in feedback inhibition of photosynthesis (Ensminger et al., 2006; Huner et al., ¨ gren, 2003; Savitch et al., 2002). To attain photostasis 1993; Hjelm and O under these conditions, over‐wintering conifers exhibit long‐term changes in the organization of the photosynthetic apparatus (Ebbert et al., 2005; Ensminger et al., 2006; Ottander et al., 1995; Savitch et al., 2002) that includes a decrease in the number of functional PSII reaction centres, a loss of light‐harvesting chlorophylls and the formation of a large thylakoid/ protein complex involving LHCII, PSII, and PSI. This aggregation is associated with increased levels of PsbS protein and increased formation of zeaxanthin coupled, at least in part, to sustained non‐photochemical quenching of excitation energy (see below; Adams and Demmig‐Adams, 1994; Ottander et al., 1995). The formation of this winter‐induced aggregated state is fully reversible upon warming in spring with the result that photosynthesis is resumed rapidly without the immediate de novo synthesis of ¨ quist and Huner, 2003). chlorophyll (Ivanov et al., 2001; O In contrast to over‐wintering evergreen conifers, over‐wintering herbaceous plants such as winter wheat, rye, Brassica and Arabidopsis, continue to grow and develop during the cold acclimation period in order to attain maximum freezing tolerance, thereby maintaining a high demand for photoassimilates. Thus, while annual cold tolerant plants show a decrease in light saturated rates of CO2 uptake in the short‐term, this decline is followed by a strong recovery of photosynthesis under cold temperatures (Huner et al., 1993; Stitt and Hurry, 2002). In the short term (hours to days), photosynthesis is inhibited because of thermodynamic eVects on enzymatic reactions. As explained in Section II.A.2., low temperature exposure results in a decrease in triose‐phosphate utilization that leads to the accumulation of

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phosphorylated metabolites, which in turn leads to a decrease in Pi availability in chloroplasts (Hurry et al., 1993; Leegood and Furbank, 1986). This Pi status of the plastids in turn reduces the availability of the electron acceptor glycerate‐1,3‐bisphosphate in the photosynthetic reduction cycle (Hurry et al., 1995; Savitch et al., 2001) and reduces the rate of ribulose‐bisphosphate regeneration (Hurry et al., 1996), limiting CO2 assimilation. In compensation for this loss in electron consumption by photosynthesis, non photochemical quenching increases in cold stressed leaves associated with increases in zeaxanthin content but, unlike over‐wintering conifers, it does so without any associated re‐organization or aggregation of the thylakoid protein complexes (Adams et al., 1995; Krol et al., 1999). Cold‐stressed and over‐wintering herbaceous plants also increase their propensity for PSII reaction centre quenching, further enhancing their energy dissipative capacity under these unfavourable growth conditions (Ivanov et al., 2008; Sane et al., 2003). Over the long‐term (days to weeks), photosynthesis recovers in cold acclimating winter annuals and this is associated with the production of new leaves better adapted to the new thermal regime (Hurry et al., 1994; Strand et al., 1997). These new leaves typically show equivalent maximum photochemical eYciency and capacity for light absorption as warm grown leaves (Gray et al., 1997). The recovery in photochemical eYciency and electron transport is associated with increases in thylakoid plastoquinone A content and a concomitant increase in the apparent size of the intersystem electron donor pool to PS I (Gray et al., 1997). Cold developed leaves also show an increase in capacity for ROS scavenging (Krol and Huner, 1985; Streb and Feirabend, 1999; Streb et al., 1999, 2003a,b), indicating that not only energy dissipation via non‐photochemical quenching and electron transport but also protection from oxidative damage is enhanced following cold acclimation. The induction of ROS scavenging systems is detailed below (Section II.B.6.). The recovery of electron transport capacity and photosynthesis following cold acclimation occurs as a result of increases in the content and activity of a number of Calvin cycle enzymes (Hurry et al., 1994, 1995, 2000; Goulas et al., 2006). In addition, cold‐tolerant, herbaceous plants grown at cold temperatures show an increase in inorganic phosphate availability (Strand et al., 1999; Stitt and Hurry, 2002), as well as in adenylates and phosphorylated intermediates, and in the capacity for the regeneration of RuBP (Hurry et al., 1994, 1996). Associated with this recovery in plastid metabolism, cold grown plants exhibit an increase in sucrose‐phosphate‐synthase (SPS) activity, SPS activation state and an increase in the cytosolic hexose‐P pool, resulting in increased sucrose biosynthesis (Hurry et al., 1994; Stitt and Hurry, 2002; Strand et al., 2003). These changes in thylakoid membrane processes coupled to increases in enzymatic content and flux capacity in both the stroma and

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the cytosol, in combination, result in recovery of photosynthesis at low temperatures to rates equivalent to plants grown under permissive warm conditions (Hurry et al., 2000; Strand et al., 2003). The induction of non‐photochemical quenching of excitation energy is a phenomenon common to evergreen conifers and to herbaceous plants submitted to cold. We will now detail the role of xanthophylls that participate to this quenching. We will also detail the importance of flavonols and anthocyanins. Even though they are not photosynthetic pigments per se, they participate in the protection of photosynthesis. b. Xanthophylls and flavonoids i. The Xanthophyll cycle. The Xanthophyll cycle is the cycle by which violaxanthin is rapidly and reversibly de‐epoxidized into zeaxanthin via the intermediate antheraxanthin. In response to low temperature, both the pool of xanthophylls (Violaxanthin þ Antheraxanthin þ Zeaxanthin) and the level of zeaxanthin increase (Krol et al., 1999).The xanthophyll cycle has two functions.The first is to convert the PSII from a state of eYcient light harvesting to a state of high thermal dissipation. Antheraxanthin and zeaxanthin are quenching pigments, unable to pass their excitation to Chla. The increase in thermal dissipation is measured as non‐photochemical quenching (NPQ) of chlorophyll fluorescence. NPQ is a protective mechanism that lowers energy delivery to PSII. The non photochemical cycle is important for short term photoadaptation of PSII and participates in the prevention of PSII photoinhibition. The npq1 mutant of A. thaliana has no xanthophyll cycle due to a lack of functional violaxanthin de‐epoxidase. Exposure for less than 2 days of whole plants to high photon flux density (1000 mol m
2 s
1) at 10 8C resulted in PSII photoinhibition that was more acute in npq1 than in the wild‐type. In the long term (10–12 days), PSII activity recovered in both npql and wild‐type, indicating that the xanthophyll cycle is not necessary for long term acclimation to chilling stress in A. thaliana (Havaux and Kloppstech, 2001). Another function of the xanthophyll cycle is to produce a powerful antioxidant. The xanthophylls have intrinsic antioxidant capacity, i.e. independently of the non‐photochemical quenching. The antioxidant capacity of zeaxanthin is higher than that of violaxanthin (Havaux et al., 2007). This antioxidant capacity is at play during cold exposure. In Arabidopsis, when exposed to high light at low temperature, wild‐type plants exhibit symptoms of severe oxidative stress: lipid peroxidation, chlorophyll bleaching, and photoinhibition. In plants that accumulate more than twice as much zeaxanthin as the wild‐type, but where the capacity of non‐photochemical quenching is not significantly diVerent from wild‐type, these symptoms are significantly ameliorated as a result of the antioxidant eVect of zeaxanthin (Johnson et al., 2007).

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ii. Flavonoids. Anthocyanins, which accumulate in leaves and stems in response to low temperature and changes in light intensity, are synthesized through the phenylpropanoid pathway that is controlled by key enzymes including phenylalanine ammonia‐lyase (PAL) and chalcone synthase (CHS). In T. arvense, a chalcone synthase, flavonol synthase, chalcone‐flavonone isomerase and a phenylalanine ammonia lyase are all induced in response to cold treatment (Sharma et al., 2007), as is a Cytochrome P450, a family known to be important for secondary metabolism. Phenylalanine ammonia lyase and chalcone synthase mRNAs accumulate in leaves of A. thaliana upon exposure to low temperature in a light‐dependent manner (Leyva et al., 1995). The flavonoid biosynthesis pathway in Arabidopsis is regulated by the MYB family transcription factor PAP2. The expression of PAP2 is strongly correlated with the acquisition of freezing tolerance of diVerent Arabidopsis accessions (Hannah et al., 2006). Here again, the fact the transcripts and/or proteins are accumulated does not necessarily means that it is biologically relevant and significant. However, the content of diVerent flavonoids and freezing tolerance have been measured in diVerent Arabidopsis accessions and in the crosses between these accessions. There was a significant correlation between heterosis eVects in freezing tolerance and the content of specific flavonols, such as kaempferol– rhamnose–rhamnose (Korn et al., 2008). This indicates a specific role for such compounds in freezing tolerance. Analysis of flavonoid‐deficient tt mutants revealed that UV/blue‐light‐absorbing flavonols have a strong protective function against excess visible radiations. In those mutants, the absence of flavonoids could not be overcome in the long term by compensatory mechanisms, leading to extensive photooxidative and photoinhibitory damage to the chloroplasts (Havaux and Kloppstech, 2001). This suggests that flavonoids are necessary for long term adaptation to photoinhibition conditions. Profiling of the leaf pigments by phase‐resolved photoacoustic spectroscopy showed that the flavonoid‐related photoprotection was due to light trapping, which decreased chlorophyll excitation by blue light (Havaux and Kloppstech, 2001). In addition to this eVect of light trapping, flavonols such as quercetin could also act as potent antioxidants (Rice‐Evans et al., 1996). Finally, flavanols, which are amphiphilic molecules, can partition into the lipid phase of membranes and it has been hypothesized that flavonols could have a role in protecting membranes during freezing (Korn et al., 2008). 6. Reactive oxygen species scavenging systems a. Enzymatic systems. Cold exposure induces an oxidative stress. ROS are either formed in the chloroplast, in the mitochondria, in peroxisome or in the cytosol. The formation of ROS in the chloroplasts has been detailed in an earlier section (see Section II.A.2). In mitochondria, ROS can be produced in

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the UQ/UQH2 cycle. At low temperature the enzymatic systems that normally ‘‘destroy’’ the ROS will be less eYcient (thermodynamically a lower temperature decreases enzymatic activity) and ROS accumulation in the cold by mitochondria has been reported (Prasad et al., 1994). To counterbalance the ROS production, plants subjected to low temperatures induce and activate scavenging systems. Catalases and superoxide dismutases are induced by cold (Guo et al., 2006; Goulas et al., 2006; Prasad et al., 1994). In A. thaliana, an increase in ascorbate is observed and is still observed after 14 days of exposure at 4 8C (Cook et al., 2004; Kaplan et al., 2004). Ascorbate is an abundant anti‐oxidant in plants (Mu¨ller‐Moule´ et al., 2002). The detoxification of hydrogen peroxide produced in the chloroplasts relies exclusively on the activity of ascorbate peroxidase bound to thylakoid membranes in the vicinity of PSI (Miyake and Asada, 1992). Monodehydroascorbate reductase catalyzes the regeneration of ascorbate in the chloroplast at the expense of NAD(P)H. Glutathione (GSH) is an intermediary in the cycle (Fig. 10). GSH is found in most tissues, cells and subcellular compartments of higher plants. GSH concentration is highest in the chloroplast but significant quantities also accumulate in the cytosol. GSH exists predominantly in the reduced form. In addition to its role in this cycle, GSH can react chemically with singlet oxygen, superoxide and hydroxyl radicals, and therefore function directly as a free radical scavenger. GSH may stabilise membrane structure by removing acyl peroxides formed by lipid peroxidation reactions (Price et al., 1990). In a proteomics study performed in rice, ascorbate peroxidases and 2‐Cys peroxyredoxin diVerentially accumulate during chilling (Yan et al., 2006). Again in rice, ferritin and glutathione‐S‐transferase (GST) are induced in H2O2

Ascorbate

Ascorbate peroxidase Superoxyde dismutase

NADPH

NADPH Monodehydro ascorbate reductase

Monodehydro ascorbate

O.− 2

GSSG

Glutathione reductase Dehydroascorbate reductase

NADP 2 GSH

H2O

Fig. 10.

Dehydro ascorbate

The ascorbate/Glutathione cycle in the chloroplast.

NADP

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response to cold. Ferritin an iron‐binding protein, is proposed to protect plants from oxidative damage induced by a wide range of stresses. GST with activity of GSH peroxidase is an enzyme catalyzing the conjugation of the glutathione to a variety of toxic substrates arising from oxidative stress, thereby reducing their toxicity (Cui et al., 2005). A similar study of the dynamics of the soluble proteome of Arabidopsis during cold acclimation also identified a number of proteins involved in redox regulation and reactive oxygen scavenging that were diVerentially regulated by cold. These included monodehydroascorbate reductase and two 2‐Cys peroxiredoxins (2‐Cys Prx A and B) that were reduced in abundance (Goulas et al., 2006). In plants, the 2‐Cys Prx proteins are localized in the chloroplast stroma where, under low H2O2 conditions, they are present as dimers and function as peroxidases (Baier and Dietz, 1999; Cheong et al., 1999; Jang et al., 2006). However, under oxidative stress eukaryotic 2‐Cys Prx proteins undergo structural and functional changes leading to the formation of high molecular mass complexes with chaperone activity (Jang et al., 2004). It is likely that the reduction in abundance of the two 2‐Cys Prx proteins in the stroma of these cold exposed Arabidopsis plants was the result of the formation of such supercomplexes. This, in turn, suggests a role for increased 2‐Cys Prx chaperone activity in response to elevated oxidative stress. A correlation can be made between chilling resistance and activation of ROS scavenging systems. Responses of antioxidative defence systems to chilling were studied in four cultivars of rice (Oryza sativa L.). Under chilling stress of 5 days at 8 8C, ‘‘Xiangnuo no. 1’’ and ‘‘Zimanuo’’, chilling‐tolerant cultivars, have much lower level of electrolyte leakage and H2O2 content than the two chilling‐ sensitive cultivars, ‘‘Xiangzhongxian no. 2’’ and ‘‘IR50’’. Activities of antioxidant enzymes (superoxide dismutase (SOD), catalase, ascorbate‐peroxidase (APX)), and contents of antioxidants (ascorbic acid and reduced glutathione) were measured during the stress treatments. All of them were greatly enhanced until 3 days after chilling stress in the two chilling‐tolerant cultivars. They all were decreased at 5 days of stress. On the other hand, activities of antioxidant enzymes and contents of antioxidants were decreased greatly in the chilling‐ sensitive cultivars after chilling stress. The results indicate that tolerance to chilling in rice is well associated with the enhanced capacity of antioxidative system under chilling condition (Guo et al., 2006). Methionine residues of proteins are major targets for oxidation by ROS. Methionine sulfoxide reductases are antioxidative enzymes that reduce methionine sulfoxide back to methionine. In Arabidopsis, a methionine sulfoxide reductase, MsrB3, has been shown to be cold responsive and msbr3 mutant plants lost the ability to become freezing tolerant after cold hardening (Kwon et al., 2007).

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b. Non enzymatic scavenging: Vitamin E and other antioxidants. Vitamin E is the collective term for a group of amphiphilic lipids, the tocopherols, and tocotrienols, which are synthesized exclusively by photosynthetic organisms. They are made of a chromanol head group and a prenyl side chain. ‐ tocopherol predominates in leaves of vascular plants, where it is found mainly in the envelope and the thylakoid membranes of chloroplasts, whereas ‐tocopherol is often the major form in seeds of many plants. It has been shown in vitro that ‐tocopherol could terminate chain reactions of polyunsaturated fatty acid free radicals generated by lipid oxidation. As a result, chromanoyl radicals are formed. These chromanoyl radicals can be reduced by ascorbate, thereby regenerating ‐tocopherol and its chain breaking anti‐ oxidant properties (Kamal‐Eldin and Appelqvist, 1996). ‐tocopherol also quenches singlet oxygen 1O2 (Di Mascio et al., 1990). The level of ‐tocopherol increases in plants exposed to stress conditions that induce oxidative stress, such as cold exposure (Fryer et al., 1998). In A. thaliana mutants deficient in tocopherol, lipid peroxidation resulting from exposure of leaf discs to high light stress at 10 8C was much higher than that of wild‐type leaf discs subjected to the same treatment. This clearly indicated an antioxidant role for tocopherols in vivo (Havaux et al., 2005). In the same mutants deficient in tocopherols, when whole plants where exposed at 8 8C under high light, PSII photoinhibition was more pronounced than with wild‐type plants, demonstrating that vitamin E protects the photosynthetic apparatus by two mechanisms: PSII protection from photoinhibition and membrane lipid protection from peroxidation (Havaux et al., 2005). This might be due to the capacity of tocopherols to quench singlet oxygen. These protective functions are integrated into a network of other protective mechanisms, such as non‐ photochemical quenching (see Section II.B.5.) and antioxidant activity of zeaxanthin (see Section II.B.5.), to fully preserve photosynthetic processes. 7. Cell architecture response a. Cell wall. Cold acclimation is accompanied by changes in the cell wall. In winter oil‐seed rape (B. napus ssp. oleifera), acclimation at 2 8C was associated with increases in leaf tensile stiVness, cell wall and pectin contents, pectin methylesterase activity, and in low‐methylated pectin content (Solecka et al., 2008). Huner et al. (1981) noted an increase in cell wall thickness associated with cold hardening in rye leaves. In T. arvense, transcripts for cellulose synthase, pectinacetylesterase, expansin, pectin methylesterase have been shown to accumulate in response to cold (Sharma et al., 2007). Xyloglucan endotransglucosylase/hydrolase (XTH) is a cell wall‐modifying enzyme. In hot pepper plants, CaXTH1, 2 and 3 mRNAs were induced at 4 8C (Cho et al., 2006). In a proteomic study performed in rice, phenylalanine

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ammonia‐lyase has been shown to be induced in response to cold (Cui et al., 2005). Phenylalanine ammonia‐lyase is a key regulatory enzyme in phenylpropanoid metabolism. The pathway produces a large amount of phenolic compounds, which are the precursor molecules of suberins and lignins, but also anthocyanins (see Section II.B.5.). In Rhododendron, cold‐induced up‐regulation of transcripts for coumarate 3‐hydroxylase, an enzyme of the lignin biosynthesis pathway, has been described (Wei et al., 2006). In addition, the up regulation of S‐adenosylmethionine synthetase already described (see Section II.B.4.c.) can be linked to the cell wall. S‐adenosylmethionine is a major methyl group donor for many cellular molecules, particularly for the methylation of several derivatives of the phenylpropanoid pathway, including the methylation of cinnamic acids, products of phenylalanine ammonia‐lyase in the lignin biosynthesis pathway. The cell wall has a major structural role and this role is all the more important during freezing: for cell dehydration to occur, cells should be able to undergo cell volume reduction or cell deformation that are dependent on the mechanical properties of the cell wall. Indeed, cell wall rigidity will prevent cell deformation and collapse as a consequence of freeze‐induced cell dehydration. Dormant stem xylem and pith tissues of river‐bank grapes (Vitis riparia) were resistant to freeze‐induced dehydration above the homogeneous nucleation temperature, and they developed cell tension reaching a maximum of 27 MPa. Similarly, extracellular freezing induced a cell tension of 16.8 MPa in the leaves of live oak (Quercus virginiana) and of 8.3 MPa in the leaves of mountain cranberry (Vaccinium vitis‐idaea) (Rajashekar and Burke, 1996). In addition, the cell wall also plays a role in excluding extracellular ice from the cell. Cell wall pore characteristics determine whether the cell wall can be an eVective barrier against ice intrusion. In suspension‐cultured cells of grapes and apple, cold acclimation resulted in an increase of cell wall strength and a decrease in ˚ to 22A ˚ . There is a correlation between the limiting cell wall pore size from 35A the decrease in cell wall pore size during acclimation and a decrease in intracellular ice formation (Rajashekar and Lafta, 1996). b. Microtubules. Exposure to low temperatures will destabilize and depolymerize microtubules. If the exposure to low temperatures is maintained, cold labile microtubules will be replaced by cold stable ones. In root‐tip cells of cucumber (Cucumis sativus L.), after low temperature (4 8C) treatment for 5 h, chilling‐stable cortical microtubules were found not only under the plasma membrane, but also as chilling‐stable punctuate microtubules in the cytoplasm, which might be the microtubules associated with organelles (Zhao et al., 2003). These changes in microtubules are due in part to changes in transcript level. The alpha‐tubulins and beta‐tubulins are the major

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constituents of microtubules. In wheat (T. aestivum L.), 15 full‐length cDNAs for the members of the alpha‐tubulin gene family were identified. During cold acclimation, 3 members of the family initially showed reduced mRNA abundance after one day of cold treatment followed by recovery at days 3 and 6. A fourth member of the gene‐family member showed increased mRNA for up to 14 days during cold acclimation and had decreased levels after 36 days of cold treatment. A fifth member of the gene family had slowly declining mRNA levels up to 36 days (Ridha Farajalla and Gulik, 2007). The importance of microtubule for chilling tolerance has been demonstrated using tobacco mutants screened either for their tolerance to aryl carbamates, a blocker of microtubule assembly, or to chilling treatment (tolerant mutants were those that could survive for several months at 3 8C). The carbamate‐tolerant mutants were cross‐resistant to chilling stress. This suggests that the cold sensitivity of microtubules limits chilling tolerance in tobacco, and that more stable microtubules leads to chilling resistant cells (Ahad et al., 2003). The stability to cold of microtubules can be enhanced by microtubule‐associated proteins, or MAPs. In A. thaliana, nine genes encode proteins of the evolutionarily conserved MAP65 family. In the presence of AtMAP65–1, microtubule bundles were more resistant to cold (Mao et al., 2005). In summary, the exposure to low temperatures induces diVerent responses that lead to cold tolerance and/or freezing tolerance. Amongst these responses, are the accumulation of hydrophilic proteins, the accumulation of sugar and compatible solutes, changes in membrane composition, induction of protein chaperones, and RNA chaperones. In addition, metabolic readjustments and the induction of ROS scavenging systems are necessary to cope with lower temperatures. It can be noted that some of these responses are not specific to cold. The accumulation of compatible solutes is also shown in response to drought stress. The induction of COR proteins is common to drought and ABA (Seki et al., 2002). The induction of ROS scavenging systems is common to many stresses (Fujita et al., 2006). It is possible to recapitulate, for each eVect of chilling and freezing stresses on plant physiology, the diVerent responses that contribute to make the plants able to cope with these eVects (Fig. 11). For freezing stress, plants have two possibilities: they can avoid freezing or tolerate freezing. In the first case, tissue freezing is delayed or prevented. In the second case, ice forms without damaging cellular structures. Freezing avoidance correlates with supercooling, i.e. the capacity of cell fluids to be cooled to a temperature lower than the freezing point without immediate freezing. Accumulation of AFP and accumulation of

COLD SIGNALLING AND COLD ACCLIMATION IN PLANTS

Effects

Chilling temperatures

73

Responses

Membrane rigidification

Lipid desaturation

ARN secondary structure stabilization

ARN chaperone

Cytoskeleton depolymerization

Induction of cold-stable microtubules Protein chaperone

Protein conformation/stability changes

Stabilization by sugar accumulation New protein synthesis

Photoinhibition

Non photochemical quenching Anthocyanins

Photosynthesis inhibition

Increase in enzyme and flux capacity

Metabolic imbalance

New enzyme synthesis

ROS accumulation

ROS scavenging systems

Freezing temperatures Antifreeze protein Ice nucleation and spreading

Sugars Cell wall pore size Membrane composition changes

Membrane disruption

Sugar accumulation COR15A accumulation

Freeze-induced dehydration

Compatible solute accumulation

Fig. 11. The eVects of low temperatures and the responses that participate to counteract them.

sugars, as well as glass formation, participate to freezing avoidance. Freezing tolerance is achieved by accumulation of sugars, of COR proteins, and changes in membrane composition. We will now see that the responses to cold are influenced by light and hormones.

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E. RUELLAND ET AL. C. LIGHT AND HORMONES IN COLD RESPONSE

1. Light The acquisition of cold hardiness by herbaceous species requires both light and CO2 (Andrews et al., 1974; Dexter, 1933; Lawrence et al., 1973; Wanner and Juntilla, 1999). Using transcriptomics it has been show in Arabidopsis that a treatment at 3 8C under light (100 mol photons m
2 s
1) up regulated twice as many genes as a treatment at 3 8C under dark (Soitamo et al., 2008). Genes that were more up regulated at 3 8C in the light included XERO2/ LTI30 (At3g50970), LTI78/RD29A (At5g52310), ERD10 (Early Response to Dehydration, At1g20450), ERD3 (At4g19120), KIN1 (At5g15960), two galactinol synthases (At1g56600 and At1g09350), and the dehydrin RAB18. Induction of a few oxidative stress related genes occurred only under the Cold/Light treatment including genes encoding iron superoxide dismutase (FeSOD) and glutathione‐dependent hydrogen peroxide peroxidases (GPX) (Soitamo et al., 2008). Light could influence cold hardening through chloroplast functions. In barley (Hordeum vulgare), the albina and xantha mutants are blocked in diVerent steps of chloroplast development. Expression of cold‐regulated genes was analyzed in control and cold‐hardened mutants. Only about 11% of the genes cold regulated in wild‐type were regulated to a similar extent in all genotypes and could be assigned as chloroplast‐independent cold‐ regulated genes. About 67% of wild‐type cold‐regulated genes were not regulated by cold in any mutant and were considered as chloroplast‐ dependent cold‐regulated genes. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold (Svensson et al., 2006). It has indeed been shown that the redox state of plastoquinone could influence cold response. Several morphological traits associated with cold acclimation, such as the development of a compact rosette growth habit in winter cereals (Fowler and Carles, 1979), are dependent on PSII excitation pressure (Gray et al., 1997), although the mechanisms through which PSII excitation pressure mediates this response have not been elucidated. Another point of interference between light and cold hardening could be ROS production in chloroplast under low temperature and light. In seedlings of chilling‐tolerant japonica rice, there is a significant overlap between genes responsive to cold and genes responsive to hydrogen peroxide (Cheng et al., 2007). Light quality has also been shown to play a role in mediating the cold response. For example, when etiolated barley (H. vulgare L.) plants were cold hardened in the dark, no COR14b accumulation was found during 7 days of

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treatment. However, if prior to the cold treatment etiolated plants were subjected to red or blue light pulses of 5 min, COR14b accumulated during the subsequent cold treatment in the dark. Neither far red nor green light pulses promoted COR14b accumulation (Crosatti et al., 1999). This suggests that phytochromes and blue light photoreceptors may be involved in control of COR14b gene expression in the cold. In Arabidopsis plants containing several copies of a cold‐responsive elements (CRT/DRE cis‐element, see Section III.A.) derived from the COR15A gene fused to uidA (GUS) gene, light was shown to enhance cold‐expression of the reporter gene. When transgenic plants were treated with 10 min pulse of red light at 3 8C and then maintained 24 h in the dark at 3 8C, the GUS mRNA level was similar to that observed with white light treatment for 24 h at 3 8C. If far red light pulse was used instead of red light pulse, the GUS mRNA was induced but at a significantly lower level than with red or white light pulse (Kim et al., 2002b). If the red light pulse was immediately followed by far‐red light pulse, the induction of the GUS mRNA level caused by red light was cancelled. This demonstrates that phytochromes mediate the light signalling of cold induction via the CRT/DRE element (CBF pathway, see Section III.B.; Kim et al., 2002b). However, at higher temperatures phytochromes activation could have a negative effect on the expression of the CBF regulon genes. When grown at 16 8C, monogenic mutants deficient in phytochromes B and D showed increased expression of COR15a (Franklin and Whitelam, 2007). 2. Abscisic acid (ABA) Abscisic acid (ABA) has long been discussed to have an important role in plant cold responses, and ABA may be required for full cold response. It has been shown that basal freezing tolerance is reduced in the Arabidopsis frs1 mutant, a plant mutagenized by EMS in ABA3 and with a resulting lower ABA content than the wild type (Llorente et al., 2000). In this mutant the capacity to cold acclimate is aVected, and after 7 days of cold acclimation, the LT50 for freezing tolerance is at
8 8C in the wild type plants but is only
5 8C in the frs1 mutant (Llorente et al., 2000). The freezing tolerance acquired by cold acclimation is also impaired in another ABA deficient mutant, aba1, and in the ABA insensitive mutant abi1 (Ma¨ntyla¨ et al., 1995). Treatments of plants with ABA results in an increase in freezing tolerance (Ma¨ntyla¨ et al., 1995), which is in agreement with the observation that many cold responsive genes are also ABA responsive (Rabbani et al., 2003; Seki et al., 2002). Cold induces the production of ABA and in A. thaliana the level of ABA increases 4‐fold after 15 h of cold exposure (4 8C day/2 8C night) but it is back to control levels by 24 h (La˚ng et al., 1994). This increase, although significant, is relatively modest when compared with a

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20‐fold increase after 3 h of drought (35% relative humidity) (La˚ng et al., 1994), providing early evidence that the role of ABA in the cold response may not be as important as it is in the drought response. Interestingly, in Arabidopsis none of the known ABA synthesis genes are regulated after 3, 6, or 24 h cold treatment at 0 8C (Lee et al., 2005). Because ABA biosynthesis is mainly regulated at the transcriptional level, this data provides support for the idea that ABA biosynthesis is not a major event in the early cold stress response. In addition, cold‐responsive genes (e.g., LTI78: La˚ng et al., 1994; ADH: de Bruxelles et al., 1996; UGPase: Ciereszko et al., 2001; AtCAMBP25: Perruc et al., 2004) are still induced in ABA deficient mutants, such as the aba1 mutant. However, some genes (e.g., RAB18 and RCI2A) are dependent on ABA for their cold induction (Capel et al., 1997; Llorente et al., 2000). Furthermore for the genes that are ABA‐dependent, cold induction is much enhanced in plants mutated in protein phosphatase 2CA. This suggests that an ABA‐dependent pathway might exist in the cold response but that it is usually inhibited by a protein phosphatase 2CA (see Section IV.B.6.; Ta¨htiharju and Palva, 2001). Thus, cold acclimation appears to be primarily independent of ABA but ABA could act later in the process and be necessary for the maximum acquisition of chilling and/or freezing tolerance. Indeed, many cold responsive genes have in their promoters cis‐acting elements, such as ABRE or CRT/DRE (see Section III.A.), which can be activated by ABAinduced proteins. Cold‐induced ABA production could therefore result in an enhancement of the cold gene response via activity on these cis‐elements. A bZIP transcription factor, ABF1 (At1g49720), that can bind to the ABA‐ responsive element in ABA‐responsive promoters is early and continually induced by cold. A related gene, ABF4/AREB2 (At3g19290), is also induced by cold (Lee et al., 2005). Thus, it is possible that cold induction of some ABA‐inducible genes might be mediated by ABF1 and ABF4. 3. Salicylic acid (SA) Cold has also been shown to increase SA level. During a metabolite profiling of Arabidopsis submitted to 4 8C, SA has been shown to increase, exhibiting a biphasic response, starting at 1 h, peaking at 4 h and 12 h, decreasing at 24 h and then continuously increasing to 96 h, the end of the experiment (Kaplan et al., 2004). Exposing seedling radicles to 0.5 mM SA for 24 h before chilling at 2.5 8C for 1–4 days reduced the chilling‐induced increase in electrolyte leakage from maize and rice leaves and cucumber hypocotyls. This chilling tolerance was associated with an increase in glutathione reductase and peroxidase activities (Kang and Saltveit, 2002). It has been proposed that the lower growth of Arabidopsis plants in chilling conditions could be related to their levels of SA. Wild‐type Arabidopsis shoots at 5 8C accumulated SA,

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particularly in glucosylated form. In plants overexpressing a SA hydroxylase (NahG), SA was accumulated at 5 8C, but at greatly lower levels due to the hydroxylase activity. Plants with the SA hydroxylase transgene grew at similar rates to wild‐type plants at 23 8C, and growth of both genotypes was slowed by transfer to 5 8C. However, at 5 8C, NahG plants displayed relative growth rates about one‐third greater than wild‐type plants, so that by 2 months NahG overexpressing plants were typically 2.7‐fold larger (Scott et al., 2004). The SA‐deficient eds5 mutant behaved like NahG. In contrast, in the mutant cpr1 that accumulated very high levels of SA at 5 8C the growth was much more inhibited than in wild‐type (Scott et al., 2004). 4. Brassinosteroids Brassinosteroids are a class of plant polyhydroxy steroids that are structurally similar to animal and insect steroid hormones. Brassinosteroids control a broad range of responses such as cell division, cell expansion, vegetative growth and apical dominance (Sasse, 2003). In Arabidopsis, CONSTITUTIVE PHOTOMORPHOGENIC DWARF/DWARF3 (CPD/DWF3, At5g05690) was down‐regulated 24 h after cold treatment (Lee et al., 2005). CPD/DWF3 encodes a member of the cytochrome P450 90A family required for C23 hydroxylation of cathasterone to teasterone in brassinolide biosynthesis. In Mung bean CYP90A2 is a gene putatively involved in brassinosteroid synthesis and it shares 77% identity with the Arabidopsis CPD gene. CYP90A2 was shown to be strongly suppressed by chilling stress (Huang et al., 2006). Thus, the growth arrest in cold might in part be due to this lowering of brassinosteroids. If so, exogenous treatment with brassinosteroids could allow the plant to recover from the inhibited growth caused by chilling. Indeed, Mung bean epicotyls whose growth was initially suppressed by chilling partly recovered their ability to elongate after treatment with 24‐epibrassinolide (Huang et al., 2006). In addition, when Arabidopsis plantlets were treated with 24‐epibrassinolide (EBR) and exposed to cold (2 8C), transcript accumulation of CBF1, LTI78, and COR47 was higher than in cold treated plantlets in absence of EBR (Kagale et al., 2007). Interestingly, rice plants that are T‐DNA‐tagged in an orthologue of the Arabidopsis brassinosteroid insensitive 2 (BIN2) gene showed enhanced tolerance to cold (Koh et al., 2006) indicating that brassinosteroids have a negative eVect on the cold response. 5. Jasmonic acid (JA) Allene oxide cyclase is the first committed enzyme of the jasmonate biosynthesis pathway. It is up regulated in response to cold in T. arvense (Sharma et al., 2007). In A. thaliana, there is a pronounced (19‐fold) increase in the Allene oxidase cyclase 2 (AOC2) protein in the stroma of leaves exposed

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10 days to cold (Goulas et al., 2006). Microarray data from Thellungiella (Wong et al., 2006) showed the cold induction of transcripts encoding a putative lipoxygenase (At1g17420) gene predicted to be involved in the biosynthesis of JA (Bell and Mullet, 1993). This further supports the conclusion of an increase of JA levels in response to cold. However, the cross‐talk between jasmonic acid signalling and cold signalling is not well documented. Transcript analysis identified as jasmonate responsive some genes encoding chalcone synthase, phenylalanine ammonialyase or dehydrins (Wasternack and Parthier, 1997). The eVects on cold tolerance of mutations in jasmonate signalling pathways would be worth studying. Other cross-talks with hormones start to be discovered. It has been shown that cold leads to the reduction of active gibberellin (GA) content in Arabidopsis. This allows the accumulation of DELLAs, a family of nuclear growth-repressing proteins, the degradation of which is stimulated by GA. Thus the repression of growth observed in plants sumitted to cold might be mediated by DELLAs protein (Achard et al., 2008). Combining metabolic study with transcriptomic study, it was possible to estimate whether the metabolic changes induced by low temperatures were dependent on transcriptomic changes (Kaplan et al., 2007). Many of the changes described above are due to transcriptome changes. We will now describe the molecular pathways triggered by cold exposure.

III. THE CBF PATHWAY IS ESSENTIAL IN TRIGGERING RESPONSE TO COLD EXPOSURE A. IDENTIFICATION OF THE CRT/DRE ELEMENT

A cis‐acting element with a 5‐bp core sequence of CCGAC, designated the C‐repeat (CRT), is present in one or more copies in the promoters of many cold‐regulated plant genes, including the Arabidopsis genes COR15a (Baker et al., 1994) and LTI78 (Yamaguchi‐Shinozaki and Shinozaki, 1994). It is also present in the promoter of the B. napus gene BN115, where it has been named Low Temperature Responsive Element (LTRE; Jiang et al., 1996). Deletion analysis of the Arabidopsis COR15a promoter indicated that the C‐repeat might be part of a cis‐acting cold‐regulatory DNA element (Baker et al., 1994), while mutation of the LTRE impaired the low temperature regulation of BN115 promoter (Jiang et al., 1996). Yamaguchi‐Shinozaki and Shinozaki (1994) showed that two 9‐bp DNA elements, TACCGACAT, containing the C‐repeat are present in the promoter of LTI78. Both these elements induced

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cold‐regulated gene expression when fused to a reporter gene. Moreover, these two DNA elements also stimulated transcription in response to dehydration stress. These C‐repeat‐containing elements were therefore given the designation: Drought Responsive Element (DRE). By promoter analysis of 26 genes, Marayuma et al. (2004) found that in A. thaliana the consensus sequence containing the CRT/DRE is A/GCCGACNT, N being any nucleotide. This sequence is statistically more present in the region
51 to
450 bp upstream of the putative transcriptional initiation. B. IDENTIFICATION OF CRT/DRE BINDING FACTORS: CBF

1. The CBF family in Arabidopsis thaliana A protein binding to this motif was first identified in Arabidopsis. This was performed by one‐hybrid assay using yeast strains that contained CRT/DRE sequences from the promoters of COR15a or LTI78 fused to a lacZ reporter gene. This protein was named CBF1 for CRT‐Binding Factor 1 (Stockinger et al., 1997). CBF1 was later found to belong to a small family, together with CBF2 and CBF3. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence (Gilmour et al., 1998). CBF1–3 are activators of transcription whose DNA binding domains are of the AP2/EREBP type (for APETALA 2/Ethylene‐Responsive Element Binding Protein) (Gilmour et al., 1998; Liu et al., 1998). The transcripts of CBF1–3 accumulate in response to cold (Medina et al., 1999) and also, to a lesser extent, in response to ABA (Knight et al., 2004). These genes are organized in tandem, on chromosome IV of A. thaliana in the following order: CBF1 (At4g25490), CBF3 (At4g25480), CBF2 (At4g25470) (Gilmour et al., 1998; Shinwari et al., 1998; Medina et al., 1999). Another protein with an AP2/EREBP domain, CBF4 (At5g51990), was isolated by Haake et al. (2002). CBF4 is 63% identical to the three CBF1–3 proteins and the identity goes up from 91% and 94% for the DNA binding domain. CBF4 is induced by drought and ABA but not by cold. CBF4 overexpression results in activation of genes that have the CRT/DRE motif in their promoter (Haake et al., 2002). This is a strong indication that CBF4 may in vivo interact with this cis‐acting element. Two other CBF homologues have been found in Arabidopsis. DDF1 (At1g12610) and DDF2 (At1g63030) share 43.5% and 45.9% identity with CBF1, respectively. DDF1 is induced by NaCl treatments, and so is DDF2 but to a lesser extent. In addition, transgenic plants overexpressing DDF1 show increased tolerance to high salinity. Seedlings overexpressing DDF1 show enhanced expression level of LTI78, KIN1, and COR47, i.e. stress

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responsive genes that possess in their promoter the CRT/DRE motif. As for CBF4, this makes a compelling argument for DDF1 being able to recognize this motif (Magome et al., 2004). Whether DDF1 and DDF2 are induced by cold is not known. In addition to the CBF proteins, Liu et al. (1998), also using a one‐hybrid strategy, identified two proteins binding to the CRT/DRE sequence of LTI78, and named these proteins DREB1A and DREB2A; DREB being for DRE‐Binding protein. DREB1A, and its homologues DREB1B, DREB1C and DREB1D, are identical to CBF3, CBF1, CBF2, and CBF4, respectively. As for DREB2A, it is also an activator of transcription whose DNA binding domains are of the AP2/EREBP type (Liu et al., 1998). Except for the DNA binding domain, the DREB2A proteins share low identity with the CBF/DREB1 proteins (see Table I). Homologues of DREB2A have been found, named DREB2B to DREB2H. DREB2A and DREB2B are highly induced by drought, NaCl or heat, while poor induction is seen in response to cold or ABA (Liu et al., 1998; Nakashima et al., 2000; Sakuma et al., 2006). Expression of DREB2C, DREB2D, and DREB2F were induced slightly by high salt treatment in leaves but not in roots. The induction of expression is much less pronounced for those genes than for DREB2A and DREB2B. Expression of DREB2E was induced slightly only by ABA in roots. No conditions have yet been shown to result in the induction of expression of DREB2G and DREB2H (Sakuma et al., 2002). There thus appears to be two kinds of DNA‐binding factors binding the same cis‐acting element and acting as transcriptional activators in at least five separate signal transduction pathways: low temperature (CBF1–3), drought stress (DREB2 proteins), saline stress (DREB2 proteins and DDF1), heat stress (DREB2A‐2B), and the ABA response (CBF4 and, to a lesser extent CBF1–3) (Fig. 12). That helps explain, at least in part, why many genes induced during a cold exposure are also induced by drought.

2. The CBF structures and signatures Subsequent to their discovery in Arabidopsis, many CBF homologues have been found in both monocots and dicots that are able to cold acclimate but also in species shown to be unable to cold acclimate. All monocot and dicot genomic CBF sequences lack introns (Skinner et al., 2005). Phylogenetic analysis revealed that all monocot homologues are separated from dicot homologues in the phylogenetic tree. This topology indicates that significant divergence from the ancestral CBF gene occurred after monocots separated from eudicots, although the conserved domains are unchanged (Xiong and Fei, 2006).

TABLE I Identities of the CBF and DREB2 Proteins from Arabidopsis thaliana Identity (%) CBF1 CBF2 CBF3 CBF4 DDF1 DDF2 DREB2A DREB2B DREB2C DREB2D DREB2E DREB2F DREB2G DREB2H

Gene index Protein sequence At4g25490 At4g25470 At4g25480 At5g51990 At1g12610 At1g63030 At5g05410 At3g11020 At2g40340 At1g75490 At2g38340 At3g57600 At5g18450 At2g40350

AAC49662 AAD15977 AAD15977 Q9FJ93 NP_172721 NP_001077764 BAA36705 BAA36706 Q8LFR2 Q9LQZ2 O80917 Q9SVX5 P61827 Q9SIZ0

CBF1 CBF2 CBF3 CBF4 DDF1 DDF2 DREB2A DREB2B DREB2C DREB2D DREB2E DREB2F DREB2G DREB2H

100

87.0 100

86.6 87.1 100

66.4 65.8 65.0 100

43.5 41.6 44.0 41.5 100

45.9 43.4 44.5 40.3 68.9 100

23.1 23.7 22.4 23.0 21.1 22.1 100

26.7 25.0 24.0 23.3 20.4 19.9 53.0 100

26.3 25.1 24.9 23.5 21.3 19.9 37.4 34.9 100

29.5 28.9 26.9 30.3 24.6 27.4 28.6 27.9 29.6 100

26.5 26.7 27.7 27.6 23.8 19.5 32.3 31.5 30.3 34.1 100

25.4 24.7 24.0 24.7 24.6 22.5 25.5 28.6 29.1 32.3 28.4 100

21.7 22.7 22.3 21.5 21.5 20.1 27.7 29.1 29.2 32.4 26.7 32.1 100

23.7 22.9 22.8 22.7 18.1 22.8 26.6 28.0 37.0 29.4 29.9 20.9 23.2 100

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ABA

Drought

CBF4

Heat stress

Low temperature

Salt stress

DREB2A-2B

CBF1−3

DDF1−2

AREB ABF

ABRE

CRT/DRE

TATA

Fig. 12. Involvement of CBF and DREB2 proteins in response to cold, ABA, heat, drought or saline stresses in Arabidopsis thaliana. ABRE is for ABA responsive element. 82% of the gene up‐regulated by cold have the ACGT‐core ABRE motif in their promoter (Suzuki et al., 2005). CRT could be a coupling element for ABRE in ABA‐dependent expression in response to dehydration and high‐salinity stresses (Narusaka et al., 2003). ABF induction by cold is shown, even though ABF is not a CBF related protein.

In some species, CBF genes are organized in tandem; for example in Arabidopsis (CBF1, 2, and 3; Medina et al., 1999), tomato (LeCBF1, 2, and 3; Zhang et al., 2004b) or rice (OsDREB1 and OsDREB1B; Xiong and Fei, 2006). Because of the phylogenetic considerations mentioned above, it is probable that those duplications occurred independently in the diVerent species (Xiong and Fei, 2006). The CBF protein contains a highly conserved ERF/AP2 domain and belongs to a large multigene family of plant specific transcription factors. These proteins are divided in three classes based on the numbers of ERF/AP2 domains. The first class includes proteins with 2 ERF/ AP2 domains; e.g. the APETALA2 (AP2) protein and AINTEGUMENTA (ANT). The second class includes proteins with one ERF/AP2 domain. CBF proteins belong to this class, together with DREB2 proteins, TINY, ERF and ABI4. The third class includes proteins that have two diVerent binding domains, ERF/AP2 and B3, such as RAV1 and RAV2 (Sakuma et al., 2002). An alignment was performed with CBF protein sequences from diVerent species together with an Arabidopsis protein representative of the other subgroups of the family of proteins with one AP2 domain, i.e. DREB2A, RAP2.10, RAP2.4, ABI4 and TINY. Five clusters of residues can be thus detected that could be considered as a CBF signature (Fig. 13).

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Fig. 13. Alignment of CBF proteins together with Arabidopsis DREB2A, RAP2.10, RAP2.4, ABI4 and TINY. The sequences used are the following. Arabidopsis TINY (CAA64359), ABI4 (AAC39489), DREB2A (BAA36705), RAP2.1 (AAC49767), RAP2.4 (AAC49770), RAP2.10 (AAC49776), DDF2 (NP_001077764), DDF1 (NP_172721), AtCBF1 (AAC49662), AtCBF2 (AAD15976), AtCBF3 (AAD15977), AtCBF4 (Q9FJ93). Eucalyptus globulus (ABB51637), Capsella bursa‐ pastoris (AAR26658), Sorghum bicolor (AAX28960) Prunus avium (BAD27123), Thalspi arvense (ABV82985), Hordeum vulgare (AAG59618), Oryza sativa (AAG59619), Zea mays (AAN76804), Vitis vitifera (AAR28673), Triticum aestivum (AAX28967), Gossypium hirsutum (ABD65473), Lycopersicon esculentum (AAS77819), Capsicum annuum (AAQ88400), Brassica napus (AAM18958), Eucalyptus gunnii (ABB51638) and Lolium perenne (AAX57275).

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Cluster I: KKPAGRKKFRETRHP. This cluster is not found in proteins outside the CBF group. This N terminal signature could play the role of nuclear location site (El Kayal et al., 2006). Cluster II: SAWR: DSAWR does not exist for proteins outside the CBF group. Cluster III: K/RX1AX2EAAL. It can be noted that X1 and X2 are very often A Cluster IV: LL X1X2MAEGMLL. In monocots, first LL is replaced by YY or YL. Cluster V: LWSY/F. This cluster is not present in VvCBF1 or O. sativa. Clusters II, III and IV appear to be more characteristics of dicots. All in all, the primary protein structure of CBF proteins has the following general amino to carboxy‐terminal features: a leader of approximately 15–40 amino acids, an AP2 DNA binding domain directly flanked by the conserved CBF subfamily signature motifs I and II, and then motifs III, IV and V. 3. The binding to CRT/DRE elements may be low temperature‐dependent Xue (2003) demonstrated via transient expression assays that a CBF from Barley, HvCBF2B, could activate a CRT/DRE‐controlled reporter gene at 2 8C, but not at 20 8C. However, this low temperature activation is not a common characteristic of CBF proteins. In rye and wheat, CBF proteins of the same phylogenetic group as HvCBF2B (HvCBF4‐subgroup members: ScCBF22, ScCBF24, ScCBF31, and TaCBF1) also failed to activate expression of the assayed COR genes in transgenic Arabidopsis plants at warm temperatures. In contrast, barley, wheat and rye CBFs of other phylogenetic groups bound CRT/DRE under both warm and cold conditions. The low temperature dependency of CRT/DRE binding might be located in the acidic C‐terminal domain of the CBF. To date, no low temperature‐dependent binding for a dicot CBF has been reported (Skinner et al., 2005). C. CBF EXPRESSION

1. Developmental expression and responses to cold in Arabidopsis In A. thaliana, an approach using promoter fusion to reporter gene makes it possible to follow the tissue specific expression of each CBF during development (Novillo et al., 2007). These experiments demonstrated that CBF1 and CBF3 genes have the same expression pattern during development and in response to low temperature. This pattern is diVerent from that exhibited by CBF2. During germination and early stages of development, at control temperature, the staining in transgenic lines containing CBF1::GUS and CBF3:: GUS fusions was restricted to roots, hypocotyls, and cotyledons. CBF2::GUS seedlings disclosed GUS activity in hypocotyls and cotyledons but also in the

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first and second pairs of leaves. However, they did not display any GUS staining in roots. GUS activity in all transgenic seedlings decreased gradually during development, completely disappearing 4 weeks after germination (Fig. 14A, Novillo et al., 2007). In fully developed plants, under control conditions, no GUS staining was detected in transgenic lines containing CBF1::GUS and CBF3::GUS, while CBF2::GUS adult plants presented GUS activity in sepals and in the abscission zone of the siliques. When exposed to low temperature, CBF1::GUS and CBF3::GUS transgenic lines disclosed A

1d

3d

5d

14 d

21 d

28 d CBF1::GUS CBF3::GUS CBF2::GUS

B

CBF1::GUS CBF2::GUS CBF3::GUS

CBF1::GUS CBF3::GUS

CBF2::GUS

C

4⬚ C

Fig. 14. Histochemical localization of GUS activity in transgenic Arabidopsis containing CBF promoters::GUS fusions. (A), GUS staining of transgenic seedlings containing CBF1 and CBF2 promoter::GUS fusions 1, 3, 5, 14, 21, and 28 days after germination. Seedlings containing the CBF3::GUS fusion exhibited identical patterns of GUS activity as CBF1::GUS transgenic seedlings. (B), GUS staining of diVerent organs (roots, stems, leaves, flowers, and siliques) from adult transgenic plants containing CBF1 and CBF2 promoter::GUS fusions grown under control conditions (C), or exposed to 4 8C for 3 h (4 8C). In all cases, CBF3::GUS adult plants displayed the same patterns of GUS activity as the transgenic plants containing the CBF1::GUS construct. From Novillo et al., 2007.

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GUS staining in leaves, sepals, and siliques (Fig. 14B). In the case of CBF2:: GUS adult plants growing under cold conditions, GUS activity was observed in leaves, sepals, siliques, and also in stems (Fig. 14B) (Novillo et al., 2007). 2. Kinetics of CBF cold expression In Arabidopsis, the CBF1–3 factors are induced at the transcriptional level very quickly (15 min) after exposure to cold; maximum accumulation is generally attained at 3 h and after 24 h transcripts are no longer detectable (Zarka et al., 2003). In addition, the half life of these transcripts was estimated to be 7.5 min upon return to normal temperatures (Zarka et al., 2003). Low temperature induction of Arabidopsis CBF1, 2, and 3 also appears to be mediated by the circadian gate, the extent to which CBF1–3 transcripts accumulate in response to low temperatures being dependent on the time of the day plants are exposed to low temperatures. The highest and lowest levels of cold induction of CBF1–3 transcripts occur 4 and 16 h after the subjective dawn, respectively (12 h photoperiod; Fowler et al., 2005). Correspondingly, it has been shown that LTI78 induction by low temperature was also circadian gated, with higher transcript abundance during subjective day and lower abundance during subjective night (Dodd et al., 2006). Similar to Arabidopsis, in O. sativa OsDREB1A accumulation is detected as soon as 20 min after cold treatment; and maximum accumulation is attained after 5/10 h, and the level decreases after 24 h. OsDRB1B accumulation is detected as soon as 40 min after cold treatment and a plateau is attained after 5/10 h of cold treatment. OsDREB1C is a constitutive gene and OsDREB1D is not detected in any plants, with any treatment (Dubouzet et al., 2003). In B. napus, BnDREBI‐5 and BnDREBII‐1 are proteins of the CBF family. The transcripts of BnDREBI‐5 are induced 2 h after cold treatment; the maximum of accumulation is seen 6 h after the beginning of the treatment and no transcript is detected after 13 h. A second wave of induction is then detected, of much lower intensity, around 18 h. The maximum of transcript accumulation of BnDREBII‐1 is detected around 13 h, and this declines back to the control level after 21 h (Zhao et al., 2006). In L. perenne, LpCBF3 transcript accumulation in leaves is very important as soon as 15 min after the beginning of the cold treatment. The level remains high for the first 2 h of exposure, and then the level goes back to control level after 8 h of treatment (Xiong and Fei, 2006). However, the cold induction of CBF genes is not necessarily followed by a return to control levels once a new steady‐state thermal environment is established. It has been found that CBF accumulation could last through chilling treatment. In Capsicum annuum, CaCBF1A transcripts accumulate in response to 4 8C, in root, stem or leaves. The transcript level is still increasing

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after 16 h, and stays stable at least for 4 days (Kim et al., 2004a). In V. riparia, the accumulation of CBF4 transcripts is maintained for 2 days (Xiao et al., 2008). In Populus spp. leaves, PtCBF3 and PtCBF4 transcript levels peak 3 h after transfer to 5 8C while PtCBF1 and PtCBF2 transcript levels peak 8 h after transfer. In stems, only PtCBF1 and PtCBF3 show a significant induction, with a peak of transcript level attained after 6 h and 3 h, respectively, after transfer at 5 8C (Benedict et al., 2006a). In Eucalyptus gunnii, there is a peak of accumulation for two EguCBF1 transcripts corresponding to 5 h at 4 8C, but under short day conditions (12 8C day/8 8C night; 8 h of day), induction is significant for all the kinetic points measured, including those measured several days after a temperature change (El Kayal et al., 2006). Thus, in conditions mimicking natural ones, with photoperiod and thermoperiod, CBF induction might be maintained. D. ROLE OF CBF IN FREEZING TOLERANCE

1. CBFs are major components of cold hardening Several studies demonstrate the importance of the CBF regulon in the cold hardening process. Using a quantitative genetic approach using crosses of two accessions of A. thaliana, ‘‘Cape Verde Island’’ and ‘‘Landsberg erecta’’ diVering in their freezing tolerance before and after cold acclimation, it was possible to show that freezing tolerance was determined by 7 quantitative trait loci. One of these loci co‐located with CBF1, CBF2, and CBF3. More specifically, the low freezing‐tolerance allele of ‘‘Cape Verde Island’’ was associated with a deletion in the promoter of CBF2, leading to low expression of CBF2 (Alonso‐ Blanco et al., 2005). In T. aestivum L., the winter cultivar ‘‘Mironovskaya808’’ exhibits a much higher level of freezing tolerance than the spring cultivar ‘‘Chinese spring’’. When exposed to low temperature, the amount of WCBF2 transcripts, a CBF orthologue, initially increases in both cultivars. But after 3 weeks at 4 8C, it decreases in the spring cultivar while it still increases in the winter cultivar. This diVerence in WCBF2 transcript accumulation in cold might explain how freezing tolerance is sustained for a longer period in the winter cultivar (Kume et al., 2005). In Triticum monococcum, a cluster of eleven CBF genes was mapped to the Frost resistance‐2 locus on chromosome 5 using a cross between frost tolerant accession ‘‘G3116’’ and frost sensitive ‘‘DV92’’. Interestingly, the transcript levels of TmCBF16, TmCBF12 and TmCBF15 were already up‐regulated by mild low temperatures (15 8C) in the frost tolerant ‘‘G3116’’, but not in the frost sensitive ‘‘DV92’’ cultivar where a temperature of 10 8C is necessary for the induction of those genes. The higher threshold induction temperatures for those CBF genes in ‘‘G3116’’ cultivar could result in earlier initiation of the cold

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acclimation process and better resistance to subsequent freezing temperatures. Besides, the sequence of TmCBF12 encoded by ‘‘DV92’’ allele contains a deletion of five amino acids in the AP2 DNA binding domain, making the protein unable to bind to the CRT/DRE element. This also could contribute to lower frost tolerance of the ‘‘DV92’’ cultivar (Knox et al., 2008). In Arabidopsis, antisense lines with reduced transcript abundance of both CBF1 and CBF3 had similar levels of freezing tolerance before cold acclimation as wild‐type plants. After cold acclimation, the antisense plants were significantly less freezing tolerant than the wild‐type ones with LT50 values around
8.1 8C, more than 1.5 8C higher than that of wild‐type plants (
9.7 8C) (Novillo et al., 2007). Conversely, the constitutive over‐expression of CBF1 or CBF3 genes in Arabidopsis plants induces an increased tolerance to freezing (Gilmour et al., 2004; Jaglo‐Ottosen et al., 1998; Kasuga et al., 1999; Liu et al., 1998). The fact that overexpressing CBF led to a constitutive freezing tolerance has been seen in other species. In Table II, the phenotypes associated with the overexpression of CBF proteins in diVerent species are displayed. A growth retardation phenotype is often observed when overexpressing CBF genes using 35S CaMV constitutive promoter. It can be overcome by the use of the cold‐inducible promoter of LTI78 gene in transgenic Arabidopsis (Kasuga et al., 1999) tobacco (Kasuga et al., 2004) and potato (Pino et al., 2007). It is reminiscent of the phenotype of ddf1. The stunted growth in this mutant is overcome by treatment with gibberellic acid; this mutant is due to the overexpression of DDF1, a member of the CBF family. Therefore the growth retardation observed might be due to a cross‐talk with gibberellic acid signalling pathway; and it might be a side eVect of constitutive expression (Magome et al., 2004). Poplar plants overexpressing AtCBF1 also have a stunting phenotype with reduced internode elongation and root growth when grown on agar. It has been shown in tobacco that GA biosynthesis can be posttranscriptionally repressed (via repression of GA20 oxidase) by the class I KNOX gene NTH15. The closest NTH15 orthologs in both Populus and Arabidopsis contain a CCGAC element in their 1500/1000bp promoter, suggesting that constitutive CBF expression may cause overexpression of NTH15, leading to repression of GA synthesis (Benedict et al., 2006b). The repression of GA content by CBF overexpression has been confirmed in Arabidopsis. Because GA represses the accumulation of DELLAs, a family of nuclear growth-repressing proteins, the repression of growth observed in plants overexpressing CBF might be mediated by DELLAs protein (Achard et al., 2008). The eVect of CBF overexpression on cold‐hardening is due to the fact that these factors can upregulate proteins or enzymes necessary for cold‐ hardening. Indeed CBF overexpression is associated with induction of cold

TABLE II Phenotypes associated with CBF overexpression in different plant species. The promoter used to drive gene expression is indicated. Gene

Source organism

Expressed in

CBF

Thalspi arvense

Arabidopsis (35S)

AtCBF1, 2, 3

Arabidopsis

Arabidopsis (35S)

AtCBF4

Arabidopsis

Arabidopsis (35S)

OsDREB1A

Oryza sativa

Arabidopsis (35S)

LpCBF3

Lolium perenne

Arabidopsis (35S)

AtCBF1, AtCBF2, AtCBF3

Arabidopsis

Brassica napus (35S)

BNCBF5, BNCBF17

B. napus

B. napus (35S)

Phenotype Constitutive freezing tolerance Enhanced expression of COR15A in control plants Growth retardation Freezing tolerance with no cold acclimation Induction of cold responsive genes in control conditions COR gene accumulation Increased freezing and dehydration tolerance Growth retardation Increased freezing tolerance Induction of cold responsive genes in control conditions Growth retardation, Flowering delay Increased freezing tolerance Induction of cold responsive genes in non stressed plants Increased freezing tolerance with no cold acclimation Induction of cold responsive genes in control conditions Increased freezing tolerance, photochemical eYciency, photosynthetic capacity

References Zhou et al. (2007) Liu et al. (1998) Gilmour et al. (2000, 2004) Jaglo‐Ottosen et al. (1998) Haake et al. (2002) Dubouzet et al., (2003)

Xong and Fei (2006)

Jaglo et al. (2001)

Savitch et al. (2005)

(continues)

TABLE II Gene

Source organism

Expressed in

CBF1

Arabidopsis

Populus (35S)

AtCBF3

Arabidopsis

Rice (Ubiquitin1)

CBF1, CBF3

Arabidopsis

Solanum tuberosum (35S)

AtCBF1, AtCBF3

Arabidopsis

AtCBF3

Arabidopsis

AhDREB1

Atriplex hortensis

S. tuberosum (LTI78 promoter) Tobacco (35S and LTI78 promoter) Tobacco (35S)

WCBF2

Triticum aestivum

Tobacco (35S)

LeCBF1

Lycopersicon esculentum Arabidopsis Arabidopsis

L. esculentum (35S)

AtCBF3 CBF1

Tomato (35S)

(continued) Phenotype

References

Increased freezing tolerance of non acclimated leaves and stems Induction of cold responsive genes in control conditions Gene induction Increase tolerance to drought and high salinity Low level of tolerance to low temperature stress No growth inhibition Smaller leaves, delayed flowering Reduction of tuber production Increased freezing tolerance Increased freezing tolerance Tuber production as in wild‐type Induction of cold responsive genes in control conditions Increased osmotic tolerance Induction of cold responsive genes in control conditions Increased freezing tolerance No growth retardation Induction of cold responsive genes in control conditions Delayed flowering No increase in freezing tolerance

Benedict et al. (2006a)

Increased chilling tolerance

Hsieh et al. (2002)

Oh et al. (2005)

Pino et al. (2007) Pino et al. (2007) Kasuga et al. (2004) Shen et al. (2003) Takumi et al. (2007)

Zhang et al. (2004b)

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responsive genes at control temperatures. Cook et al. (2004) have shown that as much as 79% of the metabolite changes elicited during cold acclimation were also found in non‐acclimated plants in response to overexpression of the AtCBF3. When a plant has no or few genes whose promoter can be bound by CBF, CBF overexpression would have little eVect. In tomato, functional CBF homologues exist, but a functional CBF regulon is lacking and very few genes are cold induced (Jaglo et al., 2001). 2. Identification of CBF target The importance of the CBF regulon in the cold response can be understood by analyzing the genes that are CBF targets. Such genes have been identified either through the overexpression experiments presented above, or by promoter analysis, i.e., by identifying genes whose promoters possess CRT/ DRE elements (Maruyama et al., 2004). A list of CBF target genes in A. thaliana is given in Table III. It can be noted that amongst those genes are genes encoding dehydrins, enzymes of sugar metabolism such as SEX1, enzymes of proline biosynthesis and enzymes involved in lipid synthesis (choline‐phosphate cytidyltransferase, stearylCoA desaturase). It appears that many aspects of the response to low temperatures are thus in part dependent on the CBF pathway. In addition, the level of CBF proteins might determine the response. The transcript levels of several well known cold‐inducible genes were investigated in lines invalidated either in CBF1 or CBF3. In response to low temperature, the levels of all messengers increased in those lines and wild‐type plants. Nevertheless, in the case of LTI78, COR15A, COR47, and ERD10, these levels were considerably lower in the CBF1‐ or CBF3‐RNAi lines than in the wild‐type, while the cold induction of KIN1, KIN2, and COR15B was similar in the CBF1‐ or CBF3‐RNAi lines and wild‐type plants. When both CBF1 and CBF3 are shut down, the cold‐induction of all CBF targets analyzed was clearly lower in those plants. This suggests that a certain level of CBF proteins would be necessary for the induction of all CBF targets and the accurate activation of cold acclimation in Arabidopsis, and that this level would be attained only when the CBF1, CBF2, and CBF3 genes are properly and co‐ordinately induced (Novillo et al., 2007). E. REGULATION OF CBF EXPRESSION

1. Autoregulation of CBF expression In Arabidopsis, the analysis of the cbf2 null mutant indicates that CBF2 is a negative regulator of the expression of CBF1 and CBF3 and plays a central part in the tolerance to stress (Novillo et al., 2004). In contrast, in

TABLE III Some CBF Target Genes in Arabidopsis thaliana Identified Either by Overexpression Experiments or Promoter Studies Gene Index

References

At2g28900 At1g01470 At1g20440 At4g23600 At2g42530 At1g09350 At2g17840 At4g33070 At5g04340 At1g10760 At1g05170 At2g24560 At3g55610

1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2 1, 2 1, 3 1, 3 1, 3 1, 3

At4g17550 At2g43620

1, 3 1, 3

At1g46768 At1g62570 At2g24560 At3g55610 At5g20830 At1g01420 At1g02820 At1g08570 At1g14580

1, 3 1, 3 1, 3 1, 3 1 1 1 1 1

Function protein translocase LEA14 COR47

Gene Index

References

Function

At4g15130 At4g27410 At4g29190 At4g34230 At4g34990 At4g40010 At5g01520 At5g12140 At5g14570 At5g17490 At5g24300 At5g24300 At5g25110

1 1 1 1 1 1 1 1 1 1 1 1 1

choline‐phosphate cytidylyltransferase RD26 transcription factor cinnamyl alcohol dehydrogenase 5 MYB32 protein kinase ubiquitin‐protein ligase CYSTATIN‐1 ATNRT2.7_ transporter, putative transcription factor starch synthase starch synthase CIPK25

CHITINASE

At5g27930 At5g37500

1 1

RAP 2.1 disulfide oxidoreductase carboxylic ester hydrolase P5CS2 sucrose synthase SUS1 UDP‐glycosyltransferase LEA3 thioredoxin transcription factor

At5g46050 At5g57110 At2g23120 At3g50970 At5g52310 At1g16850 At1g27730 At1g51090 At1g54410

1 1 2, 3 2, 3 2, 3 2 2 2 2

protein phosphatase type 2C gated outwardly‐rectifying kþ channel transporter calcium‐transporting ATPase

COR15B ERD7 pyruvate decarboxylase transcription factor SEX1 (STARCH EXCESS 1) transferase, transferring glycosyl group carboxylic ester hydrolase pyrroline‐5‐carboxylate synthetase (P5CS2) sugar porter

XERO2/LTI30 COR78 ZAT10 transcription factor/ STZ dehydrin (continues)

TABLE III (continued) Gene Index

References

At1g14580 At1g17020 At1g30360 At1g47710 At1g58360 At1g59530 At1g62710 At1g69870 At1g76580 At1g77120

1 1 1 1 1 1 1 1 1 1

At4g25570 At2g16890 At2g22590 At2g34850 At2g39030 At2g47890 At3g05660 At3g12580 At3g14680 At3g23340 At3g45260 At3g54400

1 1 1 1 1 1 1 1 1 1 1 1

Function transcription factor SRG1 ERD4 serine‐type endopeptidase inhibitor AAP1; amino acid permease transcription factor transcription factor alcohol dehydrogenase 1 rhamnosyltransferase UDP‐glycosyltransferase glycosyltransferase NAD‐dependent epimerases GCN5‐related N‐acetyltransferase transcription factor KINASE

HSP70 CYP72A14 protein kinase transcription factor pepsin A_ aspartyl protease family protein

At3g55310 1 OXIDOREDUCTASE At3g55580 1 RCC1 At3g55940 1 phospholipase C At3g57020 1 strictosidine synthase 1: Vogel et al. 2005, 2: Maruyama et al., 2004; 3: Gilmour et al., 2004

Gene Index

References

At2g21660 At2g31360 At2g33830 At2g43510 At4g14000 At4g15910 At4g24960 At4g30650 At4g35300 At4g38580

2 2 2 2 2 2 2 2 2 2

At5g17460 At5g17460 At5g58670 At4g04020 At3g50980 At1g27200 At4g21570 At1g43160 At1g65270 At5g52300 At4g12470 At4g37150

2 2 2 3 3 3 3 3 3 3 3 3

At5g15970 At5g15960 At4g12480 At1g20450

3 3 3 3

Function GRP7 stearoyl‐CoA 9‐desaturase (ADS2)

drought‐induced protein (Di21) HVA22d carbohydrate transporter AtFP6 (farnesylated protein 6) ATPLC1 dehydrin XERO1 RAP2.6 TI65/RD29B PEARL_1 hydroxynitrile lyase KIN2 KIN1 PEARL1 ERD10

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E. RUELLAND ET AL.

plants where the expression of CBF1 and/or CBF3 is impaired, the induction of CBF2 in response to low temperature is not diminished. CBF1 and CBF3 are not implicated in regulating the expression of CBF2 genes (Novillo et al., 2007). A very original regulation has been found in B. napus. Two groups of CBF genes have been isolated. These two groups were induced by low temperature, but the expression of Group I preceded that of Group II. The Group I CBFs could specifically bind the CRT/DRE cis‐acting element and activate the expression of target genes. In contrast, Group II CBFs were unable to transactivate the expression of target genes although they had the ability to bind the CFT/DRE element. Interestingly, co‐expression of Group II factors could depress the trans‐activation activity of Group I proteins in a concentration‐dependent manner. These results strongly suggested that the trans‐active Group I CBFs were expressed at the early stage of cold stress to open the CFT/DRE‐mediated signalling pathway in cold stress, whereas the trans‐inactive Group II CBFs were expressed at the later stage to close the signal pathway in a competitive manner (Zhao et al., 2006). CBF expression might be feedback down‐regulated by CBF proteins or target products. In Arabidopsis, los1–1 mutation blocks low temperature‐ induced transcription of cold‐responsive genes. However, the cold‐induced expression of CBFs is enhanced by this mutation. The LOS1 gene encodes a translation elongation factor 2‐like protein. Protein labelling studies show that new protein synthesis is blocked in los1–1 mutant plants, specifically in the cold (Guo et al., 2002). 2. Regulation of CBF expression by ICE1 a. Cloning of ICE1. The first ICE protein to be cloned was ICE1. This factor binding the promoter of CBF3 was isolated in 2003 by a genetic screen based on the cold activation of the CBF3 promoter fused with luciferase (Chinnusamy et al., 2003). ICE1 is constitutively expressed. It encodes a basic Helix‐Loop‐Helix (bHLH) protein of MYC type. This protein is located in the nucleus. In ice1 mutant plants, CBF3 cold induction is nearly totally impaired. The ice1 mutation also aVects the cold‐induction of CBF1 and CBF2; their expression is slightly reduced early in the cold, but at later time points the expression is not reduced. On the contrary, the expression of CBF2 is actually enhanced in the ice1 mutant after 6 and 12 h of cold treatment. It might be related to the fact that the expression of CBF genes is repressed by their gene products or products of their downstream target genes (Guo et al., 2002, see above). At this stage of knowledge it is supposed that CBF1,2 cold expression is not mediated by ICE1 but by ICE1‐like proteins (see Section III.E.3.). In the ice1 mutant, the chilling and freezing tolerances are impaired.

COLD SIGNALLING AND COLD ACCLIMATION IN PLANTS

95

The overexpression of ICE1 in wild plants increases the cold expression of CBF regulon and improves their tolerance to freezing. However this overexpression does not make it possible to set on the CBF regulon at ambient temperature (Chinnusamy et al., 2003). The ability of ICE1 to activate gene transcription in response to cold may be dependent on protein phosphorylation and dephosphorylation in the cytoplasm or in the nucleus. Indeed, cold stress induces phosphorylation of ICE1 (unpublished data mentioned in Chinnusamy et al., 2007). b. The ICE1 regulon. To date, ICE1 is the most upstream transcription factor in the cold signalling pathway. To find the preferred cis‐acting element used by ICE1, one approach is to consider that this motif should be overrepresented in cold induced genes whose induction is aVected in the ice1 mutant. The HACACGT and HCCACGT (H being either A or C or T) motifs, which are myc recognition motifs named ICEr3 and ICEr4, respectively, were identified bioinformatically to be overrepresented in genes less induced after 6 h of cold treatment or non responsive after 3 h of cold treatment in ice1 mutant (Benedict et al., 2006c). This left the possibility that the motifs found are not the direct targets of ICE1 but of transcription factor(s) downstream of ICE1. However, when comparing the genes with those motifs in their promoters, it was found that the ICEr3 cis‐regulon was statistically overrepresented in genes induced at 1 h, and subsequent time points of cold treatment, suggesting it might be an early cis‐acting element. The ICEr4 motif is statistically more present in promoters of genes induced at 3 h. The same approach showed that the CRT/DRE motif was overrepresented in promoters of genes induced after 6 h of cold, thus validating the idea that ICEr3 and ICEr4 are cis‐acting elements involved before the CRT/DRE cis‐ acting element. Interestingly, two ICEr3 motifs are found in CBF1 promoter and one ICEr4 motif is found in the CBF1 and CBF3 promoters, respectively (Benedict et al., 2006c). The fact that ICE1 might bind these motifs and transactivate the promoters needs to be biologically validated. c. ICE1 is regulated by sumoylation. It has been shown that in vitro and in protoplasts ICE1 could be sumoylated. This sumoylation is catalyzed by SIZ1, a SUMO E3 ligase that facilitates conjugation of SUMO to protein substrates. Interestingly, siz1–2 and siz1–3 T‐DNA insertion alleles caused freezing and chilling sensitivities. The cold‐induced expression of CBFs, particularly of CBF3, and of the regulon genes was repressed in siz1 but the expression of ICE1 was not aVected. The sumoylation of ICE1 thus appears to have a positive eVect on CBF3 cold induction and it is the sumoylated form of ICE1 that must be active to transactivate the CBF3 expression. It is confirmed

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E. RUELLAND ET AL.

by the fact that the expression of an ICE variant unable to be sumoylated in wild‐type plants repressed the cold‐induction of CBF3. It is possible that the sumoylation of ICE1 may activate and/or stabilize ICE1. Sumoylation of recombinant ICE1 reduced polyubiquitination of the protein in vitro (Miura et al., 2007). ICE1 can indeed be ubiquinated by HOS1. d. ICE1 is ubiquinated by HOS1. HOS1 is a RING E3 ligase that negatively regulates cold signal transduction. The recessive hos1 mutation causes enhanced induction of the CBF transcription factors by low temperature as well as of their downstream cold‐responsive genes. Conversely, the overexpression of HOS1 represses the expression of CBFs and their downstream genes and confers increased sensitivity to freezing stress. HOS1 is ubiquitously expressed in all plant tissues. Fusion with GFP reveals that HOS1 is in the cytoplasm at normal growth temperatures and translocates to the nucleus in response to low temperature treatments. HOS1 is an E3 ligase. It physically interacts with ICE1 and mediates the ubiquitination of ICE1 both in vitro and in vivo. Indeed, cold induces the degradation of ICE1 in plants, and this degradation requires HOS1. These results indicate that cold stress responses in Arabidopsis are attenuated by a ubiquitination/proteasome pathway in which HOS1 mediates the degradation of the ICE1 protein. The fact that not only CBF3 but also CBF1,2 cold expressions are attenuated in plant overexpressing HOS1, indicates that the ICE1‐like proteins upstream of CBF1,2 may also be degraded through HOS1‐mediated ubiquitination (Fig. 15; Dong et al., 2006; Lee et al., 2001). 3. Others ‘‘inducers of CBF expression’’ Analyses of the CBF2 promoter made it possible to identify a segment of 125 bp that is suYcient to direct the cold induction of this gene. In this segment, two sequences, ICEr1 and ICEr2 (for Inducer of CBF Expression regions 1 and 2), have been found. Each of them allows a weak response to low temperatures but, when combined, they give a strong response for the cold induction of CBF2. The ICEr1 fragment includes a consensus sequence for DNA binding of bHLH type proteins. ICE1 factor is such a bHLH protein but results obtained on the mutant ice1 suggest that ICE1 does not direct the cold induction of CBF2 (Zarka et al., 2003). By meta‐analysis of microarray data and promoter sequence analysis, it has been shown that the ICEr1 consensus is not overrepresented in cold induced genes (Benedict et al., 2006c). This is not a contradiction if one considers that it is the presence of both ICEr1 and ICEr2 motifs that direct cold expression of genes, and particularly of CBF2. The proteins binding to ICEr1 and ICEr2 have not been isolated.

97

COLD SIGNALLING AND COLD ACCLIMATION IN PLANTS Cold

ICE1 Proteolysis

P Unknown mechanism (kinase?)

Activated ICE1

HOS1 U

Proteolysis

ICE1-like

Unknown kinase

ICE1 U

S

SIZ1

S

Activated ICE1-like

U

ICE1-like

HOS1 U

MYB15

MYB15

CBF1,2 MYBRS

ICEr

CBF3 ICEr

MYBRS

Fig. 15. Cold activates CBF transcription by activation of ICE factors while MYB15 is a negative regulator of CBF cold induction. ICEr, ICE recognition motifs; MYBRS, MYB recognition sequence; U, ubiquitin protein; S, SUMO protein; P, phosphoryl group. In Arabidopsis, HOS1 and SIZ1 are RING E3 ligase and SUMO E3 ligase, respectively. ICE1 activates the transcription of CBF3. ICE1‐like proteins are suggested to activate the transcription of CBF1 and CBF2 in response to cold. Cold activates ICE1 and ICE1‐like proteins, probably via phosphorylation.

4. Negative regulators of CBF expression a. MYB15. It has been shown by yeast 2‐hybrid and pull down assays, that MYB15, a R2R3‐MYB protein, could physically interact with ICE1. MYB15 is induced by cold, with a peak of transcripts attained 12 h after the beginning of cold treatment (Agarwal et al., 2006). The expression of MYB15 might be regulated by ICE1. It has been shown that the unsumoylated form of ICE1 triggered MYB15 expression. Accordingly, cold induction of MYB15 is more rapid and intense in siz1 plants (Miura et al., 2007). The impact of ICE1 phosphorylation on MYB15 expression is not known. Interestingly, MYB15 binds to the Myb recognition sites of CBF1, CBF2, and CBF3 promoters. MYB15 overexpression led to the reduction of the expression of CBF genes, while myb15 T‐DNA knock‐out lines resulted in an increase of CBF expression. Therefore, MYB15 might be a negative regulator of CBF expression (Fig. 15; Agarwal et al., 2006; Miura et al., 2007).

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E. RUELLAND ET AL.

b. ZAT12. Vogel et al. (2005) identified ZAT12 (At5g59820), RAV1 (At1g13260), MYB73 (At4g37260), STZ/ZAT10 (At1g27730), CZF1 (At2g40140), and CZF2 (At5g04340) as transcription factors that are induced in parallel with CBF1, CBF2, and CBF3. ZAT12 is a protein with zinc‐finger domain whose role in the response to the oxidative stress has been documented (Davletova et al., 2005). The overexpression of ZAT12 induces the expression of nine genes that are also induced in response to the low temperatures. It represses the accumulation of 15 genes that are under‐regulated in response to low temperatures (Vogel et al., 2005). The overexpression of ZAT12 causes an increase in the tolerance to freezing in plants not cold‐acclimated. However, this overexpression attenuates the cold induction of CBF1, CBF2, and CBF3, suggesting that ZAT12, or a gene that it controls, negatively influences the CBF pathway (Vogel et al., 2005). It is interesting to note that ZAT12 contains an EAR domain that could function like a transcriptional repressing domain (Hiratsu et al., 2002). Interestingly, like CBF1–3, ZAT12 transcript accumulation in response to low temperatures has been shown to be gated by the circadian clock. However, the circadian eVect is reverse to that on CBF1–3: ZAT12 response to low temperature was higher at 16 and 40 h after subjective dawn, time were CBF1–3 cold response was lower (Fowler et al., 2005). Up‐regulation of ZAT12 at time 16 and 40 h might dampen cold response of CBF1–3 at those times. The ICEr3 and ICEr4 binding motifs are found in the ZAT12, promoter (see Section III.E.2.b.), suggesting that ZAT12 expression might be regulated by the ICE1 regulon (Benedict et al., 2006c). It has to be noted that STZ/ZAT10 (At1g27730), one of the transcription factors induced in parallel with the CBF genes, is also a zinc‐finger domain protein. ZAT10 is a transcriptional repressor that binds to EP2 cis‐elements. The relevance and importance on gene expression during cold response is not known (Sakamoto et al., 2004). c. STRS. In A. thaliana, STRESS RESPONSE SUPPRESSOR1, designated STRS1 (At1g31970) and STRS2 (At5g08620) are DEAD‐box RNA helicases. In the strs mutants, the cold‐expressions of CBF3 and of a CBF target gene, RD29A, are enhanced. This suggests that STRS1 and STRS2 attenuate the expression of the CBFs (Kant et al., 2007). d. FVE. In plants mutated in the FVE gene, the induction of CBF1, 2, and 3 genes is higher and earlier at 1 8C when compared to wild type plants. The induction of CBF target genes such as COR15A and COR47 is also enhanced in the fve mutant. FVE expression is not aVected by cold. FVE encodes a protein with high homology with the mammalian retinoblastoma‐associated protein (RbAp), and is predicted to contain five WD‐40 repeats. RbAp is a

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component of a histone deacetylase complex involved in transcriptional repression. A loss‐of‐function mutation in FVE could release a transcriptional repression mediated by such a histone deacetylase complex, resulting in earlier and higher induction of CBF genes and their target genes (Kim et al., 2004b). It is interesting to note that FVE is also involved in the control of flowering, where it acts by inhibiting the expression of FLC (FLOWERING LOCUS C), a repressor of flowering. COR15A expression in fve mutants is enhanced, but not in flc‐3 plants, proving that FVE eVect on expression of cold responsive genes is independent of FLC function (Kim et al., 2004b). e. FIERY2. Seven allelic mutations in the FIERY2 (FRY2) locus result in significant increases in the cold expression of CBF1–3 genes and CBF target genes such as KIN1, COR47 or LTI78. However, cold acclimation is aVected in fry2 mutants. While no diVerence in constitutive freezing tolerance was observed in non cold acclimated fry2 and wild‐type plants, after 4 days of cold acclimation, fry2 mutants did not displayed improved freezing tolerance while wild‐type plants do. FRY2/CPL1 encodes a novel transcriptional repressor harbouring two double‐stranded RNA‐binding domains and a region homologous to the catalytic domain of RNA polymerase II C‐terminal domain phosphatases found in yeast and in animals that regulate gene transcription. These data indicate that FRY2 is an important negative regulator of stress gene transcription and suggest that structured RNA may regulate cold stress responses in plants. FRY2 is expressed ubiquitously at low level and is not upregulated by cold (Xiong et al., 2002). MYB15, ZAT12 (and maybe ZAT10) negatively regulates CBF expression by specifically binding to cis‐elements in the CBF promoters, while FVE negatively regulate CBF expression via a histone deacetylase complex. The exact mode of action of FIERY 2 is not known. Contrary to the STRS, FRY2, and FVE genes, that are constitutively expressed, MYB15, ZAT12, and ZAT10 are induced by cold, in parallel to CBF. Because they negatively regulate the CBF genes, they must be seen as participating to the fine tuning of CBF expression, and therefore of the COR gene expression. F. OTHERS REGULATORS

1. SFR6 The sfr6 mutant of Arabidopsis displays a deficit in freezing tolerance after cold acclimation. In this plant, the cold induction of CBF genes is not impaired (Knight et al., 1999). Using microarray analysis, it was found that the sfr6 plants were deficient in cold‐inducible expression of many genes with

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CRT/DRE cis‐element in their promoters, such as KIN1, COR15A, and LTI78 (Boyce et al., 2003). By use of a synthetic promoter, it was shown that the CRT/DRE element is suYcient to confer the sfr6 eVect in response to cold (Boyce et al., 2003). An hypothesis expressed by the authors is that SFR6 protein might potentiate the action of CBF on the CRT/DRE elements to allow eYcient cold induction of the CBF target genes. However the exact mode of action of such a potentiation is not known. 2. LOV1 LONG VEGETATIVE PHASE 1 (LOV1) encodes a NAC‐domain transcription factor. At 4 8C the expression of COR15A is enhanced in LOV1‐overexpressing plants but reduced in loss‐of‐function lov1 mutant. The expression at 4 8C of the CBF genes does not seem aVected either by LOV1 mutation or overexpression. Loss‐of‐function lov1–4 plants are more sensitive to freezing than wild type plants both before and after cold acclimation while plants overexpressing LOV1 have a higher constitutive freezing tolerance. Interestingly, LOV1 negatively regulates the expression of CONSTANS (CO). co‐2 mutants are also freezing tolerant. This is consistent with the phenotype associated with LOV1 overexpression and suggests that CO, a well known floral promoter, may also be involved in freezing tolerance. However, whether the eVects of lov1 mutation on COR15A expression and freezing tolerance occurs through CO is not known (Yoo et al., 2007). While LOV1 positively regulates COR15A expression, other transcription factors have been identified that negatively regulate the expression of CBF target genes. 3. HOS10 In Arabidopsis, HOS10 encodes a R2R3‐type MYB transcription factor. hos10 mutants are characterized by a quicker induction of LTI78, COR15A, and KIN1 in response to low temperature. However, the expression of the CBF genes themselves is not altered by the hos10–1 mutation. The hos10 plants are extremely sensitive to freezing and are unable to cold‐ acclimate. When acclimated at 4 8C for 8 days, the majority of the wild‐ type plants tolerated freezing temperatures as low as
8 8C. However, less than 2% of the hos10–1 mutant plants survived freezing at
2 8C, and none (non‐acclimated or acclimated) survived below
2 8C. With or without cold acclimation, detached leaves of hos10–1 plants also showed more injuries than wild type ones and were unable to increase their freezing tolerance significantly when measured by electrolyte leakage (Zhu et al., 2004a). The authors suggest that this freezing sensitivity might be related to an inability to

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adjust to freeze-induced dehydration, since hos10-1 plants do not normally accumulate ABA in response to dehydration. HOS10 is therefore a negative regulator of COR gene expression that acts downstream of the CBF proteins. Another such regulator is HOS9. 4. HOS9 The HOS9 gene encodes a putative homeodomain transcription factor that is localized to the nucleus. In hos9–1 Arabidopsis mutants, the LTI78 promoter is hyper activated by low temperature but not by ABA or salinity stress. The mutants grow more slowly, and flower later, than do wild‐type plants and are more sensitive to freezing, both before and after acclimation, than the wild‐type plants. HOS9 is constitutively expressed and not further induced by cold stress. The response to cold of the CBF transcription factor genes is not altered by the hos9–1 mutation. Correspondingly, microarray analysis showed that none of the genes aVected by the hos9–1 mutation are controlled by the CBF family. HOS9 is thus thought to negatively aVect gene response to low temperatures independently of the CBF pathway (Zhu et al., 2004b). A search for the 138 available promoter sequences for the genes reported to be diVerentially induced in hos9–1 mutants in the cold versus wild‐type plants identified ACGCT(T), named HOS9r1, as the potential HOS9 binding consensus favoured in vivo. When the kinetics of gene induction or repression was followed in wild‐type plants, it was found that the HOS9r1 cis‐regulon (i.e., genes possessing this motif in their promoter) was repressed in the first 30 min of cold exposure but this repression disappeared after 1 h (Benedict et al., 2006c). HOS10 and HOS9 negatively regulate the COR gene expression by binding to cis‐elements of the promoters. The regulation of COR gene can also be aVected by modification of histone at their loci. This is shown be the hos15 mutant. 5. HOS15 HOS15, like FVE (see Section III.E.4.d.) is a WD‐40‐repeat protein. Together with histone acetyltransferase or deacetylase and many other components, WD repeat are components of protein complexes implicated in histone modification. HOS15 is localized in the nucleus and specifically interacts with histone H4. In Arabidopsis hos15 mutant plants, the cold‐activation of LTI78 promoter is increased. A microarray study allowed the identification of 136 genes with higher expression in hos15 mutant plants than in wild‐type plants when exposed to cold. The cold‐induction of CBF1–3 genes is not impaired in the mutants. HOS15 has a repressor activity. It acts by facilitating deacetylation of histone H4 associated with promoter. The level of acetylated histone H4 is higher in the hos15 mutant than in wild‐type plants and the hyperinduction of LTI78 promoter is associated with a substantially higher level of acetylated histone H4 in

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the hos15 mutant under cold stress conditions (Zhu et al., 2008). Therefore the expression of cold responsive genes can be controlled by WD‐40 proteins acting either on CBF genes (FVE) or on their gene targets (HOS15). HOS9, HOS10, and HOS15 are negative regulators of COR gene expression, acting downstream of the CBF proteins. However, there also exist some positive regulators of the COR gene expression, that act downstream of the CBF proteins. This is illustrated by the Soybean protein SCOF1. 6. SCOF1 In Soybean, SCOF1 is a nucleus‐located C2H2‐type zinc finger protein that is induced by cold stress. Constitutive overexpression of SCOF‐1 induced COR gene expression and enhanced cold tolerance of non‐acclimated transgenic Arabidopsis and tobacco plants. SCOF‐1 did not bind directly to either CRT/ DRE or ABA responsive element (ABRE) cis‐elements. However, SCOF‐1 greatly enhanced the DNA binding activity of SGBF‐1, a soybean G‐box binding bZIP transcription factor, to ABRE in vitro. In Arabidopsis leaf protoplasts, SGBF‐1 transactivated a reporter gene driven by the ABRE element. Furthermore, the SCOF‐1 enhanced ABRE‐dependent gene expression mediated by SGBF‐1. These results suggest that SCOF‐1 may function as a positive regulator of COR gene expression mediated by ABRE via a protein interaction with SGBF1, which in turn enhances cold tolerance of plants (Kim et al., 2001a). Similar to SCOF‐1 regulation, it has been shown that the elevation of the SCOF‐1 transcript level by cold stress is associated with both transcriptional activation and post‐transcriptional mRNA stability under low temperature. A secondary structure may be involved in the mRNA stability of SCOF‐1 (Kim et al., 2001b). The roles of the diVerent regulators that we have described are summarized in Fig. 16. CBF1, 2, and 3 have a central role in the cold induction of genes. CBF1–3 are very rapidly induced by cold. The cold induction of CBF1–3 expression is mediated by ICE transcription factors. CBF3 expression is for instance mediated by ICE1. ICE1 is sumoylated by SIZ1. The sumoylation of ICE1 may prevent the ubiquitination of ICE1 by HOS1. A phosphorylation of ICE1 might also be necessary to activate ICE1. CBF1,2 cold expression is attenuated in plant overexpressing HOS1, indicating the ICE1‐like proteins upstream of CBF1,2 may also be degraded through ubiquitination. CBF1–3 induction is repressed by MYB15. MYB15 is induced by cold and its induction is repressed by the sumoylated form of ICE1. The cold induction of CBF1–3 is repressed by STRS proteins (RNA helicases) and ZAT12, a transcriptional repressor. ZAT12 is itself induced by cold, may be through an ICE‐like protein. The CBF1–3 will then activate the cold expression of COR genes with CRT/ DRE cis‐element in their promoters. The induction of those genes is attenuated

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Fig. 16. Schematic representation of the CBF pathway. ICEr, ICE recognition motifs; MYBRS, MYB recognition sequence; Hos9r1, Hos9 recognition motif 1; ABRE, ABA responsive element; U, ubiquitin protein; S, SUMO protein; P, phosphoryl group. In Arabidopsis,HOS1 and SIZ1 are RING E3 ligase and SUMO E3 ligase, respectively. ICE1 activates the transcription of CBF3. ICE1‐like proteins are suggested to activate the transcription of CBF1 and CBF2 in response to cold. Cold activates ICE1 and ICE1‐like proteins. HOS9 and HOS10 and STRS1,2 are constitutively expressed. The HOS15 gene is slightly up‐regulated by cold. MYB15 is induced by cold, with a maximum at 12 h of stress. MYB15 expression is repressed by sumoylated ICE1. Induction of MYB15 by cold is more rapid and intense in siz1 mutant.

by HOS15 (a component of histone modification machinery), HOS9 (Homeobox transcription factor), and HOS10 (R2R3‐type MYB transcription factor). COR gene induction can also be activated through ABF proteins that will bind the ABRE cis‐element. ABF are indeed induced by cold. 7. More to be found All the regulators that we have presented so far act, may be not exclusively, on genes that are targets of CBFs. Most of these regulators have indeed been found in a genetic screening aiming at identifying mutants that show a higher (hos mutants) or lower (los mutants) activation of the promoter of LTI78, a CBF target gene, during cold response (Ishitani et al., 1997). However, not all cold responsive genes are members of the CBF regulon. For instance, RCI2A is a cold‐induced gene encoding a small, highly hydrophobic protein with two

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potential transmembrane domains. Cold‐induction of RCI2A is thought to be CBF‐independent, since it is not compromised in lines aVected in CBF1, CBF2 or CBF3 (Novillo et al., 2004, 2007). Using as a reporter LUC driven by the RCI2A promoter, Medina et al. (2005) isolated mutants characterized by either lower or higher RCI2A response to cold. These mutants were not aVected in the cold response of LTI78, a CBF‐driven gene. This confirms that RCI2A must be cold induced via a CBF‐independent pathway and that these mutants are impaired in elements of this CBF‐independent pathway. The genes invalidated in those mutants are not yet cloned. However, this study is a compelling argument for the existence of CBF‐independent pathways in the cold response. The existence of CBF‐independent pathways can be illustrated by the gigantea (gi) mutant. The Arabidopsis GIGANTEA (GI) gene regulates several developmental processes, including photoperiod‐mediated flowering, phytochrome B signalling, circadian clock, and carbohydrate metabolism. GI gene is induced by cold stress and loss‐of‐function gi‐3 plants showed an increased sensitivity to freezing stress. However, no significant diVerences were detected in the transcript levels of CBF1, CBF2, and CBF3 genes as well as their targeted genes RD29A, COR15A, KIN1, and KIN2 between wild‐type and gi‐3 plants in response to cold stress. These results suggest that GI gene positively regulates freezing tolerance via a CBF‐independent pathway (Cao et al., 2005). One approach to identify new pathways is to identify new cis‐regulating element in cold responsive genes. For instance, Suzuki et al. (2005) have developed a simple quantitative computational approach for objective analysis of cis‐regulatory sequences in promoters of cold‐regulated genes of A. thaliana. They could not detect significantly enriched sequences in down‐regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. In contrast, they detected significantly enriched sequences in long term up‐regulated genes. 40% had the CCGAC (CRT/DRE) motif, 21% had a CTCCCCG motif, 47% a AGTNGGTCC motif, and 74% a GATATNNT motif. The CTCCGCGT motif resembles the ICEr2 motif (ACTCCG) found in CBF2 promoters (Zarka et al., 2003, see Section III.E.3.), but with the A missing and with 3 additional significant bases. The 3 motifs CTCCGCGT, AGTNGGTCC, and GATATNNT are not detected in CBF driven genes, suggesting they might be independent of the CBF function. However, biological validation of the functionality of these motifs in cold response is still missing. In conclusion, cold exposure will trigger dramatic changes in transcript levels. The best documented genetic pathway leading to cold gene induction is the CBF pathway. This pathway has been identified in both cold resistant and cold sensitive plants. In cold resistant plants and plant able to cold acclimate, the CBF proteins will activate the transcription of genes encoding

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proteins with major role in cold tolerance and freezing resistance. The CBF pathway is not thought to be the only one triggered by cold exposure. Nevertheless, description of CBF independent pathways is scarce. Finally, in field or natural conditions it is likely that transcriptomic changes are more complex. Other phenomena, such as light or dehydration of the soil might interfere with the cold response. The fact that transcript level changes are set up after cold exposure means that cold, as a signal, has been transduced to the cell into the nucleus. We will now present the diVerent signalling events that have been identified in plant cells during a cold exposure.

IV. TRANSDUCTION OF COLD SIGNAL A. CALCIUM

1. Calcium signature An immediate increase in cytosolic calcium ([Ca2þ]cyt) is one of the major signalling events triggered by a cold exposure. In Arabidopsis guard cells, prior to stimulation the [Ca2þ]cyt was 128 nM while cold caused an immediate increase that peaked between 300 nM and 1100 nM (Dodd et al., 2006). In Arabidopsis roots, when [Ca2þ]cyt is measured by aequorin, during a fast cooling, the increase is in the form of a peak and if cooling is slower, the increase becomes biphasic, a shoulder following the peak; the peak is higher if the cold shock is fast (Plieth et al., 1999; Fig. 17A), demonstrating that there is a strong correlation between the increase in [Ca2þ]cyt and the rate of temperature decrease (dT/dt; Plieth et al., 1999). This spike pattern of [Ca2þ]cyt increase by low temperature is observed when aequorin is used to report [Ca2þ]cyt from whole seedlings, roots or cell populations (Kiegle et al., 2000). However, when cameleon is used to monitor [Ca2þ]cyt in single guard cells, low temperature induces repetitive [Ca2þ]cyt oscillations (Allen et al., 2000). A possible explanation for this apparent discrepancy is that the first peak measured using a population of cells may represent the addition of the synchronized increase of [Ca2þ]cyt occurring immediately after the low temperature treatment in individual cells. On the contrary, the second phase (shoulder) would represent the amalgamation of asynchronous [Ca2þ]cyt oscillations. A computer simulation shows that the peak and shoulder pattern can indeed result from the summation of single‐ cell [Ca2þ]cyt oscillations (Dodd et al., 2006). It is therefore likely that cold exposure induces [Ca2þ]cyt oscillations. However, it is also possible that the oscillations observed in guard cells are a feature specific to those cells.

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b

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Fig. 17. [Ca2þ]c response to cold in Arabidopsis roots. (A) Cooling experiments were conducted with a single cooling event (from T0 ¼ 18 8C down to Tf ¼ 4 8C), but with diVerent initial cooling. a: dT/dt ¼
0.44 8C/s; b: dT/dt ¼
0.39 8C/s; c: dT/dt ¼
0.26 8C/s; d: dT/dt ¼
0.05 8C/s(upper trace); dT/dt ¼
0.44 8C/s(lower trace). With high cooling rates only one single [Ca2þ]c peak is observed. Lower cooling rates reveal the biphasic response. An interesting finding here is that at very low cooling rates the [Ca2þ]c trace is lacking the first peak completely and only the second, prolonged slow response is observed. With extremely low cooling rates no [Ca2þ]c response was observed at all. (B) Desensitisation as revealed by [Ca2þ]c response in Arabidopsis roots upon repetitive cooling. (a): [Ca2þ]c response; (b) temperature monitoring. [Ca2þ]c was measured by aequorin luminescence. From Plieth et al., 1999.

During repeated applications of cold shock, it is possible to observe a reduction in the calcic answer: it is a phenomenon of desensitisation (Fig. 17B. Knight et al., 1996; Plieth et al., 1999). This increase in [Ca2þ]cyt is mainly due to calcium entry from the extracellular space. In Arabidopsis, EGTA, chelating calcium, and lanthanum, which

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blocks calcium channels of the plasmalemma, partly decrease the rise in [Ca2þ]cyt. An internal component of the [Ca2þ]cyt increase also exists. By translational fusion, it was shown that cold induced an increase in calcium near microdomains corresponding to the cytosolic face of the vacuole. The increase in [Ca2þ]cyt is sensitive to treatments with neomycin, an inhibitor of phosphatidylinositol bisphosphate‐dependent phospholipase C activities, and lithium, inhibitor of inositol‐1‐phosphatase. The inhibition by neomycin is greater when ‘‘microdomain’’ calcium is monitored, while the presence of lithium provoked a prolongation of the calcium response of the ‘‘microdomain’’. This suggests that the increase in ‘‘microdomain calcium’’ involves inositol‐tri‐phosphate (InsP3; see Section IV.C.2.) (Knight et al., 1996). The possible involvement of calcium‐induced calcium release mechanism by InsP3 is in agreement with the possible existence of calcium oscillations suggested by Dodd et al. (2006). 2. Calcium response is circadian‐gated It has been shown that the time of the day modulated the low temperature‐ induced increase of calcium signals. In guard cells, the cold‐induced [Ca2þ]cyt transients were significantly higher during the mid‐photoperiod than at the beginning or end of the subjective day. The cold‐induced [Ca2þ]cyt was lowest during the subjective night (Dodd et al., 2006) 3. Mechanisms underlying calcium signature The increase of [Ca2þ]cyt results from the activation of calcium channels (mainly located in plasma membranes but with a minor contribution of channels located in organelle membranes) that will let the calcium go into cytosol. Subsequent reestablishment of [Ca2þ]cyt to resting levels is achieved by Ca2þ pumps and antiporters. It is possible to model the calcic signature (peak followed by a bump which increases if cooling is prolonged) by considering that [Ca2þ]cyt results on one hand from a calcium entry that depends on the speed of decrease of the temperature (dT/dt) and on the other hand from a calcium exit which only depends on the temperature T itself (Plieth, 1999). The activity of the channel responsible for the calcic impulse was recently measured by patch‐clamp in protoplasts prepared out of Arabidopsis mesophyll cells (Carpaneto et al., 2007). The cold activated channel is not identified. It has been suggested that the two‐pore channel 1, an aluminium sensitive‐calcium channel, could mediate the cold response. In transgenic tobacco BY‐2 cells expressing aequorin, aluminium was indeed shown to inhibit the Ca2þ increase triggered by cold (Lin et al., 2005). However, in Arabidopsis AtTPC1 is located to the vacuolar membrane. Besides the increase in [Ca2þ]cyt is identical in aequorin‐expressing tpc1–2 knockout and

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TPC1‐over‐expressing plants, which disproves a contribution of TPC1 to cold calcium signature (Ranf et al., 2008). The Arabidopsis antiport Ca2þ/Hþ CALCIUM EXCHANGER 1 (CAX1) could be involved in the regulation of the cold response. Tobacco overexpressing CAX1 from Arabidopsis are hypersensitive to cold, and this over‐ sensitivity is removed by increasing calcium of the medium (Hirschi, 1999). CAX1 is localized to the tonoplast (Cheng et al., 2003). The expression of CAX1 was induced in response to low temperature. Two T‐DNA insertion mutants, cax1–3 and cax1–4 were identified. Their tonoplast Ca2þ/Hþ antiport activity is reduced. The mutants showed no significant diVerences with respect to the wild type when analyzed for chilling or constitutive freezing tolerance. However, they exhibited increased freezing tolerance after cold acclimation, demonstrating that CAX1 plays an important role in this adaptive response. This phenotype correlates with the enhanced expression of CBF genes and their corresponding targets (KIN1, LTI78, and COR47) in response to low temperature (Catala et al., 2003). It should be noted that these calcium movements generated by cold will be at the origin of a transmembrane change of potential (depolarization of the plasma membrane) in the cells of A. thaliana and Helianthus annus. These changes of potential are not propagated as action potentials (Lewis et al., 1997; Krol et al., 2004). Anion channels are partly responsible for the cold‐induced plasma membrane depolarization. The opening of calcium channels of the plasma membrane induces depolarizing calcium current to the cell; the increase in the cytosolic calcium concentration would open anion channels, thus reinforcing the transitory depolarization of the plasmalemma (Lewis et al., 1997). The signalling role of this depolarization in cold indication has not been investigated. 4. Calcium and gene response In parallel to measurement of calcium concentrations in plants subjected to a cold shock, various authors sought the implication of calcium in the induction of cold responsive genes. The presence of calcium‐chelating agents such as EGTA, or of agents blocking calcium channels (La3þ or verapamil) prevents the cold‐acclimation of Medicago sativa cells (Monroy et al., 1993). This is to be correlated with the fact that in these cells, the presence of La3þ, verapamil and BAPTA (another chelating agent) strongly reduced the accumulation of cold responsive genes (Monroy and Dhindsa 1995; Monroy et al., 1993). These experiments were undertaken by maintaining the plants or cells in cold. However, a cold exposure of seedlings of A. thaliana of only 1 min is enough to engage the accumulation of LTI78 transcripts. This accumulation is inhibited by EGTA or La3þ (Nordin‐Henriksson and Trewavas, 2003). This highlights the role of calcium on gene response to cold. Accordingly, when an

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artificial calcium entry is created by the addition of a calcium ionophore (A23187), the expression of cold‐responsive genes is induced at control temperature (Monroy and Dhindsa, 1995). In Arabidopsis, the growth controlled by ABA (gca2) mutant is impaired in its calcium signature, at least in response to ABA and CO2. In this mutant the cold induction of XERO2/LTI30 is greatly reduced. Even though the protein encoded by GCA2 is not identified, it supports the idea that a calcium signature is necessary for gene induction in the cold (Chung and Parish, 2008). 5. Proteins activated by calcium a. Calmodulins. Several studies have shown that calmodulins are involved in gene induction by low temperature. For example, genes encoding calmodulins are known to be induced by the low temperatures (Braam and Davis, 1990; Van Der Luit et al., 1999). Moreover, W7, an inhibitor of calmodulins, inhibits the cold induction of KIN genes (Ta¨htiharju et al., 1997). However, this inhibitor also inhibits the calcium dependent protein kinases (see Section IV.B.1). Lastly, in A. thaliana overexpression of CAM3, a calmodulin isoform, resulted in the attenuation of the cold accumulation of COR transcripts (Townley and Knight, 2002). The results by Ta¨htiharju et al. (1997) suggest a positive role of calmodulins on gene response to cold while the results by Townley and Knight (2002) suggest a negative role of calmodulins. Possibly overexpressing calmodulin, a calcium‐binding protein, will result in the buVering of calcium signal, leading to the observed phenotype. Calmodulins act through target proteins and a number of calmodulin binding proteins have been identified. Some of these calmodulin target proteins could be relevant in the cold signalling pathways. Calcium/calmodulin protein kinases are regulated by calmodulin. Their involvement in cold signalling will be detailed in Section IV.B.2. Calmodulins can interact with catalase, and thus have a role in triggering ROS scavenging systems. Calmodulins can also interact with the endoplasmic reticulum Ca2þ‐ATPase and with plasma membrane Ca2þ‐ATPase, aVecting the cold calcium signature. Calmodulin can interact with some diacylglycerol kinases. Diacylglycerol is produced through phospholipase C activation in response to cold (see Section IV.C.). In addition, some transcription factors or nuclear proteins such as TGA3 (a basic leucine zipper transcription factor) or AtSR1 (a CGCG protein) have been shown to directly interact with calmodulins (reviewed by Yang and Poovaiah, 2003). b. Calcineurin B‐like proteins (CBL). Calcineurin B‐like proteins are calcium sensors that were first identified based on their significant similarity to calcineurin B and neuronal calcium sensors from animals. CBLs have

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EF‐hand domains that can act as calcium‐binding sites. In contrast to calmodulins that can interact with unrelated eVector proteins, CBLs specifically target a group of Ser/Thr protein kinases, called CIPK for Calcineurin B‐Like‐interacting protein kinase. A family of calcineurin B‐like genes was found in the genome of A. thaliana. AtCBL1transcripts accumulate in response to cold (Kudla et al., 1999). In cbl1 null mutant, the gene induction of CBF1, CBF2, Rd29A at 2 8C is quicker than in wild‐type plants. In contrast, overexpression of CBL1 induces the expression of these genes, even under non‐stressed conditions (Albrecht et al., 2003; Cheong et al., 2003). CBL1 thus plays a part in the fine regulation of the cold expression of the CBF regulon (Cheong et al., 2003). CBL1 has been shown to potentially interact with six CIPK (AtCIPK01, 07, 08, 17, 18, 24, Kolukisaoglu et al., 2004). The partner of CBL1 in the response to cold has not been identified. In japonica rice ‘‘Zhonghua 11’’, transgenic plants overexpressing OsCIPK03 showed significantly improved tolerance to cold. Under cold, OsCIPK03‐overexpressing transgenic plants accumulated significantly higher contents of proline and soluble sugars than the wild‐type. Putative proline synthetase and transporter genes had significantly higher expression level in the transgenic plants than in the wild‐type (Xiang et al., 2007). c. Calcium‐dependent protein kinases. For the sake of clarity, this part will be developed with the other phosphorylation mechanisms involved in the cold response (see Section IV.B.1.). d. Phospholipases C and D.

These enzymes are treated separately. See IV. C.

In summary, cytosolic calcium increase is a major response to a cold shock. The calcium increase shows a dependency on the rate of temperature decrease. A desensitization phenomenon characterizes the calcium response to cold. The central role of this calcium increase in cold signalling is shown by the fact that, when it is inhibited, the response to cold (gene induction, cold acclimation) is also inhibited. This central role is explained by the fact that the calcium, itself or through calmodulin or CBL proteins, can control many signalling eVectors (Fig. 18). B. PROTEIN KINASES/PROTEIN PHOSPHATASES

Radioactive pulse‐labelling of Arabidopsis cell suspension cultures and 2‐dimensional gel electrophoresis made it possible to visualize the dynamic changes of the phosphoproteome in response to short‐term (5 min or less) exposure to 4 8C (El‐Khatib et al., 2007; Fig. 19). These data highlight the fact that during cold exposure some proteins are phosphorylated whereas

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Calcium targets in relation with cold signalling.

Fig. 19. Cold induces major changes in phosphorylation status of Arabidopsis suspension cells. Cell submitted to 4 8C for 2 min or 5 min. Proteins were labelled with 32 Pi 2 min before protein extraction. From El‐Khatib et al., 2007.

others are dephosphorylated. The activation of protein‐phosphatases and protein‐kinases by cold has also been observed in Medicago sativa suspension cells subjected to a cold stress (Monroy et al., 1993). These changes in phosphorylation status can aVect gene induction. For example, wheat Wcs120 is a cold inducible gene. Nuclear extracts from non‐acclimated plants contain multiple DNA‐binding proteins that can interact with several cis‐elements within the Wcs120 promoter. In contrast, no

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DNA‐binding activity was observed in the nuclear extracts from cold‐ acclimated plants. These binding proteins might have a negative role on Wcs120 expression under control conditions. In vitro dephosphorylation of the cold acclimated nuclear extracts with alkaline phosphatase restored the binding activity. Accordingly okadaic acid, a potent inhibitor of protein phosphatases 2A and 1, markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that some unidentified proteins can, in their dephosphorylated form, bind cis‐elements of the Wcs120 promoter. Under cold acclimation these proteins become phosphorylated, thus relieving the inhibition of Wcs120 transcription (Vazquez‐Tello et al., 1998). We will now describe the diVerent kinase or phosphatase activities that underlie these phosphoproteome changes. 1. Calcium dependent protein kinases (CDPK) a. CDPK are activated in response to cold. The calcium dependent protein kinases are serine/threonine kinases activated by calcium. As already mentioned, W7, an antagonist of the calmodulins and CDPK, inhibits some cold-induced phosphorylation events in cells of Medicago sativa and reduces their capacity to develop a tolerance to freezing (Monroy et al., 1993). Treatment with W7 also reduces cold-stimulated gene expression in A. thaliana (Ta¨htiharju et al., 1997). This action of W7 could be a consequence of an inhibiting eVect on CDPK. The barley HVA1 gene is responsive to cold and ABA (Sutton et al., 1992). Chimeric gene constructs with the HVA1 promoter and the luciferase coding sequence were generated and expressed in maize leaf protoplasts. When protoplasts were co‐transformed with constitutively active mutants of two related CDPK, CDPK1 and CDPK1a, HVA1 promoter activity was induced in non‐stressed protoplasts at control temperature. Activation is not seen with a CDPK1 mutated in the kinase domain. When the protoplasts transformed with the HVA1::LUC and CDPK are also transformed with a constitutively active protein phosphatase 2C, the activation of HVA1 promoter is diminished (Sheen, 1996). In rice (O. sativa), the activity of a 56 kDa CDPK present in membranes is increased by a treatment of 18 h at 12 8C (Martin and Busconi, 2001). The activity of a CDPK of 47 kDa is increased in the membrane fraction of rice shoots (O. sativa) subjected to a cold stress (1 h, 5 8C) (Li et al., 2003). In the rice genome, 31 CDPK genes have been identified (Ray et al., 2007). Chilling temperatures induce the transcription of OsCPK13 (OsCDPK7) but down‐ regulates that of OsCPK17 (Wan et al., 2007). The accumulation of OsCDPK13 protein was stronger in cold‐tolerant rice varieties than in cold‐sensitive ones. Furthermore, OsCDPK13 gene expression and protein accumulation were

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enhanced in response to cold. OsCDPK13 protein accumulated in 2‐week‐old leaf sheath and callus, and became phosphorylated in response to cold. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines. Transgenic rice lines overexpressing OsCDPK13 had higher recovery rates after cold stress than vector control rice (Abbasi et al., 2004). b. CDPK could be regulated via calreticulin. Calreticulin is a major calcium‐sequestering protein found in the lumen of the endoplasmic reticulum of a wide variety of eukaryotic cells, including those of higher plants. During cold exposure, it has been shown that rice calreticulin protein CRO1 was shifted from a soluble fraction to an insoluble fraction. Phosphorylation of calreticulin increased after cold treatment. Over‐expression of calreticulin enhanced the activity of a 47 kDa CDPK that had been induced by cold treatment. During cold treatment, the 47‐kDa CDPK activity increases more in the cold sensitive rice variety ‘‘IR36’’ and the sense transgenic rice than it does in other varieties. These results suggest that the calreticulin is involved in the signalling pathway leading to response to cold stress, and that it acts by activating CDPK (Li et al., 2003). Interestingly, it was shown by co‐immunoprecipitation using anti‐ calreticulin antibody that calreticulin interacted with a Calreticulin interacting protein 1 (CRTintP1) in vivo in cold stressed leaves (Sharma et al., 2004). When rice lines overexpressing either CDPK13 or CRTintP1 were incubated at 5 8C for 3 days, leaf blades of both the sense transgenic and vector control rice plants became wilted and curled. These are classical cold symptoms in rice. When the plants were transferred back to non‐stress conditions after cold treatment, the leaf blades died but the sheaths remained green in the sense transgenic rice plants, showing a positive eVect of the overexpression. Accumulation of CDPK13 or CRTintP1 in the cold‐tolerant rice variety was higher than that in rice varieties that are intermediate in their cold tolerance. Accumulation of calreticulin and CRTintP1 was not detected in cold‐ sensitive rice varieties. This indicates a role of those proteins in cold resistance, and confirms the link between CDK and calreticulin in the cold response (Komatsu et al., 2007). c. PKC‐like activities and cold response. No consensus sequences for PKC have been found in plants. However PKC‐like activities have been detected. These PKC‐like activities in plants are thought to reflect the involvement of CDPK. It is true that plant CDPKs share many characteristics of PKC activities, such as the activation by calcium and lipids. Such PKC‐like activities have been found in response to cold. For instance, in wheat, Western blot analysis using antibodies directed against protein kinase C (PKC)

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isoforms showed that a PKC homolog of 84 kDa is selectively translocated into the nucleus in response to low temperature (Vazquez‐Tello et al., 1998). 2. Ca2þ/calmodulin‐dependent protein kinases In pea, Western blot analysis showed that a protein, immuno‐counterpart of a Ca2þ/calmodulin‐dependent protein‐kinase of 72 kDa (PsCCaMK), accumulated at 4 8C, from 6 h after cold stress with a maximum at 24 h. This protein is present in the nucleus, where it can phosphorylate a protein of 40 kDa (p40). p40 is a DNA binding protein that can interact in vitro in its dephosphorylated form with a cis‐element of the CaM5 promoter of A. thaliana (Pandey et al., 2002). Therefore Ca2þ/calmodulin‐dependent protein‐kinase could be involved in the response to cold and modulate the level of calmodulin. 3. MAPK module The ‘‘mitogen‐activated kinases’’ (MAPK) are activated by phosphorylation under the action of MAPK‐kinases (MAPKK) which themselves are phosphorylated by MAPKK‐kinases (MAPKKK). In A. thaliana, expression studies showed a simultaneous accumulation of transcripts of AtMEKK1 (a MAPKKK), of AtMPK3 (a MAPK) and of AtPK19 (a S6‐kinase) (Mizoguchi et al., 1996) in the first hour of cold exposure. Using a 2‐hybrid assay, interactions have been demonstrated between AtMEKK1 and AtMKK2 (a MAPKK), between AtMKK2 and AtMPK4 (a MAPK) (Ichimura et al., 1998). In vitro and in vivo, AtMKK2 can target AtMPK4 and AtMPK6 directly. In Arabidopsis protoplasts, MKK2 is specifically activated by cold and saline stresses, and by MEKK1. Plants overexpressing MKK2 showed a constitutive activity of MPK4 and MPK6, together with a constitutive induction of cold‐stress responsive genes such as CBF2, CBF3, RAV1, FAD8, etc. Plants overexpressing MKK2 also showed a greater tolerance to freezing. In mutant mkk2 plants, MPK4 and MPK6 are not activated. These null plants are hypersensitive to a cold stress. These data suggest that MKK2 activates MPK4 and MPK6 and that these activations are required for the acquisition of cold tolerance (Teige et al., 2004). The fact that MAPK modules are upstream of the CBF pathway has also been suggested in wheat (T. aestivum L.). Ethylene‐responsive factor 1 (TaERF1) encodes a putative protein with a conserved DNA‐binding domain and a conserved N‐terminal motif (MCGGAIL). TaERF1 has a putative phosphorylation site (sequence: TPDITS) in the C‐terminal region. Transcription of the TaERF1 is induced by low temperatures. Two‐hybrid tests revealed that TaERF1 interacted with TaMAPK1 protein kinase. Deletion of the N‐terminal motif enhanced the interaction of TaERF1 with

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TaMAPK1. Interestingly, TaERF1 was capable of binding to the CRT cis‐ element in vitro, and of trans‐activating reporter gene expression in tobacco (N. tabacum L.) leaves. Furthermore, overexpression of TaERF1 in Arabidopsis activated the induction of COR genes (Xu et al., 2007). MAPK modules are also involved in the response to moderate low temperature. In rice, OsMEK1 encode a MAPKK. OsMEK1 transcripts are induced in anthers by a 12 8C treatment for 48 h. OsMEK1 induction is also observed in shoots and roots of seedlings treated at 12 8C for 24 h. No OsMEK1 induction is observed in seedlings treated at 4 8C. A 2‐hybrid approach shows that OsMEK1 interacts with OsMAP1, a MAP‐kinase. This gene is also induced at 12 8C (Wen et al., 2002). 4. STY kinase In peanut (Arachis hypogea), transcripts and the activity of a kinase with double specificity Serine/threonine kinase and Tyrosine kinase, increase in response to cold. Activation takes place at 12 h of cold, which could indicate that this kinase does not take part in the early cold response but is involved with the set up of the acclimative process (Rudrabhatla and Rajasekharan, 2002). 5. CIPK The involvement of CIPK in cold response has been detailed in Section IV. A.5.b. 6. Protein phosphatases In Brassica juncea seedlings, an in vitro phosphorylation assay on a crude protein extract from seedlings grown at room temperature revealed the phosphorylation of a 55 kDa polypeptide. This phosphorylation was inhibited by PKC inhibitors (H‐7 and staurosporine) and enhanced by the PKC activator, phorbol 12‐myristate 13‐acetate (PMA), a diacylglycerol (DAG) analogue. Interestingly, the 55 kDa peptide was not phosphorylated in seedlings exposed to 4 8C for 10 or 30 min prior to protein extraction. This suggests the activation of a phosphatase pathway by cold treatment (Deswal et al., 2004). In parallel to kinase activities, protein phosphatases are also involved in the changes of phosphoproteome triggered by low temperature stress. The Arabidopsis PP2C‐type phosphatase AP2C1 (At2g30020) is a MAPKinase Phosphatase. In vivo, AP2C1 can interact with MPK4 and MPK6 (Schweighofer et al., 2007). These MAP kinases have been found to be activated during cold stress. Besides, AP2C1 is highly induced after 3 h at 4 8C (Winter et al., 2007). The involvement of AP2C1 in cold response needs to be evaluated. In Arabidopsis, the expression of two others 2C Ser/Thr Protein Phosphatases, AtPP2CA (At3g11410) and ABI1

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(At4g26080), was induced by low temperatures: treatment at 4 8C led to an increase in AtPP2CA transcript level until it reached a steady state level after 12 h (at least till 5 days at 4 8C) whereas the expression of ABAI1 exhibited a transient increase with a maximum at 12 h followed by a rapid decrease. The freezing tolerance of transgenic antisense plants against PP2CA increased by 3 8C after one day exposure to cold‐acclimation treatment while such a freezing tolerance is only acquired after 4 days of cold‐acclimation in the wild‐type plants. Furthermore, the expression of cold‐induced genes such as LTI78, RAB18 and RCI2A was detected earlier in transgenic antisense plants against PP2CA compared to wild‐type plants while no diVerence was reported for CBF1 (Ta¨htiharju and Palva, 2001). RAB18 and RCI2A are cold‐inducible genes supposedly non‐targets of the CBF pathway but dependent on ABA (La˚ng and Palva, 1992). These results suggest that AtPP2CA is a negative regulator of cold responses, and that it acts on a CBF‐independent pathway (Ta¨htiharju and Palva, 2001). The authors suggest that it could act on an ABA‐dependent pathway. While most of the cold response seems to occur via an ABA‐independent pathway (see Section II.C.2.), an ABA‐ dependent one exists. It is normally down‐regulated by PP2CA but can be detected in plants where this negative regulator is mutated. Chung and Parish (2008) show that in the abi1 mutant, cold treatment (4 8C for 24 h) still leads to significant induction of XERO2/LTI30, a CBF target gene, which supports the idea that protein phosphatases 2C are not involved in regulation of the CBF pathway in Arabidopsis. However, the CBF pathway might be controlled by other kinds of protein phosphatases. In Arabidopsis plants transformed with a construct containing several copies of the CRT/DRE element from COR15A fused to GUS, the cold‐induced reporter expression was inhibited by okadaic acid at 1 nM, suggesting the involvement of protein phosphatases 2A (Kim et al., 2002b). C. PHOSPHOLIPASES AND PHOSPHOLIPIDS

1. Phospholipase D Phospholipases D (PLD) catalyze the hydrolysis of structural phospholipids such as phosphatidylcholine, phosphatidylethanolamine or phosphatidylglycerol into phosphatidic acid. In the presence of primary alcohols, PLDs have the ability to catalyze the transphosphatidylation reaction that will lead to the formation of phosphatidylalcohol preferentially to phosphatidic acid. Tertiary alcohols are not substrates of the transphosphatidylation reaction (Fig. 20A). In A. thaliana suspension cells, it has been shown that cold exposure leads to the accumulation of phosphatidylbutanol or phosphatidylethanol in the

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Fig. 20. Involvement of PLD in the response to cold in Arabidopsis suspension cells. (A) PLD can catalyze the transphosphatidylation reaction; (B) PtdBut is increased after 10 min at 0 8C; (C) Inhibition of PLD‐produced PA results in a decrease in gene induction after 4 h at 4 8C. RNA levels were monitored by semi‐ quantitative PCR. Unpublished results from C. Sirichandra’s M2 thesis.

presence of primary butanol and ethanol, respectively (Fig. 20B; Ruelland et al., 2002; Vergnolle et al., 2005). This clearly indicates that a PLD is activated by cold. This activation was fast, occurring in the first 10 min of cold exposure. Accumulation of transcripts was studied in presence of primary or tertiary alcohols. This allowed the identification of genes, e.g., LTI78, LTI30, HVA22, whose response to cold was inhibited by primary alcohol, but not by tertiary alcohols: these genes are likely to be regulated by

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an abrupt temperature downshift via PLD‐produced phosphatidic acid. Interestingly one of these genes was CBF3, suggesting the CBF pathway is downstream of PLD activation (Fig. 20C; Vergnolle et al., 2005). In A. thaliana plants, PLDs have been shown to be involved in the response to cold (Rajashekar et al., 2006). In Arabidopsis, 12 PLD isoforms have been identified. In plants knocked‐out in PLD1, the peak of accumulation of CBF1 takes place after 8 h of cold, compared to 2 h for wild‐type plants. However, the accumulation of raYnose after one week of cold is higher in the defective plants than in the wild-type plants (Rajashekar et al., 2006). Further studies will be necessary to unravel the exact role of PLD1 in plant responses to cold temperatures. In A. thaliana, the gene encoding PLD is induced by cold. After cold acclimation, plants knocked‐out for PLD are more sensitive to freezing while plants overexpressing PLD show a higher tolerance to freezing. Without cold acclimation, however, PLD knocked‐out and wild‐type plants behaved similarly in terms of freezing tolerance. However, the deletion in PLD gene does not aVect the induction of COR genes or the accumulation of proline in plants exposed to cold. On the other hand pld null plants have an increased sensitivity to H2O2 while plants overexpressing PLD have a decreased sensitivity to H2O2 (Li et al., 2004). This suggests that PLD is induced by cold and that this induction is necessary for resistance to cold‐ induced ROS. This role of PLDs in cold signalling should not to be confused with the role of PLD in freezing and post freezing. In A. thaliana, it has been shown by mass spectrometry that freezing at a sub lethal temperature induced a decline in many molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, while it induced an increase in phosphatidic acid. In plants where PLD1 was knocked out, the phosphatidylcholine content dropped only half as much as in wild‐type, and PA levels rose only half as high as in wild‐type plants. Therefore it appears that PLD1 is activated during freezing and is involved in freeze‐induced membrane breakdown. During freeze exposure, PLD1 is not activated to trigger a responsive pathway leading to tolerance to freezing. On the contrary, this activation leads to membrane breakdown. Such membrane breakdown is one of the many deleterious eVects of freezing on cells. Accordingly, Arabidopsis plants that are deficient in PLD1 have improved tolerance to freezing. As suggested by the authors, the greater loss of phosphatidylcholine and increase in phosphatidic acid in wild‐type plants as compared with PLD1‐deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild‐type plants (Welti et al., 2002).

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U73122, edelfosine

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Fig. 21. Involvement of PLC in the response to cold in Arabidopsis suspension cells. (A) PLD/DGK pathway; (B) PLC is very rapidly activated as seen through PA production. U73122, edelfosine, and DAGKI inhibit the PLC/DGK pathway; (C) Inhibition of PLC by U73122 (60 M) results in a decrease in gene response after 4 h at 4 8C. U73343 is the inactive analogue of U73122. RNA levels were monitored by semi‐quantitative PCR. Unpublished results from C. Sirichandra’s M2 thesis.

2. Phospholipase C Phospholipases C catalyse the hydrolysis of phosphatidylinositol‐4,5‐ bis‐phosphate into diacylglycerol (DAG) and inositol‐tris‐phosphate (InsP3) (Fig. 21A).

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In B. napus L. leaves fed with 3H‐inositol, an exposure to 0 8C led to an immediate increase in the amount of radioactivity incorporated in inositol‐ phosphates in parallel with a decrease of the radioactivity incorporated in phosphatidylinositol‐monophosphate and phosphatidylinositol‐bisphosphate (Smolenska‐Sym and Kacperska, 1994). These results make a compelling argument for the early activation of a phospholipase C in response to cold. In A. thaliana suspension cells, the accumulation of InsP3 was detected a few second after low temperature exposure. The PLC activity was coupled to a diacylglycerol kinase activity that phosphorylates diacylglycerol into phosphatidic acid. Inhibitors of the phosphoinositide‐specific PLC such as neomycin or U73122 led to a reduced amount of phosphatidic acid produced by diacylglycerol kinase (Fig. 21B; Ruelland et al., 2002). In addition, it was shown that the molecular species of diacylglycerol kinase‐produced phosphatidic acid were identical to those of phosphatidylinositol. This establishes that the diacylglycerol phosphorylated by diacylglycerol kinase comes from a phospholipase C that hydrolyzes phosphonositides and that no phosphatidylcholine‐dependent phospholipase C is in play. Besides an inhibitor of DAGK, DAGKI, inhibited the production of PA via the PLC pathway (Fig. 21B; Vaultier et al., 2006). It was also shown that EGTA and La3þ, inhibitors of calcium channels, blocked PLC activation, demonstrating that it was dependent on calcium entry (Ruelland et al., 2002). The activation of PLC is clearly an event of a signalling cascade leading to induction of cold responsive gene expression. Using U73122, an inhibitor of PLC, 58 genes were identified that were regulated by an abrupt temperature downshift via PLC activity (Vergnolle et al., 2005). Amongst those genes are transcription factors MYB73, CZF1, and TCP (At1g35560). The CBF genes do not seem to be aVected by PLC inhibition (Vergnolle et al., 2005). The challenge arising from these studies is to identify the molecules generated by the PLC pathway that have signalling activity. PLC generates DAG and InsP3. DAG can be phosphorylated into phosphatidic acid. In response to cold, no clear targets of the phospholipase‐produced lipid signals have been identified. However, some proteins activated by DAG or PA have been identified, and are known to be actors of signalling cascade. Some of them could be relevant in cold signalling pathways. It is the case of a PP2A regulatory subunit (At1g25490) or a SNF1‐related kinase (At1g60940), two PA-interacting proteins (Testerink et al., 2004). It has been shown that PLD1‐derived PA interacted with ABI1 (Zhang et al., 2004c). This 2C Ser/Thr protein phosphatase is induced by cold (see Section IV.B.6.). The exact role of ABI1 in the response to cold and its putative link to PA has not yet been deciphered. A carrot (Daucus carota) CDPK was

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shown to be activated by PA (Farmer and Choi, 1999). In Arabidopsis, 30 ‐ phosphoinositide‐dependent kinase‐1, PDK1, is also activated by PA (Anthony et al., 2004). These data make a compelling argument for PA to be able to regulate phosphatases and/or kinases in the cold response. Even though no canonical DAG are found in plants, DAG could be a signalling molecule. In addition, PLC also generates InsP3, which can be subjected to a series of phosphorylation and dephosphorylation reactions, leading to diVerent molecules that could also have a signalling role in the response to cold (Fig. 22). Lithium inhibits inositol‐1‐phosphatases and it has been shown that lithium inhibited the cold‐induced calcium increase in ‘‘microdomains’’ corresponding to the cytosolic face of the vacuole (see Section IV.A.1). This suggests that PLC‐released InsP3 might be involved in the calcium response during cold stress. For a review on InsP3 in plants, see Krinke et al., 2007. In the fiery1 mutant, the gene encoding an inositol polyphosphate 1‐phosphatase has been inactivated (Xiong et al., 2001). In this mutant, the cold responsive transcripts of LTI78, KIN1, COR15A, COR47 accumulate even under unstressed conditions. In addition, in response to cold, the accumulation of the CBF2 and CBF3 transcripts is significantly higher in this mutant than in the wild‐type. The inositol polyphosphate 1‐phosphatase encoded by FIERY1 thus has a negative eVect on the response to cold. This implies that a polyphosphoinositol having a phosphate in position 1 has a positive role in the accumulation of cold responsive transcripts. It confirms that a PLC activity producing InsP3 is involved in the response to cold. DAG

DAGK

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PLC PtdIns(4,5)P2 Ins(4.5)P2

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Ins (4)P

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Ins(3,4)P2

InsP5

InsP6

InsP6P

Fig. 22.

The diVerent molecules generated through the PLC pathway.

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When the Arabidopsis inositol polyphosphate 6‐/3‐kinase (AtIpk2) gene was constitutively overexpressed in tobacco (N. tabacum), the resulting plants exhibited improved tolerance to freezing with no acclimation period. Most transgenic plants resumed normal growth after the 2 h treatment at
20 8C, but only very few wild‐type plants did so. In the overexpressing plants, some stress responsive genes, such as NtERD10C (LEA family), were accumulated when compared to wild‐type plants in control conditions. In response to NaCl, some induced genes were more induced in the overexpressing mutants than in the wild‐type plants (Yang et al., 2008). 3. Phosphatidylinositol (4,5)‐bisphosphate and response to cold In wild‐type Arabidopsis roots, 1 h at 0 8C led to a 4‐fold increase in phosphatidylinositol‐4,5‐bisphosphate (Williams et al., 2005). In Arabidopsis, the SAC9 gene encodes a phosphoinositide‐phosphatase. In control conditions, the roots of a sac9 null mutant are characterized by a higher level (4‐fold) of phosphatidylinositol‐4,5‐bisphosphate together with a higher level (3‐fold) of InsP3. No increase of phosphatidylinositol‐4,5‐bisphosphate is seen in sac9 shoots compared to the wild‐type. However SAC9 has a role in shoots since SAC9 shoots have a phenotype characterized by lower growth and anthocyanin accumulation. In addition, in the sac9 mutant there was an accumulation of CBF1, CBF2, and COR15 transcripts in control growth conditions. It is tempting to speculate that this accumulation is either due to the increase of phosphatidylinositol‐4,5‐bisphosphate or to the increase of InsP3. The role of InsP3 would be consistent with a cold triggered activation of PLC. However, phosphatidylinositol‐4,5‐bisphosphate can have a signalling role. Many proteins possess domains such as the pleckstrin homology domain that can bind this lipid (van Leeuwen et al., 2004). Furthermore, many PLDs are activated by phosphatidylinositol‐4,5‐bisphosphate, and the eVect of the sac9 phenotype on gene response could be due to PLD. However, we cannot exclude the possibility that other enzymes activated by phosphatidylinositol‐4,5‐bisphosphate are responsible for this phenotype. In summary, an increase in cytosolic calcium is a major event that occurs very early after an abrupt decrease in temperature. Calcium is able to regulate the activity of many signalling components, including phospholipases and protein kinases, ultimately leading to the triggering of cold‐induced gene expression or repression. However, many questions remain. The proteins regulated by phospholipase‐produced messengers in the response to cold are not yet identified. It is very likely that amongst them would be found protein kinases or protein phosphatases. As for protein kinases, the genes whose expression they control are generally unknown (Fig. 23). Further, the

COLD SIGNALLING AND COLD ACCLIMATION IN PLANTS Ca2+

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Cold

PI(4,5)P2

DAG PA DAGK

PLC

PA

PC PLD

InsP3 ? ?

? MAPKKK

Ca2+

CDPK

MAPKK

CIPK

MAPKK ?

CBF-dependent pathway

?

CBF-independent pathway

Fig. 23. The missing bridges in cold signalling. Exposure to cold will trigger calcium entry into plant cells. Calcium will activate a PLC activity that will produce InsP3 and DAG. DAG is phosphorylated into PA by a DGK. In parallel, PLD activity will also lead to PA production. In response to cold, diVerent phosphorylation/dephosphorylation events will lead to changes in the phosphoproteome. Some of the kinases or phosphatases activated can be activated by calcium and/or lipid. However, the very kinases or phosphatases activated by PA or DAG in response to cold are not yet identified. Those signalling events will lead to gene induction through CBF‐dependent or ‐independent pathways.

relationship between the MAPK module and CDPK is not known. Finally, an important fact concerning cold signalling is that those signalling events occur in a special time and in a special place. Building the spatiotemporal web linking the signalling actors identified is a challenging goal.

V. SUMMARY AND CONCLUSION Cold is a major stress factor limiting the productivity and distribution of many plant and crop species. Plants may be exposed to cold stress on a daily or on an seasonal/annual basis. A drop in ambient temperature results in a number of physiological responses, including disturbing the metabolic equilibrium of the cell, leading, inter alia, to an accumulation of ROS, stabilization of RNA secondary structures and the rigidification of membranes. In some species exposure to chilling temperatures is perceived as a

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pre‐emptive signal to trigger a response leading to freeze‐tolerance. Amongst the changes contributing to freeze‐tolerance are the accumulation of AFPs, the accumulation of dehydrins and the accumulation of compatible solutes such as various sugars and proline. Because plants acclimate to cold, this means that the lowering of temperatures has been perceived by plant cells. While the specific mechanisms through which plants perceive cold are unclear, and were thus not the main subject of this review, cold‐ induced rigidification of membranes and the cold‐induced phosphate limitation have been proposed to be perception events (Stitt and Hurry, 2002; Vaultier et al., 2006). Once perceived, many of the subsequent acclimation responses rely on changes to the cellular transcriptome, meaning that the signal must be transduced to the nucleus. The best documented genetic pathway leading to changes in the transcriptome in response to cold is the ICE1‐mediated CBF pathway. The CBFs are themselves induced by cold. Their cold response is dependent on the activitation of the ICE protein. The identification of mechanisms leading to ICE1 activation is a challenging goal in the research in plant cold response. Even though the ICE/CBF pathway appears to be the major pathway activated in response to cold, others pathways are likely to be involved. However these ICE/CBF‐independent pathways remain poorly documented. Between cold perception and activation of genetic pathways, the transduction events take place. The major and very early transduction event is calcium entry from apoplastic space. The increase in calcium will then activate diVerent calcium and/or calmodulin dependent activities. Amongst such activities are protein kinases and phospholipases. It is likely that other transduction actors are still to be discovered. The relationships between the diVerent transducing pathways and actors are still to be more documented. While the aim of this review was to be comprehensive in scope, not all aspects of cold perception and signalling could be covered. For example, in A. thaliana, microarray‐based analyses have led to the identification of 19 cold regulated microRNAs (Liu et al., 2008; Zhou et al., 2008). One of these, miR398, was found to target two Cu/Zn superoxide dismutase coding genes and down‐regulation of miR398 led to improved tolerance to oxidative stress (Sunkar et al., 2006). Cold responsive miRNAs have also been found in Populus trichocarpa (Lu et al., 2008) and they are emerging as a class of gene expression regulators, acting by post‐transcriptional degradation or translational repression and understanding the role of microRNAs in cold acclimation and in crosstalk with others pathways is a challenging goal for researchers in plant signal responses. Furthermore, while much is now known of the changes in the transcriptome that occur during the early phases of cold acclimation, including eVorts to define the regulons of many

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important cold‐induced transcriptional activators, little is yet known of the additional protein factors involved in mediating recruitment of these trans‐acting factors to the promoters as well as initiating their subsequent removal or inactivation. Finding the components that comprise these transcriptional complexes will be one key future objective in this field, as eVorts are made to fine‐tune enhanced cold‐tolerance in desirable crops as well as in developing our understanding of how diVerent signalling pathways, such as cold and drought or osmotic stress, are integrated by the plant. Similarly, we still know relatively little about how cold is perceived by plants. What are the initiators of the cold acclimation signal? It seems likely that there are more than one and that ‘‘cold’’ is sensed not only at the plasma membrane but also in the organelles and perhaps the vacuole. Without this knowledge to ‘‘anchor’’ the various signal transduction pathways at both ends, unravelling these complex signalling networks (both identifying the many components and placing them in a correct temporal network) will be diYcult and time‐consuming. Lastly, much of what we now know of the cold response of plants is based on knowledge from short‐term experiments at low positive temperatures lasting only hours to days. While these short‐term responses are important and realistically cover many of the stress situations faced by plants in the field, it is also true that for many plants, including important winter crops such as wheat, the ability to over‐winter means that they must tolerate prolonged cold, numerous freeze/thaw cycles and be able to undergo second‐phase hardening in response to freezing temperatures. Understanding these longer‐term responses will be greatly enhanced by the increasing number of plant genomes that becoming available, which includes winter hardy perennial species, but it will also require a dedicated eVort to integrate transcriptome changes with changes of the proteome and the associated adjustments to cellular metabolism because long‐term survival and recovery from stress requires functional adjustments to metabolism. Developing platforms to integrate this information for a range of plant species with diVerent growth habits will be a challenging task but will bring with it much deeper insight into cold‐perception, signalling and plant survival in the field.

ACKNOWLEDGMENTS We thank Profs. Bressan, Muench, Plieth and Salinas for allowing us to use figures from their work. We thank Prof. F. Moreau for careful reading of the manuscript. Unpublished results in Figs. 20 and 21 are taken from Caroline Sirichandra’s M2 thesis.

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Genome Evolution in Plant Pathogenic and Symbiotic Fungi

GABRIELA AGUILETA,* MICHAEL E. HOOD,{ GUISLAINE REFRE´GIER,* AND TATIANA GIRAUD*

*Ecologie, Syste´matique et Evolution, CNRS, Universite´ Paris‐Sud, F‐91405 Orsay cedex, France Tatiana.Giraud@u‐psud.fr { Department of Biology, McGuire Life Sciences Building, Amherst College, Amherst, MA, USA

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Evolution of Genome Size, Content and Organization . . . . . . . . . . . . . . Chromosomal Rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene Clusters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppression of Recombination Around MAT Loci . . . . . . . . . . . . . . . . . . . . . . Evolution of Gene Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rapidly Evolving Genes and Genes Evolving Under Positive Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Horizontal Gene Transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mixing Different Genomes: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transposable Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Telomeres. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison with the Genomics of Pathogenic Bacteria. . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 49 Copyright 2009, Elsevier Ltd. All rights reserved.

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0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(08)00603-4

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ABSTRACT Approximately 100,000 species of fungi have been described so far, of which, a high percentage obtain nutrients by living in close association with other organisms (mainly plants), being pathogens or symbionts (i.e. commensalists or mutualists). At the genomic level, an association between broad‐scale genetic changes and the emergences of the parasitic and symbiotic life style in fungi has been proposed. Although comparative genomic studies in fungi are still in the early stages, they have enormous potential to improve our understanding of how such life styles evolve. In this chapter, we review the main characteristics of genome evolution in fungi, particularly in species that are pathogenic or symbiotic with plants, by focusing on the mechanisms involved in host interactions. We address the following topics in relation to the pathogenic and symbiotic lifestyles in fungi: the evolution of genome organization, chromosomal rearrangements, evolution of gene families and clusters, suppression of recombination around mating type loci, rapidly evolving genes, horizontal transfer, hybridization, transposable elements, telomeres, introns, mitochondrial genomes, and we finally compare genome evolution between pathogenic bacteria and fungi.

I. INTRODUCTION Approximately 100,000 species of fungi have been described so far, of which, a high percentage obtain nutrients by living in close association with other organisms, mainly plants. Many fungi are pathogenic and can lead to severe economic losses due to infected crops. Other associations are commensal or particularly mutualistic symbioses, which are beneficial to the host organism, including the mycorrhizal fungi that colonize the roots of many important crops and forest trees. These mutualistic fungi improve the growth of the host plants by facilitating the uptake of nutrients. Parasitic and symbiotic species are found interspersed with saprophytes in fungal phylogenies (Berbee, 2001), suggesting that transitions between these life history strategies have occurred repeatedly within the fungal kingdom. Moreover, there are numerous cases where the relative harm and benefit to the host plant is largely context dependent (e.g. Clay et al., 1993). To be a pathogen or symbiont, a fungus has to overcome the numerous physical, cellular, and molecular barriers presented by the host. To persist, they must enter another organism, then grow and replicate using nutrients from host tissues, avoid host defenses, and eventually produce propagules that lead to the infection of more than one additional new host. At the genomic level, three genetic mechanisms have been proposed to be associated with the emergences of the parasitic and symbiotic life style in fungi. First, parasitism and symbiosis are associated with the evolution of novel genes. Such genes often have specific roles during host infection and arise by horizontal gene transfer or gene duplication followed by functional divergence. Second, parasitism

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and symbiosis are often associated with gene loss and deletions. These losses can involve genes required for free‐living saprophytism and those that allow the escape from detection by host defenses. Third, adaptations to the parasitic and symbiotic habits often result from diVerences in the regulation of gene expression. Further, living in such close association with a host provides the opportunity for coevolutionary dynamics and for the invasion of novel environments through the process of host shifts. These phenomena require rapid adaptations and a more continual evolutionary progression than in response to strictly abiotic factors, which may leave footprints in the genome such as positive selection. Gene gain, gene loss, and rapid adaptation can be investigated using comparative studies. In fungi, such genomic studies are still in the early stages, being limited to a few species, but there is enormous potential to improve our understanding of the molecular mechanisms involved in host–pathogen interactions as many more genomes sequences are under construction. In this chapter, we will review the main characteristics of genome evolution in fungi, particularly plant pathogenic and symbiotic species, focusing on the mechanisms involved in adaptations to host interactions. We will address the following topics in relation to the pathogenic and symbiotic lifestyles in fungi: the evolution of genome organization, chromosomal rearrangements, evolution of gene families and clusters, suppression of recombination around mating type loci, rapidly evolving genes, horizontal transfer, hybridization, transposable elements, telomeres, introns, mitochondrial genomes, and we will finally compare genome evolution between pathogenic bacteria and fungi. General information on the species cited in this review are given in Table I.

II. THE EVOLUTION OF GENOME SIZE, CONTENT AND ORGANIZATION Compared to animals and plants, fungi exhibit streamlined and gene‐dense genomes, with an average estimated size of
37 Mb, and ranging between 6.5 Mb for Pneumocystis carinii to 795 Mb for Scutellospora castanea (Gregory et al., 2007; see the Fungal Genome Size database: http://www. zbi.ee/fungal‐genomesize/N ¼ 762). Gene densities averaged across the genome vary significantly, between 37 and 61 genes per 100 kb (Galagan et al., 2005b). For comparison, average gene density is about 10 genes per 100 kb in Drosophila and about 1 gene per 100 kb in humans and other mammals (see http://www.ornl.gov/sci/techresources/Human_Genome/faq/compgen.shtml). Chromosome numbers also vary considerably in fungi, with the smallest number at 3, in the ascomycete Schizosaccharomyces pombe, and the largest

TABLE I General Information, When Available, about Each Fungal Species Cited in the Review: Genome Statistics, Life Style, and Taxonomy Genome

Size (Mb)

Approx. gene number

Chromosome number (n)

Sequencing statusa

Life style/habitat

Ascosphaera apis Ashbya gossypii Aspergillus flavus Aspergillus fumigatus

24 9.2 36.8 29.4


8,092 1,443 13,071 9,900

7 12 8

Incomplete Complete Complete Complete

Ascomycete Ascomycete Ascomycete Ascomycete

Aspergillus niger Aspergillus oryzae Blastocladiella emersonii

33.9 36.7

11,200 12,074

14 12

Botrytis cinerea Candida albicans Candida glabrata Cryptococcus neoformans Debaryomyces hanseii

30 14.9 12.3 20 12.2

16,448 12,015 5,300 6,500 6,900

8 13 14 7

Complete Complete EST data: 16,985 Complete Complete Complete Complete Complete

Bee pathogen Plant pathogen Weak pathogen Opportunistic/ pathogen Weak pathogen Domesticated Aquatic

Ascomycete Ascomycete Ascomycete Basidiomycete Ascomycete

Giberella/Fusarium graminearum Kluyveromyces lactis Kluyveromyces waltii Laccaria bicolor

36

12,000

4

Complete

10.6 10.7 65

5,300 5,700 16,100

6 8 10, at least

Complete Complete Complete

Magnaporthe grisea Malassezia globosa

38 9

11,000 4,285

7

Complete Complete

Neurospora crassa

39

10,000

7

Complete

Plant pathogen Human pathogen Human pathogen Human pathogen Halotolerant methylotrophic Plant pathogen/ necrotroph Nonpathogenic Nonpathogenic Symbiont (mycorrhizal) Necrotroph Human pathogen (dandruV) Non pathogenic, saprofitic and pathogenic

Phylum

Ascomycete Ascomycete Chytridiomycete

Ascomycete Ascomycete Ascomycete Basidiomycete Ascomycete Basidiomycete Ascomycete

Penicillium marneVei

26

3–8

Phycomyces blakesleeanus Pichia stipitis Pisolithus microcarpus

40 15.4

14,792 5,800

Puccinia graminis Rhizopus oryzae Saccharomyces cerevisiae Sclerotinia sclerotiorum Ustilago maydis Yarrowia lipolytica Agaricus bitorquis

88.64 45.3 12.1 38.33 20.5 20.5 34.2

20,567 17,467 6,300 14,522 6,900 6,700 1,100

16 23 6 13

Agrocybe aegerita Alternaria alternata Batrachochytrium dendrobatidis Botrytis allii Cochliobolus carbonum

33.6 23.72

Candida dubliniensis

16

Coccidioides immitis

29

Cochliobolus victoriae Colletotrichum lindemuthianum Coniophora puteana

8,794

9–11 20

Nearly complete Complete Complete Incomplete

Pathogenic

Ascomycete

Non pathogenic Xylose fermenting Symbiont (ectomycorrhizal) Plant pathogen Human pathogen Non pathogenic Plant pathogen Facultative biotroph Alcane utilizer Symbiont (ectomycorrhizal) Wood decomposer

Zygomycete Ascomycete Basidiomycete

Ascomycete Chytridiomycete Ascomycete Ascomycete

Draft assembly

Plant pathogen Animal pathogen Plant pathogen Saprophyte or plant pathogen Human oral pathogen Human pathogen

Ascomycete

Incomplete Incomplete Incomplete

Plant pathogen Plant pathogen Decomposer

Ascomycete Ascomycete Basidiomycete

Complete Draft sequence Complete Complete Complete Complete Incomplete Only the mitochondrial genome is available Incomplete Complete Incomplete Incomplete Nearly complete

10,600

6, at least 9–12

Basidiomycete Zygomycota Ascomycete Ascomycete Basidiomycete Ascomycete Basidiomycete Basidiomycete

Ascomycete

(continues)

TABLE I Genome Cordyceps aka Beauveria bassiana Crinipellis perniciosa Cryphonectria parasitica Fusarium oxysporum Gibberella fujikuroi Heterobasidion annosum Leptosphaeria maculans Melampsora medusae Melampsora occidentalis Microbotryum violaceum Mycosphaerella graminicola aka Septoria tritici Nectria haematococca

Neosartorya fischeri Neotyphodium ¼ Epichloe¨ Neurospora intermedia Neurospora tetrasperma Ophiostoma novo‐ulmi Ophiostoma ulmi Phanerochaete chrysosporium Phytophthora infestans

Size (Mb)

Approx. gene number

39

Chromosome number (n)

8–10

(continued) Sequencing statusa

Life style/habitat

Incomplete

Animal pathogen

Ascomycete

Plant pathogen Plant pathogen Plant pathogen Plant pathogen Plant pathogen Plant pathogen Plant pathogen Plant pathogen Plant pathogen Plant pathogen

Basidiomycete Ascomycete Ascomycete Ascomycete Basidiomycete Ascomycete Basidiomycete Ascomycete Basidiomycete Ascomycete

Saprophytes, rhizosphere colonizers or pathogens Human pathogen Plant pathogen or mutualist Plant pathogen Plant pathogen Plant pathogen Plant pathogen Wood degrader Plant pathogen

Ascomycete

33–50 40–50

17,735

34

12,000

15–16

25 41

11,395

11–13 18

Incomplete Incomplete Complete Incomplete Incomplete Incomplete Incomplete Incomplete EST data: 40,000 Complete

40

15,707

17

Complete

32.6 29–57

10,407

8 42

Complete Incomplete

10 8–10

Incomplete Incomplete Incomplete Incomplete Complete Incomplete

12

8,000–10,000 30 237

10,048

Phylum

Ascomycete Ascomycete Ascomycete Ascomycete Ascomycete Ascomycete Basidiomycete Oomycete

Incomplete

Pleurotus ostreatus Pneumocystis carinii Podospora anserina Puccinia recondita Pyrenophora trici‐repentis Schizosaccharomyces pombe Scutellospora castanea Stachybotrys chartarum Stagonospora nodorum ¼ Phaeosphaeria nodorum Ustilago hordei a

7.7 36

10,545

7

37.8 12 50

12,171 4,800

11 3

37.24

16,597

Incomplete Incomplete

Saprophyte (Ectomyccorhizal) Animal pathogen Saprophyte Saprophyte Plant pathogen Non pathogenic Arbuscular mycorrhizal Plant pathogen Plant pathogen

Ascomycete Ascomycota

Incomplete

Plant pathogen

Basidiomycete

Incomplete Complete Incomplete Complete Complete Incomplete

Basidiomycete Ascomycete Ascomycete Basidiomycete Ascomycete Ascomycete Zygomycete

Incomplete sequencing status may be either because there is an ongoing sequencing project that is not finished yet, or because there is no sequencing project yet started.

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number at 20, in the basidiomycete Ustilago hordei and the chytrid Batrachochytrium dendrobatidis (Gregory et al., 2007). Summary statistics, including gene numbers and densities, are highly dependent on the quality of genome annotation, which is often rather poor, and therefore should be considered with caution and frequently updated. Despite common themes in fungal evolution, fungi are strikingly diverse at the genome level, showing many instances of lineage‐specific evolution. Not only can the DNA sequences of genes be highly divergent but there are also important changes in the order and localization of homologous genes among genomes. For example, the comparison of the related ascomycetes Neurospora crassa and Magnaporthe grisea, which diverged around 200 MYA, reveals that their genomes have only 74% identity at the amino acid level (a distance comparable to that between mammals and fish) and virtually no synteny (conserved gene colinearity between genomes) (Dean et al., 2005). This is perhaps not so surprising, as the comparison of similarly distant species also reveals very few traces of synteny. Another instance of synteny loss between relatively closely related species involves members of the Aspergillus genus, whose average amino acid identity is 68% (Galagan et al., 2005a). Not surprisingly, when more distant genomes are compared, it becomes evident that there is a rapid breakdown of conserved synteny over relatively short periods of time, due mostly to genome rearrangements (translocations, inversions), which frequently occur in subtelomeric regions and are associated with repetitive sequence elements (Keely et al. 2005). Other mechanisms responsible for the pervasive loss of synteny include spontaneous segmental gene duplications (Koszul et al., 2004), occasional horizontal gene transfers (Andersson, 2005; Rosewich and Kistler, 2000b), as well as extensive diVerential gene loss following duplications. A dramatic example is found among yeasts where whole‐genome duplication and the subsequent lineage‐specific sequence losses may have contributed to speciation (Giraud et al., 2008a; Kellis et al., 2004; Scannell et al., 2006). However, regions of microsynteny can be found between closely related species, such as those within the Fusarium graminearum species complex (Malz et al., 2005), and even between more distant genomes (e.g. Magnaporthe grisea and Neurospora crassa), suggesting cases for a functional role of the conserved gene order (Hamer et al., 2001). Among fungi showing the signatures of adaptations to the pathogenic life‐ style there has been a tendency for reduced genome size (Yuen et al., 2003), for instance, by losing genes or whole metabolic pathways that are no longer necessary: e.g. Hemiascomycetes have lost the genes needed to survive on the carbon source galactose that was irrelevant in the within‐host environment (Hittinger et al., 2004). Adaptations to the life style can also be seen in the

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existence of lineage‐specific genes. The comparison between the genomes of a human pathogen, Aspergillus fumigatus, and two closely related but rarely pathogenic species, Neosartorya fischeri and Aspergillus clavatus, for instance, revealed that 8–14% of the genes were species‐specific, and that many of those were involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to their specific life styles, i.e. soil or mammalian host (Fedorova et al., 2008). Interestingly, these species‐specific genes were preferentially located in subtelomeric regions, and in ‘‘genomic islands’’ enriched in pseudogenes and transposable elements (Fedorova et al., 2008). Most pathogenic fungi have also experienced the expansion of specific gene families related to functions that facilitate the infection of the host. Typically, these genes include proteases, secreted proteins, secondary metabolites, cell wall degrading enzymes, major facilitator transporters, amino acid transporters, G‐protein‐coupled receptors, and enzymes for detoxification of antimicrobial agents. An example of how the expansion of specific gene families provides pathogenic potential to an organism is given by the genome of Penicillium marneVei, the only known pathogenic fungus of the Penicillium genus. Compared with its innocuous relatives, P. marneVei has experienced reductive genome evolution (17 Mb compared to
30 Mb in other Penicillium species), and its genome is rich in secondary metabolite genes and thioester‐ mediated non‐ribosomal protein synthesis (Yuen et al., 2003). Another fungus showing peculiar genomic features related to its pathogenic lifestyle is Ustilago maydis, a biotroph basidiomycete that parasitizes maize and depends on living tissue for proliferation and development. Not surprisingly, it lacks the pathogenicity genes present in more aggressive necrotrophic fungal pathogens. However, it possesses clustered secreted protein eVectors favoring the invasion of living tissue while minimizing host damages (Kamper et al., 2006). The genomic organization of a symbiotic fungal species can now be studied with the recent availability of the genome of the basidiomycete Laccaria bicolor (Martin et al., 2008). This genome of 65 million base pairs and 20,000 predicted genes is large relative to other fungi. Only 70% of predicted genes have homologs in other fungi, and its size can be partly accounted for by a large number of transposons and repeated sequences, as well as by the presence of large lineage‐specific multi‐gene families. In particular, there is evidence for the expansion of numerous protein gene families related to the functions that make possible the symbiotic relationship between Laccaria bicolor and its tree host Populus trichocarpa. In contrast, the genome of Laccaria bicolor shows a marked reduction in the gene families coding for plant cell wall degradation enzymes, while these families are well represented in the genomes of many fungal pathogens (Martin et al., 2008).

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III. CHROMOSOMAL REARRANGEMENTS One of the main mechanisms responsible for the evolution of genome organization is chromosomal rearrangement. The relatively small genomes of fungi contain chromosomes that are very diYcult to observe by conventional microscopy. However, the chromosomes are in fact small enough to separate by pulsed‐field gel electrophoresis, providing a powerful approach to quantify any number or size diVerences across related taxa. Intra‐specific polymorphisms in electrophoretic karyotypes are extremely common in both the ascomycete and basidiomycete phyla and have their origins in mutations associated with chromosome breakage or fusion and recombination between non‐homologous loci during meiosis or parasexual cycles (Zolan, 1995). Chiasma formation between copies of repetitive DNA, such as transposable elements, has been suggested as a primary mechanism for generating chromosome length variation (Fraser et al., 2005; Zolan, 1995). In several fungal pathogens, genetic variation created by chromosomal rearrangements has been reported to favor adaptation to novel hosts or nutritional environments (Larriba, 2004). For example, in the pathogenic yeast Candida albicans phenotypic mutants derived in vitro often exhibit altered karyotypes and at mutation frequencies varying between 105 and 102, depending upon the strain (Rustchenko, 2007). Specific alterations to chromosome structure of Candida albicans have been shown to be selected for in the animal host and to result in increased virulence (Chauhan et al., 2005). Also, extensive chromosome rearrangements in Candida dubliniensis relative to its pathogenic sister species Candida albicans, may be at the origin of the diminished pathogenicity in the former species compared to the latter, possibly due to karyotypic instability (Magee et al., 2008), although there is no direct evidence for this. The biased distribution of both avirulence genes and transposable element sequences into subtelomeric regions, as suggested by genome analysis of Magnaporthe grisea (Dioh et al., 2000), may indicate a functional relationship where frequent rearrangements enhance adaptive abilities (O’Sullivan et al., 1998). However, in other pathogenic fungi, such as Cryptococcus neoformans, extensive chromosomal rearrangements have been associated with no obvious beneficial change in phenotype (Boekhout and van Belkum, 1997). The presence of variation in chromosome structure within species or even within populations (Zolan, 1995) could be expected to cause sterility of certain crosses through the failure of meiosis. It has been proposed that karyotype evolution in sexual species of fungi is constrained by this eVect, whereas asexual species may frequently be more polymorphic, i.e. the ‘meiotic maintenance hypothesis’ for karyotype stability (Kistler and Miao, 1992). Karyotypic polymorphism is for instance frequent in the asexual species Fusarium

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oxysporum and Nectria haematoccoca (Kistler and Miao, 1992). However, a number of studies have since provided evidence to the contrary, of sexual fungi generating and maintaining structurally variable chromosomes through meiotic generations, including species of Stagonospora, Crinipellis and C. neoformans (Boekhout and van Belkum, 1997; Caten and Newton, 2000; Rincones et al., 2006). In some fungi, highly controlled forms of selfing (e.g. automixis with central fusion) can preserve balanced structural heterozygosity in chromosomes, such as in Microbotryum violaceum and Saccharomycodes ludwigii (Hood and Antonovics, 2004; Yamazaki and Oshima, 1996). Support for the meiotic maintenance hypothesis may come from Colletotrichum lindemuthianum, where the presence of high variable minichromosomes appears greater in sterile lineages (O’Sullivan et al., 1998). Chromosomal rearrangements may give rise to large regions of repeated DNA sequence either by duplicative translocations or aneuploid segregation following hybridization events (Fraser et al., 2005; Greig et al., 2002). The presence of redundant copies allows for contrasting evolutionary fates for paralogous genes sequences. In the case of subtilisins in fungal pathogens of insects, ancient duplications for these essential pathogenicity genes have been retained and diversified for more than 220 million years (Bagga et al., 2004). However, the genomic loss of one paralog is a more common fate of duplicated genes, and in the case of pathogenic fungi, the genes lost may be more likely to belong to common orthologous groups involved in response to environmental stresses (Wapinski et al., 2007).

IV. GENE CLUSTERS Some genes in the genomes are kept tightly linked despite pervasive chromosome rearrangements. Growing evidence indeed shows that in fungal genomes genes that interact in the same metabolic pathway tend to be clustered together (Keller and Hohn, 1997). DiVerent hypotheses about how such clusters evolved have been proposed. One idea involves the action of horizontal gene transfer (HGT) (Walton, 2000), where whole clusters of genes are passed between organisms, as it is likely to have occurred in fungi possessing the penicillin pathway, thought to be transferred from prokaryotes (Penalva et al., 1990). Another possibility suggests that selective pressure maintains the genes together, their clustering facilitating co‐expression and co‐regulation. Examples include the regulation of secondary metabolites, such as the tricothecene (Proctor et al., 1995), the aflatoxin/sterigmatocystin clusters (Payne et al., 1993; Woloshuk et al., 1994; Yu et al., 1996) and the epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes (Patron et al., 2007b).

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In fact, several studies show that in pathogenic fungi, the families involved in pathogenicity are frequently clustered, including genes encoding host‐ specific toxins and secondary metabolites, as well as the enzymes that synthesize them (Keller et al., 2005; Sidhu, 2002). In general, genes associated with a pathogenic lifestyle can be grouped into diVerent types, according to their nature and/or function (for a review see Soanes et al. 2007): cell surface receptors like the G‐protein‐coupled receptors (GPCRs) that bind exogenous ligands and participate in signalling cascades (Cuomo et al., 2007; Dean et al., 2005); secreted proteins, which constitute a diverse group of small peptides such as toxins, proteinaceous eVectors, hydrolitic and degrading enzymes (Hane et al., 2007; Machida et al., 2005; Xu et al., 2007); protein eVectors that suppress plant defenses and alter cellular metabolism (Hane et al., 2007; Kamper et al., 2006); and secondary metabolites such as non‐ specific and host‐specific toxins, among which key families involved in the biosynthesis of toxins include polyketide synthases (PKS), non‐ribosomal peptide synthesis (NRPS), hybrid PKS‐NRPSs and cytochrome P450. Recently, PKS‐NRPS hybrids were discovered and a phylogenetic analysis suggests a single origin within ascomycetes by the fusion of a PKS and a NRPS (Bohnert et al., 2004; Kroken et al., 2003). Interestingly, hybrid PKS‐ NRPS genes have been found to encode the avirulence gene ACE1 in M. grisea, which unlike most avirulence genes is not secreted (Collemare et al., 2008); another hybrid PKS‐NRPSs has been identified in F. graminearum (GaVoor et al., 2005). All secondary metabolites (including antibiotics, potent toxins and ergot alkaloids) are produced by a few common biosynthetic pathways. The most abundant are polyketides (e.g. aflatoxin, a potent toxin with immuno suppressing eVects), which are synthesized by PKS genes: many potent toxins are polyketides used by pathogenic species such as Aspergillus, Penicillium, Fusarium, or Stachybotrys. NRPS is another important source of bioactive secondary metabolites in fungi (Reiber et al., 2005), also related to the pathogenic fungal life‐style. This key mechanism is produced by NRP synthetases, which are large multi‐functional enzymes that contain domains for adenylation, thiolation (or peptydil carrier protein PCP) and condensation. Genes encoding NRP synthetases are clustered and are typically co‐ expressed, as in Aspergillus spp., where a linked regulatory gene called LeaA, encoding a methyltransferase, is involved in secondary metabolite gene cluster regulation (Stack et al., 2007). Interestingly, some pathogenicity clusters producing host‐specific secondary metabolites are located in conditionally dispensable or supernumerary chromosomes, as in Nectria haematococca (Han et al., 2001; Temporini and Van Etten, 2002) or Alternaria alternata (Hatta et al., 2002). Note that biotroph fungi, like

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Ustilago maydis, possess biotroph‐specific gene clusters: about 70% of the predicted genes in that genome encode secreted proteins, most of which are specific to Ustilago, and one fifth of which are contained within only twelve gene clusters (Howlett, 2006; Kamper et al., 2006).

V. SUPPRESSION OF RECOMBINATION AROUND MAT LOCI Another remarkable example of gene clusters in fungal genomes corresponds to the genes determining mating compatibility, which are clustered at the mating‐type (MAT) loci (Herskowitz, 1989). MAT loci reside in genomic regions with properties similar to sex chromosomes in plants and animals. One principle among these shared traits is the suppression of meiotic recombination. In fact, the best studied models for the origin of sexes begin with haploid systems and the linkage of genes for non‐self recognition and a second trait, such as control over cytoplasmic inheritance (Hoekstra, 1987; Hurst and Hamilton, 1992). The consequences for local recombination suppression are many, including changes in gene content both for regions linked to mating type and the evolution of the genome as a whole. Because compatible gametes in fungi must diVer at the mating types, which code for haploid cell‐to‐cell recognition and post‐zygotic compatibility, these loci are always heterozygous in the diploid stage of the life cycle. In fungi that require mating prior to infection of the host, the mating type loci themselves are sometimes considered to be pathogenicity factors, such as in Ustilago hordei (Bakkeren and Kronstad, 1996). However, protein products of mating type loci additionally may serve functions for mating and virulence through common G‐protein‐mediated environmental sensing and response pathways (Bolker, 1998). Moreover, the lack of recombination at and around mating type loci can favor the diVerential fixation of alleles that impact disease interactions. For fungi that cause infections during the haploid stage, the alternate mating types may diVer in developmental traits and virulence, such as in the basiodiomycete Cryptococcus neoformans (Barchiesi et al., 2006). Among ascomycetes, reports are more limited for recombination suppression near mating type loci. However, examples do include diVerences in pathogenicity between haploids of the alternative mating types in Mycosphaerella graminicola (Zhan et al., 2007) and the contribution of heterozygosity for mating types in Candida albicans to virulence (Lockhart et al., 2005), suggesting that additional traits not directly related to sexual development may be linked to mating compatibility. The best evidence of recombination suppression in ascomycetes involves the automictic fungus Neurospora tetrasperma,

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where the majority of the mating type chromosome exhibits structural rearrangements between the homologous pair that prevent proper alignment or chiasma formation (Jacobson, 2005). This recombination suppression appears to have been built in diVerent steps, forming two ‘‘evolutionary strata’’ (Menkis et al., 2008). Among fungi with automictic reproduction (i.e. intra‐ tetrad selfing, as is common among secondary homothallic species), recombination suppression between mating type and the centromere is frequent and can facilitate the preservation of heterozygosity (Zakharov, 2005). An additional characteristic of fixed heterozygosity is the accumulation of loci with genetic load because a deleterious mutation may be permanently sheltered by the still functional allele on the homologous chromosome (Malefijt and Charlesworth, 1979). Such patterns are readily seen in non‐ self recognition systems in plants and animals, but also occur in linkage to mating types across diverse fungi. A phenomenon called ‘‘mating type bias’’ is common to pathogens in both the Ustilaginales and Microbotryales, where deleterious recessive mutations are linked to one mating type and prevent its growth during the haploid stage. Theoretical models have attempted to explain the persistence of such load due to an association with beneficial eVects in the heterozygous stage, either for the load locus itself or for tightly linked loci (i.e. hitchhiking) (Tellier et al 2007; Antonovics et al. 1998). Preservation of heterozygote advantage, continued sheltering of genetic load, or the recruitment of other mating type specific genes may contribute to the expansion of recombination suppression around the mating type locus, giving rise to large regions of compositionally divergent haploid sex chromosomes in fungi such as Cryptococcus neoformans, Ustilago hordei, and Microbotryum violaceum (Hood, 2002; Hood et al., 2004; Fraser and Heitman, 2005; Giraud et al., 2008b). Structural heterozygosities or even overall chromosome size dimorphism may be preserved in linkage to mating types of fungi, in ways reminiscent of sex chromosomes in plants and animals. Although gene composition around the mating types of the basidiomycetes remains to be fully characterized, there is a substantial contribution of repetitive DNA and transposable elements (Bakkeren et al., 2006; Hood, 2005), where it is likely that the rate of chromosomal rearrangements and divergence is enhanced among related fungal taxa.

VI. EVOLUTION OF GENE FAMILIES In addition to genomic rearrangements, genome evolution can occur via gene family evolution and gene duplication (Force et al., 1999; Ohno, 1999), or even whole genome duplications (Dujon et al., 2004). These mechanisms can

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generate redundant genetic material upon which evolution can act to generate new functions. Gene families arise and expand through functional divergence following duplication: examples in fungi include cellular motors called kinesins (Schoch et al., 2003), the ABC transporters and MFS drug eZux systems that help fungi detoxify products from the plants defenses (Howlett, 2006), the multidrug resistance transporter families (Gbelska et al., 2006), major surface glycoproteins, related proteins and proteases (Keely et al., 2005). The genes that make possible a symbiotic or a biotrophic relationship (e.g. hydrophobins and mannoproteins, adhesins, phospholipases, and transporters) between fungi and plants (e.g. endomycorrhizae and ectomycorrhizae) also seem to have evolved more extensively by gene‐family expansions. They are mostly part of specific biochemical pathways, including genes that trigger regulatory cascades (Martin et al., 2007, 2008), and are often clustered (e.g. Jargeat et al., 2003). Examples of lifestyle‐associated gene family expansions are numerous in fungi: the DandruV‐associated species Malassezia globosa, which lacks a fatty acid synthase gene and is thus lipid dependent, shows the expansion of a number of secreted lipases and phospholipases which help this fungus harvest the host’s lipids (Cornell et al., 2007; Xu et al., 2007). This lifestyle‐associated expansion also occurred in the distant species Candida albicans, which has similarly adapted to the human skin environment, but is not present in the more closely related Ustilago maydis, which is a plant pathogen. Also, host‐specific toxins have been found in Stagonospora nodorum and some of its closely related pathogenic species (Hane et al., 2007). Other gene family expansions are related to particular phenotypic traits: in a recent study, the repertoires of filamentous fungi (Pezizomycotina) and yeasts (Saccharomycotina), which do not form filaments, were compared (Cornell et al., 2007). Typically, the two classes of Ascomycota have diVerent metabolic capabilities and there is a correspondent divergence in their proteome repertoires. The authors found that in the Pezizomycotina gene families that provide greater metabolic flexibility and a broader substrate range (e.g., transport proteins, transcription factors, proteins that allow for the use of diVerent carbon sources) have expanded relative to the Saccharomycotina. Inversely, proteins involved in cell wall structure have expanded in the yeast relative to the filamentous ascomycetes. Interestingly, both ascomycete and basidiomycete filamentous fungi show an expansion of the chitosanase gene family that is involved in hyphal cell walls, an example of convergent evolution, which on the contrary is lacking in yeast cell walls. Once new genes have arisen, recombination and gene conversion further contribute to the evolution of gene families. The extent to which gene

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duplication promotes gene family evolution has been investigated in a recent study (Wapinski et al., 2007), where comparison of the genomes of seventeen ascomycetes showed that genes related to stress‐responses have undergone many duplications and losses while growth‐related genes were markedly less prone to such evolutionary dynamics. Also, duplicated genes appear to diverge through changes in regulatory sequences more often than through changes in biochemical function (Wapinski et al., 2007). The generation of repeated sequences can occur at diVerent scales, ranging from whole‐genome duplications (WGD) to short segmental or tandem duplications (Dujon et al., 2004). WGD was elegantly demonstrated to have occurred within the Ascomycete phylogeny, specifically within the yeasts (Kellis et al., 2004; Scannell et al., 2006). The WGD event was followed by massive diVerential gene losses that deleted most of the duplicated regions in lineage‐specific patterns across the yeasts group, possibly contributing to speciation. Duplication, particularly WGD, and subsequent gene loss have been identified as powerful forces shaping the genomes across the fungi kingdom, and as lineage‐splitting forces since the diVerent fate of duplicates can bring about speciation (Semon and Wolfe, 2007). Indeed, following WGD, duplicated genes can have a diVerent probability of retention: in order to be maintained, copies can either rapidly change function thus avoiding redundancy (neofunctionalization); duplicates can share a function through subfunctionalization; there can be a combination of neo‐ and subfunctionalization at diVerent stages following duplication; also, redundant copies can serve as buVers or back‐up for a given function; copies can be retained due to their slow rate of evolution; they can be maintained in order to preserve the relative stoichiometry of protein complexes or to maintain expression levels or dosage compensation (Semon and Wolfe, 2007). Interestingly, WGD can result in polyploidization (having extra sets of homologous chromosomes). This situation can explain relatively large genome sizes in, for example, mycorrhizal fungi (Glomeromycota), where it has been shown to contribute to the long‐time maintenance of high genetic variability within asexual species (Pawlowska and Taylor, 2004). Spontaneous segmental duplication also contributes significantly to the evolution of gene families and functions (Dujon et al., 2004; Koszul et al., 2004). Furthermore, gene duplication can also be mediated by the reverse transcriptase of Class I transposable elements. These act in a ‘‘copy and paste’’ fashion to create retrogenes (or ‘‘processed pseudogenes’’) that have been identified in fungi by the lack of intronic sequences (Daboussi, 1997).

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VII. RAPIDLY EVOLVING GENES AND GENES EVOLVING UNDER POSITIVE SELECTION Changes in gene functions, in particular after gene duplication, can arise either due to changes in gene expression, through the action of positive selection or by the relaxation of selective constraints (Ohno, 1999; Ohta, 2002). In the latter cases, the molecular signature of diversifying selection can be detected through the analysis of sequence data, if nonsynonymous substitution rates (amino acid‐changing) are significantly higher than synonymous substitution rates (Yang, 1997; Yang and Bielawski, 2000). The evolutionary rate of a gene can be influenced by various factors, including selective constraints (Yang and Bielawski, 2000), expression level (Hastings, 1996; Pal et al., 2001), dispensability or essentiality (Hirsh and Fraser, 2001; Krylov, 2003), and the existence of duplicated genes (Force et al., 1999). In fungal genomes, positive selection has been found to act in the evolution of functionally important gene families, in particular those that confer an adaptation to a pathogenic life‐style. These include genes coding for defense systems or for evading the host resistance mechanisms, toxic protein genes, and other virulence‐related genes (Staats et al., 2007). Particular examples of genes under positive selection in fungal genomes include the mycotoxin gene cluster in Fusarium (Cuomo et al., 2007; Ward et al., 2002), various phytotoxin genes in Botrytis (Staats et al., 2007) and Phytophthora infestans (Liu et al., 2005), the aflatoxin gene cluster in Aspergillus (Carbone et al., 2007), host specific toxin the wheat pathogen Phaeosphaeria nodorum (Stukenbrock and McDonald, 2007), antigens in Coccidioides human pathogens (Johannesson et al., 2004) and serine proteases in 10 fungal species (Hu and Leger, 2004). Positive selection in the defense R‐genes of the plant is frequently followed by coevolution in the avirulence genes of the fungal parasite (Jones and Jones, 1997; Meyers et al., 1998; Parniske et al., 1997). This gene‐for‐gene interaction with corresponding responses in both the host and the parasite genomes is referred to as an ‘‘arms‐race’’ process (Dawkins and Krebs, 1979). Some regions in the genomes appear to be rapidly evolving. In Fusarium graminearum for instance, localized and highly polymorphic genomic regions are significantly enriched with genes favouring plant infection, such as secreted proteins, major facilitator transporters and cytochrome P450s (Cuomo et al., 2007). Another example is found in the Saccharomyces strain YJM789, a human pathogen in patients with compromised immunity, where nonrandom regions with high polymorphism may be associated with the strain pathogenicity (Wei et al., 2007). These rapidly evolving regions may have been selected to be hotspots of mutations if they contained many genes

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under diversifying selection. Even in the absence of positive selection, a relaxation of selective constraints can be associated with rapidly evolving genomic regions, which provide raw material for selection.

VIII. HORIZONTAL GENE TRANSFER Besides selection for rapidly changing genes, another means to acquire novelty in genomes is via horizontal gene transfer (HGT), which has been defined as the stable transfer of genetic material between individuals, but not directly attributable to vertical transmission from a parent to a descendent cell or individual (Kidwell, 1993). Fungi are likely candidates for experiencing HGT events since they readily undergo hyphal anastomoses, and heterokaryon incompatibility is rarely completely successful in preventing cytoplasmic or nuclear exchange (Walton, 2000). For many reported cases of HGT, there is prolonged physical contact among organisms due to shared habitat and symbiotic or antagonistic interactions that may favor the eventual genetic transfer (Garcia‐Vallve et al., 2000; Rosewich and Kistler, 2000a). Moreover, types of genetic elements that are particularly prone to exchange and introgression in the classic sense of vertical transmission also provide the bulk of evidence for HGT, including transposable elements (Diao et al., 2006; Silva and Kidwell, 2000) or components of the mitochondrial genomes (Bergthorsson et al., 2004). HGT may potentially provide the recipient organisms with new genetic materials that extend or improve capabilities for adaptation (e.g., Gojkovic et al., 2004; Hall et al., 2005), and this may be particularly important for pathogens and symbionts (Oliver and Solomon, 2008). In fact, HGT between pathogens and their hosts has been reported in diverse systems, including parasitic plants (Davis and Wurdack, 2004) and between bacteria and their animal hosts (Kondo et al., 2002). For most eukaryotes, transfer mechanisms are poorly understood, and HGT has been detected by indirect evidence, usually from incongruences between the phylogeny of the suspected genetic element and the accepted phylogeny of the organism harboring it. There are diVerent types of ‘‘character‐state discordance’’ for a given genetic element that lead to the suggestion of HGT: (i) high sequence similarity between distantly related organisms, (ii) irregular, or ‘‘patchy’’ phylogenetic distribution in a variety of lineages, (iii) sequence patterns (GC content, codon usage, introns, etc.) inconsistent with respect to its genomic context (Rosewich and Kistler, 2000b). DiVerent methods, based on the sequence and phylogenetic analyses of discordances, have been proposed to detect the action of HGT (summarized by Bull et al. (1993) although none provides unequivocal evidence.

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A number of alternative explanations for observed discrepancies include phylogenetic error, use of paralogous sequences, sporadic retention of shared‐ancestral characters, introgressive hybridization and rate‐variation among lineages (Rosewich and Kistler, 2000b). Despite diYculties for proving HGT in fungi, several examples have been suggested involving diVerent types of genetic elements. The literature on HGT in fungi shows a bias towards transfer from prokaryotes to fungi (Andersson, 2005; Andersson and Roger, 2002; Hall et al., 2005), but counterexamples have also been suggested (Brown and Doolittle, 1999). Mitochondrial plasmids are widespread in filamentous fungi and are likely to be horizontally transferred, like the plasmid kalilo in Neurospora intermedia (Kempken et al., 1992). HGT has also been proposed for transposable elements in fungi (Daboussi and Langin, 1994). There are fewer examples of HGT involving nuclear genes, however, interesting cases have been suggested (Klotz et al., 1997; Li et al., 1997; Liu et al., 1997; Moens et al., 1996), especially related to pathogenic gene clusters [for a comprehensive summary see Temporini and Van Etten (2004), but also Walton (2000); Friesen et al. (2006); Richards et al. (2006)]. It is known that host‐specific toxins contribute to host‐range in plant pathogenic fungi. In a remarkable example, Friesen et al. (2006) showed how the horizontal transfer of a gene encoding the ToxA protein, from Stagonospora nodorum to Pyrenophora tritici‐repentis, gave rise to a pathogen population with a significantly enhanced virulence, thus providing the opportunity for the latter species to exploit a new niche. Note that introgression could give rise to the same pattern, and current methods to detect HGT do not necessarily distinguish between these two scenarios. HGT has been credited with the victorin toxin produced by Cochliobolus victoriae having been potentially transferred to Cochliobolus carbonum, thus giving rise to the pathogen that destroyed oats during the blight epidemic of the 1940s (ScheVer, 1991). HGT has also been supported for the transfer of ETP clusters (Patron et al., 2007a), the ACE1 avirulence gene cluster (Khaldi et al., 2008), and in a review exploring the likely association of interspecific HGT, new diseases and host‐ specific toxins (Oliver and Solomon, 2008). Interestingly, whole chromosomes seem to have been transferred horizontally in fungi. HGT may also explain the origin of supernumerary chromosomes, which are present in some but not all members of a fungal lineage (patchy distribution), and contain DNA absent in other parts of the genome (discordant sequence patterns), as they may contain repetitive elements found only in the supernumerary chromosome, and conversely, lack repetitive sequences found in the other chromosomes (He et al., 1998; Masel et al., 1996; Poplawski et al., 1997). However, it may be very diYcult to distinguish horizontal transfers from introgression as the result of hybridization events.

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IX. MIXING DIFFERENT GENOMES: HYBRIDIZATION A more conventional means of mixing genes from diVerent species is indeed hybridization. There has been growing concern during the last decade over the number of reported hybridizations in fungi, particularly among pathogenic species (see Olson and Stenlid, 2002, for a review). The increasing global transportation of plant and plant products creates new combinations of their associated pathogens. Such evolutionary events require serious attention because they may lead to the emergence of diseases with new epidemiological properties or host specificities (Brasier, 2001). Hybridization events can give rise to a variety of genomic constitutions as well as evolutionary consequences (Mallet, 2007). Here, we outline examples in fungal pathogens of hybridizations resulting in restricted introgression, homoploid hybrid speciation (emergence of a new species heterozygote at almost all loci), and allopolyploid hybrid speciation (emergence of a new species by addition of the two parental genomes, hence with higher ploidy than the parents). The origin of hybrid genotypes, always in the presence of both parental lineages, presents the opportunity for back‐crossing when hybrids are fertile. The result is often the introgression of some genes or genomic regions from one species into the predominant genetic background of the other. Theoretical expectations are that the transferred genes confer a higher fitness or that they are lost by drift. Evidence for introgressive hybridization is increasingly found with the use of multiple loci for phylogenetic reconstruction, where it is evidenced by incongruence among the individual gene trees. Important examples of introgression include Ophiostoma novo‐ulmi, one of the two ascomycete species causing Dutch elm disease. Several strains show introgression from the less virulent species Ophiostoma ulmi (Bates et al., 1993). Interestingly, one of the loci retained from O. ulmi belongs to the vegetative incompatibility system (vic #18) that prevents the spread of a virus highly deleterious to the fungus (Paoletti et al., 2006). Other suggested cases of introgression are those of the wooden rotting fungus basidiomycete fungus Coniophora puteana (Kauserud et al., 2007), the pathogen of gymnosperms Heterobasidion annosum (Gonthier et al., 2007), Fusarium graminearum, responsible for the head blight of wheat (O’Donnell et al., 2000), and the species of Microbotryum causing anther smut on Silene acaulis (Le Gac et al., 2007). In these cases, no clear fitness advantages of hybrids as compared to other natural strains have been detected, but this possibility has not necessarily been investigated. Hybridization resulting in a new and reproductively independent lineage that maintains a consistent haploid chromosome number is homoploid

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speciation, synonymous with allodiploid speciation. Frequently, such hybrids are highly heterozygous and carry alternate alleles derived from each respective parental species (Mallet, 2007). An example of homoploid hybridization event associated with ecological speciation is provided by the combination of the basidiomycetes causing poplar leaf rust: Melampsora medusae parasitizing Populus deltoides, and Melampsora occidentalis parasitizing Populus trichocarpa. Although neither of these two fungal species is able to infect the commercial host hybrid Populus deltoides x Populus trichocarpa, the fungal hybrids between the two rust species can cause disease on the hybrid host (Newcombe et al., 2000, 2001). Ancient homoploid speciation has also been suggested for the ascomycete complex Gibberella fujikuroi (O’Donnell and Cigelnik, 1997) and one of the species of M. violaceum parasitising Silene vulgaris (Devier et al., 2008). In Puccinia recondita f. sp. tritici, the leaf rust pathogen of wheat (Park et al., 1999) and Heterobasidion annosum (Garbelotto et al., 1996), alleles from two diVerent species have been found within single individuals, but lack of data impedes distinguishing between homoploid speciation and allopolyploid hybridization, described below. Addition of two diVerent genomes, thereby doubling chromosome number, is called allopolyploid speciation. One of the suggested examples in fungi is the asexual genus Neotyphodium, representing symbionts of grasses (Kuldau et al., 1999), which arose from parasitic, sexual Epichloe¨ species (Schardl et al., 1997). There is an association between allopolyploid hybridization, asexuality, and mutualism in these endophytes. One of the scenarios to explain this association relies on the meiotic infertility of the hybrids (Selosse and Schardl, 2007). Asexual reproduction of Neotyphodium is achieved by seed‐borne transmission within the host plant populations, so that the fungi may be selected for favoring mutualism because their reproduction depends entirely on that of their host; i.e. vertical transmission. Further, mutualistic hybrids would benefit from the broader combination of genes for alkaloid production, as these compounds act to protect the host plants from herbivores (Tanaka et al., 2005). Another well‐known example of allopolyploid hybridization is Botrytis allii, which is also asexual (Nielsen and Yohalem, 2001). Intriguingly, this hybrid exploits the same niche as one of his parents, suggesting that reproductive isolation alone allowed this new lineage to evolve. Polyploid hybrids of Cryptococcus neoformans, the human pathogen causing meningoencephalitis, also infect the same host as their parental lineages. Despite sterility, they are highly prevalent and hybrid vigor is invoked to account for their high prevalence (Lin et al., 2007). Allopolyploid hybrids have also been identified among saprophytic Saccharomyces species among fermentation selected strains both for wine, beer, and cider production. Their phenotypes suggest that these strains were selected for their ability in combining physical properties like good

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alcohol tolerance and fast growth owned by each of the parental species (Gonzalez et al., 2008; Masneuf et al., 1998). Several hybridization events have thus been identified among parasites and mutualists, among all clades of fungi. Some have been related to an increase in virulence or host range, a shift in host spectrum or a switch towards mutualism (Olson and Stenlid, 2002). The ongoing identification of fungal cryptic species (Arnold and Lutzoni, 2007; Hunter et al., 2006; Redecker and Raab, 2006) may boost the identification of other such events, because hybridization should be most frequent between recently separated species (Kauserud et al., 2007; Le Gac et al., 2007).

X. TRANSPOSABLE ELEMENTS To understand genome evolution, particular attention should be paid to transposable elements. They are a ubiquitous component of genetic systems and comprise a large proportion of most eukaryotic genomes. The process of inheritance lends itself to the emergence of such cheating elements that gain a transmission advantage by autonomously over‐replicating relative to the host genome and inserting copies into new chromosomal locations. In a large number of plant and animal species, transposable elements, or their degraded remnants, make up as much as half of the total nuclear DNA. In fungi, such elements typically comprise less than 20% of the genome, perhaps due to selection against the accumulation of large amounts of DNA sequences that are superfluous to the organism’s necessary functions (Wostemeyer, 2002). Still, active proliferation of transposable elements and their dispersed copies across the genome have a major influence upon fungal evolution. Transposable elements are represented by a diverse array of replication strategies whose origins remain heavily debated (Capy et al., 2000). The mechanisms of replication generally involve either a copy‐and‐paste strategy typical of Class I retrotransposons, where an RNA compliment is made by the normal transcriptional processes and then reverse‐transcribed back to a DNA copy that are inserted often randomly within the genome, or a cut‐and‐ paste strategy typical of Class II transposons, where an element is excised during genome replication from one daughter strand of DNA and inserted ahead of the advancing DNA replication fork. By virtue of the relative eYciency of copy‐and‐paste, retrotransposons elements usually make a greater contribution to total genome size (Gollotte et al., 2006). However, with the advent of genomic technologies, new types of elements are being discovered that do not fit neatly into existing classifications. For example the

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Helitron elements, present in genomes as diverse as vertebrates and the wood rot fungus Phanerochaete chrysosporium, are not reverse‐transcribed from RNA but instead use a DNA rolling‐circle replication (Poulter et al., 2003). Similar in mechanism to bacterial viruses, individual Helitrons can produce many descendant copies to proliferate within the genome. Cryptons elements, a group of tyrosine‐recombinase encoding element discovered in several pathogenic fungi (Goodwin et al., 2003), exhibit a unique combination of sequence characteristics reflecting both DNA transposons and RNA retrotransposons. Fungi possess the full spectrum of transposable element types, where their influence as powerful forces for genetic change is due to both insertional mutations and to promoting chiasma formation between non‐homologous sites (i.e. ectopic recombination). The random nature of integration for most transposable elements can lead to the disruption of coding sequences of genes or the separation of genes from necessary promoter regions. In several pathogenic fungi, such as Leptosphaeria maculans and Magnaporthe grisea, sequences coding for avirulence genes are found in genomic regions dense with transposable elements (Fudal et al., 2007; Gout et al., 2006; Kang et al., 2001; Rehmeyer et al., 2006), potentially contributing to the extreme variability of avirulence genes that is associated with host–pathogen coevolution. Fungi also have extremely high levels of structural polymorphism for karyotypes, often with chromosome length variation even within populations (Zolan, 1995). The potential contribution of recombination between transposable elements and their enzymes responsible for DNA breakage and repair has been suggested to increase rates of chromosomal rearrangements for a number of species, including Magnaporthe oryzae (Thon et al., 2006) and Fusarium oxysporum (Daboussi, 1997). Despite various reports for beneficial roles of transposable elements or their ‘‘domestication’’ to perform functions necessary to the organism (VolV, 2006), on average the increased mutations rates caused by insertion and ectopic recombination decrease individual fitness (Arkhipova and Meselson, 2005). Genomes are protected against the proliferation of transposable elements by a variety of mechanisms, and fungi possess perhaps a broader range of defenses than many eukaryotic groups. Moreover, the empirical tractability of fungi has greatly facilitated studies of co‐evolution between parasitic DNA and the host genome. A common theme to genomic defense is the detection of repeated or newly inserted DNA sequences. For example, the mechanism of Meiotic Silencing of Unpaired DNA (MSUD) in Neurospora crassa detects the hemizygosity of newly inserted elements and employs post‐transcriptional gene silencing, related to RNA‐interference, to prevent their further replication (Shiu et al., 2001). Also originally found in N. crassa, and now known to occur in a broad range of ascomycete and

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basidiomycete fungi (Galagan and Selker, 2004; Hood et al., 2005), Repeat‐ Induced Point Mutation (RIP) is a most eVective defense and one which provided the best evidence of having evolved to constrain transposable element populations. RIP is a process that hypermutates repeated DNA sequences of a few hundred base pairs, thus making transposable element copies nonfunctional by inducing non‐sense or stop codons and decreasing their sequence similarity between non‐homologous sites (i.e. reducing the risk of ectopic recombination). This defense is only known to occur in fungi, and specifically occurs during the dikaryotic stage where the continued intra‐ cellular separation of haploid nuclei may facilitate the detection of repeated DNA; whereas in diploid cells all sequences are essentially present in at least two copies. RIP is frequently described as being associated with the sexual stage of the fungal life cycle, which may be interpreted as being involved in meiosis. However, in most ascomycetes, including N. crassa, dikaryon formation occurs only briefly prior to meiosis, whereas in basidiomycetes, the dikaryotic is greatly prolonged and the potential impact on the genome defense deserves further study. The paucity of active transposable elements in lineages of N. crassa with RIP is evidence of the eYcacy of this defense system, but there may be other long‐term consequences for fungal evolution. RIP also prevents the duplication of any housekeeping or pathogenicity genes, thus greatly limiting the evolutionary potential of sequence duplications to acquire novel and adaptive functions (Brookfield, 2003).

XI. TELOMERES Telomeres are genomic regions particularly prone to transposable element accumulation and rapid evolution, thereby playing a role in host adaptation (Sa´nchez‐Alonso and Guzman, 2008). As in other eukaryotes, the ends of fungal chromosomes consist of tandem arrays of simple sequence repeats that are usually GT‐rich. The most common telomeric repeat in filamentous fungi is (TTAGGG)n, as is also found in basal metazoans and vertebrates (Traut et al., 2007). The telomere repeats are associated with a number of proteins that protect the chromosome ends from degradation and have roles in the silencing of neighboring genes in the adjacent subtelomeric region. Physical proximity during meiosis between non‐homologous chromosome ends is thought to promote ectopic recombination between shared sequences in the subtelomeric regions, leading to the amplification and diversification of telomere‐linked genes (Freitas‐Junior et al., 2000). The dynamic nature of telomeres may contribute to the variation critical for rapid evolution of

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host‐parasite interactions (Barry et al., 2003; Freitas‐Junior et al., 2000; SchaVzin et al., 1999). In several fungal pathogens of humans, subtelomeres contain gene families encoding immunogenic extracellular proteins. To avoid detection by the host, the pathogens switch gene expression among the diVerent genes of these families, taking advantage of the silencing mechanism resulting from the telomere repeat‐associated proteins (De Las Penas et al., 2003; Keely et al., 2005). Subtelomere regions of plant pathogenic fungi do not harbor families of surface protein genes, but appear to still play an important role in pathogenicity. For instance, a significant proportion avirulence genes is located very near to telomeres both in Magnaporthe oryzae (Chen et al., 2007; Rehmeyer et al., 2006) and in Phytophthora infestans (van der Lee et al., 2001). For some of these fungal avirulence genes, the importance of the position near telomeres to pathogenicity has been demonstrated, the truncation of chromosome ends allowing loss of the genes and thus the gain of virulence (Orbach et al., 2000). In contrast, silencing near telomeres does not seem to play an important role in pathogenicity in fungi. Rehmeyer et al. (2006) proposed that the recruitment of avirulence genes to subtelomeric regions may be adaptive because the subsequent instability favors the emergence of strains that can avoid detection by resistance mechanisms (by truncations of the chromosome parts harbouring the avirulence genes) and that the pathogen can thus colonize a more diverse collection of host genotypes. However, it is important to recognize that recruitment to subtelomeric regions cannot be selected for by an advantage to gene deletion: if the deletion is adaptive (i.e., allows colonizing a new host), then the selected genotype will not have the gene in telomeric position, and the parental genotype with the gene in telomeric position will not be selected for. Yet, telomeric positions can be adaptive by generating allelic variability, and therefore be selected for, and then allow further variability through deletion. Genes near telomeres indeed seem to exhibit higher sequence variability than in other chromosome regions, although it is not clear yet what mechanism is responsible for this variability. Subtelomeres of pathogenic fungi also contain large families of host adaptation genes, as do other pathogenic microbes. For instance, many fungi contain secondary metabolite gene clusters near telomeres (Rehmeyer et al., 2006), as well as putative secreted proteins that may be involved in plant infection (Rehmeyer et al., 2006). Other gene families are present in subtelomeres in fungi, that may not be directly involved in host‐parasite interactions, such as helicase gene families (Gao et al., 2002; Louis, 1995; Sanchez‐Alonso and Guzman, 1998).

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XII. INTRONS Introns are also highly dynamic in fungal genomes, and some of them exhibit traits remarkably similar to transposable elements. Fungal genomes are gene dense, with relatively simple gene structure compared to plant and animals. The number of introns per gene ranges from much less than one in S. cerevisiae (GoVeau et al., 1996), to between 1 and 2 introns per gene for other recently sequenced ascomycetes (Dean and et al., 2005; Galagan et al., 2003). The basidiomycete Cryptococcus neoformans contains an average of seven introns per gene (Loftus et al., 2005). Introns are also typically short in fungi, averaging between 80 and 150 bp in the ascomycetes and averaging 68 bp and down to 35 bp in the basidiomycete C. neoformans (Loftus et al., 2005). As in many eukaryotes, intron‐poor species of fungi have more introns in 50 positions. Although fungi appear to use alternative splicing less frequently than metazoans, the most extensive genome‐wide survey on alternative splicing in a fungus (in C. neoformans, Loftus et al., 2005) revealed evidence of alternative splicing for 4% of the genes (against 40–60% in humans for instance, Modrek and Lee, 2002). Fungal spliceosomal introns (i.e., the most common insertions found in nuclear pre‐mRNA genes) are remarkably dynamic: Nielsen et al. (2004) estimated between 150 and 250 gains and between 150 and 350 losses in each of the four Ascomycete lineages they examined (A. nidulans, F. graminearum, M. grisea and N. crassa), spanning ca. 330 millions years evolution. Fungi also carry retrotransposable elements in the form of Group I and Group II introns in their mitochondria, characterized by a distinct RNA secondary structure and self‐splicing pathway (Haugen et al., 2005). In particular, the Group I intron insertion has also been observed in the nuclear genome of parasitic fungi in the genus Cordyceps (Nikoh and Fukatsu, 2001). Frequent horizontal transfer between evolutionarily distinct lineages of fungi has been proposed based on phylogenetic distribution of Group I introns (Dujon, 1989; Feau et al., 2007; Hibbett, 1996; Holst‐Jensen et al., 1999; Mouhamadou et al., 2006) and this possibility has been demonstrated in experimental settings in vivo (Muscarella and Vogt, 1993).

XIII. MITOCHONDRIAL GENOMES Mitochondrial genomes are another component of genetic information and have their own rules of genomic evolution. Mitochondria are derived from an ancient endosymbiotic ‐Proteobacterium and have retained their own genetic system. However, in all eukaryotes, the majority of mitochondrial genes have

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been transferred to the nuclear genome and their protein products are imported into mitochondria. The reduction of the mitochondrial genome is probably due to a selection for rapid replication and to rescue conserved genes from the higher rate of mutation in organelles compared to the nuclei (Selosse et al., 2001). Genes whose expression is regulated by reduction/oxidation reactions are preferentially retained in the mitochondrial DNA (mtDNA), perhaps providing more rapid control over their activities (Allen, 2003). Currently, completely sequenced mtDNAs are available from 49 fungal species, representatives of all four fungal phyla (http://megasun.bch.umontreal. ca/ogmp/projects/other/mt_list.html). Fungal mitochondrial DNAs typically encode only 30–40 genes (Bullerwell and Lang, 2005). This is much fewer than the mtDNA of many other eukaryotes, in particular plants and protists, that can carry 50–100 mt genes (Bullerwell and Lang, 2005). This suggests that many of the mitochondrial genes have been lost early in fungal evolution, or even in the opisthokont lineage, since animals seem to have also retained few genes in their mtDNA (Bullerwell and Lang, 2005). Further genome reduction also occurred later in some fungal lineages, some chytridiomycetes having for instance completely lost their mtDNA (Bullerwell and Lang, 2005). A core set of common genes seems to be retained in most fungal mtDNAs, but they exhibit otherwise remarkable polymorphism, mostly due to variation in intron number, gene arrangement, mobile retro‐elements, the presence/absence of plasmids, and also gene number (Burger et al., 2003; Gray et al., 1999; Lang et al., 1999). Fungal mtDNAs seem to be involved in senescence, i.e. growth arrest of the mycelium that appears after some time of laboratory culture in certain filamentous fungi, such as Podospora anserina. Senescence has indeed been shown to be associated with rearrangements of the mitochondrial genome, although the causality is not completely clear (GriYths, 1992). Mutant mtDNAs, called ‘‘suppressive mtDNA’’, are however able to induce senescence when transmitted via anastomoses between hyphae from diVerent mycelia. Such suppressive mtDNA mutations can thus reduce virulence in some phytopathogenic fungi and be transmitted cytoplasmically. In the chestnut‐blight fungus Cryphonectria parasitica for instance, the syndrome associated with mutant mtDNAs and their mode of transmission resemble strikingly those of the virus responsible for hypovirulence (Bertrand, 2000). Another example of defective mitochondria is the petite mutation in Saccharomyces cerevisiae (Taylor et al., 2002). Mutant mtDNAs can persist even if they lower the fitness of the cells that carry them if they have a suYcient replication advantage compared to wild type mitochondria and if they are under the dynamics of small populations (Taylor et al., 2002): they are then selected for at the cell level and selected against at the organism level.

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A means to reduce the frequency of intra‐genomic conflicts between diVerent levels of selection is to have mtDNAs variability restricted by uniparental transmission (Aanen et al., 2004; Hoekstra, 1990;). In anisogamous eukaryotes, only the female usually transmits her mitochondria to the zygote (Hurst, 1996). Similarly, only one of the mating types passes on its mitochondria in some bipolar fungi, e.g. in Cryptococcus neoformans (Yan and Xu, 2003) and in Microbotryum violaceum (Wilch et al., 1992). In some basidiomycetes, unidirectional nuclear migration during mating allows the resulting zygote to carry a single mtDNA type (e.g. Agaricus bitorquis, Aanen et al., 2004; Hintz et al., 1988). In other basidiomycetes, mitochondria are sorted out in the young mycelium, a single mtDNA remaining after a few cell divisions, the proximal mechanism being unknown, e.g. in Agaricus bisporus (de la Bastide and Horgen, 2003) and in Agrocybe aegerita (Barrosso and Labarere, 1997). In some tetrapolar basidiomycetes with multiple mating types, however, the mycelium can remain a mosaic or even heteroplasmic (e.g., Pleurotus ostreatus, Matsumoto and Fukumasa‐Nakai, 1996), which leaves room for multi‐level selection conflicts (Aanen et al., 2004; Hoekstra, 1990). The most frequent occurrence of such conflict is seen in the S. cerevisiae yeast, where the unusual cassette system of mating type switching precludes any such control over mitochondrial inheritance and leads to the expectation for higher rates of heteroplasmy.

XIV. COMPARISON WITH THE GENOMICS OF PATHOGENIC BACTERIA The genomics of prokaryotes has seen a great development in recent years, and has had a major impact on the research of microbial pathogenesis and symbiosis. Comparative genomics of strains and species of bacteria has provided new insights into the evolution of virulence and it may be interesting to compare the genomics of fungal and bacterial pathogens. Although comparative genomics in pathogenic and symbiotic fungi is only at an early stage, it seems that bacteria and fungi have diVerent modes of host adaptation on the genomic level. Analyses of genome sequences in bacteria have demonstrated that many of the genes required for virulence are restricted to pathogenic organisms and that they have been introduced into the genomes by horizontal gene transfer. These HGT events often involve whole cassettes of genes, ranging in size from 5 to 100 kb. Their frequent integration at or near tRNA loci suggests that many were introduced via phage‐mediated transfer events (Arnold et al., 2003; Dobrindt et al., 2004; Ochman and Moran, 2001). This process is so pervasive that species‐specific chromosomal

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regions containing virulence genes are now classified under the general heading of ‘‘pathogenicity islands’’ (Arnold et al., 2003; Dobrindt et al., 2004; Ochman and Moran, 2001). In the animal pathogen Dichelobacter nodonus for instance, 20% of the genome is derived from lateral gene transfer and most of these transferred regions seem to be associated with virulence (Myers et al., 2007). Pathogenicity islands have been described in a wide range of both plant and animal bacterial pathogens, and it has become evident that their general features are displayed by a number of DNA regions with functions other than pathogenicity, such as symbiosis and antibiotic resistance, and the general term genomic islands has been adopted (Arnold et al., 2003; Dobrindt et al., 2004). Such lateral gene transfer of genes involved in host adaptation is much less pervasive in fungi, although some examples have been reported (see section on horizontal gene transfers). A large set of bacterial symbionts and pathogens have undergone massive gene loss, mostly because the host presents a constant environment rich in metabolic intermediates that renders some genes useless under a strictly symbiotic or pathogenic life‐style. Such consistent patterns of genome streamlining do not seem to have occurred across the full range of pathogenic and symbiotic fungi, in particular being influenced by whether the fungal species retains some parts of their life cycle as a free‐living stage. The ectomycorrhizal and saprophytic fungus Laccaria bicolor for instance contains a huge number of genes compared to other fungi (Martin et al., 2007). Some gene losses have been shown to be adaptive in bacteria, such as surface proteins recognized by the hosts (Nakata et al., 1993; Wren, 2000). Also, gene duplication may occur in bacterial pathogens as a mechanisms for generating variation in surface antigenic structure (Wren, 2000). These losses of avirulence genes and gene duplication in families are similar to the processes observed in the genomics of pathogenic fungi.

XV. CONCLUSION The evolution of fungi is estimated to span at least 500 million years (Taylor and Berbee, 2006), during which time these organisms have explored virtually all available ecosystems and nutritional modes of associations with other living organisms. The adaptations to such a wide range of ecological niches and lifestyles were certainly made possible by the tremendous flexibility of the fungal genomes. This flexibility is due in no small part to pervasive gene duplication at diVerent scales, including that of the whole genome, which has been shown to promote new functions. A complementary mechanism to duplication is the loss of diVerent genes, which can lead to speciation and

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specialization, as exemplified in yeasts. Chromosomal rearrangements promote genetic novelty by changing the context of elements already present and introducing new regulatory possibilities. Hybridization contributes significantly to the mixing of fungal genes and horizontal gene transfer, although diYcult to prove, is likely to have contributed to the extensive biochemical repertoire that enables fungi to colonize new niches. Genetic elements such as introns, transposable elements, repetitive sequences, supernumerary chromosomes, and the mitochondrial genome further contribute to the enormous genetic variability observed in fungi. Comparative genomic studies in plant pathogenic and symbiotic fungi, although still in the early stages and limited to a few pathogens, have already brought many insights into the evolution of the pathogenic lifestyle, in particular into the mechanisms of virulence and host adaptations. There is a marked bias in the sequencing eVorts towards pathogenic fungi, but current projects are covering the fungal genomes of biotrophs and symbiont species that will hopefully allow us to gain insight into the partnerships between fungi and plants, a critical component of terrestrial and agro‐ecosystems. Development of advanced genomics tools and infrastructure is critical for eYcient utilization of the vast wealth of available genome sequence information and will form a solid foundation for integrated studies of the biology of plant pathogenic fungi. Future research will also benefit from eVorts in the analysis of gene expression evolution through micro‐array data and other powerful techniques. Changes in gene expression are indeed diYcult to assess from genome sequences, but may play important roles in adaptations (Wapinski et al., 2007). It will also be important to understand the variation of genomes within species, in order to observe fungal evolution in action.

ACKNOWLEDGMENTS We thank Marc‐Henri Lebrun for initiating this review and an anonymous referee for helpful comments. We acknowledge the grants ANR‐06‐BLAN‐ 0201, ANR 07‐BDIV‐003, and DEB‐0747222.

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AUTHOR INDEX

Numbers in bold refer to pages on which full references are listed.

A Aanen, D. K., 178, 180 Abbasi, F., 113, 126 Achard, P., 78, 88, 126 Adams, W. W., 64–65, 126 Agarwal, M., 97, 126 Ahad, A., 72, 126 Akiyama, T., 63, 141 Albrecht, V., 110, 126 Alexandre, C. M., 38, 126 Allen, G. J., 105, 126 Allen, J. F., 177, 180 Alonso-Blanco, C., 87, 126 Alsheikh, M. K., 49, 126 Amasino, R. M., 38, 144 Andersson, J. O., 158, 169, 181 Andrews, C. J., 74, 126 Anthony, R. G., 121, 126 Antikainen, M., 50, 126 Antonovics, J. A., 161, 164, 186 Appelqvist, L. A., 70, 134 Arkhipova, I., 173, 181 Arnold, A. E., 172, 181 Arnold, D. L., 178–179, 181 Aro, E. M., 42, 127 Artus, N. N., 49, 127 Asada, K., 68, 139 B Bagga, S., 161, 181 Baier, M., 69, 127 Baker, S. S., 78, 127 Bakkeren, G., 163–164, 181 Barchiesi, F., 163, 181 Barrosso, G., 178, 181 Barry, J. D., 175, 181 Bates, M. R., 170, 181 Behzadipour, M., 53, 127 Bell, E., 78, 127 Benedict, C., 87–88, 90, 95–96, 98, 101, 127 Berbee, M. L., 152, 179, 181, 192 Bergthorsson, U., 168, 181 Bertrand, H., 177, 181 Bielawski, J. P., 167, 193 Bies-Ethe`ve, N., 48, 127 Boekhout, T, 160–161, 181

Bogdanovi, J., 60, 127 Bohnert, H. U., 162, 181 Bolker, M., 163, 182 Boyce, J. M., 100, 127 Boyer, J. S., 36, 127 Braam, J., 109, 127 Brasier, C. M., 170, 182 Bravo, L. A., 49, 127 Brookfield, J. F. Y., 174, 182 Brown, J. R., 169, 182 Browse, J., 54, 61, 148–149 Bru¨ggemann, W., 43, 128 Bullerwell, C. E., 177, 182 Bull, J. J., 168, 182 Burger, G., 177, 182 Burke, M. J., 60, 142 Busconi, L., 112, 138 C Campos. P. S., 52, 128 Cao, S., 104, 128 Capel, J., 76, 128 Capy, P., 172, 182 Carbone, I., 167, 182 Carles, R. J., 74, 130 Carpaneto, A., 107, 128 Carpenter, J. F., 59, 128 Catala, R., 108, 128 Caten, C. E., 161, 182 Cavicchioli, R., 38, 144 Charlesworth, B., 164, 188 Chauhan, N., 160, 182 Cheng, C., 74, 128 Cheng, N. H., 108, 128 Chen, Q. H., 175, 182 Cheong, N. E., 69, 128 Cheong, Y. H., 110, 128 Chinnusamy, V., 37, 94–95, 128 Choi, J. H., 121, 128 Cho, S. K., 70, 128 Chung, S., 109, 116, 128 Ciereszko, I., 76, 129 Cigelnik, E., 171, 190 Clay, K., 152, 182 Collemare, J., 162, 182 Cook, D., 55–56, 63, 68, 91, 129

196 Crosatti, C., 75, 129 Crowe, J. H., 58–59, 129 Cui, S., 63, 69, 71, 129 Cuomo, C. A., 162, 167, 182 D Daboussi, M. J., 166, 169, 173, 182 Danyluk, J., 48, 129 Davis, C. C., 168, 182 Davis, R. W., 109, 127 Davletova, S., 98, 129 Davy de Virville, J., 53, 129 Dawkins, R., 167, 182 Dean, R. A., 158, 162, 176, 183 de Bruxelles, G. L., 76, 128 de la Bastide, P. Y., 178, 183 De Las Penas, A., 175, 183 Demmig-Adams, B., 64, 126 Deswal, R., 115, 129 Devier, B., 171, 183 Dexter, S. T., 74, 129 Dhindsa, R. S., 108–109, 139 Diao, X. M., 168, 183 Dietz, K. J., 69, 127 Di Mascio, P., 70, 129 Dioh, W., 160, 183 Dobrindt, U., 178–179, 183 Dodd, A. N., 86, 105, 107, 129 Dong, C. H., 96, 129 Doolittle, W. F., 169, 182 Dowgert, M. F., 39, 44, 129 Doxey, A. C., 50, 129 Dubouzet, J. G., 86, 89, 130 Dujon, B., 164, 166, 176, 183 E Ebbert, V., 64, 130 El Kayal, W., 84, 87, 130 El-Khatib, R., 110, 130 Ensminger, I., 40–41, 64, 130 F Farmer, P. K., 121, 130 Feau, N., 176, 183 Fedorova, N. D., 159, 183 Feierabend, J., 65, 145 Fei, S., 80, 89, 149 Force, A., 164, 167, 183 Fowler, D. B., 74, 130 Fowler, S. G., 86, 98, 129 Franklin, K. A., 75, 130 Fraser, H. B., 167, 186 Fraser, J., 160–161, 164, 183 Freitas-Junioer, L. H., 174–175, 183 Friesen, T. L., 169, 183 Fryer, M. J., 70, 130

AUTHOR INDEX Fudal, I., 173, 183 Fujita, M., 72, 130 Fukumasa-Nakai, Y., 178, 189 Fukutaku, Y., 62, 130 Furbank, R. T., 41, 65, 130 G GaVoor, I., 162, 184 Galagan, J. E., 153, 158, 174, 176, 184 Gao, W. M., 175, 184 Garbelotto, M., 171, 184 Garcia-Vallve, S., 168, 184 Gibson, S., 54, 130 Gilmour, S. J., 79, 88–89, 93, 130–131 Giraud, T., 158, 164, 184 GoVeau, A., 176, 184 Gojkovic, Z., 168, 184 Gollotte, A., 172, 184 Gombos, Z., 43, 131 Gonthier, P., 170, 184 Gonzalez, S. S., 172, 185 Goodwin, T. J. D., 173, 185 Goulas, E., 43, 65, 68–69, 78, 131 Gout, L., 173, 185 Gray, C. R., 65, 74, 131 Gray, G. R., 55, 131 Gray, M. W., 177, 185 Greenberg, B. M., 42, 131 Gregory, T. R., 158, 185 Greig, D., 161, 185 GriYth, M., 41, 50, 131 GriYths, A. J. F., 177, 185 Gulick, P. J., 72, 142 Guo, Y., 94, 131 Guo, Z., 68–69, 131 Gusta, L. V., 39, 55, 58, 131 Guy, C. L., 56, 59, 131 Guzman, P., 174–175, 191 H Haake, V., 79, 89, 131 Hall, C., 168–169, 185 Hamer, L., 158, 185 Hamilton, W., 163, 186 Hane, J. K., 162, 185 Hannah, M. A., 67, 131 Han, Y. N., 162, 185 Hara, M., 49, 132 Hastings, K. E. M., 167, 185 Hatta, R., 162, 185 Haugen, P., 176, 185 Havaux, M., 43, 66–67, 70, 132 Heath, D., 55, 131 He, C. Z., 169, 185 Heitman, J., 164, 183 Hejazi, M., 57, 132 He, L., 63, 132

AUTHOR INDEX Hendrickson, L., 51, 132 Hennig, L., 38, 126 Herskowitz, I., 163, 185 Hibbett, D. S., 176, 185 Hintz, W. E. A., 178, 185 Hiratsu, K., 98, 132 Hirschi, K. D., 108, 132 Hirsh, A. E., 167, 186 Hirsh, A. G., 60, 132 Hittinger, C. T., 158, 186 Hjelm, U., 64, 132 Hoekstra, R. F., 163, 178, 186 Hohn, T. M., 161, 187 Holst-Jensene, A., 176, 186 Hood, M. E., 164, 174, 186 Horgen, P. A., 178, 183, 185 Howlett, B. J., 163, 186, 190 Hsieh, T. H., 90, 132 Huang, B., 77, 132 Huner, N. P. A., 40–41, 64–65, 70, 132–133 Hunter, G. C., 172, 186 Hurry, V. M., 41, 55, 61, 64–66, 124, 133, 145 Hurst, L. D., 163, 178, 186 Hutchison, R. S., 43, 133 I Ichimura, K., 114, 133 Imai, R., 51, 56, 63, 133 Ishitani, M., 103, 133 Ivanov, A. G., 41, 51, 64–65, 133 J Jacobson, D. J., 164, 186 Jaglo, K. R., 89, 91, 133 Jaglo-Ottosen, K. R., 88–89, 134 Jang, H. H., 69, 134 Jiang, C., 78, 134 Jiang, W., 51, 134 Johannesson, H., 167, 186 Johnson, M. P., 66, 134 Jones, D. A., 167, 186, 190 Jones, J. D. G., 167, 186, 190 Juntilla, O., 74, 148 K Kacperska, A., 120, 145 Kadpel, R., 61, 134 Kagale, S., 77, 134 Kamal-Eldin, A., 70, 134 Kamata, T., 55, 61–62, 134 Kamper, J., 159, 162–163, 187 Kang, H. M., 76, 134 Kang, S., 173, 187 Kant, P., 98, 128 Kaplan, F., 54–56, 59, 61, 68, 76, 78, 134

197

Karlson, D., 51, 135 Kasuga, M., 88, 90, 135, 138 Kasukabe, Y., 64, 135 Kauserud, H., 170, 172, 187 Keely, S. P., 158, 175, 187 Keller, N., 162, 187 Kellis, M., 158, 166, 187 Kempken, F., 169, 187 Khaldi, N., 169, 183, 187 Kidwell, M. G., 168, 187, 192 Kiegle, E., 105, 135 Kim, H. J., 75, 99, 116, 135 Kim, J. C., 102, 135 Kim, J. Y., 51, 135 Kim, S., 87, 135 Kim, T. E., 63, 135 Kim, Y. O., 51, 135 Kistler, H. C., 158, 160–161, 168, 187, 191 Kloppstech, K., 66–67, 132 Klotke, J., 54, 135 Klotz, M. G., 169, 187 Knight, H., 79, 99, 106–107, 136 Knight, M. R., 109, 147 Knox, A. K., 88, 136 Koag, M. C., 49, 136 Kodama, H., 36, 136 Koh, S., 77, 136 Kolukisaoglu, U., 110, 136 Komatsu, S., 113, 136 Kondo, N., 168, 187 Korn, M., 60–61, 67, 136 Koster, K. L., 55, 61–62, 136 Koszul, R., 158, 166, 187 Kotzabasis, K., 63, 136 Krebs, J. R., 167, 182 Krinke, O., 121, 136 Kroken, S., 162, 187 Krol, E, 108, 137 Krol, M., 65–66, 136–137 Kronstad, J. W., 163, 181 Krylov, V. N., 167, 187 Kudla, J, 110, 137 Kuldau, G. A., 171, 187 Kume, S., 87, 137 Kwon, S. J., 69, 137 L Labarere, J., 178, 181, 189 Lafta, A., 61, 142 Lang, B. F., 177, 185, 187 Langin, T., 169, 182 La˚ng, V., 48, 75–76, 116, 137 Larriba, G., 160, 187 Lawrence, T., 74, 137 Lee, B. H., 76–77, 137 Lee, C., 176, 189 Leegood, R. C., 65, 137 Lee, H., 96, 137

198 Le Gac, M., 170, 172, 188 Leger, R. J. S., 167, 186 Leshem, Y. Y., 36, 138 Leubner-Metzger, G., 38, 130 Levitt, J., 54, 137 Lewis, B. D., 108, 137 Leyva, A., 67, 137 Lin, C., 107, 138 Lin, X., 171, 188 Liu, H. H., 124, 138 Liu, H. L., 55, 138 Liu, J. H., 169, 188 Liu, Q., 79–80, 88–89, 138 Liu, Z., 167, 188 Li, W., 118, 137 Li, X. L., 169, 188 Li, Z., 112–113, 137 Llorente, F., 75–76, 138 Lockhart, S., 163, 188 Loftus, B. J., 176, 188 Louis, E. J., 175, 188 Lundmark, M., 56, 138 Lu, S., 124, 138 Lutzoni, F., 172, 181 Lynch, D. V., 55, 136 M Machida, M., 162, 188 Magee, B. B., 160, 188 Magome, H., 80, 88, 138 Malefijt, M., 164, 188 Mallet, J., 170–171, 188 Malz, S., 158, 188 Manetas, Y., 61, 140 Ma¨ntyla¨, E., 75, 138 Mao, T., 72, 138 Martin, F., 159, 179, 188 Martin, M. L., 112, 138 Maruyama, K., 79, 91, 93, 138 Masel, A. M., 169, 188 Masneuf, I., 172, 189 Matos, A. R., 53, 138 Matsumoto, T., 178, 189 McDonald, B. A., 167, 192 McKersie, B. D., 36, 138 McKown, R., 58, 139 Medina, J., 79, 82, 104, 139 Menkis, A., 164, 189 Meselson, M., 173, 181 Meyers, B. C., 167, 189 Miao, V., 160, 187 Miginiac-Maslow, M., 43, 143 Miquel, M., 53, 139 Miranda, J. A., 56, 139 Miura, K., 96–97, 139 Miyake, C., 68, 139 Mizoguchi, T., 114, 139 Modrek, B., 176, 189

AUTHOR INDEX Moens, L., 169, 189 Monroy, A. F., 55, 108–109, 111–112, 139 Moran, N. A., 178, 189 Mouhamadou, B., 176, 189 Mu¨ller-Moule´, P., 68, 139 Mullet, J. E., 78, 127 Murata, N., 62, 131 Muscarella, D. E., 176, 189 Myers, G. S. A., 179, 189 N Nakaminami, K., 51, 139 Nakano, R., 43, 139 Nakashima, K., 37, 80, 140 Nakata, N., 179, 189 Nakayama, K., 49, 140 Nanjo, T., 61, 140 Narusaka, Y., 82, 140 Navakoudis, E., 63, 140 Newcombe, G., 171, 189 Newton, A. C., 161, 182 Nielsen, C. B., 176, 189 Nielsen, K., 171, 189 Nikolopoulos, D. Y., 61, 140 Nishizawa, A., 60, 140 Niyogi, K. K., 43, 132 Nordin-Henriksson, K., 108, 140 Novillo, F., 84–86, 88, 91, 94, 104, 140 O Ochman, H., 178–179, 189 O’Donnell, K., 170–171, 190 Ogren, E., 64, 132 Ohno, S., 164, 167, 190 Oh, S. J., 90, 140 Ohta, T., 167, 190 Olien, C. R., 58, 140–141 Oliver, R., 168–169, 190 Olson, A., 170, 172, 190 ¨ quist, G., 64, 141 O Orbach, M. J., 175, 190 Oshima, Y., 161, 193 O’Sullivan, D., 160–161, 190 Ottander, C., 64, 141 P Pal, C., 167, 190 Palta, J. P., 52–53, 141 Palva, E. T., 76, 116, 137, 142, 146 Pandey, S., 114, 141 Paoletti, M., 170, 190 Papageorgiou, G. C., 62, 141 Parish, R. W., 109, 116, 128 Park, E. J., 62, 141 Park, J. J., 171, 190

AUTHOR INDEX Parniske, M., 167, 190 Parthier, B., 78, 148 Patron, N., 161, 169, 190 Pawlowska, T. E., 166, 190 Payne, G. A., 161, 190 Penalva, M. A., 161, 190 Perruc, E., 76, 141 Pillai, M. A., 63, 141 Pino, M. T., 88, 90, 141 Plieth, C., 105–107, 141 Poovaiah, B. W., 109, 149 Poplawski, A. M., 169, 191 Poulter, R. T., 173, 185, 191 Pramanik, M. H., 56, 141 Prasad, T. K., 62, 68, 141 Price, A., 68, 142 Proctor, R. H., 161, 191 Puhakainen, T., 48, 142 R Raab, P., 172, 191 Rabbani, M. A., 75, 142 Rajasekharan, R., 115, 142 Rajashekar, C. B., 60–61, 71, 118, 142, 148 Ranf, S., 108, 142 Rao, N., 61, 134 Ray, S., 112, 142 Redecker, D., 172, 191 Rehmeyer, C., 173, 175, 191 Reiber, K., 162, 191 Reyes-Dı´az, M., 58, 142 Rice-Evans, C. A., 67, 142 Richards, T. A., 169, 191 Ridha Farajalla, M., 72, 142 Rincones, J., 161, 191 Rinne, P. L., 49, 142 Roger, A. J., 169, 181 Rohde, P., 54–55, 142 Rosewich, U. L., 168–169, 191 Roughan, P. G., 54, 142 Rudrabhatla, P., 115, 142 Ruelland, E., 43, 117, 120, 143 Rustchenko, E., 160, 191 S Sagisaka, S., 55, 143 Sakamoto, H., 98, 143 Sakuma, Y., 80, 82, 143 Saltveit, M. E., 76, 134 Snchez-Alonsoa, P., 174–175, 191 Sane, P. V., 65, 143 Sasaki, K., 51, 143 Sasse, J. M., 77, 143 Sassenrath, G. F., 43, 143 Satoh, R., 62, 143 Savitch, L. V., 64–65, 89, 143 Scannell, D. R., 158, 166, 192

199

SchaVzin, J. K., 175, 191 Schardl, C. L., 171, 192 ScheVer, R. P., 169, 191 Scheller, H. V., 41, 147, 150 Schmitz, R. J., 38, 144 Schobert, B., 62, 144 Schweighofer, A., 115, 144 Scott, I. M., 77, 144 Seki, M., 72, 75, 144 Selker, E., 174, 184 Selosse, M.-A., 171, 177, 192 Semon, M., 166, 192 Sharma, A., 113, 144 Sharma, N., 63, 67, 70, 77, 144 Sheen, J., 112, 144 Shen, W., 64, 144 Shen, Y. G., 90, 144 Shikama, K., 59, 144 Shinozaki, K., 78, 149 Shinwari, Z. K., 79, 144 Shiu, P. K. T., 173, 192 Siddiqui, K. S., 38, 144 Sidhu, G. S., 162, 192 Siegel, D. P., 46, 144 Silva, J. C., 168, 192 Siminovitch, D., 54, 144 Skinner, J. S., 80, 84, 144 Smolenska-Sym, G., 120, 145 Soanes, D. M., 162, 192 Soitamo, A. J., 74, 145 Solecka, D., 70, 145 Solomon, P., 168, 190 Sonoike, K., 41, 145 Staats, M., 167, 192 Stack, D., 162, 192 Stenlid, J., 170, 172, 190 Steponkus, P. L., 39, 44, 49, 51–52, 54, 58, 60, 145 Stitt, M., 64–65, 124, 145 Stockinger, E. J., 79, 145 Storey, R., 62, 149 Strand, A., 54–55, 65–66, 145 Streb, P., 65, 145–146 Stukenbrock, E. H., 167, 192 Sulpice, R., 62, 146 Sunkar, R., 124, 146 Sutton, F., 112, 146 Suzuki, M., 82, 104, 146 Svensson, J. T., 74, 146

T Ta¨htiharju, S., 76, 109, 146 Takagi, T., 54, 146 Tanaka, A., 171, 192 Tasseva, G., 53, 146 Taylor, D., 177, 192 Taylor, J. W., 166, 179, 190, 192

200

AUTHOR INDEX

Teige, M., 114, 146 Tellier, 164 Temporini, E. D., 162, 169, 192 Testerink, C., 120, 146 Thomashow, M. F., 37, 146 Thon, M., 173, 192 Tjus, S. E., 41–42, 147 Townley, H. M., 109, 147 Traut, W., 174, 192 Tremblay, K., 50, 147 Trewavas, A. J., 108, 147 Tschesche, H., 62, 144 U Uemura, M., 45–46, 51–55, 59, 61, 134, 147 V van Belkum, A., 160–161, 181 van der Lee, T., 175, 192 Van Der Luit, A., 109, 147 Van Etten, H. D., 162, 169, 192 van Leuween, W., 122, 147 van Wijk, K. J., 43, 147 Vaultier, M. N., 120, 124, 147 Vazquez-Tello, A., 112, 114, 147 Venekamp, J. H., 62, 147 Vergnolle, C., 117–118, 120, 148 Vijayan, P., 54, 148 Vogel, J. T., 93, 98, 148 Vogt, V. M., 176, 189 VolV, J., 173, 193 W Walton, J. D., 161, 168–169, 193 Wan, B., 112, 148 Wanner, L. A., 74, 148 Wapinski, I., 161, 166, 180, 193 Ward, T. J., 167, 193 Wasternack, C., 78, 148 Wei, H., 71, 148 Wei, W., 167, 193 Welti, R., 118, 148 Wen, J. Q., 115, 148 Whitelam, G. C., 75, 130 Wilch, G., 178, 193 Williams, M. E., 122, 148 Winter, D., 115, 148 Wi, S. J., 63–64, 148 Wisniewski, M., 49–50, 148 Wolfe, K. H., 166, 192

Woloshuk, C. P., 161, 193 Wong, C. E., 78, 148 Wostemeyer, 172 Wren, B. W., 179, 193 Wurdack, K. J., 168, 182 Wyn Jones, R. G., 62, 149 X Xiang, Y., 110, 149 Xiao, H., 87, 149 Xin, Z., 61, 149 Xiong, L., 99, 121, 149 Xiong, Y., 80, 82, 89, 149 Xue, G. P., 84, 149 Xu, J. R., 162, 178, 182, 193 Xu, Z. S., 115, 149

Y Yaish, M. W., 50, 131 Yamada, Y., 62, 130 Yamaguchi-Shinozaki, K., 37, 78, 149 Yamazaki, I., 59, 144 Yamazaki, T., 161, 193 Yang, L., 122, 149 Yang, T., 109, 149 Yang, Z., 167, 193 Yano, R., 56, 58, 149 Yan, S. P., 43, 63, 68, 149 Yan, Z., 178, 193 Yohalem, D. S., 171, 189 Yoo, S. Y., 100, 149 Yoshida, S., 53, 149 Yuen, K., 158–159, 193 Yu, F. L., 161, 193

Z Zakharov, I. A., 164, 193 Zarka, D. G., 86, 96, 104, 150 Zhang, D. Q., 50, 150 Zhang, S., 41–42, 150 Zhang, X., 82, 90, 150 Zhao, J. L., 71, 150, 163, 193 Zhao, T. J., 86, 94, 150 Zhou, N., 89, 150 Zhou, X., 124, 150 Zhu, J. K., 37, 100–102, 150 Zolan, M. E., 160, 173, 193 Zuther, E., 55–56, 150

SUBJECT INDEX

A Actin filaments, 15 AFPs. See Antifreeze proteins agr mutants, 18 Allene oxide cyclase, 77–78 Allopolyploid hybrids, 171–172 Amyloplast sedimentation, 12 Antifreeze proteins (AFPs), 50 Auxin regulated transcription factors, 9 B Brassinosteroids, 18, 77 C Ca2þ/calmodulin-dependent protein kinases, 114 Calcineurin B-like (CBL) proteins, 109–110 Calcium, cold signal transduction calcium signatures, 105–107 circadian-gated, 107 and gene response, 108–109 mechanisms, 107–108 protein activation, 109–110 Calmodulins, 109 Calreticulin, 113 CBL proteins. See Calcineurin B-like proteins CNGCs. See Cyclic nucleotide-gated ion channels Cold signal transduction calcium calcium signatures, 105–107 circadian-gated, 107 and gene response, 108–109 mechanisms, 107–108 protein activation, 109–110 phospholipases and phospholipids phosphatidylinositol (4,5)-bisphosphate, 122 phospholipases C (PLC), 119–122 phospholipases D (PLD), 116–118 protein kinases/protein phosphatases calcium dependent protein kinases (CDPK), 112–113 calreticulin, 113 PKC-like activities and cold responses, 113–116 Cold stress light and hormones responses

abscisic acid (ABA), 75–76 brassinosteroids, 77 jasmonic acid (JA), 77–78 light, 74–75 salicylic acid (SA), 76–77 low temperature eVects freezing temperatures, 39 membranes, 43–47 photosynthesis, 39–43 reactive oxygen species (ROS), 38–39 low temperature responses cell architecture response, 70–73 compatible osmoticum other than sugars, 60–64 membrane composition, 51–54 photosynthesis and related pigments, 64–67 reactive oxygen species scavenging systems, 67–70 stress-related proteins, 47–51 sugars, 54–60 C-repeat-binding factors (CBF) cold expression in Arabidopsis thaliana, 84–86 kinetics of, 86–87 CRT/DRE binding factors in Arabidopsis thaliana, 79–80 low temperature-dependent, 84 structures and signatures, 80–84 CRT/DRE element identification, 78–79 freezing tolerance cold hardening components, 87–91 target identification, 91 ICE1 expression cloning, 94–95 HOS1, 96 other inducers, 96–97 regulon, 95 sumoylation, 95–96 negative regulators FIERY2, 99 FVE gene, 98–99 MYB15, 97 STRS, 98 ZAT12, 98 regulators HOS9, 101 HOS10, 100–101 HOS15, 101–102 LOV1, 100

202

SUBJECT INDEX

C-repeat-binding factors (CBF) (cont. ) SCOF1, 102–103 SRF1, 99–100 Cryptochromes, 5 Cyclic nucleotide-gated ion channels (CNGCs), 14 D Drought responsive element (DRE), 79

eal1 mutants, 12 EGY1 gene, 17

E

F Freeze-induced membrane lesions expansion-induced lysis, 44–46 hexagonal II phase and fracture-jump lesions, 46–47 Fungal spliceosomal introns, 176 G Genome evolution, fungi chromosomal rearrangements chiasma formation, 160 in chromosome structure, 160–161 DNA sequences, 158 gene clusters horizontal gene transfer (HGT), 161 pathogenicity clusters, 162–163 gene families lifestyle-associated, 165 recombination and gene conversion, 165–166 whole-genome duplications (WGD), 166 genome size, 153, 158 genomic organization, 159 horizontal gene transfer (HGT), 168–169 hybridization allopolyploid speciation, 171 meiotic silencing of unpaired DNA (MSUD), 173 origin of, 170 polyploid hybrids, 171–172 introns, 176 mitochondrial genomes, 176–178 vs. pathogenic bacteria, 178–179 pathogenic lifestyle, 158–159 rapidly evolving genes, 167–168 recombinant suppression around MAT loci, 163–164 telomeres, 174–175 transposable elements

array of replication strategies, 172–173 plant and animal species, 172 repeat-induced point mutation (RIP), 174 gil1 mutants, 21 Glycine betaine, 62–63 Gravitropic set-point angle (GSA), 10 Gravitropism definition, 10 intracellular signaling agr mutants, 18 auxin-dependent transduction pathways, 13–14 Cholodny–Went hypothesis, 17 cyclic nucleotide-gated ion channels (CNGCs), 14 ethylene, 17–18 gravitropic transduction pathway, 15 lazy1 phenotype, 19 signaling-response network, 16 light eVects in Arabidopsis, 20, 23 environmental signaling systems, 23 FHY1 and FHL proteins, 22 PIF3 (phytochrome interacting factor 3), 21 sensing gravity protoplast pressure hypothesis, 12–13 starch statolith hypothesis, 12 transient receptor channels (TRC), 13 H Horizontal gene transfer (HGT), 168–169 HOS9 regulators, 101 HOS10 regulators, 100–101 HOS15 regulators, 101–102 Hypocotyl phototropism Arabidopsis, 6 nph1 npl1 double mutants, 3–4

Introns, 176

I

L Low temperature cold eVects cell architecture response cell wall, 70–71 microtubules, 71–73 compatible osmoticum glycine betaine, 62–63 polyamines, 63–64 proline, 61–62 freeze-induced membrane lesions expansion-induced lysis, 44–46 hexagonal II phase and fracture-jump lesions, 46–47

SUBJECT INDEX general eVects chilling temperatures, 38 freezing temperatures, 39 membrane composition bulk composition, 51–52 phospholipid desaturation, 53–54 photosynthesis mechanism chilling eVects, 39, 43–44 photosystems, 42–43 ROS synthesis, 41–42 sucrose synthesis, 40–41 photosynthesis-related pigments adaptation process, 64–66 flavonoids, 67 xanthophylls, 66 reactive oxygen species scavenging systems enzymatic systems, 67–69 non-enzymatic scavenging, 70 stress-related proteins antifreeze proteins (AFPs), 50 cold shock and RNA binding proteins, 50–51 COR/LEA and dehydrins, 47–49 sugars cold acclimation, 54–56 freezing conditions, 59–60 osmotic potential, 58 protein stability, 59 ROS scavenging properties, 60 starch degradation, 56–58 M Mitochondrial genomes, 176–178 N Nonphototropic hypocotyl mutant 3 (NPH3), 7 nph1 mutant, 3 npl1 mutant, 3 P Phospholipases C (PLC) in Arabidopsis suspension cells, 119–120 fiery1 mutant, 121–122 inhibitors of, 120 Phospholipases D (PLD), 110, 116–118 in A. thaliana suspension cells, 116–117 in cold signaling, 118 Phosphorylation gradient model, 9 Phototropism blue-light-induced, 6–7 cryptochromes, 5 definition, 2–3 gravity eVects in Arabidopsis mutants, 7, 23

203

fungus phycomyces, 24 intracellular signaling Arabidopsis mutants, 7 phytochrome kinase substrate proteins, 8 light stimulus responses photomorphogenic and phototropic interplay, 10 transcript profiling, 9 unilateral irradiation, 8–9 photodetection process, 3 photoreceptors, 4 phototropic curvature, 3, 5 phytochromes, 6 in plant species, 4–5 Phytochrome interacting factor 3 (PIF3), 21 Phytochrome kinase substrate (PKS 1–4) proteins in phototrophism, 8 Phytochromes, 6 PKC. See Protein kinase C Polar auxin transport, 17 Polyamines, 63–64 Polyploid hybrids, 171 Protein kinase C Ca2þ/calmodulin-dependent protein kinases, 114 mitogen-activated kinases (MAPK), 114–115 protein phosphatases, 115–116 STY kinase, 115 Protein phosphatases, 115–116 R Repeat-induced point mutation (RIP), 174 Rha1, gravitropic mutant, 19

S STY kinase, 115 Subtelomeres, 175 Suppressive mtDNA, 177 T Telomeres, 174–175 Thermal hysteresis. See Antifreeze proteins (AFPs) Transient receptor channels (TRC), 13

U Unilateral irradiation, 8–9

W WD-40-repeat protein, 101–102