Fruit development and seed dispersal (Annual Plant Reviews, Volume 38)

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ANNUAL PLANT REVIEWS VOLUME 38 Fruit Development and Seed Dispersal

Edited by

Lars Østergaard John Innes Centre, Norwich Research Park, Norwich, UK

A John Wiley & Sons, Ltd., Publication

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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This edition first published 2010
C 2010 Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing programme has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom Editorial office 9600 Garsington Road, Oxford, OX4 2DQ, United Kingdom 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. 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, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Fruit development and seed dispersal / edited by Lars Østergaard. p. cm. – (Annual plant reviews ; v. 38) Includes bibliographical references and index. ISBN 978-1-4051-8946-0 (hardback : alk. paper) 1. Fruit–Development. 2. Seeds–Dispersal. I. Østergaard, Lars. II. Series: Annual plant reviews; v. 38. SB357.283.F78 2010 631.5–dc22 2009020417 A catalogue record for this book is available from the British Library. Annual plant reviews (Print) ISSN 1460-1494 Annual plant reviews (Online) ISSN 1756-9710 R Set in 10/12 pt Palatino by Aptara
Inc., New Delhi, India Printed in Singapore

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Annual Plant Reviews A series for researchers and postgraduates in the plant sciences. Each volume in this series focuses on a theme of topical importance and emphasis is placed on rapid publication. Editorial Board: Prof. Jeremy A. Roberts (Editor-in-Chief), Plant Science Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK; Dr David Evans, School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, OX3 0BP; Prof. Hidemasa Imaseki, Obata-Minami 2419, Moriyama-ku, Nagoya 463, Japan; Dr Michael T. McManus, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand; Dr Jocelyn K.C. Rose, Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA. Titles in the series: 1. Arabidopsis Edited by M. Anderson and J.A. Roberts 2. Biochemistry of Plant Secondary Metabolism Edited by M. Wink 3. Functions of Plant Secondary Metabolites and their Exploitation in Biotechnology Edited by M. Wink 4. Molecular Plant Pathology Edited by M. Dickinson and J. Beynon 5. Vacuolar Compartments Edited by D.G. Robinson and J.C. Rogers 6. Plant Reproduction Edited by S.D. O’Neill and J.A. Roberts 7. Protein–Protein Interactions in Plant Biology Edited by M.T. McManus, W.A. Laing, and A.C. Allan 8. The Plant Cell Wall Edited by J.K.C. Rose 9. The Golgi Apparatus and the Plant Secretory Pathway Edited by D.G. Robinson 10. The Plant Cytoskeleton in Cell Differentiation and Development Edited by P.J. Hussey 11. Plant–Pathogen Interactions Edited by N.J. Talbot 12. Polarity in Plants Edited by K. Lindsey 13. Plastids Edited by S.G. Moller 14. Plant Pigments and their Manipulation Edited by K.M. Davies 15. Membrane Transport in Plants Edited by M.R. Blatt

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16. Intercellular Communication in Plants Edited by A.J. Fleming 17. Plant Architecture and Its Manipulation Edited by CGN Turnbull 18. Plasmodeomata Edited by K.J. Oparka 19. Plant Epigenetics Edited by P. Meyer 20. Flowering and Its Manipulation Edited by C. Ainsworth 21. Endogenous Plant Rhythms Edited by A. Hall and H. McWatters 22. Control of Primary Metabolism in Plants Edited by W.C. Plaxton and M.T. McManus 23. Biology of the Plant Cuticle Edited by M. Riederer 24. Plant Hormone Signaling Edited by P. Hadden and S.G. Thomas 25. Plant Cell Separation and Adhesion Edited by J.R. Roberts and Z. Gonzalez-Carranza 26. Senescence Processes in Plants Edited by S. Gan 27. Seed Development, Dormancy and Germination Edited by K.J. Bradford and H. Nonogaki 28. Plant Proteomics Edited by C. Finnie 29. Regulation of Transcription in Plants Edited by K. Grasser 30. Light and Plant Development Edited by G. Whitelam 31. Plant Mitochondria Edited by David C. Logan 32. Cell Cycle Control and Plant Development Edited by D. Inz´e 33. Intracellular Signaling in Plants Edited by Z Yang 34. Molecular Aspects of Plant Disease Resistance Edited by Jane Parker 35. Plant Systems Biology Edited by Gloria M. Coruzzi and Rodrigo A. Guti´errez 36. The Moss Physcomitrella Patens Edited by Celia Knight 37. Root Development Edited by Tom Beeckman 38. Fruit Development and Seed Dispersal Edited by Lars Østergaard

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CONTENTS

Contributors Preface 1 Carpel Evolution Aur´elie C.M. Vialette-Guiraud and Charlie P. Scutt 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11

The importance of having carpels Hypotheses of carpel origin A phylogenetic framework for studies of carpel evolution A morphological portrait of the ancestral carpel The genetic control of carpel development in the first flowering plants A major role for the E-function in the origin of the carpel? Carpel specification in monocots Gene duplication and carpel evolution in the core eudicots The A-function finds a role in fruit development The multiple origins and mechanisms of syncarpy in the angiosperms A fruit by any other name: evolutionary convergence between angiosperms and gymnosperms References

2 Gynoecium Patterning in Arabidopsis: A Basic Plan Behind a Complex Structure Eva Sundberg and Cristina Ferr´andiz 2.1 Introduction 2.2 The basic plan in lateral organs 2.3 The Arabidopsis gynoecium 2.4 Genetic and hormonal factors controlling gynoecium development 2.5 Conclusion Acknowledgements References 3 The Ins and Outs of Ovule Development Raffaella Battaglia, Monica Colombo and Martin M. Kater 3.1 Introduction 3.2 Origin of the ovule

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Contents 3.3 3.4 3.5 3.6 3.7 4

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Ovule development in Arabidopsis Sporophytic tissues Gametophytic tissue Interaction between the female gametophyte and the maternal sporophyte Ovule identity determination References

Fertilisation and Fruit Initiation Sara Fuentes and Adam Vivian-Smith 4.1 Introduction 4.2 Pollination 4.3 Female receptivity and the cessation of gynoecial growth 4.4 Additional restraints on flower development and fruit initiation 4.5 Fertilisation 4.6 Hormonal cues during fruit initiation 4.7 RNA silencing during fruit initiation 4.8 Signal transduction from ovule to carpel and vascular canalisation 4.9 Current models of fruit initiation 4.10 Concluding remarks Acknowledgements References Arabidopsis Fruit Development Antonio Mart´ınez-Laborda and Antonio Vera 5.1 Introduction 5.2 Morphology of the Arabidopsis silique 5.3 Determining the boundary between valve and replum: valve margin genes 5.4 The making of valves and replum requires repression of valve margin genes 5.5 Suppressors of the rpl phenotype: setting up territories 5.6 A model for patterning the mediolateral axis of the Arabidopsis silique 5.7 Auxin: a signaling molecule for the mediolateral axis? 5.8 A biotechnological view Acknowledgements References Long-Distance Seed Dispersal Frank M. Schurr, Orr Spiegel, Ofer Steinitz, Ana Trakhtenbrot, Asaf Tsoar and Ran Nathan 6.1 6.2

Introduction Six generalizations on LDD mechanisms

72 73 81 91 93 97 107 107 110 113 115 117 123 141 145 147 150 151 151 172 172 174 177 179 182 185 192 195 196 196 204

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6.3 6.4

A vector-based perspective on the evolution and predictability of long-distance seed dispersal Future directions Acknowledgements References

7 Seed Dispersal and Crop Domestication: Shattering, Germination and Seasonality in Evolution Under Cultivation Dorian Q. Fuller and Robin Allaby 7.1 Introduction 7.2 Loss of natural seed dispersal in wheat and barley: archaeobotanical evidence 7.3 Non-shattering in other cereals: rice, pearl millet and maize 7.4 The genetics of non-shattering cereals 7.5 Reduction in seed dispersal aids 7.6 Non-cereal alternative: appendage hypermorphy in fibre crops 7.7 Loss of natural seed dispersal in pulses and other crops 7.8 Germination traits in domestication: the importance of loss of dormancy 7.9 The genetic basis for dormancy and germination 7.10 Germination and seedling competition: changes in seed size 7.11 The genetics of seed size 7.12 Seasonality controls: photoperiodicity and vernalization 7.13 Discussion: evolution and development of domesticated seed traits References 8 Factors Influencing the Ripening and Quality of Fleshy Fruits Cornelius S. Barry 8.1 Introduction 8.2 Control of fruit ripening 8.3 Transcription factors serve as master regulators of fruit ripening 8.4 Hormonal control of fruit ripening 8.5 The influence of light on fruit quality 8.6 The discovery of aroma and flavour genes in fruit 8.7 Cell wall changes influence fruit quality 8.8 The cuticle influences fruit quality and postharvest longevity

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Contents 8.9 8.10

9

Genomics Resources Conclusions and future perspectives Acknowledgements References

Parthenocarpy in Crop Plants Tiziana Pandolfini, Barbara Molesini and Angelo Spena 9.1 Introduction 9.2 Parthenocarpy 9.3 Auxin-synthesis parthenocarpy 9.4 Parthenocarpy via auxin signal transduction 9.5 Parthenocarpy via gibberellin signal transduction 9.6 Aucsia-silencing parthenocarpy 9.7 Auxin sensitivity and parthenocarpy 9.8 Apetalous parthenocarpy and the role of other floral organs 9.9 Stenospermocarpy 9.10 Parthenocarpy in perennial crop plants 9.11 Parthenocarpy and fruit crop breeding 9.12 From green plants to fruit crop plants References

Index Color plate section appears at the start of the book, before Chapter One

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CONTRIBUTORS

Robin Allaby Warwick HRI, University of Warwick, Wellesbourne, Warwick, United Kingdom Cornelius S. Barry A32 Plant and Soil Sciences, Michigan State University, East Lansing, MI, USA Raffaella Battaglia Dipartimento di Biologia, Universit`a degli Studi di Milano, Via Celoria 26, Milano, Italy Monica Colombo Dipartimento di Biologia, Universit`a degli Studi di Milano, Via Celoria 26, Milano, Italy Cristina Ferr´andiz Instituto de Biolog´ıa Molecular y Celular de Plantas (UPV-CSIC), Campus de la Universidad Polit´ecnica de Valencia, Avda de los Naranjos s/n, Valencia, Spain Sara Fuentes Department of Crop Genetics, John Innes Centre, Norwich, United Kingdom Dorian Q. Fuller Institute of Archaeology, University College London, London, United Kingdom Martin M. Kater Dipartimento di Scienze Biomolecolari e Biotecnologie, Universit`a di Milano, via Celoria 26, Milano, Italy Antonio Mart´ınez-Laborda ´ de Gen´etica, Universidad Miguel Hern´andez, Campus de San Division Juan, Ctra. de Valencia s/n, 03550-San Juan de Alicante, Spain

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Contributors Barbara Molesini Dipartimento Scientifico e Tecnologico, Universit`a di Verona, Strada Le Grazie 15, Verona, Italy Ran Nathan Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Tiziana Pandolfini Dipartimento Scientifico e Tecnologico, Universit`a di Verona, Strada Le Grazie 15, Verona, Italy Frank M. Schurr Plant Ecology and Conservation Biology, University of PotsdamMaulbeerallee 3, Potsdam, Germany Charlie P. Scutt Reproduction et Developpement des Plantes, ENS de Lyon, 46 allee d’Italie, Lyon Cedex, France Angelo Spena Dipartimento Scientifico e Tecnologico, Universit`a di Verona, Strada Le Grazie 15, Verona, Italy Orr Spiegel Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Ofer Steinitz Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Eva Sundberg Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden Ana Trakhtenbrot Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel Asaf Tsoar Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel

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Contributors
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Antonio Vera ´ de Gen´etica, Universidad Miguel Hern´andez, Campus de San Division Juan, Ctra. de Valencia s/n, 03550-San Juan de Alicante, Spain Aur´elie C. M. Vialette-Guiraud Reproduction et Developpement des Plantes, ENS de Lyon, 46 allee d’Italie, Lyon Cedex, France Adam Vivian-Smith Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, AL Leiden, The Netherlands

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PREFACE

The reproduction system of flowering plants (angiosperms) provides an extraordinary advantage, which has led to the massive dominance of this division over any other plant division on the planet. At the centre of angiosperm reproduction is the carpel that protects and nurtures the seeds developing inside until they are ready for dispersal. Different species have developed different strategies for efficient seed dispersal involving both short-range and long-range aims allowing widespread colonization. While angiosperms represent an evolutionary success story by themselves, fruit and the seed that they produce have been crucial for human civilization and particularly for crop domestication during the Neolithic revolution. Plant research over the last couple of decades has beautifully demonstrated how fruit provide excellent model systems to move crop improvement even further to increase the yield. Such advances are required now more than ever in order to maintain a sustainable production while meeting the increasing demands from an expanding population. Fruit are, however, not only interesting from an agronomic point of view. Both flowers and fruit are also ideal systems to study cell differentiation processes and tissue-specific development that can answer general questions regarding the development of multicellular organisms. In this book, the aim is to cover the recent impressive advances of research into fruit development and seed dispersal. Moreover, the objective is also to review the history of crop domestication as well as the most novel discoveries in optimizing the development of fruit of significant agricultural importance. The opening chapter describes the evolution of carpels and fruit among the flowering plants and how this has led to a tremendous biodiversity of forms in different angiosperm lineages. The following chapters describe in detail the genetic, hormonal and molecular knowledge on the different stages of development from the unfertilized flower till the mature fruit including gynoecium development, ovule development, fruit initiation and the postfertilization fruit development. Chapter 6 reports on exciting new insight to long distance seed dispersal, while Chapter 7 emphasizes the importance of controlling seed dispersal for the establishment of agriculture and human civilization. The eighth chapter provides a compilation of the most recent discoveries in development and ripening of the agricultural important fleshy fruit and finally, Chapter 9 shows how precision technology can be employed to create seedless varieties in certain crop plants.

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Preface
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The topics that are covered in this volume are intended to provide in-depth knowledge of a wide scope of aspects relating to fruit biology. As such, this book contains information that should be of interest to scientists ranging from experimental biologists to agronomists pursuing technology to exploit the knowledge for crop improvement purposes. Lars Østergaard

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Plate 1 The Arabidopsis gynoecium. The different axes of polarity and morphological terms are indicated (a) scanning electron micrograph of the mature gynoecium at anthesis. (b) Chloral hydrate cleared anthesis gynoecium to reveal vascular patterns. Primary and secondary bifurcations of the medial veins are indicated with arrows. (c) Cross section of the ovary at anthesis.

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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Plate 2 Confocal laser scanning microscopy (CSLM) images of unfertilized and fertilized ovules of Arabidopsis expressing the synthetic auxin-responsive reporter gene DR5rev::eGFP. (a) Unfertilized anthesis ovule with minimal GFP expression. (b) Post-fertilized ovule at 5 h. The first nuclear endosperm division has occurred and GFP expression is observed in the endothelium, the chalazal domain and adjacent to the funiculus vascular strand. (c) Ovule after the third endosperm division (9 h post-fertilization) with eight endosperm nuclei and an elongated zygote. Strong GFP expression occurs in the endothelium, the chalaza and funiculus. Weaker expression is observed in the outer integument. (d) Treatment of detached pistils with NAA (50 µM) for 1 h, with subsequent washing for 7 h, induces strong GFP activation in the funiculus and chalaza, and moderate activation in the inner integument and weaker expression in the outer integument. a, antipodal cells; cc, central cell; cr, chalazal region; e, egg cell; en, endosperm; f, funiculus; fv, funiculus vascular tissue; g, generative cell; ii, inner integument; m, micropyle; p, pollen tube; pn, polar nucleus; oi, outer integument; rv, replum vascular tissues; s, synergid cell; sp, sperm cells; t, endothelium; z, zygote.

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Spatial scale, x (m) Plate 3 (a) The total dispersal kernel (TDK, thick black line) for a hypothetical plant population dispersed by four vectors (different orange tints). The inset shows the unequal distribution of seed loads (Q) on a log scale. The lines at the bottom indicate the range of distances of all seeds dispersed by each vector. (Figure modified from Nathan et al., 1071.) (b) The relative contribution of each vector to the TDK as a function of spatial scale x, showing that the vectors dominating long-distance dispersal of seed can be very different from those dominating seed dispersal at the typical scales of empirical studies.

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Plate 4 In vitro development of parthenocarpic DefH9-iaaM tomato fruit. (a) (Left panel) Flower buds from auxin-synthesis parthenocarpic plants collected at pre-anthesis and cultivated in medium not supplemented with auxin. (Middle panel) Ovaries growth after 10 days of in vitro cultivation. (Right panel) Mature fruits after approximately 30 days of in vitro cultivation. (b) (Left panel) Ovaries present in pre-anthesis (stage a) wild-type (wt) flower buds as compared with the ovaries present in DefH9-iaaM flower buds. (Right panel) DefH9-iaaM pre-anthesis flower bud showing enlarged ovary.

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Annual Plant Reviews (2009) 38, 1–34 doi: 10.1002/9781444314557.ch1

www.interscience.wiley.com

Chapter 1

CARPEL EVOLUTION Aur´elie C.M. Vialette-Guiraud and Charlie P. Scutt Laboratoire de Reproduction et D´eveloppement des Plantes (CNRS UMR 5667-INRA-ENSL-UCBL), Universit´e de Lyon, Lyon Cedex, France

Abstract: The carpel is the progenitor organ to the fruit and a defining feature of the flowering plants, or angiosperms. This organ has evolved in the angiosperms to generate a wide diversity of forms, often related to breeding strategies and seed distribution mechanisms. In this chapter, we focus on a number of key stages in the evolution of the carpel and fruit, about which something can be said of the molecular mechanisms underlying evolutionary change. In particular, we describe hypotheses for the evolutionary origin of the carpel in the first flowering plants and attempt to reconstruct the history of its structural diversification in various major angiosperm groups. In doing so, we concentrate on the genes and mechanisms whose presence can be deduced at key evolutionary stages in the angiosperms, and on molecular-evolutionary processes such as neo- and sub-functionalization, which have moulded these genes and the developmental processes they regulate. We also review the literature on the evolution of syncarpy – a phenomenon of enormous adaptive significance in the angiosperms. Lastly, we describe some examples of convergent evolution that have led to the development of fruit-like structures both within and outside the flowering plants. Keywords: carpel; fruit; evolution; development; angiosperm; syncarpy

1.1 The importance of having carpels The carpel is the female reproductive organ that encloses the ovules in the flowering plants, or angiosperms. By contrast, the ovules of the remainder of the seed plants, the gymnosperms, are most frequently naked structures borne in the axils of leaf-like organs. The carpel is thought to confer several major selective advantages on the flowering plants. Firstly, carpels protect, both physically and biochemically, the ovules within them: many classes of carpel-specific genes encode proteins associated with defence against insects or micro-organisms (Scutt et al., 2003). Secondly, highly efficient systems have evolved to facilitate pollen capture and pollen tube guidance in angiosperm carpel tissues, which probably represent considerable improvements over Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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Fruit Development and Seed Dispersal the mechanisms which bring about fertilization in gymnosperms. Thirdly, during the phase of pollen germination and growth, the carpel provides a site for the operation of self- and inter-specific incompatibility mechanisms which may confer important evolutionary advantages. Accordingly, self-incompatibility prevents close inbreeding, while inter-specific incompatibility prevents too wide hybridizations that may lead to infertile offspring. Fourthly, after fertilization, carpel tissues undergo further developmental changes to form fruits, which protect the developing seeds within them and, at maturity, contribute to the dissemination of these by a wide variety of mechanisms. Carpels and fruits have evolved to generate a tremendous biodiversity of forms in different angiosperm lineages. The novel carpel and fruit structures thus generated are often linked to diversification in factors including pollinators, breeding systems, seed structure and seed dispersal mechanisms. In numerous angiosperm groups, carpels have fused together to form a syncarpic pistil. A syncarpic arrangement provides a single stigmatic surface, giving a common point of access to all the ovules in the flower. Syncarpy may also allow for heavier pollination vectors and larger fruits with more elaborate seed dispersal mechanisms. For all of the above reasons, carpels and fruits were almost certainly of key importance in the evolutionary success of the angiosperms, which arose from an unknown common ancestor living some 160 MYA (million years ago) (Davies et al., 2004) to generate over 300 000 species alive today. The immense biodiversity of carpel and fruit development in the extant angiosperms means that no thorough or comprehensive treatment of this subject can realistically be undertaken. In this chapter, we will therefore concentrate on a few key stages in the evolution of carpels and fruits, about which something can be said of the molecular mechanisms underlying evolutionary change. Accordingly, we first describe a number of hypotheses for the evolution of the first carpels and fruits in the flowering plant lineage and review the literature on the likely state of the female reproductive structures in the last common ancestor of the extant angiosperms. We then describe the molecular and morphological differences between carpel development which have arisen following speciation events at two key stages in angiosperm evolution: the last common ancestors of the euangiosperms (including monocots and eudicots) and of the core eudicots (including rosids, asterids and Caryophyllales). We particularly concentrate on such molecular-evolutionary processes as sub- and neo-functionalization, as these apply to the genes of carpel and fruit development. As syncarpy represents an evolutionary change within the angiosperms that was clearly of enormous adaptive significance, we also review the literature on this subject, again concentrating on examples on which something can be said of the molecular mechanisms involved. Lastly, we look briefly at fruit-like structures that, to a botanist, are not fruits. Examples of convergent evolution that has generated structures resembling fruits can be found both within and outside the flowering plants.

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Carpel Evolution
3

1.2 Hypotheses of carpel origin The origin of the carpel is intimately linked to that of the flower – a structure for which several technical definitions have been proposed. For example, Arber and Parkin (1907) suggest ‘an ampisporangiate strobilius of determinate growth and with an involucrum of modified bracts’. According to this definition, the flower equates to a single, compacted developmental axis, or strobilus, rather than a multiply branched system in which each floral organ would correspond to a reduced strobilus. The male and female reproductive organs of the flower are separated into two zones: a central gynoecium, containing the carpels, is typically surrounded by an androecium, containing the stamens. A number of hypotheses have been formulated to account for the unique features of the flower (Fig. 1.1). As our interest here centres on the carpel, we will concentrate on what these hypotheses have to say about that organ type. It should first be mentioned that a hypothesis proposed by Goethe, the German philosopher, poet and dramatist (von Goethe, 1790), which is now well supported by molecular genetic evidence (Honma and Goto, 2001), regards all plant lateral organs, including carpels, as mutually homologous. Such lateral organs, which form on the flanks of the stem apical meristem or floral meristems, can accordingly be regarded as variants of a basic leaf-type developmental ground plan. Though carpels may be homologous to leaves, these reproductive organs are almost certainly more directly related to leaflike organs in the reproductive structures of gymnosperms. On this subject, the existing hypotheses for flower origin divide conceptually into two types, depending on whether they regard the carpel as derived by the modification of male or female structures in the presumed gymnosperm-like ancestor of the flowering plants. The Mostly Male Theory (MMT) (Frohlich and Parker, 2000; Frohlich, 2003) postulates the flower to be mainly derived from the male strobili, or male cone-like structures, of a gymnosperm-like ancestor. According to this hypothesis (Fig. 1.1a), the ancestor of the flowering plants would first have generated ectopic ovules on (male) microsporophylls, which would thereby have become bisexual. The MMT postulates that ectopic ovules were concentrated on sporophylls near the apex of the strobilus, and that the sporophylls bearing these ovules subsequently lost their ability to produce microsporangia, thus becoming female. These newly female sporophylls would then have closed around the ovules to form, in effect, the first carpels. In subsequent evolutionary steps, the residual, entirely female strobili of these proto-flowering plants would have been lost, leaving only bisexual reproductive axes containing apical carpels and basal microsporophylls which would later become stamens. The MMT is based on evidence from a number of sources, including molecular evidence linked to the LEAFY (LFY) gene, which acts upstream of genes that specify the identities of floral organs in typical angiosperm flowers. In at

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Figure 1.1 Hypotheses for the origin of the flower and its carpel. (a) According to the Mostly Male Theory (Frohlich, 2003), ectopic ovules developed on previously male sporophylls. In a second step, these sporophylls lost their (male) microsporangia and closed around the ovule to form the carpel. The outer integument of the angiosperm ovule (thick line) was formed from a pre-existing female cupule structure. (b) According to the Out-of-Male hypothesis (Theissen et al., 2002), the basipetal movement of male-determining, MADS-box B-function gene expression (shaded area) in a male strobilus left female structures at the apex, which later became carpels. (c) According to the Out-of-Female hypothesis (Theissen et al., 2002), the acropetal movement of MADS-box B-sister gene expression (shaded area) in a female strobilus left male structures at the base, which later became stamens. Female structures at the apex became carpels. (d) According to the Baum and Hileman hypothesis (Baum and Hileman, 2006), a temporal switch in the transcriptional regulation of B- and C-function MADS-box genes by LEAFY (LFY) occurred in an ancestor of the flowering plants. This change generated high concentrations of C-function-rich MADS-box protein complexes at late developmental stages, causing the patterning of the strobilus into apical female and basal male reproductive structures, which later became carpels and stamens, respectively.

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least certain gymnosperm taxa, a paralogue of LFY, termed NEEDLY (NLY), appears to be expressed principally in female cones (Mouradov et al., 1998). The orthologue of NLY seems to have been lost from the angiosperm lineage, after its separation from that of the living gymnosperms. The MMT postulates that the loss of NLY was accompanied by a more extensive loss of female-specific developmental programmes during the evolution of the flower. Hence, the MMT regards the carpel and the rest of the flower, with the exception of the ovule, as historically male. It should be noted that several studies have brought into question the proposed sex-specific expression of LFY and NLY in gymnosperms (Carlsbecker et al., 2004; Dornelas and Rodriguez, 2005). Vazquez-Lobo et al. (2007) have recently postulated a rather different partitioning of functions between these genes into early and late roles in reproductive development. However, it should also be noted that the hypothesized sex-specific expression of LFY and NLY in the (unknown) ancestor of the flowering plants is not an absolute requirement for the MMT. Rather, the loss of NLY is correlative evidence for the MMT, of which the key postulate is that of ectopic ovule development, which leads to the evolution of carpels from previously male sporophylls. An attractive feature of the MMT is that it provides an explanation for several unique aspects of reproductive development in angiosperms. For example, this hypothesis is formulated with fossil gymnosperms of the extinct order Corystospermales in mind as potential ancestors of the flowering plants. Corystospermales produced ovules enclosed within cupules, which were borne on unisexual female axes. According to the MMT, such female cupules would have become the outer integument of the ovule – a developmental feature which, like the carpel, is specific to the angiosperms. Several further hypotheses of flower origin have been proposed, which differ from the MMT in that they postulate the bisexuality of the flower to have arisen by a spatial or temporal change in factors governing the sex of reproductive organs. Hence, these hypotheses do not, in contrast to the MMT, postulate the extensive loss of female developmental programmes during angiosperm evolution. It follows that these various hypotheses would regard the carpel as homologous to the female reproductive structures in gymnosperms. The Out-of-Male (OOM) hypothesis (Theissen et al., 2002) proposes the bisexual flower to have evolved by the basipetal movement of male-promoting, B-class MADS-box gene expression in a previously male strobilus, leaving female structures at the apex (Fig. 1.1b). A sister hypothesis to the OOM hypothesis, the Out-of-Female (OOF) hypothesis (Theissen et al., 2002), postulates a sex-determining role for B-sister MADS-box genes, whose expression is proposed to have moved acropetally in a female strobilus to leave male structures in basal positions (Fig. 1.1c). The identification of the function of a B-sister gene in Arabidopsis thaliana, which proves to determine coloration in the outer integument (Nesi et al., 2002), has hardly provided support for the OOF variant of the above two hypotheses. However, it is certainly possible that B-sister genes played a much more central role in

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Fruit Development and Seed Dispersal female organ identity in early stages in the evolution of the flowering plants, and may continue to do so in basal flowering plant lineages. In general, therefore, the OOM and OOF hypotheses look below the level of LFY and NLY in the hierarchical control of gene expression, and postulate a spatial change in MADS-box gene expression, forming a boundary of B- or B-sister class gene expression in a previously unisexual strobilus, which would thereby have become bisexual. Baum and Hileman (2006) have formulated a further, nameless, hypothesis (we will call it the B&H hypothesis), to account for the evolution of the first flowers (Fig. 1.1d). Like the MMT, the B&H hypothesis proposes a central role for LFY in the origin of the flower, but postulates that the origin of floral bisexuality was caused, not by the loss of female-specific developmental programmes, but by a temporally generated switch in the response to LFY. According to this hypothesis, LFY protein builds up over time in the meristems of developing reproductive axes and, at a certain threshold of LFY concentration, these meristems switch from the production of (male) microsporoplylls to (female) megasporophylls. This hypothesized switch may involve the action of LFY cofactors, such as the ancestors of the Arabidopsis F-box protein UNUSUAL FLORAL ORGANS (UFO), and transcription factor WUSCHEL (WUS). Whatever the precise mechanism, the B&H hypothesis proposes that a difference occurred in the relative response to LFY of Band C-class MADS genes during early flower evolution. Accordingly, C-class proteins are proposed to have predominated at high LFY concentrations, encountered at the apex of the strobilus at late developmental stages, resulting in MADS-box complexes that were rich in C-class proteins. These proteins would have formed C-rich complexes which would then have specified the development of megasporophylls at the apex of the strobilus. The above hypotheses are, to some extent, testable. Baum and Hileman (2006), for example, propose a list of predictions that could be tested in basal angiosperm and gymnosperm lineages to support or refute their hypothesis. The MMT stands out from the other hypotheses in proposing the extensive loss of female developmental programmes during early flower evolution. This prediction might provide a means to eliminate either the MMT, or the other contending hypotheses, from consideration. Essentially, if the MMT were correct, we might expect to find numerous classes of genes with femalespecific expression patterns in gymnosperms, whose orthologues have apparently been lost from the angiosperm lineage. Gymnosperm genes with male-specific expression patterns should not be affected in this way. The fullscale testing of the MMT by this method has yet to be performed. However, one question mark concerning such a test relates to the degree to which male and female developmental programmes in gymnosperms might be based on different sets of genes, rather than on subtle differences in the expression patterns of a common set of genes. If the latter is predominately the case, this relatively simple method of hypothesis testing may be unavailable. All of the hypotheses discussed above for the evolution of the flower concentrate to a large extent on the origin of bisexuality, with only the MMT

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explicitly accounting for the origin of the carpel. Additionally, it must be admitted that even the MMT does not go into detail on the molecular mechanism of carpel closure. Thus, even if substantial evidence were to favour one of the above hypotheses, elevating it to the status of a theory, many of the mechanistic gaps would still have to be filled in concerning carpel evolution.

1.3 A phylogenetic framework for studies of carpel evolution Evolutionary studies of the carpel and other unique features of the flowering plants are possible in part due to the contribution of molecular data to phylogenetic analyses of the flowering plants and their relatives. Studies in the 1990s (Goremykin et al., 1996; Winter et al., 1999) indicated the extant seed plants to divide into the two monophyletic groups of the extant angiosperms and gymnosperms, respectively (Fig. 1.2). These findings replaced an earlier hypothesis, based on morphological data, that the small gymnosperm order Gnetales might form a sister group to the flowering plants, as discussed by Donoghue and Doyle (2000). Rather, it would seem that the living gymnosperms, including Gnetales, are monophyletic and share a more distant common ancestor with the flowering plant lineage, which would have lived some 300 MYA (Savard et al., 1994; Goremykin et al., 1997). According to the current consensus view of seed plant phylogeny, the living gymnosperms can be divided into five groups, with Cycadales in the most basal position, followed by the monotypic Ginkgoales, represented only by Ginkgo bioloba. In the crown group of living gymnosperms, most molecular phylogenetic studies split the conifers into two, placing Pinaceae in a sister position to Gnetales, as discussed by Kuzoff and Gasser (2000). This split leaves a clade of remaining conifers (Fig. 1.2) that, following the classification of Page (1990), would be composed of Araucariaceae, Cephalotaxaceae, Cupressaceae, Phyllocladaceae, Podocarpaceae, Sciadopityaceae, Taxaceae and Taxodiaceae. Molecular phylogenetic analyses have also clearly identified the firstdiverging lineages within the angiosperm clade, as reviewed by Kuzoff and Gasser (2000). According to these studies, three extant lineages, Amborellales, Nymphaeales and Austrobaileyales, collectively known as the ANA grade, would have diverged from a remaining common lineage at an early stage in the evolution of the flowering plants (Fig. 1.2). Amborellales contains the single species Amborella trichopoda, a small tree which is endemic to the tropical island of New Caledonia in the Southern Pacific. Nymphaeales contains three families of herbaceous aquatic plants: Nymphaeaceae, Cabombaceae, and the recently added Hydatellaceae (Saarela et al., 2007). Austrobaileyales contains the four families of Austrobaileyaceae, Illiciaceae, Schissandraceae and Trimeniaceae, which are mostly shrubs, climbers or small trees. There is good evidence, both from phylogenetic analyses and from INDEL (insertion/deletion) mutations (Aoki et al., 2004; Stellari et al., 2004), that Amborellales

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Figure 1.2 The phylogeny of the seed plants, based on a consensus of molecular phylogenetic studies. The numbers of species in major clades are given in parentheses, while approximate dates of divergence are taken from Davies et al. (2004), based on a calibration of the molecular clock using fossil data. The positions of some of the taxa referred to in the text are indicated as follows: Am, Amborella trichopoda; An, Antirrhinum majus; Ar, Arabidopsis thaliana; Ca, Cabomba; Cr, Catharanthus roseus; Il, Illicium; Ze, Zea mays (maize); Pe, Petunia hybrida; Or, Oryza sativa (rice).

and Nymphaeales diverged from the remaining angiosperm lineage before Austrobaileyales. However, the relative order of divergence of Amborellales and Nymphaeales remains unclear. Most molecular phylogenies have placed Amborellales alone in the most basal position (e.g. Zanis et al., 2002), while others have grouped it together with Nymphaeales in a first-diverging clade (Qiu et al., 2001). The remaining angiosperm lineage, after the divergence of the ANA grade, diversified to give five groups with living representatives: Cerratophyllum, Chloranthanceae, eumagnoliids, monocots and eudicots (Fig. 1.2). Of these,

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the eudicots and monocots together account for over 95% of the estimated 300 000 extant angiosperm species, while some 6500 species of eumagnolids are known (Davies et al., 2004). The short internal branches connecting the five groups of euangiosperms, compared to their long terminal branches, mean that even very large DNA data sets have failed to convincingly resolve their relative points of divergence within the flowering plants (Moore et al., 2007). Within the eudicots, molecular phylogenetic analyses have clearly identified a number of early-diverging lineages, of which Ranunculales occupies the most basal position. The divergence of these basal lineages leaves a crown group eudicots that includes the major clades of the rosids, asterids and Caryophyllales (Fig. 1.2). The last common ancestor of these ‘core eudicots’ seems to have been a critical stage in angiosperm evolution, perhaps of equal evolutionary significance as the origin of the flowering plants itself. Indeed, many of the genes controlling flower development in model eudicots seem to be derived from a large-scale or whole-genome duplication that occurred in an ancestor of the core eudicots (Litt and Irish, 2003; Vandenbussche et al., 2003; Kramer and Hall, 2005). Phylogenetic studies do not of themselves explain the origin of the flower and its carpel. However, these studies provide an essential framework in which we may attempt to answer these questions, which Charles Darwin famously described as an ‘Abominable Mystery’. Part of the mystery surrounding the origin of the flower is the evolutionary distance of the flowering plants from their nearest living relatives, the extant gymnosperms, with no continuum of intermediate forms known from the fossil record. Molecular clock estimates, calibrated from well-documented fossil divergences within the angiosperms, suggest the last common ancestor of this group to have lived some 160 MYA (Davies et al., 2004). This date is in reasonable agreement with the first appearance of angiosperm fossils, corresponding to the Lower Cretaceous period, some 130 MYA (Friis et al., 2005). We must therefore conclude that the origin of the carpel, among other unique angiosperm features, occurred some 140 MY after the last common ancestor of the seed plants. It seems likely that the flower resulted from a combination of molecular and ecological or environmental factors. On the molecular level, a large-scale gene or whole-genome duplication seems to have preceded the radiation of the angiosperms (De Bodt et al., 2005), from which many pairs of paralogous genes that function in reproductive development have been retained. This hypothesized duplication may therefore have provided the raw material for neo-functionalization events on a large scale, which may have been necessary to generate such a novel structure as the flower. However, the generation of polyploids, corresponding to whole-genome duplications, is relatively common in plants (much more so than in animals). It therefore seems probable that the appearance of flowering plants in the Late Jurassic or Early Cretaceous, rather than at any earlier time since the last common ancestor of the seed plants, occurred not only because of a whole-genome duplication, but also in response to specific ecological or environmental conditions. Among such

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Fruit Development and Seed Dispersal ecological factors, co-evolution with novel groups of insects that acquired roles as pollinators seems likely to be of significance (Barrett and Willis, 2001). Certain novel features that contributed to the success of the early flowering plants may have been external to the flower itself. It has been proposed, for example, that the first flowering plants were shrubs or small trees, with a short lifespan compared to their gymnosperm-like ancestors (Feild et al., 2003, 2004). These somewhat ephemeral species are proposed to have found a successful ecological niche in disturbed soils along shaded riverbanks. Other suggestions for ecological factors that may have helped select for the flowering plants include a change in the feeding behaviour of herbivorous dinosaurs, related to a change in their jaw anatomy, and a change in atmospheric carbon dioxide concentration, as discussed by Barrett and Willis (2001). These conjectures serve to illustrate that the fullest possible explanation for the origin of the angiosperms will be achieved only by combining molecular data with the results of morphological and ecological studies and, if possible, with key evidence from fossil groups. In the absence of a living, close relative to the flowering plants, at least some novel fossil evidence would seem to be essential if we are to fill in the exasperating gap of 140 MY of evolution which preceded the origin of the flower.

1.4

A morphological portrait of the ancestral carpel

Morphological comparisons of ANA grade angiosperms (Fig. 1.3a–f) have enabled a number of conclusions to be made on the likely state of the flower, and of the carpel, in the last common ancestor of the living flowering plants (Endress and Igersheim, 2000; Endress, 2001). Accordingly, the flowers of this ancestral species were probably small, bisexual and protogynous. Its carpels were likely to have been simple (apocarpic), rather than fused together into a syncarpic pistil. The stigmatic tissues that permitted the capture of pollen grains in the angiosperms’ ancestor were likely to have been covered in multicellular protrusions and would probably have secreted a sticky liquid to hold and supply water to pollen grains during germination. Pollen tubes would then have grown towards the ovary through a canal or aperture in the carpel, which would have contained substances secreted from the carpel margins. Self-incompatibility (SI) systems operating between female tissues and pollen grains are present in some ANA angiosperms, including Austrobaileya scandens (Prakash and Alexander, 1984) and Trimenia moorei (Bernhardt et al., 2003). However, it is not yet clear whether these SI systems in distantly related Austrobaileyales are homologous, and still less certain that such a system would be ancestral in the entire angiosperm clade. Interestingly, Amborella, the only representative of the likely most basally diverging angiosperm lineage, Amborellales, avoids inbreeding by dieocy, rather than through an SI mechanism. However, female Amborella flowers contain a nonfunctional stamen, or staminode, which would seem to indicate Amborella to be descended from a bisexual ancestor.

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Figure 1.3 A comparison of carpel structures in ANA grade and core eudicot taxa. (a) Longitudinal section of an Amborella trichopoda (ANA grade, Amborellales, Amborellaceae) carpel (cr) showing its single anatropous ovule (o) and stigmatic crest (sc) of ridged tissue, which harbours an aperture through which pollen tubes may grow. (b) Transverse section of an A. trichopoda female flower bud showing the five separate carpels (cr) of the apocarpic gynoecium. (c) Longitudinal section of a Cabomba aquatica (ANA grade, Nymphaeales, Cabombaceae) flower bud showing two anatropous ovules (o) attached to the placenta (p) of one of the three carpels (cr) present. (d) Longitudinal (slightly oblique) section of a C. aquatica flower bud showing the secretion-filled canal (ca) in the style, through which pollen tubes may grow. (e) Transverse section of a C. aquatica flower bud showing the ovary tissues of the three separate carpels (cr). One or both ovules (o) are visible in each carpel. (f) Transverse section of a C. aquatica flower bud showing the secretion-filled canal (ca) in the style of each carpel. (g) Longitudinal section of the syncarpic gynoecium (gy) of Petunia hybrida (core eudicots, asterids, Solanaceae) showing many ovules (o) attached to an axile placenta (p). (h) Transverse section of the P. hybrida gynoecium showing its two fused carpels, axile placentation (p) and many ovules (o). (i) Transverse section of the syncarpic gynoecium (gy) of Arabidopsis thaliana (core eudicots, rosids, Brassicaceae), which is divided into two locules by the post-genital development of a vertical septum (vs). Placentation (p) is parietal. All scale bar represent 250 µm.

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Fruit Development and Seed Dispersal The carpel of the ancestral angiosperm probably contained a single ovule of anatropous placentation. This ovule would probably have been covered by two integuments that enclosed an embryo sac and a large nucellus (for more on ovule development, see Chapter 3 of this issue). Double fertilization, leading to the production of an embryo and a bi-parental endosperm, also appears to be a pleisiomorphic feature of the angiosperms. However, the likely cellular arrangement of the embryo sac in the last common ancestor of the angiosperms, and the ploidy of its endosperm tissue, remain open to question. In the majority of flowering plants, the embryo sac arrangement is of the Polygonum type, which contains seven cells, one of which, the central cell, is binucleate (Fahn, 1982). The two nuclei of the central cell combine with one sperm nucleus on fertilization of the Polygonum-type embryo sac to generate a triploid endosperm. In Nuphar (Williams and Friedman, 2002) and Hydatella (Friedman, 2008) of Nymphaeales and in Illicium (Williams and Friedman, 2004) of Austrobaileyales, the embryo sac contains only four cells, including a uninucleate central cell. Double fertilization in these ANA grade species generates a diploid embryo and a diploid, rather than a triploid, endosperm. However, studies of Amborella, which represents the likely most basally diverging ANA grade lineage, indicate a different embryo sac arrangement. The Amborella embryo sac contains eight cells, including a binucleate central cell that produces a triploid endosperm after fertilization (Friedman, 2006). The extra cell in the Amborella embryo sac, by comparison to the Polygonum type, is in the egg apparatus, which thus contains four cells in Amborella. Interestingly, Hydatella produces a perisperm, or embryo-nourishing tissue derived from maternal cells, which develops significantly prior to fertilization (Friedman, 2008). This feature resembles the arrangement in gymnosperms, in which entirely maternal tissues fulfil the role of nourishing the embryo, and in which substantial reserves are laid down before fertilization is effected. In the absence of a clear conclusion on the embryo sac arrangement in the last common ancestor of the extant angiosperms, the multiplicity of arrangements in ANA grade angiosperms has been interpreted as a sign of early diversification, prior to the selection of features which became standard in the majority of flowering plant groups (Friedman, 2006).

1.5

The genetic control of carpel development in the first flowering plants

Using molecular techniques to compare ANA grade angiosperms with model plants, we can now begin to describe the mechanisms likely to have controlled carpel development in the ancestor of the living flowering plants. Carpel development in the model taxa Arabidopsis and Antirrhinum is specified, according to the ABC model of flower development, through the expression of Cfunction MADS-box transcription factors, in the absence of B-function factors (Coen and Meyerowitz, 1991). The molecular genetic ABC model has more

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recently been extended to an ABCE model, in which E-function MADS-box factors form various higher order complexes with those of the A-, B- and C-functions (Pelaz et al., 2000; Honma and Goto, 2001; Theissen and Saedler, 2001). According to this model, carpel development is specified by tetramers containing two molecules of E-function, and two molecules of C-function, MADS-box transcription factors (Fig. 1.4a). The unisexual reproductive structures of gymnosperms appear to be specified by the expression of orthologues of B- and C-clade MADS-box genes (Tandre et al., 1995; Becker et al., 2000; Jager et al., 2003; Zhang et al., 2004), certain of which show similar activities to their Arabidopsis orthologues in transgenic Arabidopsis plants (Tandre et al., 1998; Winter et al., 2002; Zhang et al., 2004) (Fig. 1.4b). These data strongly suggest that some basic elements of the regulation of flower development have been conserved since the last common ancestor of the living seed plants, well before the origin of the flower. Phylogenetic analyses of the MADS-box family in ANA grade angiosperms and in gymnosperms clearly indicate that a duplication event took place in the C-function MADS-box lineage prior to the last common ancestor of the living flowering plants (Kim et al., 2005). As a result of this duplication, the ancestors of the clade-defining genes AGAMOUS (AG) from Arabidopsis thaliana, and FLORAL BINDING PROTEIN7 (FBP7) from Petunia hybrida (reviewed by Kramer et al., 2004) were generated. The AG clade contains angiosperm C-function genes, whereas the FBP7 clade contains genes involved in ovule development in diverse taxa, including Petunia, Arabidopsis and rice. The role of FBP7-like genes in ovule development has been defined as the D-function (Angenent et al., 1995; Colombo et al., 1995). This function is postulated to be necessary for ovule development, and its inactivation leads to supernumerary carpels that develop ectopically in the place of ovules. Interestingly, the FBP7 (D-function) clade appears to have been lost from Ranunculales (Kramer et al., 2004) and, as will be discussed later, has evolved to share its ovule development function with genes of the AG clade in some eudicots, including Arabidopsis. Both of these observations suggest a degree of functional fluidity between MADS-box genes of the related C- and D-clades. In addition to the C- and D-functions, two clades of SEPALLATA (SEP) genes, which encode E-function MADS-box proteins, have been found in basal angiosperms. The genes SEP1, SEP2 and SEP4 from Arabidopsis appear to be orthologous to one of these ANA grade SEP-clades, while SEP3 appears to be orthologous to the other (Zahn et al., 2005). The expression of C-function genes in ANA grade angiosperms is mostly limited to the third and fourth floral whorls, while the E-function genes of these species are expressed in all floral organs (Kim et al., 2005) (Fig. 1.4c). These expression patterns closely resemble those of the corresponding genes in Arabidopsis, suggesting important elements of the control of carpel identity to have been conserved in distinct lineages throughout angiosperm evolution. Kim et al. (2005) did however note some expression of C-function genes in the perianth organs of the ANA grade angiosperms Amborella (Amborellales)

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Figure 1.4 The ABCE model of flower development in Arabidopsis, and its derivatives in other taxa. (a) In Arabidopsis, A-, B-, C- and E-function floral homeotic genes, expressed in overlapping domains (horizontal bars) of the floral meristem, control the identities of floral organs in a combinatorial manner: A + E specifies sepal development in the first whorl, A + B + E specifies petal development in the second whorl, B + C + E specifies stamen development in the third whorl, and C + E specifies carpel development in the fourth whorl. In addition, the C-function causes an arrest of organ proliferation (the ‘STOP’ function) in the fourth whorl. AG, AGAMOUS; AP1, APETALLA1; AP2, APETALA2; AP3, APETALA3; PI, PISTILLATA; SEP1–4, SEPALLATA1–4. (b) In gymnosperms, B- and C-clade MADS-box genes are expressed in a combinatorial manner in male (B + C) and female (C alone) reproductive structures, resembling the expression of their Arabidopsus orthologues in male and female floral organs. (c) In ANA grade angiosperms, B- and C-clade MADS-box gene expression resembles that of the respective Arabidopsis orthologues, though with less well-defined boundaries. Strong B-clade gene expression is generally detected in the outer floral whorl of ANA grade angiosperms, possibly reflecting an absence of developmental differentiation between whorls 1 and 2. A-clade MADS-box gene expression differs radically between ANA grade angiosperms and Arabidopsis, extending throughout the flower and into leaves. (d) In rice flowers, A-, B- and E-function genes are expressed in similar patterns to those of their Arabidopsis orthologues to specify specialized perianth organs (paleas, lemmas and lodicules) and stamens. Two paralogous C-clade MADS-box genes show a partial sub-functionalization between the third and fourth whorls, with one paralogue playing a major role in stamen development in the third whorl, while the other plays a major role in the ‘stop’ function in the fourth whorl (thick arrows, major roles; thin arrows, minor roles). The YABBY gene DROOPING LEAF (DL) plays a major role in carpel development that is independent of C-clade MADS-box gene expression. DL may act directly on carpel development (solid arrow), or indirectly by limiting the inner boundary of B-function gene expression (dashed arrow), or both of these.

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and Illicium (Austrobaileyales), in contrast to the more strictly delimited expression patterns of C-function genes in model eudicots. As pointed out by these authors, this observation may reflect the rather gradual transition of floral organ types that is apparent in ANA grade angiosperms, rather than a more fundamental difference in the regulation of carpel development between early and later diverging plant lineages. In addition to MADS-box genes, the expression patterns of several YABBY transcription factors have been analyzed in ANA grade angiosperms. In Arabidopsis, these factors participate in the specification of abaxial cellular identity in lateral organs by defining the side of these organs that faces away from the developmental axis (Bowman, 2000). CRABS CLAW (CRC) is a YABBY gene that is expressed only in the abaxial tissues of the gynoecium and in the nectaries of Arabidopsis flowers (Bowman and Smyth, 1999). AmbCRC, a putative CRC orthologue from Amborella, shows a similar pattern of expression in carpels to that of CRC in Arabidopsis (Fourquin et al., 2005), suggesting these genes to have conserved a common developmental role since the speciation event that separated their lineages at the base of the flowering plants. CRC is a direct target of AG in Arabidopsis (Gomez-Mena et al., 2005), though it is not yet known whether such a direct control relationship exists between the Amborella orthologues of these two genes. INNER NO OUTER (INO) represents a further YABBY gene with a very specific role in female reproductive development in Arabidopsis. INO is expressed in the outer ovule integument, and its inactivation causes the loss of this angiosperm-specific tissue (Villanneva et al., 1999). A putative INO orthologue from the ANA grade angiosperm Nymphaea alba is expressed in both ovule integuments and in the suspensor (Yamada et al., 2003). The broadly similar expression patterns of INO orthologues between the ANA grade angiosperm Nymphaea and the eudicot Arabidopsis suggest the conservation of a role in integument development since the last common ancestor of the flowering plants. CRC and INO are unusual in the YABBY family in showing very specific expression profiles: the other members of this family in Arabidopsis are more generally expressed in the abaxial zone of both vegetative and reproductive plant lateral organs (Bowman, 2000). The carpel and outer integument, in which CRC and INO are respectively expressed, represent pleisiomorphic features of the angiosperms. The relationships of these structures to reproductive organs in gymnosperms could therefore be highly informative of the mechanism by which the flower evolved. Hence, it would be extremely interesting to know whether CRC and INO orthologues exist in gymnosperms, and if so, to determine their exact expression patterns. In general, the search for carpel development genes in ANA grade angiosperms has highlighted several instances of the broad conservation of gene functions since the common ancestor of the last flowering plants. In particular, mechanisms involving C- and E-function genes, that specify carpel development at a high level in gene hierarchies, seem to be conserved. However,

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Fruit Development and Seed Dispersal much work remains to be done in this field. Many families of transcription factors of known importance to carpel development in Arabidopsis, including the auxin response factor (ARF), basic Helix–Loop–Helix (bHLH), MYB and NAC families, have yet to be examined in any detail in ANA grade angiosperms. As major elements of the ABCE model appear to be conserved between angiosperms and gymnosperms, it is at a lower level in regulatory hierarchies, perhaps involving the families listed above, that we might discover the molecular changes that were responsible for the evolution of the carpel in early flowering plants. EST resources are proving very useful for the analysis of flower development orthologues in numerous seed plant groups (Brenner et al., 2003; Albert et al., 2005; Brenner et al., 2005; Pavy et al., 2005, 2007). However, in any given investigation, the use of such database resources must frequently be complemented by onerous approaches such as cDNA library screening and reverse-transcriptase PCR. With the advent of novel, high-throughput sequencing technologies, the sequencing of the Amborella genome from the ANA grade, which has a c-value of 0.89 pg (Leitch and Hanson, 2002), has recently been proposed (Soltis et al., 2008) (The c-value indicates the mass of DNA in one haploid chromosome complement and is thus a convenient measure of genome size.) The sequencing of the Amborella genome and other genomes from the ANA grade would certainly provide an invaluable resource for studies of the origin of the flowering plants. The complete sequences of one or more gymnosperm genomes would also be extremely useful in this regard, though this seems still some way off due to the large genome sizes of this group. Gymnosperm genomes range in size from the relatively modest (for a gymnosperm) c-value of 2.25 pg for Gnetum ula (Ohri and Khoshoo, 1986) to the very large value of 36.00 pg for Pinus ayacahuite (Grotkopp et al., 2004). In particular, the cycads, which are of special interest for their basal position in the gymnosperms, all have large genomes. For example, Cycas revoluta has a c-value of 12.75 pg (Ohri and Khoshoo, 1986), some 80-fold larger than that of Arabidipsis thaliana (Bennett et al., 2003).

1.6

A major role for the E-function in the origin of the carpel?

E-function MADS-box genes play fundamentally important roles in flower development in Arabidopsis. The transcription factors encoded by these genes are hypothesized to act together with combinations of A-, B- and C-function proteins in quaternary complexes that specify the type of floral organ that develops in each whorl of the flower (Pelaz et al., 2000; Honma and Goto, 2001; Theissen and Saedler, 2001). As discussed above, two E-function clades seem to have been present in the last common ancestor of the extant flowering plants (Zahn et al., 2005), perhaps not very long after the origin of the flower. Interestingly however, no SEP-like genes have been found in gymnosperms

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(Becker and Theissen, 2003). It might therefore seem reasonable to postulate that the origin of the E-function, leading to the generation of quaternary MADS-box complexes, was of fundamental importance for the origin of the flower. Theissen and Melzer (2007) have discussed the possibility that, before the flower, dimers of C-function genes may have specified the development of female reproductive organs, and that the evolution of quaternary MADSbox protein complexes, incorporating both C- and E-function proteins, may thus have represented a key step in the evolution of the carpel. More precisely, the evolution of such quaternary complexes is hypothesized to have caused transcription factor binding to two distinct MADS-box binding motifs, which are termed CArG boxes, in the cis-acting control regions of their downstream target genes. According to this hypothesis, the newly evolved binding behaviour of quaternary MADS-box complexes, also involving Aand B-function proteins in different combinations, would have generated the necessary multiplicity of interactions to specify at least three organ types in early flowers, carpels, stamens and tepals, with the possible later division of tepals into distinct whorls of petals and sepals. Quaternary complexes might also have led to a positive cooperativity of binding to multiple sites in target gene promoters, which might in turn have generated a steeper gradient of transcriptional response between MADS-box genes and their direct targets. Such a steep gradient of response could have generated sharper transitions between the reproductive organs and produced the concomitant compaction of the axis that is apparent in the angiosperm flower. In discussing the evolutionary role of the E-function, it should be noted, however, that the origin of this function is, like that of the angiosperms, rather mysterious. The four SEP genes of Arabidopsis form an E-function clade that is closely related to the Arabidopsis gene AGAMOUS-LIKE6 (AGL6). Orthologues of AGL6, rather than those of the combined AGL6 + SEP-clade, appear to be present in gymnosperms (Carlsbecker et al., 2004). If this phylogenetic interpretation is correct, the SEP-clade would seem to have been lost from the extant gymnosperm lineage, rather than generated by a gene duplication event in the angiosperm lineage. In this case, the origin of the angiosperm SEP-clade could not have correlated with that of the flower. Even if the above phylogenetic interpretation is incorrect, and SEP genes did originate specifically in the angiosperm lineage, the presence of two distinct SEP-lineages in ANA grade angiosperms suggests the SEP-clade to have existed for some time prior to the origin of the flower. If indeed the SEP-clade considerably predates the flower, the formation of quaternary MADS-box complexes involving SEP proteins may also be far more ancient than the flower itself, and hence perhaps not a key factor in flower origin. Furthermore, the formation of quaternary MADS-box complexes may not involve SEP proteins in all plant groups. For example, AGL6 proteins, as close relatives of SEP proteins, might participate in quaternary MADS-box complex formation in gymnosperms. Careful attention should now be paid to the formation of complexes of MADS-box proteins in both angiosperms and gymnosperms

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Fruit Development and Seed Dispersal to attempt to address these questions. At present, it is not clear whether the origin of quaternary MADS-box protein complexes correlated with the origin of the flower.

1.7

Carpel specification in monocots

The monocots form a large, monophyletic group of angiosperms whose lineage diverged after the divergence of the ANA grade lineages, probably around 145 MYA (Davies et al., 2004). The functional comparison of carpel development genes in monocot and eudicot models provides evidence of differences in the molecular mechanisms specifying carpel development in these two groups. Genes controlling floral organ identity have been analyzed at a functional level in rice and maize of the Poaceae or grass family. Phylogenetic analyses suggest at least one major gene duplication event to have occurred in the MADS-box C-clade prior to the separation of the rice and maize lineages, with an additional duplication in one of the two sub-clades generated in that duplication, specifically in the maize lineage. Accordingly, the rice C-clade gene OsMADS58 appears orthologous to the maize gene ZAG1, while OsMADS3 from rice appears orthologous to both ZMM2 and ZMM23 from maize (Yamaguchi et al., 2006). The phenotypes associated with mutations in C-clade genes have been investigated in both rice and maize, though more thoroughly in the former of these species. The inactivation of OsMADS58 in rice leads to defects in carpel development, though it does not eliminate carpels (Yamaguchi et al., 2006) (Fig. 1.4d). In addition to abnormal carpels, osmads58 mutants show reduced floral determinacy, indicating a major contribution of this gene to the so-called ‘stop’ function, which arrests the proliferation of organs in the fourth whorl. Whereas OSMADS58 appear to act mainly in the fourth floral whorl, the inactivation of its paralogue OsMADS3 has little or no effect on either carpel development or floral determinacy. Instead, stamen development is eliminated in osmads3 mutants (Kang et al., 1998; Yamaguchi et al., 2006). Rice plants in which both OsMADS3 and OsMADS58 have been inactivated produce aberrant carpels, similar to those of the osmads58 mutant, suggesting OsMADS3 to make no unique contribution to carpel development (Yamaguchi et al., 2006). In maize, zag1 mutants show a defect in floral determinacy, indicating functional conservation of ZAG1 with its rice orthologue OsMADS58. It seems, therefore, that C-clade genes in the grass family have undergone significant sub-functionalization, following a monocot-specific gene duplication. The multiple roles of the single Arabidopsis gene AG in carpel development, stamen development and floral determinacy are thus shared in a whorl-specific manner between two and three C-clade genes in rice and maize, respectively. Additionally, in these monocot species, carpel development can occur independently of C-clade MADS-box genes, suggesting that some other factor may be involved in the specification of carpel development. The functions of a paralogous pair of D-clade genes, OsMADS13 and OsMADS21, have been investigated in rice (Dreni et al., 2007). Of these,

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OsMADS13 shows ovule-specific expression and appears to play a classical D-function role in ovule development: inactivation of OsMADS13 results in the ectopic conversion of ovules into internal carpelloid organs. Interestingly, OsMADS21 appears to make no significant contribution to the D-function, and is expressed more widely in female reproductive tissues. It is thus tempting to speculate that OsMADS21 might show genetic redundancy in the control of carpel development with rice C-clade MADS-box genes. Double and triple knockout mutants between osmads21, osmads58 and osmadsS3 would be required to address this question. Though the inactivation of C-clade genes does not lead to the loss of carpels in rice flowers, carpels are entirely replaced by ectopic stamens in mutants in which the YABBY gene DROOPING LEAF (DL) has been inactivated (Yamaguchi et al., 2004) (Fig. 1.4d). dl mutants also show a developmental defect in leaves, which consequently lack a mid-rib. DL is the likely rice paralogue of CRC, though it clearly shows a considerable functional difference from CRC in Arabidopsis. DL expression is maintained in the carpels of rice plants in which both OsMADS3 and OsMAD58 have been inactivated (Yamaguchi et al., 2006), demonstrating its action to be independent of these C-function genes. This may represent a difference from the situation in Arabidopsis, in which CRC is a direct target of AG (Gomez-Mena et al., 2005). It is not yet clear whether carpel development depends on DL expression per se, or whether DL is mainly responsible for preventing B-function gene expression in the fourth floral whorl. Experiments that combine B-clade, C-clade and dl mutations in rice might help to evaluate the role of DL in floral patterning, and/or in carpel specification. The conservation of expression patterns of CRC orthologues between Arabidopsis and ANA grade angiosperms (Fourquin et al., 2005), as discussed above, suggests the distinct roles of DL in carpel identity and leaf development (Yamaguchi et al., 2004) to have arisen specifically in the monocot lineage. Experiments in which Arabidopsis crc-1 mutants were rescued by transformation with CRC orthologues from various species (Fourquin et al., 2007), however, showed the DL coding sequence to be capable of restoring near wild-type carpel development when expressed from the Arabidopsis CRC promoter. These experiments indicate the CRC and DL coding sequences to show similar activities in carpel development, suggesting that upstream factors may be largely responsible for the novel function shown by DL in carpel specification, or in the definition of the inner limit of the third whorl, in rice. It is not yet clear whether changes to the DL coding sequence, in addition to changes to its regulatory region, may have been necessary for the evolution of the leaf development function of DL in rice. Transformation of dl mutants with constructions containing eudicot CRC coding sequences may help to answer this question. SEP genes, which are necessary for carpel development in eudicots, are also known from monocots. OsMADS1 from rice corresponds to the LEAFY HULL STERILE1 locus, and groups within the same clade as SEP1, SEP2 and SEP4 from Arabidopsis (Zahn et al., 2005). Outer whorl floral organs in

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Fruit Development and Seed Dispersal osmads1 loss-of-function mutants take on a leaf-like appearance, whereas the inner whorl floral organs of these mutants are partially converted to paleas and lemmas, which are normally found only in the outer two whorls of rice flowers (Agrawal et al., 2005). These results suggest OsMADS1 to be a principal component of the E-function in rice (Fig. 1.4d), while the functions of the four remaining rice SEP genes, OsMADS5, OsMADS7, OsMADS8 and RMADS217 (Zahn et al., 2005), remain to be determined. In general, carpel and ovule development in the highly derived Poaceae of the monocots seem to depend on the orthologues of regulatory genes that are known to play key roles in these processes in Arabidopsis and other eudicots. However, duplications have taken place in several MADS-box gene lineages in Poaceae, including the C-, D- and E-function lineages, in some cases leading to sub-functionalization events between paralogous genes. The precise limits of this sub-functionalization have not yet been defined, which might explain the currently hidden component of the specification of carpel identity in monocots.

1.8

Gene duplication and carpel evolution in the core eudicots

The core eudicots form a monophyletic group that contains several very successful molecular genetic model species, including Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida. This group is estimated to descend from a last common ancestor that lived around 110 MYA (Davies et al., 2004). Analysis of the Arabidopsis genome sequence has provided evidence of a largescale duplication event that probably occurred not long before the divergence of the main core eudicot lineages, the rosids, asterids and Caryophyllales (De Bodt et al., 2005). As discussed above, several classes of MADS-box genes in core eudicots contain pairs of paralogues that are orthologous to single genes in basal eudicots, such as Ranunculales, and which may therefore have been generated in the hypothesized core eudicot genome duplication event. The retention of many of these pairs of paralogues over long periods of evolutionary time would appear to be a clear indication that sub- and neofunctionalization processes have occurred, rendering both copies of each pair essential or advantageous to survival. In the core eudicots, two C-clades are present in place of an ancestral paleoAG clade in basally diverging eudicot lineages. In Arabidopsis, the euAG clade contains the AG gene itself, while the PLENA (PLE) clade (Fig. 1.5), contains a pair of paralogous genes termed SHATTERPROOF1 and SHATTERPROOF2 (SHP1/2), which resulted from a more recent duplication in the Arabidopsis lineage. In Antirrhinum majus, the probable orthologue of AG is termed FARINELLI (FAR), while that of SHP1/2 is the clade-defining gene PLE. Interestingly, the non-orthologous genes AG and PLE are responsible

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(a) Arabidopsis thaliana AG sub-clade (AG)

PLE sub-clade (SHP1 and SHP2)

Stamen Carpal identity identity

(b) Antirrhinum majus AG sub-clade (FAR)

Fruit dehiscence zone development

PLE sub-clade (PLE)

Carpel identity Stamen Pollen identity fertility STOP function

STOP function

Ovule identity Funiculus and seed abscission layer development

FBP7 sub-clade (STK)

(c) Petunia hybrida AG sub-clade (PMADS3)

PLE sub-clade (FBP6)

FBP7 sub-clade (not characterized)

(d) Phylogeny of C-clade MADS-box genes

STOP

Stamen function identity Carpel identity

FBP-clade (D)

AG-clade (C) PLE-clade (C) Ovule identity

Pre-angiosperm duplication FBP7 sub-clade (FBP7 and FBP11)

Core-eudicot duplication

Figure 1.5 Fluidity in the functionalization of C- and D-function MADS-box genes in the core eudicots. (a–c) Venn diagrams representing the functions of genes from the MADS-box clades AG (C-function), PLE (C-function) and FBP7 (D-function) in three species of core eudicots. Overlapping regions represent functional redundancy between genes in wild-type genetic backgrounds. AG, AGAMOUS; FAR, FARINELLI; FBP, FLORAL BINDING PROTEIN; PLE, PLENA; SHP, SHATTERPROOF. (d) The sequence of duplications that generated of the eudicot AG, PLE and FBP7 MADS-box gene clades.

for specifying the C-function in Arabidopsis and Antirrhinum, respectively (Davies et al., 1999; Kramer et al., 2004) (Fig. 1.5). FAR, by contrast, is redundantly involved in stamen development and is required for pollen fertility in Antirrhinum. In an example of neo-functionalization, the paralogues SHP1 and SHP2 play a novel role in Arabidopsis fruit development (Liljegren et al., 2000). In Petunia hybrida, which, as a member of the asterids, is more closely related to Antirrhinum than to Arabidopsis (Fig. 1.2), a further case of sub-functionalization is apparent. The Petunia AG orthologue, PMADS3, is principally responsible for stamen development (Kapoor et al., 2002), though it also plays a redundant role with the PLE orthologue FLORAL BINDING PROTEIN6 (FBP6) in carpel development and floral determinacy (Kramer et al., 2004).

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Fruit Development and Seed Dispersal Though sub-functionalization between the paralogous AG and PLE clades in Arabidopsis has left AG playing the major C-function role, elegant experiments involving multiple mutants show that the SHP genes have retained a capacity for C-function activity, perhaps reflecting the C-function role of the common ancestor, prior to the radiation of the core eudicots, which these genes share with AG. Ectopic carpelloid organs develop in the first floral whorl of Arabidopsis ag mutants, conditionally on the inactivation of APETALLA2 (AP2), a gene which contributes to the A-function (Bowman et al., 1991). This effect is thought to occur because AP2 is responsible for downregulating C-clade MADS-box genes in the outer floral whorls of wild-type plants. In the case of ag/ap2 double mutants, the C-function activity responsible for specifying ectopic carpel development in the first whorl is provided by SHP1 and SHP2, as evidenced by the fact that first whorl organs of quadruple ap2/ag/shp1/shp2 mutants are devoid of all carpelloid features (Pinyopich et al., 2003). These data indicate a subtle effect of functional overlap between paralogous gene clades, which does not equate to simple genetic redundancy: the SHP genes adopt novel C-function-like roles in ap2/ag mutants which they do not play in wild-type plants. The fluidity of functions among duplicated genes in the core eudicots is further illustrated by an exchange of function between C- and D-clade MADSbox genes. Two paralogous D-function genes in Petunia, FBP7 and FBP11, are redundantly essential for ovule development (Angenent et al., 1995). The probable Arabidopsis orthologue of these genes, SEEDSTICK (STK), is also involved in ovule development, though STK shares this role redundantly with the C-clade genes SHP1 and SHP2 (Fig. 1.5). Accordingly, the Arabidopsis stk/shp1/shp2 triple mutant (Pinyopich et al., 2003), like the Petunia fpb7/fpb11 double mutant (Angenent et al., 1995), produces supernumerary carpels in the place of ovules within the gynoecium. In addition to its redundant role in ovule specification, STK plays non-redundant roles in the development of the funiculus and in seed abscission in Arabidopsis (Pinyopich et al., 2003). The combined C+D-clade in the eudicots, whose members were derived from duplication events that occurred both before and after the radiation of the angiosperms, therefore represents a complex situation in which diverse evolutionary processes have taken place. These processes include subfunctionalization between paralogous genes, exchanges of function between paralogous genes, exchanges of function between non-paralogous genes, and, finally, neo-functionalization to generate novel fruit shattering mechanisms (Fig. 1.5).

1.9

The A-function finds a role in fruit development

A further likely consequence of the hypothesized genome duplication at the base of the core eudicots is the generation of a second sub-clade of MADS-box genes within the A-clade (Litt and Irish, 2003). The A-function MADS-box gene APETALLA1 (AP1) plays roles in floral meristem patterning and in the

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specification of perianth (petal and sepal) organ identity in Arabidopsis. This latter role corresponds to the A-function, as defined by the ABC and ABCE models. However, gene duplications in the core eudicots have provided further A-clade sequences, one of which appears to have been recruited to carpel and fruit development somewhere along the lineage leading to Arabidopsis. Accordingly, the Arabidopsis A-clade MADS-box gene FRUITFULL (FUL) is involved in the patterning of the gynoecium and fruit wall (Gu et al., 1998). FUL is known to act in a network involving a large number of genes (Roeder et al., 2003; Liljegren et al., 2004), including the MADS-box genes SHP1 and SHP2 (Ferrandiz et al., 2000) that also function redundantly with STK in ovule development, as described above. Gene duplication in the A-function clade of MADS-box genes has thus resulted in novel fruit shattering mechanisms in the Brassicaceae by the process of neo-functionalization. An interesting feature of gene-duplication in the A-clade is the evolution of a distinct C-terminal protein motif in AP1 genes, apparently produced by a frame-shift mutation that occurred towards the 3
-extremity of the coding sequence in an ancestor of the core eudicots (Litt and Irish, 2003). This frameshift created a farnesylsation site in the encoded protein that is known to be post-translationally modified in vivo in Arabidopsis and which is required for wild-type AP1 protein activity (Yalovsky et al., 2000). Other frame-shift mutations in duplicated genes are present in the B- and C-function MADS-box clades of the eudicots (Vandenbussche et al., 2003). However, the conserved motifs generated in these cases are distinct from that of the AP1 lineage and do not contain farnesylation sites. The novel C-terminal motifs present in certain lineages within the eudicot A-, B- and C-clades of MADS-box genes have been conserved over a long period, clearly indicating their functional significance. However, it is not yet known whether the functions of these novel motifs are connected with biochemical processes in common, such as the higher order assembly or sequestration of MADS-box transcription factor complexes (Vandenbussche et al., 2003).

1.10

The multiple origins and mechanisms of syncarpy in the angiosperms

Most angiosperm flowers possess more than one carpel. As discussed above, the carpels of species from the early-diverging ANA grade lineages are typically separate structures that occur in a spiral arrangement at the centre of the flower (Fig. 1.3a–f). Such an arrangement, with separate carpels, is termed apocarpic and, from its presence in early-diverging lineages, appears to represent the pleisiomorphic condition of the angiosperms. However, more than 80% of extant angiosperm species are syncarpic: their carpels are fused into a single female structure in the centre of the flower (Endress, 1982) (Fig. 1.3g–h). Various morphological sequences have been described which lead to carpel fusion in syncarpic species (Verbeke, 1992). These developmental processes

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Fruit Development and Seed Dispersal can be conceptually divided into two types, based on the timing of the fusion event involved. Accordingly, in cases in which carpels are fused from the earliest emergence of carpel primordia, the organ fusion is termed ‘congenital’, whereas in cases in which carpels are observed to fuse together during development, the fusion is termed ‘post-genital’. Congenital carpel fusion is the most common type, whereas post-genital carpel fusion is known from only a handful of angiosperm families (Lolle and Pruitt, 1999). Arabidopsis, which typifies gynoecium development in the Brassicaceae, is a good example of congenital carpel fusion. At developmental stage 6 of Arabidopsis flowers (Smyth et al., 1990), a central dome emerges from the flower meristem which will become the syncarpic gynoecium. By developmental stage 7, this dome begins to invaginate by a reduction in growth rate at its centre to generate a slot, which forms in line with the pair of lateral stamen primordia of the Arabidopsis flower bud. The tissues on either side of this slot, perpendicular to the lateral stamen primordia, then grow out to meet in the middle, eventually forming a vertical septum that divides the ovary of the Arabidopsis gynoecium into two locules. The gynoecium wall undergoes considerable differentiation at the extremities of the vertical septum to define the dehiscence zones that will permit pod shattering in the mature fruit. Interestingly, though Arabidopsis is a clear example of a species showing congenital syncarpy, it is not absolutely clear how many carpels are fused together in the Arabidopsis gynoecium. Most recent authors (Okada et al., 1989) have interpreted this structure as containing two carpels, corresponding to the two locules of the (secondarily) divided ovary. This view is to some extent supported by molecular genetic studies, which have succeeded in isolating a number of mutants in which carpel fusion is affected, resulting in the division of the Arabidopsis gynoecium into two separate carpelloid organs. However, the ovary wall contains four vascular traces in Arabidopsis, two of which occur at either extremity of the vertical septum, and two in positions corresponding to the valves of the ovary. Hence, it is entirely possible that there are four carpels in the wild-type Arabidopsis gynoecium (Lawrence, 1951). According to this view, the valve carpels would have become sterile, whereas the carpels at either extremity of the vertical septum, corresponding to the positions of the placentae, would have remained fertile. Postgenital syncarpy has been best characterized in Catharanthus roseus (The Madagascar Periwinkle) of the Apocynaceae. In this species, two separate carpel primordia are initiated and grow until their inner surfaces come into contact (Walker, 1978; Siegel and Verbeke, 1989; Verbeke, 1992). The already differentiated epidermal cells of these surfaces then begin to interlock and redifferentiate into parenchyma. This redifferentiation is dependent on diffusible, water-soluble substances produced by the carpels themselves and takes a total of about 9 h (Siegel and Verbeke, 1989). Even before epidermal cell redifferentiation has terminated, plasmodesmata can be observed to form between the surface layers of the fusing carpels (Vanderschoot et al., 1995). Walker (1978) has shown that the pollen tubes are then able to cross between

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the carpels, thus fulfilling one of the principal theoretical reasons for the evolution of syncarpy, which are explained in greater detail below. The major evolutionary advantage of syncarpy is probably to allow a regular repartition of pollen tubes within the gynoecium by the formation of a ‘compitum’, which consists of tissues that permit pollen tube transfer between carpels (Endress, 1982). Accordingly, a visit to a syncarpic by a single pollinator might result in full seed set. Another potentially important advantage of syncarpy results from the enhanced competition between pollen tubes that this phenomenon produces, which may act as a filter of fitness by selecting for vigorous male parents. Syncarpy also allows the production of larger fruits, with potentially more complex and efficient seed dispersal mechanisms (Walker, 1978; Endress, 1982; Armbruster et al., 2002). A further advantage of syncarpy may stem from its potentially lesser requirement for cell wall synthesis, compared to an apocarpic gynoecium of similar total size. Though most representatives of basally diverging angiosperm lineages are morphologically apocarpous, Endress (1982) has noted several unusual constructions that confer a degree of functional syncarpy. In Nymphaea (Nymphaeaceae), for example, large quantities of mucilage are secreted from the stigmatic tissues of the separate carpels. Pollen tubes are able to grow through the resulting mucilage layer, which is sufficiently extensive as to form a bridge between the carpels. In Tambourissa of the eumagnolid family Monimiaceae (Laurales), a ‘hyperstigma’ is generated by the entire female flower, which forms a cup that fills with mulicage into which pollen grains may fall and germinate (Endress, 1982). The separate carpels of Tambourissa develop in this floral cup, and are thus potentially accessible to any pollen grain present. Functional syncarpy in the ANA grade genus Illicium (Austrobaileyales) is achieved by the growth of pollen tubes in a groove around a central axis which connects the separate carpels (Williams et al., 1993). By mapping different arrangements of morphological and functional syncarpy onto a phylogeny of the angiosperms, Armbruster et al. (2002) estimated that there have been at least 17 independent transitions from apocarpy to syncarpy during angiosperm evolution. Only two instances, by contrast, of a likely change from syncarpy to morphological apocarpy were noted. As syncarpy seems to have arisen several times independently in the angiosperms, it is possible that distinct molecular mechanisms have been recruited to bring about carpel fusion in distantly related syncarpic groups. In Arabidopsis, numerous mutations, including aintegumenta, crabs claw, ettin, leunig, spatula and tousled, generate various degrees of carpel separation, either singly or in double mutant combinations (Sessions and Zambryski, 1995; Roe et al., 1997; Alvarez and Smyth, 1999; Liu et al., 2000). It is not, however, clear whether these genes played any role in the evolution of syncarpy in the Arabidopsis lineage. Rather, the inactivation of such key regulators of carpel development might disrupt the delicate and labile process of carpel fusion, which perhaps evolved through other, unrelated molecular changes. One genetic system that may potentially have been responsible for the generation of

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Fruit Development and Seed Dispersal congenital syncarpy, however, does seem worthy of particular mention. The genes CUP-SHAPED COTYLEDON1 and 2 (CUC1/2) (Aida et al., 1997) are members of the NAC transcription factor family which promote, in a partially redundant manner, organ separation in a number of positions in the plant, including the cotyledons, floral meristems and leaf margins. Both CUC1 and CUC2 are, together with five other NAC genes, negatively regulated by the MIR164 family of microRNAs, which contains three genes in Arabidopsis. Interestingly, both the triple mutant mir164abc (Sieber et al., 2007), and plants transformed with a miR164-resistant allele of CUC2 (Nikovics et al., 2006), show complete carpel separation, thus implicating the negative regulation of CUC2 by miR164 in the process of carpel fusion in Arabidopsis. The CUC and MIR164 system may represent a conserved developmental module that has been recruited many times independently over the course of angiosperm evolution to modify certain highly variable traits in the angiosperms, such as leaf dissection (Nikovics et al., 2006). It is therefore possible that this developmental module has also been independently recruited to generate syncarpy in distinct angiosperm lineages. The transformation of diverse syncarpic species with miR164-resistant alleles of their own native CUC genes could help to shed some light on this question. A clue to the potential molecular mechanism of post-genital carpel fusion in C. roseus and other taxa comes from the Arabidopsis mutant fiddlehead (fdh). In fdh mutants, all above-ground organ types, including leaves, stems, sepals, petals and stamens, tend to fuse together on contact (Lolle et al., 1992). In addition, wild-type Arabidopsis pollen can readily germinate and emit pollen tubes into leaf and other non-carpel tissues of fdh mutants (Lolle and Cheung, 1993). FDH encodes an enzyme necessary for the generation of the waxy cuticle (Pruitt et al., 2000). Inactivation of FDH makes the cuticle much more permeable to small molecules, and this effect also seems to be conserved in Antirrhinum majus (Efremova et al., 2004). Interestingly, the transfer of small water-soluble molecules is known to be involved in post-genital carpel fusion in C. roseus (Siegel and Verbeke, 1989). It thus seems plausible that carpel fusion in C. roseus and other taxa showing post-genital fusion might depend on the down-regulation, specifically in the contacting surface of developing carpels, of a gene such as FDH that is necessary for cuticle formation.

1.11

A fruit by any other name: evolutionary convergence between angiosperms and gymnosperms

The fruit is a pleisiomorphic character of the angiosperms, and to use this term to describe non-angiosperm seed bearing structures might be regarded as botanical heresy! However, it is interesting to note that the reproductive structures of several groups of living gymnosperms have evolved to superficially resemble angiosperm fruits. For example, the aril of the yew, Taxus baccata (Taxaceae), is red and fleshy at maturity, and contributes to

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seed dissemination by forming a food source for birds. Arils in Taxaceae and Taxodiaceae develop from ovuliferous scales that grow around the seed after pollination. These structures are thus quite distinct from angiosperm fruits, which are formed by the modification of pre-existing carpel tissues. Similarly, in Gnetum of the gymnosperm order Gnetales, a fruit-like structure is generated from two extra tissue layers that form around the unitegmetic ovule. In the case of Gnetales, the production of fruit-like structures represents just one of a long list of evolutionary convergences with angiosperms (Donoghue and Doyle, 2000). For example, a form of double fertilization is present in this group, though this leads to the production of a second, inviable embryo, rather than endosperm tissue (Friedman, 1998). Other evolutionary convergences of Gnetales with angiosperms include an apical meristem that is divided into a tunica and corpus, vessel elements in xylem tissues, a lack of archegonia, net-veined leaves (in Gnetum), morphologically bisexual male strobili (in Gnetum and Welwitschia), and even insect pollination (in some Gnetum spp.) (Kato et al., 1995). Structures playing the role of fruits, but which are not derived from carpels, have also evolved in some angiosperm groups. Examples of such false fruits include the ‘pome’, which develops from the receptacle in Rosaceae including Malus and Pyrus, and the false berry of Vaccinium spp.

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Fruit Development and Seed Dispersal Yalovsky, S., Rodriguez-Concepcion, M., Bracha, K., Toledo-Ortiz, G. and Gruissem, W. (2000) Prenylation of the floral transcription factor APETALA1 modulates its function. Plant Cell 12, 1257–1266. Yamada, T., Ito, M. and Kato, M. (2003) Expression pattern of INNER NOOUTER homologue in Nymphaea (water lily family, Nymphaeaceae). Development Genes and Evolution 213, 510–513. Yamaguchi, T., Lee, D.Y., Miyao, A., Hirochika, H., An, G.H. and Hirano, H.Y. (2006) Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa. Plant Cell 18, 15–28. Yamaguchi, T., Nagasawa, N., Kawasaki, S., Matsuoka, M., Nagato, Y. and Hirano, H.Y. (2004) The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa. Plant Cell 16, 500–509. Zahn, L.M., Kong, H., Leebens-Mack, J.H., Kim, S., Soltis, P.S., Landherr, L.L., Soltis, D.E., de Pamphilis, C.W. and Ma, H. (2005) The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout Angiosperm history. Genetics 169, 2209–2223. Zanis, M.J., Soltis, D.E., Soltis, P.S., Mathews, S. and Donoghue, M.J. (2002) The root of the angiosperms revisited. Proceedings of the National Academy of Sciences of the United States of America 99, 6848–6853. Zhang, P.Y., Tan, H.T.W., Pwee, K.H. and Kumar, P.P. (2004) Conservation of class C function of floral organ development during 300 million years of evolution from gymnosperms to angiosperms. Plant Journal 37, 566–577.

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Annual Plant Reviews (2009) 38, 35–69 doi: 10.1002/9781444314557.ch2

www.interscience.wiley.com

Chapter 2

GYNOECIUM PATTERNING IN ARABIDOPSIS: A BASIC PLAN BEHIND A COMPLEX STRUCTURE Eva Sundberg1 and Cristina Ferr´andiz2 1

Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden 2 Instituto de Biolog´ıa Molecular y Celular de Plantas (UPV-CSIC), Campus de la Universidad Polit´ecnica de Valencia, Avda de los Naranjos s/n, Valencia, Spain

Abstract: The Arabidopsis gynoecium consists of two congenitally fused carpels that, at maturity, form a bilocular chamber protecting the ovules and placentae produced by the meristematic regions of the carpel margins. This meristematic region also gives rise to a style capped with stigmatic papillae at the apical end of the developing gynoecia and a transmitting tract that connects the stigma to the ovule-bearing chambers. Most data point towards a common evolutionary origin of leaves and carpels and suggest that leaves can be transformed to carpels by expressing only a few carpel identity genes. In this review, we have therefore approached the carpels from the leaf-like organ hidden within by stressing the parallels between leaf and carpel development. Many of the genes with a role in leaf development were first identified by the effect their mutations cause in carpel development, suggesting that the regulatory networks may be more robust in leaves than in the more complex and evolutionary younger carpels. Similar genetic networks ensure the maintenance of adaxial–abaxial, proximal–distal and medial–lateral dichotomies in leaves and carpels. Data have emerged showing that crosstalk and redundancies are characteristics of these pathways, as well as a general interplay of hormonal balances, with auxin as a major morphogen. Keywords: gynoecium; fruit; patterning; hormones; auxin; development

2.1 Introduction In 1735, the Swedish botanist Carolus Linneaus published the first version of Systema Naturae where he presented a sexual system for classifying Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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Fruit Development and Seed Dispersal flowering plant species according to the number of male and female organs, a system both easily accessible and useful, although not completely correct in evolutionary terms. Linnaeus became fascinated by the complexity, beauty and the romantic aura of the plant reproductive system and in an essay, shockingly to some of his contemporaries, he portrayed the more or less enclosed space formed by the petals as a bridal chamber and the gynoecium, the female reproductive structure and most complex organ of the flower, as a bride. This extraordinary bride consists of one or more ovule-bearing organs, so-called carpels, whose structures have contributed to their evolutionary success and have been used to define the group of flowering plants, which are termed angiosperms in Greek, meaning ‘seed enclosed in a vessel’ (angion, vessel and sperma, seed). The ingenious bridal organ protects the ovules, assists in the selection of male gametophytes and aids pollen tube growth for successful pollination. After fertilization of the ovules, the gynoecium develops into a fruit that initially protects and subsequently disperses the seeds. All present data are consistent with Goethe’s early hypothesis that floral organs originate from ancestral vegetative leaf-like organs (Goethe, 1790). For example, ectopic expression of a set of floral organ identity genes is sufficient to convert leaves to floral organ-like structures (Pelaz et al., 2001; Castillejo et al., 2005), and, accordingly, loss of function of SEPALLATA 1 (SEP1), 2, 3 and 4 results in the transformation of all floral organs into vegetative leaves (Honma and Goto, 2001; Ditta et al., 2004). The hypothesis that carpels have evolved from leaves, probably spor-producing sporophylls, is therefore widely accepted. However, it is still debated which ancestral type of sporophyll was recruited and how it evolved into a closed gynoecium (reviewed in Scutt et al., 2006 and Chapter 1 in this book). Molecular and genetic research in the model plant Arabidopsis thaliana has provided us with basal knowledge of the frameworks controlling gynoecium development. Reviews picturing what is known about the regulation of carpel and fruit development in Arabidopsis have recently been published (Dinneny and Yanofsky, 2005; Roeder and Yanofsky, 2005; Balanz´a et al., 2006). Here, we aim to complement those reviews and to summarize the recent progress in the field. As in Balanz´a et al. (2006), we have attempted to approach the carpels from the leaf-like organ hidden within by stressing the parallels between leaf and carpel development. We have also highlighted the role of hormones in gynoecium morphogenesis now that the view on hormone action in relation to development has rapidly become more comprehensive.

2.2

The basic plan in lateral organs

Most data point towards a common evolutionary origin of leaves and floral organs. It is likely that the pathways regulating the basic organ plan inherited from the leaf-like ancestral organs are active still during floral organ

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development, although perhaps in a slightly modified manner downstream of the floral identity genes. In strong support of this hypothesis, the majority of floral organ patterning mutants are also defective in the development of the basic plan of leaves. So, how is patterning of lateral organs, formed at the flanks of the shoot apical meristem (SAM), regulated? Huge progress has recently been made in discerning genetic networks promoting leaf initiation and patterning. Several in-depth reviews have recently been published in this field of plant development (Tsukaya, 2003, 2006; Byrne, 2005, 2006; Fleming, 2005; Piazza et al., 2005; Aida and Tasaka, 2006; Kepinski, 2006; Shani et al., 2006; Barkoulas et al., 2007) and the aim in this section is only to give a brief overview of how the basic plan of lateral organ develops in Arabidopsis, which will be used in later sections as a framework to present the gynoecium. 2.2.1 Lateral organ initiation The first questions we would like to address is: How are lateral organs initiated? And what changes are required at the initiation zone to allow for organ primordia to be formed? Lateral organ primordia are established in the peripheral zone (PZ) of the SAM, a process preceded by the local downregulation of SAM promoting factors (Fig. 2.1a). Maintenance of the undifferentiated state of the meristematic cells in the SAM requires the activity of transcription factor class I KNOTTED1-like homeobox (KNOX) family members (Hake et al., 2004) partially acting by modulating hormonal balances (Jasinski et al., 2005; Yanai et al., 2005). High cytokinin levels induce CyclinD-mediated cell division and low gibberellin represses cell expansion, two characteristics important for undifferentiated cells (Riou-Khamlichi et al., 1999; Dewitte et al., 2007). KNOX proteins directly regulate the level of these hormones by binding the promoter and repressing the expression of the gibberellin biosynthesis gene GA20-OXIDASE (ga20ox; Hay et al., 2002; Chen et al., 2004) and by activating the cytokinin biosynthesis gene ISOPENTENYL TRANSFERASE7 (IPT7) in Arabidopsis (Yanai et al., 2005). Prior to lateral organ initiation, KNOX gene activity becomes repressed at the initiation sites, and auxin has been proposed as a major factor influencing this process. In the PZ, auxin flux is directed to local sink positions by the action of auxin efflux facilitators such as PINFORMED (PIN), resulting in high local auxin concentrations, pinpointing the location of lateral organ primordium emergence (Reinhardt et al., 2000, 2003; Heisler et al., 2005). In these auxin peak positions, KNOX gene activity is silenced by the converging activities of auxin and a transcriptional repressor complex consisting of the MYB protein ASYMMETRIC LEAVES1 (AS1) and the LOB domain protein AS2 which results in the promotion of leaf development (Hay et al., 2006; Guo et al., 2008). The AS1/AS2 complex may also serve to maintain KNOX silencing during leaf development by recruiting the chromatin-remodelling factor HIRA to the KNOX gene (Phelps-Durr et al., 2005).

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Fruit Development and Seed Dispersal (a) HD-ZIPIII ta-siRNA AS

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Figure 2.1 (a) Interactions between transcription factors, small RNAs and hormones for SAM maintenance, lateral organ initiation and establishment of adaxial–abaxial polarity in the leaf. (b) Hormonal and genetic determination of boundaries between SAM and lateral organs. (c) Genetic factors involved in the establishment of distal–proximal polarity of the leaf.

2.2.2

Establishment of boundaries

Next, we will tackle the question how boundaries between the SAM and the newly initiated lateral organ are established? Once the new organ primordium has been initiated, auxin flux in the SAM is redirected to a new position for initiation of the next lateral organ (Heisler et al., 2005). This results in the depletion of auxin and a concomitant change in gene expression in the cell layers immediately adjacent to newly formed primordia, thus creating distinct cell types with reduced cell division activity which forms a morphological boundary that separates the primordia from the rest of the meristem (Fig. 2.1b; Heisler et al., 2005; reviewed by Aida and Tasaka, 2006). Growth repression in the boundary between the SAM and lateral primordia appears to be mediated by the NAC domain transcription factors CUP-SHAPED COTYLEDON (CUC1), CUC2 and CUC3 (Vroemen et al., 2003; Hibara et al., 2006; Sieber et al., 2007) and the JAGGED LATERAL ORGANS (JLO) LBD

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domain protein, which promotes the boundary function by activating KNOX genes and repressing PIN activity (Borghi et al., 2007). 2.2.3 Establishment of the adaxial–abaxial asymmetry Outward growth of the delimited and initially radial leaf primordium is immediately accompanied by the establishment of an adaxial–abaxial (dorsoventral) asymmetry required for the expansion of a flattened lamina. Cells adjacent to the SAM develop adaxial characteristics typical of the photosynthetic upper part of the leaf, whereas cells on the opposite side become abaxialized and form the lower part carrying out gas exchange (for recent reviews see: Golz, 2006; Chitwood et al., 2007; Kidner and Timmermans, 2007; Xu et al., 2007). Establishment of the adaxial–abaxial axis is dependent on the conversion of positional signals provided by the SAM and likely also other surrounding areas, into differential expression of mutually antagonistic transcription factors (e.g. HD-ZIPII and KAN, see below) in the adaxial or abaxial domains, respectively (Fig. 2.1a). Adaxial identity in Arabidopsis leaves is primarily specified by the partially redundant class III homeodomainleucine zipper (HD-ZIPIII) genes PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV) (McConnell and Barton, 1998; McConnell et al., 2001; Emery et al., 2003; Prigge et al., 2005) and exclusion of activity of these genes from the abaxial domain is mediated, at least in part, by miRNA165/166 via posttranscriptional cleavage and/or chromatin modifications (Emery et al., 2003; Kidner and Martienssen, 2003; Tang et al., 2003; Bao et al., 2004a; Mallory et al., 2004; Williams et al., 2005b; Alvarez et al., 2006; Zhou et al., 2007). Adaxial fate also appear to be promoted by AS1 and AS2 via the repression of abaxial identity in the adaxial domain (Lin et al., 2003; Xu et al., 2003; Fu et al., 2007). Abaxial fate, on the other hand, is cooperatively specified by the redundant KANADI (KAN) transcription factors belonging to the GARP family and the auxin response factors ARF3/ETTIN (ETT) and ARF4 (Eshed et al., 2001, 2004; Kerstetter et al., 2001; Pekker et al., 2005). The activity of the two ARF genes is restricted by ta-siRNAs encoded by TAS3 and recent work shows that the ta-siRNAs accumulate on the adaxial side of the leaf primordium where they repress the activity of the ARFs and other abaxial factors (Vazquez et al., 2004; Williams et al., 2005a; Adenot et al., 2006; Fahlgren et al., 2006; Garcia et al., 2006; Hunter et al., 2006; Nogueria et al., 2007). YABBY transcription factor encoding genes that appear to act downstream of both KANs and the ARFs are sufficient to specify abaxial fate and promote leaf lamina expansion in Arabidopsis (Sawa et al., 1999; Siegfried et al., 1999; Eshed et al., 2001, 2004). The mutually antagonistic activity of the KAN and HD-ZIPIII genes is not only required during leaf blade formation but also to establish polarity during embryogenesis and the development of the vasculature (Eshed et al., 2001; Emery et al., 2003; Izhaki and Boman, 2007). How is the polarity information obtained? Microsurgical separation of emerging leaf primordia from the shoot apex results in abaxialized radial

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Fruit Development and Seed Dispersal structures that fail to expand laterally (Sussex, 1954; Reinhardt et al., 2005). It has therefore been suggested that a meristem-derived signal (the Sussex signal) specifies adaxial identity. This signal, the nature of which still is unknown, must continuously enter the primordium during the early developmental stages in order to suppress abaxial identity (Reinhardt et al., 2005). The HD-ZIPIII START domain appears capable of lipid/sterol binding and has therefore been suggested to be a putative target of an unknown SAM-derived lipid/sterol signal (McConnell et al., 2001). Recently, the ta-siRNA encoded by TAS3 has also been suggested as a Sussex signal that could repress the activity of abaxial determinants in the adaxial domain (Garcia et al., 2006). In contrast to microRNAs, which appear to be cell autonomous, siRNA-induced gene silencing in Arabidopsis spreads from cell to cell (Tretter et al., 2008). Mobile signals specifying abaxial identity have also been suggested. The involvement of auxin response factors in abaxial cell fate specification introduces auxin as a possible abaxial polarizing signal (Pekker et al., 2005). The auxin influx facilitator AUX1 localizes specifically to the abaxial epidermal layer, suggesting that auxin flows into the abaxial half of the incipient primordium and establishes an auxin gradient across the primordium (Reinhardt et al., 2003). 2.2.4

Patterning of leaf shape and vasculature formation

How is leaf shape controlled? Auxin gradients appear to coordinate processes determining the final shape and size, as well as venation patterning of the developing leaf. It has been proposed that the tip of the developing leaf primorida is established as an auxin sink via directed auxin transport (Reinhardt et al., 2003), thereby generating a distal–proximal auxin gradient with its maximum at the tip of the developing primordium (Benkov´a et al., 2003). This gradient could provide positional information for morphogenesis and has been suggested to be important for midvein development (Mattsson, 1999; Zgurski et al., 2005). As they develop, primordia would become auxin sources, synthesizing auxin first at the leaf tip and then at the symmetrically positioned hydathodes at the leaf margins. This proposed sink–source transition coincides with the lateral growth of primordia and the formation of the secondary veins (Avsian-Kretchmer et al., 2002; Aloni et al., 2003; Sawchuk et al., 2007). Eventually, gradients of cell proliferation and differentiation/expansion become established in the distal–proximal and medial–lateral (from midvein to the margin of the leaf lamina) axes. Cell division rates gradually decline in a gradient from the tip of the leaf to the base as cells begin to differentiate (Donnelly et al., 1999; Nath et al., 2003), whereas cell divisions cease in the mid-region of the leaf slightly ahead of divisions at the margins (Byrne, 2005). Although no comprehensive view of growth regulation in lateral organs has emerged so far, the activity of JAGGED (JAG), ARGOS, AINTEGUMENTA (ANT), ANGUSTIFOLIA3 (AN3), GROWTHREGULATING-FACTOR5 (GRF5), PEAPOD (PPD), LEAFY PETIOLE (LEP) and class I TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) encoding genes have been shown to prolong the period during which proliferative

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cell divisions occur during leaf organogenesis (Fig. 2.1c; Misukami and Fischer, 2000; Van Der Graaff et al., 2000; Hu et al., 2003; Horiguchi et al., 2005; Li et al., 2005; Dinneny et al., 2006; Ingram and Waites, 2006; White, 2006; reviewed in Anastasiou and Lenhard, 2007). The auxin-inducible ARGOS acts through ANT, and the class I TCP proteins bind to and activate the promoters of cyclin and ribosomal protein genes. Class II TCP proteins appear to have an opposite function and promote growth cessation and tissue differentiation from the tip to the leaf base (Fig. 2.1c; Nath et al., 2003; Palatnik et al., 2003; Ori et al., 2007; Efroni et al., 2008). Thus, modulations in activity of these factors may constitute the evolutionary basis for variations in leaf size and shape. Apart from a gradual polarity in cell proliferation, the Arabidopsis leaf also shows a distinct distal–proximal asymmetry. The leaf blade develops in the distal part whereas the very proximal end is occupied by a narrow petiole lacking leaf blade. Recently, two genes specifying petiole identity by the repression of cell proliferation and growth have been identified; BLADE-ON-PETIOLE1 (BOP1) and BOP2, both encoding BTB/POZ domain proteins. These are expressed in the proximal part of the leaf where they promote petiole differentiation by repressing JAG activity as well as, indirectly via AS2/LOB, several KNOX genes (Ha et al., 2003, 2004, 2007; Hepworth et al., 2005; Norberg et al., 2005), resulting in early cellular determination. 2.2.5 Marginal shapes Fine-tuned regulation of cell proliferation and differentiation also determines the shape of the leaf margins (Palatnik et al., 2003; Dinneny et al., 2004). In leaves with smooth edges, auxin responsiveness is evenly distributed along the margins (Aloni et al., 2003) whereas serrations with hydathodes at their tips, both producing and strongly responding to auxin, are formed in a specific pattern during leaf serration. Leaf serration is associated with repression of the meristem and boundary gene CUC2 at the teeth site, and an activity balance of CUC2 and miRNA164A between the teeth determines the depth of the sinus (Nikovics et al., 2006). These data exemplify a common reuse of processes related to meristem function during lateral organ development. In summary, robust and complex genetic networks ensure the maintenance of the meristem–primordia, adaxial–abaxial, proximal–distal and medial–lateral dichotomies (Fig. 2.1). Crosstalk, redundancies and common regulators are characteristics of these pathways, as well as a general coordination provided by the interplay of hormonal balances, with auxin as a major morphogen.

2.3 The Arabidopsis gynoecium The gynoecium in Arabidopsis consists of two carpels congenitally fused that, at maturity form a bilocular chamber with parietal placentation that contains

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Fruit Development and Seed Dispersal

Figure 2.2 The Arabidopsis gynoecium. The different axes of polarity and morphological terms are indicated (a) scanning electron micrograph of the mature gynoecium at anthesis. (b) Chloral hydrate cleared anthesis gynoecium to reveal vascular patterns. Primary and secondary bifurcations of the medial veins are indicated with arrows. (c) Cross section of the ovary at anthesis. (For a colour version of this figure, please see Plate 1 of the colour plate section.)

around 50–80 ovules. Gynoecium primordium is first visible at stage 5 of flower development (according to Smyth et al., 1990) as an oval-shaped bulge at the centre of the floral primordium surface. At stage 6, it becomes clearly distinct, as an oval platform that subsequently (stages 7–8) develops a central invagination and grows into an open hollow tube. At later stages, the internal medial regions bulge giving rise to two opposing meristematic ridges, which in turn produce the placentae and ovules laterally and fuse in the centre to form the septum. At stage 9, the epidermis at the open apical end starts to differentiate stigmatic cells and the style becomes distinct. At stage 11, the gynoecial tube closes and different cell types are clearly visible. At anthesis, all tissues required for fertilization have fully developed and those required for fruit maturation and dehiscence are already specified, although they will become functional after fertilization (Bowman et al., 1999; Alvarez and Smyth, 2002; Roeder and Yanofsky, 2005). In Fig. 2.2, the different regions of a mature gynoecium are shown. Along the apical–basal axis: the stigma, composed of a single layer of papillar cells; the style, a solid structure that externally shows characteristic crenellated cells and is rich in stomata; the ovary, comprising most of the length of the gynoecium, which houses the ovules; and the gynophore, a short basal stalk-like structure which connects the ovary to the base of the flower. In a transverse section of the ovary (Fig. 2.2c), the patterning along the medial–lateral axis is shown: the valves correspond to the two carpel walls and are placed in lateral positions; in the valve margins 2–3 columns of smaller cells are visibly forming narrow furrows. The medial positions are formed by the

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fused margins of the carpels: the replum corresponds to the external medial domain; the septum, which forms postgenitally and divides the ovary longitudinally; and the ovules, derived form the placentae. The transmitting tract, a specialized tissue that facilitates pollen tube growth, runs along the entire length of the gynoecium from the stigma and through the centre of the style and the septum. All these medial tissues, including the apical style and stigma, are called marginal tissues, since they all derive from the margins of the two fused carpels. Both lateral and medial tissues show adaxial–abaxial polarity, with different cell types and tissues in the abaxial (external) and the adaxial (internal) positions. The vasculature in the gynoecium at anthesis is formed by four major veins, two lateral and two medial, that longitudinally span the entire gynoecium (Fig. 2.2b). The lateral veins are ontogenetically related to the middle vein in the leaf, and run through the centre of the valves terminating close to their apical end. The medial veins extend through the repla, bifurcating extensively when they reach the style. The mature Arabidopsis fruit is a dehiscent silique, and therefore, specialized tissues develop to facilitate the shattering process and seed dispersal. The valve margins develop the so-called dehiscence zones, comprising a separation layer of small cells, which defines a longitudinal plane of rupture at both sides of the replum, and a patch of adjacent lignified cells. These lignified cells, together with the lignification of the valve internal subepidermal layer, are critical for the dehiscence process, since, when the mature fruit dries the harder lignified regions provide mechanical tensions that facilitate pod opening (Ferr´andiz, 2002).

2.4 Genetic and hormonal factors controlling gynoecium development Many mutations that affect gynoecium development in Arabidopsis, and their corresponding genes, have been identified in the last few years, and the list is still growing. As we will see, only one of them causes a complete loss of carpel identity, few others can be related to the development of specific tissues within the gynoecium, and many others contribute to the distribution of territories and the establishment of polarity axes. As expected, most of the genes in this later group also have a role in leaf patterning. Thus, the genetic networks that direct lateral organ development and which we have described previously also work at patterning the two carpels that constitute the Arabidopsis gynoecium. Interestingly, many of the genes with a role in lateral organ development were first identified by the effect that their mutations caused in carpel development, suggesting that these genetic networks may be more robust in leaves than in the more complex and evolutionary younger carpels.

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Fruit Development and Seed Dispersal 2.4.1

From the leaf to the carpel: identity genes

Two decades after the original formulation of the ABC model, which explained floral organ identity based on the phenotypes and genetic interaction of floral homeotic mutants (Coen and Meyerowitz, 1991), it has been extensively validated. Intense research carried out in many labs during these years has confirmed the ABC models original postulates and, moreover, it has revealed much of the underlying molecular mechanisms, integrating all this information in the nowadays widely accepted ‘floral quartet model’ for organ identity (Krizek and Fletcher, 2005; Theissen and Melzer, 2007). We have previously introduced how, according to this model, leaves can be transformed into floral organs by expressing the corresponding combination of organ identity genes. In Arabidopsis, to make a carpel out of a leaf, we need to express the C-function gene AGAMOUS (AG) and one of the SEP genes (Honma and Goto, 2001). AG stands alone at the top of the hierarchy of carpel identity genes because ag single mutants are unique in lacking carpels completely: carpels are replaced by a reiteration of the sequence sepals–petals–petals. This is because, in addition to C-identity function, AG is required to prevent floral meristem indeterminacy. Also, as the ABC model predicted, A and C functions are mutually exclusive and in ag mutants, A-function expands to all whorls, causing the homeotic transformation of stamens into petals. Because of its central role in reproductive development, much is currently known about AG regulation, mechanisms of action and targets. AG is a member of the MADS-box gene family of transcription factors, like SEP and all but one of the ABC genes. Both AG and SEP are already expressed at the inception of the carpel primordia, before any morphological sign of differentiation (Yanofsky et al., 1990; Savidge et al., 1995; Mandel and Yanofsky, 1997). AG is activated in the floral meristem by the joint action of the products of the floral meristem identity gene LEAFY (LFY) and the meristem maintenance homeobox gene WUSCHEL (WUS). Once present, AG negatively feeds back on WUS through currently unknown mechanisms, terminating WUS expression and thus, floral meristem activity (Lenhard et al., 2001; Lohmann et al., 2001). While the LFY–WUS pathway appears to play a major role, independent mechanisms for AG activation must exist, as inferred from the phenotypes of lfy mutants, where AG is still expressed and carpelloid organs develop, or the normal stamens found in wus mutants (Weigel and Meyerowitz, 1993; Laux et al., 1996). Little is known about these alternative routes, although it has been shown that in 35S::SEP3 plants, AG is ectopically expressed, suggesting that SEP3, likely in combination with other MADS-box genes, could participate in these LFY-independent pathways (Castillejo et al., 2005). The A-function gene APETALA2 (AP2) prevents AG expression in the two outer whorls of the flower. In addition, many other factors have been described that refine the AG spatial and temporal pattern of expression, both at the transcriptional and posttranscriptional levels (Liu and Meyerowitz, 1995;

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Sieburth and Meyerowitz, 1997; Franks et al., 2002; Cheng et al., 2003; Bao et al., 2004b). The prominent role of AG in establishing carpel identity is not, however, unique in providing carpelloid features to floral organs. This fact was revealed by the phenotypes of some ag mutant combinations like the ap2 ag double mutants, where most carpel tissues still develop in the first whorl organs, even in the absence of AG function (Bowman et al., 1991). This observation led to the proposal of an AG-independent pathway for carpel identity, but in fact, it is better explained by the functional redundancy of AG with two other highly related MADS-box genes, SHATTERPROOF1 and SHATTERPROOF2 (SHP1, SHP2), primarily involved in the specification of valve margin identity (Liljegren et al., 2000). All carpelloid features disappear in the ap2 ag shp1 shp2 quadruple mutant. Moreover, complementation studies have shown that SHP and AG proteins are largely equivalent at the functional level, and that their distinct roles mostly derive from their different expression patterns (Pinyopich et al., 2003). In the wild-type flower, SHP1 and SHP2 are activated in a broad domain at early stages of carpel development, and later they become restricted to placental tissue, valve margins and ovules. SHP expression, as well as these tissues, is absent in ag mutants, and this observation, together with additional molecular studies, places the SHP genes downstream of AG (Savidge et al., 1995). However, this appears to be only partially true since, as seen in ap2 ag mutants, they can be activated independently of AG and are also under negative regulation by the A-function. Filamentous Flower (FIL), YABBY3 (YAB3) and JAG, present in both leaves and floral organs, have been shown to jointly activate SHP in the valve margins (Dinneny et al., 2005; see below), but their possible roles in activating SHP in the first whorl organs of ap2 ag mutants are unexplored at this point. As we will see later, when discussing the development of the different territories, AG seems to be absolutely required for valve identity, but the development of most of the other tissues depend on two factors that appear to act downstream of AG/SHP. SPATULA (SPT) encodes a bHLH transcription factor that is widely expressed in different specific tissues throughout vegetative and reproductive development (Heisler et al., 2001). CRABS CLAW (CRC) belongs to the YABBY family but is specifically expressed in nectaries and carpels (Bowman and Smyth, 1999). spt mutants show defects in the development of most carpel specific tissues (see below), whereas crc gynoecia are shorter and wider than wild type and partially unfused at the top. The crc spt gynoecium develops as two unfused organs with a very reduced amount of ovules and of stigmatic and stylar tissue. Furthermore, loss of SPT and CRC function in the ap2 ag background mimics the phenotype of ap2 ag shp1 shp2 mutants (Alvarez and Smyth, 1999). CRC has been identified as a direct target ´ of AG (Gomez-Mena et al., 2005), and less is known about SPT activation, although both genes are still expressed in the ectopic carpels of ag ap2 mutants (Bowman and Smyth, 1999), suggesting that their expression is driven by SHP. Thus, CRC and SPT appear to mediate AG/SHP specification of many

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Fruit Development and Seed Dispersal carpel tissues, although expression of CRC or SPT outside the gynoecium context does not appear to be sufficient to induce ectopic carpelloid features, suggesting that they could require AG/SHP activities to exert their functions. Supporting this idea, CRC and SPT are also involved in other aspects of AG function, such as the control of meristem determinacy (Alvarez and Smyth, 1999). In this sense, it is also noteworthy that in rice, for example the CRC orthologue DROOPING LEAF (DL) has a prominent role in C-function and, while mutants in the rice AG orthologue only have mild floral defects, dl mutant flowers show complete homeotic conversions of carpels into stamens and loss of floral determinacy (Yamaguchi et al., 2004). 2.4.2

Partitioning the gynoecium

At inception (stage 6 of flower development), the gynoecium primordium appears as a small mound of cells. Early patterning events act to partition the growing primordium, first specifying abaxial (outer) versus adaxial (inner) domains and medial versus lateral domains, and only later, as the primordia grows into a tube-shaped organ, to define apical–basal polarity. Adaxial–abaxial patterning basically follows the plan at work in all lateral organs that we have discussed above. Already at stage 6, all types of abaxial factors are expressed in the external domains of the gynoecium: KAN genes (Sessions et al., 1997; Kerstetter et al., 2001; Pekker et al., 2005), the auxin response factors ETT and ARF4 and the YABBY genes like FIL or YAB3 are also expressed abaxially, together with CRC, which is not found in leaves or other floral organs. As previously discussed, genetic networks directing adaxial–abaxial polarity are complex and rely on redundancy to provide robust patterning along this axis. Most single mutants in any of the ‘abaxial’ loci only show weak phenotypes, but mutant combinations can cause strong polarity defects that can turn the gynoecium inside out. Thus, for example crc kan1, kan1 kan2 or ett arf4 double mutants exhibit dramatic phenotypes with ovules and transmit tract tissues developing on the external side of the gynoecium (Eshed et al., 1999, 2001; Pekker et al., 2005). Adaxial HD-ZIP factors in the wild-type gynoecium primordium are expressed in their expected internal domains. Although the effect of HD-ZIP mutations in gynoecium development has not been documented in detail and appears to be less conspicuous than in leaves, it has been described that gain-of-function mutations in PHB or REV cause phenotypes similar to loss of function mutations in the abaxial KAN1 (McConnell and Barton, 1998; Zhong and Ye, 2004; Dinneny et al., 2006). In addition, mutants in the small RNA processing machinery show polarity defects in the carpels as well (Bohmert et al., 1998). All these data indicate that the same antagonistic relationships between KAN/YAB and HD-ZIP found in leaves are maintained in the gynoecium. However, not only a new abaxial player such as CRC is incorporated to the scheme, but also additional factors appear to contribute to specify adaxial fate in the carpels. NUBBIN (NUB) is a C2H2 zinc-finger transcription factor closely related to

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JAG (Dinneny et al., 2006). JAG is expressed in lateral organs, including floral organs, in a non-polar manner, and its role in lateral organ development has been related to promote cell proliferation and differentiation (Dinneny et al., 2004; Ohno et al., 2004). However, NUB expression is restricted to the adaxial domain of leaves, stamens and carpels and the double jag nub mutants display abaxialized stamens and carpels. The expression of polarity genes in jag nub carpels is not significantly affected and thus, NUB/JAG activities have been placed downstream or in parallel to the KAN/YAB/HD-ZIP network. However, it is still unclear whether the role of NUB/JAG in promoting adaxial fate is direct or reflects a requirement for correct organ growth to maintain organ polarity (Dinneny et al., 2006). Medial and lateral domains within the gynoecium are also specified in early stages of development. Since we have been stressing parallelisms between leaves and carpels to understand the common mechanisms that direct their development, we need to point out here their major differences. Medial–lateral symmetry in leaves (Fig. 2.1c) cannot be directly translated into the medial–lateral domains in the gynoecium. As previously described, the Arabidopsis gynoecium develops from two congenitally fused carpels and, in such a structure, medial domains correspond to the fused margins of the two carpels and will give rise to the marginal tissues, while the lateral domains are the ‘blade’ of these organs and later will develop into the valves. Medial domains in the gynoecium differentiate adaxially a new meristem, the medial ridge, which shares the typical three-layered structure of the SAM although instead of dome shaped, develops as an elongated ridge along the gynoecial tube (Hill and Lord, 1989; Azhakanandam et al., 2008). Thus, within the emerging gynoecium primordium, the dichotomy of meristematic versus differentiated lateral domains appears along the medial–lateral axis. Accordingly, several genes involved in SAM maintenance like the KNOX genes SHOOT MERISTEMLESS (STM) or BREVIPEDICELLUS (BP), or other homeobox genes like REPLUMLESS (RPL; a.k.a. PENNYWISE, BELLRINGER and VAAMANA) are expressed in the medial domain, as well as the ‘boundary’ genes CUC1 and CUC2 (Long et al., 1996; Aida et al., 1997; Ori et al., 2000; Pautot et al., 2001; Byrne et al., 2003; Smith and Hake, 2003; Bhatt et al., 2004; Alonso-Cantabrana et al., 2007). In contrast, genes that repress the undifferentiated state in the primordia of lateral organs and promote their development (like AS1/AS2, the YAB genes, the HD-ZIP genes or JAG/NUB) are restricted to lateral domains (Bowman and Smyth, 1999; Siegfried et al., 1999; Dinneny et al., 2006; Alonso-Cantabrana et al., 2007). Since most mutants in meristem maintenance genes do not make flowers, strategies to overcome this problem have been used to determine their specific role in carpel development. Thus, for example flowers produced out of calli regeneration of cuc1 cuc2 mutants fail to fully develop marginal tissues (Ishida et al., 2000). A significant contribution has been recently made by Scofield et al. (2007). Using inducible STM-RNAi, the effect of reduced STM activity on gynoecium development was studied. Phenotypes ranged from total absence of carpel

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Fruit Development and Seed Dispersal development to gynoecia that appeared completely unfused and lacked all signs of marginal tissue development. Conversely, overexpression of STM or the related gene KNAT2, was able to induce ectopic carpel formation in a largely AG-dependent manner, indicating a prominent role of KNOX genes in both gynoecium specification and patterning (Pautot et al., 2001; Scofield et al., 2007). As discussed above, genes expressed in emerging leaf primordia that direct adaxial–abaxial polarity in both leaves and floral organs are generally excluded from the medial domains of the gynoecium primordium. Thus, the KAN/YAB/HD-ZIP network appears to work essentially the same to specify the adaxial–abaxial axis in the gynoecium, but, surprisingly, at least the KAN genes are also required to maintain the medial–lateral dichotomy, since in the heavily adaxialized kan1 kan2 double mutants, adaxial marginal tissues such as ovules and transmitting tract develop all over the external side of the gynoecium in a radial manner (Eshed et al., 2001). While these radialized phenotypes could just reflect the requirement of abaxial–adaxial polarity to maintain the correct growth of lateral domains, they could also point to a possible role of KAN genes in the coordination of both adaxial–abaxial and medial–lateral patterning, although the mechanisms involved in such coordination are still unclear. KAN1 and KAN2 expression patterns in the developing gynoecium have not been extensively documented, although it appears that they change dynamically from some compartments to others during development. Likewise, the effect of kan mutations on the expression of gynoecium patterning genes has only been limitedly addressed (Eshed et al., 2001; Kerstetter et al., 2001). The auxin response factors ETT and ARF4, as in leaves and other lateral organs, appear to work together with the KAN genes in the specification of abaxial fate in the gynoecium, as revealed by the almost identical phenotypes of kan1 kan2 and ett arf4 double mutants and the nature of their genetic interactions (Pekker et al., 2005). ETT expression in the young gynoecium primordia is abaxial, and spans the whole perimeter of the gynoecial tube (Sessions et al., 1997) yet, in the ett arf4 double mutant, radialization of the primordium occurs. In addition, in mutants with reduced activity of the YUCCA (YUC) genes, which encode enzymes in the auxin biosynthetic pathway, as in yuc2 yuc4, or mutants in the PINOID (PID) kinase, a regulator of auxin transport, gynoecia develop as a solid radial structure capped by a ring of stigma (Bennett et al., 1995; Cheng et al., 2006). Taking all these data together, it is tempting to speculate that auxin could play a role not only in abaxial–adaxial polarity but also in medial–lateral compartmentation and be part of this hypothetical coordination. 2.4.3

Development of the lateral domains

The lateral domains differentiate into the valves, including the valve margins that eventually will develop into the dehiscence zones. Several factors involved in the specification of carpel identity also play major roles in the

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development of these tissues (reviewed in Ferr´andiz, 2002; Dinneny and Yanofsky, 2005; and Chapter 5 of this book). Thus, AG is required for valve identity specification, as deduced from the phenotype of ap2 ag mutants, where most of the gynoecium tissues still form in the first whorl organs but they lack the typical cell layer structure and some specific cell types of the valves (Alvarez and Smyth, 1999). CRC, expressed in the lateral domains at early stages, also influences valve growth and development, and in crc mutants, vascular development in the carpels shows an abnormal pattern of branching resembling that of leaves or sepals and suggesting a partial loss of carpel identity. Conversely, SHP1 and SHP2 are placed at the top of the hierarchy directing valve margin development, as shp1 shp2 double mutants fail to differentiate valve margins and develop siliques unable to open. Downstream additional factors involved in valve margin specification are INDEHISCENT (IND) and ALCATRAZ (ALC), two members of the bHLH family of transcription factors whose mutations also result in indehiscent phenotypes. IND is exclusively expressed in the valve margins, while SHP and ALC are expressed in the margins and in additional domains that, notwithstanding, do not appear to be affected in the corresponding mutants (Liljegren et al., 2000; Rajani and Sundaresan, 2001; Liljegren et al., 2004). SHP, IND and ALC expression is tightly confined to the valve margin by two antagonistic activities, the MADS-box factor FRUITFULL (FUL) and the homeodomain protein RPL, that operate in the valves and the replum, respectively (Gu et al., 1998; Roeder et al., 2003). In ful mutants, SHP, IND and ALC are ectopically expressed in the valves which, as a consequence, develop the small lignified cells typical of dehiscence zones and are unable to elongate and break prematurely. Conversely, SHP, IND and ALC are not expressed in 35S::FUL carpels, which in turn give rise to indehiscent fruits (Ferr´andiz et al., 2000; Liljegren et al., 2004). In rpl mutants, the replum is indistinct and the lignified cells of both dehiscence zones appear merged (Roeder et al., 2003). FUL and SHP appear to be the major factors directing cell fate in the lateral domains, namely the valves and the valve margins, respectively. It has been shown that both are under the regulation of some of the ‘lateral factors’ that we have described before (Fig. 2.3). Thus, the cooperative activities of FIL, YAB3 and JAG positively regulate the transcription of FUL and the SHP genes in the valve and the valve margin, respectively. It has been proposed that, while a higher FIL/YAB3/JAG activity in the valves would turn on FUL expression in this domain, SHP genes would only be activated by the weaker FIL/YAB3/JAG activity present in the valve margins (Dinneny et al., 2004). Then, FUL would repress SHP expression, neatly delimiting the two domains of FUL-expressing valve and SHP-expressing valve margin territories (Ferr´andiz et al., 2000). In the replum, RPL represses the expression of FIL/YAB3/JAG genes, therefore preventing SHP activation (Roeder et al., 2003; Dinneny et al., 2005). Thus, FUL and RPL restrict SHP expression to a narrow domain spanning 3–4 cell layers between the valve and the replum. In this domain, SHP activates IND and ALC, which are ultimately in charge

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Fruit Development and Seed Dispersal

STM BP

AS FIL/YAB3 JAG

RPL SHP IND ALC

FUL

Valve

Replum Margin

Figure 2.3 Model for medial–lateral patterning of the gynoecium. Genetic interactions of the factors directing the development of valves, valve margins and repla. A hypothetical model based on two opposing gradients of valve factors and replum factors explains the positioning of the valve margins at the overlapping domain of both activities.

of directing the differentiation of the tissues in the dehiscence zone (Liljegren et al., 2000, 2004; Rajani and Sundaresan, 2001). Both ful and rpl show extreme phenotypes of ectopic dehiscence zone formation. However, a change in dehiscence zone position, and therefore, of the proportions of valve/replum territories in the fruit can be observed in other genetic backgrounds, generally related to the establishment of the medial–lateral boundaries discussed previously. This supports the idea of medial–lateral dichotomy as equivalent to meristematic-differentiated. Thus, as1 or as2 mutants show enlarged medial regions resulting in expanded repla and narrow valves, as do fruits overexpressing the class I KNOX gene BP (Alonso-Cantabrana et al., 2007). KNAT6, another class I KNOX gene, which in wild-type fruits is expressed in the valve margins, is ectopically expressed in the repla of rpl mutants and this expression appears to be crucial for the rpl phenotypes, as in rpl knat6 double mutants, the replum is restored to a wild-type size (Ragni et al., 2008). YABBY genes have been described to downregulate KNOX genes in lateral organs (Kumaran et al., 2002), thus providing a link between the AS/KNOX antagonism to control replum size and the

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FIL/JAG activities to direct valve regionalization. A hypothetical but attractive model has been proposed to explain how the position of the boundaries between lateral and medial domains, and therefore the dehiscence zones, is established. In this model, a gradient of FIL/YAB3/JAG and AS valve factors with a lateral maximum would coexist with a gradient of KNOX/RPL replum factors with a medial maximum. Both gradients would antagonize each other and only overlap in a narrow domain, where the valve margin would be formed (Fig. 2.3; Alonso-Cantabrana et al., 2007; Mart´ınez-Laborda and Vera (Chapter 5 of this book)).

2.4.4 Marginal tissue development Early during gynoecium development, the meristematic medial ridge develops on the internal, adaxial part of the medial domain, localized at the boundary between the two fused carpels. Genetic data and gene expression patterns strongly suggest the medial ridge to coordinately generate all tissues required for the reproductive competence of the gynoecium; the placenta, ovules, septum and associated transmitting tracts, as well as portions of the style and stigma. The lateral organ boundary genes CUC1 and CUC2 are expressed in the medial ridge from very early stages of development (Fig. 2.4; Ishida et al., 2000; Takada et al., 2001). In cuc1 cuc2 mutant gynoecia, there is an

LUG/SEU/ANT/YAB SPT/HEC

SHI/STY TOP

CUC KNOX HD-ZIP Auxin

ETT

Figure 2.4 Model for apical–basal patterning of the gynoecium. According to the auxin gradient hypothesis (Nemhauser et al., 2000), auxin levels are proposed to be high in the apical regions promoting development of style and stigma whereas intermediate levels determine the ovary and low levels the gynophore. Auxin synthesis in the apical end could be under the control of STY/SHI and NGA genes, while SPT and ETT may be involved in translating the auxin gradient into differentiation programmes.

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Fruit Development and Seed Dispersal apical gradient of decreasing medial tissue production, sometimes resulting in an unbroken cylinder of valve tissue (Ishida et al., 2000). It has therefore been suggested that the CUC genes initially act to maintain an undifferentiated state of the medial cells and subsequently promote the expansion and fusion of the medial ridge resulting in septum formation (Alvarez and Smyth, 2002). As discussed previously, class I KNOX meristem maintenance genes are also expressed in the medial ridge from early in gynoecium development (Fig. 2.4) and, in the few stm mutants that flower, carpels exhibit reduced fusion and medial tissue growth (Endrizzi et al., 1996; Long et al., 1996; Pautot et al., 2001). Activity of class I KNOX genes has also been detected in the meristematic placenta, as well as in the ovules and the style suggesting multiple roles of this class of genes during development of marginal tissues (Ori et al., 2000; Venglat et al., 2002; Alonso-Cantabrana et al., 2007; Scofield et al., 2007). As mentioned in an earlier section, expression of adaxial identity genes, such as the HD-ZIP genes, within the adaxial margin has been suggested to be required for development of the placenta and for ovule initiation (Alvarez and Smyth, 2002). As in the SAM, all data so far suggest that the pluripotent medial cells represent a low auxin level/response zone, whereas high auxin responses are detected in some of the tissues subsequently formed by the medial cells. Several genes required for carpel fusion and development of the medial ridge-derived structures have been identified through mutant screening, but it is still unclear if they are involved in determining marginal tissue identity or merely growth of the medial ridge required for further development. Many of these genes share some functional redundancy, and although the contribution of each gene is rather limited, their collective activity is essential for the development of marginal tissues. It has been suggested that LEUNIG (LUG), SEUSS (SEU), ANT and FIL may be part of a multimeric complex important for the development of the medial domain of the gynoecium (Fig. 2.4; Azhakanandam et al., 2008). LUG and SEU encode transcriptional coregulators, with sequence similarity to the Tup1/Groucho and Ssdp/Chip protein families, and to the LIM-domain-binding protein family, respectively (Conner and Liu, 2000; Franks et al., 2002). LUG and SEU act as repressors of AG activity in the first two floral whorls, can interact physically and have been proposed to coregulate common targets throughout numerous stages of plant development (Liu and Meyerowitz, 1995; Franks et al., 2002; Sridhar et al., 2004). The lug mutant gynoecia are unfused at the apical end, and exhibit defects in the septum fusion and ovule development, consistent with its expression pattern (Liu and Meyerowitz, 1995; Chen et al., 2000; Conner and Liu, 2000). Gynoecia of seu mutants are also often split at the apical end whereas mutations in both SEU and LUG completely abolish the development of fourth whorl organs, and the homeotic carpelloid organs that form in whorl one show no trace of marginal tissues (Franks et al., 2002). The Arabidopsis LUG homologue LUH shares redundant functions with LUG, including interactions with SEU, and overlapping functions among SEU-like

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(SLK) genes has also been suggested (Conner and Liu, 2000; Franks et al., 2002; Azhakanandam et al., 2008; Sitaraman et al., 2008). Like lug and seu, ant mutants exhibit partially unfused carpels (Elliot et al., 1996). Additional functional similarities of LUG, SEU and ANT include promotion of leaf growth as well as repression of AG in outer floral whorls (Liu and Meyerowitz, 1995; Chen et al., 2000; Misukami and Fischer, 2000; Franks et al., 2002; Krizek et al., 2000; Cnops et al., 2004). Marginal tissue development is fully dependent on the cooperative activities of ANT and LUG, as revealed by the complete lack of replum, style, septum and placental tissues in the gynoecia of ant lug double mutants (Liu et al., 2000). ANT encodes an AP2/ERF domain DNA-binding transcriptional activator expressed in the medial ridge and medial-derived tissues, as are also several ANT-like (AIL) genes (Krizek, 2003; Nole-Wilson et al., 2005; Krizek and Sulli, 2006). Knock-down of individual AIL genes has no effect on medial tissues, suggesting functional redundancy within the gene family. The defects in medial tissue development in seu ant double mutant gynoecia are also severe. seu ant gynoecia are split in the apical part, fail to initiate ovule primordia, produce reduced amounts of other medial tissues and display a reduced activity of the adaxial identity genes PHB and REV suggesting that ANT and SEU may potentiate the expression of the adaxial fate determinants (Azhakanandam et al., 2008). Apart from ANT, LUG and SEU, some YABBY genes may also be important for medial tissue formation. First, gynoecia of fil lug and fil ant double mutants show similarities to seu ant or even lug ant double mutants (Chen et al., 2001; Nole-Wilson and Krizek, 2006). Second, evidence that SEU and LUG may physically cooperate with FIL comes from analysis of orthologous genes in Antirrhinum majus (Navarro et al., 2004). Finally, LUG, SEU and ANT appear to be required for maintaining abaxial–adaxial polarity by positively regulating FIL expression and/or activity (Franks et al., 2006; Nole-Wilson and Krizek, 2006). Apart from the HD-ZIP genes, additional putative downstream effectors of the LUG/SEU/ANT/YAB proteins in gynoecia have been suggested, which all appear auxin related. The Antirrhinum orthologue of LUG, STYLOSA, was shown to have a role in auxin responses and in Arabidopsis the auxin response genes ARFX15 and ETT/ARF3 were recently identified as potential downstream targets of LUG action (Navarro et al., 2004; Gonzales et al., 2007). Furthermore, SEU physically interacts with ETT/ARF3 and it has been suggested that ETT/ARF3 may recruit SEU, and perhaps thereby also LUG, to auxin response genes to regulate their transcription (Pfluger and Zambryski, 2004). Members of the SHI/STY gene family, encoding zinc-finger transcriptional activators expressed in the apical end of gynoecium primordia, style and ovule primordia, have also been suggested as downstream effectors of LUG because lug mutations are epistatic over sty phenotypes and STY1 expression is reduced in lug mutants (Kuusk et al., 2006). STYLISH1 (STY1) shows only a subtle defect in style development, and mutations in additional members of the family gradually enhance the sty1 phenotype in a redundant and dose-dependent manner. Multiple mutant lines reveal that at least six of

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Fruit Development and Seed Dispersal the SHI/STY-related genes contribute to carpel fusion as well as to the formation of all marginal tissues in the apical part of the gynoecium; the style, the stigma and the internal septum with placenta and ovules. These lines also show similar leaf defects as mutants in LUG/SEU/ANT/YAB; highly serrated leaf margins and partial loss of adaxial–abaxial polarity (Fridborg et al., 1999; Kuusk et al., 2002, 2006). One of the direct downstream targets of STY1 is the auxin biosynthesis gene YUC4, suggesting SHI/STY family members positively regulate local auxin biosynthesis (Sohlberg et al., 2006; Eklund et al., 2008). Indeed, exogenous application of auxin on the apical end of young sty1 sty2 double mutant gynoecia induced wild-type-like style and stigma morphogenesis, suggesting high local auxin levels to be required for style development and proliferation. Because ectopic expression of STY1 can rescue the style defects not only of lug and seu, but also of crc, jag and spt mutant gynoecia, STY1 most likely induces a pathway far downstream in the medial tissue programme (St˚aldal et al., 2008). Members of the NGATHA (NGA) gene family encode four partially redundant B3 domain proteins required for apical fusion and marginal tissue development of gynoecia (Weigel et al., 2000; Alvarez et al., 2006. Interestingly, the activity of these genes are necessary for YUC2 and YUC4 expression in the style, and mutations in SHI/STY and NGA genes show synergistic interactions resulting in dramatic reduction in apical fusion and marginal tissue development. It has been suggested that SHI/STY and NGA either act in same pathway or co-operatively (Alvarez et al., 2009; Trigueros et al., 2009). How TOUSLED (TSL) relates to the LUG/SEU/ANT/YAB complex is still unresolved. TSL encodes a protein kinase required for growth and fusion of the apical gynoecial tissue and has been suggested to play a role in the maintenance of transcriptional gene silencing, thereby controlling, for example cell division (Roe et al., 1993, 1997; Ehsan et al., 2004; Wang et al., 2007). SPT is required for transmitting tract development, and for supporting development of the septum, style and stigma. The absence of transmitting tract cells in spt mutants results in poor pollen tube growth and fertilization. SPT encodes a bHLH transcriptional activator and is expressed in all medial tissues, including ovule primordia and funiculi (Alvarez and Smyth, 1999; Heisler et al., 2001; Alvarez and Smyth, 2002; Groszmann et al., 2008). Three other bHLH genes, HECATE (HEC) 1, 2 and 3, are expressed in the SPT domain, and multiple knock-down of all three HEC genes results in an unfused septum and loss of transmitting tract, signifying overlapping function of all four bHLH genes. HEC proteins heterodimerize with SPT in yeast cells, suggesting these proteins cooperate in the control of marginal tissue development (Gremski et al., 2007). Another gene, NO TRANSMITTING TRACT (NTT), appears important for the final stages of transmitting tract development and the two auxin response factors, ARF6 and ARF8, expressed within the stigma, style and transmitting tract, have been suggested to regulate the production of some component necessary for pollen tube germination or growth (Wu et al., 2006; Crawford et al., 2007). SPT is likely to be involved in

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auxin patterning, both as a target of auxin regulation and as a mediator of auxin effects (Nemhauser et al., 2000). Interestingly, when the transcriptional activation capacity of SPT is enhanced via fusion to the VP16 activation domain, ectopic STY2 expression is induced in the valves suggesting SHI/STY family members act downstream of SPT. Although strong synergistic interactions of spt and shi/sty mutations have been observed, spt mutations are epistatic over some shi/sty phenotypes, suggesting that they may, at least in part, act in the same genetic pathways (Kuusk et al., 2002, 2006). However, the STY2 expression in spt is unaffected implicating that STY2 is not a direct target of SPT activation, or alternatively, other factors redundantly regulate its expression (Groszmann et al., 2008). SPT has also been connected to other hormonal processes and was identified as a repressor of a key gene in the GA biosynthetic pathway, GA3ox, during seed dormancy (Penfield et al., 2005). Although a corresponding function in the gynoecium has not yet been established, the data suggest a possible role of SPT in mediating a balance between auxin and gibberellin signalling pathways. Interestingly, ectopic expression of STY/SHI family genes appears to repress GA responses (Fridborg et al., 2001; Sundberg, unpublished). Although mutant analysis has so far not implicated GA in patterning of the gynoecium, we find it tempting to speculate that the antagonistic relationship between auxin and GA in the SAM may also be acting in the ‘meristematic’ medial domain of the gynoecium. It would thus be interesting to investigate if SPT and STY/SHI genes may cooperate in stimulating auxin responses, while simultaneously repressing GA signalling, and if this balance is required for gynoecium medial tissue development. 2.4.5 Apical–basal patterning Several of these genes required for marginal tissue development, for example SPT, STY/SHI and TOP/NGA, may also be considered as major factors promoting the proliferation of the apical end of the gynoecium. Besides the mutations resulting in disturbed development of apical tissues, additional mutants display an altered partitioning of tissues along the apical–basal axis. Mutants defective in polar auxin transport (PAT), such as pin and pid, show severe defects in apical–basal patterning with enlarged apical and basal regions and strongly reduced ovaries (Okada et al., 1991; Bennett et al., 1995), implicating auxin as a major morphogen directing patterning in this axis. Accordingly, mutations in the auxin response factor genes MP/ARF5 and ETT/ARF3 result in similar apical–basal defects (Sessions and Zambryski, 1995; Przemeck et al., 1996; Sessions et al., 1997) and it has been shown that ETT promotes ovary formation largely through the repression of SPT in this domain (Heisler et al., 2001). Based on the majority of these results, Nemhauser et al. (2000) proposed a model for apical–basal patterning of the gynoecium relying on a proposed auxin gradient spanning the gynoecia primordia. This model predicts high auxin levels in the apical region to promote

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Fruit Development and Seed Dispersal differentiation and proliferation of the style and stigma, and intermediate and low levels to specify ovary and gynophore, respectively (Fig. 2.4). Reduced PAT results in apical shifts in the boundaries between the different tissues because of high accumulation of auxin in the source tissues, hypothesized to be the most apical parts, and depletion of auxin in the cells normally downstream of the transport (Nemhauser et al., 2000). Further support for the model comes from the recent finding that STY1 and TOP/NGA genes, expressed in the apical end of the gynoecia, promote the activity of the auxin biosynthesis gene YUC4 (Sohlberg et al., 2006; Trigueros et al., 2009). Because several YUC genes are active in the distal gynoecial tip and share partially redundant functions, multiple mutants were required to reveal a common role in patterning of the gynoecia (Cheng et al., 2006). Inhibition of PAT, as well as overexpression of STY1, can restore apical tissue proliferation in various mutant lines affected in apical tissue development, for example lug, seu, ant, sty1, spt, crc and jag, suggesting auxin to act downstream of, or in parallel with, the corresponding apical tissue promoting factors during style/stigma development (Chen et al., 2000; Nemhauser et al., 2000; Sohlberg et al., 2006; St˚aldal et al., 2008). At the other end of the gynoecium, KNUCKLES (KNU) encodes a zinc-finger protein and may restrict development of the gynophore by establishing or maintaining the basal ovary boundary (Payne et al., 2004). Although the auxin gradient model provides a nice framework to explain the apical–basal patterning, the evidence for the auxin gradient model is only based on mutant data and has not yet been supported by auxin level measurements along the length of the gynoecia. We also need to further understand the mechanisms of action of the genes involved. Generally, auxin biosynthesis (yuc1, yuc4) and response mutants (axr1-3, ett/arf3 and tir1) are hyper-responsive to chemically – or genetically inhibited PAT (Nemhauser et al., 2000; Cheng et al., 2007; St˚aldal et al., 2008). So are also gynoecia carrying mutations in any of the medial tissue genes LUG, SEU and STY, suggesting that these genes may support apical–basal patterning by promoting auxin gradient or auxin response pathways (Pfluger and Zambryski, 2004; Sohlberg et al., 2006; St˚aldal et al., 2008). In contrast, spt mutants are less sensitive to decreased PAT (Nemhauser et al., 2000) and it is tempting to speculate that the SPT/HEC bHLH genes normally may act to modulate PAT or to sense parts of the auxin gradient. In summary, we propose a model placing SHI/STY-controlled YUCmediated auxin synthesis at the apical end of the developing gynoecium (Fig. 2.4). The SPT/HEC genes could direct stigma, style and transmitting tract differentiation by mediating the response to high levels of auxin and/or by participating in the formation of the auxin gradient by regulating its polar transport. ETT may establish the size of the ovary by responding to intermediate levels of auxin and by repressing the activity of SPT/HEC from this region. ETT may also restrict stigma and style differentiation to the apical, high auxin level region. However, whether an auxin gradient is established,

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and what is the nature of such a gradient, are important questions to address in the near future.

2.5 Conclusion The impressive progress in elucidation of genetic pathways regulating Arabidopsis gynoecium development has given us a good platform for modelling and has pointed out some clear directions for future work in order to understand how gynoecium patterning is established. Because the present knowledge is still very fragmented, the interrelationship between identified genetic factors must be developed and their downstream targets identified. Although the role of hormones in establishing gynoecium morphogenesis has also become clear, very little is known about the hormone dynamics and localization during gynoecium development, and even less about the regulation of the dynamics. Using modern technology for isolation of small amounts of tissue together with high quality hormone measurements, or immunolocalization methods, we will be able to address these questions in the near future. Establishing the localization of auxin, GA and cytokinin peak sites and the response dynamics of these hormones would greatly improve our understanding of the regulatory pathways required for specific tissues or developmental stages. Another intriguing question is how common or specific are the complex genetic and hormonal pathways directing lateral organ patterning. A large number of parallels between the developmental programmes in leaves and gynoecia have already been discovered, and it remains to be seen if some of the pathways still seemingly specific to gynoecia also are active during leaf development and vice versa.

Acknowledgements We would like to thank Desmond Bradley for critical reading and careful corrections to the manuscript. Our work is supported by research grants ´ y Ciencia to CF and from the BIO206–10358 from Ministerio de Educacion Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning to ES.

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Annual Plant Reviews (2009) 38, 70–106 doi: 10.1002/9781444314557.ch3

www.interscience.wiley.com

Chapter 3

THE INS AND OUTS OF OVULE DEVELOPMENT Raffaella Battaglia,1 Monica Colombo2 and Martin M. Kater1 1 Dipartimento di Scienze Biomolecolari e Biotecnologie, Universit`a degli Studi di Milano, Via Celoria Milano Italy 2 Dipartimento di Biologia, Universit`a degli Studi di Milano, Via Celoria Milano Italy

Abstract: In the past few years, ovule development has been widely studied in different plant species, both from a morphological and a more molecular point of view. At early stages of flower development, the placenta becomes specified inside the carpel and ovules develop from this tissue as meristematic protuberances. Shortly after, a complicated genetic network regulates ovule patterning controlling the differentiation of three regions named funiculus, chalaza and nucellus. In the past decade, genes playing important roles during ovule development have been identified, and in a few cases, genetic models that could explain the molecular relationship among these genes have been proposed. Here we focus our attention on the molecular genetic mechanisms that stand at the base of ovule development in the model species Arabidopsis thaliana and we report an updated description of the molecular networks controlling both sporophytic and gametophytic tissue development in the ovule. Furthermore, we observe that the mechanisms controlling ovule development seem to be evolutionary conserved, even in a distantly related species such as rice. Keywords: ovule; female gametophyte; embryo sac; haploid generation; ovule identity determination

3.1

Introduction

Ovule development has been addressed in different species since many years, first from a morphological point of view and later from a molecular point of view. The great interest towards the comprehension of the genetic and molecular aspects controlling ovule development relies not only on the fact that ovules can be considered as model organs to study differentiation processes 70

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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in plant but also on the fact that they are essential sites for sexual plant reproduction where megagametogenesis, fertilization and embryogenesis take place. Recently, the combination of advanced morphological analysis techniques together with molecular data and mutant characterization allowed a deeper understanding on the genetics at the base of ovule development. As expected, the availability of information concerning ovule development in the model species Arabidopsis thaliana has somehow facilitated the investigation in other model species such as Petunia but also in non-related species such as rice. More data are therefore now published about ovule identity determination and differentiation in different species; not only this is of great interest for applicative projects in cultivated species but also it will help in tracing the evolutionary steps of ovule ontogenesis. Due to the large amount of data concerning ovule development in Arabidopsis, this chapter will focus on this model species in order to offer a complete scenario regarding the molecular control of ovule formation. An intriguing characteristic of this organ is the close relation between sporophytic and gametophytic tissues which suggest the existence of molecular signals regulating the coordinate development of these tissues. Different aspects concerning the molecular control of sporophytic and gametophytic tissue development are reported and a paragraph focused on the molecular signals between these tissues is presented. Moreover, the most recent findings concerning ovule identity determination in Arabidopsis, Petunia and rice are described at the end of the chapter. As it will be illustrated, the characterization of ovule homeotic mutants in different species has been particularly helpful to highlight some aspects concerning the evolutionary conservation at the base of ovule identity determination.

3.2 The origin of ovule Ovules represent the salient feature of gymnosperms and angiosperms; as precursors of seeds, these organs are therefore an integral aspect of the plant life cycle. Different theories have been proposed in order to explain the origin of the ovule. Based on fossil evidence, the telomic theory seemed to be the most plausible. It proposes that the nucellus is derived from a megasporangium that retained a single megaspore. Moreover, according to this theory, the integument(s) are derived from the fusion of sterile branches around the megasporangium (De Haan, 1920). In seed plants, the megaspore is designed as energy transfer-megaspore since the female gametophyte develops and matures with the energy derived from the nucellus over the entire course of megagametophyte development. On the contrary, megaspores in non-seed plants are defined as energy-storage megaspores since the nutrients required for female gametophyte development are accumulated prior the first nuclear division. Many other theories have been proposed and the main difference

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Fruit Development and Seed Dispersal relies on the nature of the integument(s) which are proposed to derive from fertile branches (Herr, 1995). More than a century ago, Charles Darwin defined the origin and early evolution of flowering plant as the ‘abominable mystery’ (Charle’s Darwin letter to J.D. Hooker in the year 1879). Indeed, Cenozoic extinctions are responsible for the absence of fossils which could highlight the morphology of ancient non-flowering seed plants and early angiosperms. The identification of ancient lineages of flowering plants are fundamental to understand which are the evolutionary steps that created the extant flowering plants. In order to study the early evolution of the gametic structures, Friedman (2006) reported the developmental analysis of megagametophyte development in Amborella trichopoda, the appearance of which predates the establishment of all the other angiosperms (monocotyledons, eumagnoliids and eudicotyledons). He showed that this ancient female gametophyte is composed of eight cells and nine nuclei; nevertheless, more studies on other ancient species are necessary to understand the evolutionary origins of the Polygonum-type angiosperm embryo sac. Concerning integument(s) evolution in the angiosperm, Impatiens species have been recently studied (McAbee et al., 2005). Most basal angiosperms are bitegmic (i.e. ovule possess two integuments) and the earliest angiosperms were likely bitegmic while many extant angiosperm are unitegmic. Therefore, the idea is that reduction in integument number has occurred several times among different angiosperms.

3.3

Ovule development in Arabidopsis

In Arabidopsis, the morphology of the ovule has been described in detail and the process that leads to the formation of mature ovules has been subdivided into six distinct stages (Modrusan et al., 1994). At stage 8 of flower development (Smyth et al., 1990), ovule primordia arise from the placental tissue as finger-like protrusions with a radial symmetry (Figs. 3.1a and 3.1b). During the subsequent stages (9 and 10), three distinct regions can be distinguished along the proximal–distal (PD) axis: a proximal part, named funiculus, that connects the ovule to the placenta; a medial part, the chalaza, where the inner and outer integuments emerge; and a distal part, the nucellus, that contains the megaspore mother cell. The inner and outer integuments develop from the chalaza at stage 11. The outer integument displays asymmetrical growth through increased cell divisions on the side facing the central septum (Fig. 3.1c). During mid-stage 12, the growth of inner and outer integument continues upwards enclosing the nucellus, and at maturity, the outer integument completely overgrows the inner integument leaving only a small opening, the micropile, through which the pollen tube will pass. The faster growth of the integuments on the abaxial side forces a curvature in the developing ovules.

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Figure 3.1 Ovule development in Arabidopsis thaliana. The upper panel shows the microscopic analysis of ovule development in Arabidopsis while a schematic representation is reported below. (a) At stage 8 of flower development, ovule primordia develop from the placenta. (b) At stage 9, ovule primordia elongate as finger-like structures. The megaspore mother cell is visible in the nucellus. (c) At stage 11, the integuments elongate in the chalaza. The outer integument displays asymmetrical growth. (d) At stage 12, integuments grow to cover the nucellus where the meiotic division takes place. (e) DIC microscopy image of mature ovule. The integuments completely cover the mature embryo sac. In the embryo sac, the egg cell, the two synergids, the central cell and the three antipodal cells are visible. op, ovule primordia; MMC, megaspore mother cell; oi, outer integument; ii, inner integument; f, funiculus; es, embryo sac.

In the nucellus, megasporogenesis is initiated at early stage 11 when the megaspore mother cell (MMC) undergoes a meiotic division that originates a tetrad of haploid megaspores, three of which degenerate (Fig. 3.1d). At stage 12, the distal cell in the tetrad, the functional megaspore, proceeds in megagametogenesis. Three mitotic divisions originate the embryo sac, which is surrounded by the integuments. At the beginning of stage 13, the ovule is ready to be fertilized: the nucellus has degenerated and the medial part of it is occupied by the mature embryo sac, which is composed of eight nuclei and seven cells (Fig. 3.1e). Mature ovules are therefore composed of sporophytic diploid tissues like the funiculus and the integuments while the embryo sac represents the gametophytic haploid tissue.

3.4 Sporophytic tissues 3.4.1 Specification and formation of the placenta Arabidopsis ovules arise from specialized meristematic regions of the internal surface of the carpels, referred to as the placenta (Robinson-Beers et al., 1992), which belong to the carpel medial domain. The Arabidopsis gynoecium is derived from two congenitally fused carpels that appear as a single

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Figure 3.2 Adaxial–abaxial polarity establishment in the developing carpel and ovule. In general, adaxial refers to being situated towards an axis of an anatomical structure while abaxial refers to being situated away from an axis of an anatomical structure. (a) SEM image of a developing carpel. At stage 5, the carpel appears as a open hollow cylinder. If an axis passes through this cylinder, adaxial refers to those structures which are close to the axis while abaxial refers to structures which are located away from the axis. (b) Schematic representation of a transversal section of a carpel at stage 8 of flower development. Ovule primordia arise from the placenta. At this stage, the septum is not completely fused. The adaxial face of the carpel is indicated in black while the abaxial side is grey. (c) Developing ovule at stage 12 of flower development. The faster growing outer integument forces a curvature in the developing ovule. Adaxial sides of the developing integuments are indicated in black and abaxial sides are indicated in grey. cr, carpel; st, stamen; s, septum; p, placenta; op, ovule primordium; ii, inner integument; oi, outer integument; ad, adaxial; ab, abaxial.

primordium in the centre of the flower. Subsequently, a central invagination forms and the primordium elongates as an open hollow cylinder (Fig. 3.2a) (Smyth et al., 1990). During gynoecium development, two opposing internal meristematic outgrowths (the medial ridges) form at medial positions and protrude into the centre of the cylinder where they fuse, giving rise to the septum. Placental tissue differentiates along the length of the septum adjacent to the lateral walls (Fig. 3.2b) (Bowman et al., 1999). Early patterning events divide the gynoecial primordium into distinct domains that will give rise to specific tissues (Bowman et al., 1999; Balanza et al., 2006). Along the apical–basal axis, there are, from top to base, the stigma; a short, solid style; the ovary, which contains the ovules; and basally the gynophore, which attaches the ovary to the flower base. The lateral domains develop into the valves, corresponding to the ovary walls. In medial position, the fused margins of the carpels are found. Several medial tissues (replum, septum, placenta and ovules, transmitting tract) arise from these fused margins. All these medial tissues, together with the apical style and stigma, are collectively termed marginal tissues. Therefore, the gynoecium medial domain is really important since it gives rise to the placenta and the ovules, and several other structures critical for reproductive competence. Despite the importance of the placenta for ovule development, the molecular events that lead to the development of this tissue remain still not well understood. Many mutations that affect the development of the medial domain have been identified (Bowman et al., 1999; Ferrandiz et al., 1999; Sessions, 1999;

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Balanza et al., 2006). However, extensive redundancies between the pathways and genes involved in the formation of the medial tissues have made genetic analysis of this region problematic. So far, no single mutant has been identified that completely blocks marginal tissue formation. However, severe developmental alterations have been reported in a variety of double mutants. Good examples are AINTEGUMENTA (ANT), LEUNIG (LUG), SEUSS (SEU) and FILAMENTOUS FLOWER (FIL), which show to have overlapping and partially redundant functions in marginal tissue development. For instance, the gynoecia of the double mutants lug ant (Liu et al., 2000) and fil ant (NoleWilson and Krizek, 2006) lack almost completely the structures derived from the medial domain, including the placenta and ovules. The double mutant lug seu shows severe developmental phenotypes, including first whorl carpelloid organs that develop as filamentous organs with no traces of marginal tissues (Franks et al., 2002; Sridhar et al., 2004). The seu ant mutant gynoecia fail to initiate ovule primordia and show reduced growth of other medialridge derived tissues (Azhakanandam et al., 2008). A possible model is that these proteins form a multimeric complex, with the SEU/LUG corepressor complex physically interacting with FIL and/or ANT, that is involved in supporting medial domain development and ovule initiation in wild-type gynoecia (Nole-Wilson and Krizek, 2006; Azhakanandam et al., 2008). 3.4.2 Primordium outgrowth In Arabidopsis, approximately 50 ovules develop inside a gynoecium. The outgrowth of ovule primordia requires a signal to direct cells in the placenta to expand and divide out of the plane of the developing septum. Some genes have been identified that seem to play a role in ovule primordium initiation, for instance the CUC genes. Before ovule initiation, indeed, CUC2 is detected in the placenta and, in many cases, the gynoecium of cuc1 cuc2 double mutants form fewer ovules than wild-type, cuc1 or cuc2 (Ishida et al., 2000). Moreover CUC genes may play a role in the location of primordium initiation, as suggested by their expression pattern: CUC2 and CUC3 are expressed at the boundaries of ovule primordia (Ishida et al., 2000; Vroemen et al., 2003). A putative role in ovule primordium initiation seems to be played by ANT. The ANT gene encodes for a transcription factor required for proper initiation and growth of plant lateral organs. It is expressed in carpel primordia and reaches very high levels in the placenta before and during ovule initiation. Moreover, ANT is strongly expressed in the developing ovule primordium and becomes restricted to the chalaza region where the integuments will arise (Elliott et al., 1996). ANT plays important and multiple roles in placenta and ovule formation and development. Concerning the primordium, loss of the ANT function leads to the formation of fewer and more distantly spaced ovule primordia that continue to develop until integument initiation (Elliott et al., 1996; Klucher et al., 1996).

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Fruit Development and Seed Dispersal Another gene that seems to be involved in placenta and ovule primordium initiation is CRABS CLAW (CRC). crc mutant carpels are wider and shorter than wild-type and contain fewer ovules. Moreover, early during carpel development, CRC is expressed in a longitudinal strand in the internal region of the carpel, adjacent to where the placenta will develop (Alvarez and Smyth, 1999; Bowman and Smyth, 1999), suggesting a possible role for this gene in ovule primordia formation. Ovule primordium outgrowth seems to be dependent on metabolic energy, necessary to sustain growth and cell division, as demonstrated by the huellenlos (hll) mutant, which exhibit defects in ovule growth and development (Schneitz et al., 1998). In hll mutant ovule primordia, the integuments are highly reduced or absent and the cells in the distal regions often collapse. The HLL gene encodes for a mitochondrial ribosomal protein (Skinner et al., 2001). Mitochondria perform processes essential for the cell; in addition to energy production, they are required for carbon backbone synthesis and the final steps of some amino acid biosynthesis pathways; thus, loss of HLL may slow growth and cause cells to collapse. Double mutant analysis of hll in combination with either short integuments2 (sin2) or ant, both promoters of ovule growth, has shown to result in an even more severe, synergistic reduction in ovule primordium outgrowth (Schneitz et al., 1998; Broadhvest et al., 2000). Thus, the effects of reduction in growth-promoting activity (as in the ant and sin2 mutants) are enhanced by a reduction in metabolic competence, resulting in a dramatic alteration in growth and form. 3.4.3

Patterning the ovule primordium

As already mentioned, ovule primordia appear as finger-like protrusions which show radial symmetry. Along the PD axis, three distinct regions can be recognized: the funiculus, the chalaza and the nucellus. At stage 11 of flower development, the appearance of integument primordia in the chalaza visibly marks the switch from PD polarity towards the adaxial–abaxial (Ad–Ab) polarity. A key regulator of pattern establishment is the NOZZLE (NZZ) gene (Schiefthaler et al., 1999; Yang et al., 1999; Balasubramanian and Schneitz, 2000, 2002; Sieber et al., 2004). nzz mutant ovules show many defects; the funiculus appears longer than wild-type ovules due to a problem in the establishment of the chalaza region. Integuments do not present the typical asymmetrical growth and the nucellus is extremely reduced. Furthermore, the MMC does not differentiate. The molecular characterization of the nzz single mutant together with other ovule defective mutants (Balasubramanian and Schneitz, 2002; Sieber et al., 2004) led to the hypothesis that NZZ plays a pivotal role in the temporal regulation of pattern development. The lack of NZZ activity seems to cause a precocious establishment of the Ad–Ab polarity when the PD axis has not been completed yet (Fig. 3.3a).

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n (b)

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Integuments development

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Figure 3.3 Schematic representation of ovule pattern establishment. (a) The NZZ gene plays an important role in the coordination of pattern formation along both the distal–proximal and the adaxial–abaxial axes. nzz mutant ovule displays pleiotropic defects during development. The distal region is reduced or missing and the MMC does not develop. Integuments are variably shortened and the funiculus is longer due to extra cell proliferation. (b) Funiculus development. The MADS-box gene STK regulates funiculus growth. stk single mutant ovules develop a longer and thicker funiculus compared to wild-type ovules. (c) Integuments develop from the chalaza region. Integument initiation is controlled by the ANT and WUS genes. ant and wus mutant ovules show a very similar phenotype since integuments do not develop. During the next stages, the PHB, INO and ATS genes play important roles in the determination of inner and outer integument development. The PHB gene is specifically expressed in the inner integument. Interestingly, the phb mutant shows a phenotype very similar to a weak ino allele since the outer integument growth is blocked. INO loss of function results in the lack of outer integument development. The ATS gene is expressed at the boundary between the outer and inner integuments. The ats mutant ovules are characterized by a single integument which derives from the fusion of the two integuments. (d) Nucellus development. n, nucellus; ch, chalaza; f, funiculus.

3.4.3.1 Funiculus The funiculus connects the ovule to the placenta and it hosts the vascular tissue. One of the genes that has been shown to control funiculus development is the MADS-box gene SEEDSTICK (STK) (Pinyopich et al., 2003). In the stk mutant, defects in the differentiation of the seed abscission zone cause the lack of seed dispersion. Moreover, the funiculus appears thicker and longer than in wild-type ovules suggesting that STK is required to control cell expansion and division in this structure (Fig. 3.3b).

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Fruit Development and Seed Dispersal Also in the nzz mutant, the funiculus is longer when compared to wildtype ovules (Balasubramanian and Schneitz, 2000). As already discussed, NZZ plays an important role in the coordination of the pattern formation along both axes. The longer funiculus of the nzz mutant ovules seems to be a consequence of the precocious onset of the Ad–Ab axis instead of a specific role of NZZ in the regulation of funiculus development. The lack of NZZ activity interferes with PD pattern formation in the primordium resulting in the absence of a nucellus and the presence of a longer funiculus (Balasubramanian and Schneitz, 2002). 3.4.3.2 Chalaza 3.4.3.2.1 Integument development: initiation and elongation. The formation of integument primordia in the chalaza is linked to the determination of Ad–Ab polarity (see Fig. 3.2 for adaxial–abaxial definition). Nevertheless, it seems that at a molecular level, the Ad–Ab polarity is established before the asymmetrical initiation of the outer integument (Sieber et al., 2004). A key regulator of adaxial cell fate in Arabidopsis is the transcriptional regulator PHABULOSA (PHB) (McConnell and Barton, 1998; McConnell et al., 2001). In the developing ovules, PHB mRNA is detected at very early stages of development and it is restricted to the adaxial side of the ovule. Later, at the time of integument development, PHB expression is restricted to the distal chalaza where the inner integument develops (Sieber et al., 2004). Outer integument formation occurs immediately after the polar expression of INNER NO OUTER (INO) on the abaxial side of the proximal chalaza (Villanueva et al., 1999; Balasubramanian and Schneitz, 2000; Meister et al., 2002). INO belongs to the YABBY family and it is necessary for outer integument initiation since ino mutant ovules are characterized by the absence of the outer integument (Villanueva et al., 1999). The presence of PHB and INO mRNAs can therefore be considered as molecular markers for inner and outer integument formation, respectively. Moreover, it was reported that these genes do not regulate each other during ovule development (Sieber et al., 2004). The isolation and characterization of the aberrant testa shape (ats) mutant led us to better understand how adaxial and abaxial identities are established both in the inner and outer integuments. The ATS gene belongs to the KANADI (KAN) gene family of putative transcription factors. In ovules, ATS is specifically expressed at the boundary between the inner and outer integuments (McAbee et al., 2006). The lack of ATS activity results in the formation of a single integument which derives from the fusion of inner and outer integuments which did not separate. McAbee et al. (2006) proposed that, like in the leaf, ovule integuments, adaxial and abaxial identities and growth direction are controlled by the expression domain of adaxial and abaxial identity factors. In both these organs, the expression of the PHB gene is correlated to adaxial identity while the expression of KAN and YABBY members (like ATS and INO, respectively) is linked to the determination of abaxial domain. The

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direction of growth is strongly correlated to the juxtaposition of adaxial and abaxial factors (Fig. 3.3c). The ANT gene plays a fundamental role during integument development since ant mutant ovules completely lack integuments (Elliott et al., 1996; Klucher et al., 1996). A few years after the characterization of ant ovules, another mutant that fails to form integuments was described. It was shown that ovules that lack WUSCHEL (WUS) activity do not develop integuments (Fig. 3.3c) (Gross-Hardt et al., 2002). In the shoot apical meristem, WUS is part of a feedback loop with the CLAVATA3 (CLV3) gene to regulate size homeostasis of the stem-cell population (Laux et al., 1996; Brand et al., 2000; Schoof et al., 2000). A similar feedback loop occurs at the end of flower development between WUS and AGAMOUS (AG). AG is required to terminate WUS expression and therefore floral meristem activity (Lenhard et al., 2001; Lohmann et al., 2001). In wild-type ovules, the WUS homeobox gene is specifically expressed in the nucellus and it is sufficient to promote integument initiation in the neighbouring chalaza in a non-cell autonomously way (Gross-Hardt et al., 2002). In situ hybridization experiments demonstrated that PHB mRNA is not detected neither in the chalaza of the ant mutant nor in the chalaza of wus mutant ovules, indicating that both ANT and WUS act upstream of PHB during inner integument formation (Sieber et al., 2004). Furthermore, INO expression is temporally delayed in the ant mutant background and is not sufficient to induce outer integument development (Sieber et al., 2004). Information about the different roles that WUS and ANT play during integument development came from the expression analysis of INO in wus mutant ovules and from the molecular characterization of ANT::WUS ovules (Gross-Hardt et al., 2002; Sieber et al., 2004). In situ hybridization experiments showed that INO mRNA is still present in wus mutant ovules but it is spatially deregulated since it can be detected in the distal chalaza instead of the proximal chalaza as observed in wild-type ovules. Moreover, when WUS is ectopically expressed in the chalaza under the regulation of the ANT promoter, it is sufficient to induce the formation of ectopic integuments (Gross-Hardt et al., 2002). In situ hybridization experiments performed on these ectopic integuments showed that PHB is expressed in the distal integument while INO is expressed in all the ectopic integuments that develop proximally but its expression cannot be maintained (Sieber et al., 2004). These data suggest that WUS can be considered a patterning gene which plays an important role in the establishment of the chalaza since it is necessary for the proper PHB expression in the distal chalaza and for the restriction of INO expression in the proximal chalaza. Moreover, WUS seems not to be sufficient for the maintenance of INO expression and outer integument development suggesting that integument development requires additional region-specific factors. Sieber et al. (2004) suggested that ANT can be considered as such a factor promoting primordia growth and cell proliferation (Elliott et al., 1996; Mizukami and Fischer, 2000).

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Fruit Development and Seed Dispersal INO expression is deregulated also in the nzz mutant background, since nzz mutant ovules show early initiation of the outer integument suggesting that NZZ behaves as a negative temporal regulator of INO expression. The early INO expression in nzz mutants interferes with PD development. Ovules of nzz mutants develop a smaller nucellus, but form a hyperplasic funiculus. Therefore, the temporal control of NZZ prevents INO from being active too early, leaving enough time for the completion of PD axis development. NZZ seems to act redundantly with ATS to spatially restrict INO expression in the adaxial chalaza (Balasubramanian and Schneitz, 2000). Moreover, NZZ is also required to restrict PHABULOSA (PHB) expression to the distal chalaza, from where the inner integument initiates, since in nzz ino double mutant ovules, PHB is ectopically expressed throughout the nucellus. In situ hybridization experiments performed in the nzz mutant background showed that WUS expression was strongly reduced suggesting that NZZ might also play as a positive regulator of WUS in the nucellus. Taken together, these results indicate that INO, PHB, and WUS all act downstream of NZZ during the switch from PD to Ad–Ab development (Sieber et al., 2004). Another regulator of INO is the zinc finger transcription factor SUPERMAN (SUP) (Sakai et al., 1995). SUP acts as a spatial repressor of INO since it interferes with the maintenance of INO on the adaxial side of the ovule primordium (Balasubramanian and Schneitz, 2002; Meister et al., 2002). INO therefore extends in the chalaza of sup mutant ovules resulting in the equal growth of the outer integument on the adaxial and abaxial side causing the absence of the typical curvature that is observed in wild-type ovules (Gaiser et al., 1995; Balasubramanian and Schneitz, 2002; Meister et al., 2002). 3.4.3.2.2 Integument elongation. Despite the fact that inner and outer integument initiation seems to be regulated independently, the subsequent elongation of both the integuments is somehow correlated. Different genes involved in the regulation of cell division and/or expansion participate in the coordinated regulation of ovule integument extension. The SIN2 gene encodes for a mitochondrial GTPase which is necessary for cell division specifically in the ovule integuments (Hill et al., 2006). sin2 mutant ovules are in fact characterized by the presence of short integuments which do not cover the nucellus (Broadhvest et al., 2000; Hill et al., 2006). More often, mutations in cell division controlling genes result in pleiotropic effects. For instance, the SEU and LUG genes encode for components of a putative transcriptional regulatory complex which is necessary for the regulation of floral homeotic genes. Both in seu and lug single mutants, a reduction in the ovule outer integument growth and an over-proliferation of the inner integument, which is supposed to be a consequence of the reduced outer integument growth, were observed (Franks et al., 2002). The specific role of SEU and LUG during cell expansion has recently been studied in petals. SEU and LUG seem to regulate petal polarity along the Ad–AB axis and they are supposed to enforce

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the key polarity genes PHB (Franks et al., 2006). The same mode of action can therefore be supposed during ovule development. Cell proliferation in developing ovules is impaired in the sin1 mutant which is female sterile due to the uncoordinated growth of the integuments and over-proliferation of the chalazal nucellus (Robinson-Beers et al., 1992; Lang et al., 1994). The mutant phenotype is not restricted to ovule integument growth since SIN1 also plays a role in the maternal sporophytic control of embryo pattern formation (Ray et al., 1996). Cloning the SIN1 gene showed that it encodes a RNA III/RNA helicase important for the translational regulation of developmental genes (Golden et al., 2002). The emerging scenario seems to indicate that there are only few specific integument growth regulators. Whereas the majority of genes that control integument growth seem to be general regulators that also control the growth in other tissues. 3.4.3.3 Nucellus The nucellus is derived from the distal part of the ovule primordium and hosts the MMC. In the nzz mutant, nucellus development is impaired since the MMC does not differentiate and the nucellus appears shorter than in wild-type (Yang et al., 1999; Balasubramanian and Schneitz, 2000). Recent work supports the hypothesis that the lack of MMC formation and the reduced size of the nucellus in the nzz mutant is a consequence of the temporal deregulation of pattern establishment instead of a specific role for NZZ in the control of MMC differentiation and nucellus formation. This hypothesis comes from the observation that the WUS gene is still expressed in the nucellus of the nzz mutant indicating that nucellus determination is independent from MMC differentiation. Moreover, in the nzz ino double mutant, the MMC is visible in the nucellus leading to the conclusion that the mutant phenotype of nzz can be mostly ascribed to the lack of temporal regulation of INO by NZZ (Balasubramanian and Schneitz, 2002; Sieber et al., 2004). As already mentioned, the meristem maintenance gene WUS is specifically expressed in the nucellus of developing ovules. wus mutant ovules are characterized by the absence of both integuments indicating that in the ovule WUS expression is a marker for nucellus determination while the WUS protein acts non-cell autonomously to regulate integuments growth (Gross-Hardt et al., 2002).

3.5 Gametophytic tissue 3.5.1 Development of the female gametophyte Female gametophyte development occurs over two phases referred to as megasporogenesis and megagametogenesis. More than 15 different patterns of female gametophyte development have been described, which arise mainly

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Fruit Development and Seed Dispersal from variations in cytokinesis during meiosis, number and pattern of mitotic divisions, and pattern of cellularization. The developmental pattern exhibited by most species is the Polygonum type, which was first described in Polygonum divaricatum (Drews and Yadegari, 2002). The Polygonum-type female gametophyte is found in more than 70% of flowering plants and is the pattern found in many economically and biologically important groups, including Brassicaceae (e.g. Arabidopsis, Capsella, Brassica), Gramineae (e.g. maize, rice, wheat), Malvaceae (e.g. cotton), Leguminoseae (e.g. beans, soybean) and Solanaceae (e.g. pepper, tobacco, tomato, potato, petunia). Polygonum-type female gametophyte development has been well described in Arabidopsis by Schneitz et al. (1995) (Fig. 3.4). In the nucellus, shortly after ovule initiation, a single subepidermal cell, directly below the nucellus apex, enlarges and differentiates into the MMC. During megasporogenesis, the diploid MMC undergoes meiosis to produce four haploid megaspores (Fig. 3.4, stage 2). The megaspore closest to the chalaza survives and enlarges. The other three non-functional megaspores undergo cell death, degenerate and are eventually crushed by the expanding functional megaspore (Fig. 3.4, stage 3-I). The female gametophyte is generated from the functional megaspore via a process termed megagametogenesis (described in Schneitz et al. (1995), Christensen et al. (1997)). The megaspore undergoes three sequential mitotic nuclear divisions to form a coenocytic embryo sac with eight haploid nuclei. After the first mitosis, the two nuclei migrate to opposite poles and the smaller vacuoles coalesce into a large central vacuole (Fig. 3.4, stage 3-III). Each of the two nuclei then divides two more times, resulting in an eight-celled coenocytic megagametophyte (Fig. 3.4, stage 3-V). Two nuclei (the polar nuclei), one from each pole, then migrate towards the centre of the cell. In Arabidopsis, the polar nuclei fuse, forming the secondary endosperm nucleus. During polar nuclei migration, the embryo sac cellularizes to form a seven-celled structure consisting of the egg cell (the future zygotic embryo after fertilization) closely associated with two supporting cells called synergids at the micropylar end, a large central cell with a diploid secondary nucleus (the future endosperm after fertilization), and at the chalazal end three cells of undetermined function, the antipodals that disintegrate prior to fertilization (Fig. 3.4, stage 3-VI). In some species, this is the final form: the Polygonum female gametophyte is typically a seven-celled structure at maturity. However, this structure may be modified by cell death or cell proliferation events in various species. For example, in Arabidopsis, the antipodal cells undergo cell death immediately before fertilization, whereas in grasses (e.g. maize), the antipodal cells proliferate to form a cluster of up to 40 cells (Kiesselbach, 1949). 3.5.2

Regulation of female gametophyte development

In the pathway leading to female gametophyte development, the first step is the differentiation of a single subepidermal cell at the tip of the ovule

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Figure 3.4 Schematic representation of wild-type female gametophyte development in Arabidopsis thaliana. Stages according to Schneitz et al., 1995. The grey areas represent cytoplasm, the white areas represent vacuoles and the black points are the nuclei. ac, antipodal cells; cc, central cell; ec, egg cell; m, megaspore; sc, synergid cell; sn, secondary nucleus.

primordium as the archesporial cell. In Arabidopsis, the archesporial cell directly functions as the megasporocyte or MMC (Reiser and Fischer, 1993). So far, little is known about the molecular mechanisms underlying archesporial cell fate determination. In maize, the multiple archesporial cells (mac1) mutation results in the formation of multiple archesporial cells in ovules

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Fruit Development and Seed Dispersal and anthers produce excessive numbers of sporogenous cells (Sheridan et al., 1996, 1999). The rice gene MSP1 (MULTIPLE SPOROCYTES1) also controls early sporogenic development by restricting the number of cells entering into male and female sporogenesis (Nonomura et al., 2003). MSP1 encodes a leucine-rich repeat receptor kinase, which is orthologous to EXCESS MICROSPOROCYTES1 (EMS1)/EXTRASPOROGENOUS CELLS (EXS) in Arabidopsis (Canales et al., 2002; Sorensen et al., 2002; Zhao et al., 2002). Like mac1 and msp1 mutants, exs/ems1 mutants produce excess microsporocytes in the anther instead of a tapetum, causing male sterility, whereas no effect has been observed on the female side. The exs/ems1 phenotype is also observed in mutants of TAPETUM DETERMINANT1 (TPD1) (Yang et al., 2003), which encodes a small protein that serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction (Jia et al., 2008). The control of meiosis is a key step in the transition from the sporophytic to the gametophytic phase in the plant life cycle. Several meiotic mutants affecting key stages in meiosis (chromosome cohesion, recombination, synapsis, chromosome segregation, and cell cycle regulation) have been identified in Arabidopsis using a combination of forward genetic (screening for plants exhibiting reduced fertility or sterility) and, more recently, using a reverse genetics approach (reviewed in Bhatt et al., 2001; Mercier et al., 2001; Caryl et al., 2003; Hamant et al., 2006). The use of reverse genetics has proven to be very powerful and led to the characterization in Arabidopsis of many homologues of yeast genes, implying that several of the basic mechanisms underlying meiotic functions are conserved between higher plants and fungi (Caryl et al., 2003). For instance, the Arabidopsis dmc1 mutant has been shown to be defective in bivalent formation (Couteau et al., 1999). A mutation in an Arabidopsis homologue of the SPO11 gene, which encodes a type II topoisomerase responsible for generating double-strand breaks in meiosis in yeast, has been shown to reduce meiotic recombination and bivalent formation (Grelon et al., 2001). Functioning downstream of AtSPO11-1, the Arabidopsis homologue of the RAD51 gene is essential for chromosome pairing and synapsis at early stages in meiosis (Li et al., 2004). The Arabidopsis asynaptic (asy1) mutant is defective in chromosome synapsis. This phenotype is due to a mutation in an Arabidopsis homologue of the yeast HOP1 gene which is required for homologous pairing (Caryl et al., 2000). Gametophytic mutations affect those aspects of female gametophyte development and function after meiosis, including megagametogenesis, fertilization and seed development. Embryo sac-expressed genes therefore play an important role in the control of embryo sac development as well as seed development. Although extensively studied at the morphological and cytological level, relatively little was known about the genes and the pathways involved in gametophytic development (Schneitz et al., 1995; Christensen et al., 1997). However, this is changing since in the last few years several

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studies have been carried out to identify genes that control female gametophyte development. Gametophytic mutants, typically, are identified using two criteria: reduced seed set and segregation distortion. Reduced seed set in a plant heterozygous for a female gametophyte mutation is due to the fact that half of the female gametophytes are mutant and non-functional and therefore they fail to undergo normal seed development. The segregation distortion that is observed in female gametophyte mutants is due to the fact that these mutations affect the haploid gametophyte phase of the plant life cycle, and therefore, in the case of female gametophyte mutants, the mutant allele is only transmitted through the male gametes. As a consequence, they are transmitted to subsequent generations at reduced frequency and exhibit non-Mendelian segregation patterns. The consequence is that these mutations can only be transmitted from generation to generation as heterozygotes (Drews et al., 1998; Drews and Yadegari, 2002). Many genes required for female gametophyte development and function have been identified and analysed using reverse genetic approach; among them are cell cycle genes (e.g. NOMEGA (Kwee and Sundaresan, 2003) and RETINOBLASTOMARELATED1 (Ebel et al., 2004)), transcription factors (e.g. MYB98 (Kasahara et al., 2005), AGL80 (Portereiko et al., 2006a) and AGL23 (Colombo et al., 2008)) and several others (including FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) (Ohad et al., 1996), MEDEA (MEA) (Chaudhury et al., 1997; Grossniklaus et al., 1998), FERTILIZATION-INDEPENDENTSEED2 (FIS2) (Chaudhury et al., 1997), GFA2 (Christensen et al., 2002), DEMETER (Choi et al., 2002), ARABINOGALACTAN PROTEIN18 (Acosta-Garcia and Vielle-Calzada, 2004), SLOW WALKER1 (Shi et al., 2005), CHROMATIN REMODELLING PROTEIN11 (Huanca-Mamani et al., 2005) and LPAT2 (Kim et al., 2005)). The screening of Arabidopsis T-DNA or transposon insertion lines has led to the identification of mutants defective in almost all stages of female gametophyte development (e.g. Christensen et al., 1998; Pagnussat et al., 2005). Drews and Yadegari (2002) identified five phenotypic mutant categories corresponding to key developmental events involved in the formation of a mature female gametophyte. Category 1 mutants are affected at the earliest possible step and fail to progress beyond the one-nucleate stage, suggesting that expression of the haploid genome is required very early in megagametogenesis. Category 2 mutants have defects during the nuclear division phase of megagametogenesis and fail to cellularize. These mutants have defects in nuclear number and/or positions, and developmental arrest at stages 3-III to 3-V (Fig. 3.4). Category 3 mutants become cellularized but have defects in cellular morphology, including abnormal nuclear positions within cells, misshapen cells, and unusual cell features. Category 4 mutants have defects in fusion of the polar nuclei. They migrate properly, come to lie side by side, but fail to fuse. Category 5 mutants have phenotypically wild-type female gametophytes at the terminal developmental stage (stage 3-V; Fig. 3.4), which suggests that

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Fruit Development and Seed Dispersal megagametogenesis is not affected. However, they show alteration at the level of female gametophyte’s reproductive functions, like pollen tube guidance, fertilization, induction of seed development and maternal control of seed development. Pagnussat and co-workers (2005) reported the results of a large-scale mutant screen of Ds transposon insertion lines. They identified 130 Arabidopsis mutants with defects in female gametophyte development and function and a wide variety of mutant phenotypes were observed, ranging from defects in different stages of early embryo sac development to mutants with apparently normal embryo sacs, but exhibiting defects in processes such as pollen tube guidance, fertilization or early embryo development. The successive sequence identification of the genes disrupted in these mutants revealed genes involved in several different processes, that is secondary metabolism, protein degradation, cell death, signal transduction and transcriptional regulation, therefore providing a wide-ranging assessment of the genes and functions required for embryo sac development, fertilization and early embryogenesis. Despite the progresses made with the identification of female gametophytic mutants, the total set of genes expressed in the embryo sac was poorly defined due to the fact that the embryo sac is embedded within the sporophytic tissues of the ovule, making it difficult to directly isolate embryo sac tissue for gene expression analysis. In the last few years, different attempts have been made to identify female gametophyte-expressed genes. Hennig and co-workers (2004) analysed the expression profiles of thousands of genes present on the Arabidopsis ATH1 microarray, focusing on developmental stages of the female gametophyte. They found that more than 2300 genes are specifically regulated during these developmental transitions or are expressed preferentially in the tested samples. Furthermore, they observed that several members of the YABBY, MADS-box and Myb transcription factor families are significantly over-represented, suggesting an important role for these families during reproduction. Interestingly, among the specifically expressed MADS-box genes, several of them belong to the recently discovered type I family (Alvarez-Buylla et al., 2000). Recently more information has become available through the comparison of expression profiles of ovules with and without an embryo sac, which led to the isolation of several gametophytically expressed genes. Yu et al. (2005) performed a screen for genes with reduced expression in sporocyteless/nozzle mutant ovules in Arabidopsis and identified a set of 249 genes. As a first step towards dissecting the gene regulatory networks of the female gametophyte, Steffen et al. (2007) identified, using determinant infertile1 (dif1) ovules, a large collection of genes expressed in specific cells of the Arabidopsis female gametophyte. Johnston et al. (2007) identified hundreds of genes expressed or enriched in the embryo sac, using as approach a genetic subtraction and microarray-based

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comparative profiling between the wild-type and two sporophytic mutants lacking a functional embryo sac, coatlique (coa) and sporocyteless (spl). They also identified a set of genes that are over-expressed in the maternal sporophyte in the absence of a functional embryo sac, suggesting that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac. Jones-Rhoades et al. (2007) used two recently developed technologies, whole-genome tiling microarrays and high-throughput cDNA sequencing, to identify hundreds of genes expressed in embryo sacs of Arabidopsis. Most of these embryo sac dependent genes have unknown function, and include entire families of related genes that are only expressed in the embryo sac. The fact that so many paralogous genes have overlapping domains of expression in the embryo sac suggests that there will be a high degree of functional redundancy between embryo sac genes and may explain why genes from these families have not been identified previously in mutant screens. Moreover, many of these embryo sac dependent genes are not expressed at high levels in tissues other than ovules, suggesting that they may be specialized for roles in female reproductive development and function. Furthermore, most embryo sac dependent genes encode small proteins that are potentially secreted from their cells of origin, suggesting that they may act as intracellular signals or to modify the extracellular matrix during fertilization or embryo sac development. 3.5.3 Function and patterning of the female gametophyte The four different cell types that form the gametophyte are distinct with respect to morphological and molecular attributes and perform unique functions that are essential for the reproductive process. The two gametic cells, the egg cell and the central cell, are fertilized by one sperm cell each to form the embryo and the endosperm, respectively. These two gametic cells are flanked by accessory cells, the synergids and the antipodal cells, that aid in the fertilization process. Soon after pollen is transferred from anther to stigma, the male gametophyte forms a pollen tube that grows through the carpel to reach the female gametophyte. Pollen tube guidance is mediated by multiple signals emitted by both sporophytic female cells and the embryo sac which, using short-range attractants, guides the final stage of tube growth (reviewed in Higashiyama and Hamamura, 2008). Several lines of evidence indicate that the synergid cells are the source of one or more pollen tube attractants (reviewed in Punwani and Drews, 2008). Laser ablation of the synergid cell in T. fourieri abolished the pollen tube guidance process (Higashiyama et al., 2001, 2003). Moreover, in Arabidopsis, it has been shown that loss of function of synergid-expressed genes also abolished the ovule ability to attract pollen tubes. For instance, in the Arabidopsis myb98 mutant, pollen

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Fruit Development and Seed Dispersal tube guidance and the development of the filiform apparatus are affected. The filiform apparatus is an elaborated cell wall present at the synergid micropylar pole that likely plays a role in pollen tube guidance and reception (Kasahara et al., 2005). Punwani et al. (2007) demonstrated that MYB98 functions as a transcriptional regulator within the synergid cells and that many of the downstream genes encode proteins that are secreted into the filiform apparatus, suggesting that they play a role in the formation or function of this structure. In maize (Zea mays), RNA interference knockdown of the ZmEA1 gene, initially isolated from egg cells and expressed in the synergid cells, abolished micropylar pollen tube guidance (Marton et al., 2005). The expression of ZmEA1 in egg cells suggested that in addition to the synergids, other cells of the female gametophyte may be involved in pollen tube guidance. Recently, this hypothesis was strengthened by Chen et al. (2007) who demonstrated that the central cell also plays a critical role in this process. They identified the central cell guidance (ccg) mutant, which is defective in micropylar pollen tube guidance. The CCG gene is expressed in the central cell of the female gametophyte and its specific expression in the central cell alone is sufficient to restore the pollen tube guidance defect in the mutant. Many other Arabidopsis mutants with synergid cell defects function have been identified, for example feronia (fer), which is allelic to sirene (srn) (Huck et al., 2003; Rotman et al., 2003; Escobar-Restrepo et al., 2007) and gametophytic factor2 (gfa2) (Christensen et al., 2002). In wild-type plants, when the pollen tube arrives to the female gametophyte, it enters into one of the synergid cells, ceases to grow and bursts to release its two sperm cells. The synergid cell that interacts with the pollen tube typically undergoes cell death. Degeneration appears to occur only upon contact with the pollen tube but before tube discharge (Sandaklie-Nikolova et al., 2007). In the feronia (fer) mutant, when a wild-type pollen tube enters the receptive synergid of a mutant female gametophyte, it fails to arrest its growth and does not rupture to release the sperm cells, so it continues to grow inside the female gametophyte. Thus, the fer mutation disrupts the female control of pollen tube reception. FER encodes a synergid-expressed, plasma membrane localized receptor-like kinase that might interact with a ligand from the pollen tube (Escobar-Restrepo et al., 2007). gametophytic factor2 (gfa2) female gametophytes fail to undergo synergid cell death following pollination (Christensen et al., 2002). GFA2 encodes a J domain containing protein required for mitochondrial function, suggesting a role for mitochondria in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. Finally, the two sperm cells migrate to the egg cell and central cell, and the gametes fuse to accomplish double fertilization (Lord and Russell, 2002). In the cdka;1 (also called cdc2a) mutant, double fertilization does not occur because the central nuclei are never fertilized. Indeed in cdka;1 mutant pollen only one sperm cell, instead of two, is produced and it fertilizes exclusively

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the egg cell. This shows that egg and central cell are not equally likely to be fertilized. However, it is not clear whether this preferential fertilization is due to the egg cell closeness to the sperm cell, to an active signalling or to predetermination (Iwakawa et al., 2006; Nowack et al., 2006). A series of mutants affecting central cell development and function are known. Several of them show defects in fusion of the polar nuclei (e.g. Pagnussat et al., 2005) and, interestingly, among them, several of the mutated genes encode mitochondrial proteins or predicted mitochondrial proteins, such as GFA2 (Christensen et al., 2002), RPL21M (Portereiko et al., 2006b) and SDH1-1 (Leon et al., 2007), suggesting that the karyogamy defect is a consequence of an impairment in basic mitochondrial functions. On the contrary, in the lachesis (lis) mutant, defects in polar nuclei fusion appear to be a secondary consequence of the fact that the central cell differentiates egg cell attributes at the expenses of central cell fate (Gross-Hardt et al., 2007). In the agl80 mutant, polar nuclei fuse but central cell development is compromised; nucleolus and vacuole fail to mature properly and, after fertilization, endosperm is not formed (Portereiko et al., 2006a). Within the ovule and seed, AGL80 is expressed exclusively in the central cell and during early endosperm development, suggesting that this transcription factor is required for late steps of central cell differentiation. Moreover, central cell-expressed gene products control the activation of endosperm development (Grossniklaus et al., 1998; Luo et al., 1999; Ohad et al., 1999). Until now, no clear function has been assigned to antipodal cells. Christensen et al. (1998) identified some mutants where antipodal cells fail to degenerate, but these mutants also showed other defects in female gametophyte development. Gross-Hardt et al. (2007) showed that in lis-1 mutant, antipodal cells can adopt a central cell fate and eventually disintegrate their membrane allowing nuclei fusion. It is therefore possible that the antipodal cells might function as a backup in case of gametic failure. Despite the large number of mutants showing defects during megagametogenesis isolated in Arabidopsis, the mechanisms underlying the establishment of cell identity in the female gametophyte are not known. Mutant analysis suggests that the differentiation of the distinct cell fates is tightly controlled and appears to follow regional cues, with a pivotal role for cell–cell communication during the patterning process. Important aspects of gametophyte development are gametophytically regulated. Moreover, the female gametophyte seems to be a flexible structure with enormous respecification potential suggesting that the status of distinct cell fates is constantly monitored. In the lis-1 mutant, synergids and central cells adopt egg cell fate, whereas antipodal cells behave like central cells (Gross-Hardt et al., 2007). Therefore, accessory cells of lis-1 mutant gametophytes frequently adopt a gametic cell fate, suggesting that all the cells in the female gametophyte are competent to differentiate gametic cell fate and that this competence is repressed in accessory cells of wild-type gametophytes. Intriguingly, shortly after

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Fruit Development and Seed Dispersal cellularization, expression of LIS, which is homologous to the yeast splicing factor PRP4, is highly up-regulated in gametic and down-regulated in accessory cells. A lateral inhibition model has been proposed based on these results: the gametic cells upon differentiation generate a LIS-dependent signalling molecule that is transmitted to the adjacent accessory cells to inhibit their gametic cell competence, thereby not only preventing excess gametic cell formation but also assuring that the accessory cells can act as a backup if required. Moreover, the central cell in lis-1 gametophytes additionally adopted egg cell fate, suggesting that a further, LIS-dependent mechanism suppresses egg cell fate in the central cell. Thus, the lis-1 mutant phenotype reveals two levels of cell fate regulation, one between gametic and accessory cells, and the other between egg and central cell. Pagnussat et al. (2007) observed that, although this mechanism of lateral inhibition can explain the maintenance of only one egg cell in the embryo sac after the initial specification of cell fate, the mechanisms involved in the early establishment of different cell fates in the embryo sac are not addressed by the model. They proposed that a positional mechanism would explain how cell fate is specified in early megagametophyte development. The migration and position of nuclei during megagametogenesis in Arabidopsis have been shown to be highly regular (Webb and Gunning, 1990; Webb and Gunning, 1994), and previous studies suggested that cytoplasmic domains may determine the fate of cells during cell partitioning (Brown and Lemmon, 1991, 1992). According to this positional model, the nuclei that move towards the micropylar or chalazal poles acquire accessory cell fates and, upon cellularization and egg cell formation, the lateral inhibition mechanism maintains their cell fates. In this way, superimposition of a gametic module onto existing micropylar and chalazal domains can generate four distinct cell types (Kagi and GrossHardt, 2007; Pagnussat et al., 2007). The idea of a positional mechanism is also supported by other studies. In the maize indeterminate gametophyte1 (ig1) mutant, the female gametophyte has a prolonged phase of free nuclear divisions, which results in a variety of embryo sac abnormalities, including the presence of extra egg cells, extra polar nuclei, and extra synergids. The phenotypes of the ig1 mutant embryo sacs suggest a position-based determination of cellular identity. The ability of the extra cells and nuclei to function as egg cells or polar nuclei, for example, appears to depend on their position in the embryo sac (Guo et al., 2004; Evans, 2007). Another mutant supporting the idea that a positional mechanism might be directing establishment of cell fates in early megagametophyte development is the eostre mutant which shows a synergid to egg cell fate switch (Pagnussat et al., 2007). The eostre-1 mutant embryo sac appears to contain two functional egg cells and only one synergid. As eostre-1 embryo sacs also show nuclear migration abnormalities, it has been proposed that the cell fate switch might be due to the unusual position of the nuclei within the embryo sac. This makes them exposed to different cytoplasmic domains compared to what happens in wild-type embryo sac.

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3.6 Interaction between the female gametophyte and the maternal sporophyte A feature of ovule morphology is that diploid sporophytic tissues such as the funiculus and the integuments and the haploid tissue of the embryo sac develop very close to each other. Despite the increasing number of information about the molecular events that control ovule development in Arabidopsis, an intriguing aspect that is still not completely understood at the molecular level is the relation between the sporophytic and gametophytic tissues. It is still not clear which are the molecular signals that coordinate the development of both these tissues. More than 10 years ago, Schneitz et al. (1997) classified the ovule defective mutants in three different classes. Mutants that show defects in both sporophytic and gametophytic tissues are named sporophytic and megagametogenesis defective mutants (smd). When the defects are restricted to the development of the gametes, these mutants are named megasporogenesis defective (msd) and embryo sac defective mutants (emd). The characterization of several smd mutants provides information about the interdependency between sporophytic and gametophytic tissues during ovule development. A strong smd mutant is the nozzle (nzz) mutant. In fact, when NZZ activity is missing, both sporophytic and gametophytic tissues are impaired (Schiefthaler et al., 1999; Yang et al., 1999). Regarding many other smd mutants characterized until now, a common feature seems to be the fact that the meiotic division proceeds normally while a block at different stages of megagametogenesis occurs later. Based on this observation, it was proposed that genes which regulate megasporogenesis act independently from those that regulate sporophyte development, while megagametogenesis is strongly influenced by the ovular context. Until now, many smd mutants have been isolated. The bel1 single mutant and the stk shp1 shp2 triple mutant have been classified as smd mutants (Schneitz et al., 1997; Battaglia et al., 2008). Aniline blue staining performed on these mutant backgrounds marked the cells resulting from meiosis meaning that megasporogenesis is not affected; later in development, the homeotic conversion of integuments into carpelloid tissue is accompanied by a block during megagametogenesis. Together with the bel1 single mutant and the stk shp1 shp2 triple mutant, the majority of integument-defective mutants can also be classified as smd mutants. Among them, a few examples are ino (Baker et al., 1997; Schneitz et al., 1997), sup (Sakai et al., 1995) and most of the ant alleles (Klucher et al., 1996). Since smd mutant defects are to a large extent region-specific, it is likely that funiculus, chalaza and nucellus develop independently for a certain period. Later, the sporophytic tissue influences embryo sac ontogenesis. In particular, it seems that megasporogenesis is independent from integument primordia

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Fruit Development and Seed Dispersal development in the chalaza while megagametogenesis is strongly influenced by the morphogenesis of the inner and outer integuments. The characterization of the bel1 stk shp1 shp2 quadruple mutant allowed not only to better understand the molecular regulation of ovule identity determination but this mutant also represents an example of an integumentdefective mutant which is blocked during megasporogenesis (Brambilla et al., 2007; Battaglia et al., 2008). Despite the fact that at early stages the bell stk shp1 shp2 quadruple mutant resembles the bel1 ovules in the chalaza, surprisingly, in the nucellus, differences with the wild-type ovules appear more precociously when compared with the stk shp1 shp2 triple mutant and the bel1 single mutant. DIC microscopy analysis using aniline blue staining showed that in this quadruple mutant, the MMC never goes into meiotic division. Furthermore, callose accumulation indicates that cells in this region are degenerating (Battaglia et al., 2008). Until now, it is still not clear if the ovule identity factors STK, SHP1 and SHP2 together with BEL1 directly regulate target genes involved in the control of megasporogenesis. Another possibility is that a cross talk between nucellus and chalaza coordinates ovule ontogenesis during the early stages of development. So far, the characterization of megasporogenesis-affected mutants led to the identification of genes that regulate meiosis in both the female and male megaspore/microspore mother cells or exclusively during female or male meiotic divisions. For instance, the sterile apetala (sap) mutant is both male and female sterile due to a missing meiotic division (Byzova et al., 1999). A different example is the dyad mutation which affects exclusively the female germ line while pollen development and male fertility are normal (Siddiqi et al., 2000). Interestingly, in both the sap and dyad mutants, the remainder of the sporophyte is normal, indicating that the activity of these genes is independent from the neighbouring tissues. In the attempt to identify embryo sac specific transcripts, Johnston et al. (2007) used a genetic subtraction and microarray-based comparative profiling approach. The authors compared the transcriptome of wild-type pistils with those of the spl (nzz) and cotilique (coa) mutant pistils (Vielle-Calzada, Moore and Grossniklaus, unpublished data). These mutants are characterized by the absence of the embryo sac and they can therefore be used as a powerful tool for the identification of genes which regulate the female gametes development. The results obtained from the microarray analysis allowed them to identify two groups of genes: (i) those that are specifically expressed during female gametogenesis and (ii) those that appear to be over-expressed in both the spl and coa mutants. These genes are deregulated in the maternal sporophyte probably due to the absence of the embryo sac meaning that not only the diploid sporophytic tissues influence megagametogenesis but also the presence of a mature embryo sac controls the sporophytic transcriptome. Among this group of genes, transcriptional regulators such as MYB genes,

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homeobox genes (including SHOOT MERISTEMLESS (STM)) and zinc-finger genes (including SUP) were identified and signalling factor encoding genes were deregulated (Johnston et al., 2007). The emerging scenario indicates that the development of the female gametophyte is strictly controlled by the development of the neighbouring maternal sporophytic tissues and vice versa. Nevertheless, the molecular nature of this interaction is still unknown.

3.7 Ovule identity determination 3.7.1 Arabidopsis Information about ovule identity determination came available through the characterization of mutants which showed transformations of ovules into carpelloid tissues. A good example is the bell1 (bel1) mutant reported by Robinson-Beers et al. (1992). The loss of the BEL1 function results in the formation of a single integument-like structure which in the strong bel1-4 allele develops into carpelloid-tissue bearing ectopic ovules (Robinson-Beers et al., 1992; Ray et al., 1994). It was suggested that this change in identity was due to the ectopic expression of the AGAMOUS (AG) gene in the developing integuments. AG belongs to the well-characterized Arabidopsis MADS-box family of transcription factors and it controls stamen and carpel identity (Bowman et al., 1989; Yanofsky et al., 1990). A similar conversion was later described by Pinyopich et al. (2003) in the stk shp1 shp2 triple mutant. A more recent detailed morphological analysis of this mutant showed that ovule development proceeds like in wild-type plants until stage 12 of flower development. Subsequently, in most of the ovules, integuments grow abnormally and do not cover the nucellus, with some of them converted into carpel-like tissue (Brambilla et al., 2007). SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2 also encode for MADS-box transcription factors and in a protein maximum likelihood tree, they all group together with AGAMOUS (Parenicova et al., 2003). The fact that AG also plays a role in ovule development was demonstrated by the comparison of the apetala2 (ap2) single mutant with the ap2ag double mutant. In the ap2 single mutant, petals are mostly absent and sepals are converted into carpel structures on which ectopic ovules develop, of which some have been converted into carpelloid structures. Interestingly, the first whorl organs of the ap2ag double mutant still had carpel identity; however, the number of ovules that lost their identity and were converted into carpel structures was significantly increased indicating that AG activity also contributes to ovule identity (Western and Haughn, 1999; Pinyopich et al., 2003). Protein interaction studies allowed a better understanding of the mode of action of these factors. Yeast two-hybrid experiments have shown that STK,

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Fruit Development and Seed Dispersal SHP1, SHP2 and AG cannot directly interact but higher order protein complexes can be formed when the SEPALLATA SEP3 MADS-box factor is added in yeast three-hybrid experiments (Favaro et al., 2003). Interestingly, AG is not only able to interact with SEP alone to regulate carpel identity but it can also participate in the formation of the ovule identity complex (Favaro et al., 2003). The genetic relationship between the ovule identity genes BEL1, AG, STK, SHP1 and SHP2 has been very recently elucidated with the characterization of the bel1 stk shp1 shp2 quadruple mutant (Brambilla et al., 2007). This mutant shows an enhancement of the bel1 phenotype since the single integument is transformed into carpel-like structure. Furthermore, on this structure and on the funiculus, new ectopic ovules develop. In situ hybridization experiments allowed to study the identity of the developing tissues in the ovules of this mutant. The loss of integument identity in the bel1 stk shp1 shp2 mutant was demonstrated using the ovule identity marker STK and the carpel identity marker CRC as probes. This analysis showed that until stage 12 of flower development, integument identity is maintained. After this stage, STK expression disappeared while CRC starts to be expressed in the mutant chalaza clearly showing the change from integument to carpel identity (Brambilla et al., 2007). BEL1, STK, SHP1 and SHP2 loss of function is also responsible for WUS deregulation in the developing ovules. In fact, starting from stage 10, WUS transcripts were not only detected in the distal part of the ovule primordia but also in the chalaza, in structures developing from the chalaza region and in the funiculus. Moreover, WUS expression was still clearly detectable in mutant ovules at late stage 12 and stage 13, while in wild-type ovules, its expression weakens from stage 11 to become undetectable at later stages (Gross-Hardt et al., 2002). These results suggest that the cell proliferations observed from the chalaza and the ectopic ovule-like structure formation from the funiculus, as observed in the quadruple mutant, might be due to a combination of ectopic WUS expression and the absence of integument identity gene activity. Interestingly, deregulation of WUS was only observed when the BEL1 gene was inactive, since in the stk shp1 shp2 triple mutant, WUS expression was not changed relative to wild-type plants. Protein interaction experiments allowed the authors to interpret the genetics results (mutant ovule phenotypes) and the molecular data obtained through in situ hybridization analysis and to suggest a model for integument identity determination (Fig. 3.5). Brambilla et al. (2007) demonstrated that BEL1 strongly interacts with the AG–SEP dimer. Since in the bel1 mutant ovules, integuments are converted into carpel tissue, it means that the interaction between BEL1 and the AG–SEP dimer is necessary to prevent AG carpel identity activity during ovule integument development. Whenever the BEL1 protein is missing, the free carpel identity AG–SEP dimer induces carpel development in the chalazal region (Fig. 3.5). Moreover, the WUS gene is ectopically expressed in the chalaza of the bel1 mutant indicating that the AG–SEP–BEL1 complex also plays a role in the restriction of the WUS expression domain.

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BEL1

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Figure 3.5 Schematic representation of integument identity determination in Arabidopsis. (a) In wild-type plants, the BEL1 factor interacts with the carpel identity complex. The ovule identity complex seems to stabilize the CIC–BEL1 complex. (b) In the bel1 single mutant, the lack of BEL1 protein results in an increasing amount of free carpel identity complex which induces carpel development in the chalaza. (c) In the stk shp1 shp2 triple mutant, the absence of the ovule identity complex probably causes the destabilization of the BEL–CIC interaction which results in an increased carpel identity activity in the chalaza region. (d) In the case that the amount of AG protein is reduced, like in the stk shp1 shp2 ag3/+ mutant, integument development is impaired but differently from the stk shp1 shp2 triple mutant, they do not longer develop in a carpel structure. (e) In the bel1 stk shp1 shp2 quadruple mutant, the presence of only the carpel identity complex strongly induces carpel tissue instead of integuments. CIC, carpel identity complex (AG-SEP); OIC, ovule identity complex; ii, inner integument; oi, outer integument; f, funiculus; cls, carpel-like structure.

In the stk shp1 shp2 triple mutant, ovule integuments are transformed into carpel tissue. This phenotype suggests that the MADS-box ovule identity complex somehow stabilizes the AG–SEP–BEL1 protein complex. The lack of STK, SHP1 and SHP2 activity results once again in the ectopic carpel activity of the AG–SEP dimer (Fig. 3.5). The role of the AG–SEP dimer in promoting carpel development in the stk shp1 shp2 mutant background has been further confirmed by the phenotype of the stk shp1 shp2 ag3/+ quadruple mutant which is characterized by a less extension of the carpel-like structures that developed instead of integuments (Fig. 3.5). The enhanced ovule phenotype of the bel1 stk shp1 shp2 quadruple mutant is due to the fact that only the AG–SEP dimer is available, so that it strongly induces carpel development in the chalaza of this quadruple mutant (Fig. 3.5).

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Fruit Development and Seed Dispersal 3.7.2

Petunia and rice

The first results towards the understanding of the molecular mechanisms controlling ovule identity determination came from studies performed in Petunia (Angenent et al., 1995; Colombo et al., 1995). In Petunia plants in which two putative STK orthologues, FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, were cosuppressed, carpel structures developed directly from the placenta replacing the ovules. The carpel identity of these structures was confirmed molecularly by showing that the carpel marker genes pMADS3 and FBP6 were active in these structures. Furthermore, FBP7 and FBP11 also interact with SEP-like proteins, which are FBP2 and FBP5 (Ferrario et al., 2003; Immink et al., 2003). These SEP-like proteins are, like in Arabidopsis, essential for correct ovule development since in the fbp2 fbp5 double mutant instead of ovules, leaf-like structures are directly formed from the placenta (Vandenbussche et al., 2003), suggesting that the ovule identity complex cannot be formed without SEP-like proteins and therefore the ovule identity pathway cannot be initiated. Another example that shows that correct ovule identity determination is dependent on the same basic principle and evolutionary well-conserved factors comes from the more distantly related species rice. In rice, it has been shown that OsMADS13, which is orthologous to STK, determines ovule identity (Dreni et al., 2007). In the osmads13 mutant, ovules are converted into carpelloid structures. Furthermore, in this mutant, a reiterated set of carpels develops in the place of the ovule (Dreni et al., 2007). Interestingly, the complexes that control ovule identity also seem to be conserved between grasses and core eudicot plants since OsMADS13 is also able to interact with the SEP-like rice proteins OsMADS24 and OsMADS45 (Favaro et al., 2002). The conservation of the interactions of the Arabidopsis, Petunia and rice ovule identity proteins is further illustrated by the fact that OsMADS13 interacts with the SEP proteins of Arabidopsis and Petunia, and STK and FBP7 with OsMADS24 and OsMADS45. Although this suggests that OsMADS24 and OsMADS45 are also involved in ovule identity determination, functional analysis of these genes has still to be done to prove this hypothesis. Phylogenetic analysis of the AGAMOUS-subfamily revealed that in the angiosperm lineage, an ancient gene duplication occurred that produced the ovule specific group to which STK, FBP7, FBP11 and OsMADS13 belong (Kramer et al., 2004; Yamaguchi and Hirano, 2006). This group further contains a large variety of angiosperm species and it will be interesting to analyse if all these genes are still controlling ovule development. One of the differences that can be observed in ovule identity defective mutants in Arabidopsis, Petunia and rice is that while in the Arabidopsis stk shp1 shp2 triple mutant, ovule integuments are converted into carpelloid tissue, in Petunia and in rice, carpel structures develop instead of complete ovules. Another important difference is the origin of the placenta. In Petunia and rice, the floral meristem is maintained after carpel primordia development, and

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placenta and ovules subsequently arise directly from the inner part of the floral meristem. By contrast, in Arabidopsis, both the placenta and the ovules differentiate from the inner ovary wall and the floral meristem is already completely ‘consumed’ by the carpel primordia. Related to this difference in the origin of the placenta is the regulation of floral meristem activity through the action of stem-cell maintenance genes such as WUS (Lenhard et al., 2001; Lohmann et al., 2001). In Arabidopsis, appearance of the carpel primordia is accompanied by the repression of WUS by the carpel identity gene AG (Sablowski, 2007). In rice and Petunia, where floral meristem identity is maintained after the development of the carpel primordia, the ovule identity genes seem to be involved in the determinacy of the floral meristem (Ferrario et al., 2006; Dreni et al., 2007). However, in rice, indeterminacy due to the loss of OsMADS13 activity is different in respect to Arabidopsis since it results in a reiteration of carpels and not in a complete reiteration of the floral meristem, as observed in the Arabidopsis ag mutant (Yanofsky et al., 1990). It will be interesting to study the rice orthologue of WUS and investigate whether the ovule identity gene OsMADS13 controls this stem-cell maintenance gene. In Petunia, simultaneous ectopic expression of FBP2 (which encodes a SEP orthologue in Petunia) and the ovule identity gene FBP11 caused an early arrest in development at the cotyledon stage. Molecular analysis of these transgenic plants revealed a possible combined action of FBP2 and FBP11 in the repression of the Petunia WUS homologue named TERMINATOR (TER) (Ferrario et al., 2006). The role of SEP orthologues in floral meristem determinacy in Petunia was already suggested previously by the observation that in the fbp2 mutant, determinacy is lost in the centre of the flower (Angenent et al., 1994; Vandenbussche et al., 2003). However, cosuppression of both FBP7 and FBP11 does not result in the loss of meristem determinacy. This could be explained by the presence of a third still-unidentified ovule identity gene that is not silenced in the cosuppressed plants. In fact, in the cosuppression plants, some of the ovules develop normally. It will be interesting to investigate whether the meristem determinacy function is maintained for this class of ovule identity genes in all plants that develop the placenta directly from the floral meristem.

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Annual Plant Reviews (2009) 38, 107–171 doi: 10.1002/9781444314557.ch4

www.interscience.wiley.com

Chapter 4

FERTILIZATION AND FRUIT INITIATION Sara Fuentes1 and Adam Vivian-Smith2 1

Department of Crop Genetics, John Innes Centre, Norwich, United Kingdom Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg, Leiden, The Netherlands

2

Abstract: Angiosperms have evolved the unique processes of double fertilization and fruit development as key steps of their survival and dispersal strategies. In this chapter, we will examine fertilization and fruit initiation as central restriction points to fruit and seed development. Pollination and fertilization appear essential for fruit initiation, since angiosperm flowers universally enter terminal senescence and abscission phases if pollination is prevented. We review key developmental processes, pathways and genes that were recruited to control and restrict the reproductive growth of the carpel and ovule until fertilization is achieved. Ever since the discovery that exogenous application of phytohormones results in the development of seedless fruit without fertilization (termed parthenocarpy), most research has concentrated on the role of endogenous phytohormones as triggers for fruit initiation after fertilization. We will highlight how uncoupling of fruit initiation from fertilization through mutational studies can further contribute to the understanding of these complex processes. Initial analysis shows that strict local control of auxin signalling, through a transcription factor network, forms one of the decisive and primary events that leads to the hierarchical control over gibberellin metabolism and perception. Testing and challenging these assumptions will provide further knowledge indispensable for controlling fruit set and yield in agriculture. Keywords: double fertilization; ovule; female receptivity; fruit initiation; vascular development; phytohormonal signalling cascades

4.1 Introduction From a purely biological standpoint, the success of higher plants hinges on reproduction strategies and the dissemination of viable progeny into

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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Fruit Development and Seed Dispersal habitable environmental niches. Fertilization and fruit initiation are essential processes in angiosperm plant reproduction and play a part in maximizing the success and variation. This is evident since the conquest and rise of the angiosperm plant lineage in the dominance of land environments can be directly attributed to the developmental innovations of the flower, ovule, carpel and fruit as vital components of propagation and dispersion. Selection pressure has lead to large variation in fruit development; however, the control on fruit initiation appears to be a universally conserved mechanism amongst all angiosperms. Without pollination and successful fertilization, the ovary and sometimes the accessory tissues, cease to grow and the flower begins a terminal phase of senescence that ends in floral abscission. Alternatively, when pollination and fertilization take place, a cascade of events is triggered, leading to growth and development of the fruit and seed. Speculation about the evolutionary origin of the fruit has largely been focused on the role of the carpel in initiating and providing protection around the developing ovules and seeds. The development of the carpel and integuments presumably also played a role together with the stigma and style in the selective discrimination of male gametes during pollination and, thus, constitutes an important mechanism in the control of outbreeding (Mulcahy, 1977, 1979). The development of the fleshy fruit most likely coevolved with extinct megafauna and avifauna where the function was to enable vectorial dispersion. The whole seed would be swallowed at the end of fruit development, and subsequent excretion would ensure a fertile environment for seeds to germinate and colonize. Various mechanisms arose to strictly manage and restrain carbon partitioning to optimal levels in developing flowers and fruits in order to match fitness with success of the zygote in the environment. From an economical standpoint, fruit initiation and fruit set are essential processes in many horticultural and agricultural cropping systems. Shortly after fruit initiation commences, large diversions in plant resources often occur and the fruit actively recruits photoassimilates and nutrients into the reproductive tissues. Plant breeders, both past and present, have sought to maintain and stabilize high yields and prevent premature fruit drop, while on the other hand, they concentrated selection on plant varieties which maximize their resource allocation into fruits and seeds to provide larger and more various fruit forms (Paran and Van der Knaap, 2007). The economic relevance of pollination and fertilization is clear if we consider the economic costs associated with their potential loss. In 1998, reduced pollination of crops and harvest loss, examined in a combination of 30 crops, comprised US $54.6 billion, a total loss of 46% in harvest yield (Kenmore and Krell, 1998). Broader estimates in 2005, placed the worldwide economic value of pollination alone at €153 billion, although no loss in harvest yield was calculated (Gallai et al., 2008). More recent studies did not support a hypothesis that pollinator decline has yet affected crop yield at a global scale (Ghazoul, 2005; Aizen

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et al., 2008); it has been proposed that the increase in pollinator-dependent crops may result in important ecological and economic consequences if pollinator decline is to further continue (Aizen et al., 2008). On the other hand, fruit initiation and fruit development are also strong determinants of plant weed invasiveness which has detrimental effects on cropping systems and the natural environment. In Australia, the economic cost of plant weed invasiveness, facilitated by fruit development and seed dispersal, was estimated at an annual cost of AUD $ 4.039 billion (Sinden et al., 2004; Keller et al., 2007). Thus, it is easy to envisage how mechanisms of altering fruit initiation and retention may offer a way to stabilize and increase yields in crops or, alternatively, become an Achilles’ heel for the manipulation and control of invasive plant species. Several different pillars of research characterize the published knowledge about fruit initiation and how it relates to fertilization. Early research focused on induced parthenocarpy (Noll, 1902), whereby fruit was artificially stimulated independent of fertilization by application of plant-growth regulators (PGRs; Gustafson, 1936) or by various pollination treatments that restricted fertilization or compatibility (Noll, 1902; Yasuda, 1930, 1935). As success with artificial growth regulators gained momentum (Gustafson, 1939a,1939b, 1942; Nitsch, 1952), another area of research was directed at quantifying and localizing phytohormones in specific tissues of the fruit based on the hypothesis that fruit development was initiated and sustained by the developing seeds (Talon et al., 1990a, 1992; Kim et al., 1992; van Huizen et al., 1995; Fos et al., 2000, 2001). Both areas of research now cover a vast number of agricultural and horticultural crops (Schwabe and Mills, 1981), but often the relationship remained obscure between phytohormonal activity and fruit initiation. In contrast, heritable parthenocarpy that occurs naturally or through induced genetic lesions has provided valuable breakthroughs in crops and several genetic loci involved in fruit initiation are now known (Lin, 1984; Rotino et al., 1997; Vivian-Smith et al., 2001; Yao et al., 2001; Bassel et al., 2008; Marti et al., 2008). Recent advances in understanding fruit initiation and the intrinsic linkage to fertilization are now being completed by genetic analysis and transcriptome profiling. In this chapter, we examine the role of female receptivity in fruit set and the key pathways and genes that control fruit initiation together with their complex relationship with fertilization, and with flower maturation. We present data that reinforce the idea that fruit initiation occurs in a very short period of time, characterized by hours and minutes, and not necessarily days. The contribution of various phytohormones such as auxin and gibberellins is also examined, as is the molecular genetic study of parthenocarpy as a tool to interrogate the early and immediate steps in fruit initiation. Through the course of understanding the molecular basis of fruit initiation, the evolution of the angiosperm fruit structure is also addressed, since extensive conservation of candidate regulatory genes exists.

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4.2 4.2.1

Pollination Pollen-stigma recognition and interaction

The landing of pollen on a compatible stigma marks the beginning of pollination. The pollen will then adhere, hydrate and germinate to produce the pollen tube, a structure specialized in the delivery of the sperm cells to the ovule (Fig. 4.1). Stigmas can be divided into two broad types, wet and dry, depending on the amount of exudate present (Heslop-Harrison and Shivanna, 1977). It has been suggested that in plants with dry stigmas (e.g. crucifers), the pollen coat plays a more active role in the adhesion and hydration of the pollen grain (Heslop-Harrison and Shivanna, 1977; Heslop-Harrison, 1992 ). The role of the pollen coat in hydration has been further elucidated thanks to the grp17-1 Arabidopsis mutant, where the loss of a single oleosin protein from the coat resulted in a significant delay in pollen hydration (Mayfield and Preuss, 2000). Pollen coat substances are also involved in early pollenstigma recognition events such as the Brassicaceae-type self-incompatibility system (for more detailed reviews on this subject see Lord and Russell, 2002; Takayama and Isogai, 2005). In the stigma surface, aquaporin-like proteins have been suggested to play a major role in the control of pollen hydration (Tyerman et al., 2002) as well as in pollen acceptance (Lord and Russell, 2002). Additionally, the female determinants of various self-incompatibility systems have also been identified on the stigma surface (Takayama and Isogai, 2005). Pollen-stigma recognition is an active process which subsequently leads to pollen-tube germination (Fig. 4.1). Nevertheless, there is no evidence suggesting that recognition alone is sufficient to trigger fruit initiation (Zhang and O’Neill, 1993). The time period between pollen landing and pollen-tube germination varies greatly among plants. In Phalaenopsis orchids, pollen germinates 4 days after landing (Duncan and Curtis, 1942) providing a unique system for the study of the effect of pollen landing on fruit initiation. Orchids are also unusual among flowering plants in that the ovary and ovules of many orchid species mature after pollination (Withner, 1974). Zhang and O’Neill (1993) showed that physical contact of pollen alone is sufficient to trigger ovary maturation in Phalaenopsis orchids. Nevertheless, this interaction failed to induce fruit initiation (Zhang and O’Neill, 1993). 4.2.2

Pollen germination and pollen-tube growth

After pollen hydration, germination occurs which results in the emergence of the pollen tube (Fig. 4.1). The pollen tube is formed by a generative cell which contains the two sperm cells and the vegetative nucleus. Both pollen germination and pollen-tube growth are subjected to gibberellic acid (GA)mediated control. It has recently been shown in rice that de novo synthesis of

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3

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(a)

X

(b)

(c)

p g

p

sp

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rv

p

g sp

fv

e

s

z pn

cr

cc oi

a

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Figure 4.1 Pollination and fertilization in Arabidopsis and Brassicaceae. (a) Flower at anthesis stage (left) and during pollination (right) showing several rows of ovules within the pistil. During pollination, the stages of adhesion and hydration (1), recognition and pollen tube emergence (2), tube growth (3) and guidance (4) are shown (right). When anthers dehisce, the inhibitory stimulus for fruit development is removed (see the cross symbol). (b) A pollen tube containing two sperm cells is guided to the ovule micropyle by signals emanating from a fertile female gametophyte and the surrounding sporophytic tissue. (c) The tube tip enters the micropyle of the ovule and unites with a synergid cell that degenerates upon fusion. Two sperm cells migrate to combine with the egg cell and polar nuclei of the central cell, respectively. (d) Synergid cells degenerate and the diploid zygote and triploid endosperm begin development. The outer and inner integuments undergo cell expansion and division to form the seed testa. a, antipodal cells; cc, central cell; cr, chalazal region; e, egg cell; en, endosperm; f, funiculus; fv, funiculus vascular tissue; g, generative cell; ii, inner integument; m, micropyle; p, pollen tube; pn, polar nucleus; oi, outer integument; rv, replum vascular tissues; s, synergid cell; sp, sperm cells; z, zygote.

GA in the pollen grains is required for pollen germination (Chhun et al., 2007). Similarly, gibberellins are also necessary for pollen-tube growth across different plant species (Singh et al., 2002; Cox and Swain, 2006; Chhun et al., 2007). The pollen tube grows by tip growth and periodic callose deposition (for a detailed review on this subject see Krichevsky et al., 2007). Directional tube

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Fruit Development and Seed Dispersal growth towards the egg cell is guided by several mutual signalling processes in the pistil, style, funiculus, ovules and female gametophyte (Fig. 4.1). Several studies have focused on the role played by the pollen tube in fruit initiation. It was first observed by Hildebrand (1865) that in certain orchids, where self-incompatible pollinations arose, pollen tubes grew only a small amount but this appeared to stimulate the development of parthenocarpic fruit. Fruit initiation has also been related to the degree of penetration into the style by the pollen tube. It was concluded that to stimulate fruit initiation in cucumbers and eggplants, the base of the style had to be reached by the pollen tube (Yasuda, 1936). These early observations led to the hypothesis that pollen-tube growth resulted in the transfer of growth-promoting substances from the pollen to the ovary and ‘stimulated’ the growth of the fruit (Gustafson, 1939b). Later measurements of growth-promoting hormones (namely auxin) in pollen grains and pollen tubes ruled out these as the possible sources of growth hormones. However, it was suggested that pollen tubes may secrete an enzyme responsible for the activation of auxin precursors in the style and ovary (van Overbeek et al., 1941; Muir, 1942). Recently, Schijlen et al. (2007) provided further evidence of the possible contribution of pollen-tube growth on fruit initiation. In this case, the downregulation of the flavonoid biosynthesis pathway genes, CHALCONE SYNTHASE 1 and 2 (CHS1/CHS2), by RNA interference led to parthenocarpic tomato fruit development (Schijlen et al., 2007). Downregulation of the flavonoid pathway arrested pollen-tube development in self-pollinated gynoecia. Although the pollen tubes failed to reach and fertilize ovules, their initial growth appeared to be sufficient in triggering fruit set and produce stimulatory parthenocarpy (Schijlen et al., 2007). The role of the flavonoid pathway in the gynoecium and the stimulatory effect of pollen-tube growth in tomato fruit initiation require further examination since several interactions between polar auxin transport and flavonoids have been identified. Most notably the loss of flavonoid biosynthesis in Arabidopsis led to increased polar auxin transport (PAT; Murphy et al., 2000; Brown et al., 2001; Lazar and Goodman, 2006; Santelia et al., 2008), leading to the hypothesis that PAT could stimulate fruit initiation. Roles for PAT in fruit initiation are considered in sections below. The use of ionizing radiation has also provided data about the role of the pollen tube in fruit initiation. Fruit initiation can occur even when pollen samples are treated with high doses of ionizing radiation (Denissen and Den Nijs, 1987; Knox et al., 1987; Sniezko and Visser, 1987; Polito, 1999; Peixe et al., 2000). This technique, commonly known as ‘prickle pollination’, stimulates parthenocarpic fruit growth and has been documented in various crops including Cacao, Cotton, Pistacio, Capsicum and other Solanaceaous species. High-irradiation treatments do not always impair pollen-tube growth but prevent fertilization or steps immediately after fertilization (Speranza et al., 1982; Denissen and Den Nijs, 1987; Peixe et al., 2000). Although the degree and precise stage of impairment requires clarification, arrest is characteristically earlier and different from the late post-zygotic arrest observed in

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stenospermocarpic seedless grapes (Cain et al., 1983; Emershad and Ramming, 1984; Hanania et al., 2007) or in Capsicum fruit development triggered by interspecific crosses (Tong and Bosland, 2003). The results from prickle pollination suggest that pollen-tube growth and/or processes immediately after fertilization but prior to zygotic development are able to trigger fruit initiation. Pollen-tube guidance towards the ovules depends on a complex signalling network which involves signals from the female gametophyte and components of the style and ovary (Higashiyama et al., 1998; Higashiyama, 2002; Dresselhaus, 2006; Palanivelu and Preuss, 2006; Escobar-Restrepo et al., 2007; Rotman et al., 2008). Disruption to this complex signalling network can lead to the disorientation of pollen tubes and, thus, prevent fertilization. For example, specific fruit initiation mutants can affect pollen-tube polarity and reduce seed set. This is the case in the parthenocarpic pat-2 tomato mutant where the distorted pollen-tube growth has been related to the high proportion of defective ovules present in the pistil (Mazzucato et al., 2003) and associated with increased levels of endogenous gibberellins (Olimpieri et al., 2007). Altered seed set patterns as a result of defective pollen-tube growth are also found in Arabidopsis plants overexpressing the GA2ox2 gene (Singh et al., 2002; Cox and Swain, 2006). In the light of previous observations, it is tempting to suggest that the impaired pollen-tube growth could also play a part in enhancing parthenocarpic fruit development in the pat-2 background. On the other hand, even though stimulatory parthenocarpy has been observed in a few crops, it has not been detected yet in Arabidopsis mutants where pollen or ovules have subtle defects disrupting fertilization (Vivian-Smith, 2001; Vivian-Smith et al., unpublished data).

4.3 Female receptivity and the cessation of gynoecial growth Often in horticulture, two or more varieties are planted together in a specific planting or orchard design to maximize cross-pollination for hybrid production or yield. The female receptive period is an important final component of the floral maturation process and has a direct bearing on fruit set and initiation, since viable female and male components must both exist in space and time, while the plant sufficiently conserves essential resources. The receptive period has also been referred to as the effective pollination period (EPP) and is the mutual or partial sum of the longevity for the stigma, style and ovule, while taking into account the time taken for the pollen tube to grow and fertilize the ovule (Williams, 1966; reviewed Sanzol and Herrero, 2001; Page et al., 2006). During the maturation and receptive periods, specific molecular pathways restrict the growth of the pistil and accessory tissues and, thus, stop them from developing into fruit.

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Fruit Development and Seed Dispersal Despite the wealth of data in crop species and extensive analysis of female gametophyte development, female receptivity has not been the subject of any significant genetic analysis. Female receptivity can be quantified by emasculating flowers before anthesis and allowing a sample of flowers to be pollinated on successive days post-anthesis (Williams, 1966). Final seed set reflects the period when pistils were most receptive to pollination. As a rule, pollen-tube growth on floral tissues is temperature dependent and different results would be expected if pistils were incubated at sub-optimal temperatures for stigmatic receptivity, pollen-tube development or ovule longevity (Sanzol and Herrero, 2001). Often the optimum for pollen-tube elongation and the optimum for support on the female component do not coincide, and the optima are usually higher for pollen-tube growth (Hedhly et al., 2005a,b). In Arabidopsis, female receptivity, as assessed by seed set, lasts up to 3 days post-anthesis and effectively correlates with the integrity of the female gametophyte which deteriorates shortly thereafter (Christensen et al., 1998; VivianSmith and Koltunow, 1999; Vivian-Smith and Offringa, unpublished). There are marked differences in female receptivity duration between the ecotypes Landsberg and Columbia (Vivian-Smith and Koltunow, 1999). Nevertheless, the receptivity periods in Arabidopsis ovules are significantly shorter than the period the pistil remains receptive to exposures of 10 nmol GA3 that stimulate fruit development (Vivian-Smith and Koltunow, 1999). A longer period of gibberellin perception suggests that the viability of the gametophyte and ovule is completely independent to the perception and signalling of a GA3 mediated growth in the pistil and that the gibberellin-mediated restriction maybe directly occurring in the carpel. Mutations in the Auxin Response Factor 8 gene (ARF8), which lead to parthenocarpic fruit initiation, dramatically shorten the duration of female receptivity and lead to reduced seed set (Vivian-Smith et al., 2001). ARF8 mutants also initiate fruit development precociously and the pistil protrudes far enough to prevent proper contact between the stigma and anthers to effect proper self-pollination (Vivian-Smith et al., 2001). Taken alone, however, the arf8 mutant data may suggest an indirect link with female receptivity. On the contrary, mutations in ARF8 together with the related gene ARF6 lead to complete sterility and dramatically prevent flower maturation in numerous aspects (Nagpal et al., 2005; Wu et al., 2006). This data suggest a global role for both genes in flower maturation, female receptivity and pollen-tube growth. Distinct genetic pathways halt further development of the egg cell and the central cell at maturity and this has been demonstrated with the use of Arabidopsis gametophytic and sporophytic mutants (see sections below). Evidence that the female gametophyte reciprocally exerts control over the developing sporophyte comes from transcriptional profiling studies where mutants lack a viable gametophyte (Johnston et al., 2007). Significant modulation of the sporophytic genes has been observed for SUPERMAN (SUP), Small Auxin Upregulated RNA (SAUR), C3HC4-type RING finger proteins, the homeobox gene SHOOT MERISTEMLESS (STM) and the STYLISH2 (STY2)

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transcription factor. However, these genes are just a few examples amongst 527 genes identified (Johnston et al., 2007). Many of these genes could be candidates linking female receptivity, the female gametophyte and the regulation of fruit initiation. Other studies have also implicated the phytohormone cytokinin in gametophyte development and maintenance of receptivity. Pischke et al. (2002) and Hejatko et al. (2003) demonstrated that CKI is expressed in the female gametophyte until fertilization and it is essential for gametophyte viability. Previous research showed that overexpression of CKI results in cytokinin independency in somatic tissues (Kakimoto, 1996; Glover et al., 2008). If CKI functions in a similar manner in the female gametophyte, it may play a significant role in maintenance of gametophyte viability via a cytokinin-related pathway. Female receptivity can also be positively influenced by the application of nitrogen fertilizer (Williams, 1965; Tromp et al., 1994), and by stigmatic secretions induced by pollination, that can help release carbohydrates from the transmitting tissue and prolong embryo sac viability (Herrero, 1992). While the beginning of female receptivity is demarcated by the period when pollen tubes can grow on the stigma, style and transmitting tissue (Kandasamy et al., 1994), the end of female receptivity is onset by an irreversible initiation of floral senescence (O’Neill, 1997; O’Neill and Nadeau, 1997; Lewis et al., 2006).

4.4 Additional restraints on flower development and fruit initiation Prior to pollination, the floral whorls surrounding the pistil may play a role in repressing or slowing ovary growth (Vivian-Smith, 2001; Vivian-Smith et al., 2001; Fig. 4.1). Accordingly, the specific removal of stamens in Arabidopsis thaliana has been shown to promote pistil growth slightly in wild-type plants, but moreover, the effect is significantly pronounced in genetic backgrounds that display parthenocarpy (fwf/arf8) or fertilization-independent seed development (fis2-2; Vivian-Smith, 2001). Combinations of these mutants with the conditional male sterile pop1/cer6-1 mutant do not alleviate the retardation in silique growth and emasculation of pop1/cer6-1 flowers is still required to achieve full comparative silique elongation (Vivian-Smith, 2001; Vivian-smith et al., 2001). From these experiments, stamens and pollen have been pinpointed as being fully responsible for the retardation in fruit initiation (Fig. 4.1; Vivian-Smith and Offringa, unpublished). The basis of both the FWF/ARF8 and anther dehiscence pathways is to ensure that wildtype plants are successfully synchronized in dehiscence, self-pollination and fruit initiation, but taken separately, the anther acts independently to prevent precocious pistil growth.

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Fruit Development and Seed Dispersal The restraint on ovary growth during female receptivity could be mediated in part by anthers producing high concentrations of free auxins (Aloni et al., 2006), since auxin flow in anther filaments and high auxin levels in the tapetal tissues are critical for pollen development (Feng et al., 2006; Cecchetti et al., 2008). High auxin levels appear to be mediated in part by the auxin biosynthesis genes YUCCA6 and YUCCA2 (Feng et al., 2006; Cecchetti et al., 2008) which are regulated by the SPOROCYTLESS/NOZZLE gene (SPL/NZZ) that controls gametophyte development (Li et al., 2008). Before the female receptive period, a strong auxin maximum is formed in stamens as judged by the auxin transcriptional response reporter, DR5::GUS (Aloni et al., 2006; Feng et al., 2006; Cecchetti et al., 2008; Li et al., 2008). Prior to wild-type pollen dehiscence, the auxin maximum declines and is absent upon dehiscence, providing a natural mechanism to decrease the growth of the anther filaments through reduced PAT (Aloni et al., 2006; Cecchetti et al., 2008). The strongest evidence for pathways facilitating the restraint of Arabidopsis fruit growth from the anther comes from double mutant analysis where several genes have been isolated (Vivian-Smith and Offringa, unpublished). Serendipitously, one was found during the map-based cloning of the fwf-1/arf8-4 mutant (VivianSmith, 2001; Vivian-Smith et al., 2001). Mutations in the aberrant testa shape-1 (ats-1) mutant, also known as kanadi4-1 (kan4-1; McAbee et al., 2006), were observed to enhance silique development in the pop1/cer6-1 ats-1/kan4-1 fwf1/arf8-4 background independently of anther emasculation (Vivian-Smith, 2001; Vivian-Smith et al., 2001). Defects in ATS/KAN4 cause incomplete separation and growth of the ovule integuments. ats-1/kan4-1 mutant ovules consist of three cell layers that have a shared unitegmic identity, as opposed to two outer and three inner integuments in wild type (L´eon-Kloosterziel et al., 1994; McAbee et al., 2006). Importantly, total mesocarp cell counts from fully developed siliques of pop1/cer6-1 ats-1/kan4-1 fwf-1/arf8-4 and wild-typepollinated siliques were the same (Vivian-Smith et al., 2001) suggesting that together ATS/KAN4 and FWF/ARF8 control a large portion of the fruit initiation pathway. The identity or reduced integumentary cell layers in ats1/kan4-1 appear to disrupt a key parallel signalling pathway that does not alone trigger fruit initiation but does link signalling with the restriction of fruit growth facilitated by the anther (Vivian-Smith et al., 2001). The study of MADS box gene mutants has further contributed to the understanding of the restraint imposed by the other floral whorls in ovary growth. For example, loss of function mutation in the MdPI (apple PISTILLATA homologue) causes parthenocarpic fruit development in apple (Yao et al., 2001) which could also be attributed in part to the disappearance of the restraint imposed by the third whorl organs (namely stamens) and to the replacement of ovule identity. Similarly, the parthenocarpic fruit development observed in tomato transgenic plants with low expression levels of TM29 (tomato SEPALLATA homologue) could also be linked to the disruption of petal and stamen identity (Ampomah-Dwamena et al., 2002). However, this phenotype can also be attributed to the altered expression levels of TM29 in the ovaries

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(Ampomah-Dwamena et al., 2002) and, thus, the correlation between outer floral whorls disruption and the observed ovary growth remains to be further clarified in these transgenic plants. In Arabidopsis, carpelloid identity replaces ovule primordia in alleles of the bel1-1 mutants (Reiser et al., 1995; Western and Haughn, 1999; Brambilla et al., 2007) and in the knuckles mutant (Payne et al., 2004). Both mutants develop fruit independent of fertilization but the length is completely correlated with the total number of carpelloid ovule structures produced in the carpel (Vivian-Smith, 2001). Similar occurrences are observed in Capsicum and tomato species (Gray-Mitsumune et al., 2006; Tiwari et al., 2006).

4.5 Fertilization In the vast majority of angiosperms, the mature female gametophyte consists of a seven-cell, eight-nucleate ‘Polygonum-type’ structure, bounded by a membrane that lacks a plant cell wall (Fig. 4.1; Christensen et al., 1998; Yadegari and Drews, 2004). This type of gametophyte has two synergid cells and an egg cell located at the micropylar pole, thus comprising the three-celled egg apparatus (Fig. 4.1, note that one synergid is hidden behind the other). Three antipodal cells are positioned at the chalazal pole of the ovule. Two nuclei of the central cell form the polar nuclei that locate adjacent to the egg cell (Fig. 4.1b; Yadegari and Drews, 2004). Considerable variation exists on the general architecture of the female gametophyte, however, the basal angiosperm Amborella has a similar structure to higher angiosperms and consists of an eight-celled, nine-nucleate female gametophyte (Friedman, 2006). In Amborella, an egg cell is derived from a division of one of the three synergid cells to form a four-celled egg apparatus, unlike the Arabidopsis female gametophyte where the egg cell is specified from a designated nucleus and remains in association with the two synergids. Other basal angiosperms frequently contain a four-celled ‘Nuphar/Schisandra-type’ gametophyte that contains an egg cell, two synergids and a uninucleate central cell at maturity (Friedman and Williams, 2003; Williams and Friedman, 2002; Friedman, 2008). However, the majority of higher angiosperms presents a seven-cell polygonum-type gametophyte and, thus, this type of gametophyte is used as a reference point for the remainder of this review. Further information on gametophyte development is extensively covered elsewhere (Drews and Yadegari, 2002; Punwani and Drews, 2008). 4.5.1 Signal transduction before fertilization The delivery of two sperm cells to the mature female gametophyte by the ¨ pollen tube relies on a robust mutual communication (Fig. 4.1; Hulskamp et al., 1995). Palanivelu et al. (2003) showed that pollen-tube growth in stigma,

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Fruit Development and Seed Dispersal style and ovule is guided by gradients in ␥ -aminobutryic acid (GABA). Pollen tubes utilize a GABA-transaminase, encoded by the POP2 gene, to provide pollen directionality by degrading the GABA stimulant. Guidance towards the female gametophyte occurs since the GABA gradient peaks at the micropylar integument cells. Degradation of GABA is probably involved in ensuring that only a single tube enters the micropyle (Fig. 4.1). As such, GABA maybe a contact-mediated guidance mechanism (Palanivelu and Preuss, 2006). Palanivelu and Preuss (2006) also defined two other processes regulating pollen-tube guidance. These include diffusible ovule-derived attractants from unfertilized ovules and repellents from fertilized ovules. The male gametophytic tepitzin1 mutant indicates a requirement for the auxininducible homeobox gene WOX5 for Arabidopsis pollen-tube growth (Gonzali et al., 2005; Dorantes-Acosta and Vielle-Calzada, 2006). Auxin, together with calcium produced in synergids, had long been hypothesized as pollen-tube chemotropic attractants (Van Went and Willemse, 1984; Chaubal and Reger, 1990; Raghavan, 2003). The synergids and the central cell also play a role in the guidance of the pollen tube prior to fertilization (Higashiyama et al., 2001; Kasahara et al., 2005; Chen et al., 2007; Rotman et al., 2008). In contrast to the GABA gradient, these appear to be short-range recognition and developmentally regulated (Palanivelu and Preuss, 2006). For instance, the plasma membrane-associated GEX3 protein is expressed in the female gametophyte and required for micropylar pollen-tube guidance (Alandete-Saez et al., 2008). Boisson-Dernier et al. (2008) also show that the AMC gene, that encodes peroxisomal protein, functions at short range in both female and male gametophytes through potential diffusible signals. Mutations in the MYB98 gene specifically prevent proper differentiation of the synergid cells which fail to differentiate the structural filiform apparatus that facilitates the reception of pollen tubes (Kasahara et al., 2005). As a consequence, most myb98 ovules fail to attract pollen tubes, suggesting that MYB98 plays a role in the transcriptional activation of the network of genes involved in signalling or in the structural differentiation required for signalling (Kasahara et al., 2005). Transcriptional profiling has validated transcriptional networks regulated by MYB98 and identified small secreted peptides/proteins as MYB98 targets (Jones-Rhoades et al., 2007; Punwani et al., 2007). Our reanalysis of supplementary data from myb98 (Jones-Rhoades et al., 2007) and female gametophyte transcriptional profiles (Yu et al., 2005) shows that the cytochrome P450 CYP78A9 gene is upregulated in myb98 female gametophytes. This is of potential interest since overexpression of CYP78A9 with the 35S promoter provides strong sterility and fruit initiation (Ito and Meyerowitz, 2000). However, the fact that viable myb98 homozygotes are generated indicates double fertilization per se is not defective (Kasahara et al., 2005). In the context of pollen-tube signalling and fruit initiation, Arabidopsis contrasts with reports of stimulatory parthenocarpy in horticultural crops.

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From Arabidopsis research alone, one could be convinced that pollen-tube development does not produce a stimulus for fruit initiation. Further analysis of the many Arabidopsis mutants that avoid fertilization (Pagnussat et al., 2005) should illuminate this aspect.

4.5.2 Double fertilization Double fertilization, first described by Guignard in 1899, involves one sperm cell uniting with the egg cell, while the second sperm cell fuses with the central cell and undergoes karyogamy with the polar nuclei (Fig. 4.1; Weterings and Russell, 2004; Yadegari and Drews, 2004). It has been extensively reviewed (Raghavan, 2003; 2006; Weterings and Russell, 2004; Dresselhaus, 2006) and recently the technical limitations of live visualization have been resolved by confocal laser scanning microscopy (CLSM; Ingouff et al., 2007; Fig. 4.2; Vivian-Smith and Offringa, unpublished data). Double fertilization begins upon fusion of the tube tip with a synergid cell (Fig. 4.1c; Higashiyama et al., 2000; Weterings and Russell, 2004; Sandaklie-Nikolova et al., 2007). At this point, the pollen tube stops growing and discharges the two sperm cells. In some species, synergid degeneration occurs well before pollen tube arrival (Raghavan, 2003) while in Arabidopsis it has been reported that synergid cell death occurs upon pollen tube contact (Sandaklie-Nikolova et al., 2007). Bidirectional communication can however increase cell permeability well before contact, since preferential propidium iodide (PI) staining occurs in the selected synergid before the pollen tube has arrived (Vivian-Smith and Offringa, unpublished data). This is also the case in ∼15% of ovules from the pop1/cer6-1 male sterile mutant, suggesting that a long-range pollen-synergid signalling triggers events prior to synergid cell death and double fertilization. Upon discharge of the sperm cells into the degenerating synergid, migration of sperm cells to the egg and central cells occurs (Faure et al., 2002; Weterings and Russell, 2004; Ingouff et al., 2007). Movement towards their respective nuclei is facilitated by remnant F-actin coronas and microtubles (Ye et al., 2002; Raghavan, 2003). Following double fertilization, development of a diploid zygote and triploid endosperm is initiated (Fig. 4.1; Faure et al., 2002). The remaining synergid eventually deteriorates (Kasahara et al., 2005) and the integuments expand and divide to accommodate the developing embryo and endosperm. In Arabidopsis and other angiosperms, the integuments differentiate post-fertilization to form the seed coat or testa that protects the seed and facilitates the transfer of nutrients and photoassimilates to the seeds (Fig. 4.1; Bowman, 1993; Wittich, 1998). Many steps are required to enable gamete fusion and karyogamy (Jensen, 1964; Faure et al., 2002), but in Arabidopsis this occurs within 2–3 h after the arrival of the pollen tube (Berger et al., 2008; Fig. 4.1c).

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Fruit Development and Seed Dispersal (a)

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Figure 4.2 Confocal laser scanning microscopy (CSLM) images of unfertilized and fertilized ovules of Arabidopsis expressing the synthetic auxin-responsive reporter gene DR5rev::eGFP. (a) Unfertilized anthesis ovule with minimal GFP expression. (b) Post-fertilized ovule at 5 h. The first nuclear endosperm division has occurred and GFP expression is observed in the endothelium, the chalazal domain and adjacent to the funiculus vascular strand. (c) Ovule after the third endosperm division (9 h post-fertilization) with eight endosperm nuclei and an elongated zygote. Strong GFP expression occurs in the endothelium, the chalaza and funiculus. Weaker expression is observed in the outer integument. (d) Treatment of detached pistils with NAA (50 µM) for 1 h, with subsequent washing for 7 h, induces strong GFP activation in the funiculus and chalaza, and moderate activation in the inner integument and weaker expression in the outer integument. a, antipodal cells; cc, central cell; cr, chalazal region; e, egg cell; en, endosperm; f, funiculus; fv, funiculus vascular tissue; g, generative cell; ii, inner integument; m, micropyle; p, pollen tube; pn, polar nucleus; oi, outer integument; rv, replum vascular tissues; s, synergid cell; sp, sperm cells; t, endothelium; z, zygote. (For a colour version of this figure, please see Plate 2 of the colour plate section.)

4.5.3

Signal transduction during fertilization

After compatible fertilization, rapid changes in membrane-bound calcium occur and the female gametophyte changes cellular polarity, forming the zygote and endosperm (Russell, 1993). A complex signalling network is involved in the coordination of double fertilization (for detailed reviews see Dumas and

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Gaude, 2006; Berger et al., 2008). An early signalling event is the generation of a calcium influx following the fusion of the sperm and egg cell (Antoine et al., 2000). This Ca2+ influx will subsequently spread throughout the egg cell and it is believed to contribute, at least partially, to the transient elevation of cytosolic Ca2+ observed shortly after (Antoine et al., 2001). The release of high concentrations of Ca2+ during the synergid cell degeneration (Chaubal and Reger, 1990, 1992a,b) is also likely to contribute to this process (Digonnet et al., 1997). The increase in cytosolic Ca2+ concentration has been proposed to trigger egg cell activation (Digonnet et al., 1997; Antoine et al., 2000). Kranz (1999) also reported that isolated gametes that were fused using in vitro fertilization techniques required pulses of auxin (25–40 mg L–1 2,4-D) to initiate cell division in the newly formed zygote. Characterization of the Arabidopsis aca9 mutant has further contributed to the understanding of the role played by calcium signalling during fertilization. ACA9 encodes a Ca2+ pump that is primarily expressed in pollen (Schiott et al., 2004). Mutant aca9 pollens not only display reduced pollen-tube growth but are also defective in sperm cell release (Schiott et al., 2004). Disruption in sperm cell release was also previously reported in the sirene (Rotman et al., 2003) and feronia (Huck et al., 2003) female gametophyte mutants. In both sir´ene/feronia and aca9 mutants, pollen tubes fail to initiate the release of sperm cells into the synergid and, consequently, pollen tubes continue to grow inside the female gametophyte (Huck et al., 2003; Rotman et al., 2003; Schiott et al., 2004; Escobar-Restrepo et al., 2007). However, while sirene/feronia are female gametophyte mutants (see section below), aca9 mutation affects the male gametophyte (Huck et al., 2003; Rotman et al., 2003; Schiott et al., 2004). More recently, a similar phenotype to that observed in sir´ene/feronia and aca9 mutants was also described in the Arabidopsis amc mutant (Boisson-Dernier et al., 2008). In this mutant, sperm cell release is only impaired when an amc pollen tube reaches an amc female gametophyte, resulting in the pollen-tube outgrowth previously described (Boisson-Dernier et al., 2008). AMC functions as a peroxin in reproductive tissues and it has been postulated that mutations in this gene may result in the loss of a molecule originating from the peroxisome required for female and male gametophyte communication (Boisson-Dernier et al., 2008). Undoubtedly, coordination of an intrinsically complex and costly process such as double fertilization must rely on a robust regulatory network, and miscommunication during female gametophyte development can lead to fertilization-independent fruit initiation. 4.5.3.1 Roles of the egg cell and central cell in fruit initiation The role of the endosperm and egg cell in the control of seed development has been extensively reviewed (Pien and Grossniklaus, 2007) and several mutants have been isolated that uncouple fruit initiation from fertilization. Autonomous endosperm development in the absence of fertilization was first observed in the FERTILIZATION-INDEPENDENT ENDOSPERM (FIE)

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Fruit Development and Seed Dispersal mutant (Ohad et al., 1996) and in the FERTILIZATION-INDEPENDENT SEED (FIS) mutant class (Chaudhury et al., 1997). FIE, FIS2 and MEDEA/FIS1 (MEA; Grossniklaus et al., 1998) are all part of the FIS mutant class (Ohad et al., 1996; Grossniklaus et al., 1998; Luo et al., 1999) and derived from a genetic screen that searched for silique elongation in a male sterile background (Chaudhury and Peacock, 1994). The screen additionally produced the fwf mutant (Vivian-Smith et al., 2001). The fis class mutants encode members of the Polycomb group (PcG) proteins and together with MSI1 and RBR1 form various multiprotein complexes involved in transcriptional regulation through chromatin remodelling (Spillane et al., 2000; Sørensen et al., 2001; Kohler et al., 2003; Guitton et al., 2004; Ingouff et al., 2005; Jullien et al., 2008). Mutations in FIS1/MEA, FIS2, FIE, MSI1 or RBR1 in the female gametophyte result in fertilization-independent endosperm development which triggers fertilization-independent silique elongation (Ohad et al., 1996; Grossniklaus et al., 1998; Luo et al., 1999; Kohler et al., 2003; Jullien et al., 2008). Additionally in fis and msi mutants, autonomous endosperm development can also give rise in some cases to seed-like structures containing aborted embryos arrested at an early stage (Chaudhury et al., 1997; Guitton and Berger, 2005), suggesting that the central cell plays an important role in the control of both fruit and embryo initiation. Recently, autonomous endosperm development was also observed in sir`ene (srn) and scylla (syl) mutants (Rotman et al., 2008). ` receptor-like kinase is expressed in the two synergids The FERONIA/SIRENE and it is involved in the control of the release of the sperm cells (Huck et al., 2003; Rotman et al., 2003; Escobar-Restrepo et al., 2007). In fis loss of function mutants, endosperm proliferation in the absence of fertilization is caused by the relief of the restraint imposed by FIS genes in the central cell; however, the origin of endosperm proliferation in srn and syl mutants remains to be clarified (Rotman et al., 2008). The study of cdka:1/cdc2a mutants has also provided useful insights into autonomous endosperm and fruit initiation (Nowack et al., 2006). In cdc2a mutant pollen, a single sperm cell is produced and is able to fertilize the egg cell (Nowack et al., 2006). Although selfing of heterozygous mutant plants as well as reciprocal crosses with wild-type plants showed that cdc2a pollen caused seed abortion (Nowack et al., 2006), initial egg cell fertilization was able to promote autonomous endosperm proliferation (Nowack et al., 2006). It is widely accepted that upon fertilization auxin originating from the seed is generated (Fig. 4.2). CLSM images have shown that initial endosperm division is enough to trigger the auxin signal (Fig. 4.2b). It would be interesting to investigate whether fertilization of the egg cell by cdc2a pollen can indeed trigger auxin responsiveness to the same extent that double fertilization does. 4.5.3.2 Roles of the integuments in fruit initiation Apart from selection of male gametes arriving at the ovule and providing nutritive support to the developing zygote and endosperm, the integuments have a role in signal transduction that directly stimulates fruit initiation.

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Analysis of the cell cycle marker PCycB1;2 :GUS in integuments indicates that cells remain mitotically active throughout the female receptive period (Ingouff et al., 2006). However, soon after fertilization, the integument cells begin expansion and the mitotic index further increases (Ingouff et al., 2006). Significantly, the expression of auxin-responsive DR5rev::eGFP marker also begins in the integuments around 5 h post-fertilization (Fig. 4.2b; VivianSmith and Offringa, unpublished data). Considering that it takes 2–3 h for green fluorescent protein (GFP) expression from the DR5rev::eGFP reporter to become visible by CLSM (Sauer et al., 2006), auxin responsiveness must at least occur 2–3 h post-fertilization. Another indication that integument development can trigger fruit initiation independent of fertilization comes from two other sources. The knockout of the MET1 DNA methyltransferase enzyme, in the met1-3 mutant, stimulated both the differentiation of the seed coat testa and also that of fruit development without fertilization (FitzGerald et al., 2008). These results imply control of DNA methylation in integument morphogenesis. In another case, the genetic analysis of the bel1-1 mutant also uncouples fruit initiation from fertilization and ovule development suggesting that in wild type, the control of integument development, possibly together with the nucellar identity, is linked to the control fruit initiation (Western and Haughn, 1999; Vivian-Smith, 2001). Associated pseudo-integument development has also been reported in parthenocarpic tomatoes (Mazzucato et al., 2003; Goetz et al., 2007; De Jong et al., 2008) and in the Arabidopsis fwf/arf8 mutants, albeit much weaker than in tomato (Vivian-Smith et al., 2001).

4.6 Hormonal cues during fruit initiation In 1936, Gustafson discovered that application of synthetic auxins to emasculated flowers of several different plant species resulted in parthenocarpic fruit development and, thus, established the initial linkage between fruit initiation and plant-growth regulators (Gustafson, 1936). At present, three main types of plant-growth regulators are recognized as having phytohormonal properties that can potentially induce fruit setting and fruit development (Gillaspy et al., 1993). Application of auxin, gibberellins or cytokinin, either alone or in combination, has been shown to trigger parthenocarpy across a wide variety of plant species (Gustafson, 1936; King, 1947; Srinivasan and Morgan, 1996; Vivian-Smith and Koltunow, 1999; Ozga et al., 2002, 2003). Application of optimal combinations of plant-growth regulators to emasculated pistils can often promote elongation to the extent observed in fully seeded fruits (Vivian-Smith et al., 2001). These results have led to a long standing belief that fruit initiation is sustained by phytohormone biosynthesis occurring during the stages of seed development, although often this assertion remains unchallenged. Understanding the genetics and molecular genetics behind natural parthenocarpic mutants permits further investigation of the phytohormonal

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Fruit Development and Seed Dispersal signalling and the relationship to pollination, fertilization and fruit initiation. Uncoupling fruit initiation from fertilization, with parthenocarpy, offers a unique method to examine these relationships. For example, a role for the ovule in fruit initiation during parthenocarpy has been uncovered (VivianSmith, 2001; Koltunow et al., 2002). However, many questions remain to be answered. To what degree does the ovule contribute in signalling? How are phytohormonal responses initiated and then propagated through the entire flower? Which are the sites of biosynthesis and signal transduction? 4.6.1

Auxin

Auxin plays a crucial role in plant development by directing basic processes such as division, elongation, phyllotaxy, organ primordia differentiation, apical dominance, tropic responses and response to shading (Sauer et al., 2006; for a more detailed review see Benjamins and Scheres, 2008). Auxin also appears to have a primary role during fruit initiation since the genetic analysis of wild-type Arabidopsis fruit initiation with gibberellin biosynthesis and perception mutants shows that auxin-mediated differentiation underlies other signalling pathways (Vivian-Smith and Koltunow, 1999; Vivian-Smith et al., 2001). Furthermore, transcriptional profiling during fruit initiation also shows directionality in phytohormonal responses with auxin preceding gibberellin responses at 12–14 h period post-fertilization (Vriezen et al., 2007). The use of the transgenic DEFH::iaaM construct in a broad range of species (Rotino et al., 1997; Ficcadenti et al., 1999; Mezzetti et al., 2004; Yin et al., 2006) additionally suggests a universal role for auxin in triggering fruit set. Accordingly, auxin-mediated signalling is an early response in the Arabidopsis ovule (Fig. 4.2). Auxin-responsive reporters show transcriptional activation 2–3 h post-fertilization expression, when the nuclear endosperm has undergone only one division (Fig. 4.2). Auxin responses continue to increase after the third endosperm nuclear division, primarily in the integument tips, the endothelium and the chalazal region. Activation at the base of the funiculus is observed less than 12 h after fertilization (not shown). Apart from expression data, portrayed in Fig. 4.2, parthenocarpic mutants and quantitative trait loci (QTLs) have also been characterized and these clearly support roles for auxin as a primary fruit initiation cue (Vivian-Smith et al., 2001; Wang et al., 2004; De Jong et al., 2008; Gorguet et al., 2008). 4.6.1.1 Auxin-mediated transcriptional activation Several genetic lesions in the auxin pathway conferring autonomous fruit initiation have been isolated. Each of these mutants appears to work within the auxin-mediated transcriptional network. The Arabidopsis genome encodes 22 functional auxin response transcription factors (ARFs) and 29 Aux/IAA interacting proteins, and each gene appears to have strong sequence conservation in other plant genomes (Remington et al., 2004; Okushima et al., 2005).

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The ARFs are a family of Aux/IAA interacting proteins that contain a DNA-binding domain (DBD) which recognizes auxin response elements (AuxREs) in DNA sequences (Figs. 4.3 and 4.4; Kim et al., 1997; Ulmasov et al., 1999a,b). Information about the role of ARF8 in fruit initiation came from the map-based cloning of the FWF locus (Vivian-Smith, 2001). All nullomorphic lesions in the Arabidopsis FWF/ARF8 gene induce parthenocarpy, but surprisingly the homeologous expression of the arf8-4 gDNA allele can also provide parthenocarpy in tomato and Arabidopsis (Vivian-Smith, 2001; Vivian-Smith et al., 2001; Goetz et al., 2006, 2007). FWF/ARF8 transcripts are naturally downregulated within 24 h post-pollination (Goetz et al., 2006, 2007), unambiguously similar to the nullomorphic phenotype, suggesting that the removal of the ARF8 mRNA induces fruit initiation. QTL mapping and microsynteny between Arabidopsis and tomato has also uncovered a potential link between natural variations in parthenocarpic tendency and the SlARF8 locus located on chromosome 4 (Gorguet et al., 2008). Recently, the related SlARF7 locus has also been implicated in fruit initiation (Vriezen et al., 2007; De Jong et al., 2008). SlARF7 was initially identified through cDNA AFLPs technology used for transcriptional profiling. This methodology was applied to wild type and GA3 -induced fruit initiation and revealed that SlARF9 and the Aux/IAA proteins SlIAA2 and SlIAA14 are upregulated within 24 h post-fertilization, but that the SlARF7 transcript was downregulated within 24 h in ovule and placental tissues (Vriezen et al., 2007). On the basis that SlARF7 may function as a repressor, like AtARF8, De Jong et al. (2008) specifically silenced SlARF7 by RNAi. Indeed SlARF7 silenced lines produced fruit initiation without fertilization, although the morphology was not entirely similar to pollinated fruit. RNAi has also been used to silence the tomato Aux/IAA protein SlIAA9 (Wang et al., 2005). Silencing SlIAA9 also caused parthenocarpy, but it also resulted in the expression of simple leaves and altered leaf vascular differentiation (Wang et al., 2005). Single base deletions in SlIAA9 recapitulated the RNAi phenotype confirming the specificity of the SlIAA9 RNAi phenotype (Zhang et al., 2007). While the expression of SlIAA9 is not known, the expression of SlARF7 in the placenta and ovules (Vriezen et al., 2007) and the expression of AtARF8 in the endothelium, the female gametophyte, the funiculus and the chalaza (Goetz et al., 2006) clearly indicate a strong involvement with the ovule. The expression of the synthetic auxin-responsive reporter gene shown in Fig. 4.2 correlates precisely with ARF8 reporters in the unfertilized Arabidopsis ovule (as observed in Goetz et al., 2006). Initial expression occurs in the chalaza at <5 h post-fertilization, the endothelium and the integument tips (Fig. 4.2b). Stronger expression occurs with each nuclear endosperm division (Fig. 4.2c). Auxin treatment to the unfertilized Arabidopsis pistil, which presumably overrides ARF8-mediated restriction, recapitulates a similar post-fertilization expression pattern (Fig. 4.2d). Many nullomorphic and antimorphic Aux/IAA mutants have been characterized in Arabidopsis (Overvoorde et al., 2005). Mutations in conserved

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Figure 4.3 Auxin-responsive gene regulation. (a) The structure and function of ARF and Aux/IAA proteins which regulate expression of auxin-responsive genes. ARF proteins contain a DNA-binding domain (DBD) and carboxy-terminal domains (CTD) III and IV. The CTD regions facilitate hetero- and homodimerization amongst other ARF and Aux/IAA proteins as well as binding with MYB77. (b) Repression of downstream coding regions occurs when an Aux/IAA protein interacts with an ARF protein that is bound to auxin response elements (AuxREs). Downstream target genes are often Aux/IAA proteins and GH3 genes creating a loop of auxin transcriptional responsiveness. (c) Possible transcriptional activation by ARF of auxin response genes containing an AuxRE after free auxin induces lability of the Aux/IAA protein through the TIR1/AFB proteolysis pathway. Free IAA is sandwiched between domain II of the Aux/IAA protein. This enables interaction of the TIR1/AFB auxin receptors with Aux/IAA proteins that shunt Aux/IAAs into the ubiquitin-proteasome pathway. Transcription of the downstream gene occurs once the ARF is depressed by the lability of the Aux/IAA protein, and enhanced by MYB77-ARF interaction with the transcriptional response elements adjacent to the AuxRE. (d) The synthetic auxin-responsive reporter, DR5rev::eGFP, consists of multimerized AuxREs within a miminal promoter. This allows the activity of ARF and Aux/IAA proteins to be monitored at the cellular level by observing the output of the eGFP protein.

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Figure 4.4 (a) Neighbour-joining phylogeny of Arabidopsis ARF protein sequences. Activator and repressor ARFs are represented on the left side and right side, respectively. ARFs with non-canonical middle regions (MRs) are centrally located. miR167 targets are highlighted by full circles (ARF6 and ARF8), miR160 targets by broken circles (bottom ARFs) and tasi-RNA targets by short-dashed circles (top-right). Distances were determined using the Juke–Cantor algorithm and displayed using the TreeView programme (Page, 1996). (b) Expression profiles of ARF and Aux/IAA genes from Arabidopsis ovules at female gametophyte (FG) stages 1–4 and 5–7 derived from microarray data (adapted from Yu et al., 2005).

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Fruit Development and Seed Dispersal domain II often produce antimorphic behavior due to increased stability of the IAA protein (Muto et al., 2007) and several mutants provide insight into their functions during fertilization and fruit development. BDL/IAA12, for instance which interacts with MP, has a crumpled fruit phenotype and fails to elongate properly (Hamann et al., 2002), suggesting that auxin-induced development is impaired. In the dominant mutants AXR5/IAA1, IAA28, IAA19/MSG2 and CRANE/IAA18 sterility is observed (Rogg et al., 2001; Yang et al., 2004; Muto et al., 2007; Uehara et al., 2008). So far, construction of double and triple mutant nullomorphs has provided only very subtle phenotypes, underscoring heavy redundancy amongst the 33 proteins in this family (Overvoorde et al., 2005). In fact, iaa8-1 iaa9-1 and iaa5-1 iaa6-1 iaa19-1 mutants appear wild type (Overvoorde et al., 2005). By contrast, promoter and domain swap experiments amongst Aux/IAA proteins have uncovered redundancy, but importantly promoters and Aux/IAA proteins themselves provide specific interactions that control plant development (Muto et al., 2007). The specific spatial and temporal distribution, that has been determined for some of the ARFs and IAA pairs, suggests that specific ARF–IAA complexes mediate developmental responses to auxin, but that Aux/IAA proteins tend to be promiscuous (Weijers et al., 2005; Muto et al., 2007). This contrast to some extent with the result in tomato for SlIAA9 where a single knockout specifically induces fruit initiation (Wang et al., 2005). The reduction of SlIAA9 in either the carpel or the ovule may be specific enough to perturb auxin responses and induce parthenocarpy, and this result may signify specialized function for SlIAA9 in Solanaceous flower and fruits. Experiments whereby antimorphic proteins are specifically expressed within the ovule could provide data on the local modulation by auxin signalling, thereby overcoming redundancy. Another level of regulation occurs at the level of translation of ARF proteins. Nishimura et al. (2005) describe the ribosomal protein L24/SHORT VALVE 1 (RPL24/STV1), which has a role in assisting the re-initiation of translation from small upstream open reading frames (uORFs) and is required for auxin-mediated gynoecium patterning. The ability to regulate translation was demonstrated by the removal of upstream uORFs which led to significantly increased expression of the mORF for MP and ETTIN (ETT/ARF3) (Nishimura et al., 2005; Tran et al., 2008). At this stage, widespread conservation for uORFs in ARF 5 UTR regions has not been described, although an examination of rice and Arabidopsis has been performed and this shows numerous uORFs in most ARF mRNA transcripts (Nishimura et al., 2005; Tran et al., 2008). AtARF8 has 10 AUGs, or potential uORFs, in the 627 bases comprising the 5 UTR (Goetz et al., 2007). Expression of the arf8-4 gDNA allele, which was used to verify the cloning of ARF8 (Vivian-Smith, 2001), also produces parthenocarpic phenocopies of null fwf/arf8 alleles. Thus, the action of RPL24/STV1 may potentially account for the antimorphic impact of fwf-1/arf8-4 gDNA in both Arabidopsis and tomato (Vivian-Smith, 2001; Goetz et al., 2007) since RPL24/STV1 may be recruited to the full length mutated fwf-1/arf8-4 transcripts, thereby reducing transcription upon the native ARF8 5 UTRs.

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ARF8 expression is also presumably regulated at many other levels. This can be highlighted by the fact that a conserved and functional alternative splice acceptor sequence introduces 5 bp deletion and a premature stop codon within the DBD giving rise to shorter splice variant mRNAs in both Arabidopsis and tomato (Goetz et al., 2007). 4.6.1.2 Auxin biosynthesis and transport Three important processes that regulate auxin action in the flower and fruit are its biosynthesis, transport and catabolism. The role of de novo auxin biosynthesis is certainly vital during fruit initiation and fruit development. However, few studies have considered auxin biosynthesis in ovules or developing fruits in detail. Direct proof of the significance for localized auxin biosynthesis in fruit initiation was first obtained by Rotino et al. (1997). In this experiment, the iaaM gene from Pseudomonas syringae pv savastonoi was placed under the control of the placental and ovule-specific promoter DEFH9. Eggplants and tobacco transformed with this construct had parthenocarpic fruit development. Since iaaM is involved in the conversion of tryptophan to indole-3-acetamide, which is then hydrolyzed to IAA, this results in increased levels of localized IAA (Gaudin and Jouanin, 1995) suggesting that a rate-limiting step during fruit initiation could be the production of free auxin. Many other species have now been successfully transformed and all develop parthenocarpic fruit (Ficcadenti et al., 1999; Mezzetti et al., 2004; Yin et al., 2006; Chapter 9 in this book). Although these results highlight the importance of the ovule during fruit development, the potential use of this chimeric transgene in obtaining additional data regarding the control of wild-type fruit initiation is limited. Additionally, DEFH9 specificity to the ovule and or placenta is often not reported for these other species. Nevertheless, experiments where iaaM expression is specifically targeted to different parts of the ovule or female gametophyte may further clarify the role that these tissues play during fruit set. Auxin biosynthesis in Arabidopsis is partly carried out by flavin monooxygenases encoded by the YUCCA gene family (Zhao et al., 2001; Cheng et al., 2006). Dramatic reductions in vascular tissue occur when multiple members are knocked out, consistent with a reduction in auxin biosynthesis and redundancy amongst family members. While yuc2 yuc6 mutants develop normal vascular and floral morphologies, the yuc1 yuc2 yuc6 triple mutant develops severe vascular deficiencies (Cheng et al., 2006). Significantly, floral morphology and fruit development could be restored by a YUC1::iaaM chimeric construct (Cheng et al., 2006). YUC1, YUC2 and YUC4 are apparently expressed in the carpel, while YUC6 is expressed in pollen (Cheng et al., 2006), however, examination of ovule expression data suggests YUC specificity in anthesis ovules (supplemental data, Yu et al., 2005). Experiments with PAT inhibitors show that flower morphology and apical–basal carpel polarity can be severely disrupted by PAT inhibitor application (Nemhauser et al., 2000). Timed applications of PAT inhibitors can

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Fruit Development and Seed Dispersal also induce parthenocarpy, suggesting that the anthesis ovules and pistil may exert control over PAT, either directly or indirectly, and this has an impact on the regulation of fruit initiation (Beyer and Quebedeaux, 1974; Schwabe and Mills, 1981; Kim et al., 1992). This appears to contradict the proposed mechanism for auxin action in fruit initiation, since PAT is required to transport auxin from the ovule to the carpel to trigger growth and development. Therefore, the mechanism by which PAT inhibitors triggers fruit initiation may rely on a balance between PAT and AuxRE transcriptional activation. To explain the apparent paradox, one must consider that auxin is also prevalent in the target cells. Therefore, the end concentration in target cells is important and may normally be restricted to a low level through potentiation of PAT and a reduction in transcriptional activation. Once PAT is inhibited, AuxRE would be activated due to the increased net auxin accumulation within the cell that would trigger fruit initiation through cross-talk with other phytohormonal pathways. To explain PAT, a chemiosmotic model was proposed in which the noncharged, lipophilic IAA molecule enters the cell through diffusion, or through the action of a saturable auxin import carrier (Rubery and Sheldrak, 1974; Raven, 1975). Once inside the cell, the IAA molecule is deprotonated at the higher cytoplasmic pH and only can exit through active export by auxin efflux carriers (AECs) and endosomal/vessicle transport (Dhonukshe et al., 2008). The specific location of AECs at the basal side of the cell was hypothesized to be the rate-limiting transport step of PAT (Lomax et al., 1995). The PIN1 gene encodes a transmembrane protein that has similarity to bacterial-type transporters and data suggest that PIN proteins function as AECs (G¨alweiler et al., 1998). The PIN family in Arabidopsis comprises eight members six of which have been functionally characterized through genetic analysis (Blilou et al., 2005; Wisniewska et al., 2006). Genetic analysis has also uncovered partial redundancy and functional compensation amongst PIN family members (Friml et al., 2003). As a functional AEC, the PIN1 protein is localized to the basal end of xylem parenchyma and cambial cell files in the Arabidopsis inflorescence and root axis (G¨alweiler et al., 1998). Microarray analysis also shows that PIN1, PIN2, PIN3 and PIN6 are the most predominant AECs in the anthesis ovule (supplementary data, Yu et al., 2005). Important new insights into flower development have been obtained through molecular characterization of the Arabidopsis pin-formed (pin1), pinoid (pid) and the weak mp mutants (Benjamins et al., 2001; Hardtke et al., 2004). In contrast to mutations in PIN2, PIN3 and PIN6, pin1 mutants severely disrupt ovule development at early stages (Sauer et al., 2006), implying an essential role for these proteins in ovules. The mutant also develops pin-like inflorescences, with few infertile flowers, a characteristic phenotype of wild-type plants grown in the presence of high levels of PAT inhibitors (G¨alweiler et al., 1998; Nemhauser et al., 2000). Phenocopies have also been observed in the weak mp mutant (Hardtke et al., 2004) and the pid protein kinase mutant (Benjamins et al., 2001), suggesting a commonality in pathway components.

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In this aspect, PINOID (PID) regulates subcellular PIN polarity (Friml et al., 2004) and is rapidly induced by auxin (Benjamins et al., 2001). Intriguingly, PID is expressed in chalazal and funiculus during fruit development (Benjamins et al., 2001). This overlaps directly with the areas where FWF/ARF8 activity restricts fruit initiation (Vivian-Smith, 2001; Koltunow et al., 2002) and the area where vascular development between the ovule funiculus and carpel is later observed (see Section 4.8). Organization of PAT appears to occur also through KANADI (KAN) proteins since PIN1-dependent patterning and subcellular PIN1 polar localization during early Arabidopsis embryogenesis are dependent on redundant actions of the KAN protein class (Izhaki and Bowman, 2007). This is of interest since strong parthenocarpy is elicited by the introduction of the kan4-1/ats-1 mutant into the fwf/arf8 background (Vivian-Smith et al., 2001). KAN4/ATS could set up, maintain or directly act to control PAT in specific tissues of the ovule. KAN1 appears to act through ETT and ARF4 to control laminar growth and polarity in lateral organs (Pekker et al., 2005) and a model has been presented for the control of apical–basal carpel patterning by PAT and ETT (Nemhauser et al., 2000). It is tempting to speculate that the KAN4 protein in ovules works in a similar manner to KAN1 in support of this model, it has been observed that the gene trap expression for ETT does indeed show increasing restriction to ovules and the replum vasculature as the flower approaches anthesis (Nakayama et al., 2005). 4.6.1.3 Auxin signalling and feedback regulation Auxin rapidly induces early auxin response genes including two other major classes, SAURs and GH3s (Hagen and Guilfoyle, 2002). The GH3 gene family in Arabidopsis represents an important network component in feedback signalling, auxin homeostasis and in the homeostasis of jasmonic acid (Liu et al., 2005; Terol et al., 2006). GH3 enzymes catalyze bidirectional conjugations of indolic compounds and jasmonic acids with amino acids (Liu et al., 2005). Many GH3 genes are responsive to environmental stimuli, but most are also primary auxin response genes and are induced in response to auxin treatments to pistils and have altered expression in fwf/arf8 and arf6 mutant backgrounds (Nagpal et al., 2005). The genes GH3.5, GH3.6 and GH3.17 appear to be targets of ARF8 and ARF6 (Tian et al., 2004; Nagpal et al., 2005) and plants overexpressing ARF8 showed a decrease in free IAA content possibly due to GH3 expression (Tian et al., 2004). Regulation through ARF8 and GH3 genes at anthesis may therefore be a mechanism that constrains auxin responses further since free auxin would be removed and this would potentiate Aux/IAA proteins to form inactive complexes with ARF proteins. Auxin is involved in many crucial roles in plant development and there are many interconnected processes with auxin signalling. The challenge with investigating auxin-mediated fruit initiation will be to break up the signalling in specific tissues and to independently interrogate these tissues with either mutational genetics, in vivo reporters or through transcriptional profiling that

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Fruit Development and Seed Dispersal reveals transcriptional networks. Alternatively, one could employ chemical genetics to perturb specific auxin signalling pathways related to fertilizationinduced fruit initiation. 4.6.2

Gibberellins

Gibberellins form a large family of tetracyclic diterpernoid compounds of which only a small number are active PGRs. Bioactive gibberellins are known to be involved in diverse developmental processes including stem elongation, seed germination, leaf expansion, trichome development, de-etiolation and flower and fruit development (Olszewski et al., 2002; Swain and Singh, 2005). Several lines of evidence have shown that fertilization results in increased levels of GA in the ovary (Eeuwens and Schwabe, 1975; Mapelli et al., 1978; Ozga et al., 1992; van Huizen et al., 1995; Ben-Cheikh et al., 1997; Serrani et al., 2007b, 2008; Vriezen et al., 2007). Due to their high gibberellin content, fertilized ovules have long been considered the source of growth-promoting and fruit-setting compounds (Garc´ıa-Mart´ınez et al., 1991). However, a far more complex GA-mediated control of fruit initiation and development is being unveiled, partially as a result of the characterization of the GA biosynthesis and signalling pathways. 4.6.2.1 Gibberellin biosynthesis In higher plants, GA biosynthesis can be divided into three stages (Olszewski et al., 2002). The first stage results in the synthesis of ent-kaurene by the action of two cyclases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS; Fig. 4.5), which in anthesis flowers is located adjacent to vascular bundles, such as the funiculus, medial and lateral vasculature bundles (Silverstone et al., 1997). ent-Kaurene is also highly volatile suggesting mobility at this point of synthesis (Otsuka et al., 2004). In the second stage, GA12 and/or GA53 are produced as a result of the action of ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO) in the case of GA12 and an extra 13-hydroxylation step in the case of GA53 . The final stage results in the synthesis of active GAs through two parallel pathways (Fig. 4.5): the non13-hydoxylation pathway (leading to GA4 ) and the early 13-hydoxylation Geranylgeranyl CPS diphosphate

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Figure 4.5 The GA biosynthesis pathway in higher plants. The pathway is shown from the common precursor geranylgeranyl diphosphate to the active gibberellins GA1 and GA4 . The names of the enzymes catalyzing each step are shown in italics.

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pathway (leading to GA1 and GA3 in some cases). Enzymes involved in this final stage include 2-oxoglutarate-dependent dioxygenases, GA 20-oxidases (GA20ox) and GA 3-oxidases (GA3ox). Active GAs can be converted to inactive forms by the action of GA 2-oxidases which catalyze the introduction of a hydroxyl at the 2␤ position (for more detailed review on GA biosynthesis see Olszewski et al., 2002). Our present understanding of the role of GA biosynthesis in fruit initiation and fruit development is based mainly on the study of the final steps of the biosynthetic pathway. Several studies have shown that pollination/fertilization results in increased expression levels of enzymes catalyzing the last steps of GA biosynthesis (Ozga et al., 1992; Ben-Cheikh et al., 1997; Ngo et al., 2002; Olimpieri et al., 2007; Serrani et al., 2007b; Hu et al., 2008). It was first shown in pea that removal of seeds reduced the activity of GA 20-oxidase in the pericarp (Ozga et al., 1992). In tomato, the study of the early 13-hydroxylation pathway (the main GA metabolic pathway in this species) has also shown that pollination/fertilization results in increased expression levels of GA 20-oxidases on the ovary (Serrani et al., 2007b). GA 20-oxidases are responsible for the conversion of GA19 to GA20 which will then be metabolized to GA1 (the main active GA in tomato) by GA 3-oxidases. Serrani et al. (2007b) showed that while transcript levels of GA 3-oxidases remained constant in both unpollinated and pollinated ovaries, a marked increase in GA 20-oxidase transcript levels was detected upon pollination/fertilization which suggested that fruit initiation in unpollinated tomato ovaries is perhaps partially limited by the low activity of GA 20-oxidases. This is in accordance with the studies carried out in pat tomato mutants where the parthenocarpic phenotype can at least partially be explained by the constitutive expression of GA20ox1 (Fos et al., 2000; Olimpieri et al., 2007). Although in wild-typepollinated tomatoes as well as in parthenocarpic pat tomatoes GA 20-oxidases were found to be expressed throughout the ovary, higher expression levels were observed in the seeds and ovules, respectively (Olimpieri et al., 2007; Serrani et al., 2007b). Therefore, it is possible that seeds (in wild-type plants) and ovules (at least in some parthenocarpy conferring mutations) may be the origin of growth promotion. Similarly, a recent study of GA3ox genes in A. thaliana has also concluded that developing seeds are likely to be sites of GA biosynthesis (Hu et al., 2008). Analysis of the expression pattern of GA3ox genes in developing siliques showed that GA3ox1 expression is limited to the replum, funiculi and silique receptacle while GA3ox2, GA3ox3 and GA3ox4 are expressed in developing seeds (Hu et al., 2008). Despite these expression patterns, further mutant analysis concluded that only GA3ox1 and GA3ox4 are likely to be involved in the control of fruit initiation and fruit development in Arabidopsis (Hu et al., 2008). Furthermore, it was shown that GA3ox1 and GA3ox4 gene expression increased after anthesis indicating that pollination/fertilization is required to induce GA biosynthesis and, thus, fruit growth promotion (Hu et al., 2008). It has been suggested that GA3ox1 may promote fruit initiation by acting in maternal tissues while the

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Fruit Development and Seed Dispersal activity of GA3ox4 is thought to be limited to the seed endosperm (Hu et al., 2008). The role of maternal tissues as potential sites for GA biosynthesis during fruit initiation has also been underlined by Rieu et al. (2008a,b) who showed that pollination of the Arabidopsis ga20ox1 ga20ox2 double mutant with wild-type pollen fails to restore normal silique elongation, while downregulation of the main GA inactivation pathway (C19 -GA 2-oxidation pathway) induced parthenocarpic fruit development. These findings are in agreement with those by Ozga et al. (1992), Olimpieri et al. (2007), Serrani et al. (2007b) and Hu et al. (2008) and, taken together, suggest that fertilization results in the upregulation of GA biosynthesis both in the ovary and ovules. 4.6.2.2 Gibberellin signalling Similarly to the GA biosynthesis pathway, GA response and signalling pathways have also been intensively studied in the past few decades and many molecules involved in these pathways have been characterized. However, few components of the GA response and signalling pathways have been studied in the context of fruit initiation and fruit development. The SPINDLY (SPY) locus of Arabidopsis was one of the first molecular players of the GA signalling pathway shown to be involved in the control of fruit initiation and fruit development (Jacobsen and Oleszewski, 1993). Although doubts still persist about the precise role played by SPY in the control of GA-mediated responses, particularly in relation to other components of the signalling pathway (Silverstone et al., 1998), it is generally considered to be a negative regulator of the GA response pathway (Jacobsen and Oleszewski, 1993). During the initial characterization of the SPY locus, it was reported that emasculation of spy mutant pistils resulted in parthenocarpic silique elongation. This and other phenotypes of the spy mutants were suggested to be the consequence of the constitutive activation of the GA perception and/or GA signal transduction. Nevertheless, attempts to repeat the parthenocarpic silique elongation observed by Jacobsen and Oleszewski (1993) have failed and, thus, the role of SPY in the control of fruit initiation and/or fruit development remains to be clarified (Vivian-Smith et al., 1999). DELLA proteins are probably the most intensively studied components of the GA signalling pathway. They are part of the GRAS transcription factors family and, within this larger family, DELLA proteins are characterized by a conserved N-terminal amino acid sequence which appears to be essential for the regulation of GA responses. It has been shown that DELLA proteins act as growth repressors and GA-mediated degradation of these proteins through the 26S proteosome pathway is required in order to promote growth (Fig. 4.6) (for a more detailed review on this subject see Alvey and Harberd, 2005). The restraint in growth imposed by DELLA proteins has recently been considered in the context of fruit initiation by Marti et al. (2008) who showed that silencing of the only DELLA protein in tomato (SlDELLA) is sufficient to trigger parthenocarpic fruit development. Earlier studies have indicated that DELLA proteins regulate the expression of GA biosynthesis genes in a

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Figure 4.6 (a) Simplified model of GA-mediated DELLA degradation. In the presence of GA, nuclear-localized DELLA proteins associate with the complex form by GA and the GID1 receptor. This association enables the further interaction with the SCFSLY/GID2 complex which results in DELLA protein poly-ubiquitination and, ultimately, in growth promotion by the degradation of the DELLA proteins through the 26S proteasome. (b) DELLA mRNA silencing causes facultative parthenocarpic fruit development in tomato. From left to right: pollinated wild-type fruit, parthenocarpic asSlDELLA transgenic fruit and hand-pollinated asSlDELLA fruit (adapted from Marti et al., 2007). Used with permission of the publisher and authors.

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Fruit Development and Seed Dispersal feedback manner (Peng et al., 1997; Silverstone et al., 1997). In accordance with these observations, reduced levels of GA biosynthesis genes were recorded in SlDELLA tomato fruits (Marti et al., 2008). The role played by endogenous GA levels in SlDELLA parthenocarpic fruit set can therefore be dismissed, the simplest explanation being that SlDELLA parthenocarpy is due to release of the restraint in growth imposed by DELLA proteins (Marti et al., 2008). 4.6.3

Cytokinin and ethylene perception

The role of cytokinins in fruit initiation and fruit development has been studied to a considerably lesser degree than other phytohormonal processes (for a detailed review of cytokinin signalling see To and Kieber, 2008). Like numerous other phytohormones, cytokinins have also been identified as potential phytohormonal components produced in developing seeds that could promote fruit development (Burrows and Carr, 1970; Garc´ıa-Mart´ınez et al., 1991). Indeed, exogenous cytokinin application to pistils can result in parthenocarpic fruit development in Arabidopsis (Vivian-Smith et al., 1999), Brassica napus (Srinivasan and Morgan, 1996) and in pea (Garc´ıa-Mart´ınez and Carbonell, 1980), amongst many other species (Bangerth and Schroder, 1994; Yu et al., 2001). Additionally, stimulation of cytokinin biosynthesis by using fruit and ovary-specific expression of the ipt gene from Agrobacterium produces parthenocarpy in tomato (Li, 2001; Zichao et al., 2002). Strong linkage of cytokinins to the control of cell cycle progression has led to the speculation that cytokinins could be responsible for stimulating carpel cell division post-fertilization (Yu et al., 2001; Li et al., 2003; Vriezen et al., 2007). At this time point, it is impossible to conclude whether fertilized ovules constitute a significant source of cytokinins which either triggers fruit initiation or alters fruit growth. In spite of this, genetic and systems analysis illustrate strong cross-talk between cytokinin, auxin and ethylene signalling pathways. In microarray experiments carried out on Arabidopsis seedlings, cytokinin downregulated ARF8, PIN2 and an auxin biosynthesizing nitrilase gene (Brenner et al., 2005). These three genes are characterized as late responders to cytokinin treatment since the modulation of expression occurred after 120 min (Brenner et al., 2005). This matches well with data that cytokinin treatment to pistils can trigger an auxin response in unfertilized Arabidopsis ovules (Vivian-Smith and Offringa, unpublished data). Unfertilized pistils that were treated with benzyl adenine (BA; 1 nmol pistil−1 ) had a similar auxin response to that observed following pollination (Figs. 4.6b, 4.6c), yet this occurred after a 12 h period. This is significantly greater than the auxin treatment described earlier (<2 h) or even that induced by fertilization (<3 h). Since the cytokinin-induced auxin response occurs outside these timeframes this suggests indirect mechanisms of triggering fruit initiation, or at least that BA treatment had reduced mobility when compared to auxin. The use of a cytokinin-responsive reporter after fertilization (such as TCS cytokinin

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reporter, Muller and Sheen, 2008) could contribute to clarify whether cytokinin is indeed a primary trigger for the post-fertilization fruit initiation. Several lines of evidence suggest a role for cytokinin perception in gametophyte development and during stages related to post-fertilization development of the ovule. For example, the CKI gene is expressed after the FG5 stage and then it is transiently expressed in the zygote and endosperm until 72 h post-fertilization (Pischke et al., 2002; Hejatko et al., 2003). Evidence for the involvement of the type-A ARR22 in fertilization and fruit initiation comes from the studies of Gattolin et al. (2006) and Horak et al. (2008). ARR22 is expressed in the chalazal region of the ovule shortly after fertilization and may be linked to vascular development since cytokinin negatively regulates protoxylem specification (Mahonen et al., 2006). In contrast to cytokinin, ethylene has been typically associated with floral and fruit abscission and in fruit ripening (Kendrick and Chang, 2008). Associations of ethylene with fruit initiation have not been intensively investigated, however ethylene precursor molecules, like 1-aminocyclopropane1-carboxylic acid (ACC), have been associated with actions that stimulate fruit growth (O’Neill, 1997; O’Neill and Nadeau, 1997). Proof of the definitive involvement of the ethylene precursor molecule, ACC, in fruit initiation has been provided by Tang (2003). In these experiments, exogenous application of ACC to unpollinated pistils induced fruit elongation (Tang, 2003). Furthermore, genetic analysis proved that ACC induction was completely dependent on AXR1, a ubiquitin-activating enzyme E1 involved in the auxin response pathway (Leyser et al., 1993). The genetic analysis revealing the involvement of AXR1 in ACC induced fruit elongation, potentially implicates that the degradation of Aux/IAA proteins is involved in parthenocarpic fruit development triggered by the ACC response. These results together with the experiments that show that radio-labelled ACC transport readily occurs in carnation gyneocia (Reid et al., 1984; O’Neill, 1997) may suggest that ACC is an important player in fruit set. The involvement of ethylene as a positive trigger in fruit initiation has also been demonstrated via other genetic analyses. Combinations of the ethylene perception mutant ctr1-1 with ovule defective mutations nzz-2 and ino-2, ats1/kan4-1 produce a parthenocarpic fruit when emasculated (Vivian-Smith, 2001; Koltunow et al., 2002). These experiments indicate that the ctr1-1 mutation, which constitutively activates downstream ethylene signalling, can provide a positive stimulus for fruit initiation, but only when components of the ovule that are perceived as repressor elements are removed (Vivian-Smith, 2001). Since the combination of ctr1-1 ats-1/kan4 gave the strongest elongation when emasculated, PAT within ovule integuments maybe involved (VivianSmith, 2001). The ethylene receptor gene ETR2 is also expressed in the ovule (Sakai et al., 1998) and ETR2 transcripts appear to be female gametophyte enriched (microarray of Yu et al., 2005; Johnston et al., 2007). This further suggests that ethylene signalling via ETR2 is a potential component in ethylene signal transduction at the time of fertilization.

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Fruit Development and Seed Dispersal 4.6.4

Hormonal cross-talk

Treatment of unpollinated ovaries with auxins, gibberellins or cytokinins alone does not result in normal fruit development across different plant species (Srinivasan and Morgan, 1996; Vivian-Smith et al., 1999; Ozga et al., 2002) and application of specific hormonal combinations is required to trigger fruit development to the extent observed in fully seeded fruit. For example in Arabidopsis, application of gibberellins together with either cytokinins or auxins is required to restore silique length to that of pollinated siliques (VivianSmith et al., 2001). These observations suggest that a hormonal interplay is necessary for normal fruit development. Gibberellin and auxin cross-talk has been the most widely studied hormonal interaction in fruit initiation and fruit development. Growing evidence suggests that the stimulation of fruit initiation and growth by seed origin auxin can at least be partially attributed to the upregulation of gibberellin metabolism. van Huizen et al. (1995) showed that the conversion of GA19 to GA20 in pea pericarp is seed regulated and that application of the auxin 4-Chloroindole-3-acetic acid (4-Cl-IAA) can substitute for the seeds in the promotion of this conversion. Similar conclusions were reached by Ngo et al. (2002) who found that treatment of deseeded pea pericarps with 4-Cl-IAA increased GA 20-oxidase gene expression. Recent experiments have also shown that auxin-induced parthenocarpy can be blocked by GA-specific inhibitors (mainly paclobutrazol) (Serrani et al., 2008). On the other hand, analysis of the ovary and ovule transcriptomes induced after pollination or by GA3 treatment provided data that auxin-induced transcripts were unaffected by GA3 treatment (Vriezen et al., 2007). These results together with the GA-biosynthesis upregulation observed upon auxin treatment (Van Huizen et al., 1995; Ngo et al., 2002; Serrani et al., 2008) and the lost of auxin-induced parthenocarpy upon PCB treatment (Serrani et al., 2008) suggest that auxin stimulation of fruit set is partially mediated by gibberellins while the opposite appears to be improbable. However, several lines of evidence have also implied that auxin is likely to act independently to gibberellin in many aspects of fruit initiation. For example, clear morphological differences are observed at the tissue level in fruits treated with gibberellins or auxin (Vivian-Smith and Koltunow, 1999; Serrani et al., 2007a). Furthermore, simultaneous application of gibberellin and auxin has an additive effect on fruit development (Vivian-Smith et al., 2001; Serrani et al., 2008). Finally, application of auxin to unpollinated gai dominant (gai-1d) Arabidopsis mutants results in silique growth promotion (Vivian-Smith and Koltunow, 1999). GAI encodes one of the five DELLA proteins in Arabidopsis (Peng et al., 1997). In gai-1d mutants, the GAI mutant protein lacks 17 amino acids critical for GA-mediated degradation but retains its growth repression function (Peng et al., 1997; Harberd et al., 1998). Consequently, gai-1d mutants appear to be unable to respond to GA-mediated growth promotion (Peng et al., 1997) which will suggest that the growth promotion observed upon auxin application can be attributed to the

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independent effect of auxin in fruit initiation (Vivian-Smith and Koltunow, 1999). Another level of interaction between gibberellin and auxin during fruit initiation and fruit development can be found in A. thaliana ga1-3 mutants. These mutants are impaired in an early step of GA biosynthesis (Sun and Kamiya, 1994) and, consequently, produce very low levels of active gibberellins. Vivian-Smith and Koltunow (1999) showed that application of the auxin ␣-naphthalene acetic acid to ga1-3 mutant pistils did not cause parthenocarpic fruit development. Similarly, upregulation in the ga1-3 mutant background of the auxin signal required for fruit initiation by the introduction of the arf8-4 mutation (formerly fwf ) did not result in parthecarpic silique elongation either (Vivian-Smith et al., 2001). However, parthenocarpic fruit development was promoted in other GA-biosynthesis mutants such as ga4-1 and ga5-1 upon auxin treatment (Vivian-Smith and Koltunow, 1999). Only one of the highly redundant enzymes catalyzing the later steps of GA biosynthesis is impaired in ga4-1 and ga5-1 mutants (Talon et al., 1990b; Chiang et al., 1995; Phillips et al., 1995; Sponsel et al., 1997) and, thus, they are weaker GA-biosynthesis mutants than the ga1-3 mutant. Based on these results, Vivian-Smith and Koltunow (1999) concluded that a threshold of endongenous gibberellins may be required for auxin-induced fruit initiation. The study of the hormonal regulation of fruit set has mainly focused on the role played by gibberellin and auxin and, thus, relatively little is known about the cross-talk between other hormones during fruit initiation. It has been suggested that SPY may play a pivotal role in the integration of gibberellin and cytokinin pathways by acting as both a repressor of GA responses and as a positive regulator of cytokinin signalling (Greenboim-Wainberg et al., 2005). Nevertheless, without a better understanding of the role of SPY in fruit set (see Section 4.6.2.2), it is difficult to draw any further conclusions regarding the importance of such findings in the context of fruit initiation and fruit development. A recent study has also shown that application of brassinosteroids can induce parthenocarpic cucumber fruit development (Fu et al., 2008). This is in agreement with previous results by Montoya et al. (2005) which showed that Br C-6 oxidase, an enzyme catalyzing what it is thought to be a rate-limiting step in brassinosteroid biosynthesis, is highly expressed in developing seeds in tomato. Both studies certainly point towards a role of brassinosteroids in fruit initiation and fruit development. Furthermore, brassinosteroids have also been shown to act synergistically to auxin in the regulation of several target genes (Goda et al., 2002; Nakamura et al., 2003; Nemhauser et al., 2004; Vert et al., 2008). For example, the brassinosteroid-regulated BIN2 kinase is able to phosphorylate auxin response factor 2 (ARF2) which results in the loss of ARF2 activity (Vert et al., 2008). In the model proposed by Vert et al. (2008), brassinosteroids release the repression activity of ARFs (such as AFR2) while auxin increases the expression of activator ARFs. Thus, brassinosteroids and auxin would coregulate gene expression through ARFs activity (Vert et al.,

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Fruit Development and Seed Dispersal 2008). This is in agreement with previous data by Nemhauser et al. (2004) who showed that transcript levels of ARF4 and ARF8 are negatively regulated by brassinosteroid treatment. Although none of these studies were performed in the context of fruit initiation or fruit development, it is easy to envisage that a similar cross-talk mechanism may be operative during fruit initiation. In addition, ARF2 interacts with the ethylene pathway (Li et al., 2004) and it is expressed in the integument tip cells (Schruff et al., 2006). Mutations alter flower, seed and fruit development in Arabidopsis which could further support the involvement of brassinosteroids and ethylene in fruit initiation (Vivian-Smith et al., 2001; Okushima et al., 2005; Goetz et al., 2006). A number of studies have also considered the interplay between auxin and other hormones such as cytokinins, ethylene and abscisic acid (ABA) in fruit initiation. Application of cytokinins to unpollinated Arabidopsis pistils increased seed origin auxin, suggesting that cytokinin stimulation of fruit initiation is at least partially mediated by auxins (Vivian-Smith and Offringa, unpublished data). On the other hand, analysis of the tomato ovary transcriptome showed that fruit set either by pollination or by gibberellin application resulted in the downregulation of ethylene and ABA biosynthesis (Vriezen et al., 2007). Based on these results, it was concluded that ABA and ethylene might play an antagonistic role to that of auxin and gibberellin in fruit initiation, possibly by keeping the ovaries protected and/or dormant prior to pollination and fertilization (Vriezen et al., 2007). Although many different phytohormones and phytohormonal precursors appear to trigger fruit initiation, how this maze of connections is integrated during fruit initiation remains to be further clarified. One particular signalling component which has common elements in ethylene, cytokinin and gibberellin signalling is the MAP kinase pathway. Marcote and Carbonell (2000) described the PsMAPK3 gene from pea which is an orthologue to the Arabidopsis AtMPK3. Interestingly, PsMAPK3 is upregulated within 30 min of GA3 and 45 min of cytokinin treatment to unpollinated pea pistils, though no experiments were performed to understand the post-pollination control of PsMAPK3 transcription (Marcote and Carbonell, 2000). PsMAPK3 is expressed in the ovule, mesocarp and carpel vascular tissues upon GA and cytokinin treatment. In Arabidopsis, much is now known about the role played by AtMPK3 and the redundant partner protein AtMPK6 (Ouaked et al., 2003; Takahashi et al., 2003; Miles et al., 2005; Wang et al., 2008; Yoo et al., 2008). Mutations in AtMPK3 and AtMPK6 show short integuments when double mutants are constructed (Wang et al., 2008), suggesting that the two proteins support the growth of the integuments during ovule development. It has recently been shown that the two proteins act in a central but pervasive role including signalling for glucose, ethylene, jasmonic acid, reactive oxygen species, floral, abscission, and in both abiotic and biotic stresses. Yoo et al., 2008 showed that AtMPK3 and AtMPK6 proteins translate information from these pathways and provide a distinct output on the EIN3 protein, which is a component of the ethylene signalling pathway. The ACS2 and ACS6 proteins

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are also directly phosphorylated by this cascade which leads to ACS stabilization and enhanced ethylene signalling (Liu and Zhang, 2004). Therefore, the MPK3 and MPK6 appear to be critical in determining ethylene-signalling specificity amongst many other integrated signals. In the light of the results by Marcote and Carbonell (2000), further analysis of the MPK3 and MPK6 genes in fruit development is now needed.

4.7 RNA silencing during fruit initiation Many ARF mRNAs are subjected to post-transcriptional regulation via endogenous RNA silencing through either miRNA or tasi-RNA regulation (Figs. 4.4 and 4.7; Hunter and Poethig, 2003; Mallory et al., 2005; Williams et al., 2005; Fahlgren et al., 2006; Yang et al., 2006; Liu et al., 2007; Wu et al., 2007). The small RNA molecules generated through these pathways usually encode short 21 nucleotide RNAs that either guide cleavage or translationally inhibit a targeted mRNA transcript via the ARGONAUTE (AGO) proteins (Fig. 4.7; Chapman and Carrington, 2007; Brodersen et al., 2008; for more detailed information about RNA silencing in plants please see Brodersen and Voinnet, 2006). Many miRNAs are under strict spatio-temporal regulation (Valoczi et al., 2006; Wu et al., 2006). For example, miR160 is expressed in the funiculus vasculature and placental tissue (Valoczi et al., 2006), while miR167 is expressed within developing and mature ovules (Valoczi et al., 2006; Wu et al., 2006). The impact of small RNA regulation on auxin signalling pathway is highly significant both in terms of the number of auxin-related targets as well as in the wide variety of developmental processes affected. Moreover, miRNAARF post-transcriptional regulation appears to be key in flower maturation and reproductive development in angiosperms and gymnosperms (Ru et al., 2006; Wu et al., 2006; Fujioka et al., 2008; Oh et al., 2008). ARF2, ETT/ARF3 and ARF4 transcription factors are targeted by miR390 (Montgomery et al., 2008) while the auxin receptors, TIR1, AFB1, AFB2 and AFB3 are under the regulation of miR393 (Navarro et al., 2006). Two key ARF transcription factors in fruit initiation, ARF6 and ARF8, are targets of one of the most abundant miRNAs, miR167 (Lu et al., 2006; Rajagopalan et al., 2006). Undoubtedly, RNA silencing provides the means for relatively rapid mRNA turnover and removal of key transcription factors. All targeted ARFs have short mRNA half-lives (Narsai et al., 2007), particularly when compared to the 3–4 h halflife reported for ARF1 protein turnover (Salmon et al., 2008). Elimination of ARFs through transcript clearance could leave other ARFs, such as MP, free to activate auxin responses in ovules (Fig. 4.7). The formation and biogenesis of miRNAs and ta-siRNAs occur through defined pathways that are dependent on the transcription of precursor noncoding RNAs (pri-miRNAs) from unique MIR loci by processes consistent with PolII-driven transcription (Fig. 4.7; Xie et al., 2005). Often the number

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of MIR loci outnumber the target mRNA locus. For example, miR167 has four loci in Arabidopsis (miR167a–d; Wu et al., 2007) and larger numbers in other sequenced genomes (Populus, 10 miR167 loci; Barakat et al., 2007). In this capacity, site specific and developmental control of miRNA action is achieved. After transcription, precursor molecules are then folded into a hairpin structures (Fig. 4.7) and presented to a complex that includes DICERLIKE1 (DCL1), SERRATE (SE) and the double-stranded RNA-binding protein HYPONASTIC LEAVES1/DRB1 (HYL1/DRB1; reviewed, Chapman and Carrington, 2007; Mallory and Bouche, 2008). Processing by DCL1 trims the arms and loop of the miRNA to generate a 5 phosphorylated 21-nucleotide dsRNA (Fig. 4.7). HUA ENHANCER1 (HEN1), which encodes 2 -O-methyltransferase, stabilizes miRNAs by a 3 methylation (Li et al., 2005; Yang et al., 2007). Following stabilization, this pre-miRNA is loaded into the AGO protein where the sequence is presumably cleaved. This allows the mature miRNA free to guide translational repression or slicing (Fig. 4.7, Brodersen et al., 2008). Translational repression requires the specialist action of other pathway components such as the microtubule-severing enzyme katanin and the mRNA decapping component VARICOSE (VCS)/Ge-1, which is also involved in vascular biogenesis (Brodersen et al., 2008). AGO proteins therefore are key participants regulating gene expression at the post-transcriptional level and possess both the capability of an irreversible miRNA-guided mRNA slicer or a dynamically reversible translational repressor (Brodersen et al., 2008). The later process may allow rapid changes in gene expression. ←--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Figure 4.7 Biogenesis of miRNAs and post-translational regulation of mRNA targets in the auxin response pathway. (a) miRNAs are generated from non-coding RNA loci and first undergo folding of primary pre-miRNA (top left). The transcript with hairpin foldback structure undergoes processing by the SER/HYL1/DCL1 complex. The CBC complex binds to the mRNA cap and the mRNA is acted upon by the HYL1/DRB protein and DCL1. This generates a 21 nucleotide double-stranded RNA with 5 phosphorylated two base overhangs and some internal mismatches. The dsRNA is methylated by HEN1 which adds a 2 -methyl group to the 3 end, increasing the stability of the miRNA. Double-stranded miRNAs are loaded into AGO1 proteins and the miRNA* strand is lost, thereby creating an active miRNA–AGO complex. AGO1 and AGO10/ZWILLE miRNA RISC complexes identify miRNA targets. Either target cleavage or translational inhibition occurs. The Arabidopsis ARF6 and ARF8 mRNAs are miR167 cleavage targets and are degraded by an EIN5/XRN4-independent mediated decay. ARF10, ARF16 and ARF17 are targeted by miR160 and are degraded by an XRN4-dependent process. The ta-siRNA targets ARF2, ARF3 and ARF4 are processed by a second order miRNA processing mechanism started by miR390. miR390 is preferentially loaded into AGO7 and targets a TAS3 mRNA. Cleavage generates a phased dsRNA priming site. This is enacted upon by RDR6 and SGS3 to generate long dsRNA which is then processed by the DCL4/DRB4 complex into 21 nucleotide siRNAs. These are loaded into AGO7 and processed to target a variety of mRNAs including ARF2, ARF3 and ARF4. (b) Representation of relative levels of AGO mRNA transcripts in ovules at female gametophyte (FG) stages 1–4 (left) and 5–7 (anthesis; adapted from Yu et al., 2005, supplementary data).

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Fruit Development and Seed Dispersal The strict spatio-temporal accumulation of miR167 in ovules and marginal tissues (Valoczi et al., 2006; Wu et al., 2006; Gifford et al., 2008), combined with the shear miR167 abundance, potentially underlines the importance of miR167 in developmental fate as well as in the control of ARF6 and ARF8 and, thus, potentially in the control of fertilization and fruit initiation. In fact, weak dcl1 alleles cause severe developmental and reproductive defects, including short integuments, incomplete carpel closure and pleiotropy, whereas strong alleles are embryonic lethal (Schauer et al., 2002). Several of these phenotypic effects are recapitulated when genomic versions of the ARF8 and ARF6 genes that have mutated miR167-binding sites are introduced into plants (Wu et al., 2006). Since ARF8 is downregulated 24 h post-anthesis (Goetz et al., 2006), one may conclude that this occurs either through transcriptional downregulation or by the function of miR167 loci. Although transcriptional downregulation in ovules pre- and post-fertilization cannot be ruled out, a strong case is made for the action of miR167 in triggering target cleavage and clearance, rather than simply refining patterns of ARF6 and ARF8 transcript distribution during development. For example, overexpression of miR167 recapitulates many of the phenotypes observed in fwf/arf8 mutants (Ru et al., 2006; Wu et al., 2006). Overexpression studies involving individual miR167 loci also appear to underscore the additional specificity of the miR167 sequence. miR167a phenocopies the arf6 arf8 null mutants (Wu et al., 2006), while miR167b appears to act specifically on ARF8 alone (Ru et al., 2006). Furthermore, the expression patterns of individual miR167 loci in Arabidopsis (Ru et al., 2006; Wu et al., 2006) provide spatio-temporal information that suggests that miR167 loci regulate the clearance of ARF8 mRNA transcripts in specific regions of the ovule during development and flower maturation (Wu et al., 2007). Examining miRNA loci expression pre- and post-fertilization should clarify the roles of each loci as to whether they (a) merely maintain transcriptional mRNA lability; (b) they refine expression patterns of transcription factors or (c) take part in triggering development. ARF8 is apparently self-regulated (Goetz et al., 2006) leading to an alternative theory in which miR167 resolves this function. miRNA mobility may also be important in the context of fruit initiation and fruit development. Experiments by Tretter et al. (2008) have shown that ta-siRNAs, targeting ARF2, ARF3 and ARF4, indeed appear to be mobile from cell to cell in vegetative tissues, but miRNAs appear to be limited to cell autonomous action in the same area (Tretter et al., 2008). Given that miR160 and miR167 are located near vascular tissues, evidence for miRNA mobility is less unambiguous. miRNAs, along with various other types of RNAs and RNA-binding proteins, have been isolated and localized in phloem sap (Yoo et al., 2004; Omid et al., 2007; Buhtz et al., 2008; Deeken et al., 2008; Pant et al., 2008). Experimentation with miR399, along with micro-grafting experiments, categorically shows that miR399 is transported in phloem sap of diverse plant species and acts as a systemic molecule that regulates phosphate homeostasis (Deeken et al., 2008). Therefore, although miRNAs mobility in the ovule and

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gametophyte has not yet been addressed, it is possible thet the mobility of miR160 and miR167 may be an important factor in their function. miRNAs and ta-siRNAs regulating ARFs are highly conserved plant gene families (Fahlgren et al., 2007) and a non-neutral selection in miR167 stemloop structures has occurred with Arabidopsis accessions in contemporary times (Ehrenreich and Purugganan, 2008). Strongest purifying selection conserves the short nucleotide sequence of miR167 and the binding sites of exon 13 from both ARF6 and ARF8 to gymnosperm and angiosperm lineages (Axtell and Bartel, 2005; Axtell et al., 2007; Axtell and Bowman, 2008). Functional miR167 activity against ARF8 in gymnosperm female gametophytes and zygotic embryos (Oh et al., 2008) also supports the conservation of the DCL1-miR167-ARF8 pathway as well as its association with the control of seed development. Several hypotheses about miRNA evolution have been formulated (Chapman and Carrington, 2007) and the roles of miR167-ARF8 in both seed and fruit development could be clarified through the analysis of ancestral miR167 and ARF8 families in basal angiosperms and gymnosperms. Often phenotypes of Arabidopsis miRNA pathway mutants are affected by the genetics of the ecotype background and this may have a specific bearing on fruit initiation. For instance, there are dramatic modifications of the weak dcl1 phenotype between the Columbia and the Landsberg erecta background (Schauer et al., 2002). Similarly, there are also ecotype-specific modifiers of the ago1 and ago10/zwille mutations (Vaucheret, 2008). Moreover, Landsberg erecta shows dramatically increased sensitivity to auxin, parthenocarpy or the fwf/arf8 mutations compared to the Columbia background (Vivian-Smith, 2001; Vivian-Smith et al., 2001). This leads to the hypothesis that the ecotypespecific modifiers of AGO1, AGO10, DCL1 and ARF8 pathways might be linked to miRNA biogenesis, action or RNA metabolism. The modifier or modifiers, effecting auxin responsiveness and fruit initiation could therefore be dependent on miRNA or AGO-related activities. Given the important interactions of the non-coding RNA regulation with the auxin response pathway, and the evolutionary conservation of miR167 in seed plants, together with the specific expression of miR160 and miR167 in ovules, one can expect significant new findings concerning non-coding RNA regulation and fruit initiation. This is the case for the GA2ox2 gene that could be targeted by miR390 activity (Adai et al., 2005). Notably GA2ox2 provides parthenocarpy when mutated (Rieu et al., 2008a), but conclusive activity by miR390 on GA2ox2 has not yet been proven, and as such this link must be considered with caution.

4.8 Signal transduction from ovule to carpel and vascular canalization Fruit initiation involves coordinated intra- and inter-organ signalling between the ovule and carpel. The data by Vriezen et al. (2007) highlight that numerous signalling components in the ABA, ethylene, cytokinin, GA and

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Figure 4.8 Post-fertilization canalization of vascular development in Arabidopsis ovules. (a) Development of vascular networks in unfertilized ovules. Unfertilized ovules have a vascular strand that is separated from the replum vascular strand (arrow). (b) Upon treatment with 2–4 D or (c) pollination, vascular biogenesis occurs, thereby joining the replum vascular network with the ovule vascular network. Note that 2–4 D treatment also induces isolated vascular elements throughout the funiculus (arrows). c, chalaza; f, funiculus; p, placental tissue; rv, replum vascular bundle.

auxin pathways are already modulated within 24 h post-pollination. In a short time period, developmental changes also occur and carbon partitioning is established. The development of vascular networks in the ovule, carpel and pedicel of the flower facilitates this process. At anthesis, the vascular network in Arabidopsis is separated between the unfertilized ovule and carpel (Fig. 4.8a). A connection is initiated within 54 h post-pollination between the fertilized ovule, which contains a single vascular strand, and the carpel (Fig. 4.8c). This time frame is similar to the Zinia in vitro system where tracheid biogenesis occurs within 48 h after auxin and cytokinin treatment (Pesquet et al., 2005). In Arabidopsis, treatment with 10 nmol pistil−1 2,4-D (a synthetic auxin) alone was able to induce vascular development in unfertilized ovules (Fig. 4.8b) suggesting that PAT may facilitate the development of this critical vascular junction. In fact, vascular development at this junction occurs precociously in the fwf/arf8 mutant background supporting the idea that FWF/ARF8 restricts auxin responses within the ovule (Vivian-Smith, 2001; Vivian-Smith et al., unpublished data). This is compatible with experiments where combinations of the fwf-1/arf8-4 mutant with mutations in ovule

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development show that a signal is generated in the ovules of the fwf-1/arf8-4 mutant that stimulate the growth of the carpel into a fruit (Koltunow et al., 2002; Vivian-Smith et al., unpublished data). Vascular biogenesis and development within the ovule may ultimately alter carbon partitioning and source-sink relationships. The processes of vascular biogenesis may not only be restricted to the ovule–carpel vascular junction, but also elsewhere in the flower, since vascular biogenesis, or its absence, in the pedicel has been linked to growth and abscission in citrus (Bustan et al., 1995), Prunus (Else et al., 2004) and apple (Drazeta et al., 2004), respectively. Importantly, these observations appear to pinpoint a time when discrimination occurs. Competition begins between fruit and flowers, and causes the fruit abscission often observed in the first week of many important commercial tree crops (Sedgley and Griffin, 1989). Competition also occurs between parthenocarpic fruit and seeded fruit, which are usually stronger (McConchie et al., 1994). Vascular biogenesis presumably reinforces nutrient and photoassimilate allocation to developing fruit affecting retention. Interestingly, the fruit weight locus (fw2.2) from tomato is highly expressed in ovules at anthesis and is responsive to adjacent fruit loads (Baldet et al., 2006). This suggests an early role for fw2.2 in fruit retention. The Arabidopsis SUC2, on the other hand, is expressed in the funiculus of the ovule and is required for phloem loading and unloading (Truernit and Sauer, 1995). However, no studies have yet documented SUC2 expression in enough detail during the pre- and post-fertilization stages to determine whether SUC2 expression is related to phytohormonal responses in the ovule.

4.9 Current models of fruit initiation The utilization of PAT inhibitors has provided an excellent understanding into the basis of auxin-mediated vascular network biogenesis in leaves and inflorescences (Mattsson et al., 1999; Sieburth, 1999; Scarpella et al., 2004; Sauer et al., 2006). In leaves, auxin is thought to be synthesized in the marginal tissue and transported away via PAT that is dependent on MP and PIN efflux carriers (Scarpella et al., 2004; Sauer et al., 2006). However, transport also selfreinforces and stimulates the development of provascular strands that would act as efficient drainage canals, thereby developing the observed vascular networks. In Arabidopsis ovules, an analogy can be drawn, with the integuments of the ovule regulating the provision of auxin to the chalaza and funiculus and then to carpel margin. Auxin synthesis after fertilization in the integuments, or integument tips, would not only stimulate the formation of the provascular network in the carpel margin, but also the growth of the carpel into the fruit (Fig. 4.9). Prior to fertilization, ARF8 is expressed in the female gametophyte, the endothelium of the inner integument and in the chalaza/funiculus regions (Goetz et al., 2006). At this stage, the activity of ARF8 would restrict the auxin response, possibly through self-reinforcement (Goetz et al., 2006) and through the interaction with Aux/IAA proteins, presumably IAA9 and IAA28, which are expressed in Arabidopsis ovules (Fig. 4.9). KAN4 may function during

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Figure 4.9 Integrated model of fruit initiation. (a) In the absence of fertilization, FIS class genes actively restrain central cell growth and autonomous endosperm proliferation. The primary auxin response is also restricted by the activity of ARF8 in the ovules. Specific ovule cells in the pathway shutdown the intracellular auxin response and communication via ARF–ARF and ARF–IAA protein interactions. PID may not play a direct role in this communication but might become important later in the post-fertilization PAT processes. KAN4 may contribute to the synchronization of pistil development before anther dehiscence via control of PAT. In the carpel, GA response remains blocked by the restraint in growth imposed by DELLA proteins. (b) Following double fertilization, zygote and endosperm development is initiated. Concomitant upon the first nuclear division in the endosperm 3–5 h post-fertilization, the primary sporophytic auxin response is initiated in the chalaza and endothelium. The restraint upon auxin response is also eliminated possibly by DCL1-mediated ARF8 removal. Upregulation of the PAT results in the auxin growth response being transmitted to the carpel which in turn, triggers the GA biosynthesis pathway and vascular development. Increased levels of gibberellins cause growth stimulation by DELLA protein degradation.

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female receptivity by altering PAT in the ovule (Pekker et al., 2005; Izhaki and Bowman, 2007; Fig. 4.9) and thereby functioning in the pathway that synchronizes pistil growth before anther dehiscence (Fig. 4.9). The stimulation of GH3 transcription by ARF8 and possibly ARF6 (Wu et al., 2007) would further remove free auxin from the ovule through conjugation with amino acids (Woodward and Bartel, 2005) and together with the above mentioned processes restrict vascular biogenesis between the ovule and carpel (Fig. 4.9). miR167 could either refine ARF8 expression during the pre-fertilization period or actively stimulate the destruction of ARF8 transcripts post-fertilization. Alternatively, miR167 could act as a ‘circuit breaker’ by removing any selfreinforcement of ARF8 self-activation during post-fertilization stages. The expression pattern described by Golden et al. (2002) for DCL1, however, suggests a role in post-anthesis ARF8 clearance in the female gametophyte and funiculus vasculature. Since MP is also expressed in the funiculus vasculature (Hardtke and Berleth, 1998), a role for MP would be in the direct activation of auxin responses post-fertilization in this region (Fig. 4.9). PAT and vascular biogenesis would also positively stimulate GA metabolism since both the KS enzyme are localized with vascular tissues (Fig. 4.9; Silverstone et al., 1997; Vivian-Smith and Koltunow, 1999; Vivian-Smith et al., 2001) and PAT has been documented to stimulate the destruction of DELLA proteins (Fu and Harberd, 2003). GA2ox2, which would restrict active GAs prior to fertilization (Rieu et al., 2008a), could come under the control of miR390 driven clearance postfertilization (Adai et al., 2005). miR160 may also have roles during this since this miRNA is localized in the placental and funiculus regions. ZWL/AGO10 and VCS may participate in vascular biogenesis. The roles of ethylene, ethylene precursor molecules, cytokinin and other phytohormones are difficult to place within an integrated model of fruit initiation at this particular point in time, but they may function in the female gametophyte (Fig. 4.9). The evolution of fruit initiation has largely been absent from review or research. Yet, the fundamental nature of fruit initiation is central and possibly underpins primary characteristics of the angiosperm: closed carpel and ovule development. Processes of flower maturation and synchronization maybe of recent origin, but the aspects relating to canalization of auxin responses and organ initiation are likely to be of older origin and relevant to the ontogeny of fruit initiation. Deducing homologies in structure between non-angiosperms and angiosperms is difficult and often angiosperm and gymnosperm taxonomy is heavily reliant on vascular morphologies of the ovule integuments and the carpel (Frohlich, 2003). The conserved nature of fruit initiation may be elucidated in part by comparable treatments of models relating vascular biogenesis and canalization together with the control of ovule and carpel identity through the transcription factors studied by Alvarez and Smyth (1999) and Herr (1995). It is worthy to note that expression of ARF8 and miR167 does actually occur in the integument primordia, and in the leaf and carpel margins (Wu et al., 2006; Goetz et al., 2007). These are the precise locations where auxin is proposed to be biosynthesized (Mattsson et al., 1999; Sieburth, 1999; Scarpella et al., 2004; Sauer et al., 2006).

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Fruit Development and Seed Dispersal A model where ARF8 restricts auxin responses in key marginal tissues to control organ development and canalization may renew interest in vascular biogenesis as a taxonomic morphological marker, while underscoring the relevance for ARF8 and miR167 expression analysis. Scrutinizing the wide conservation of ARF8 and ARF6, and miR167 families which are restricted to the seed plants, along with the potential derivation of miR167 from an ancestral ARF6 or ARF8 gene may also potentially provide crucial pieces of the missing fruit initiation model while providing information on pathways that were recruited during angiosperm flower evolution.

4.10

Concluding remarks

Many studies have contributed to the better understanding of the complex regulatory system controlling fertilization and fruit initiation. Nevertheless, vital questions remain to be answered. Are hormones the initiators or just systemic components of the signalling cascade? Can we isolate the first step triggering fruit set? What are the signals upstream of phytohormonal signalling that are activated directly after fertilization? Which are the sites of endogenous hormone biosynthesis during fruit initiation and are they regulated in the first steps of fruit initiation? What is the nature of the communication events between the female and male gametophyte? The study of fruit initiation in Arabidopsis and other species will undoubtedly help to clarify these and other unknowns. Finally, a number of publications have stated that Arabidopsis and Brassicaceae appear to be far from optimal models for fruit development and have suggested that plant species bearing large fleshy fruit offer superior advantages for understanding the molecular basis of fruit initiation. However, genes controlling fruit initiation are likely to be conserved throughout angiosperm plant lineages. Furthermore, many commercial fruit crops have been domesticated over thousands of years and show strong selection for consumer traits (e.g. tomato, Nesbitt and Tanksley, 2002; Bai and Lindhout, 2007; Cong et al., 2008; Xiao et al., 2008; Apple, Harris et al., 2002; Capsicum, Paran and Van Der Knaap, 2007; Phaseolus, Curcubitaceae). This is highlighted by the finding that parthenocarpic figures were intentionally planted as early as 11 200–11 400 years ago in the Jordan Valley (Kislev et al., 2006). As a consequence, many of these crops also show a degree of latent parthenocarpy that does not exist in wild accessions. For example in tomato, 23 commercial cultivars were recently tested all of which displayed certain degree of latent parthenocarpy (Goetz et al., 2007). In contrast, Arabidopsis is comparatively free from selected potentiation and latent parthenocarpy and, thus, it is likely to provide a more truthful picture of fruit initiation. Further research together with the transfer of mutant trait loci into crop species will undoubtedly lead to an acceptance of Arabidopsis as a tractable model for fruit initiation.

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Acknowledgements S.F. acknowledges the Marie Curie Early Stage Researcher Training Fellows Project and John Innes Foundation for funding. A.V-S. was supported by the Dutch STW grant LB06822. The authors apologize to colleagues whose work could not be cited due to space constraints.

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Annual Plant Reviews (2009) 38, 172–203 doi: 10.1002/9781444314557.ch5

www.interscience.wiley.com

Chapter 5

ARABIDOPSIS FRUIT DEVELOPMENT Antonio Mart´ınez-Laborda and Antonio Vera ´ Area de Gen´etica, Universidad Miguel Hern´andez, Ctra. de Valencia s/n, San Juan de Alicante, Spain

Abstract: The Arabidopsis fruit is a dehiscent silique that basically consists of a mature ovary. Along the mediolateral axis, the ovary develops as a cylindrical species that externally comprises two valves with lateral polarity separated at the medial plane by two repla. Another tissue with lateral polarity, the valve margin, develops between valve and replum and will differentiate into the dehiscence zone. Our knowledge of the molecular mechanisms and genetic networks involved in the formation of these three pattern elements has increased greatly over the past few years. The present view on the establishment of this pattern is that the ovary reproduces the antagonistic interactions between meristem- and leaf-expressed genes. Thus, the replum displays meristematic properties and expresses meristem genes (replum factors), whereas valves express genes that work to make leaves (valve factors) and are more related to these lateral organs. Following this line of argument, a recent model puts forward that the antagonistic activities of the opposing gradients of replum and valve factors determine the territories of the ovary where replum, valve margin and valves will form. Undoubtedly, understanding these mechanisms will contribute to optimize harvest yield in crops with dehiscent fruits, providing control of seedpod shatter. Keywords: gynoecium; fruit development; mediolateral axis; pattern formation; positional information; dehiscence

5.1

Introduction

To our eyes, the flower is very likely the most appealing structure of angiosperms. Flowers usually show loud colours and attractive shapes that have been sculpted throughout the evolution to facilitate pollination of the gynoecium, the female reproductive part of the plant. The gynoecium consists of one or more lateral organs named carpels, which are organized either in monocarpelate (only one carpel) or pluricarpelate (more than one carpel) 172

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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mesocarp enb

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Figure 5.1 The silique of Arabidopsis thaliana. On the left, scanning electron micrographs of pistil and fruit showing pattern elements along the apical–basal (upper case letters) and the mediolateral (lower case letters) axes of symmetry. Scale bars represent 200 µm. On the right, cross-sections through the ovaries of two fruits.

pistils. Along the apical–basal axis of the pistil, the most basal pattern element is the gynophore, the stalk that attaches the pistil to the floral receptacle. Above the gynophore, the ovary encloses the ovules, and the apical stigma favours the hydration and germination of the pollen grain, as well as pollen tube penetration into the style, the last pattern element, which connects the stigma with the ovary, allowing the growth of the pollen tube through the transmitting tract (Fig. 5.1). The structures and tissues of these pattern elements help the mature male gametophyte, the germinated pollen grain with its pollen tube, to find its way to the embryonic sac or female gametophyte, ending up the process with the typical double fertilization of angiosperms (Russell, 1992; Raghavan, 2003; Dresselhaus, 2006). Once double fertilization has taken place, the ovule begins to develop into a seed, with the concomitant increase in size, and consequently, the pistil must also increase its volume to appropriately house the seeds, developing into a fruit. To this end, fertilized ovules release signals that determine fruit set and growth, in such a way that seed and fruit development occur as coordinate processes (Gillaspy et al., 1993; O’Neill and Nadeu, 1997; Raghavan, 2003).

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Fruit Development and Seed Dispersal The fruit is an organized structure that protects the seeds as they mature and ensures their dispersal by diverse mechanisms. Thus, fleshy fruits have succulent and tasty pulps for animals that eat them to subsequently disperse the seeds, whereas seedpods are dehiscent fruits that split when ripe, releasing the seeds located inside. This chapter will focus on the Arabidopsis thaliana fruit, which deserves attention not only because this crucifer is a model organism with a myriad of genetic and molecular tools available that allow the study of developmental phenomena in plants (Page and Grossniklaus, 2002; Salinas and S´anchez-Serrano, 2006), but also because it is a standard silique, the dehiscent fruit that characterizes more than 3000 species of the Brassicaceae family (Ferr´andiz et al., 1999), which shows certain degree of similarity to the seedpod of legumes (Giovannoni, 2004). Therefore, understanding the mechanisms involved in the development of the Arabidopsis silique should contribute to the improvement of crops with dehiscent fruits (Salentijn et al., 2007).

5.2 5.2.1

Morphology of the Arabidopsis silique Pistils outline the fruit pattern elements

In Arabidopsis, the gynoecium consists of a solitary pistil that comprises two congenitally fused carpels, namely, a bicarpelate pistil that develops from a single primordium (Sessions and Zambryski, 1995; Bowman et al., 1999). At anthesis, the period just prior to fertilization in which the flower opens and anthers shed pollen grains, some tissues have reached complete maturity. This is what happens to tissues involved in pollen germination or in the guidance and growth of the pollen tube. However, at this stage, many other pistil tissues remain in a predifferentiated state, exhibiting a species of prepattern that outlines the pattern elements that will be subsequently distinguished in the silique. Thus, the fruit shows the same four pattern elements along the apical–basal axis that have been described above for pistils: the stigma, the style, the ovary and the gynophore (Ferr´andiz et al., 1999; Fig. 5.1). Among all these pattern elements, this chapter will pay particular attention to the ovary, since the Arabidopsis silique is basically a mature ovary that has enlarged to enclose the seeds (Vivian-Smith and Koltunow, 1999). Pistils already show the main territories that will be present in the mature ovaries of siliques. Externally, the ovary wall exhibits two valves with immature stomata, which are separated by a ridge of cells that form the replum (Fig. 5.1). According to floral symmetry, valves and replum are pattern elements along the mediolateral axis, with the valves in lateral positions and the replum in the medial plane of the flower. Transverse sections exhibit a conspicuous bilateral symmetry, showing a post-genitally fused septum that joins both repla and divides the internal cavity into two locules. Inside

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these, the ovules arise from the placenta, a tissue with meristematic properties located along the septum in positions immediately adjacent to the inner side of the ovary walls. Replum, septum and placenta comprise marginal tissues, since they derive from the margins of the fused carpels, whereas the six cell layers of the valves correspond to the carpel walls that enclose and protect the ovules (Sessions and Zambryski, 1995; Bowman et al., 1999; Ferr´andiz et al., 1999). Additionally, although morphologically indistinguishable from other tissues, the valve margin contains a few files of cells located between replum and valves that express a specific set of genes (Ferr´andiz, 2002; Dinneny and Yanofsky, 2005; Robles and Pelaz, 2005; Lewis et al., 2006; Roeder and Yanofsky, 2006). 5.2.2 Structure of the ovary along the mediolateral axis of the silique Fruit set and development are strictly dependent on ovule fertilization and seed set (Ohad et al., 1996; Chaudhury et al., 1997), so that unpollinated pistils show a very limited growth and rapidly senesce (Vivian-Smith and Koltunow, 1999; Vivian-Smith et al., 2001). Post-fertilization events release signals that trigger the differentiation of fruit tissues from those of the pistil, giving rise to an increase in size and the maturation of stomata in the outer epidermal layer of valves (Gu et al., 1998; Vivian-Smith and Koltunow, 1999). These signals, still not completely understood, very likely involve plant hormones emanating from the seeds (Barendse et al., 1986; Vivian-Smith and Koltunow, 1999; Goetz et al., 2006), in such a way that the size of the silique will ultimately depend on the number of developing seeds. The final size is attained by cell divisions in the apical–basal axis and by cell expansion in length and width, with no divisions along the mediolateral axis perhaps with the exception of the innermost layer of the valves (Spence et al., 1996; Ferr´andiz et al., 1999; Vivian-Smith and Kultunow, 1999). Signals that initiate fruit development stimulate different responses in the cells, depending on their positions along the axes of polarity. Thus, in the mediolateral axis, cells in the outer layer of valves acquire irregular shapes and large sizes, while cells in the outer layer of the replum manifest less expansion in width and appear thinner (Gu et al., 1998). Valve margin cells, initially indistinguishable from those of the valves, remain small because of their slower expansion rates (Liljegren et al., 2000; Rajani and Sundaresan, 2001; Fig. 5.1). The valves of fruits, in addition to the outer epidermis, also named exocarp, display other five cell layers. Immediately adjacent to the exocarp, there are three mesocarp layers with photosynthetic cells, and facing inside, two endocarp layers, the endocarp a (ena) layer or inner epidermis and the endocarp b (enb) layer or inner subepidermal layer. Cells of the ena layer become large and display very thin walls, whereas those of the enb layer show thick walls (Spence et al., 1996; Ferr´andiz et al., 1999; Vivian-Smith and Koltunow, 1999; Fig. 5.1). During ripening, the enb layer lignifies and subsequently the

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Figure 5.2 Cross-sections of wild type and shp1 shp2 siliques. The wild-type fruit exhibits a normal dehiscence zone with separation and lignified layers. The dehiscence zone is absent in the shp1 shp2 fruit, which does not form the separation and lignified layers.

ena layer disintegrates (Spence et al., 1996; Ferr´andiz et al., 1999; Rajani and Sundaresan, 2001; Ferr´andiz, 2002). At the boundary between valve and replum, the dehiscence zone (the territory in which the ripe silique will dehisce) develops from the valve margin, where small-sized cells form a constriction on the fruit surface (Liljegren et al., 2000). In transverse sections, the dehiscence zone appears as a V-shaped region approximately four- to six-cell-wide that consists of two main tissues. Close to a row of parenchyma cells in the replum, the separation layer comprises non-lignified and isodiametric cells, and adjacent to the valve, the lignified layer is made up of thick-walled cells (Spence et al., 1996; Rajani and Sundaresan, 2001; Wu et al., 2006; Fig. 5.2). This layer will form a continuity of lignified tissues with the enb layer by the time the silique reaches its final length. During dehiscence, the fruit desiccates and the lignified tissues of the valve margin and the enb layer create a spring-like tension that contributes to separate the valve from the replum. Silique shattering occurs by the action of both these mechanical forces and degrading enzymes, which produce breakdown of the middle lamella between adjacent cell walls at the separation layer, giving rise to a fracture line that results in valve detachment (Spence et al., 1996; Rajani and Sundaresan, 2001; Ferr´andiz, 2002). Therefore, pattern elements and tissues of fruits are previously preformed in the pistil from which they all derive. The development of the pistil basically consists of generating a complex structure from a morphologically uniform group of cells that initially form a dome-shaped primordium. A relevant issue in this developmental process is the determination of polarity along the main axes of symmetry. The polarity is then interpreted by cells to acquire positional information that results in the organization of a precise pattern, which involves the development of territories with specific tissue types, that is to say, the development of specific pattern elements. Genes playing crucial roles in the establishment of territories in the fruit are already expressed from

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early stages of pistil development, when different sets of genes are switched on in specific domains to produce the three main pattern elements along the mediolateral axis: the valve, the valve margin and the replum.

5.3 Determining the boundary between valve and replum: valve margin genes 5.3.1 On top of the network: the SHATTERPROOF genes Isolated because of their high homology to AGAMOUS, a homeotic gene that determines the C floral identity function (Yanofsky et al., 1990; Coen and Meyerowitz, 1991), and formerly named AGL1 and AGL5, SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) encode two closely related MADS-box proteins, sharing 87% identity at the amino acid level. Both display a very similar expression pattern in the pistil, which includes a noticeable expression at the valve margin. Their mRNAs are initially detected throughout the developing pistil. Later on, the expression is first restricted to medial domains, which include the presumptive repla, and is finally resolved to the placenta, ovules and four narrow stripes just in the positions where the valve margin develops (Ma et al., 1991; Savidge et al., 1995; Flanagan et al., 1996; Liljegren et al., 2000). Single mutants for loss-of-function alleles in either of the two genes display a wild-type phenotype, suggesting that SHP1 and SHP2 determine redundant activities. This was confirmed after the analysis of shp1 shp2 siliques, which show reduction of lignification in cells of the valve margin and defects in separation layer formation. Consequently, the valve margin does not properly form in double mutant fruits, which are indehiscent (Liljegren et al., 2000; Fig. 5.2). 5.3.2 Two additional activities to make valve margin The abnormal development of valve margin tissues in shp1 shp2 fruits goes along with changes in the expression profiles of several molecular markers. One of these, the GT140 reporter, is expressed at the valve margins of developing wild-type siliques (Sundaresan et al., 1995), whereas its expression is completely absent in shp1 shp2 fruits, suggesting that the SHP genes are upstream positive regulators of the gene corresponding to GT140 (Liljegren et al., 2000; Ferr´andiz et al., 2000b). The reporter is inserted in INDEHISCENT (IND), another gene with key roles in dehiscence zone development, which codes for a basic helix-loop-helix (bHLH) transcription factor (Liljegren et al., 2004). Mutants lacking IND function show a large effect on dehiscence zone formation, since both the lignified layer and the separation layer do not develop because of the absence of unequal cell divisions that originate the distinct cell types of these layers (Liljegren et al., 2004; Wu et al., 2006). As a result, ind siliques

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Fruit Development and Seed Dispersal do not show the constriction that identifies the dehiscence zone and display a strong indehiscent phenotype. The effect of ind alleles on dehiscence zone development is more dramatic than that of the shp1 shp2 double combination, indicating that IND and the SHP genes also perform independent activities, and suggesting the presence of other pathways to activate IND independently of the SHP genes. This is additionally supported by the phenotype of shp1 shp2 ind triple mutant in which dehiscence zone development is more severely affected than in either shp1 shp2 or ind mutants (Liljegren et al., 2004). The ALCATRAZ (ALC) gene, whose name comes from the San Francisco Bay federal prison, was identified by loss of dehiscence caused by mutant alleles of the gene, which also encodes a bHLH transcription factor (Rajani and Sundaresan, 2001) and is positively regulated by the SHP genes (Liljegren et al., 2004). Its expression is detected in a broad domain in the valve and valve margin of the pistil, though with more intensity at the valve margin, and is limited to this region after fruit set. According to this expression, alc siliques have altered dehiscence zones in which cells of the separation layer fail to differentiate, exhibiting abnormal morphology. In fact, inner cells in this layer show ectopic lignification and create a continuous bridge between the lignified replum vasculature and the enb layer of the valve. Hence, when the outer layers of the valve margin tear down due to cell rupture, dehiscence is prevented by this lignified bridge that can be broken by the simple application of manual pressure (Rajani and Sundaresan, 2001). Indeed, the dehiscence zone of alc mutants is quite different from those of ind and shp1 shp2 fruits, which show alteration of the whole dehiscence zone and cannot be opened so easily, so that the alc phenotype, in which only the separation layer is affected, can be considered as a subset of the phenotypes produced in the valve margin either by ind alleles or by the shp1 shp2 double combination. 5.3.3

A working model for valve margin genes

Despite the positive regulation of SHP genes on ALC expression, the triple shp1 shp2 alc shows a loss of valve margin definition with respect to shp1 shp2 and alc fruits. The same as with IND, some roles of ALC on dehiscence zone differentiation must be accomplished independently of the SHP genes. As a matter of fact, the strongest valve margin mutant phenotype is obtained in the shp1 shp2 ind alc quadruple mutant. Therefore, all these genes form a regulatory network in which there are three activities with both unique and overlapping functions in dehiscence zone formation (Liljegren et al., 2004). Nevertheless, the mutant phenotype of ind fruits do not increase in the ind alc double mutant, which suggests that ALC function is also carried out by IND, at least in a wild-type SHP background. ALC and IND code for bHLH transcription factors that can form heterodimers, and indeed, both proteins interact in a two-hybrid yeast assay (Liljegren et al., 2004). All the aforementioned results suggest a model in which the SHP genes are upstream activators of IND and ALC, and these two genes account for most of the

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SHP1 SHP2

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IND

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IND

IND/ALC

SL LL Valve margin

Replum

Figure 5.3 Genetic network controlling dehiscence zone development. SHP1 and SHP2 activate the expression of IND and ALC in the dehiscence zone, although additional factors should be involved in activating low levels of expression of IND and perhaps also of ALC. SHP1, SHP2 and IND determine the development of the entire dehiscence zone, while ALC is only involved in separation layer formation. IND and ALC possibly form a heterodimeric complex to specify the separation layer. LL, lignified layer; SL, separation layer.

functions required to make the dehiscence zone. The protein products of ALC and IND dimerize to regulate the differentiation of the separation layer, whereas the IND protein has an additional role in the differentiation of the lignified layer. Despite this, the redundant SHP genes, ALC and IND also show independent activities, so that the strongest mutant phenotype is only observed when the four genes are impaired. Finally, although the SHP genes activate the expression of IND, as ind and shp1 shp2 ind mutants show a stronger phenotype than shp1 shp2 plants, and IND is weakly expressed in the valves of shp1 shp2 ful fruits (see next section), there must be additional factors involved in the activation of low levels of IND expression (Liljegren et al., 2004). Possibly, this is also true for ALC (Fig. 5.3).

5.4 The making of valves and replum requires repression of valve margin genes 5.4.1 FRUITFULL represses valve margin genes in valves The first factor identified to participate in patterning the mediolateral axis of fruits was FRUITFULL (FUL; Gu et al., 1998), which codes for a MADS-box transcription factor closely related to the proteins encoded by the floral meristem identity genes APETALA1 (AP1; Mandel et al., 1992) and CAULIFLOWER (CAL; Kempin et al., 1995). Formerly named AGL8 (Mandel and Yanofsky, 1995), FUL has a key role in valve development and also collaborates with AP1 and CAL in conferring floral identity to meristems (Gu et al., 1998; Ferr´andiz et al., 2000a). In ful mutants, siliques fail to differentiate valve cells and do not elongate after pollination, showing a reduced size with seeds tightly

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ful

Wild type

rpl

as1

Figure 5.4 Fruit phenotypes of ful, rpl and as1. In ful siliques, the valves are small as a consequence of the reduced cell size, whereas the replum appears broad and its cells adopt a zigzag pattern. rpl fruits do not show a normal replum due to the small size of the cells. The as1 silique exhibits large repla. Arrowheads indicate the positions of the valve margins. Scale bars represent 100 µm.

compressed inside. The valves of these fruits lack stomata and display small and rounded cells that have not expanded properly, as well as lignification of the three mesocarp layers (Gu et al., 1998; Liljegren et al., 2004). However, cells of the outer replum expand and adopt a much larger size than valve cells, so that the replum acquires a zigzag appearance in order to accommodate its length to that of the small valves (Fig. 5.4). As the gene is expressed in the six layers of valves, but not in the replum and valve margins, these results were interpreted as FUL playing a crucial role in valve cell differentiation and expansion after fertilization, and being responsible for providing competence to valve cells to respond to signals that trigger fruit set and development (Gu et al., 1998). An important question is whether FUL confers valve identity to tissues. The answer is that not really, but the fruit phenotype of 35S::FUL plants, which express constitutively FUL, seems to agree with this hypothesis (Ferr´andiz et al., 2000b). The outer replum and valve margin of these siliques are transformed into valve, suggesting that the ectopic expression of FUL is enough to determine valve fate. Interestingly, the expression of the SHP genes was completely abolished in these fruits, so that FUL downregulates these valve margin genes. Another important hint came from the study of gain-of-function phenotypes in 35S::SHP1 35S::SHP2 plants, whose siliques show a ful-like appearance (Liljegren et al., 2000), pointing out to the alternative hypothesis that FUL functions by repressing valve margin genes in the valves. These 35S::SHP1 35S::SHP2 siliques exhibit expression of FUL in its normal lateral domain, showing that the SHP genes do not negatively regulate FUL

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transcription (Ferr´andiz et al., 2000b), and express ectopically IND (Liljegren et al., 2000), in accordance with the positive regulation exerted by the SHP genes on IND expression. In fact, fruits of plants overexpressing IND are also reminiscent to those of ful mutants (Liljegren et al., 2004), and ful siliques also display ectopic expression of IND, which is even detected in the valves of shp1 shp2 ful fruits, though more weakly (Ferr´andiz et al., 2000b). Therefore, a key function of FUL is to prevent valve cells from adopting a valve margin fate by the repression of valve margin genes. The results above pose a question. In which degree do ectopic activities of valve margin genes account for the whole ful phenotype? To uncover the relative contribution of these genes to the phenotype, multiple mutants were obtained that carried a ful allele and one or more mutant alleles in valve margin genes (Liljegren et al., 2004). Fruits of shp1 shp2 ful, alc ful and ind ful plants show partial suppression of the ful phenotype and, according to the more severe valve margin phenotype caused by ind alleles, the rescue is more evident in ind ful siliques, in which the ectopic lignification of mesocarp cells is eliminated and some stomata differentiate in the exocarp. Furthermore, the most evident suppression of the ful phenotype is observed when the three valve margin activities are compromised in the shp1 shp2 alc ind ful quintuple combination, which practically restores the wild-type appearance (Liljegren et al., 2004). Hence, the phenotypes of these multiple mutants favour again that the main role of FUL in valve development is to prevent the ectopic expression of valve margin genes in the valves. However, the rescue is not complete, even in the quintuple mutant, suggesting that there are additional factors ectopically expressed in the valves of ful siliques or that FUL has additional roles in valve cell differentiation (Liljegren et al., 2004). Alternatively, the loss of enb layer lignification, which is only seen in quintuple mutant siliques (Liljegren et al., 2004), could account for the altered phenotype of these fruits (Dinneny and Yanofsky, 2005). 5.4.2 REPLUMLESS represses valve margin genes in the replum Is there a similar process to exclude valve margin gene expression from the replum? Although mechanistically different from the repression of FUL on valve margin genes, the answer to this question is yes. A key factor to maintain valve margin activities out of the replum is the homeobox gene REPLUMLESS (RPL), which is expressed in medial tissues of the ovary (the presumptive replum) from early stages of pistil development (Roeder et al., 2003). RPL codes for a homeodomain protein of the BELL (BEL1-like) family of transcription factors, members of which are known to interact with proteins of the class I KNOX (KNOTTED1-like homeobox) homeodomain proteins to regu¨ late specific developmental programmes (Bellaoui et al., 2001; Muller et al., 2001; Smith et al., 2002). The gene was identified following a second-site mutagenesis strategy on a ful background, so taking advantage of the large replum of ful siliques, which is visible with the naked eye. In ful rpl mutants, the

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SHP1 SHP2

FUL Valve

Valve margin

RPL Replum

Figure 5.5 FUL and RPL limit the expression of the valve margin genes. FUL in valves and RPL in the replum repress valve margin genes, whose expressions are limited to narrow stripes between the valves and the replum.

replum is absent and the entire surface of the ovary is covered by small cells, suggesting that replum cells have also adopted a valve margin cell fate. After getting rid of the ful allele, fruits of rpl single mutants look quite normal, but a careful inspection reveals that they lack replum. In fact, the replum does not completely disappear, but instead its cells have a reduced size, taking on a valve margin identity (Fig. 5.4). Accordingly, SHP gene expression is ectopically detected in the replum of rpl pistils and completely surrounds the ovary in ful rpl plants. Implication of valve margin genes in the cell replum phenotype of rpl mutants is further demonstrated with the triple rpl shp1 shp2 and the quadruple rpl ful shp1 shp2, in which the shp alleles restore the wild type and the ful replum phenotypes, respectively (Roeder et al., 2003). In conclusion, the valve margin, initially recognized by the expression of specific genes in the pistil, marks the limit between valve and replum, developing into the dehiscence zone after fruit set. FUL in the valves and RPL in the replum negatively regulate valve margin genes to restrict their expressions to the valve margin, ensuring in this way the development of valves and replum (Fig. 5.5). Therefore, neither FUL nor RPL is strictly necessary to make these pattern elements, as indicated by the suppression of mutant phenotypes by mutations in valve margin genes, raising the question of which selector genes are acting in the mediolateral axis to establish the specific patterns of gene expression, as well as to determine the fate of valves and replum.

5.5

5.5.1

Suppressors of the rpl phenotype: setting up territories Loss-of-function in either FILAMENTOUS FLOWER or JAGGED suppresses the rpl phenotype

FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3) encode two closely related proteins belonging to the YABBY family of zing-finger transcription factors involved in adaxial–abaxial polarity. Adaxial makes reference to the side of the organ nearest to the apical meristem, the inner layers in pistils and fruits, and abaxial to the side facing away from it, the outer layers of pistils

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and fruits (Eshed et al., 1999). Both genes are expressed in the abaxial region of lateral organs, conferring this identity to tissues (Sawa et al., 1999; Siegfried et al., 1999). In the ovary of developing pistils, FIL and YAB3 display a similar expression pattern, including valves and cells that will become valve margin, but their products are never detected in the replum. Interestingly, FIL expression in the valve is not only restricted to the exocarp and mesocarp layers, both of abaxial identity, but is also seen in an adaxial tissue, the enb layer (Sawa et al., 1999; Siegfried et al., 1999; Dinneny et al., 2005). Although fil and yab3 single mutants have dehiscent siliques, the double fil yab3 exhibits an indehiscent phenotype, indicating that FIL and YAB3 play a crucial role in valve margin development. However, the phenotype is somehow more complicated, and the double mutant also shows valve regions with ectopic valve margin-like tissues, which is reminiscent of the ful mutant valve phenotype (Dinneny et al., 2005). In accordance with these results, FUL expression is absent from the valves of double mutant siliques, whereas that of the SHP genes is lost from the valve margins, indicating that FIL and YAB3 promote the transcription of FUL and SHP genes in valves and valve margins, respectively. Nevertheless, SHP expression is also ectopically detected in the valves of fil yab3 fruits, which explains the differentiation of ectopic valve marginlike cells (Dinneny et al., 2005). Are perhaps FIL and YAB3 acting as inducers of the SHP genes in the valve margins and as repressors in the valves? How can this dual behaviour on SHP expression be explained? The answer to these questions is that there is another player in the game. JAGGED (JAG) encodes a transcription factor with a single C2H2 zincfinger motif that regulates lateral organ development by promoting growth and suppressing the premature differentiation of tissues (Dinneny et al., 2004; Ohno et al., 2004). The gene is expressed in all tissue layers of valves and valve margins in developing pistils, and cooperates with FIL and YAB3 to regulate gene expression along the mediolateral axis (Dinneny et al., 2004, 2005; Ohno et al., 2004). Thus, valves of fil jag fruits show an ectopic stripe of valve margin cells in which FUL expression is not detected, and after elimination of a single copy of YAB3 from the fil jag background the fruit phenotype is strikingly similar to that of ful plants. Accordingly, in these jag/jag; fil/fil; YAB3/yab3 fruits, FUL expression is restricted to small patches in valves, whereas the SHP genes are ectopically expressed in this territory. The ful phenotype disappears in the triple jag fil yab3 in which FUL expression is completely abolished and that of the SHP genes is extremely reduced. As a whole, these results show that JAG redundantly works with FIL and YAB3 to promote the expression of FUL and valve margin genes, so that the activity of these three genes in ovaries has been named JAG/FIL activity (Dinneny et al., 2005). FUL expression is more sensitive than that of the SHP genes to the loss of JAG/FIL activity, suggesting that FUL and SHP are activated by different levels of this activity. Therefore, a convincing model emerges in which JAG/FIL activity would act as a gradient of morphogen, activating downstream genes depending on its concentration along the mediolateral

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Fruit Development and Seed Dispersal axis of the ovary. According to the model, high levels of JAG/FIL activity in lateral positions activate FUL expression, whereas this activity decreases towards medial positions. Valve margin genes are activated in places close to the presumptive replum where there are lower levels of JAG/FIL activity (Dinneny et al., 2005). Ectopic expression of JAG in plants carrying the gain-of-function jag-5D allele (Dinneny et al., 2004) gives rise to a phenotype reminiscent to that of rpl fruits (Dinneny et al., 2005). In these plants, the SHP genes are expressed in medial regions of the pistil corresponding to the presumptive replum, so that the outer replum is transformed into valve margin tissue. In addition, the same as in rpl mutants, the replum phenotype is rescued after removing the SHP activity in the triple jag-5D shp1 shp2. This suggests that the groundwork for the phenotype caused by rpl alleles could be ectopic JAG/FIL activity in the replum, which would activate SHP expression in abnormal medial positions. In fact, rpl mutants exhibit a clear expansion of FIL expression into the replum, and replum development is restored after loss of either FIL or JAG function. Therefore, the obvious outcome is that the negative regulation of RPL on SHP expression is mediated by repression of the JAG/FIL activity (Dinneny et al., 2005). Nevertheless, this indirect effect does not rule out a more direct repression of SHP expression by RPL. Actually, RPL directly represses the expression of AG, a close paralog of the SHP genes with which it shares some functions, by binding to specific motifs in regulatory regions of the gene (Bao et al., 2004). 5.5.2

Loss-of-function in ASYMMETRIC LEAVES1 suppresses the rpl phenotype

The RPL gene has also been identified in several independent works because of its role in meristem function (Byrne et al., 2003; Smith and Hake, 2003; Bhatt et al., 2004). These works have shown that RPL (aka BELLRINGER, PENNYWISE and VAAMANA), as happens with other genes of the BELL family, interacts with several class I KNOX genes, particularly with BREVIPEDICELLUS (BP), SHOOT MERISTEMLESS (STM) and KNAT6, which suggests that these interactions could also be taking place in the replum. Really, this is what seems to occur, since the rpl bp double mutant exhibits a very strong replumless phenotype, showing a synergistic interaction between both mutations. Congruently, BP is expressed in medial tissues of the pistil that will give rise to the replum. However, loss-of-function mutations in BP do not produce any mutant phenotype in the replum (Alonso-Cantabrana et al., 2007; Ragni et al., 2008), probably because of the redundant activities of other class I KNOX genes also expressed in the presumptive replum, such as STM (Long et al., 1996; Ragni et al., 2008). Null stm mutants lack an apical meristem and, hence, do not form flowers, yet plants that carry an inducible RNA interference construct against STM transcripts show a range of pistil phenotypes in which severely affected flowers lack carpels, indicating that STM has an essential

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function in pistil initiation. Interestingly, in flowers with weaker phenotypes, carpels fail to fuse and exhibit strong reduction of marginal tissues, showing that the gene determines an important function for proper development of these tissues, including the replum (Scofield et al., 2007). Class I KNOX genes are known to be expressed in indeterminate cells of the meristem, being downregulated at positions of leaf initiation (Lincoln et al., 1994; Dockx et al., 1995; Long et al., 1996; Semiarti et al., 2001), in which loss of STM expression activates the transcription of the ASYMMETRIC LEAVES genes (AS1 and AS2), whose function is to promote the differentiation of leaf cells. AS1 and AS2 encode a myb transcription factor (Byrne et al., 2000; Sun et al., 2002) and a protein with the LATERAL ORGAN BOUNDARIES domain (Iwakawa et al., 2002; Shuai et al., 2002), respectively, which physically interact to maintain the repression of BP, KNAT2 and KNAT6 in leaves (Byrne et al., 2000; Ori et al., 2000; Semiarti et al., 2001; Xu et al., 2003; Guo et al., 2008). Thus, these three class I KNOX genes become misexpressed in leaves of as1 and as2 mutants. AS1 transcripts have also been detected in pistils (Byrne at al., 2000; Sun et al., 2002), with high expression levels in valves and lower levels in the replum (Alonso-Cantabrana et al., 2007), suggesting that the AS genes could also downregulate class I KNOX genes in the pistil. Certainly, as1 mutants express BP ectopically in valves, show higher expression levels of this gene in the presumptive replum, and exhibit the same subtle phenotype as loss-of-function mutants in AS2, consisting of larger repla and valves slightly smaller, which can be explained by an increased number of replum cells and a reduction of cell numbers in valves (Fig. 5.4). The likely cause of this silique phenotype is the overexpression of class I KNOX genes, since the gain-of-function 35S::BP construct generates the same fruit appearance (Alonso-Cantabrana et al., 2007). Furthermore, loss of AS1 function suppresses the effect of rpl alleles on replum development, which has been explained by increased transcription of BP in medial regions of as1 pistils (Alonso-Cantabrana et al., 2007).

5.6 A model for patterning the mediolateral axis of the Arabidopsis silique 5.6.1 A variation on the French flag model for pattern formation One of the crucial questions in development is how cells perceive and interpret positional clues to differentiate properly. A well-established mechanism for pattern formation involves the propagation of morphogens, small diffusible molecules that determine different patterns of gene expression in cells according to their concentrations in developing fields. The classical French flag model described by Lewis Wolpert (1968) shows a simple way to

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Fruit Development and Seed Dispersal represent the action of a morphogen to provide cells with positional information, which is interpreted in the model to rule the formation of three territories, that is, the three colours of the French flag: red, white and blue. Cells initially have the same potential to acquire any of the three fates but they will differentiate in response to a gradient of morphogen, with the highest concentration at one end of the developing field and the lowest at the other end. The concentration of morphogen in each specific position of the developing field provides positional information, so that a French flag pattern can be obtained whenever cells respond according to two thresholds. Then, above the highest morphogen concentration threshold cells adopt a blue fate, and below that and above the lowest threshold cells become white. Finally, below this second threshold cells are red (Fig. 5.6). Thresholds can represent either concentrations of a diffusible chemical, as retinoic acid or auxin, or activity of proteins (transcription factors, ligands, etc.). While two thresholds give rise to a French flag, the presence of a single threshold would generate a pattern with only two territories. This last example is more similar to the pattern generated by the JAG/FIL activity. Above a threshold of this activity cells would acquire a valve fate, and below the threshold cells that still perceive the activity would adopt a valve margin fate (Fig. 5.6). However, one of the aspects less understood in the contribution of morphogenetic gradients to pattern formation is how to establish the positions of thresholds with precision. An informative gradient can be further refined by the contribution of an antagonistic activity, which helps to produce more precise thresholds. Thus, considering again the example with only one threshold that generates two territories, we can add an opposing and antagonistic gradient of a second morphogenetic agent to form a third territory. At one end of the developing field there is a maximum of the first activity, whereas the second morphogenetic agent shows its highest activity at the other end. This creates a region of conflict in which both morphogens are present and each one tends to inhibit the other, resulting in low levels of the two activities that determine the precise positions of the thresholds and ensure that the middle territory is generated properly. Finally, a new French flag is created in which the size of the white (middle) territory is more accurately determined (Fig. 5.7). 5.6.2

Pistils as modified leaves

Pistils are composed of carpels that have evolved from leaves bearing spores (Scutt et al., 2006), in such a way that carpels are basically modified leaves (Friedman et al., 2004; Balanz´a et al., 2006). Thus, under the different morphologies and attributes that display both, carpels must have underlying leaf properties. The role in pistil development of genes such as AS1, AS2, FIL, YAB3 and JAG, all of them involved in producing normal leaves, gives evidence for this reasoning. In this way, AS genes, FIL and YAB3, which have important roles in valve development (Dinneny et al., 2005; Alonso-Cantabrana et al.,

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(a)

(b)

Figure 5.6 Specification of positional information by morphogens. (a) French flag model. Initially all the cells in the developing field are equivalent and have the same developmental potential, but two thresholds in the concentration of a morphogen determine the acquisition of distinct fates, which are denoted by the three colours of the French flag. (b) The JAG/FIL activity works like a morphogen with only one threshold concentration. Below the threshold, low levels of the JAG/FIL activity induce the expression of valve margin genes, and above the threshold, higher levels of the activity induce FUL expression. FUL determines the valve territory by repressing the expression of valve margin genes.

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Figure 5.7 Specification of positional information by two antagonistic activities. One of the morphogenetic agents shows its maximum activity at one end of the developing field, while the other agent displays its highest level of activity at the other end. This results in low activities of both agents in the region in which their expressions overlap and refine the positions of the two thresholds in the developing field to establish three territories. Threshold A and B indicate the thresholds of the morphogenetic activities A and B, respectively.

2007), prevent ectopic expression of class I KNOX genes in leaves (Byrne et al., 2000; Ori et al., 2000; Semiarti et al., 2001; Kumaran et al., 2002). In pistil tissues, the AS genes also downregulate class I KNOX genes, which have been suggested to play a crucial role in replum development (Alonso-Cantabrana et al., 2007). Really, replum and valves seem to parallel the antagonistic relationships between meristem and leaves. Accordingly, genes with essential functions in the meristem as STM, BP and RPL are expressed in the replum, whereas FIL, YAB3 and JAG are expressed in the valves and have crucial roles in leaf development. The phenotype of double mutants carrying lossof-function alleles in the CUP-SHAPED COTYLEDON genes CUC1 and CUC2 also supports this view. CUC1 and CUC2 are boundary-specifying genes that encode two related proteins of the NAC family of putative transcription factors (Aida et al., 1997; Takada et al., 2001; Duval et al., 2002). The function of these genes is essential to make the apical meristem, since the cuc1 cuc2 double mutant dies at the seedling stage because of its inability to form shoot meristem (Aida et al., 1997). Fortunately, the important roles of these genes in pistil development were revealed after regeneration of shoots from calli of the cuc1 cuc2 double mutant. The flowers generated from these shoots contained many pistils in which marginal tissues like replum and septum did not form

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properly (Ishida et al., 2000), showing that two genes required to generate the meristem also have an essential role in replum development. Nevertheless, although the replum displays meristem attributes and valves seem to be more related to leaf blades, there are necessary exceptions to these parallelisms. For instance, the AS1 gene is never expressed in the meristem, but its transcripts are detected at low levels in the replum where the AS1 product contributes to maintain BP expression below certain levels (AlonsoCantabrana et al., 2007), and KANADI1 (KAN1), an abaxial identity gene whose ectopic expression causes meristem arrest (Eshed et al., 2001), is also expressed in medial regions of the pistil (Kerstetter et al., 2001). In any case, these medial domains exhibit some kind of meristematic properties, probably necessary to produce the internal extensions that will give rise to placenta and septum. Interestingly, loss of YABBY function in fil yab3 leaves causes the production of ectopic meristems on the adaxial surface, near the leaf margin (Kumaran et al., 2002), and the placenta is precisely a marginal tissue that emerges on the adaxial side of pistils. Thus, one could envision the evolution of carpels from leaves related with, among other events, loss of YABBY activity at the margins that caused the acquisition of meristematic identities, and finally, after the fusion of carpels at their margins, marginal tissues as replum and placenta would have maintained these meristematic properties. 5.6.3 Antagonism between valves and replum In addition to the different set of genes that are expressed in valves and replum, several data also point out to the antagonism between these two pattern elements. Overexpressing class I KNOX genes in pistils, either in as mutants or in 35S::BP plants, increases the size of the replum and reduces the size of valves (Alonso-Cantabrana et al., 2007). Accordingly, it has been suggested that an unknown replum factor downregulates FUL expression (Liljegren et al., 2004). On the other hand, in 35S::FUL plants, the ectopic expression of FUL transforms the valve margin and the outer replum into valve tissue (Ferr´andiz et al., 2000b), at the same time as the expressions of BP and RPL vanish (J.J. Ripoll and M.F. Yanofsky, personal communication). Other genes expressed in valves as AS1, FIL and YAB3 also downregulate replum genes like class I KNOX genes (Byrne et al., 2000; Ori et al., 2000; Semiarti et al., 2001; Kumaran et al., 2002). Combining BP overexpression and loss of FUL function in the same background produces a synergistic fruit phenotype in which repla acquire very large sizes and valves are extremely reduced (Alonso-Cantabrana et al., 2007). Finally, the class I KNOX genes STM and KNAT6 interact positively in organ separation with the CUC genes (Aida et al., 1999; Takada et al., 2001; Belles-Boix et al., 2006), whose functions are also required for replum formation, whereas the AS genes and JAG cooperatively repress CUC1 and CUC2. This repression has been described in sepals and petals in which the AS genes and JAG determine the extent of the organs by downregulating boundary-specifying genes (Xu et al., 2008).

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Fruit Development and Seed Dispersal 5.6.4

The antagonistic activities of valve and replum factors pattern the mediolateral axis of the ovary

A recent model has been put forward with the purpose of clarifying how these antagonistic relationships are involved in patterning the fruit (AlonsoCantabrana et al., 2007; Fig. 5.8). The model considers that pattern elements along the mediolateral axis develop depending on the opposing activities of two antagonistic factors: valve factors represented by the previously defined JAG/FIL activity (Dinneny et al., 2005), and the activity of class I KNOX genes, named as replum factors (Alonso-Cantabrana et al., 2007). According to the model, replum and valves develop in territories with high activity of replum and valve factors, respectively, whereas the valve margin appears in a narrow stripe in which both factors are expressed. The JAG/FIL activity, as indicated above, is proposed to work through a gradient (Dinneny et al., 2005), so that high levels of the activity in lateral positions activate FUL, whose main function is to prevent ectopic expression of valve margin genes in valves (Ferr´andiz et al., 2000b), while only low levels are required to activate the SHP genes at the valve margin. Although not strictly necessary, class I KNOX genes also have been proposed to function by a gradient, with high expression levels in the presumptive replum and lower levels at the valve margin in which the JAG/FIL activity would downregulate these replum factors. Class

AS genes JAG

AS genes

FIL YAB3

FUL

Valve factors (JAG/FIL activity)

Valve

STM BP KNAT6 KNAT2

Valve margin genes

Valve margin

RPL

Replum factors (Class I KNOX genes)

Replum

Figure 5.8 A model for patterning the mediolateral axis of the ovary. The opposing gradients of two antagonistic factors determine the formation of the three pattern elements along the mediolateral axis. The FIL/JAG activity (valve factors) specifies valve formation, class I KNOX genes (replum factors) determine the replum and the valve margin develops in a narrow stripe in which valve and replum factors are expressed at low levels. Replum factors determine a complex activity with antagonistic interactions between class I KNOX genes. STM and BP are transcribed in the replum, where they and RPL repress KNAT6 and KNAT2, whose expressions are restricted to the valve margin. AS1 and AS2 repress class I KNOX genes, preventing the induction of these genes in valves and maintaining their expression below certain levels in the replum and valve margin. Dashed lines denote hypothetical interactions.

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I KNOX genes positively regulate the expression of RPL (Alonso-Cantabrana et al., 2007), whose function is to prevent the ectopic expression of valve margin genes in the replum by downregulating the JAG/FIL activity (Roeder et al., 2003; Dinneny et al., 2005), and indeed, both class I KNOX genes and RPL probably collaborate in this function, since the overexpression of BP strongly reduces the expression of FIL (J.J. Ripoll and M.F. Yanofsky, personal communication). In the model, AS genes prevent the ectopic expression of class I KNOX genes in the valves and maintain these genes below certain levels in the replum (Alonso-Cantabrana et al., 2007). Thus, by a simple shift of the valve margins towards more lateral positions, because of an increase in the activity of replum factors, this model explains why the overexpression of BP produces larger repla and smaller valves. Here, the magnitude of the shift depends on the position in which the antagonistic valve factors counteract the increase in the activity of replum factors. Besides, provided that FIL is not expressed in the presumptive replum, a similar explanation accounts for the large replum of fil mutants, since a fall in the JAG/FIL activity would cause the expansion of the expression domain of replum factors and a consequent shift in the position of valve margins. There are conceptually similar models whereby two gradients of antagonistic factors determine positional information and pattern formation. For instance, during vertebrate development, two antagonistic gradients of retinoic acid and fibroblast growth factors provide positional information along both the proximo-distal axis of limbs and the main axis (Mercader et al., 2000; Diez del Corral et al., 2003). 5.6.5 Replum factors display a complex activity with two components Class I KNOX genes have been thought to function with partial redundancy (Byrne et al., 2002). However, a recent work shows a series of antagonistic interactions between members of this family of genes (Ragni et al., 2008). Thus, the altered inflorescence phenotype of bp is partially suppressed by loss of the KNAT6 function, and further rescued with the homozygosis of knat2 null alleles in bp knat6 knat2 plants, while the inflorescence pny (rpl) phenotype is completely eliminated in rpl knat6 plants. Congruently, the expression domains of KNAT2 and KNAT6 are enlarged in bp, rpl and bp rpl mutants, indicating that BP and RPL cooperate to restrict the expression of KNAT2 and KNAT6. Hence, the mutant inflorescence phenotypes of bp and rpl mainly result from ectopic KNAT6 expression and to a lesser extent from KNAT2 misexpression. Interestingly, this also comes true for the replum phenotype of rpl and rpl bp mutants, since the double rpl knat6 and the triple rpl bp knat6 exhibit completely normal repla, showing that the mutant replum phenotype requires KNAT6 activity. Ragni and coworkers have also determined the expression patterns of class I KNOX genes in fruits. While STM and BP are effectively expressed in the replum, KNAT2 and KNAT6 expressions are restricted to the valve margin and are ectopically detected in medial

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Fruit Development and Seed Dispersal regions of rpl and rpl bp fruits. Therefore, class I KNOX genes (replum factors) represent a complex activity with at least two components, STM and BP on the one hand, perhaps determining replum identity, and KNAT2 and KNAT6 in the valve margin on the other (Fig. 5.8). Although, neither of the single mutants nor the double knat2 knat6 produce indehiscent fruits, suppression of the replumless phenotype by knat6 suggests a role for the gene in valve margin development.

5.7 5.7.1

Auxin: a signalling molecule for the mediolateral axis? AS1 and auxin cooperate to repress BP in leaves

Our knowledge on the genetic network that determines the development of different pattern elements along the mediolateral axis of pistils and fruits has increased notably during the last decade (Ferr´andiz, 2002; Dinneny and Yanofsky, 2005; Robles and Pelaz, 2005; Roeder and Yanofsky, 2006). However, nothing is known about the nature of the signalling mechanisms that activate replum and valve factors in their precise domains. Fortunately, we can take advantage of the underlying foliar nature of carpels and figure out what signalling molecules might be at work in the pistil. Dynamic gradients of auxin that determine local maxima appear as an essential step to produce leaves and, in more general terms, to generate all the plant organs (Benkov´a et al., 2003). Thus, leaves develop from founder cell populations at the flanks of the apical meristem, according to positions specified by local auxin maxima (Reinhardt et al., 2000, 2003; Kuhlemeier and Reinhardt, 2001; Heisler et al., 2005). These peak levels are produced by polar transport of auxin, a process in which PINFORMED1 (PIN1), a gene that codes for an efflux auxin carrier protein, plays an outstanding role (G¨alweiler et al., 1998; Paponov et al., 2005). Interestingly, auxin seems to be involved in the downregulation of class I KNOX genes in places of leaf inception, as well as in later stages of leaf development (Scanlon, 2003; Hay et al., 2006). Loss-of-function mutants in AXR1 are affected in the response to auxin (Leyser et al., 1993; del Pozo et al., 2002), and show ectopic expression of BP in leaves (Hay et al., 2006). The as1 axr1 double mutant exhibits both an enhanced leaf phenotype and higher levels of BP expression in leaves as compared with either of the two single mutants, indicating that auxin and AS1 cooperate to exclude BP expression from leaves. Auxin transport is also involved in this repression of BP, since pin1 mutants and plants treated with transport inhibitors display ectopic leaf expression of the gene. In addition, bp and rpl alleles partially rescue the failure of pin1 mutants to produce leaves and flowers, showing that this phenotype is mediated, at least in part, by the activities of BP and RPL and that these activities antagonize the role of PIN1 in promoting leaf initiation. In fact, this antagonistic relationship is

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further supported by the repression of PIN1 in the distal lamina of leaves from 35S::BP pants (Hay et al., 2006). Since these processes seem to operate also in other plant organs, it is tempting to imagine a similar antagonistic interaction of AS1 and auxin in valves, on the one hand, versus class I KNOX genes and RPL in the replum, on the other. 5.7.2 Auxin as a patterning molecule in pistils Several mutants in genes that pattern the mediolateral axis of pistils do not show a completely homogeneous phenotype but they display regional differences. The shp1 shp2 double mutant exhibits a reduction in valve margin lignification, which is more evident at the base of the fruit, whereas there are lignified valve margin cells at the apex. Accordingly, these fruits occasionally open at the apical margin (Liljegren et al., 2000). The expression of YJ80, an enhancer trap that marks the valve margin, is lost at the basal region of shp1 shp2 and alc fruits, but is still detected at the apical region of these siliques (Liljegren et al., 2004). Fruits of fil yab3 also show regional differences, since they lack valve margin at the apex, with a consequent loss of SHP expression, whereas they display ectopic transcription of these genes at the base along with formation of valve margin tissue in the valve territory (Dinneny et al., 2005). These regional phenotypic heterogeneities suggest that there may be an interplay between the mediolateral axis and the apical–basal axis, possibly with the presence of common elements involved in patterning both axes. In this respect, it has been proposed that a gradient of auxin established during pistil development rules the formation of pattern elements along the apical–basal axis (Nemhauser et al., 2000). According to the model, peak levels of auxin at the apex give rise to stigma and style, whereas the lowest levels at the base produce the formation of the gynophore. Moderate levels in intermediate positions promote ovary development. Hence, auxin would behave as a morphogen involved in patterning the apical–basal axis of pistils and might well be a common element shared with the mediolateral axis. The ETTIN (ETT) gene has been proposed to mediate the intermediate levels of auxin that determine ovary development (Nemhauser et al., 2000), a role carried out through the negative regulation of SPATULA (SPT), a bHLHencoding gene implicated in the development of stigma and style, as well as other marginal tissues (Alvarez and Smyth, 1999; Heisler et al., 2001). ETT codes for ARF3, a member of the auxin response factor (ARF) family of transcriptional regulators (Sessions et al., 1997), so its protein product is a good candidate to interpret the auxin gradient in terms of gene regulation. The expression of ETT becomes restricted to the abaxial domain of leaves by the adaxially expressed ta-siRNA gene TAS3, which targets the ETT mRNA for degradation (Garcia et al., 2006). In agreement with the expression pattern, ETT and its paralogous gene ARF4 specify abaxial identity to the tissues in which they are expressed, linking auxin gradients and polarity along the adaxial–abaxial axis (Pekker et al., 2005). The same as ETT, FIL also determines

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Fruit Development and Seed Dispersal abaxial cell fate (Sawa et al., 1999; Eshed et al., 2004), whereas the AS genes specify adaxial identity and repress FIL expression (Lin et al., 2003; Xu et al., 2003; Iwakawa et al., 2007). In fact, ETT positively regulates FIL, whose expression is excluded from the adaxial domain by the action of the AS genes and the indirect regulation of TAS3 through the repression of ETT (Garcia et al., 2006). Thus, a hypothetical auxin signalling along the mediolateral axis of developing pistils might also be interpreted by ETT, which is also expressed abaxially in the ovary where it could be responsible to activate FIL transcription in valves, whereas the antagonistic activities of class I KNOX transcription factors and RPL would prevent the activation of this abaxial gene in the replum. Interestingly, although gradients of auxin have not yet been detected in the developing pistil, several members of the YUCCA gene family, involved in auxin biosynthesis, are expressed at the apex of pistils, and YUC2 is additionally expressed in lateral domains of the ovary (Cheng et al., 2006), so that the biosynthesis in these regions could establish auxin gradients along both the apical–basal and the mediolateral axes of pistils. 5.7.3

Auxin inhibits dehiscence

Once the fertilization of ovules has occurred, the tissues of the pistil have to respond to the signals released from seeds to generate the silique. The biochemical nature of these signals is complex and must involve several plant hormones (Vivian-Smith and Koltunow, 1999), including auxin (Vivian-Smith et al., 2001; Goetz et al., 2006). Experimental work in Brassica napus pods has shown that fruit shattering requires the maintenance of low levels of auxin in the dehiscence zone, since an increase in cellulase activity correlates with a decrease in auxin content in this territory, and treatments of siliques with auxin give rise to retardation of both the cellulase activity and dehiscence, indicating that auxin exerts a negative regulation on shattering (Chauvaux et al., 1997). In Arabidopsis, SPT might indicate the existence of a connection between dehiscence zone development and low levels of auxin. Although spt mutants do not show a role of the gene in dehiscence, SPT exhibits an intriguing expression at the valve margin and codes for a bHLH transcription factor with close homology to ALC (Heisler et al., 2001; Rajani and Sundaresan, 2001). Thus, it could play a redundant role with ALC and IND in the development of the dehiscence zone. Interestingly, several clues point towards a connection between auxin and SPT, as this gene is negatively regulated by ETT (Heisler et al., 2001) and the spt mutant phenotype is suppressed by inhibitors of the auxin polar transport (Nemhauser et al., 2000). This has led to propose that the role of SPT in patterning the apical axis of the pistil is carried out by inhibition of the polar auxin transport (Dinneny and Yanofsky, 2005), so that the SPT protein would cause auxin to accumulate at the apex to generate the development of stigma and style tissues. Following a similar function, bHLH proteins in the valve margin could inhibit the polar transport of auxin causing its depletion in the dehiscence zone. Indeed, a minimum

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of auxin response has been detected in valve margin cells of fully elongated siliques, coinciding with the formation of the separation layer and the thickening of cell walls in the enb layer, whereas ectopic synthesis of auxin in the valve margin completely abolishes the formation of the dehiscence zone. In fact, IND appears to be implicated in creating low auxin levels at the valve margin, possibly by inhibition of the polar auxin transport, since ind mutants do not show the auxin response minimum at the valve margin and IND ectopic expression drastically distorts cellular localization of the auxin efflux carrier PIN1. Therefore, a possible scenario for fruit development could involve the function of valve margin genes to generate low auxin levels at the dehiscence zone, the repression of these genes by FUL perhaps to produce high levels of auxin in valves, and the mutually antagonistic relationship between auxin and class I KNOX genes to develop replum.

5.8 A biotechnological view The work with model organisms is a powerful tool to gain a better understanding of biological phenomena, and as a plant model, Arabidopsis has allowed us to get new insights on how a dehiscent fruit is built. Nevertheless, the ultimate challenge is to translate our knowledge on the genetic networks operating in fruit dehiscence from Arabidopsis to crop species. Dehiscence is an important agronomical trait and great efforts are dedicated to optimize harvest yield, as seen in oilseed crop plants, in which considerable losses are due to early pod shattering. This is the case for canola and other Brassicaceae, which must share many gene regulatory networks with Arabidopsis and, therefore, should be amenable to genetic manipulation in order to get control of seedpod shatter. Really, shattering is a rather general problem, and also includes losses for seed shattering in cereals. Wild relatives of rice show the ‘easy-to-shatter’ trait, which results in low yield. Thus, loss of seed-shattering habit is possibly one of the most significant events in rice domestication by humans. In Arabidopsis, SEEDSTICK (STK), a gene closely related to the SHP genes and AG, is essential for seed abscission (Pinyopich et al., 2003). However, loss of seed shattering in cultivars of rice is due to a mutation that affects the expression in the seed abscission layer of a gene with homology to RPL. Hence, although the dehiscence zone in the Arabidopsis silique and the abscission layer in rice have different botanical origins, mutations in the same gene produce loss of shattering in both species (Konishi et al., 2006). Many Arabidopsis lines show indehiscent phenotypes as a result of genetic alterations that we should be able to apply in crops with dehiscent fruits, since the genetic pathway involved in valve margin specification is conserved between Arabidopsis and Brassica species (Østergaard et al., 2006). B. napus has two orthologues of IND, which are able to complement ind alleles in Arabidopsis, indicating that their protein products perform the same biochemical function as IND, so that several strategies have been proposed to use both

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Fruit Development and Seed Dispersal genes to control dehiscence in this crop (Yanofsky and Kempin, 2006). Ectopic expression of FUL in Arabidopsis causes the transformation of valve margin and replum into valve tissues, producing indehiscent fruits (Ferr´andiz et al., 2000b). This approach has been used in Brassica juncea in which the ectopic expression of the Arabidopsis FUL gene gives rise to non-shattering fruits (Østergaard et al., 2006). However, eliminating IND function or overexpressing FUL prevents the formation of the dehiscence zone and completely blocks dehiscence, seriously hindering the processes to harvest seeds. A way to solve this drawback is using ALC as a target for gene manipulation, since loss of ALC activity only affects cells of the separation layer in such a way that alc siliques can be easily opened. This strategy has been used to obtain indehiscent Arabidopsis plants with reduced ALC function through overexpression of either an antisense construct or a dominant-negative form (Rajani and Sundaresan, 2001). An Arabidopsis mutant with indehiscent phenotype has been obtained by upregulation of the GARGOYLE (GGL) gene through activation tagging. GGL is allelic to JAG and same as for the dominant jag-5D allele, the ggl mutant shows ectopic lignification in the replum resulting in the complete encircling of the silique with lignified cells and a significant reduction in silique shattering (Dinneny et al., 2005; Aharoni and Pereira, 2006). A promising strategy to obtain fruits with defective dehiscence in B. napus is to overproduce the JAG/GGL protein in the replum with specific promoters (Aharoni and Pereira, 2006). In conclusion, following these approaches, or others like the study of quantitative trait loci (QTL) affecting dehiscence in crops, we can imagine a promising future in which it will be possible to prevent losses by premature opening of fruits and, therefore, improve harvest yield.

Acknowledgements We would like to express our gratitude for the useful comments provided by Juan Jos´e Ripoll and Santiago Gonz´alez-Reig. Our work on fruit development is generously supported by research grant BIO2006–04502 from the Spanish ´ Ministerio de Ciencia e Innovacion.

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Annual Plant Reviews (2009) 38, 204–237 doi: 10.1002/9781444314557.ch6

www.interscience.wiley.com

Chapter 6

LONG-DISTANCE SEED DISPERSAL Frank M. Schurr,1,2 Orr Spiegel,3 Ofer Steinitz,3 Ana Trakhtenbrot,3 Asaf Tsoar3 and Ran Nathan3 1

Plant Ecology and Conservation Biology, Institute of Biochemistry and Biology, University of Potsdam, Maulbeerallee 2, 14469 Potsdam Germany 2 Equipe G´en´etique et Environnement, Institut des Sciences de l’Evolution de Montpellier, UMR-CNRS 5554, Universit´e Montpellier II, 34095 Montpellier cedex 5 France 3 The Movement Ecology Laboratory, Department of Evolution, Systematics and Ecology, Alexander Silberman Institute for Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem 91904, Israel

Abstract: Most seeds of most plant species are dispersed over distances shorter than a few dozen metres, and only very few seeds travel over long distances. While the long-distance dispersal (LDD) of seeds is, thus, typically rare, it has disproportionately large effects on the long-term and large-scale dynamics of plants. Here, we first highlight the importance of LDD for various aspects of plant biology, discuss problems with quantifying LDD, and advocate a new vector-based framework for LDD research that may help to overcome some of these problems. We then present six generalizations about LDD mechanisms that can be derived using this framework. While the framework and the generalizations are also highlighted in Nathan, R., Schurr, F.M., Spiegel, O., et al. (2008) Mechanisms of long-distance seed dispersal. Trends in Ecology and Evolution 23, 638–647, this chapter provides a more in-depth derivation of the framework and additional evidence for the generalizations. In particular, we present here a new meta-analysis validating an innovative model for the allometry of seed dispersal by animals. In the second part of the chapter, we extend Nathan et al.’s (2008) discussion of the implications of the framework and generalizations for understanding LDD evolution and forecasting large-scale dynamics of plants. In particular, we use the vector-based framework to address two fundamental questions about LDD: can we identify all relevant LDD vectors and can plant traits influence LDD? We conclude by suggesting directions for future research on LDD. Keywords: body size; dispersal vectors; extreme events; functional traits; humanmediated dispersal; mechanistic model for seed dispersal by animals

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6.1 Introduction 6.1.1 The importance of long-distance seed dispersal Because most (terrestrial) plants are mobile only as seeds, the spatial dynamics of plants is largely driven by seed dispersal (Nathan and Muller-Landau, 2000). Short-distance seed dispersal shapes the local dynamics of plant populations and communities and provides a template for subsequent local processes such as predation, competition and mating (Nathan and MullerLandau, 2000). The long-distance dispersal (LDD) of seeds, on the contrary, is of primary importance for the large-scale dynamics of plant species and communities. For a plant species to persist in a fragmented landscape, local extinction from occupied habitat fragments has to be balanced by the colonization of unoccupied fragments, which requires LDD to these fragments (e.g. Higgins and Cain, 2002; Levin et al., 2003). LDD is also critical for the rates at which transgenes spread in plants that are perennial and/or form feral populations (Williams and Davis, 2005; Kuparinen and Schurr, 2007), for the velocity at which plant species can migrate in response to climate change (Clark et al., 1998; Higgins et al., 2003a) and for the speed at which invasive plants expand their range (Hastings et al., 2005). Because of its importance for range expansion, LDD is an important determinant of biogeographical and macroevolutionary dynamics, a fact that was already recognized by ˜ Darwin (1859) and has recently received renewed attention (Munoz et al., 2004; de Queiroz, 2005; Alsos et al., 2007). Moreover, LDD is not only important for the dynamics of single species but also shapes community structure by determining migration rates between local communities and the regional species pool (or metacommunity) (Hubbell, 2001; Levine and Murrell, 2003). LDD is thus crucial both for fundamental ecological, biogeographical and evolutionary processes, as well as for applied questions of biodiversity conservation under environmental change (Levin et al., 2003; Levine and Murrell, 2003; Trakhtenbrot et al., 2005; Nathan, 2006). An overview of the key terms relevant for the study of LDD is given in Table 6.1.

6.1.2 Defining long-distance seed dispersal A review of LDD research is complicated by the fact that both absolute and proportional definitions of LDD are used in the literature (Cain et al., 2000; Nathan, 2005; Fig. 6.1). The absolute definition identifies LDD events as those dispersal events that are longer than a specified threshold distance (e.g. 1 km). The alternative to this absolute definition is a proportional definition, which defines LDD events as those that exceed a certain high quantile of dispersal distance (e.g. the 99% quantile as the distance exceeded by only 1% of all seeds). It is important to note that the proportional definition actually identifies extreme dispersal events rather than LDD events: in a species

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Fruit Development and Seed Dispersal Table 6.1 An overview of the key terms used in this chapter (modified from Nathan et al., 2008) Term

Definition

Dispersal kernel

A probability density function characterizing the spatial distribution of seeds dispersed from a common source. The ‘dispersal distance kernel’ more precisely describes the distribution of seed dispersal distances (and hence the distribution of the product of displacement velocity, V, and seed passage time, P).

Dispersal vector

An agent transporting seeds or other dispersal units. Dispersal vectors can be biotic (e.g. birds) or abiotic (e.g. wind).

Vector displacement velocity (V)

The speed at which a dispersal vector moves a seed away from the uptake location (Fig. 6.3).

Fat-tailed dispersal kernel

A dispersal kernel with a tail that drops off more slowly than that of any negative exponential kernel. Seed dispersal by a single dispersal vector.

Haplochory Irregular dispersal vector

A dispersal vector acting in such a haphazard way that it cannot be predicted whether the vector will be involved in the seed dispersal of a given plant species.

Non-standard dispersal vector

A dispersal vector different from the one inferred from traditional morphological classifications (Higgins et al., 2003b).

Polychory

Seed dispersal by multiple dispersal vectors.

Regular dispersal vector

A dispersal vector that is regularly (but potentially rarely) involved in the seed dispersal of a given plant species.

Seed passage time (P)

The time for which a seed is retained by a dispersal vector (Fig. 6.3).

Seed

In a strict sense, the fertilized ovule of spermatophytes consisting of embryo, endosperm and testa. We follow here the typical use of this term in the plant ecological literature as a synonym for a reproductive propagule.

Seed dispersal

The movement of seeds from the mother plant to a potential establishment site.

Vector seed load (Q)

The number of seeds dispersed by a particular dispersal vector (Fig. 6.3).

Standard dispersal vector

A dispersal vector inferred from the phenotypic characters of the plant (e.g. the morphology of the dispersal unit) (Higgins et al., 2003b). Typically, this is the vector dispersing most seeds.

Total dispersal kernel (TDK)

The dispersal kernel generated by all vectors dispersing a certain plant species (Nathan, 2007).

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(b) log probability density

(a) log probability density

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log distance (x)

Figure 6.1 Application of absolute (black solid arrows) and proportional (grey hatched arrows) definitions of long-distance dispersal (LDD) of seed to two different dispersal distance kernels. Note that the proportional definition identifies extreme dispersal events rather than LDD events. Hence, in a species with highly restricted seed dispersal (a), the proportional definition will include dispersal distances of only a few metres that typically are not considered as ‘long’. On the other hand, in a species whose seeds are frequently dispersed over long distances (b), only the most extreme dispersal events meet the proportional definition, although somewhat less extreme distances might be long enough to influence large-scale dynamics.

with highly restricted seed dispersal, the 99% quantile of dispersal distance might be located at only a few metres, a distance that typically will not be considered as ‘long’ (Fig. 6.1a). On the other hand, in a species whose seeds are frequently dispersed over long distances, only the most extreme dispersal events will meet the proportional definition, even though somewhat less extreme dispersal distances might also be long enough to affect the largescale dynamics of this species (Fig. 6.1b). For studies interested in LDD rather than extreme dispersal, we thus recommend applying the absolute definition. The threshold distance for the absolute definition should be chosen based on the question studied: when studying the dynamics of a plant species in a fragmented landscape, such an LDD threshold could be the typical distance between neighbouring habitat fragments. For certain questions (e.g. to predict the spread of populations, Clark et al., 2001), it can be important not only to quantify the proportion of dispersal events exceeding this threshold, but also to report the frequency distribution of dispersal distances above the threshold. Note that it is impossible to derive a single all-encompassing absolute threshold for LDD, since the LDD threshold depends on the specific objectives of a study and may range from 102 m (for landscape-scale studies) to 107 m (for global-scale biogeographical studies). 6.1.3 The challenge of studying long-distance seed dispersal The importance of LDD for the large-scale dynamics of plants has long been recognized (e.g. Darwin, 1859). Nevertheless, LDD research has long been based on the compilation of anecdotal observations and has lacked a

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Fruit Development and Seed Dispersal rigorous theoretical background. LDD has also rarely been examined experimentally, and there are very few attempts at quantitatively synthesizing observational data on LDD. This paucity in quantitative LDD research can be explained through the concurrence of several circumstances. First, direct empirical observations of LDD are difficult, if not impossible, due to the severe methodological problems encountered both in tracking seeds during LDD and in finding those seeds that have been long-distance dispersed (Cain et al., 2000; Nathan et al., 2003). Second, even if LDD events can be observed, the fact that these events are typically rare, severely limits our ability to empirically estimate their probability of occurrence (and other properties). Third, two different approaches are commonly used to define LDD (see Section 6.1.2), meaning that there is no general currency to compare different studies. Fourth, the ‘seed-centred’ way in which seed dispersal has been studied traditionally makes it very difficult to detect and quantify LDD mechanisms. In our opinion, a detailed examination of this fourth point is the basis for the formulation of a new quantitative ‘vector-centred’ framework for LDD research. In the following section, we thus describe the traditional approaches to dispersal research and examine why these approaches fail for LDD. Box 6.1

Polychory and the study of long-distance seed dispersal

The fact that most plant species are not dispersed by a single vector (haplochory) but by multiple vectors (polychory) has profound consequences for the study of LDD. To illustrate this, we consider the hypothetical case of a plant species producing a large number of seeds (108 ) that are dispersed by four different dispersal vectors (Fig. 6.2a). The four vectors are arranged in order of decreasing seed load Q and increasing fat-tailedness of the dispersal kernel they produce (Fig. 6.2a). For a plant species with elaiosome-bearing seeds, these four vectors could for example be (1) ants (the standard dispersal vector), (2) large animals, (3) strong floods and (4) transportation by humans. Vector 1 transports the vast majority of seeds (95%), but its thin-tailed dispersal kernel causes it to generate only short dispersal distances (Fig. 6.2a). Vectors 2 through 4 generate increasingly larger maximum dispersal distances, although they disperse successively fewer seeds (Fig. 6.2a). In fact, vector 4 disperses only a tiny fraction (0.0125%) of all seeds, but the maximum dispersal distance it generates is more than five orders of magnitude greater than that generated by the standard vector (vector 1). When plotting the relative contribution of each vector to the total dispersal kernel (TDK), it becomes apparent that different vectors dominate seed dispersal over different scales (Fig. 6.2b). In particular, the vectors dominating seed dispersal at the scales typically investigated in empirical studies are very different from the vectors dominating LDD. This hypothetical example illustrates the limited ability of small-scale studies to detect LDD vectors, and the fallacy of extrapolating from the small-scale behaviour of the standard vector to LDD. The traditional seed-centred approach will almost inevitably miss the rare seeds dispersed by non-standard LDD vectors. We thus advocate a vector-centred approach that specifically targets those vectors that can transport seeds at the scales of interest.

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6.1.4 The traditional framework of seed dispersal research The traditional framework of seed dispersal research can be described as ‘seed centred’: it focuses on a set of seeds (e.g. all seeds produced by a mother plant) and asks by which mechanisms and over which distances these seeds

Seed load × dispersal kernel Q × F(x)

(a) 10

Q 108

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6 4 Vector 1 Vector 2 Vector 3 Vector 4

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(b) Typical scale of empirical studies

Contribution of vector TDK (%)

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100 80 60 40 20 0 10

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2

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Figure 6.2 (a) The total dispersal kernel (TDK, thick black line) for a hypothetical plant population dispersed by four vectors (different orange tints). The inset shows the unequal distribution of seed loads (Q) on a log scale. The lines at the bottom indicate the range of distances of all seeds dispersed by each vector. (Figure modified from Nathan et al., 2008.) (b) The relative contribution of each vector to the TDK as a function of spatial scale x, showing that the vectors dominating long-distance dispersal of seed can be very different from those dominating seed dispersal at the typical scales of empirical studies. (For a colour version of this figure, please see Plate 1 of the colour plate section.)

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Fruit Development and Seed Dispersal are dispersed. This seed-centred framework typically assumes that a plant species has a single ‘standard’ dispersal vector which disperses most seeds and whose identity can be inferred from specific morphological attributes of the dispersal unit (e.g. the fact that seeds are equipped with an elaiosome suggests that ants are their standard dispersal vector) (Van Der Pijl, 1982; Higgins et al., 2003b). According to this framework, the LDD potential of a plant species can be predicted from its standard dispersal vector which in turn can be predicted from seed and plant morphology. Consequently, plant species with fleshy fruits are classified as being ‘bird dispersed’ and having high LDD potential, whereas species with explosive discharge of seeds are classified as having ‘ballistic dispersal’ and no LDD potential (Ridley, 1930; Van Der Pijl, 1982; Berg, 1983). While the seed-centred framework with its focus on standard dispersal vectors might produce reliable results for short-distance dispersal (which depends on the majority of dispersal events), it frequently fails for LDD (Box 6.1; Fig. 6.2). The case of the woodland herb Asarum canadense provides a good example for such failure (Cain et al., 1998). A. canadense has been classified as ant dispersed because its seeds bear elaiosomes. However, empirically parameterized models for range expansion by ant dispersal suggest that since the last glacial maximum A. canadense should only have spread by 10–11 km, whereas the species actually covered hundreds of kilometres during this time (ca. 16 000 years). This suggests that occasional LDD by vectors other than ants dominated the postglacial expansion of this species. The application of the seed-centred framework to LDD is thus problematic because the vectors that transport seeds over long distances are typically not those which disperse the majority of seeds (Higgins et al., 2003b; Box 6.1). Consequently, a research approach focusing on the majority of seeds is likely to miss those seeds that are long-distance dispersed.

6.1.5

A vector-centred framework for research on long-distance seed dispersal

Recent years have seen a shift from the traditional seed-centred framework to a vector-centred framework for LDD research (e.g. Nathan et al., 2002; ˜ et al., 2004; Levey et al., 2005; Jordano et al., 2007). This vector-centred Munoz framework focuses on a dispersal vector capable of generating LDD and asks how many seeds this vector disperses over which distance. This permits to concentrate research efforts on those processes that cause LDD rather than having to study a large number of seeds of which at best a few will be longdistance dispersed. The vector-centred approach is by no means new: as many other branches of ecology, this field was pioneered by Charles Darwin who conducted quantitative experiments on seed dispersal by ocean currents (Darwin, 1859). Berg (1983) pointed out two important elements of the vector-based

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framework: the fact that most plant species are polychorous (dispersed by multiple vectors) and that generalist non-standard vectors can disperse many plant species (see Nathan et al., 2008 for details). Recent studies adopting a vector-based approach found that many plant species have multiple LDD vectors (Ozinga et al., 2004), that generalist vectors such as white-tailed deer (Odocoileus virginianus) can cause LDD of plants with a wide variety of dispersal morphologies (Myers et al., 2004), and that LDD can result from unusual behaviour of standard dispersal vectors (e.g. strong turbulent updrafts can cause long-distance transport of wind-dispersed seeds, Nathan et al., 2002; Kuparinen et al., 2007). In summary, the evidence available from vector-based studies shows that the LDD is largely independent from morphological dispersal syndromes and the associated standard dispersal vectors and mechanisms (Higgins et al., 2003b; Box 6.1; Fig. 6.2). To identify those vectors and mechanisms that are capable of generating LDD, we here use a general model for passive dispersal (Fig. 6.3; Nathan

(a)

(b)

Figure 6.3 (a) A general mechanistic model for passive dispersal (adapted from a general model for aerial transport, Isard and Gage, 2001) that describes three phases of passive dispersal (boxes) through three key parameters (given below the boxes). (The figure is modified from Nathan et al. (2008).) (b) Illustration of the general model through the comparison of two seed lots (circles) that are dispersed by two different vectors (A and B). In this example, vector A has higher seed load Q, but lower displacement velocity V and shorter passage time P than vector B. Hence, vector B produces a longer dispersal distance (which arises as the product of V and P).

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Figure 6.4 Examples for the three general phases of passive dispersal (see Fig. 6.3). (a) Initiation phase: seeds of sugarcane (Saccharum spp.) depart from their mother plants in a strong wind (© Ran Nathan). (b) Transport phase: a floating island composed of papyrus (Cyperus papyrus L.) transports seeds, entire plants and other organisms across Lake Malawi (Oliver and McKaye, 1982, © Kenneth McKaye). (c) Termination phase: after secondary wind dispersal over a burnt area in the South African fynbos, a seed of the common sugarbush (Protea repens, L.) is trapped in burnt vegetation remains (© Frank Schurr).

et al., 2008). This model (adapted from Isard and Gage, 2001) describes three main phases of vector-mediated dispersal (Figs. 6.4a–c): in the initiation phase, seeds are removed from the mother plant by a dispersal vector; in the transport phase, the vector transports the removed seeds away from the source; and in the termination phase, these seeds are deposited. This chain can be repeated and may involve different vectors in the transport of a single seed (Vander Wall and Longland, 2004). Each of the three phases is characterized by a single key parameter and the interplay of these parameters determines the extent of LDD. The initiation phase is characterized by the vector seed load (Q), the number of seeds taken by a dispersal vector in a certain time period. This number Q is a function of plant fecundity, of the plant’s fruiting schedule in relation to the behaviour of the vector and of the vector’s loading ability. The transport phase is characterized by the vector displacement velocity (V) after seed uptake, which depends primarily on the movement properties of the vector, such as its travel velocity, directionality

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and intermittence. Finally, the termination phase is characterized by the seed passage time (P), the duration of seed transport by the vector, a parameter that is affected by seed traits, vector traits and their interaction. All three key parameters also depend on external factors such as landscape structure and climatic conditions. The distributions of displacement velocity (V) and seed passage time (P) together define the shape of the dispersal distance kernel, whereas seed load (Q) defines how many seeds the vector disperses. General considerations (Box 6.2; Fig. 6.5) show that important LDD vectors are those which generate a relatively high per-seed probability of LDD, because retention time (P) and/or displacement velocity (V) are at least occasionally high. Such vectors can be important for LDD even if they have a small seed load (Q).

Box 6.2

How to identify LDD vectors

We can use the general model for passive dispersal shown in Fig. 6.3 to identify vectors that are likely to cause LDD of a given plant species (see also Nathan et al., 2008). To illustrate this, we first consider the dispersal of a single seed. A single seed’s dispersal distance is the product of its retention time at the vector (P) and the vector’s displacement velocity during this time (V). For a set of seeds dispersed by the same vector, V and P are two random variates that follow statistical distributions (Fig. 6.5a). The dispersal distance as the product of these two random variates again follows a statistical distribution which is called the vector’s dispersal distance kernel (Fig. 6.5b). The area under the dispersal distance kernel that lies to the right of the LDD threshold gives the per-seed probability of LDD. Formally, this per-seed LDD probability is
∞ P (LDD) =

f (x) dx = 1 − F (xLDD ) xLDD

where x LDD is the LDD threshold, f is the probability density function and F the cumulative density function of dispersal distance. The per-seed LDD probability thus depends on the dispersal distance kernel which in turn depends on the distributions of V and P. The conditions under which a vector generates a high per-seed probability of LDD fall into two broad categories: in the rarer case, the median dispersal distance is close to or above the LDD threshold so that dispersal events frequently exceed the LDD threshold (hatched grey line in Fig. 6.5b). This arises if the product of typical values for V and P is high (hatched grey line in Fig. 6.5a). An example for this rarer case might be provided by large animals for which both typical displacement velocities and typical times of endozoochorous seed retention are high (see Box 6.3). In the commoner case, the LDD threshold is far greater than the typical dispersal distances, and a high per-seed LDD probability can arise only if the dispersal kernel has a fat tail (Fig. 6.5b). A dispersal kernel is called fat-tailed if its tail drops off more slowly than the tail of a negative exponential kernel (Table 6.1). In a plot of log probability density against dispersal distance (Fig. 6.5b), a fat tail curves away from the x-axis, whereas a thin (or exponentially bounded) tail bends towards the x-axis. How does the fat-tailedness of the dispersal kernel depend on the distributions of V and P? To examine this, we conduct a ‘crossing experiment’ with the two typical

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Fruit Development and Seed Dispersal Box 6.2

(Continued)

distributions of V and P plotted in Fig. 6.5a: the solid black (lognormal) distribution has a lower mean than the solid grey (truncated normal) distribution, but it is leptokurtic and therefore has a higher probability of generating high values. The dispersal kernel as the product of V and P is fat-tailed when one or both of V and P follow the leptokurtic distribution (Fig. 6.5b). In contrast, if both V and P conform to the mesokurtic solid grey distribution, the resulting dispersal kernel has a thin tail (Fig. 6.5b). Our example thus shows that a dispersal vector causes a high per-seed probability of LDD if V and/or P typically or occasionally reach high values (hatched and black solid lines in Figs. 6.5a and 6.5b). Figure 6.5b also shows that – as the LDD threshold increases – the per-seed LDD probability of thin-tailed kernels drops far more quickly than that of fat-tailed kernels: if the LDD threshold was set to 1500 m, the two fat-tailed kernels would generate a higher per-seed LDD probability than the hatched grey kernel. To calculate the expected number of LDD events for a given vector, the per-seed probability of LDD has to be multiplied with the vector’s seed load (Q). Figure 6.5c shows how Q and LDD probability interact: for low per-seed probabilities of LDD (thin-tailed dispersal kernels with typical distances below the LDD threshold), the expected number of LDD events is essentially zero even if Q is very high. However, for high per-seed probabilities of LDD (dispersal kernels that are fat-tailed or have typical distances above the LDD threshold), the number of LDD events increases steeply with Q. Important LDD vectors are thus those which generate a high per-seed probability of LDD because the product of retention time (P) and displacement velocity (V) is at least occasionally high. Such vectors can have a considerable contribution to LDD even if their seed load (Q) is small. On the contrary, vectors for which the product of V and P has a thin tail and typically lies below the LDD threshold hardly contribute to LDD.

6.2

Six generalizations on LDD mechanisms

We used the vector-centred framework outlined above to search the seed dispersal literature for mechanisms promoting LDD. This led us to formulate six generalizations on LDD mechanisms (Nathan et al., 2008) which we arranged according to the spatial scale for which they are most relevant (Fig. 6.6). These generalizations state that LDD is more frequent in open terrestrial landscapes (G1), and it is likely to be caused by large animals (G2), migratory animals (G3), extreme meteorological events (G4), ocean currents (G5) and human transportation (G6). The first generalization (G1) thus concerns the environmental conditions affecting LDD, whereas the remaining generalizations (G2–G6) identify five major LDD vectors. For each generalization, we, in the following, summarize the underlying mechanisms, review the available evidence and discuss the scope of the generalization in terms of the plant species for which it is most relevant. In addition to the information given in Nathan et al., 2008, we provide here additional evidence for the generalizations, in particular for the importance of large animals (G2; Box 6.3).

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(a)

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Expected number of LDD events

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Vector seed load (Q)

Figure 6.5 Effects of vector seed load (Q), displacement velocity (V) and retention time (P) on the expected number of long-distance dispersal (LDD) events caused by different vectors dispersing a given plant species (different line types). (a) Distributions of V and P for different vectors. (b) Dispersal distance kernels that are the product of these distributions of V and P (note that the y-axis has log scaling). The solid black, solid grey and hatched grey kernels result from multiplying the respective distributions in (a) with themselves, and the black/grey line shows a kernel which is the product of the solid black and the solid grey distribution in (a). The black arrow indicates the LDD threshold chosen for this example. (c) The relationship between the expected number of LDD events and Q. Note that the hatched grey line increases so steeply that for Q = 100 000, the expected number of LDD events exceeds 12 000.

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Fruit Development and Seed Dispersal G1: Open landscapes (V,P ) G2: Large animals (V,P) G3: Migratory animals (V ) G4: Extreme meteorological events (V ) G5: Ocean currents (Q,P ) G6: Human transportation (Q,V,P )

10

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Figure 6.6 A representation of six major generalizations about long-distance dispersal mechanisms indicating the range of dispersal distances for which each generalization is most relevant. The brackets give the key parameters of the general dispersal model (Fig. 6.3) that are predominantly affected by each mechanism.

6.2.1

G1: Open landscapes

6.2.1.1 Mechanisms Open landscapes are regions of no, sparse or very short vegetation. Examples of such open landscapes are grasslands, arid steppes and arctic tundra, as well as landscapes in which vegetation has been removed by disturbances such as fire or hurricanes. In open landscapes, LDD is facilitated by the low density of obstacles to the movement of seeds and their vectors. In comparison to closed landscapes (such as forests), open landscapes enable the vectors moving through them to have higher displacement velocity V and longer retention time P. On the other hand, seed load Q is not necessarily larger in open landscapes. 6.2.1.2 Evidence Mechanistic models show a clear positive effect of landscape openness on LDD for both primary (Nathan and Katul, 2005) and secondary (Schurr et al., 2005) seed dispersal by wind. The low density of obstacles to seed movement on smooth playa surfaces (Fort and Richards, 1998), on snow (Greene and Johnson, 1997) and in post-fire environments (Bond, 1988) means that in these environments dispersal distances of wind-dispersed seeds are much longer than in closed landscapes. A lack of vegetation also seems to promote LDD by watercourses: cleared channels and relatively wide rivers function as corridors for the LDD of seeds and vegetative fragments of many plant species (Boedeltje et al., 2003; Truscott et al., 2006). Further support for this generalization comes from a study of 123 Dutch plant communities which found that

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the number of potential LDD vectors increases with light availability as an index for landscape openness (Ozinga et al., 2004). Clearly, different dispersal vectors may require different degrees of landscape openness: for instance, a grassland may lack obstacles for the movement of large mammalian seed dispersers, but it holds many obstacles to the wind dispersal of seeds starting from within the grass canopy. 6.2.1.3 Scope All plants. It should, however, be noted that LDD is not restricted to open landscapes. In closed forests, highly mobile animals such as cassowaries (Casuarius casuarius) and spider monkeys (Ateles paniscus) can transport seeds over long distances (Westcott et al., 2005; Russo et al., 2006). Explicit analyses of how landscape openness affects the V and P of seed dispersing animals are still lacking. 6.2.2 G2: Large animals 6.2.2.1 Mechanisms Animals with larger body mass tend to have larger home ranges, higher travel velocities, larger gut capacities and longer gut retention times, compared to smaller animals within the same taxonomic group (Calder, 1996; Box 6.3). The larger home ranges and higher travel velocities of large animals lead to higher V, and their longer gut retention times imply longer P for internally dispersed seeds. The relationship between animal body mass and Q is less obvious: on the one hand, the larger food intake rate of large animals means that for internally dispersed seeds Q is higher per individual animal; on the other hand, smaller animals may compensate by visiting fruiting plants more frequently and by having higher population densities (e.g. Spiegel and Nathan, 2007). 6.2.2.2 Evidence Several quantitative studies have demonstrated that large animals can cause LDD. The majority of these studies refer to internal seed dispersal (e.g. ˜ Vellend et al., 2003; Westcott et al., 2005; Russo et al., 2006; Calvino-Cancela et al., 2006; Spiegel and Nathan, 2007), but there is also evidence that large animals can externally disperse seeds over long distances (Couvreur et al., 2008). In addition, large animals such as white-tailed deer and cassowaries have been shown to take up seeds with a wide range of dispersal morphologies (e.g. Myers et al., 2004; Westcott et al., 2005). The few available comparisons of seed dispersal by animals of different body mass suggest that larger animals generate more LDD (Dennis and Westcott, 2007; Jordano et al., 2007; Spiegel and Nathan, 2007). Here we add to this evidence by presenting a meta-analysis of data on endozoochorous dispersal by birds which shows that seed dispersal

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Fruit Development and Seed Dispersal Box 6.3

The allometry of endozoochorous seed dispersal

Allometric relationships between the body mass and various other quantities of organisms are ubiquitous (Calder, 1996). Here we use such allometric relationships to build a simple general model that relates the body mass of animals to the mean dispersal distance of the seeds they disperse endozoochorously. The dispersal distance of a seed transported internally by an animal is the product of the gut retention time P (the passage time through the animal gut) and the animal displacement velocity V (Fig. 6.3; Box 6.2). Because the movement velocity U of animals, rather than their displacement velocity V, is usually measured, we incorporated a straightness factor c which is 1 if the animal moves in a straight line and decreases towards 0 as the movement path becomes less positively autocorrelated. We also accounted for the fact that animals do not always move, by assuming that the time allocated to movement is a constant fraction f of the gut retention time P. Hence the displacement velocity is V = fcU Both the mean movement velocity U¯ and the mean gut retention time P¯ have strong allometric relationships to animal body mass M (Robbins, 1993; Calder, 1996). These relationships can be expressed as U¯ = U 0 M b1 and P¯ = P0 M b2 where U0 and P 0 are the allometric constants and b1 and b2 are the allometric exponents for U and V, respectively. The expected mean dispersal distance x¯ of seeds dispersed by an animal with body mass M is thus x¯ = V¯ P¯ = f cU¯ P¯ = f cU 0 M b1 P0 M b2 = ( f cU 0 P0 )M b1 +b2

(6.1)

When parameterizing this general mechanistic model with comparative data for the allometry of flight velocity and gut retention time in flying birds (Tucker, 1973; Robbins, 1993; Calder, 1996), we obtain x¯ = 90 432 f c M 0.5 in standard units of x¯ [m] and M[kg]

(6.2)

as a general mechanistic prediction for the mean distance of endozoochorous seed dispersal. To test this prediction, we extracted data from studies of seed dispersal by flying frugivorous birds (Fig. 6.7). All these studies calculated seed dispersal kernels by combining empirical data on V and P, both estimated directly, and independently of M. The ten bird species included vary in body mass by two orders of magnitude and inhabit various ecosystems around the globe. A linear regression of log x¯ against log M for these studies (Fig. 6.7) estimated a scaling exponent of 0.502 that is very close to the independent expectation of 0.5 (although the estimate has a broad 95% confidence interval ranging from 0.15 to 0.78). The allometric constant (f c U0 P 0 , see Eq. (6.1)) is estimated to be 1198.3 m, which together with Eq. (6.2) implies that the product of the proportion of time for which birds fly (f ) and the straightness factor of their movement (c) is on average 0.013. Our simple allometric model highlights the importance of large animals for LDD (G2). For large flying birds, even the mean distance of endozoochorous seed dispersal exceeds 1 km (Fig. 6.7) showing that these species generate LDD frequently, as part of their ordinary behaviour. The wide confidence interval on the allometric exponent indicates that large deviations from the mean allometric trend are not uncommon. Positive deviations from the mean trend represent cases of particular importance for plant LDD.

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1 000 4

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10 0.001

0.01

0.1 Mean body mass of flying frugivorous birds, M (kg)

1

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Figure 6.7 Mean distance of endozoochorous seed dispersal versus disperser body mass in six field studies on ten flying frugivorous bird species. The line shows the fit of a regression of log mean dispersal distance (¯x ) against log mean body mass (M). The estimated scaling exponent (the regression slope = 0.502) closely matches the value predicted by a generic allometric model (0.5). Numbers besides the points indicate the data source – 1: Westcott and Graham (2000), 2: Ward and Paton (2007), 3: Weir and Corlett (2007), 4: Spiegel and Nathan (2007), 5: Sun et al. (1997), 6: Holbrook and Smith (2000).

distance increases with the body mass of the seed-dispersing animal as predicted from allometric relationships for P and V (Fig. 6.7; Box 6.3). 6.2.2.3 Scope Plant species dispersed by animals, especially such dispersed endozoochorously (Jordano et al., 2007). The relationship does not necessarily hold for inhomogeneous taxonomic and/or dietary groups of animals. Although larger body mass is associated with higher V in both epi- and endozoochorous dispersal, it is currently unclear whether the allometric relationship between body mass and P found for endozoochorous dispersal (Fig. 6.7; Box 6.3), also holds for epizoochory. 6.2.3 G3: Migratory animals 6.2.3.1 Mechanisms During migration, animals show relatively fast and directed movements. Migratory animals therefore have considerably higher V than equivalent sedentary animals. In addition, migratory animals are more likely to move

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Fruit Development and Seed Dispersal seeds across dispersal barriers such as mountain chains and oceans. Also, Q could be very high given the large number of migratory animals. However, general conclusions about the magnitude of Q and P for migratory animals still seem premature. 6.2.3.2 Evidence A wealth of anecdotal evidence for LDD by migratory animals (reviewed, e.g. in Carlquist, 1981) has been collected since Darwin (1859) pointed out the possibility of seeds being long-distance dispersed on the feet of migratory seabirds. Quantitative studies have mostly focused on measuring seed viability and passage time (P) (e.g. Charalambidou and Santamaria, 2002), and the expected dispersal distances were then calculated under the assumption that V can be estimated from observed migratory flight speeds (Charalambidou et al., 2003). This assumption is likely to lead to gross overestimation of LDD, because it does not account for delays between seed uptake and the start of migratory flights as well as pauses in stopover sites (S´anchez et al., 2006). Laboratory estimates of P for captive animals may fail to represent effects of pre-migratory fasting and reduction of the digestive system (Clausen et al., 2002). The only available experiment incorporating such effects suggests that seeds dispersed by migrating ducks have relatively long P (Figuerola and Green, 2005). However, despite the recent upsurge in research on this topic (Clausen et al., 2002; Charalambidou and Santamaria, 2002; Figuerola and Green, 2002; Charalambidou et al., 2003; Figuerola and Green, 2005; S´anchez et al., 2006), quantitative estimates of V, P and Q are still largely missing for migratory animals. 6.2.3.3 Scope Plants fruiting during migration periods (Hanya, 2005). Fleshy-fruited plants are likely to be dispersed by migrating passerines (Jordano, 1982), while waterfowl mostly disperse small-seeded plants (Figuerola and Green, 2002; Soons et al., 2008). Since studies have largely focused on aquatic plants dispersed internally by waterbirds, there is virtually no quantitative information on other plants, on other groups of migrating animals and on the importance of epizoochorous dispersal by migrating animals.

6.2.4

G4: Extreme meteorological events

6.2.4.1 Mechanisms As extreme meteorological events we denote phenomena that cause highly energetic wind or water flow. These events are important for LDD because they have exceptionally high V and are likely to exhibit relatively long P (storms/floods carry seeds for longer periods than normal conditions) and high Q (storms/floods induce mass release of seeds). However, extreme meteorological events can also damage plant seeds, thereby lowering effective

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seed load (Q) and reducing the establishment probability of long-distance dispersed seeds. 6.2.4.2 Evidence Extreme meteorological events have long been recognized as potential LDD vectors that may even act at intercontinental scales (e.g. Visher, 1925). Indirect evidence from genetic analyses and studies of plant recruitment suggests causal relationships between extreme meteorological events and LDD: after severe storms, for example, Reed et al. (1988) observed a high frequency of kelp (Pterygophora californica) recruitment at distances up to 4 km from the nearest propagule source. At far larger scales, a biogeographical analysis ˜ (Munoz et al., 2004) found that the floristic similarities of moss, liverwort, lichen, and pteridophyte floras for 27 locations across the southern hemisphere conform better with how well these locations are connected by ‘wind highways’ than with their geographic proximity. This study thus suggests that wind can be an important LDD vector at the global scale. However, it does not permit to assess the relative contribution of standard and extreme conditions to LDD by wind. While quantitative analyses that directly link extreme events to plant LDD are critically missing, some insights can be drawn from a long-term data set on various objects that have been found as fallout debris from tornadic thunderstorms and could be traced back to their source location (Snow et al., 1995). Most plant seeds will fall into Snow et al.’s (1995) ‘paper’ category, for which the mean transport distance was 111.2 km and the maximum (of 48 records) was 338 km. Even ‘heavy’ objects (>450 g) were transported over a mean distance of 31.6 km and a maximum distance of 177 km (n = 30 records). Although Snow et al. (1995) did not trace back seeds, their data set demonstrates the ability of tornadoes to cause LDD (Nathan et al., 2008). 6.2.4.3 Scope Extreme meteorological events probably disperse seeds (and larger diaspores) irrespective of taxonomic and morphological classifications (Nathan et al., 2008). However, LDD by this mechanism is more likely in plant species inhabiting regions where such extreme events are comparatively frequent (e.g. ocean coasts and islands). 6.2.5 G5: Ocean currents 6.2.5.1 Mechanisms Surface ocean currents can cause long-distance transport of seeds that float on the water surface and of rafts that transport seeds or entire plants. The displacement velocity V of ocean currents is low (in the order of 0.1–0.3 m s−1 ), but their potential seed load Q is essentially limitless. For seeds with good floating capacity, the passage time P in ocean currents amounts to several

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Fruit Development and Seed Dispersal tens of days (exceptionally extending to 19 years), and for rafts, P tends to be one order of magnitude longer (Thiel and Haye, 2006). 6.2.5.2 Evidence A large number of floating and rafting records suggest that transoceanic LDD of terrestrial plants is a recurrent phenomenon that is relevant for biogeographical dynamics at macro-evolutionary timescales (Darwin, 1859; Ridley, 1930; Carlquist, 1981; de Queiroz, 2005; Thiel and Gutow, 2005; Thiel and Haye, 2006). But even at much shorter timescales, ocean currents can regularly cause LDD of terrestrial plant seeds with non-specialized dispersal morphologies: of 48 plant species dispersed to the volcanic island Surtsey (35 km from Iceland) in the first decade after its emergence, 78% arrived by ocean currents, although only 25% had apparent morphological adaptations for seed dispersal by water (Higgins et al., 2003b). For marine species, the frequency of LDD by ocean currents is likely to be even higher than that for terrestrial plants: mechanistic models predict that seeds of eelgrass (Zostera marina) contained in floating reproductive shoots are regularly transported ¨ et al., 2008). over more than 100 km (Erftemeijer et al., 2008; K¨allstrom 6.2.5.3 Scope Terrestrial plants, sea grasses and macroalgae (Thiel and Gutow, 2005; Thiel and Haye, 2006). The Surtsey example shows that seeds do not have to have obvious morphological adaptations for floating to be long-distance dispersed by ocean currents (Higgins et al., 2003b). Moreover, a wide range of plant species has been recorded as rafting. These species mostly originate from islands and coastal habitats (e.g. estuaries and saltmarshes) (Thiel and Gutow, 2005; Thiel and Haye, 2006). 6.2.6

G6: Human transportation

6.2.6.1 Mechanisms Seed transportation by humans causes LDD from landscape to global scales. Intercontinental trade and traffic has much higher V, P and Q than any non-human dispersal vector and regularly disperses seeds over past biogeographical barriers. 6.2.6.2 Evidence An early example of human-mediated LDD is the spread of agricultural crops that originated in the Fertile Crescent and rapidly expanded across Eurasia (Diamond, 2002; Chapter 7). The onset of global-scale trade and traffic in the last centuries then led to massive human-mediated LDD and caused the naturalization of many plant species outside their native ranges (Hodkinson and Thompson, 1997; Mack and Lonsdale, 2001). In fact, ‘only humans could have transported scores of western European species to Australia, Argentina and western North America, and vice versa, in less than 500 years’ (Novak

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and Mack, 2001). For some of these species, the available evidence points to particular modes of human-mediated dispersal. Of the 67 species of Southern African Iridaceae that have become naturalized outside their native range, 93% are known to be used in horticulture, whereas this percentage is only 30% for the entire set of 1036 species (Van Kleunen and Johnson 2007). This indicates horticultural trade as the main means of global-scale dispersal in Iridaceae (Van Kleunen and Johnson 2007). Similarly, the invasion of Europe by the South African ragwort (Senecio inaequidens), started independently in at least five centres of wool industry, suggesting that wool transports were the dominant mechanism of intercontinental dispersal for this species (Heger ¨ and Bohmer, 2005). At smaller spatial scales, it is even easier to identify specific human-mediated LDD mechanisms: experiments and observations demonstrated that LDD is caused by cars (Von der Lippe and Kowarik, 2007), agricultural machinery (Bullock et al., 2003) and livestock herds (Manzano and Malo, 2006). 6.2.6.3 Scope Probably most plants, especially those closely associated with human activities (e.g. crop and ornamental plants, weeds and ruderals).

6.3 A vector-based perspective on the evolution and predictability of long-distance seed dispersal The importance of the above generalizations for LDD evolution and our ability to understand and predict the large-scale dynamics of plants will depend on the answers to two fundamental questions: 1. Is it possible to identify all important LDD vectors? 2. How important are plant and seed traits for LDD? In the following, we first address these two questions from a vector-based perspective and then discuss their implications for the evolution and predictability of LDD. 6.3.1 Is it possible to identify all important LDD vectors? The above generalizations certainly do not provide an exhaustive list of LDD mechanisms. But the potential of the vector-based framework to advance our understanding of LDD will depend on whether it is possible to identify all important LDD vectors for a given plant species. It seems obvious that we cannot identify all vectors that might potentially disperse seeds over long distances. This is because the number of vectors that could potentially cause LDD is essentially limitless. For instance, seeds of temperate plant species

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Fruit Development and Seed Dispersal could be long-distance dispersed by an escaped circus elephant, and the shock wave following a major asteroid impact could cause LDD of those seeds that are not destroyed by the impact. Such ‘freak events’ occur in such an irregular manner that it seems impossible to assess whether or not they will be involved in the LDD of a given plant species. The notion that LDD is driven by ‘chance dispersal’ (Carlquist, 1981) seems to suggest that these freak events dominate LDD. However, as pointed out by Berg (1983), we need to distinguish two types of ‘chance dispersal’: LDD can either arise from ‘an unusually favourable combination of the regular dispersal factors’ or from ‘an unusual coincidence involving a dispersal factor not normally operating together with the taxon in question’. The key distinction between these two forms is whether LDD results from the rare but essentially predictable behaviour of a regular dispersal vector (such as extremely turbulent wind conditions), or whether it results from the rare and unpredictable involvement of an irregular dispersal vector (such as an escaped elephant). The distinction between regular and irregular LDD vectors is important, because it marks the boundary between what can and what cannot be predicted by a vector-based approach to LDD research. We can hope to obtain a quantitative understanding of regular LDD vectors (such as the ones in G2–G6), but research into the mechanisms of LDD by irregular LDD vectors seems futile because of the sheer number of irregular vectors. Hence, the overall contribution of regular vectors to LDD defines an upper limit of what can be explained by a vector-based approach (Fig. 6.8). The success of the vector-based approach will thus depend on the composition of a plant’s LDD vector spectrum: LDD can – at least in principle – be explained if it is dominated by a limited number of regular vectors (Figs. 6.8a and 6.8b). On the contrary, the vector-based approach will fail if LDD results from the joint action of an essentially unlimited number of irregular vectors (Figs. 6.8c and 6.8d). The vector-based approach thus directs our attention to the shape of LDD vector spectra. But what do these LDD vector spectra look like for particular plant species and what is the relative contribution of regular and irregular vectors to the LDD of these species? While these are clearly very difficult questions, we are starting to gather information that helps to answer them. An important new direction in dispersal research combines biotelemetry, mechanistic modelling and genetic analyses to directly quantify the ‘TDK’ generated by a set of vectors dispersing the same plant species (Jordano et al., 2007; see Nathan, 2007 for a review). Another approach is to compare mechanistic predictions for regular LDD vectors with data on large-scale plant distributions. One exam˜ et al. (2004) (see ple for this approach is the biogeographical analysis of Munoz Section 6.2.4) which suggests that wind as a regular LDD vector dominates the floristic similarity between cryptogam communities across the southern hemisphere. A second study (Schurr et al., 2007) found that the combination of mechanistic models for LDD by primary and secondary wind dispersal (Tackenberg, 2003; Schurr et al., 2005) explains a substantial proportion of the

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Regular vectors dominate LDD (a)

(b) 100 Cumulative contribution to LDD (%)

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Figure 6.8 Two idealized types of dispersal vector spectra and their consequences for the predictability of long-distance dispersal (LDD). Regular vectors are shown in grey and irregular vectors in white. (a) and (c) depict the two dispersal vector spectra by showing the relative contribution of the 20 most important vectors to overall LDD. Note that the number of involved vectors is essentially unlimited. (b) and (d) show the respective cumulative contribution of vectors to overall LDD, with grey horizontal lines indicating the proportion of LDD explained by all regular vectors. If LDD is dominated by regular vectors, it can – at least in principle – be predicted through the quantification of these vectors. However, if LDD is dominated by irregular vectors, it is largely unpredictable.

amount to which 37 species of South African Proteaceae fill their potential range. In these two studies, LDD thus seems to be dominated by one regular dispersal vector. The fact that both studies focus on wind dispersal is not a coincidence: the quantitative understanding of LDD mechanisms – as a prerequisite for the comparison of mechanistic predictions to large-scale patterns – is most advanced for seed dispersal by wind (Kuparinen, 2006). Further quantitative analyses that also include mechanistic predictions for other regular dispersal vectors are needed to assess how frequently LDD is dominated by one or a few regular vectors. The limited evidence available to date does, however, suggest that LDD is not always dominated by irregular

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Fruit Development and Seed Dispersal vectors and that we do not always have to consider many regular vectors to adequately describe LDD. If LDD is dominated by a limited number of regular vectors, the vector-based framework will help to identify these vectors, to measure their Q, V and P and hence to quantify their contribution to LDD. 6.3.2

How important are plant and seed traits for LDD?

Another critical question for our understanding of LDD evolution and the large-scale dynamics of plants is to which extent plant and seed traits control LDD. For instance, LDD can only evolve adaptively if it is to some extent controlled by phenotypic traits, and if these traits have in part a genetic basis. A similar question can be asked within an ecological context: do interspecific differences in LDD depend in part on species traits or is LDD essentially identical for all species inhabiting a given environment with a given set of LDD vectors? If LDD depends on species traits, an understanding of interspecific variation in biogeographical distributions and dynamics requires the quantification of these traits. If, on the other hand, species traits are unimportant for LDD, all species co-occurring in a given environment with a given set of LDD vectors should have the same rates of LDD. In this case, the assumption of the neutral theory of biogeography (Hubbell, 2001) that all species in a metacommunity have the same migration rate might be a good description of reality. Moreover, if species traits are unimportant for LDD, efforts to predict range dynamics under environmental change can neglect variation in these traits. Instead, these efforts should then focus on interspecific differences in demographic processes (population growth rates, probability of establishment) and on how environmental change will affect the joint spectrum of LDD vectors shared by all species. To assess the consequences of the generalizations of G1–G6 for LDD evolution, for the large-scale dynamics of plants and for our ability to predict these dynamics under environmental change, the critical question is thus to which extent plant and seed traits control LDD. As outlined above, the traits used in traditional morphological classifications have no or little effect on LDD. However, this does not necessarily mean that traits in general are irrelevant – traits other than those traditionally considered may well control LDD by particular vectors. For instance, the LDD of Trillium grandiflorum seeds does not depend on the fact that they are equipped with an elaiosome, but rather on their ability to survive ingestion by whitetailed deer (Vellend et al., 2003). The high importance of human-mediated dispersal at very large scales (G6) makes an entirely different suite of traits relevant for LDD; for example, the attractiveness of Iridaceae species to gardeners seems to strongly determine their spread at global scales (Van Kleunen and Johnson 2007). These examples show how a vector-based approach can identify LDD-relevant traits which escaped the traditional seed-centred approach. Moreover, they show that plant and seed traits can exert some control on LDD by at least certain dispersal vectors.

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Therefore, the question does not seem to be whether phenotypic traits can in principle affect LDD, but rather how much of the variation in LDD they explain. It seems likely that different LDD mechanisms differ in the amount of trait control (for instance, phenotypic traits might have a bigger effect on LDD by large animals than on LDD by tornadoes). It also seems likely that, for a given LDD mechanism, the amount of trait control decreases with dispersal distance. However, to give definite answers to these questions, we need a quantitative understanding of multiple LDD mechanisms which currently is still lacking. 6.3.3 Evolution of long-distance seed dispersal A necessary condition for LDD to evolve in response to natural selection is that phenotypic traits of plants and seeds must exert a certain degree of control over LDD. As discussed above, this condition seems to be fulfilled at least for certain plant species and certain LDD vectors. Yet, in comparison to environmental effects, phenotypic traits are likely to have only a small effect on the LDD probability of an individual seed. Because phenotypic traits themselves are also affected by environmental variation, this could be taken to mean that the heritability of LDD (as the proportion of LDD variation explained by additive genetic variation) is even smaller. However, when quantifying the heritability of LDD, one has to carefully distinguish between different levels at which environmental variation acts: if we assume that the relevant phenotypic traits are mostly determined by the genotype of the mother plant, the heritability of LDD should not be calculated from between-seed variation in LDD, but rather from between-mother plant variation in the LDD of their offspring. At the mother plant level, the environmental stochasticity involved in the LDD of individual seeds will to some extent average out, with environmental stochasticity becoming less important the more fecund individuals are. Hence, while the importance of environmental effects for LDD means that the heritability of LDD is unlikely to be very high, it also seems premature to conclude that LDD inevitably has a heritability close to zero. Because of the difficulties of directly observing LDD (see Section 6.1.3), it seems virtually impossible to quantify its heritability from direct empirical observations (as was done for short-distance dispersal by Donohue et al., 2005). Instead, it seems more promising to estimate the heritability of LDD by using mechanistic models of LDD vectors that propagate observed phenotypic variation in dispersal traits and represent realistic levels of environmental variation. The fact that LDD can have strong fitness consequences means that it is likely to be exposed to strong directional selection pressures. Evolutionary models show that LDD can be selected for in species that expand their geographic range (Travis and Dytham, 2002), are exposed to specialized pests (Muller-Landau et al., 2003), face frequent catastrophic extinction from local habitat fragments (e.g. Van Valen, 1971; Hamilton and May, 1977; Comins

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Fruit Development and Seed Dispersal et al., 1980; see also Ronce et al., 2000; Poethke et al., 2003) or inhabit spatially autocorrelated landscapes (Hovestadt et al., 2001). Selection against LDD may occur in island situations where LDD carries high costs because long-distance dispersers are likely to end up in hostile environments (Cody and Overton, 1996). In metapopulations, the costs and benefits of LDD may interact so that between-population selection for colonization favours increased LDD in young populations, whereas within-population selection favours reduced LDD as populations age (Olivieri et al., 1995). The short-term evolutionary response of LDD depends on the product of its heritability and the strength of selection acting on it (Lynch and Walsh, 1998). As discussed in the previous paragraphs, LDD is likely to have rather low heritability, but it may also be exposed to strong directional selection. A priori we can thus exclude neither the possibility that LDD might respond quite quickly to natural selection nor the possibility that it might hardly react at all. To assess the speed at which LDD can evolve adaptively, we instead have to quantify both its heritability and the selective forces acting on it. New avenues for studying the evolution of multivariate dispersal phenotypes are suggested by the realization that most plant species have multiple LDD vectors. Seed movement over different spatial scales is exposed to different selective pressures (Ronce et al., 2001), which raises the question whether LDD and short-distance dispersal can evolve independently, or whether they are linked through positive or negative genetic correlations (Ronce, 2007). The fact that LDD vectors are often different from the standard vectors driving short-distance dispersal (Higgins et al., 2003b; Box 6.1) suggests that LDD and short-distance dispersal might be able to evolve independently. However, seed dispersal by different vectors can still be linked through phenotypic correlations between the relevant dispersal traits. For instance, for seeds of a given mass, increased pappus size both reduces the seed’s terminal falling velocity and increases its attachment to animal furs (Couvreur et al., 2005; Tackenberg et al., 2006), which should lead to a positive correlation between wind dispersal and epizoochorous dispersal. Such interactions between seed dispersal by different vectors are likely to be mediated by quantitative, continuously varying traits (such as pappus size). However, they can also arise from single mutations: the domestication of wild wheat and barley might have been triggered by the emergence of single-gene mutants that did not shatter their seeds and thus had reduced probabilities of abiotic seed dispersal but increased human-mediated LDD (Diamond, 2002; Chapter 7). Changes at individual loci might thus have profound consequences for the composition of TDKs and can substantially promote LDD. 6.3.4

Forecasting the large-scale dynamics of plants

To forecast the large-scale dynamics of plants under global change, we need a vector-based understanding of TDK composition. Firstly, this is because

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different LDD vectors play differential roles in the response of plant species to global change (Nathan et al., 2008). Many European plants will have to migrate northeastwards under climate change (Bakkenes et al., 2002) but they fruit in autumn when birds migrate southwards (Hampe et al., 2003). Hence, their ability to expand in response to climate change may depend more on LDD by extreme southwesterly storms (which are common in autumn) than on LDD by migratory birds. Secondly, a vector-based understanding of TDK composition is important because different LDD vectors may be differentially affected by environmental change. For instance, climate change is likely to increase the intensity of tropical cyclones (IPCC, 2007, p. 15), is very likely to slow down ocean currents such as the Atlantic meridional overturning circulation (Meehl et al., 2007) and may shorten or lengthen bird migration routes (Fischlin et al., 2007, p. 239). The effect of climate change on the LDD of a particular plant species will thus depend on the relative importance of migrating birds (G3), extreme storms (G4) and ocean currents (G5) for the tail of this species’ TDK. A quantitative description of LDD combined with knowledge on the probability of population establishment after LDD (Nathan, 2006) is thus the basis for forecasting the large-scale dynamics of plants (e.g. Clark et al., 2001). However, LDD forecasts will inevitably involve uncertainty that arises from three main sources, namely from model, parameter and inherent uncertainty (Higgins et al., 2003a). Model uncertainty is caused by uncertainty about the identity of dominant LDD vectors and by uncertainty about the exact mechanisms by which these dominant vectors move seeds. This component of uncertainty can be greatly reduced by research into LDD mechanisms and by the adequate modelling of these mechanisms (Higgins et al., 2003a). However, the proportion of LDD accounted for by irregular LDD vectors (Fig. 6.8) will limit our ability to reduce model uncertainty (see Section 6.3.1). Parameter uncertainty is the result of imprecise knowledge about the key parameters (Q, V and P) for each LDD vector. For most LDD vectors, we seriously lack quantitative knowledge about these key parameters (see Section 6.2) meaning that parameter uncertainty can be substantially reduced through the collection of empirical data. Finally, inherent uncertainty is the consequence of stochasticity in LDD processes or in the models used to describe them. For vectors with high seed load (Q) and fat-tailed dispersal kernels, the inherent uncertainty about the location of the furthest dispersal distance is high. This leads to high uncertainty about population spread rates that cannot be reduced by an improvement of models or parameter estimates (Clark et al., 2003). Despite inevitable uncertainty in LDD predictions, informative quantitative forecasts of range dynamics seem possible at least for certain species: the fact that LDD mechanisms can explain interspecific variation in biogeographical distributions of South African Proteaceae (Schurr et al., 2007, see above) suggests that at least for these species it may be possible to mechanistically forecasts future range dynamics (Thuiller et al., 2008).

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6.4

Future directions

In this chapter, we have pointed out some key questions for future research on LDD of plants: 1. Can we identify all important LDD vectors for a given plant species? 2. What is the shape of LDD vector spectra (Fig. 6.8) for different plant species? 3. Which phenotypic traits of plants and seeds affect LDD? 4. How important are these phenotypic traits for LDD? 5. How large are the heritability of LDD and the strength of selection acting on it, and how fast can LDD evolve? 6. To what extent are LDD forecasts limited by inherent uncertainty that cannot be reduced through improvement of model structure and parameter estimation? We believe that our ability to answer these questions can be greatly advanced through the wide adoption of a vector-based framework for LDD research that defines the spatial scales of interest for a particular problem, identifies the dispersal vectors operating at these scales and quantifies the key parameters Q, V and P (Fig. 6.3) for these vectors. To quantify how Q, V and P are determined by the interaction of environmental conditions (e.g. landscape openness, G1) and phenotypic traits, we need to develop and/or refine mechanistic models for specific LDD vectors, notably for those mentioned in G2–G6. Mechanistic models for seed dispersal by animals (G2 and G3) are so far restricted mostly to specific animal species. However, the allometric model presented in Box 6.3 demonstrates that it is possible to formulate more generic models for seed dispersal by animals. Models for LDD by abiotic vectors such as extreme meteorological conditions (G4) and ocean currents (G5) can build on existing models for fluid dynamics in meteorology and oceanography (e.g. Erftemeijer et al., 2008). Finally, it seems possible to construct mechanistic models for LDD by human transportation (G6) that use data on traffic and commodity flows. The development of mechanistic models for LDD vectors will have to go hand in hand with the collection of data on relevant environmental variables, vector properties and phenotypic traits. For instance, the development of large databases on functional species traits (e.g. Knevel et al., 2003; Poschlod et al., 2003) should interact closely with the development of LDD models to ensure that the trait databases provide parameter estimates necessary to forecast large-scale dynamics under environmental change. To maximize the value of trait databases for conservation, it will furthermore be important to ensure that they cover the geographical regions and the species that are most affected by environmental changes. The development of mechanistic models for LDD vectors will also have to be integrated with the collection of relevant data at large spatial scales.

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Large-scale data can serve both to validate mechanistic LDD models and to assess whether models for regular LDD vectors provide an adequate description of overall LDD (see Section 6.3.1). Large-scale information on LDD can be collected by regional-scale sampling of long-distance dispersed seeds (Shields et al., 2006), through technological advances in the tracking of seeds and their LDD vectors (Wikelski et al., 2007) and through the combination of ˜ large-scale genetic (Jordano et al., 2007) and biogeographical (Munoz et al., 2004) data with information on LDD mechanisms. In conclusion, we believe that the combination of new quantitative tools in a vector-centred approach holds great promise for LDD research. The pace at which this field has been developed in recent years lets us hope that further advances in our understanding of long-distance seed dispersal are not far away.

Acknowledgements We thank Jordi Figuerola, Daniel Garc´ıa, Arndt Hampe, Anna Kuparinen, Wim Ozinga, Oph´elie Ronce, Sabrina Russo, Louis Santamar´ıa and Steve Wagstaff for helpful comments and suggestions. This study has been supported through the Israeli Science Foundation (ISF 474/02 and ISF-FIRST 1316/05), the International Arid Land Consortium (IALC 03R/25), the Israel Nature and Parks Authority, the US National Science Foundation (IBN9981620 and DEB-0453665), the German Ministry of Education and Research (BMBF) in the framework of Biota Southern Africa (FKZ 54419938), the European Union through Marie Curie Transfer of Knowledge Project FEMMES (MTKD-CT 2006-042261), the Minerva short-term fellowship program, the Simon and Ethel Flegg Fellowship and the Friedrich Wilhelm Bessel Research Award of the Humboldt Foundation.

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www.interscience.wiley.com

Chapter 7

SEED DISPERSAL AND CROP DOMESTICATION: SHATTERING, GERMINATION AND SEASONALITY IN EVOLUTION UNDER CULTIVATION Dorian Q. Fuller1 and Robin Allaby2 1 2

Institute of Archaeology, University College London, London, UK Warwick, HRI, University of Warwick, Wellesbourne, Warwick, UK

‘The Angiosperm seed had a double significance. It not only gave command of dry land to plant life, but it provided the means by which mankind has been able to obtain an ample and assured food supply. To the Angiosperm seed, perhaps more than to any other structure, the economic evolution of the human race is due.’ Oakes Ames (1939, p. 5) Abstract: The transition between wild plant forms and domesticated species can be considered an evolutionary adaptation by plants in response to a human driven ecology. Evidence from archaeobotany and genetics is providing deeper insight into this evolutionary process in terms of its scale, mechanism and parallelism between species. The evidence indicates that the timescale of this evolution was considerably longer than previously supposed, raising questions about the mode of human mediated selection pressure and increasing the importance of the role of pre-domestication cultivation. Different selection pressures were chronologically separated into at least three stages, each important at different points of the evolutionary process affecting different traits. Early selection pressures were ultimately driven by the pre-domestication sowing activities affecting the polygenically controlled germination and seed size traits. Later, in the second stage, release of natural selection pressures of dispersal requirements led to modification of architecture such as awns loss of awns and increase in dispersal unit size. The loss of dispersal requirement combined with positive pressure through harvesting practice led

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to the typically monogenically controlled non-shattering phenotypes. At the tertiary stage new selection pressures were imposed with changing climate caused by movement of the crops into different latitudes, resulting in typically monogenically controlled aseasonal phenotypes. The genetic evidence shows in most cases that genetically similar mechanisms have been affected in different plant species implying an evolutionary convergence in response to adaptation to human ecology. These adaptations can be considered various types of heterochrony; a mechanism of major importance generally in plant evolution. Keywords: archaeology; genetics; cereals; legumes; convergent evolution; dehiscence; dormancy

7.1 Introduction In the long-term view of human history, the beginnings of agriculture was one of the great turning points, and a central part of this turning point was the evolution of new plant forms, domesticated crops. Anthropologists and botanists alike have argued about how precisely to demarcate ‘domestication’ from non-domesticated wild species (e.g. Higgs and Jarman, 1969, 1972; Harris, 1996, 2008; Zeder, 2006). But, in general, all agree that domestication implies an increased interdependence between human cultivators and the plants they cultivate, and that this can be considered a case of symbiotic coevolution (Higgs and Jarman, 1969; Reed, 1977; Rindos, 1980). It is certainly true that different kinds of crops have experienced different selective pressures and show different adaptations for domestication; thus fruit trees and vines differ fundamentally from tuber crops or seed crops. In the present contribution, we will focus on seed crops and review the role of changes in seed dispersal, broadly interpreted, and how these have been essential aspects of the domestication process. By seed crops, we mean those species which are cultivated primarily for their harvested seeds or fruits and which are plants grown from seed. As such, this category not only includes all cereals, pulses (grain legumes) and oilseeds, but also some fibre crops. We will assess examples of changes in dispersal traits and how their evolution can be studied through archaeological plant remains (archaeobotany) as well as through genetics. While we will not attempt to provide a comprehensive list of seed crops and domestication traits, we will draw from a selection of examples from across different regions of origin, taxonomic families and degrees of current knowledge. Domestication, as we use it here, is a quality of plants in which morphological (and genetic) changes are found amongst cultivated populations by comparison to free-growing wild populations. These changes represent adaptations to systems of cultivation and human harvesting, and as such evolved by frequency changes of key alleles in the genomes of cultivated populations. These changes would have first appeared during a period of pre-domestication cultivation when human behaviours modified the

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Fruit Development and Seed Dispersal environments and reproductive cycles of plants, especially by human intervention in the dispersal of seeds (Harris, 1989; Hillman and Davies, 1990, 1999; Fuller, 2007a). One of the key changes often regarded as the characteristic of domesticated grain crops was a shift from natural seed dispersal through shattering mechanisms (pod dehiscence, or spikelet shedding of grass ears/panicles) to obligate dispersal by people (Zohary, 1969; Harlan et al., 1973; Hillman and Davies, 1990, 1999). Populations can be regarded as fully domesticated when they are dominated by such non-dispersing genotypes, and the term ‘semi-domesticated’ has been proposed for populations which show other changes associated with domestication prior to fixation of the non-shattering traits (Fuller, 2007a). Some of these other changes include loss of wild-type germination inhibition and changes in seed size, which are also linked to successful early growth of seeds planted in cultivated fields (Harlan et al., 1973; Harlan, 1992: Ch. 6; Smith, 2006a; Fuller, 2007a). All of these changes were essential to the success of domesticated plants, and archaeobotanical studies are providing increased evidence for the process and timing of their evolution. Another important set of changes in many crops, which is broadly related to dispersal, is seasonality control, through processes of photoperiodicity and vernalization. Changes in the control of seasonality of growth and flowering played an important role in the dispersal of some domesticated plants by farmers into new geographical zones, which differed in climate or environment. The genetic dissection of these traits is providing new insights into the history of this process. In the sections that follow, we will review these traits and their study, drawing selected examples from those species that have been best documented.

7.2

Loss of natural seed dispersal in wheat and barley: archaeobotanical evidence

‘. . . wild wheats and barley have fragile spikes, and their ears disarticulate immediately upon maturity. The fragility of the spike is, in fact, the main diagnostic character that serves for distinction of wild cereals from their cultivated counterparts. But what is less emphasized is that brittleness is only the most conspicuous reflection of one of the major adaptations of these wild cereals to their wild environment: their specialization in seed dispersal.’ Daniel Zohary (1969, pp. 57–58)

This is often regarded as the single most important domestication trait (‘domestication’ sensu stricto) because it makes a species dependent upon the human farmer for seed dispersal. In cereals, this occurs by the loss of abscission at the abscission scars, such as the rachis attachment points in wheat or barley ears or the rachilla to spikelet base attachment in panicled cereals (rice and millets). The result is that instead of shedding seeds when they are mature, a plant retains them, and they are then usually separated

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(a)

(b)

Figure 7.1 Comparisons between wild and domesticated plants in terms of seed dispersal. (a) Comparison between a wild shattering wheat ear (left) and domestic wheat ear with a tough rachis, which requires pounding to break apart (right). The form of rachis segments that can be recovered archaeologically is shown in the middle. (b) Generalized wild bean with pod that twists and opens, dispersing seeds (left) compared with a domestic pod that remains closed (middle) and must be split open by human force (right).

by the addition of human labour (threshing and winnowing) (Fig. 7.1). For farmers, this increased the efficiency of harvest and thus yields. Higher yields can be produced because the farmer could wait until all, or most, of the grains on a plant have matured, whereas earlier harvesting would have had to balance loss of grain through shedding, as they matured, with reduced yields through grains harvested immature (i.e. before spikelets have filled entirely). This would have been a particular problem with cereals such as

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Fruit Development and Seed Dispersal wild rice which has a long period of grain maturation, and which may have grown in wetland environments in which shed grains were lost (Fuller et al., 2007a; Fuller and Qin, 2008). The evolution of non-shattering would have occurred as a result of particular methods of harvesting that favoured non-shattering (tough rachis) mutants in harvested populations which were then sown (Hillman and Davies, 1990, 1999). Archaeologists have long attributed this to the use of sickles for harvesting (e.g. Wilke et al., 1972; Hillman and Davies, 1990, 1999; Bar-Yosef, 1998; Willcox, 1999), although recently Fuller (2007a) has questioned this on the grounds that non-shattering cereals appear to have evolved slower than what sickle harvesting models have predicted; in some regions such as the Near East, sickles precede domestication by many thousands of years, while in other regions such as the Yangtze valley of China, sickles or stone harvesting knives were introduced to artefact toolkits after rice was already domesticated. This is currently an area of debate and discussion amongst archaeobotanists (see Balter, 2007). What is clear is that other methods of harvesting might not have selected for this domestication trait. Ethnographically gatherers of wild seeds have often used paddle and basket harvesting methods (Harris, 1984; Harlan, 1989, 1992) and some harvest by uprooting immature grasses (Allen, 1974). It has been suggested that early hunter–gatherer groups in the Near East could have gathered wild wheat and barley spikelets from the ground after shedding, which is also one method that would not be expected to select for domestication traits (Kislev et al., 2004). The methods of harvesting, together with their timing in relation to spikelet maturity, created some level of selection for non-shattering domestic-type mutants. While archaeologists may continue to debate what those human behaviours were, archaeobotanical studies are beginning to provide hard evidence, at least for a few species, for the proportions of wild (shattering) and domesticated (non-shattering) morphotypes in populations at particular times and places and thus we are able to document that rate at which domestication evolved. Wheat and barley have the best documented record of domestication which took place in the Near East (Fig. 7.2). In these cereals, the distinction between shattering and non-shattering forms is clearly manifest in the attachment scars on the rachis segments, which are part of the spikelet base in wheats. Therefore, a clear distinction between wild and domesticated plants, and documenting the transition between them, should involve a study of rachis remains. While this was already clear to Helbaek (1959), data were limited, mainly to a few impressions in mud-brick. Early flotation in the Near East did recover rachis remains but large assemblages in which wild and domesticated morphtypes were distinguished did not begin to be published until the mid-1980s, with Van Zeist’s study of the barley rachis from Tell Aswad (Van Zeist and Bakker-Heeres, 1985). It is only in the past few years that studies have directly examined the time gap between the beginnings of cultivation, and the initial appearance of non-shattering cereal ears, and the end of the

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Figure 7.2 Map of Southwest Asia, showing the locations of sites with archaeobotanical evidence that contribute to understanding the origins and spread of agriculture (after Fuller, 2007a). Sites are differentiated on the basis of whether they provide evidence for pre-domestication cultivation, enlarged grains, mixed or predominantly domestic-type rachis data. Note that these sites represent a range of periods, and many sites have multiple phases of use, in which case the earliest phase with significant archaeobotanical data is represented. Shaded areas indicate the general distribution of wild progenitors (based on Zohary and Hopf, 2000 with some refinements from Willcox, 2005). It should be noted that wild emmer (Triticum diococcoides) occurs over a subset of the wild barley zone, and mainly in the western part of the crescent.

domestication process marked by the predominance or fixation of domestictype non-shattering cereals (Tanno and Willcox, 2006; Fuller, 2007a). Although theoretically it could have happened very quickly, as demonstrated under ideal experimental conditions (Hillman and Davies, 1990, 1999; see also Zohary, 2004), this no longer appears to be the case. As already indicated, there is now growing recognition of a long period of pre-domestication cultivation. In a compilation of data from five representative sites, three with einkorn wheat and two with barley, Tanno and Willcox (2006) suggested that cereal domestication might take millennia, perhaps as long as 3000 years, while Weiss et al. (2006) accepted at least a 1000-year period. A comprehensive compilation of nearly 5000 barley rachises (Hordeum spontaneum/vulgare) and 1800 einkorn wheat (Triticum boeitucm/monococcum) spikelet bases (Fuller, 2007a) similarly indicated slow domestication with an estimated 1500–2000 years for the transition to predominantly non-shattering morphotypes, starting from ca. 9500 BC (Fig. 7.3). But if weed flora evidence for pre-domestication cultivation is accepted for Abu Hureyra and Mureybit (Colledge, 1998; Hillman et al., 2001; Willcox et al., 2008) and assumed to be continuous with later cultivation and domestication, then it should be

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Figure 7.3 Domesticate rates in barley and einkorn wheat modelled from archaeobotanical data (after Fuller, 2007a). Proportion of domesticated type for each site is plotted by a box against a median estimate of site age. A margin of error is indicated by the line which connects the sum of domesticated and uncertain types (indicated by a cross or x). Trend lines are shown based on the lower estimate. (a) Barley domestication rate model, on which period averages are also plotted for the PPNA, Early PPNB and Late PPNB, in which the diamond indicates the proportion of domesticated types and the circle the sum of domesticated and uncertain types. (b) Einkorn domestication rate model; trend line does not consider the much later Kosak Shamali.

assumed that cultivation began a further 1000–1500 years earlier, bringing the estimate to 3000–3500 years. The recognition of pre-domestication cultivation together with a slow domestication process reopens the question as to just how early some cultivation might have begun. It also dissociates the beginnings of cultivation from subsequent domestication leaving an open question

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as to how many centres of origin there were for cultivation and whether all of these were equally involved in selection for domesticated plants. Most archaeologists now assume that there were multiple independent centres of early cultivation and eventual domestication in the Near East (Nesbitt, 2004; Willcox, 2005; Weiss et al., 2006; Fuller, 2007a; Zeder, 2008) but further research is still needed to delineate these. The overall regional pattern is the replacement of entirely/predominantly morphologically wild barley with predominantly domesticated barley by the end of the Pre-Pottery Neolithic periods. How we explain this long domestication period, however, remains uncertain. Willcox et al. (2008) have suggested that continued collecting from wild stands to replenish stores would slow down the rate at which domesticated types were selected, and harvesting of ears somewhat immature would also act against strong selection for domesticated types (as already noted by Hillman and Davies, 1990, 1999; see also Fuller, 2007a; and parallel issues with Asian rice, Fuller et al., 2007a). Certainly, even in the late Pre-Pottery Neolithic B (PPNB) period of the Near East, there is intersite variability in the proportion of wild barley rachis, which may relate to different degrees of continued reliance on gathering from wild stands. As suggested by Ladizinsky (2008), local bottlenecks may have been caused by drought or disease and forced cultivation of additional stock from wild populations. Another alternative is to reconsider the presumption that hunter–gatherers would have sickle-harvested wild cereals, which has long been the basis for our models of the evolution of non-shattering domesticates, but which should have led to rapid domestication (Hillman and Davies, 1990, 1999). It can be suggested that the sickle was transferred to harvesting crops after non-shattering ears were a significant component of crops (Fuller, 2007a). Sickle harvesting of crops was an exaptation as sickles were developed earlier as a technology for cutting basketry or building materials (Sauer, 1958; Sherratt, 1997; Kislev et al., 2004; and note the cut wild straw as bedding material at 23 000 bp Ohalo II: Nadel et al., 2004). Hillman and Davies (1999) had discussed how harvesting by cultivators would be expected to maximize yield per unit area (cultivated plots) rather than unit of time, as expected for hunter–gatherers (also Bar-Yosef, 1998). Fuller (2007a) proposes that multiple harvests over time of a single crop, which would increase total yields from a crop that matured unevenly, would lead to the latest harvests favouring domesticates, even if sickles were not used. Variation between households in terms of whether first or last harvests were stored for sowing could create very weak selection at the community level for domestic morphotypes from those late harvests. Another emerging issue is whether full cereal domestication (fixation of tough-eared mutants) took place first outside the area of the wild progenitors and earliest cultivation. While the predominance of domesticated type barley on most Near Eastern sites may have waited until ca. 7500–7000 BC, by this period crops had dispersed towards Europe, reaching mainland Greece and Crete by ca. 7000 BC (see Colledge et al., 2004, 2005), where fully domesticated

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Fruit Development and Seed Dispersal forms dominate. Even earlier by 8000 BC, cereals had been transferred to Cyprus, where domesticated chaff remains also dominate (although assemblages are very small) (Colledge, 2004). This may suggest that local bottleneck effects when crops were carried away from their centres of origin sped up domestication within the translocated population. The recent genetic diversity study of einkorn wheat indicates that early domestics retained high levels of genetic diversity (Kilian et al., 2007), and this is to be expected where proximity allowed continued introgression between cultivated and free-growing populations, especially since the domestication that differentiated them was slow to evolve. By contrast, major genetic diversity bottlenecks can be expected with dispersal events beyond the wild range when farming spread to new regions, such as Cyprus or Europe. It must also be kept in mind that in centres of origin, because there remained wild populations, natural selection for wild-type adaptations continued alongside artificial selection amongst cultivars. The invasion of crop fields of weedy, wild-types, as well as the abandonment of old fields, would have provided contexts that favoured persistence of the wild, shattering morphotype. This could have been further reinforced by cross-pollination with wild populations. All of this would have bolstered the wild-adapted genetic diversity amongst early crops, which may have provided degrees of resistance to the ‘artificial’ selection of cultivated populations (Allaby, 2008; Allaby et al., 2008). Those crops which were dispersed in small founding populations to Cyprus, Crete and Greece would have been removed from conflicting selection for wild-type adaptations.

7.3

Non-shattering in other cereals: rice, pearl millet and maize

No other cereals are as well documented archaeologically as einkorn wheat and barley, although there are growing data sets for rice (Oryza sativa), pearl millet (Pennisetum glaucum) and maize (Zea mays). For most crops early archaeobotanical evidence documents use. Meanwhile, domestication is inferred by other traits, such as grain size (see below) or else from associated archaeological context or changes in distribution that suggest dispersal outside of the wild habitat. For example in Sorghum bicolor, early Holocene finds in the Western Desert of Egypt at Nabta Playa indicate that wild-type shattering spikelets were harvested together with other wild grasses by ca. 7500 BC (Wasylikowa et al., 1995, 1997, 2001), as also in central Sudan by ca. 4000 BC (Magid, 1989, 2003; Stemler, 1990). A single possible non-shattering specimen is reported from the Sudan (Stemler, 1990). Subsequent evidence for sorghum comes from grains that appear domesticated in size and shape that had been translocated from Africa to India around 2000 BC (Fuller, 2003). But there remains no data from which to infer when selection for the domesticated

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forms began or ended. Some have hypothesized that domesticated forms first appeared outside Africa in South Asia aided by the separation from wild populations (Haaland, 1999), although the archaeobotanical record in eastern Africa remains so depauperate during the period between 4000 BC and 1000 BC that this hypothesis cannot yet be tested. Rice shattering is controlled by abscission layers where the spikelet attaches to the rachilla (Li et al., 2006) and this can be studied archaeologically from the spikelet base which preserves the scar (Thompson, 1996, 1997; Zheng et al., 2007; Fuller and Qin, 2008). These spikelet bases are very small and until recent changes in how archaeological sites were sampled for plant remains, they were not recovered from early sites in either China or India where rice domestication events have been postulated. It is now clear that these can preserve in quantity, and current research programmes in the Yangtze valley are quantifying the proportions of wild, domesticated and potentially immature harvested spikelet base types (Fig. 7.4). Work by the author and others will

Figure 7.4 Archaeological remains of rice spikelet bases that allow the distinction between wild and domesticated types. At left are the drawings of three spikelet base types from the archaeological sites of Caoxieshan, Jiangsu, China ca. 4000 bc (after Fuller and Qin, 2008), showing domesticated, non-shattering scar (top), smooth scar of shattering wild mature type (middle), and protruding scar of probable immature type (bottom). At right are the scanning electron micrographs of archaeological spikelet bases from Neolithic Tian Luo Shan, ca. 4700 bc, Zhejiang Province China (see Fuller et al., 2009): at top right is domesticated type and at lower right is wild-type. Reproduced with permission from Antiquity Publications Ltd.

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Figure 7.5 Diagram of differences between a domesticated (top) and wild (bottom) spike of pearl millet (Pennisetum glaucum). In domesticated type, non-shattering involucres are born on a stalk, and often include more than one grain, whereas in wild pearl millet sessile spikelets, normally one-grained, are shed by dehiscence (based on Poncet et al., 2000; Fuller et al., 2007b). Images provided by University of Groningen, Groningen Institute of Archaeology.

soon provide some quantitative results from which to estimate how quickly non-shattering rice evolved and when domestication was completed, but current estimates suggest this process began sometime before 6000 BC and was completed by ca. 4000 BC (cf. Zheng et al., 2007; Fuller et al., 2007a; Fuller and Qin, 2008; Fuller et al., 2009). Pennisetum glaucum, pearl millet, is the only African cereal for which existing archaeobotanical evidence provides some indicators of the domestication process, but this is still limited and hampered by an absence of data sets of ancient wild-type pearl millet prior to the start of domestication. The involucres, which contain spikelets and bristles, change from being sessile and shed when mature to being non-shedding and stalked in the domesticated form (Fig. 7.5; also, Brunken et al., 1977; Poncet et al., 2000; D’Andrea et al., 2001; Zach and Klee, 2003; Fuller et al., 2007b). Evidence for the early occurrence of domesticated, stalked involucres comes from impressions in ceramics of pearl millet chaff that had been mixed with clay during pottery production (Amblard and Pernes, 1989; MacDonald et al., 2003; Klee et al., 2004; Fuller et al., 2007b). These impressions can preserve the threshed involucre stalks (Fig. 7.6), of which the earliest are now from 2500 BC to 2200 BC at Karkarichinkat (unpublished data of Fuller and K. Manning; cf. Finucane

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Figure 7.6 Examples of archaeological pearl millet remains of domesticated type. At left is a cast (in polyvinylsiloxane) of an impression of pearl millet chaff used to temper pottery from Neolithic Djiganyai Mauretania (1500–1700 bc) (after MacDonald et al., 2003; Fuller et al., 2007b); at right is an image of charred macro-remains of pearl millet involcure from Cubalel, Senegal (ca. ad 500) (after Murray et al., 2007). Images provided by University of Groningen, Groningen Institute of Archaeology.

et al., 2008). Chaff can also sometimes be preserved carbonized although we still lack very early assemblages (Fig. 7.6; cf. Murray et al., 2007). The most important cereal domesticate in the New World was maize which also differs from its wild progenitor in terms of being non-shattering. While the small alternating involucres of wild teosinte ears (Zea mexicana) shatter at maturity, the cobs of maize do not and grains must be forcibly removed from their cupules (Iltis, 2000). The presence of this trait is apparent from the earliest preserved maize cobs from dry caves in southern Mexico that date back about 6200–6300 years (Benz, 2001; Long and Fritz, 2001; Piperno and Flannery, 2001; Smith, 2001). However, the beginnings of cultivation is inferred to be much earlier based on evidence from phytoliths and starch grains, including evidence that maize had dispersed already towards South America before this time, ca. 7000 BC (Dickau et al., 2007). The beginnings of cultivation remain obscure and there is no significant early archaeological record for wild teosinte use, or how quickly this was transformed into the small non-shattering cobs of maize.

7.4 The genetics of non-shattering cereals Breeding experiments have long shown that the genetic control of seed shattering is simple in that the trait is usually governed by a single locus as evidenced by simple Mendelian inheritance ratios of brittle and tough rachis phenotypes. The tough rachis alleles have been found to be loss of function

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Fruit Development and Seed Dispersal genes that are recessive. The synteny of the grass genomes and initial quantitative trait loci analyses led to the initial supposition that the same genes might be responsible in the different grass lineages in a neat example of evolutionary convergence (Paterson et al., 1995). While this was a reasonable hypothesis given the evidence at the time, the picture has subsequently developed into something more complex. It now appears to be the case that different grass lineages have had different genes modified to produce the loss of seed shattering (Li and Gill, 2006). The number of genes responsible that have been characterized by DNA sequence still remains low, with rice leading the way. Two different genes have been identified in rice, qSH1 (Konishi et al., 2006) and sh4 (Li et al., 2006), which carry mutations leading to the non-shattering phenotype. The qSH1 gene codes for a homeodomain protein highly similar to REPLUMLESS (RPL) in Arabidopsis that is responsible for down-regulating two other homeodomain proteins (SHP1 and SHP2) involved in developing a dehiscent zone in silique maturation (Roeder et al., 2003). It seems likely that the qSH1 is also involved in an interaction between MADS-box homeodomain genes in dehiscence regulation, although it should be noted that the dehiscence zones between the two plants are not homologous and the SHPs of Arabidopsis have no known orthologues in rice. In this case, the change of function at the qSH1 gene is associated with just one single nucleotide polymorphism (SNP), although the mechanism of action is as yet unclear. The second gene identified in rice to produce a non-shattering phenotype sh4 is different to qSH1 and has only low levels of similarity to genes found in Arabidopsis, or elsewhere in the rice genome. It has a Myb3 DNA binding domain, which suggests it is a transcription factor. Again a single SNP, leading to a single conserved amino acid change, results in the change in function, which appears to result in either the incomplete formation of the abscission zone (Li et al., 2006), or the failure to initiate cell degradation (Lin et al., 2007). In this case, these two studies have found allelic variations of the sh4 gene, suggesting an allele of some antiquity and perhaps with interesting phylogeography. In the case of rice then, two independent genetic pathways to non-shattering have occurred. Haplotype analysis including one of these, qSH1, suggests that the non-shattering phenotype came after other mutations associated with an increase in grain size and the waxy phenotype (Shomura et al., 2008), supporting the idea that the mutation arose in the ‘domesticated’ population of rice. Interestingly, although the sh4 non-shattering genotype was found in the wild progenitor Oryza nivara, this was thought to be due to an introgression from domesticated rice, as the alleles that were most closely related to the non-shattering type in the wild were found in O. rufipogon rather than O. nivara. Since O. rufipogon is generally regarded as the ancestor of domesticated japonica rices in East Asia, while O. nivara has closer affinities in general with indica cultivars (Cheng et al., 2003; Fuller, 2006; cf. Sweeney and McCouch, 2007; Vaughan et al., 2008), this evidence implies that sh4 evolved during japonica rice domestication, and entered indica rice

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through a more complex process involving hybridization between lineages (Sang and Ge, 2007). Two alternative hypotheses exist: either there were high levels of introgression from wild South Asian rices as domesticates spread from a single origin in the Yangtze valley, the ‘snowball model’ (Sang and Ge, 2007; Vaughan et al., 2008), or else there were separate origins of cultivation and subsequent hybridization of cultivars, the ‘combination model’ (Sang and Ge, 2007; preferred by Kovach et al., 2007; and consistent with the archaeobotany of South Asia, cf. Fuller, 2006). Similarly the mutation for a white pericarp evolved once in early japonica and was introgressed into South Asian rices (Kovach et al., 2007; Sweeney and McCouch, 2007; Sweeney et al., 2007; Vaughan et al., 2008). In barley, two closely linked loci btr1 and btr2 have long been implicated in tough rachis formation on two independent occasions in the evolution of domesticated varieties (Takahashi, 1955). While these two genes have yet to have their sequences characterized, sequence analysis of closely linked biomarkers found through amplified fragment length polymorphism (AFLP) have supported this suggestion strongly by clearly showing two clades of brittle rachis origin for the two respective loci (Azhaguvel and Komatsuda, 2007). Based on mapping positions, it seems unlikely that the two Btr loci in barley are orthologous to the Br1 locus responsible for tough rachis in lower wheats (Li and Gill, 2006). Two loci have been identified in wheat to confer tough rachis formation in different types of disarticulation, W (wedge type) and B (barrel type), respectively. The W-type disarticulation occurs in the A, B, S, G and T genomes and is governed by Br1 located on the short arm of chromosome 3. Einkorn, emmer and Iranian spelt wheats have this disarticulation type, for instance. The B-type shattering originates from A. tauschii and is found on the long arm of chromosome 3D, and gives rise to the shattering found in European spelt. Comparison with QTLs derived from maize also suggests that these loci are not orthologous between maize, rice and wheat (Li and Gill, 2006). It should be noted that a number of other loci have been identified to be involved with rachis fragility (Janatasuriyarat et al., 2004), through the pleiotropic action of glume tenacity (Tg) on chromosome 2D, and the free threshing gene Q on chromosome 5A. Interestingly, the Q gene has been characterized by sequence, and found to be similar to APETALA 2 (AP2) of Arabidopsis, an important transcription factor in floral development (Simons et al., 2006). What makes the Q gene especially interesting is that the free threshing allele, q, is a gain of function mutation. A single amino acid change from Q to q has resulted in the ability of the protein to form homodimers, which has had consequences on its transcription regulation activity. There is little information on the remaining principal panicoid grasses maize, sorghum and Pennisetum. A discrete shattering locus was identified through QTL in sorghum (Paterson et al., 1995), which was syntenic to a high scoring region for the trait in maize, which also had further seven regions associated with shattering. Pennisetum is thought to have oligenic control of

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Fruit Development and Seed Dispersal shattering in a tightly linked cluster (Poncet et al., 1998, 2000), but further characterization has not yet been published. Finally, another crop in which tough rachis loci have been identified is buckwheat (Matsui et al., 2003, 2004). In this case, two independent loci have been identified (Sht1 and Sht2), which are not closely linked but both can confer the non-brittle phenotype independently. The sht1 allele that confers the first non-brittle phenotype is fixed within the cultivated population, but the sht2 is not. On this basis, the authors argue that the sht2 mutant may have occurred after the crop was domesticated and has not been subject to strong selection. Further phylogeographic evidence will help to elucidate this story.

7.5

Reduction in seed dispersal aids

Accompanying the loss of natural seed dispersal was the reduction of appendages that aid dispersal. De Candolle (1885, p. 460) summarized this as changes in the ‘form, size, or pubescence of the floral organs which persist round the fruits or seeds.’ Plants, and especially grasses, have a range of structures that aid seed dispersal, including hairs, barbs, awns and even the general shape of the spikelet in grasses. Thus, domesticated wheat spikelets are less hairy, have shorter or no awns and are plump, whereas in the wild, they are heavily haired, barbed and aerodynamic in shape. All of these tend to be greatly reduced in the domesticated form. While this is connected to the loss of shattering, we expect it to have evolved by a different process (Fuller, 2007a). Instead of being positively selected for by human activities, as the tough rachis was, this probably came about by the removal of natural selection for effective dispersal. The recent study by Elbaum et al. (2007), demonstrated how the awns in wild wheat function mechanically to help the spikelet work its way into the soil by daily cycles of humidity. Dispersal by wind and by sticking to animal fur may be co-selected (see Schurr et al., this volume), and the wild progenitors of several cereals include bristly diaspore units for such dispersal, such as in Setaria spp. and Pennisetum glaucum. Once natural selection was removed to maintain such dispersal aids, smaller and fewer appendages may have developed by genetic drift, in which case we would expect to find a great deal of diversity in early cultivars. Certainly, there remains a great deal of variation in this regard: some cultivated rices have awns while others do not; there are ‘bearded’ and ‘unbearded’ wheats. However, it may also be the case that selection operated by reducing metabolic ‘expenditure’ creating a parallel trend towards less barbed and hairy cereal spikelets, which can be observed across species. Unfortunately, there is little archaeological evidence on this evolutionary trend, as hairs and awns survive poorly in the archaeological record. Some remains of early rice from China have been examined in this regard (e.g. Sato, 2002; Tang et al., 1996), and the reduction of the number of spikelets with awns, the density of hairs on the awns, and the length of those hairs can

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Figure 7.7 Comparison of awn hair (bristle) density and bristle length on wild and domesticated rice awns, together with a few archaeological specimens from Hemudu, Zhejiang Province China, ca. 4800 bc (after Tang et al., 1996).

potentially be studied (Fig. 7.7). Evidence for variation in both the number of unicellular trichomes (bristles) on awns and the length of these trichomes suggests that shorter trichomes are typical of domesticated rices that have awns at all (Fig. 7.7), and indeed many domesticated rices have lost their awns altogether. Evidences from four archaeological rice awns examined from the site of Hemudu (5000–4500 BC) place these amongst the wild scatter (Tang et al., 1996), a situation in agreement with arguments from grain size data from the region (Fuller et al., 2007a), and more recent evidences from spikelet bases (Zheng et al., 2007; Fuller and Qin, 2008; Fuller et al., 2009) that indicate populations of rice from the Lower Yangtze of that period were dominated with wild-type morphological adaptations. To date, too few samples of archaeological rice awns have been studied for any temporal trends in such evidence; nor has comparable data from other taxa been examined. A related trait is the shift from single-grained wild dispersal units to the multiplication of grains under domestication. The best studied example is that of barley, in which wild Hordeum spp. normally have a single grain with two lateral sterile florets that contribute to an overall aerodynamic shape of the diaspore. In domesticated barley, six-row varieties have evolved by the

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Fruit Development and Seed Dispersal removal of inhibition of lateral florets. This not only led to production of more grains (by ca. 150%) per year, but it also created difficult-to-disperse grouped triplets of spikelets (Harlan, 1992, p. 120; Zohary and Hopf, 2000, p. 60). Genetic studies indicate that the six-row condition evolved three times, by three distinct mutations, across different parts of Eurasia (Komatsuda et al., 2007). Archaeobotanically, this trait can be inferred from the form of grains as well as the form of well-preserved rachis segments. Such evidence indicates that this trait evolved very early indeed. Asymmetrical grains, typical of six-row forms, and rachis segments with widened apices are reported in the Near East from the Early PPNB (8800–8000 BC) (Zohary and Hopf, 2000, p. 68), prior even to the fixation of non-shattering rachises (see discussion above). Similarly, the earliest barley remains from Pakistan at Mehrgarh, ca. 7000 BC, include evidence for six-row forms (Costantini, 1983). Possible parallel trends are indicated for the New World little barley (Hordeum pussilum), for which some twisted grains, and possible naked–grained varities have been reported, but remains debated (cf. Bohrer, 1984; Asch and Asch, 1985, p. 194; Hunter, 1992). This species has long been argued to be an indigenous cultivar in prehistoric North America on the basis of finds of large quantities, mainly from the First Millennia BC and AD (Asch and Asch, 1985, pp. 191–195; Dunne and Green, 1998). Such evidences require confirmation and further documentation, but it would imply domestication in terms of being released from the need to maintain wild dispersal aids. A similar development occurred with the domestication of pearl millet (Pennisetum glaucum). Wild Pannisetum, normally has a single grain in each bristly involcure, while domesticated forms often have multiples grains. The study by Godbole (1925) of Indian peal millet suggests ∼70% of involucres include two spikelets (each with a grain), while ∼20% are single grained. The other ∼10% includes more than two grains, with as many as nine grains reported from a single involucre. Archaeologically, early impressions of pearl millet preserved in pottery, indicate not only the presence of the non-shattering stalked forms, but also the presence of paired spikelets indicating that this trait had evolved in cultivated populations certainly by ca. 1700 BC (see Fuller et al., 2007b).

7.6

Non-cereal alternative: appendage hypermorphy in fibre crops

In the case of at least a few fibre crops, selection under cultivation has favoured increases in appendage size, as human selection has worked on what were adaptations for dispersals and caused exaptation for fibre production. This is most clear in the cases of cottons, in which four domesticated species are cultivated for seed coat hairs, which are extensions of testa cells. In the wild, such hairs may aid dispersal by wind or attachment to animal fur (see Ridley, 1930,

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p. 158; Fryxell, 1979, pp. 142–143; Hovav et al., 2008), but in wild tetraploid cottons domesticated in the New World, lint seems to have been exapted to dispersal by water to littoral habitats (Fryxell, 1979, pp. 143–147, 164–165), and oceanic drift is hypothesized to have brought A-genome cotton from Africa to America (Phillips, 1976). In domesticated cottons, however, hairs have become so heavy, long and tangled as to preclude such dispersal methods. In addition, domesticates have lost the hard impermeable seed coats which allowed survival in salt water. Early cultivators seem to have chosen those wild Gossypium species with the longest hairs, but it is also true that all cultivated cottons have significantly longer hairs than their wild relatives indicating selection. Hutchinson (1970, p. 271) reported an apparently spontaneous single gene mutation controlling this in wild G. barbadense. Fryxell (1979, p. 173) argues that selection for increased lint probably preceded selection for increased fruit size, at least in domesticated G. hirsutum. Unlike the loss of wild-type seed dispersal which is regarded as having evolved from unconscious selection on the part of farmers, we might expect hair enlargement to have been intentional. As such, conscious selection might be expected to exert a stronger selection pressure on genes involved in seed coat hair formation than that typical of most domestication traits. Another example comes from the Devil’s claw (Proboscidea parviflora), which has also been cultivated for its fibres in the American Southwest since prehistoric times (Nabhan et al., 1981; Bretting, 1982, 1986; Nabhan and Rea, 1987). The claws of this species represent extensions of seed capsule apices (rostra). These apical claws bend such that they can serve as hooks to cling to animal hair for long-distance dispersal. Human use of this species involves softening and pounding of capsules to separate the fibres that make up the capsule. These are used to make cords and basketry type products. It is suggested that it has only been cultivated in recent centuries, and the earliest finds are ca. AD 1150 (Nabhan and Rea, 1987, pp. 59–60). The enlarged capsules and much longer claws of domesticated forms provide for more extensive fibres. This is therefore also likely to have been a product of conscious selection. In addition, domesticated devil’s claw has evolved white seeds, rather than the black seeds of the wild form, probably indicative of typical domesticate-type loss of germination inhibition (see below).

7.7 Loss of natural seed dispersal in pulses and other crops Other seed crops have also evolved non-dispersing fruit types with domestication, although these remain largely undocumented archaeologically. Members of the Fabaceae have been domesticated in parallel in most world regions which had early cereal domestications (Harris, 1981, 2004; Smartt, 1990). Natural seed dispersal in wild legumes, including the wild progenitors of

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Fruit Development and Seed Dispersal domesticated pulses, is normally by pod dehiscence. That is, seeds are physically shed by pods that twisted and split as they dried after maturity. In domesticated species, this is removed or delayed (see Fig. 7.1), although various observations suggest that the degrees of reduction in this trait vary across taxa (e.g. Fuller and Harvey, 2006, p. 223, for South Asian species). In contexts where pulse pods are preserved, such as by desert conditions, it may be possible to determine the presence of this domestication trait by examination of the pod layering: the inner layer that causes dehiscence should be reduced. Pods of Phaeseolus lunatus and P. vulgaris from Guitarerro Cave in Peru show that this non-shattering trait was present (Kaplan et al., 1973), although these may be intrusive finds in deposits attributed to ca. 8000 BC (cf. Lynch et al., 1985), since direct dates on P. lunatus and P. vulgaris seeds go back to 3500 and 2300 years ago, respectively (Fritz, 1994, p. 307; Kaplan, 2000). Within some pulses, genetic loci involved in non-deshicent pod formation have been identified, such as Dpo1 and Dpo2 in Pisum (Weeden et al., 2002; Weeden, 2007), and the different loci v and p were selected in common bean, Phaseolus vulgaris (Koinange et al., 1996). Interestingly, by contrast to non-shattering in cereals, this trait appears to be controlled by more than one locus in some of the above studied Fabaceae domesticates (Phaseolus spp., Pisum). This presumably accounts for the degrees of pod dehiscence reported from some species and may suggest that this was a less central part of the early domestication syndrome in many pulses than it was in cereals. Nevertheless, in other pulse species, there appears to be one key gene mutation involved in non-shattering. Such evidence comes from Lens (Ladizinsky, 1979), and from azuki bean, Vigna angularis (Kaga et al., 2008). In these species, this trait is thus comparable to the cereal rachis in that respect, especially with regards to processes of selection on a population level. As argued by Ladizinsky (1987, 1993), pulse domestication may be fundamentally different from cereal domestication, contradicting Zohary and Hopf (1973; Zohary, 1989), in that loss of germination inhibition may have been the key and prerequisite trait that made early cultivation efficient. Some other seed crops show parallel trends towards non-dehiscent morphologies, such as flax (Linum usitatissimum) which has non-shattering capsules in the domesticated state (Zohary and Hopf, 2000, p. 123). Early evidence suggesting domesticated flax comes from the Near East from fragments of probable capsules at Pre-Pottery Neolithic Jericho (8400–7500 BC) and larger than wild seeds at Tell Ramad (7500–6500 BC) (Zohary and Hopf, 2000, p. 130). On the other hand, a few crop species appear to not have evolved this, perhaps due to differences in the genetic architecture of this trait. Thus, in sesame (Sesamum indicum), for example, capsules still dehisce in most domesticated forms, and this constitutes a persistent issue for plant breeders (Day, 2000; Fuller, 2003). In this case, non-dehiscent forms produce much lower yields and are unattractive. Because pods and capsules tend to be light, and are therefore unlikely to survive contact with fire, they are exceedingly rare in archaeological contexts. It is therefore the case that we have little

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direct archaeological evidence on the evolution of these traits in pulses or oilseeds.

7.8 Germination traits in domestication: the importance of loss of dormancy In the wild, many seeds will only germinate after certain conditions have passed, such as conditions of day length, temperature, or after the seed coat is physically damaged. Crops tend to germinate as soon as they are wet and planted. This is selected for simply by cultivation, and sowing from harvested yield, as those seeds that do not readily germinate will not contribute to the harvest. As such, the selective forces and mechanisms involved are expected to differ from those involved in the loss of wild-type seed dispersal. Germination differences between crops and their wild progenitors come in a range of severity. The study of changes in dormancy with domestication is complicated in many crops; this is due to limited morphological visibility of dormancy-related traits, and limited knowledge of the factors that govern dormancy. Dormancy and germination are traits that are controlled in a highly complex manner involving one or a combination of morphology, physiology and physical structures (Baskin and Baskin, 2001; Finch-Savage and LeubnerMetzger, 2006). Not least of the complications of dormancy is its definition. Dormancy can be described as a block to germination, which is to say that a non-germinating seed may not be dormant, but merely awaiting induction of germination. Finch-Savage and Leubner-Metzger (2006) define dormancy classes by distinguishing morphological, physiological deep, physiological non-deep and physical dormancy. Morphological dormancy refers to seeds that have an underdeveloped embryo and require time to grow and germinate. Physiological dormancy, the most prevalent form of dormancy, appears to broadly involve abscissic acid (ABA) and gibberellins (GA) metabolism. Physical dormancy (coat dormancy) involves the development of a waterimpermeable seed coat, and is typically broken by scarification. Such physical dormancy is typical of the wild progenitors of cultivated legumes, and is one of the key traits that has been modified with domestication (Zohary and Hopf, 1973, 2000, p. 93; Ladizinsky, 1987; Plitman and Kislev, 1989; Kaplan, 2000). It is interesting to note that morphological dormancy is more typical of the less-derived flowering plants; physiological dormancy is found throughout flowering plants (and gymnosperms), while physical dormancy occurs amongst the most derived families, most notably the Fabaceae (see Finch-Savage and Leubner-Metzger, 2006). In crops from several dicotyledonous families, dormancy traits can be seen in the seed coat. In particular, wild-type seeds tend to have thicker seed coats, often of a different colour (black or dark brown, or mottled) and often with

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Figure 7.8 Comparisons between wild and domesticated seeds, showing seed coat colour, surface texture and thickness differences with domestication. At left are wild and domesticated Sesamum indicum, wild above and domesticated below; at right are wild and domesticated Chenopodium album, wild above and domesticated below. Seeds from Institute of Archaeology, UCL collections: Seasmum malabaricum from coastal sanddune, Sindhudurg district, Maharashtra, India (coll. D Fuller 9/2004); Domesticated S, indicum black variety from Pune (8/2000); white S. indicum from India PI164384 01 SD; Chenopodium album, wild from European seed reference collection, Institute of Archaeology, UCL; Chenopodium album, domesticated, collected by E. Takei from the Rukai tribe, Taiwan (5/2007) (sample in UCL, courtesy of E. Takei).

additional surface ornamentations. Domestication has resulted in the thinning of seed coats, the lightening of seed coat colour and the loss of rugae or papillae. Such traits have evolved in parallel across families and genera, and world regions. For illustration, examples of modern wild and domesticated seed pairs are shown from Sesamum indicum and Chenopodium album (Fig. 7.8). Pigmented seed coats (or pericarps), which have long been associated with functional dormancy in wheats (Flintham and Humphry, 1993), may also be linked to dormancy in wild rice, which has evolved white pericarps only once after domestication (Sweeney et al., 2006, 2007). Nevertheless, it is also the case that physical changes are not always evident from visible morphology. Morphological indicators of pericarp colour change are not detectable in the charred grains recovered by archaeologists. Even in other families, this may prove difficult to document archaeologically. In Near Eastern pulse crops, for example Butler (1989, 1990) was able to document clear morphological differences in the seed coats of wild and domesticated peas, but not of lentils, chickpeas or Vicia spp., where morphological variation falls along a spectrum from thicker (and sometimes ornamented) seed coats in wild populations and some cultivars, to thinner, smooth forms in other cultivars. This physical spectrum may relate to a functional spectrum in germination

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inhibition. Weeden (2007), for example, has documented a spectrum of variation in germination between wild Pisum sativum subsp. elatius, with virtually no immediate germination within 1 year of seed formation, Pisum abyssinicum (the Ethiopian pea), which shows partial breakdown of germination inhibition (seeds germinate in 3–12 months), and modern Pisum sativum subsp. sativum which readily germinates (cf. Weeden, 2007). A wide range of germination rates is reported from Lablab purpureus, but with significantly higher proportions of faster germination in cultivated populations (Maass, 2005). A wide range of variation is especially noted amongst wild accessions of Lablab, which may suggest that domestication drew upon existing genetic variation in this species. Ladizinsky (1987) argued that the very low germination rates in wild pulses, in particular Lens, would have precluded successful cultivation on the basis of very low yields from planted seeds. He therefore suggested that hunter–gatherers must have recognized favourable wild mutants with ready germination from which to begin cultivation, that is there was a form of ‘pre-cultivation domestication’. This hypothesis, however, received critiques (Zohary, 1989; Blumler, 1991). Ladizinsky’s (1987) argument for Lens cultivation contrasts to cereal cultivation in that he reasons that the domestication syndrome phenotype of a lack of dormancy would have to arise in the wild rather than the cultivated gene pool because cultivator pressures would not have been sufficient to break dormancy. Ladizinsky argued that this is also supported by genetic diversity data based on isozymes, which show different cultivated groups of Lens appear to be most closely related to different wild groups of Lens, indicating a multiple domesticated origin, despite a single mutation responsible for dormancy breaking (Ladizinsky, 1987, 1993). He argued that the most parsimonious explanation is that such a mutant may have been persisting in the wild. The ‘comparable’ tough rachis mutant in cereals is postulated to have arisen in the cultivated population rather than the wild where it has been believed that the tough rachis mutant would not persist. Zohary (1989) argues that the lack-of-dormancy mutant also would not survive in the wild. More recently, Kerem et al. (2007) have suggested that even low yields from wild-type pulses (in particular chickpea, Cicer arietinum) may have been favoured because the presence of particular micronutrients (the amino acid tryptophan) were the target of early pulse consumption rather than overall protein or carbohydrate (also Abbo et al., 2007, on Pisum). The extent to which any of these hypotheses might apply across pulse domestications from difference subfamilies and different regions is unclear. More research is needed. So far, archaeobotanical evidence has contributed little to the documentation of the earliest processes of pulse domestication and the evolution of these domestication traits. Evidence for the loss of germination inhibition may be preserved archaeologically, although detailed studies are only available for a few species. One challenge is preservational: seed coats are often not preserved on charred pulses. This is clearly the case with Indian Vigna spp., for example (Fuller

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Fruit Development and Seed Dispersal and Harvey, 2006). Those species which have been best documented are New World Chenopodium domesticates (e.g. Smith, 1989, 1992, 2006bb; Bruno and Whitehead, 2003; Bruno, 2006). In a classic case of the fossil record (archaeobotany) identifying an extinct crop, Smith (1989, 1992, 2006b) tracked a marked decrease in seed coat thickness in Chenopodium berlanderii seeds from sites in the Eastern Woodlands of the United States between ca. 2500 BC and 1500 BC. In addition to thinning seed coats, presumably linked to loss of wild-type germination inhibition, seeds tended to change shape and size, although wild-type forms persisted as weeds alongside the Chenopodium crop (Gremillion, 1993). Bruno (2006) has developed a similar approach to studying South American Chenopodium domestication, although variation in seed coat thickness amongst wild species makes this more difficult requiring the use of additional size and shape characters. Although modern material suggests a similar change has occurred in Old World Chenopodium album, at least amongst East Asian domesticated populations (see Fig. 7.8), archaeobotanical evidence tracking such changes has not been gathered. Given suggestions that Chenopodium was formerly a crop of Iron Age Europe (Helbaek, 1954; cf. Henriksen and Robinson, 1996, pp. 9–19; Stokes and Rowley-Conwy, 2002) or of Bronze Age Gujarat, India (‘intential collection’ inferred by Weber, 1991, p. 121), studies along these lines are warranted.

7.9

The genetic basis for dormancy and germination

A large number of genes may be directly or indirectly involved with dormancy. For instance, developmental genes can be expected to be involved in morphological and physical dormancy, while abscissic acid (ABA) and giberellic acid (GA) make up two of the most common plant hormones, which are expected to involve numerous loci across the genome. Some progress is being made with understanding the molecular basis of dormancy with regards to non-deep physiological dormancy, typical of the cereals. Physiological dormancy is largely governed by the ratio of ABA to GA. When this ratio is high, dormancy prevails, and when GA levels become high enough relative to ABA, then germination is initiated (White et al., 2000; Kucera et al., 2005). Two other hormones also known to have roles are ethylene and the brassinosteroids, both of which act similar to GA to promote germination and counter the effects of ABA (Kucera et al., 2005). Dormancy is a necessary part of seed development during which ABA promotes maturation pathways that govern storage compound deposition and dessication of the grain. GA synthesis is actively inhibited in maize during this time (White and Rivin, 2000). It is thought that dormancy release is due to ABA breakdown and this is the primary hormone. After breakdown, the presence of GA in sufficient concentrations relative to ABA can promote germination. This is supported by work with Avena fatua, which demonstrates that GA is involved in dormancy loss (although it can be used to break

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dormancy in this case), but is involved in initiating germination (Fennimore and Foley, 1998). In cereals, QTL studies have been used to try and track down important loci for dormancy. One gene of great significance is VP1 (McCarty et al., 1991). VP1 is a transcription factor that promotes dormancy in the presence of ABA (Cao et al., 2007). Mutations in this gene lead to a loss of function that results in vivipary where seeds will germinate while still on the plant (also known as preharvest sprouting). This gene has been identified in sorghum, wheat, rice, barley and oats (Hattori et al., 1994; Jones et al., 1997; Bailey et al., 1999; Carrari et al., 2003; Osa et al., 2003). In wheat, the incorrect assemblage of exons (mis-splicing) of VP1 messenger RNAs is responsible for the nonfunctional types leading to vivipary (McKibbin et al., 2002). Interestingly, these mutants in hexaploid wheat appeared to have been inherited from their tetraploid ancestors, thus implying that they were established early on in the development of agriculture. The VP1 gene is up-regulated by ABA, and has the function of promoting dormancy (Cao et al., 2007). The functional VP1 gene also activates an anthocyanin pathway resulting in pigmented seed coats, which have long been associated with functional dormancy in wheats (Flintham and Humphry, 1993). The VP1 and seed coat colour (R) genes are loosely linked which may also partly explain the correlative effect to dormancy – efforts are being made to increase dormancy of white-grained wheats (Kottearachchi et al., 2006). There are several other genomic regions, which are important in dormancy, which have been identified by QTL analysis, but these genes’ loci have yet to be identified (e.g. Wan et al., 2005; Vanhala and Stam, 2006; Hori et al., 2007; Gao et al., 2008). The physical dormancy imposed by seed coats is thought to be largely associated by ␤-1,3-glucans (callose) which are deposited in cell walls (the neck regions of plasmodesmata) during maturation (Finch-Savage and LeubnerMetzger, 2006). Increased callose deposition is associated with increased dormancy in a number of species. The ␤-1,3-glucanases which break the callose down are associated with the dormancy release. It is likely that mutations in genes associated with these pathways are involved in the domestication processes that are associated with weaker dormancy by seed coat thinning as seen in pulses. This may also be true in other families of domesticates such as Amaranthaceae, Chenopodiaceae and Pedaliaceae/Martyniaceae (Sesamum, Proboscidea). In the case of lentil, a single dominant gene is reported to be related to the hard seed coat in non-germinating wild-types (Ladizinsky, 1985).

7.10

Germination and seedling competition: changes in seed size

‘. . . we must conclude that man cultivated the cereals at an enormously remote period, and that he formerly practiced some degree of selection, which in itself is not improbable. We may, perhaps, further believe that, when wheat was first

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Fruit Development and Seed Dispersal cultivated the ears and grains increased quickly in size, in the same manner as the roots of the wild carrot and parsnip are known to increase quickly in bulk under cultivation.’ Charles Darwin (1883, p. 338)

Changes in size are one of the most widely commented on and obvious differences between domesticated and wild varities (e.g. Darwin, 1883; De Candolle, 1885, p. 460; Helbaek, 1960). While this is one trait that might be suggested to be under conscious and intentional selection on the part of humans, what Darwin termed ‘methodical selection’, there remains no clear evidence that this was the case in prehistory when seed sizes changed under cultivation. Heiser (1990, p. 199) concluded that it is ‘more likely that large seeds result from unconscious selection over a period of time,’ because it was unlikely to be easy to select for as it is thought to be the product of the interaction of many genes. In a recent comparative review, it was shown that changes in seed size did not happen at a consistent rate or timing in relation to the beginnings of cultivation or other domestication traits (Fuller, 2007a). This suggests in turn that certain factors in cultivation regimes interact with the inherent variability and genetic architecture of seed size traits within particular taxa in ways that are not uniform across taxa. Changes in size are not qualitative traits of domesticates, like non-shattering is, or to a certain extent that even ease of germination tends to be. Thus, it has been suggested that the trait be regarded as ‘semi-domestication’ as it is a quantitative population level trait that is selected at some stage during human cultivation but not necessarily linked directly to key domestication traits. Seed size and other such traits constitute a kind of soft selection in relation to the cultivated environment and probably a high degree of population variability built on a multi-genic basis. In this regard, it is of interest to document the extent to which grain-size increases precede hard-selected domestication traits, like non-shattering or loss of germination inhibition, as seems to be the case in wheat, barley and possibly rice; or whether size increase is later as appears to be the case in pulses and Pennisetum glaucum (Fuller, 2007a). These differences may point towards the underlying selective pressures in the soil environment. The arable field has been called a ‘botanical battleground’ (Jones, 1988), and this is true not only between crops, farmers and weeds but also within species in the form of seedling competition. Well-tilled and cleared fields offer nutrients, abundant sunlight and normally plenty of water, and thus competition should be expected to favour seeds that not only germinate rapidly but also establish rapidly and even overtop competition. This tilled field competition, including factors of both general disturbance and depth of burial, can be expected to select for larger seed size (Harlan et al., 1973; Harlan, 1992, p. 122; Fuller, 2007a). As studies of weed seed ecology have shown, there is a variation between species in terms of ideal and tolerable depths of germination (King, 1966, pp. 138–140), and this means that weed communities have been

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heavily influenced by human tillage practices that have established differing average depths of burial. This is indicated by studies within species and between species that suggest a correlation between larger seeds and larger seedlings (Krishnasamy and Seshu, 1989; Harlan, 1992, p. 122; Baskin and Baskin, 2001, p. 214). Comparative ecology indicates that larger seeds generally have competitive advantages over smaller seeds under certain kinds of competition including deeper burial (Maranon and Grubb, 1993; Westoby et al., 1996; but there are some apparent exceptions amongst grasses (Baskin and Baskin, 2001, pp. 212–213). Oka and Morishima (1971) showed in experimental cultivation of wild rice that some increase in average grain weight could be measured within just five generations, in the cultivation of wild perennial rice (O. rufipogon). An old agronomic rule of thumb is that seeds germinate best at depths up to four times the diameter of the seed (King, 1966, p. 140). Since archaeology mainly recovers the seeds of crops, archaeobotanical evidence lends itself to studies of variation and changes in sizes through time. Nevertheless, caution is warranted in interpreting such evidence as domesticated crops often include a much greater range of grain size variation than is found in wild species (Harlan et al., 1973; Vaughan et al., 2008). While study of seed size is the most readily available domestication trait in archaeological evidence, it is complicated by some confounding factors. Preserved seed size may be affected by the state of archaeological preservation: most archaeological seeds are preserved carbonized, by exposure to fire, and this has been shown to distort seed shape but especially to lead to shrinkage (e.g. Helbaek, 1970; Van Zeist and Bakker-Heeres, 1985; Lone et al., 1993; Braadbaart et al., 2004). Nevertheless, if it is assumed that most archaeological seeds have been affected in a similar manner, then real trends can be inferred from the data. Experiments provide some general guidance on the probable range of correction factors for comparing modern seeds, although the usual 10–20% shrinkage that is suggested is by no means a given. Another potential problem is that past crops may have been harvested before all seeds were mature and immature seeds may resemble smaller versions of the more mature seeds. For example, on the basis of growing experiments with a number of pulses, Butler (1990) concluded that seed size could be misleading: ‘If harvesting is confined to one episode, the seeds constituting the crop are not all in the same state of maturity. This may be reflected in their size; smaller, slightly immature seeds may be present together with the full-sized ripe ones. Commonly, it seems, the number of fruiting nodes per branch is two, which bear seeds at two stages of maturity at any one time. If these are harvested together, the impression may be formed that the seeds have been derived from two different populations or even different taxa. This could lead to erroneous identifications such as the seeds of a cultigen occurring together with those of its wild relative.’ E. A. Butler (1990, p. 350)

Similar concerns over the likelihood of immature harvesting of wild rice and early rice crops were discussed by Fuller et al. (2007a; also Fuller, 2007a).

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Fruit Development and Seed Dispersal While such concerns make it dubious to identify single archaeological grains as domestic or wild only on the basis of size, it nevertheless still appears useful to examine the metrical traits of populations (site assemblages and assemblages from across a region), as these appear to show real evolution trends over time. There is a growing morphometric database for wheat and barley from the Near East (Colledge, 2001, 2004; Peltenberg et al., 2001; Willcox, 2004). This indicates that wheat and barley grains increased in size starting in the Pre-Pottery Neolithic A (PPNA) and earliest PPNB. This is before clear and widespread evidence for tough rachises and loss of natural seed dispersal. It is well known that wild and domesticated cereal grains differ in size and this has been used to infer the domesticated status of cereals, already in the PPNA and the earliest PPNB, including sites from the Jordan Valley, the upper Euphrates in Syria, and the first settlements on Cyprus (Colledge, 2001, 2004). This evolutionary shift can be illustrated from evidence from individual site sequences, such as at Jerf el Ahmar (Willcox, 2004), in which a contrast is seen between the barley grains from the early phase at Jerf el Ahmar (9500–8800 BC) and the later phase at Jerf el Ahmar, ca. 8500 BC (Fig. 7.9). The grains of the later phase are comparable to those from the Chalcolithic Kosak Shimali (ca. 5500 BC). If such data are plotted as means and standard deviations against time, the long-term trend is clear (Fig. 7.10): an early increase in grain thickness and breadth followed by a remarkable stable grain size from 6000 BC onwards. Nevertheless, an explanation of these data remains controversial. We take this to indicate evolution towards larger grain size during the occupation of this site (Fuller, 2007a; also, Nesbitt, 2004, p. 39), whereas Willcox (2004), by contrast, queries whether this is not just a product of better tended cultivars or the introduction of larger grained varieties from elsewhere (see also Willcox et al., 2008). This early change is indicated in seed width and thickness, but not in seed length. While Willcox (2004) argues that this does not fit with evolution of larger grains under cultivation, we think that a comparative perspective indicates quite the opposite. As was hypothesized by Harlan et al. (1973), grain size should increase as a product of soil disturbance and deeper burial with cultivation, and this has an established observational and experimental basis in seed ecological studies (e.g. Krishnasamy and Seshu, 1989; Maranon and Grubb, 1993; Baskin and Baskin, 2001, p. 214; see also experimental cultivation by Oka and Morishima, 1971). However, rather than seeing this as a single directional process, we must consider the likelihood that there were differing selective thresholds that acted on grain size (and multiple contributing genetic loci) at different times. This is suggested by comparative examples, such as West African pearl millet in which an initial grain thickening occurred, but increase in grain size (mainly in length and, allometrically, in width) only happened much later, in regions and periods with more intensive agriculture.

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Figure 7.9 Scatter plots of archaeological grain measurements showing the increase in grain size under early pre-domestication cultivation (after Willcox, 2004). (a) Barley grain measurements, comparing early Pre-Pottery Neolithic A Jerf el Ahmar with the much later domesticated material from Kosak Shimali. (b) Comparing early and late Jerf el Ahmar, indicating that shift towards larger grain size had already occurred. (c) Similar comparison of einkorn grains (probably including some rye grains) at early Jerf el Ahmar and Kosak Shimali. (d) Trend towards larger grain sizes over the course of Jerf el Ahmar occupation.

In the case of pearl millet, we have some metrical data from West Africa from which to examine grain size change during and after domestication, with some comparative data from ancient India (Figs. 7.11 and 7.12). Data sets for looking at morphometric traits of past African populations of pearl millet have only been published recently, since 2000. As already noted, pearl millet domestication is evident from ceramic impressions of pearl millet chaff that include the stalk, which are present by ca. 2500 BC in northeast Mali (unpublished data), and 1700–1500 BC in Mauretania (Amblard and Pernes, 1989; MacDonald et al., 2003; Fuller et al., 2007a), and slightly later in Nigeria (Klee et al., 2000, 2004). Early grain assemblages of similar date show the subtle change in grain shape, becoming apically thicker and more club-shaped

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Figure 7.10 Time series of archaeobotanical metrical data on charred barley grains. Data plotted on the basis of a median age estimate for each site in calibrated radiocarbon years. Lines indicate standard deviation and minimum and maximum outliers are also indicated. Sites (in chronological order): Jerf Early, ZAD 2, Jerf Late, Djade, Ganj Dareh (no thickness data), Ramad, Bouqras, Erbaba, Kosak Shamali, Selenkhiye, Hadidi, Rosh Hiyat. Where standard deviations were not provided in published sources, these have been estimated after the normal distribution following Pearson and Hartley (1976).

(D’Andrea et al., 2001; Zach and Klee, 2003). However, a major increase in seed size appears delayed (D’Andrea et al., 2001, p. 346; Fuller, 2007a). Of note is that early West African populations, from the second and first millennia BC, have their averages firmly in the wild size range, although there are long tails of variation that extend into the larger size range (e.g. at Birimi, Ghana).

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Figure 7.11 A map of archaeobotanical sites with important pearl millet data in Africa in relation to probable West African domestication zones. Later ‘historical’ sites post-date 100 bc, and represent only a selection with metrical data used in Fig. 7.12 (Indian sites not shown). Site numbers are as follows: 1. Dhar Tichitt sites; 2. Dhar Oualata sites; 3. Djiganyai; 4. Winde Koroji; 5. Karkarichinkat; 6. Ti-n-Akof; 7. Oursi; 8. Birimi; 9. Ganjigana; 10. Kursakata. Historical sites with pearl millet metrical data: 11. Arondo; 12. Jarma; 13. Qasr Ibrim. (Primary data sources compiled in Fuller, 2007a.)

One of the earliest finds of pearl millet from India comes from Surkotada, Gujarat, ca. 1700 BC, which can be seen to fall with these early domesticated African populations. By contrast, rather later seeds of a North Indian (Gangetic) population from Narhan are markedly larger, suggesting selection for larger grained pearl millet. On basis of Vigna pulse size increase in the same horizon, it was suggested that selection for larger grains may be driven by deeper seed burial through the use of ard tillage (Fuller and Harvey, 2006; Fuller, 2007a). However, the continued small-grained populations in Early Historic South India (Nevasa) suggests that there may be factors that work against gigantism in pearl millet, and in the absence, reinforcing selection populations may retain or even revert to smaller size ranges. In Africa, larger grained populations appear only in the First Millennium AD, represented by finds from Nubia and Libya, as well as Medieval Senegal. This raises questions about the selection pressures involved in largegrained Pennisetum, and in seed crops generally. While initial cultivation

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Figure 7.12 Metrical data for archaeological pearl millet, and expectations from modern reference material. Clockwise from lower left: Modern population averages for wild (W) and domesticated (D) pearl millet, showing population minima for domesticates and maxima for wild, with all measurements reduced by 10% to account for expected shrinkage in charred specimens (data from Brunken et al., 1977 and Zach and Klee, 2003); this division is indicated in other graphs by dashed box. Plots of archaeological site averages and ranges for early West African sites (Birimi, 1700–1500 bc; Kursakata, 1500–800 bc), medieval Senegal at Arundo, and Qasr Ibrim, Nubia (preserved by dessication and thus reduced by 10%); plots of early measurements from India (Surkotada, approximately 1700 bc) are close to wild or African Neolithic, as are Early Historic (200 bc–ad 300) Nevasa in southern India. North Indian Narhan (1400–800 bc) shows a marked shift towards larger sizes comparable with modern domesticates; plot of measured grains from Jarma in Southwest Libya may show an apparent shift towards somewhat larger grains during the early first millennium CE, but Later Medieval Jarma has shifted back towards to near wild size range. (Primary archaeological data sources compiled in Fuller, 2007a.)

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may have selected for non-shattering, and slight changes in grain weight and shape (the club shape), serious gigantism may have required a stronger selection pressure and therefore evolved later: a millennium or more later in India, and two millennia later in Africa. As both Libya and South India lack wild populations, this cannot be attributed to cross-pollination with wild-types. There may be some constraints particular to this crop, as one experiment indicates that optimal germination occurred under higher temperatures that result in lower average grain weights (Mohamed et al., 1985). In addition, pearl millet involucres are polymorphic in grain count with the vast majority producing two grains, a large minority with one larger grain, and a further minority producing three to nine grains, which are necessarily smaller (Godbole, 1925). Thus selection for higher grain counts, and more reliable germination, might conflict with selection for larger seed sizes. Nevertheless, as a working hypothesis, it is proposed that there is a deeper burial threshold that selected for gigantism in pearl millet in some times and places (Fuller, 2007a). If so, then large-grained varieties evolved under plough systems and then dispersed back to West Africa at a later date. In that regard, it might be noted that the larger grain populations in Libya and Nubia, like that in Gangetic India, are associated with more intensive plough cultures. This suggests separate events of grain enlargement in India and northeastern Africa. Beyond informing us about pearl millet, this case provides useful comparison to other cases of plant domestication, including that in the Near East. It suggests that we need to consider different aspects of the domestication syndrome separately, even different aspects of grain shape and size change. A lag between domestication and any appreciable seed size increase appears to be the case in several tropical pulses, including Indian Vigna (Fuller and Harvey, 2006; Fuller, 2007a), and West African Vigna unguiculata (D’Andrea et al., 2007). This may suggest that a higher selective pressure was needed to cross the threshold into big-seeded pulses; a threshold inferred by Fuller and Harvey (2006) to be ploughing (ard tillage). Perhaps, a similar effect created a lag time between initial grain thickening in cereals, associated with the earliest cultivation, and more marked grain size increase in all dimensions, including length. In Near Eastern lentils, size change appears to have been much slower and more gradual than in the cereals, without a clear levelling off after the Neolithic (Fig. 7.13). This may suggest an initially weaker selection, but may also indicate that seed size is more plastic in pulses. The genetics of seed/grain size is still poorly understood, but it is presumed to be under polygenic control. This has been documented in Lens (Abbo et al., 1991) and Pisum (Weeden, 2007). In the case of rice domestication, the utility of grain measurements is hotly debated (Thompson, 1996; Crawford and Shen, 1998; Fuller et al., 2007a; Liu et al., 2007). As modern comparative data indicate, there is a vast range of metrical variation in domesticated rice (Fuller et al., 2007a; Vaughan et al., 2008), and some of this variation seems to be correlated with climatic conditions

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Figure 7.13 Time series of archaeobotanical metric data on charred lentil seeds. Data plotted on the basis of a median age estimate for each site in calibrated radiocarbon years. Lines indicate standard deviation and minimum and maximum outliers are also indicated. Where standard deviations were not provided in published sources, these have been estimated after the normal distribution following Pearson and Hartley (1976). (Compiled from a large number of primary archaeobotanical reports by Jupe, 2003: From left to right, sites include Qermez Dere, Murreybit, Jericho, Aswad, Jericho, Ganj Dareh, Yiftah’el, Aswad, Ain Ghazel, Basta, Ramad, Ali Kosh, Ras Shamra, Jericho, Tepi Sabz, Beth Shean, Jericho, Lachish, Arad, Tell Bazmosian, Jericho, Hadidi (some sites occur more than once representing different phases))

such as altitude or latitude (Oka, 1988; Kitano et al., 1993): more northerly temperate japonica landraces are short grained, while tropical varieties (the javanica race rices) are massively long; in East Asia, upland rices tend to be longer grained versus shorter grained lowland forms (Nitsuma, 1993). Such problems are further compounded by variation in grain measurements that may relate to maturity, especially as wild rice and early cultivars are likely to have been harvested somewhat immature to increase total harvests and because of uneven ripening (Fuller, 2007a; Fuller et al., 2007a). For this reason, it is probably safest to focus on changes through time within a fairly restricted region, or even within individual stratigraphic sequences (Fuller et al., 2008). On the left hand side of Fig. 7.14 is a time series of grain width data from the Lower Yangtze region (Chinese provinces of Zhejiang and Jiangsu), whereas on the left hand side later are the data from the Yellow River valley further north where climatic conditions may have both selected for smaller rice and reduced the reliability of harvests and yields (thus causing the incorporation of more immature or poorly formed grains). While the metrical data vary between regions, within a particular region (the Lower Yangtze), often postulated as a probable centre of domestication and trajectory of increasing grain size is visible. Changes in grain size have played an important role in documenting past domestications in oilseed crops as well, including an extinct species form North America. Achene size is an important domestication trait of the sunflower (Helianthus annus) and archaeological documentation of this indicates domestication taking place by ca. 2000–1500 BC in North America (Asch and Asch, 1985; Smith, 2006b; cf. Heiser, 2008). The most extensively documented

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Figure 7.14 Time series of archaeobotanical metrical data on charred rice grains. Data plotted on the basis of a median age estimate for each site in calibrated radiocarbon years. Lines indicate standard deviation and minimum and maximum outliers are also indicated. Grey arrows indicate suggested selection trends with domestication; over the same period, selection for non-shattering is indicated by spikelet base data. (Huang and Zhang, 2000; Tang, 2003; Zheng et al., 2004; Liu et al., 2007; Tian Luo Shan: D. Fuller, unpublished.)

increase in seed/fruit size in North America is that associated with marsheldar, Iva annua. Although this species is not known to have been cultivated within historically documented periods, it was a major domesticate of the eastern North America from ca. 2000 BC, alongside the native Chenopodium, Hordeum pussilum and Helianthus annus. Indeed, it is the documentation of potential morphological indicators of domestication traits related to germination that has allowed the reconstruction of indigenous cultivation in Eastern North America (Smith, 1989, 1992, 2006b).

7.11

The genetics of seed size

The quality of seed size is a trait that is affected by many factors, and so can be thought of as polygenically controlled. Moreover, the regulatory networks involved with governing seed size, either directly or indirectly, are not at all well known. This is borne out by the many QTL analyses that have been carried out which have shown seed size to be associated with many loci of varying effect (Gupta et al., 2006). Again, it is rice that is leading the way to elucidation of what these loci might be doing. At the time of writing, three genes derived from principal QTLs have been identified in rice that directly influence grain size (Fan et al., 2005; Song et al., 2007; Shomura et al., 2008). All three are loss of function mutations. Two result in an increase in the number glume cells, thereby giving the grain milk a larger cavity to fill, resulting in larger grains which are wider (Song et al., 2007; Shomura et al., 2008). The first of these, GW2,

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Fruit Development and Seed Dispersal occurs on chromosome 2 and encodes an ubiquitin ligase that may possibly be involved with negative regulation of the cell cycle. The larger grained phenotype is associated with an allele that has a mutation causing a premature stop codon resulting in a non-functional truncated protein, which may result in an inability to down-regulate cell division in the glume. The second, qSW5, occurs on chromosome 5. In this case, the large grain phenotype is associated with a large deletion in the gene, again resulting in more cells in the glume. It remains to be seen whether these two loci are actually interacting with the same process of grain development, probably at different stages. The third locus, GS3, affects grain length and size rather than width (on which it has a small effect). This gene occurs on chromosome 3 and encodes a transmembrane protein of unknown function. The protein normally has four domains: a PEBP-like (phosphatidylethanolamine-binding protein) domain, a transmembrane region, TNFR/NGFR (tumour necrosis factor receptor/nerve growth factor receptor), cysteine-rich domain and a VWFC (von Willerbrande factor C) domain. The PEBP-like domain is partially deleted in proteins associated with longer and heavier grains. The authors assert that the gene could be involved with regulating grain growth, which is to say they suppose this might be a direct influence rather than the more indirect consequences on grain size that glume cell number has in the previous two loci. These early glimpses into grain size regulation confirm the expectations that grain size is influenced by many factors that may only be indirectly concerned with grain size itself, and part of as yet uncharacterized networks of interaction. In the case of barley, which has QTLs affecting grain mass across all seven chromosomes, the largest effect is linked to the Vrs-1 locus, which determines the row architecture (Marquez-Cedillo et al., 2001; Komatsuda et al., 2007). In this case, the genetic control of grain size is quite indirect. The grains of two-row barley are fatter than those of six-row, most likely because they have more space in which to develop. Similarly, wheat and maize have QTLs associated with increasing grain mass on all chromosomes (Gupta et al., 2006), and pleiotropic effects of loci being involved with both grain weight and ¨ plant height are evident from recent studies (Maccaferri et al., 2008; Roder et al., 2008). Undoubtedly, many of these will be tracked down to genes in the coming years. Gupta et al. (2006) cite only one gene in wheat, three in barley and two in maize to be involved with grain size. As with the elucidation of rice outlined above, these are both directly and indirectly involved with grain development. Examples of directly involved genes include the crinkly4 (Becraft et al., 1996) and mn1 (Carlson et al., 2000) in maize. The pseudoresponse regulator ppd1 in both wheat and barley is also associated with grain size, but is likely to be an indirect effect through day-length sensitivity altering the developmental time of grain maturation. The number of loci governing seed size appears to be equally large in legumes as with cereals with as many as ten QTLs governing the trait in peas (Blair et al., 2006), and between three and nine QTLs in azuki bean (Kaga et al., 2008). Only four

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such QTLs were identified in chickpea (Cho et al., 2002), but very smallseededness has been shown to result from interaction of two recessive genes (Upadhyaya et al., 2006). Again, very little is currently known about which genes are responsible.

7.12

Seasonality controls: photoperiodicity and vernalization

Another set of key changes with domestication are the controls over the seasonality of crop harvests and planting: photoperiodicity and vernalization. In many plant species, the seasonality of flowering and hence of seed set is controlled by environmental cues such as day length, thus species can be divided into long-day and short-day plants. As discussed by Willcox (1992), there tends to be an important difference between the crops domesticated in the Near East and those of the Old World tropics, such as those of India or the African savannas. The tropical crops tend to be grown in summer and adapted to monsoon rainfall, flowering as days shorten after summer. By contrast, those of the Near East were originally tied to winter rains. The importance of seasonality of cultivation and the changes in seasonality between different regions has been a focus of much discussion in archaeological circles, since differences in seasonal potential of different regions might serve to create environmental frontiers that limited the spread of certain crops into certain regions (e.g. Sherratt, 1980; Halstead, 1989; Bogaard, 2004, pp. 160–164; Kreuz et al., 2005; Fuller, 2007b, p. 405; Conolly et al., 2008). On morphological grounds, there is no basis for distinguishing the seasonality of archaeological crop remains, although archaeobotanists have made some progress in inferring seasonality from associated non-crop weed remains. Nevertheless, there remain debates, for example as to whether or not the earliest agriculture in central Europe was autumn sown, and grown over the winter, or spring sown and grown in the summer (e.g. Jacomet and Behre, 1991, p. 86; Bogaard, 2004, pp. 160–164; Kreuz et al., 2005). As crops spread northward into new latitudinal bands, with longer and colder winters, cultivation may have become increasingly difficult over the traditional winter season. Wetter and cooler summers may also have precluded certain species, as appears to be the case with lentils and chickpeas (Conolly et al., 2008). As a result, crops either had to evolve adaptations to surviving cold winters, such as through vernalization in which autumn sown crops have a pause in growth during the frost months and then resume growth in the warming of spring; or else their original seasonality of flowering had to be switched off, allowing them to be planted in spring and grown through the summer, thus flowering during shortening days rather than long days. Recent years have seen substantial developments in understanding the genetic basis of vernalization and photoperiod sensitivity in cereals.

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Figure 7.15 Diagrammatic representations comparing the regulatory gene networks involved in vernalization and photoperiodicity, as inferred for wheat/barley, rice and Arabidopsis. For sources, see text.

The emergent picture of the genetics of seasonality in various plant groups gives a fascinating insight into regulatory network evolution. This example shows how fluidly regulatory networks change over time often retaining core components, but not necessarily with the same functionality. The scientific community has been of the opinion for some time that the vernalization response evolved in parallel in grasses and in eudicots as exemplified by Arabidopsis thaliana. This opinion is borne out by phylogenetics which supports the idea that the ancestral grass type was more like the panicoid group which is adapted to tropical conditions being short-day plants with no vernalization response (Kellogg, 1998). However, recent identification of the major components of the vernalization response in grasses shows that much of the same molecular apparatus is utilized between the groups (Fig. 7.15). In wheat and barley, three principal components to the vernalization system have been discovered, named VRN1, VRN2 and VRN3 (Yan et al., 2003, 2004a, 2004b, 2006). In an unfortunate clash of nomenclature, it should be noted that these are not directly comparable components to the similarly named VRN1 and VRN2 in A. thaliana. While the latter two work in synergy at a different point in the vernalization pathway, VRN1 and VRN2 in cereals work antagonistically. All these genes have been named so because of their direct effects on phenotype through which they were originally discovered. As with many of the relationships shown in the network diagrams of

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Fig. 7.15, the interactions have been correlatively determined between genes in the cereals; consequently, they may be either direct or indirect through other as yet undetermined factors. That some of these interactions at least are likely to be indirect, or an incomplete description of the regulation due to other factors, is evident from some inconsistencies in the network relative to observations that will be pointed out below. In barley and wheat, VRN2 represses VRN3 and VRN1. VRN3 acts to promote VRN1, which in turn promotes flowering and further down-regulates VRN2 that would otherwise resume action in the absence of short days or cold temperatures. Short days or cold temperatures inhibit the action of VRN2 (Dubcovsky et al., 2006). It is not likely that VRN2 is involved directly in sensing such environmental cues. The inhibition of VRN2 is not in itself enough to cause up-regulation of VRN3 and subsequently VRN1. After VRN2 inhibition, VRN3 up-regulation is induced by long days, through the action of a fourth important intermediary Ppd1. Through this set of interactions, temperate cereals have evolved a system in which they must experience either short days or cold followed by longer days before flowering so ensuring that flowering is delayed through the winter and initiated in the spring. This winter habit is the ancestral condition, and suited to the climate of biogeographical range of the wild progenitors. The spring habit has evolved several times and in several ways from this regulatory network involving degenerative mutations at each of the three main loci. The action of VRN2 is to repress VRN1 and VRN3 through the action of a CCT domain in VRN2, a domain type found in the CO-like group of genes in A. thaliana. A mutation causing a R/W amino acid change at a conserved position in this domain results in a lack of repression (Robson et al., 2001; Cockram et al., 2007a) causing a phenotype in which flowering is triggered by long days without the need for vernalization. This mutation makes a recessive allele (vrn2) because in the heterozygous condition the functioning allele will still achieve repression. A similar phenotype is also caused by naturally occurring deletions at the VRN2 locus (Dubcovsky et al., 2005). A spring phenotype is also caused by mutations at the VRN1 locus (Yan et al., 2004b; Fu et al., 2005). There appear to be two sites at the VRN1 locus that are involved with repression of the gene by acting as receptors to the VRN2-mediated repressors. One is in the promoter region, and the second within the first intron. Deletions at either of these sites result in a lack of repression of VRN1 by VRN2. This time the mutant allele is dominant (Vrn1), because in the heterozygous condition, even though the wild-type responsive allele is repressed successfully by VRN2, the receptor region deleted allele will still initiate flowering. The resulting phenotype is similar to that obtained with the vrn2 allele in that vernalization is not required for long days to initiate flowering. Interestingly, a range of large deletions have occurred in intron 1 both in barley and wheat, which indicate that both cereals have achieved the spring phenotype independently, but through the same underlying mechanism (Fu et al., 2005). There are now known to be a large range of

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Fruit Development and Seed Dispersal combinations of VRN1 and VRN2 alleles possible, with 17 haplotypes occurring in the European barley germplasm of which only one winter and two spring types dominate 79% of varieties (Cockram et al., 2007b). The VRN3 locus also has a regulatory element in its promoter through which VRN2 represses its action. In this case, a dominant mutant allele Vrn3 occurs in wheat that has a retrotransposon in the promoter rendering it insensitive to VRN2 repression, again resulting in a similar phenotype to the mutants described above (Yan et al., 2006). Although VRN3 completes the network of interaction, as described in Fig. 7.15, it is not likely to be the complete story. The current understanding shown in the diagram implies that the clear association between Vrn1 and spring phenotypes could not have been discovered unless the Vrn3 genotype was also in place. Indeed, germplasm surveys have already indicated that wherever Vrn3 occurs Vrn1 is also found. However, Yan et al. (2006) argue that a mutation in the regulatory region of either gene is enough to initiate the flowering cascade. A fourth important locus in the seasonality of temperate cereals is Ppd1, which builds on the spring phenotype produced by mutations at the vernalization loci. Ppd1 is responsible for initiating signal cascades in response to long days (Turner et al., 2005). There are differing Ppd1 mutants in barley and wheat, respectively. In barley, a SNP causes the ppd-H1 (recessive) mutant, which is insensitive to long days resulting in delayed flowering. In most of the wild biogeographical range, this phenotype is selected against, since the growing season is short followed by a hot dry summer that the late flowering plants would find difficult to survive in. However, further north, the potential growing season is much longer with wetter summers. Spring varieties grown in northern temperate latitudes benefit from the ppdH1 mutant that has a longer vegetative phase resulting in more resource sequestration and so in larger grain yields. This mutation appears to have arisen within the domesticated barley gene pool east of the Fertile Crescent as crops moved in to more northern latitudes (Jones et al., 2008). The known Ppd1 mutants in wheat result in a different phenotype to that of barley, probably due to different underlying mutations which appear to involve large promoter region deletions (Beales et al., 2007). In the case of wheat, the Ppd1 mutants are dominant resulting in floral initiation regardless of day length. These early flowering types appear to do well under conditions in southern Europe, but less well in more northerly latitudes (Worland et al., 1998). The regulatory networks for vernalization between the temperate cereals and Arabidopsis thaliana are remarkably similar. Orthologous components are utilized in each – VRN3 in wheat and barley is orthologous to FT, and VRN1 is orthologous to AP1. Wheat and barley also have versions of the GI and CO genes which are established to act upstream of FT in A. thaliana, and are likely to prove the same in cereals. However, there is no identifiable gene orthologous to FLC in cereals, nor is there anything like the cereal VRN2 in A. thaliana. However, these two genes act in a highly similar way in terms of the network of relationships. It seems that a similar network solution has

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been converged upon in temperate cereals and the eudicot lineage represented by A. thaliana. In the cereals, VRN2 appears to have evolved at least in part from a CO-like ancestor, whereas in eudicots, FLC originated from another MADS-box gene (Zhao et al., 2006). However, there may be more to discover in the cereals. A. thaliana has a second ‘FLC independent’ vernalization pathway in which VIN3 is up-regulated during cold spells and activates AGL24 which initiates the flowering cascade (Michaels et al., 2003). VIN3 also has the function of repressing FLC. Curiously, a well-conserved version of the VIN3 gene has also been shown to be up-regulated by vernalization in wheat (Fu et al., 2007). There is also a group of genes orthologous to AGL24 in wheat (Zhao et al., 2006). Rice and the panicoid grasses such as maize do not have vernalization adaptations. These grasses are naturally adapted to the tropics to flower under short days. Very little is known yet about maize, although mutations associated with early flowering have been identified (Chardon et al., 2005). It is likely that the regulation of flowering time in this group of grasses is well represented by rice about which a great deal has emerged in recent years. The emergent picture of regulatory interactions that govern rice flowering shows striking similarities and differences to the cereal network. Under conditions of long days, both Hd1, orthologous to CO, and Ghd7 repress Hd3a, which is orthologous to FT (Yano et al., 2000; Kojima et al., 2002; Hayama et al., 2003; Xue et al., 2008). The function of Hd1 is surprising in this instance, because it acts in the opposite way to its orthologous counterpart in Arabidopsis. However, it appears that Hd6, which is also known to have a repressive effect on flowering time under long days, may be acting in conjunction with the Hd1 complex to repress Hd3a (Yamamoto et al., 2000; Takahashi et al., 2001; Ogiso et al., 2007). Ghd7, most closely related to VRN2 in cereals (Xue et al., 2008), also represses Ehd1 that would otherwise initiate flowering (Doi et al., 2004), resulting in a regulation reminiscent of VRN2 and FLC. Interestingly, Ghd7 also appears to have pleiotropic effects on plant size and grain number, leading to increased growth, cell proliferation and differentiation. As days become shorter, during the monsoon season, the actions of both Hd1 and Ghd7 change. Ghd7 promotes Ehd1 (Xue et al., 2008) and Hd1 promotes Hd3a (Yano et al., 2000). Possibly, this apparent return of Hd1 to a function more normally associated with a CO orthologue may represent a release of action by Hd6. Once Hd3a has been promoted, the flowering cascade is initiated. Photoperiod sensitive rice (and maize) crops are restricted to tropical areas, because the growing season further north is too short. Photoperiod insensitive varieties of rice that can be grown in more temperate conditions further north are associated with less or non-functioning alleles, and the earliest rice varieties actually have Ghd7 deleted, resulting in a strong latitudinal cline of Ghd7 alleles (Xue et al., 2008). Consequently, not only is the resemblance between the wheat/barley and rice regulation striking, the point at which mutations causing loss of functionality to induce seasonal insensitivity also coincide in VRN2 and Ghd7.

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7.13

Discussion: evolution and development of domesticated seed traits

The domestication of crop plants represents numerous trajectories of parallel evolution, with some instances of true convergence as plants adapted to the selective pressures brought to bear by human farmers. The major selective pressures were associated with the dispersal and establishment of the next generation of seeds, from dispersal mechanism (the shift to human harvesting), seed germination (shifts away from dormancy and changes in seasonal controls on germination), and seedling establishment. In most cases, these selective pressures were initially unconscious on the part of people, as recognized by Darwin and his successors (e.g. Darwin, 1883, Chapter 20; Zohary, 1969; Darlington, 1973, p. 155; Harlan, 1992). Since human food production as a behaviour follows similar patterns for similar aims, the selection pressures with domestication have often been similar. There has been some debate, and perhaps, confusion over whether to regard the similar domestication outcomes as products of convergence or parallelism. As clarified by the definitions of Niklas (1997, pp. 303–305) and Gould (2002, pp. 81–82, 1076–1089), convergence is a case of analogy when unrelated organisms produce similar morphological ends (adaptations) in different ways. By contrast, parallel evolution is when related organisms evolve similar adaptations from the same ancestral mechanism or underlying developmental/genetic architecture: this represents selection working on existing developmental constraints that are shared across species. Parallelism like phylogenetic/historical homology is a form of syngeny (generative homology) (as defined in Butler and Saidel, 2000). Seen in these terms, there are clear cases of both parallelism and convergence in domestication: orthologous loci, such as some loci regulating flowering show parallel evolution (e.g. Vrn3 in Hordeae, Hd3a in rice, FT in Arabidopsis; see Fig. 7.15); by contrast, the loci involved in cereal shattering differ between wheat/barley and rice such that they have evolved along multiple non-orthologous genetic paths, and thus represent allogeny or generative homoplasy (as defined in Butler and Saidel, 2000). Such nonorthologous means of achieving similar adaptations are convergent in the sense used here (but note that Paterson et al., 1995 argued for ‘convergence’ in the sense of parallelism as used here; a hypothesis now falsified by further work: Li and Gill, 2006; and see above). A key area of evolution under domestication involved changes in seed dispersal, taken broadly to include the timing of fruiting, the mode of dispersal, and patterns of germination. In recent years, much scientific progress has been made in understanding these evolutionary processes through the efforts of archaeobotany and through genetics. Archaeological plant remains (archaeobotany) can provide hard fossil evidence for the rate and extent of evolution in those morphological traits which are prone to archaeological preservation, especially in charred seeds or cereal chaff (rachises and spikelet bases). Genetics, starting from QTLs and moving onto sequencing studies,

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allows the identification of the coding gene regions and the developmental pathways involved in these domestication traits, how many different ways and times these have evolved and potentially provides a framework for inferring aspects of the geographical history of these traits. We expect that soon association mapping studies will provide important new insights about linked syndromes of genetic adaptation, as suggested by recent work in Arabidopsis (Aranzana et al., 2005), and the extent to which genetic linkage evolved during the domestication process (cf. D’Ennequin et al., 1999) or was part of pre-existing wild variation. In both archaeology and genetics, there is much further research to do. At present, there are still relatively few species which have been studied, and there are limited cases of potential comparisons from which recurrent patterns of the evolution of domesticated seed dispersal can be studied. Nevertheless, there are a few aspects that can be highlighted. The fact that available data point to rather slow processes of evolution towards fixation in traits such as non-shattering in cereals and grain size increase, taking place on the order of 1000 generations or more, was unexpected by earlier theorists. For example Harlan (1992, p. 124) concludes that ‘cultivated plants have the capacity to evolve rapidly,’ and experimental inferences of Hillman and Davies (1990, 1999) suggested that 20–100 generations of self-pollinating cereals should be sufficient. As reviewed above, archaeobotanical data now suggest a much slower process (also Fuller, 2007a; Allaby, 2008; Allaby et al., 2008), perhaps more akin to cases of natural selection, as seed dispersal and seedling traits became adapted to human ecology. As with many cases of natural selection, genetic changes involved with domestication have operated through changes in the regulation of seed and fruit development, with several known domestication genes representing mutations to regulatory transcription factors (cf. Doebly et al., 2006; Burger et al., 2008). It may be possible to consider these changes in an ontogenetic framework of heterochrony. As discussed by Niklas (1994, pp. 262–274; also Nikalas1997, pp. 101–104), there are different ways in which the timing of development may change in heterochronic evolution. One that is often discussed is paedomorphosis, in which the ancestral juvenile form shows more resemblance to the derived mature form. A subcategory of this is neoteny, in which some vegetative traits are arrested and some ancestral juvenile character states are retained longer in relation to overall organismal development. The loss of shattering appears to represent an example of this as formation of wild-type adult abscission layers is arrested, that is sexual maturation is delayed. Other domestication traits may be regarded as peramorphosis or pre-displacement in which development is accelerated in vegetative traits and completed before all of the ancestral adult reproductive traits have formed: this may be applied to germination, loss of photoperiodicity functions, or the loss of appendages. Finally, acceleration may be applied to the expanded and exaggerated traits of domesticates, such as increases in grain size or the expanded appendages associated with a few fibre crops (cotton, devil’s claw), in which vegetative

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Fruit Development and Seed Dispersal growth is simply increased prior to reproductive maturity. Indeed, the prolonged development programme of cotton testa cells involved in fibre production has been molecularly characterized (Hovav et al., 2008). Because the domesticate often differs in shape and proportion (e.g. domesticated cereals are wider and thicker but may not be longer), and not merely size, this is a not a simple case of gigas (gigantism), when size is increased along a fixed allometric relationship (cf. Niklas, 1994). In some cases of domesticated size increase, such as in cucurbit fruits, simple gigantism may apply (see Sinnott, 1936, 1939). Further research documenting when during seed development certain traits, which have been modified by domestication, are expressed may provide further insights into the evolution of the domestication syndrome. What remains unclear is why the rates and ordering of domesticated traits have varied across some taxa and differences between families (cf. Fuller, 2007a), and an ontogenetic perspective on these traits may offer a framework for understanding the nature of these parallel or convergent evolutions. As Ames (1939) recognized the angiosperm seed has been central to human economic evolution, but it is the changes to seed dispersal and establishment that made this possible, giving human populations sources of growing surpluses, and particular species in domesticated form evolved unprecedented fitness across a range of environments. The further investigation of the evolution of seed crop domestication has much to contribute to our understanding of the processes of parallel and convergent evolution and the intertwined history of a limited range of plant species and Homo sapiens.

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Annual Plant Reviews (2009) 38, 296–325 doi: 10.1002/9781444314557.ch8

www.interscience.wiley.com

Chapter 8

FACTORS INFLUENCING THE RIPENING AND QUALITY OF FLESHY FRUITS Cornelius S. Barry Department of Horticulture, Michigan State University, East Lansing, MI, USA

Abstract: Fleshy fruits have a dual function in the reproductive strategies of plants. Initially, fleshy fruits protect the developing seeds from predation and then, once the seeds are mature, they facilitate dispersal of the enclosed seeds. Plants have evolved numerous chemical and physical barriers that discourage seed predation from fleshy fruits. Similarly, the ripening of fleshy fruits occurs through a range of coordinated biochemical processes that convert an unpalatable unripe fruit into a fruit that is nutritious and desirable to seed-dispersing fauna. The biochemical changes that occur at the onset of ripening are species specific but several general processes occur that are common to many fruits, suggesting that the mechanisms that control ripening may be evolutionarily conserved. For example, fruit ripening is often accompanied by the accumulation of brightly coloured pigments, the synthesis of aroma volatiles and the conversion of complex carbohydrates into sugars. These changes facilitate seed dispersal strategies. The genetic and biochemical pathways that lead to fruit ripening are not fully understood. However, significant progress has been made in identifying some of the components of these pathways. This review highlights recent research that has contributed to the understanding of the ripening process at the molecular level and outlines the development of genomics-based resources for fleshy fruit-bearing species. Keywords: fleshy fruits; seed dispersal; fruit ripening; hormone signalling; light signalling; aroma

8.1

Introduction

Fleshy fruits are important sources of nutrition for humans and animals providing energy, vitamins, minerals, antioxidants and fibre to the diet. In return for the energy that the plant invests in the production of the fruit, its consumption ensures that seeds are dispersed in a nutrient rich media, 296

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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often at a distance from the parent plant therefore facilitating survival and colonization. In order for frugivore-assisted seed dispersal to occur, dramatic biochemical changes are initiated that convert an unpalatable immature fruit into a ripe fruit that is attractive and nutritious. These ripening-associated changes are conserved in many diverse fruit species and include the conversion of starch to sugars, alterations in texture, the accumulation of brightly coloured pigments, and the synthesis of volatile aroma compounds. Plant secondary metabolites, including aroma volatiles, appear to be key in the interaction of frugivores and ripening fruits, serving as both feeding attractants and deterrents (Tewksbury, 2002; Foley and Moore, 2005). In tomato, the precursors of many aroma volatiles are essential nutrients in animal diets including carotenoids which serve as the precursors of pro-vitamin A, essential amino acids and essential fatty acids, leading to the hypothesis that these aroma compounds serve as nutritional cues for seed dispersers (Goff and Klee, 2006). However, evidence suggests that the interaction between volatiles and frugivores may in some instances be more complex. For example, fermentation, as a result of yeast infection, can increase as fruits become overripe. Fermentation leads to the production of ethanol, which at concentrations greater than 1%, can act as a feeding deterrent to both primates and fruit bats but at lower concentrations may act as a feeding stimulant (Milton, 2004; Sanchez et al., 2004, 2006). Similarly, mammalian frugivores are deterred by capsaicin in chilli peppers, yet birds, that are believed to be more efficient dispersers of the pepper seeds, show no aversion to fruits with high capsaicin content (Tewksbury and Nabhan, 2001). In addition, it has been hypothesized that the evolution of these thick fleshy fruits primarily occurred as a mechanism to prevent seed predation and that ripening may have evolved as a secondary phenomenon to aid in dispersal of the mature seeds (Mack, 2000). This hypothesis does have some credence when considering that many fleshy fruits contain toxic compounds, particularly those of the Solanaceae family that can contain high levels of glycoalkaloids which can be fatal if ingested and serve as a feeding deterrent (Cipollini and Levey, 1997). These few examples clearly illustrate that the interaction between frugivores and plants and the mechanisms that have evolved to serve as attractants and deterrents for seed dispersal and predation are complex. However, it is well established that the ripening of fruits serves as a key positive stimulus for seed-dispersing animals. This review focuses on some of the recent advances in the biology of fruit ripening and the genetic determinants that control this process and contribute to fruit quality.

8.2 Control of fruit ripening Fleshy fruits display a broad range of phenotypic and chemical diversity. However, despite this diversity there are several common features that accompany the onset of fruit ripening. For example, ripening often results in

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Fruit Development and Seed Dispersal changes in colour, increased softening, the conversion of starch into sugars, the production of flavour and aroma compounds and an increased susceptibility to pathogen infection. This suite of common biochemical changes suggests that diverse fruits may share common pathways that mediate ripening. Although the factors that control the onset of ripening are not fully understood, this represents an area of intense research activity. The last decade has seen tremendous progress in the molecular identification of mutant loci of tomato that alter fruit ripening and quality (Table 8.1). In addition, the development of genomics-based resources for fleshy fruit-bearing species, including the generation of large numbers of expressed sequence tags (ESTs) and gene expression profiling, coupled with functional analysis, has provided valuable insight into the identity of the genes that are expressed during fruit ripening, their regulation and contribution to the overall fruit phenotype.

8.3

Transcription factors serve as master regulators of fruit ripening

The characterization of tomato mutants with impaired fruit ripening has proven to be an effective strategy for gaining insight into the mechanisms that control ripening. The ripening inhibitor (rin), non-ripening (nor) and Colourless non-ripening (Cnr) display severe inhibition of fruit ripening manifest through inhibited ethylene synthesis, greatly reduced carotenoid synthesis and reduced fruit softening. These phenotypes cannot be alleviated by exogenous ethylene treatments although the expression of ripening-related genes can be induced by ethylene application suggesting that these loci act upstream of ethylene and control the competency of the fruit to ripen (Robinson and Tomes, 1968; Tigchelaar et al., 1973, 1978; Yen et al., 1995; Thompson et al., 1999). The rin locus maps to the long arm of tomato chromosome 5 and is tightly linked to the macrocalyx (mc) locus that causes the production of large sepals. The lack of separation between these traits led to the hypothesis that the rin locus may be caused by a deficiency (deletion) (Robinson and Tomes, 1968). This hypothesis was confirmed following the isolation of the rin locus using a positional cloning approach revealing the presence of a 1.7 kb deletion (Vrebalov et al., 2002). The deletion was found to remove the last exon of the RIN gene and regulatory sequences upstream of the MC gene that are required for normal expression levels in wild-type sepals. Both RIN and MC encode members of the MADS-box transcription factor family of tomato. MADS-box proteins constitute a large family in plants that act coordinately and often redundantly to control many developmental processes including floral organ identity, meristem determinacy and flowering time (Ng and Yanofsky, 2001). RIN is a member of the SEPALLATA (SEP) subfamily of MADS-box genes and is most similar to Arabidopsis SEP4/AGL3 (Malcomber and Kellogg, 2005).

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Locus

Gene product/ function

Fruit phenotype

Apricot (at)

Unknown

Beta (β)

Chromosome

Reference

Altered carotenoids

5

Lycopene β-cyclase

Altered carotenoids

6

Jenkins and Mackinney, 1955 Ronen et al., 2000

Colourless epidermis (y)

Unknown

Reduced flavonoids in peel

1

Rick and Butler, 1956

Colourless non-ripening (Cnr) Cuticular water permeability (Cwp)

SBP-box transcription factor Novel, unknown function

Severe ripening inhibition

2

Manning et al., 2006

Fruit cracking and shrivelling

4

Hovav et al., 2007

Delayed fruit deterioration (dfd)

Unknown

Altered fruit cuticle properties

Unknown

Saladie et al., 2007

Delta (Del)

Lycopene epsilon Altered cyclase carotenoid profile

12

Ronen et al., 1999

Dwarf (d)

Cytochrome P450, brassinosteroid biosynthesis

Delayed fruit ripening, pleiotropic effects

2

Lisso et al., 2006

Green-flesh (gf )

STAY-GREEN homologue

Altered fruit pigmentation

8

Barry et al., 2008

Green-stripe (gs) Unknown

Striped fruit epidermis

7

Larsen and Pollack, 1951

Green-ripe (Gr)

Ethylene signalling

Reduced ethylene responsiveness

1

Barry and Giovannoni, 2006

High-pigment-1 (hp-1)

DDB1 homologue

Enhanced fruit pigmentation

2

Liu et al., 2004

High-pigment-2 (hp-2)

DET1 homologue

Enhanced fruit pigmentation

1

Mustilli et al., 1999

High-pigment-3 (hp-3)

Zeaxanthin epoxidase

Enhanced fruit pigmentation

2

Galpaz et al., 1999

Lecer6

β-ketoacyl-CoA

Shrivelling on the vine

Unknown

Vogg et al., 2008

Reduced ethylene responsiveness

9

Wilkinson et al., 1995

synthase Never-ripe (Nr)

Ethylene receptor

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Fruit Development and Seed Dispersal Table 8.1

(Continued) Gene product/ function

Fruit phenotype

Non-ripening (nor) Ripeninginhibitor (rin) Tangerine (t)

Transcription factor MADS-box transcription factor Carotenoid isomerase

Yellow-flesh (r)

Phytoene synthase

Locus

Chromosome

Reference

Severe ripening inhibition Severe ripening inhibition

10

Giovannoni, 2001 Vrebalov et al., 2002

Altered carotenoid profile

10

Isaacson et al., 2002

Altered carotenoid profile

3

Fray and Grierson, 1993

5

In Arabidopsis, the four SEP genes act redundantly to specify floral organ identity and maintain floral meristem identity with single mutants having little or no altered phenotypes but quadruple mutants forming only leaf-like structures in place of the normal floral organs (Pelaz et al., 2000; Ditta et al., 2004). The severity of the rin mutant allele indicates that the redundancy observed in SEP gene function in Arabidopsis is not fully conserved in other species. MADS-box transcription factors regulate floral development through the formation of ternary and quaternary protein complexes suggesting that RIN may well function with other MADS-box genes to facilitate fruit ripening (Egea-Cortines et al., 1999; Honma and Goto, 2001). Indeed, several other MADS-box genes are expressed in tomato fruit and some, like RIN, display a ripening-related increase in expression and are therefore good candidates for additional regulators of ripening although functional analysis to confirm this hypothesis is currently lacking (Fei et al., 2004; Giovannoni, 2004; Hileman et al., 2006). The Cnr mutant has a particularly striking phenotype that is distinct from that observed in the rin and nor mutations (Thompson et al., 1999). The fruit pericarp of mature Cnr fruit is white and exhibits reduced cell–cell adhesion resulting in fruits with a mealy texture. Analysis of the cell wall properties of the Cnr mutant revealed several changes when compared to wild type including stronger pericarp cell walls in mature Cnr fruit, a 50% increase in intercellular spaces, reduced calcium-binding capability of homogalacturonan in the middle lamella and altered deposition of (1 → 5)-␣-L-arabinan in mutant fruit cell walls (Orfila et al., 2001). Together with the altered physical and chemical properties of the cell wall in Cnr fruit, there is extensive alteration in the expression and activity of several enzymes involved in cell wall modification during ripening and many other ripening-related genes display reduced or altered expression in Cnr fruit (Eriksson et al., 2004). Interestingly, there is increased expression of genes associated with stress responses and pathogen

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infection in Cnr fruit including chitinases and PR proteins. The appearance of these classes of genes is characteristic of abscission and dehiscence zones (Roberts et al., 2002). Together with the altered cell wall properties, the gene expression profile of Cnr fruit raises the intriguing possibility that the pericarp cells in mutant fruit are undergoing separation processes similar to those that occur during the dehiscence of dry fruits. The Cnr locus was mapped to within a 13 kb interval on the long arm of chromosome 2. No sequence differences were observed between wild type and Cnr alleles across this region although the expression of a gene encoding a member of the SBP-box (SQUAMOSA Promoter-Binding Protein-like) gene family designated LeSPL-CNR was reduced in the Cnr mutant background (Manning et al., 2006). Virus-induced gene silencing of this gene in fruit resulted in sectors that failed to fully ripen therefore mimicking the Cnr mutant phenotype. Bisulphite sequencing of the promoter region of LeSPL-CNR revealed hypermethylation of cytosine residues in the Cnr mutant background. Hypermethylation of promoter regions can lead to alteration in gene expression and has been confirmed as the cause of several higher plant epigenetic mutations affecting plant development. In addition, approximately one Cnr plant per thousand produced a fruit with normal ripening sectors. These rare revertants are typical of epigenetic mutations. Together these data indicate that the Cnr mutant phenotype is the result of a stably inherited spontaneous epigenetic mutation that leads to reduced expression of LeSPL-CNR during fruit development and ripening (Manning et al., 2006). LeSPL-CNR is most closely related to the Arabidopsis SPL3 gene that has been implicated in regulating floral development via possible regulation of AP1 expression. Expression profiling of Cnr mutant fruit revealed altered expression of several MADS-box genes compared to wild type, in particular TDR4 expression was significantly reduced in Cnr (Eriksson et al., 2004). TDR4 is a member of the FRUITFUL/APETALA1 lineage of MADS-box genes and is a closely related homologue of the Arabidopsis FRUITFUL (FUL) gene (Litt and Irish, 2003). Fruit of the Arabidopsis ful mutant displays severe growth defects due to a lack of valve cell expansion following fertilization (Gu et al., 1998). FUL is expressed throughout the valve in wild-type fruit and acts together with REPLUMLESS (REP) to restrict the expression of four transcription factors, SHATTERPROOF (SHP) 1 and 2, INDEHISCENT (IND) and ALCATRAZ (ALC) to the valve margin (Roeder et al., 2003; Liljegren et al., 2004). These four transcription factors are required to specify valve margin identity and in ful mutants are ectopically expressed in valve cells that subsequently appear to adopt the fate of valve margin cells including lignin deposition during the later stages of fruit development (Liljegren et al., 2004). The altered phenotypes of Cnr fruit are indicative of a change in cell identity within the fruit pericarp and it may be possible to exploit the increasing body of knowledge on Arabidopsis fruit development to explain the biology of Cnr. The identification of RIN and CNR as transcription factors provides a remarkable opportunity to investigate the regulation of the pathways that

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Fruit Development and Seed Dispersal control fruit ripening in more detail. Obvious experimental directions include defining the in vivo targets and interaction partners of these proteins and, given that the expression of both RIN and CNR are up-regulated during fruit ripening, it will be informative to define the factors that regulate their expression. In addition, defining the genetic interaction of RIN and CNR with each other and in the context of the nor mutation may also provide insight into the mechanisms that regulate fruit ripening. For example, can overexpression of RIN in a nor genetic background restore aspects of the ripening process? Of additional importance will be determining whether the action of these transcription factors is conserved across species boundaries to control ripening in other climacteric as well as non-climacteric fruits.

8.4

Hormonal control of fruit ripening

All of the major plant hormones have been shown to influence aspects of fruit development and ripening. Hormone levels change during cell division following fertilization, cell expansion during fruit growth and at the onset of ripening, influencing the expression of a multitude of genes implicated in these processes (Srivastava and Handa, 2005). Discussion of hormonal regulation in this chapter will focus purely on the effects of hormones on ripening and fruit quality. 8.4.1

Ethylene

Fleshy fruits have traditionally been classified based upon their ripening behaviour. Climacteric fruits, including tomato, avocado, apple and banana produce a burst of respiration and display increased ethylene synthesis at the onset of ripening whereas in non-climacteric fruits such as grapes, strawberries and citrus these changes are not evident (McMurchie et al., 1972). The role of ethylene in regulating the ripening of climacteric fruits is well defined. Chemicals that block either ethylene synthesis or perception can inhibit fruit ripening (Hobson et al., 1984; Yang and Hoffman, 1984; Watkins, 2006). Similarly, genetic control of ethylene synthesis through reduction of ACC levels or ACC oxidase activity also leads to inhibition of ripening (Klee et al., 1991; Oeller et al., 1991; Picton et al., 1993; Ayub et al., 1996; Schaffer et al., 2007). Mutations or transgenic approaches that disrupt the function of genes involved in the ethylene-signalling pathway also disrupt ripening. For example, the Never-ripe (Nr) mutant of tomato displays dominant ethylene insensitivity due to an amino acid substitution in an ethylene receptor, leading to inhibition of ripening, and overexpression of a mutated Arabidopsis ETR1 ethylene receptor in tomato also leads to inhibition of ripening (Lanahan et al., 1994; Wilkinson et al., 1995, 1997). Ethylene receptors are encoded by a family of at least six genes in tomato and act as negative regulators of ethylene responses (Tieman et al., 2000; Kevany et al., 2007). Recent evidence suggests that

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ethylene exposure leads to degradation of the receptors, resulting in enhanced ethylene responsiveness and earlier fruit ripening (Kevany et al., 2007). Mutation and manipulation of ethylene receptors in tomato leads to reduced ethylene responsiveness throughout the whole plant, however, recent characterization of the Green-ripe (Gr) mutant of tomato has provided insight into factors that mediate tissue-specific control of ethylene responses. Gr fruit fails to fully ripen due to reduced ethylene responsiveness and also displays reduced rates of ethylene-induced floral senescence and abscission. However, Gr mutant seedlings retain normal ethylene responsiveness (Barry et al., 2005). Using a positional cloning strategy, the Gr mutation was shown to result from a deletion within the promoter and 5 -untranslated region of a gene that resulted in its ectopic expression in the Gr mutant background (Barry and Giovannoni, 2006). Overexpression of GR under the control of the CaMV35S promoter recreated the mutant phenotype but did not lead to plants that displayed a whole-plant reduction in ethylene responsiveness, indicating that GR modulates tissue-specific ethylene signalling via an unknown mechanism. GR encodes a novel protein that is predicted to be membrane localized and is conserved in plants, metazoans and protozoa. In a separate study, a GR homologue from Arabidopsis, designated REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1) was isolated in a genetic screen to identify suppressors of the weak ethylene-insensitive mutant receptor allele, etr1-2 (Resnick et al., 2006). However, unlike the tissue specificity observed in the Gr mutant allele, rte1 mutant alleles display ethylene-related phenotypes throughout the whole plant suggesting different ethylene signalling mechanisms operate in tomato and Arabidopsis. In addition to targeted transgenic approaches and mutant analysis in tomato, variation in climacteric ethylene production and ethylene responsiveness has also been observed in several fleshy fruit species including apple, melon, peach, plum, pepper and, Asian pear, leading to different fruit quality and ripening characteristics (Itai et al., 1999; Sunako et al., 1999; Villavicencio et al., 1999; Zuzunaga et al., 2001; Perin et al., 2002; Tatsuki et al., 2006; Yamane et al., 2007; Itai and Fujita, 2008). In apple, peach and Chinese pear, the reduced ethylene biosynthesis observed in cultivars with extended shelf life has been linked to reduced expression levels of ACC synthase isoforms that are typically expressed during ripening (Sunako et al., 1999; Tatsuki et al., 2006; Yamane et al., 2007). Furthermore, in apple and Asian pear cultivars structural differences have been observed in different ACS isoforms and these correlate with ethylene production and postharvest shelf life (Itai et al., 1999; Sunako et al., 1999; Itai and Fujita, 2008). Tremendous variation for fruit morphology and ripening traits also exists within the Cucurbitaceae family and in particular, melons display both climacteric and non-climacteric ripening behaviour. Cantaloupe melons display a climacteric ripening phenotype, they have a netted skin, orange flesh, are aromatic and undergo abscission as they reach maturity and ripen. In contrast, honey dew melons produce little ethylene, have low respiration rates,

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Fruit Development and Seed Dispersal have a smooth skin, reduced aroma volatiles and fail to abscise. Transgenic cantaloupe melons with reduced ethylene synthesis due to the expression of an ACC oxidase antisense transgene gene can partially suppress some of the typical ‘cantaloupe-type’ phenotypes resulting in plants that display phenotypes typically associated with honey dew melons (Ayub et al., 1996; Guis et al., 1997; Flores et al., 2002). Furthermore, genetic analysis of a recombinant inbred population generated from a cross between a non-climacteric, ethylene-insensitive melon, PI161375 and a climacteric cantaloupe melon revealed that the non-climacteric character was controlled by two recessive loci (Perin et al., 2002). Fruits that have traditionally been classified as non-climacteric have recently been re-evaluated for the possible role of ethylene in regulating various aspects of the ripening process. The role of ethylene in regulating de-greening in citrus is well established but recent evidence has also been presented that suggests the possibility that citrus fruits may display climacteric-like behaviour following harvest (Goldschmidt et al., 1993; Jacob-Wilk et al., 1999; Katz et al., 2004). Similarly, ethylene has been shown to stimulate the accumulation of anthocyanin, and the expression of ripening-related genes in grape berries and the differential expression of components of the ethylenesignalling pathway have been demonstrated in several non-climacteric fruits including strawberry, citrus and grape (El-Kereamy et al., 2003; Chervin et al., 2004; Tesniere et al., 2004; Trainotti et al., 2005; Fujii et al., 2007). These experiments provide correlative evidence of a role for ethylene in different aspects of the ripening of non-climacteric fruits. The introduction of dominant ethylene receptor mutants into transgenic strawberry and grape, possibly under the control of fruit-specific promoters, is now technically feasible and should provide conclusive evidence as to the role of endogenous ethylene in these non-climacteric fruits. 8.4.2

Brassinosteroids

Although the role of ethylene in enhancing the ripening of climacteric fruits is well established, additional factors that promote ripening, particularly in non-climacteric fruits are less well defined. However, several lines of evidence point to a role for brassinosteroids (BRs) as potential promoters of fruit ripening in grape (Symons et al., 2006). BRs were shown to increase in grapes at the onset of ripening and this increase is mirrored by changes in the expression of genes involved in BR biosynthesis. Furthermore, exogenous application of BRs to grape berries accelerated ripening whereas the application of the BR inhibitor, brassinazole, delayed the onset of ripening. These data provide correlative evidence for a role of BRs in stimulating ripening although a more definitive genetic or transgenic approach will be needed that disrupts either the synthesis or action of BRs in grape berries to truly verify this interesting discovery. Exogenous application of BRs has also been reported to stimulate ripening of tomato fruit discs suggesting that this phenomenon is not

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restricted to grape (Vardhini and Rao, 2002). Genetic evidence of a role for BRs in mediating ripening and quality attributes in tomato has come from characterization of the dwarf (dx) mutant of tomato. The dwarf mutation lacks a functional cytochrome P450, required for the synthesis of castasterone and exhibits delayed fruit ripening and altered quality attributes including reduced levels of starch and sugars and elevated amino acids (Lisso et al., 2006). 8.4.3 Auxin In tomato, auxin levels increase early during fruit development during the cell division phase and subsequently decline before increasing at the onset of ripening (Srivastava and Handa, 2005). Many auxin responses are controlled by two different classes of transcription factors, the auxin response factors (ARFs) that bind to targets in the promoters of auxin-regulated genes and the Aux/IAA proteins that bind to the ARFs and act as repressors of auxin responses (Guilfoyle, 2007). Under low auxin conditions, the Aux/IAA proteins bind to the ARFs and inhibit transcription. In contrast, when auxin levels increase, binding of auxin to the receptor, an F-box protein designated SCFTIR1, targets the Aux/IAA proteins for degradation and releases their inhibitory effect on auxin-inducible gene expression (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). The expressions of several Aux/IAA and ARF genes are induced at the onset of ripening in both tomato and peach and show regulation by ethylene, suggesting possible interplay between ethylene and auxin in mediating ripening-related gene expression (Jones et al., 2002; Trainotti et al., 2007). Transgenic manipulation of the ARF gene DR12 in tomato causes a range of pleiotropic phenotypes that includes dark green immature fruits that when mature exhibit a blotchy ripening pattern. Pericarp cells also possess an unusual pattern of cell division with a higher number of small cells in the outer pericarp that contributes to an increased overall thickness of the pericarp. DR12 suppressed fruit displays enhanced firmness that may result from the increased thickness of the pericarp or subtle changes that are observed in pectin composition in the transgenic lines (Jones et al., 2002; Guillon et al., 2008). Evidence for auxin playing a role in early fruit development in tomato has also been obtained through transgenic manipulation of the AUX/IAA gene, IAA9, leading to cell expansion prior to fertilization and the development of parthenocarpic fruit (Wang et al., 2005). 8.4.4 Polyamines Polyamines (PAs) are ubiquitous aliphatic organic compounds that are reported to have a range of phenotypic effects including an ability to delay fruit ripening and senescence (Galston and Sawhney, 1990; Cohen, 1998). PAs are synthesized from basic amino acids. Putrescine can be formed from either arginine or ornithine by the action of either arginine or ornithine decarboxylase. Putrescine together with a decarboxylated S-adenosylmethionine (SAM)

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Fruit Development and Seed Dispersal moiety can then be utilized to form spermidine and subsequently spermine by the action of spermidine and spermine synthases, respectively (Cohen, 1998). In an experiment designed to assess the role of PAs in fruit ripening, Mehta et al. overexpressed a yeast SAM decarboxylase (ySAMdc) gene under the control of the strong ripening-specific E8 promoter in transgenic tomatoes (Mehta et al., 2002). When compared to segregating control fruit, the transgenic lines with elevated PA levels exhibited a range of beneficial phenotypes including increased vine life, higher carotenoid levels and increased juice viscosity. The transgenic fruit also produced elevated levels of ethylene. This result was unexpected given that both PAs and ethylene share SAM as a common precursor as it was anticipated that diverting SAM towards PA biosynthesis would reduce ethylene levels. However, the elevated ethylene levels in the PA overproducing lines indicate that SAM is not a limiting substrate for either pathway. Together these data suggest that the PA accumulating transgenic fruit may exhibit delayed senescence, remaining metabolically active for a longer period of time than fruits from non-transgenic controls allowing for increased synthesis of phytonutrients.

8.5

The influence of light on fruit quality

The accumulation of brightly coloured pigments in fruits is one of the most dramatic events that accompanies ripening and serves as a key signal to seed-dispersing fauna of the ripeness and palatability of the fruit. Fleshy fruits predominantly accumulate carotenoids, anthocyanins and flavonoids and the de novo synthesis of these compounds at the onset of ripening is preceded by, or occurs concomitantly with, the degradation of chlorophyll (Seymour et al., 1993). An exception to this generalization occurs in banana where the degradation of chlorophyll at the onset of ripening leads to the unmasking of the yellow-pigmented xanthophylls that are already present in immature fruit (Seymour et al., 1993). The biochemical pathways that lead to the synthesis of carotenoids, anthocyanins and flavonoids are well established and mutations in various steps in these pathways or regulators of these pathways lead to the colour variants witnessed in many fruit species (Hirschberg, 2001; Takos et al., 2006; Bogs et al., 2007; Chagne et al., 2007; Espley et al., 2007). In tomato, mutants that disrupt ethylene signalling have reduced carotenoid content indicating that ethylene plays a significant role in regulating carotenoid synthesis in this species (Lanahan et al., 1994; Alba et al., 2005; Barry et al., 2005). However, physiological and genetic studies have also implicated light-signalling pathways in influencing carotenoid, flavonoid and anthocyanin accumulation during ripening in several species including tomato, apple and grape (Alba et al., 2000; Solovchenko et al., 2006; Takos et al., 2006; Ristic et al., 2007). For example, carotenoid synthesis in tomato can be stimulated by a short red light pulse and this is reversed by a far-red light pulse indicating that this effect is mediated by phytochrome (Alba et al., 2000).

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Transgenic and mutant analysis in tomato has also provided several lines of evidence implicating light-signalling pathways in regulating pigment accumulation during fruit ripening. For example, overexpression of cryptochrome 2 and the oat phytochrome A gene in tomato results in plants that display light hypersensitive phenotypes characterized by shortened internodes and increased pigment accumulation including higher levels of lycopene and flavonoids in fruits (Boylan and Quail, 1989; Giliberto et al., 2005). The manipulation of downstream components of the light-signalling pathway can also elicit changes in the pigment content of tomato fruit. Silencing of LeHY5, a positive regulator of light responses and LeCOP1-like, a negative regulator of light signalling, resulted in fruits that accumulated lower and higher levels of carotenoids than non-transformed controls, respectively (Liu et al., 2004). A light hypersensitive phenotype is also observed in the high pigment 1 (hp-1) and high pigment 2 (hp-2) mutants of tomato leading to increased levels of pigments in ripe fruits as well as altered plant morphology (Soressi, 1975; Peters et al., 1989; Yen et al., 1997; Cookson et al., 2003). Using a combination of map-based cloning and candidate gene approaches the hp-1 and hp-2 loci have been identified as tomato homologues of Arabidopsis UV-DAMAGE DNABINDING PROTEIN 1 (DDB1) and DE-ETIOLATED 1 (DET1), respectively (Mustilli et al., 1999; Lieberman et al., 2004; Liu et al., 2004). These proteins are conserved in eukaryotes and form complexes with a RING-finger protein, RBX1, CONSTITUTIVE PHOTOMORPHOGENESIS 10 (COP10) and Cullin 4 and have been implicated in a range of physiological processes including protein turnover during photomorphogenesis and modification of chromatin structure (Benvenuto et al., 2002; Schroeder et al., 2002; Yanagawa et al., 2004; Bernhardt et al., 2006). The hp-1 and hp-2 loci have been restricted in their use in breeding programmes because of detrimental traits associated with reduced plant vigour. However, fruit-specific expression of HP2 in transgenic tomato led to beneficial accumulation of pigments in the transgenic fruits without the associated alteration in plant growth (Davuluri et al., 2005). These diverse experiments indicate that the manipulation of light-signalling components appears to be an effective strategy to modify the phytonutrient content of fleshy fruits.

8.6 The discovery of aroma and flavour genes in fruit The characteristic aromas imparted by fleshy fruits as they begin to ripen probably represent the most complex and species-specific aspect of the ripening process and together with colour represent one of the key attractants for frugivores. Individual species and varieties within species all have very unique aroma profiles consisting of hundreds of individual compounds that can be classified into many chemical groups including alcohols, aldehydes, ketones, esters, terpenes, furans, phenolics and sulphur-containing compounds (Buttery and Ling, 1993; Zabetakis and Holden, 1997). For

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Fruit Development and Seed Dispersal example, ripening tomato fruits have been documented to produce over 400 volatiles although a subset of approximately 30 of these compounds have been determined to define the ‘characteristic aroma’ of tomato (Buttery and Ling, 1993; Tieman et al., 2006b). The pathways that contribute to aroma production in these diverse species are not fully understood but in recent years significant progress has been made in identifying some the genes and enzymes that synthesize aroma volatiles in fleshy fruits. Biochemical and genetic evidence has demonstrated that volatile components are intimately linked to ripening-associated colour changes in fruits that accumulate carotenoids as the principle pigment (Lewinsohn et al., 2005; Tieman et al., 2006b). A number of the aroma volatiles are derived from fatty acids that are presumably formed as a result of membrane breakdown and the reorganization that occurs during the transition of the chloroplast to the chromoplast or are directly formed from the breakdown of carotenoids. Genes involved in the production of some of these compounds have been defined. For example, silencing of a plastid-targeted lipoxygenase gene, designated TOMLOX-C, in transgenic tomato led to a reduction in the production of C6 volatiles in ripening fruit (Chen et al., 2004). Similarly, silencing of the tomato CAROTENOID CLEAVAGE DIOXYGENASE 1 gene of tomato was found to result in a reduction of the carotenoid volatiles, ␤-ionone, pseudoionone and geranylacetone in ripening fruits (Simkin et al., 2004). Most domesticated fruit crops have been bred for long shelf life and tolerance to postharvest handling whereas flavour and aroma traits have been a lower priority for plant breeders. The use of wild species germplasm has proven to be an excellent source of natural variation for breeding of quality traits and for fundamental studies to assess biological phenomena (Fernie et al., 2006). In tomato, a set of introgression lines (ILs) that contain defined segments of the Solanum pennellii genome within a cultivated, Solanum lycopersicum, genetic background have been utilized for identifying quantitative trait loci (QTLs) controlling a number of fruit quality traits including those contributing to aroma production (Lippman et al., 2007). Utilizing this population, variation in a number of volatile compounds derived from fatty acids, amino acids and carotenoids has been identified (Tadmor et al., 2002; Tieman et al., 2006a, 2006b; Matsui et al., 2007). The power of the IL population is that the variation detected in specific metabolites can be immediately assigned to specific chromosomal segments aiding map-based strategies for gene identification. Similarly, natural variation in aroma volatiles occurs between cultivated strawberry, Fragaria ananassa and wild strawberry, Fragaria vesca with the latter displaying increased diversity in monoterpene profiles while lacking the sesquiterpene, nerolidol (Pyysalo et al., 1979; Aharoni et al., 2004). This variation in terpenoid accumulation has been attributed to expression and structural differences in two genes, NEROLIDOL SYNTHASE 1 (NES1) and PINENE SYNTHASE (PINS) (Aharoni et al., 2004). NES1 is highly expressed in cultivated strawberry but is expressed at low levels in F. vesca and is differentially localized in the two species. In contrast, PINS is highly

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expressed in wild species but not in cultivated species and furthermore a two base pair insertion exists in the gene from the cultivated species that results in a frame shift and a corresponding premature stop codon suggesting a nonfunctional enzyme. These examples illustrate the utility of harnessing natural variation for elucidating biochemical pathways in ripening fruits.

8.7 Cell wall changes influence fruit quality The softening of fleshy fruits as they ripen is one of the key determinants of quality leading to alterations in texture and is also a primary target for manipulation to enhance postharvest storage and handling characteristics. The processes that constitute the changes in cell wall structure and their contribution to fruit softening have recently and comprehensively been reviewed by Brummell (2006) and I refer readers to this article for an in depth discussion of this topic. In general terms, fruit softening is brought about by a combination of structural changes to the cell wall and also a reduction in turgor pressure. One of the initial changes in cell wall architecture at the onset of ripening is the dissolution of the pectin matrix that composes the middle lamellae that form a connective layer between adjacent cells and this is subsequently followed by alteration in the structure of the cell wall polysaccharides (Jarvis et al., 2003). An interesting aspect of cell wall dissolution during fruit ripening is the diversity in polysaccharide metabolism that occurs during softening of different species. For example, pectin de-polymerization is absent in strawberry, banana, apple and pepper but occurs at moderate to high levels in tomato, peach and avocado (Brummell, 2006). Similar species variation is also apparent for other polysaccharide components highlighting the complexity of the softening process in fleshy fruits and implying that manipulation of softening by a single method is unlikely to be successful in multiple species. Transgenic approaches to manipulate the activity of different classes of cell wall hydrolases and wall loosening enzymes, with the aim of reducing fruit softening have been undertaken with a range of success (Brummell and Harpster, 2001). In some cases, these studies have revealed a modification of polysaccharide structure following silencing or overexpression of particular enzymes, but these modifications have typically resulted in either small or no alterations in fruit softening (Giovannoni et al., 1989; Brummell et al., 1999; Brummell and Harpster, 2001; Smith et al., 2002; Powell et al., 2003). However, pyramiding of some transgenes has led to increased firmness, particularly in ripening and ripe fruit. For example, transgenic tomato lines suppressed for both polygalacturonase and expansin activity show increased firmness and were less susceptible to the postharvest pathogen Botrytis cinerea, suggesting that the pathogen may require certain cell wall modifications to occur within the fruit prior to being able to establish an infection (Cantu et al., 2008). Further pyramiding of cell wall-associated transgenes may yield further insight into the co-operative nature of cell wall disassembly during fruit ripening.

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8.8

The cuticle influences fruit quality and postharvest longevity

The plant cuticle is a layer formed by cutin and wax and is associated with the outer wall of epidermal cells. The cuticle is instrumental in maintaining water content of fleshy fruits and water content can be high for extended periods of time following harvest. The cuticle also acts as a barrier to pathogen infection and in the case of fruits can act as a reflective surface to enhance colour signals. The tomato fruit cuticle is approximately 10 ␮m thick and is astomatous and is composed of various waxes and cutin (Wilson and Sterling, 1976; Petracek and Bukovac, 1995; Bauer et al., 2004; Bargel and Neinhuis, 2005). The genetic factors that control the composition and permeability of fruit cuticles have remained largely undefined but recent identification of loci in tomato that alter water permeability in fruit promises to provide new insight into this horticulturally important phenomenon. For example, a tomato mutant deficient in a very long chain fatty acid ␤-ketoacyl-CoA synthase displays enhanced water loss, cuticle permeability and fruit shrivelling due to a reduction in the content of n-alkanes and aldehydes with chain lengths of longer than C30 (Vogg et al., 2004). Similarly, the Cuticular water permeability 1 (Cwp1) locus of tomato results in microfissues on the fruit surface and causes water loss through the cuticle leading to fruit shrivelling both on and off the vine (Hovav et al., 2007). The Cwp1 locus maps to the long arm of chromosome 4 and was isolated following the introgression of a chromosomal segment from the wild species Solanum habrochaites into a cultivated tomato background. CWP1 encodes a protein of unknown function that is expressed in fruit of the IL, designated Cwphir, but is not expressed in the wild-type control line, Cwpesc. Confirmation of gene identity was provided through overexpression of CWP1 in cultivated tomato under the control of the CaMV35S promoter which resulted in fruits that phenocopied the S. habrochaites IL. Interestingly, CWP1 is not expressed in red and orange-fruited wild species of tomato but is expressed in green-fruited species including Solanum peruvianum, S. habrochaites and Solanum chmiliewskii. However, the microfissure and water loss phenotype is only observed when the alleles from these green-fruited species are introgressed into cultivated tomato suggesting that there is an interaction between CWP1 and as yet unidentified components from cultivated tomato. Analysis of chemical composition of homozygous Cwp1hir and Cwpesc lines failed to reveal any differences in wax and cutin components between the genotypes and the mechanism of CWP1 action remains unknown although it is possible that this gene may play a role in structural organization of the cuticle or in the interaction of the cuticle with the epidermal cell wall. The delayed fruit deterioration (dfd) locus of tomato has an opposite phenotypic effect to that of Cwp1 in that the cuticle is less permeable to water loss than wild type and had increased cell turgor leading to fruits displaying an extended shelf life (Saladie et al., 2007). No visible differences were evident in the surface or thickness of dfd cuticles although they exhibited altered

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biomechanical properties. However, large differences in wax and cutin monomers were observed in dfd compared with control fruits. For example, total wax content is >30% higher in dfd than control fruits with the largest increases observed in the content of the alkadienes. Similarly, cutin monomer content was increased by 84% in dfd fruits. Cloning of dfd should prove interesting, particularly within the context of the CWP gene.

8.9 Genomics resources As in other areas of plant biology, the development of high-throughput ‘omics’ resources has revolutionized research in fruit biology. This has led to the generation of large data sets and the subsequent need for database infrastructure to curate, store and disseminate this data. Several family or speciesspecific databases now exist for many fruit crop species (Table 8.2). These databases contain a wealth of annotated data including genetic and physical maps and associated marker information, EST and genomic sequences, data sets from expression, metabolite and proteome profiling experiments and germplasm information. These databases are invaluable to researchers working on particular crops but are also extremely useful for accessing data for performing comparative genomics of fruit development and ripening. The availability of large EST collections sequenced from fruit cDNA libraries has enabled the in silico comparison of gene expression during fruit development and ripening and the development of microarray resources for the majority of the major fruit crop species including tomato, peach, grape, strawberry, apple and citrus. Profiling experiments using these gene expression arrays have provided insight into the identities of the genes that are expressed during fruit ripening and their regulation by hormone treatments and genetic mutations. These experiments have led to the identification of co-regulated genes that are expressed during the ripening process and have provided a platform for comparative gene expression analysis between fruit crop species (Aharoni et al., 2000, 2002; Alba et al., 2004, 2005; Fei et al., 2004; da Silva et al., 2005; Lemaire-Chamley et al., 2005; Newcomb et al., 2006; Park et al., 2006; Trainotti et al., 2006; Deluc et al., 2007; Schaffer et al., 2007). The availability of large EST collections has also directly led to the recent identification and characterization of small RNAs from tomato and apple fruit which will undoubtedly lead to new opportunities for examining genetic regulation of fruit development and ripening (Pilcher et al., 2007; Gleave et al., 2008; Itaya et al., 2008). Advances in chromatographic separation techniques coupled with sensitive mass spectrometry have facilitated the ability to simultaneously detect many primary and secondary metabolites in single small-scale extractions (Schauer and Fernie, 2006; De Vos et al., 2007; Last et al., 2007). The application of these metabolomic techniques to the study of fruit ripening, particularly when combined with genetic diversity is proving to be a powerful approach for identifying loci that control metabolite levels and fruit quality. A survey

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Fruit Development and Seed Dispersal Table 8.2 species

Databases containing genomics-based resources for fleshy-fruited crop

URL

Represented species

International grape genome programme

http://www.vitaceae.org

Grape

Vitis gene expression database

http://cropdisease.ars. usda.gov/vitis at/mainpage.htm

Grape

Sol genomics network

http://www.sgn.cornell.edu/

Tomato, pepper, eggplant, coffee

Tomato expression database Tomato metabolite database Metabolome tomato Database

http://ted.bti.cornell.edu/ http://tomet.bti.cornell.edu/ http://appliedbioinformatics. wur.nl

Tomato Tomato Tomato

http://www.icugi.org/

Melon, cucumber, watermelon

http://www.bioinfo.wsu. edu/gdr/

Apple, pear, peach, cherry, apricot, raspberry, strawberry

International citrus genomics consortium

http://int-citrusgenomics.org/

Multiple citrus species

The Hawaii papaya genome project

http://cgpbr.hawaii.edu/ papaya/

Papaya

Global musa genomics consortium

http://www.musagenomics. org/

Banana

PineappleDB

http://genet.imb.uq.edu.au/ Pineapple/

Pineapple

Database Vitaceae

Solanaceae

Cucurbitaceae Cucurbit genomics database Rosaceae Genome database for rosaceae Miscellaneous

of fruit from cultivated and wild tomato species revealed significant differences in the levels of several classes of primary metabolites including sugars, organic acids and amino acids, highlighting the diversity that exists between these species (Schauer et al., 2005). In a refinement of this study, 74 metabolites were surveyed in an IL population of 76 individuals comprising S. pennellii chromosomal segments in an S. lycopersicum genetic background. This experimental approach localized 889 QTLs associated with fruit metabolite content onto the tomato genome (Schauer et al., 2006). A number of these loci display

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significant variation between the IL and the M82 parental cultivar raising the possibility that their identity may be resolved through genetic mapping and gene cloning. Liquid chromatography–mass spectrometry (LC–MS)-based metabolomics has also been utilized for identifying a range of semipolar metabolites in tomato fruit, including revealing the presence of compounds not previously identified in fruit (Moco et al., 2006). Further analysis revealed distinct spatial and temporal localization of metabolites in specific tissues at different stages of tomato fruit development and ripening (Moco et al., 2007). These pioneering studies open the door to incorporate large-scale analysis of metabolites into the functional genomics toolbox of biologists investigating fruit ripening. 8.9.1 Genome sequencing of fleshy-fruited species Several genomes from higher and lower plants have now been sequenced revealing information on gene content, genome structure and evolution. Recently, draft genome sequences of two fleshy-fruited species, grape and papaya have been released (Jaillon et al., 2007; Ming et al., 2008). An 8.4-fold draft genome sequence has been produced for grape, revealing a 487 Mb genome containing 30 484 predicted genes (Jaillon et al., 2007). Annotation of the grape genome revealed an increased prevalence of genes encoding enzymes involved in secondary metabolites associated with aroma production and wine quality. For example, the terpene synthase (TPS) family, which is important for the synthesis of oils, resins and aroma compounds, is more than twice as large in grape compared to Arabidopsis. Furthermore, monoterpene synthases make up greater than 40% of the TPS family in grape whereas in Arabidopsis they constitute approximately 15% of the total TPS complement. The monoterpene synthases are specifically involved in the formation of C10 terpenoids that are present in grape aroma volatiles such as linalool and geraniol. Similar results were found for stilbene synthases (STSs) that are involved in the synthesis of the phytoalexin resveratrol suggesting diversification and specialization of gene complements in species that produce a range of diverse secondary metabolites. The papaya genome was sequenced to 3X coverage by whole genome shotgun sequencing, revealing 372 Mbp containing 24 746 predicted genes (Ming et al., 2008). Similar to the grape genome, annotation of the papaya genome revealed an increased number of genes involved in the production of aroma volatiles compared to those present within the Arabidopsis genome. The sequencing of the grape and papaya genomes represents significant milestones in plant biology. The increased prevalence within these genomes of genes involved in aroma volatile production is congruent with the evolution of fleshy fruits and ripening processes as seed dispersal mechanisms and likely also reflects selection during crop domestication for varieties with enhanced organoleptic properties. The vast majority of the species listed in Table 8.2 have genome projects that are in progress and at various stages of completion,

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Fruit Development and Seed Dispersal and it is likely in the coming years that additional fruit crop species will be sequenced, opening the way to examine comparative fruit genomics across plant families with divergent ripening behaviours, fruit morphologies and chemical compositions.

8.10

Conclusions and future perspectives

The ripening of fleshy fruits imparts desirable characteristics on an otherwise unpalatable product. The biochemical changes that occur during fruit ripening serve as attractants and important quality determinants for both humans and other animals that ultimately aid in seed dispersal. In recent years, research on the factors that mediate fruit ripening and quality has undergone a renaissance with large advances in our knowledge of the genes involved in these processes. The sequence of the grape and papaya genomes represents a milestone in fruit biology research and the emerging sequence of the tomato genome and that of other fleshy fruit-bearing species will similarly have a tremendous impact on future research directions and will create hitherto unavailable opportunities for comparative biology of fruit crops on a genomic scale. However, functional analysis of large numbers of genes in fleshy fruitbearing species remains a challenge due to the time and resources required to generate mutants or transgenic lines. This is particularly problematic in tree and other woody fruit-bearing crop species, although the development of transient-based assays for gene function analysis in fruits may be a useful technology to overcome this bottleneck (Fu et al., 2005; Hoffmann et al., 2006; Orzaez et al., 2006). In addition, the generation of stable genetic populations utilizing exotic germplasm to harness natural variation will greatly enhance our future ability to isolate genes required for determining fruit ripening and quality from a variety of species and to utilize this information for breeding varieties with enhanced quality traits.

Acknowledgements Research in the author’s laboratory is supported by start-up funds from Michigan State University and the Michigan Agricultural Experiment Station.

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Villavicencio, L., Blankenship, S.M., Sanders, D.C. and Swallow, W.H. (1999) Ethylene and carbon dioxide production in detached fruit of selected pepper cultivars. Journal of the American Society for Horticultural Science 124, 402–406. Vogg, G., Fischer, S., Leide, J., Emmanuel, E., Jetter, R., Levy, A.A. and Riederer, M. (2004) Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase. Journal of Experimental Botany 55, 1401–1410. Vrebalov, J., Ruezinsky, D., Padmanabhan, V., White, R., Medrano, D., Drake, R., Schuch, W. and Giovannoni, J. (2002) A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus. Science 296, 343–346. Wang, H., Jones, B., Li, Z., Frasse, P., Delalande, C., Regad, F., Chaabouni, S., Latche, A., Pech, J.-C. and Bouzayen, M. (2005) The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell 17, 2676–2692. Watkins, C.B. (2006) The use of 1-methylcyclopropene (1-MCP) on fruits and vegetables. Biotechnology Advances 24, 389–409. Wilkinson, J.Q., Lanahan, M.B., Clark, D.G., Bleecker, A.B., Chang, C., Meyerowitz, E.M. and Klee, H.J. (1997) A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants. Nature Biotechnology 15, 444–447. Wilkinson, J.Q., Lanahan, M.B., Yen, H.-C., Giovannoni, J.J. and Klee, H.J. (1995) An ethylene-inducible component of signal transduction encoded by Never-ripe. Science 270, 1807–1809. Wilson, L.A. and Sterling, C. (1976) Studies on cuticle of tomato fruit. 1. Fine structure of cuticle. Zeitschrift Fur Pflanzenphysiologie 77, 359–371. Yamane, M., Abe, D., Yasui, S., Yokotani, N., Kimata, W., Ushijima, K., Nakano, R., Kubo, Y. and Inaba, A. (2007) Differential expression of ethylene biosynthetic genes in climacteric and non-climacteric Chinese pear fruit. Postharvest Biology and Technology 44, 220–227. Yanagawa, Y., Sullivan, J.A., Komatsu, S., Gusmaroli, G., Suzuki, G., Yin, J.N., Ishibashi, T., Saijo, Y., Rubio, V., Kimura, S., Wang, J. and Deng, X.W. (2004) Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes. Genes & Development 18, 2172–2181. Yang, S.F. and Hoffman, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 35, 155–190. Yen, H.-C., Lee, S., Tanksley, S.D., Lanahan, M.B., Klee, H.J. and Giovannoni, J.J. (1995) The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene. Plant Physiology 107, 1343–1353. Yen, H.C., Shelton, B.A., Howard, L.R., Lee, S., Vrebalov, J. and Giovannoni, J.J. (1997) The tomato high-pigment (hp) locus maps to chromosome 2 and influences plastome copy number and fruit quality. Theoretical and Applied Genetics 95, 1069–1079. Zabetakis, I. and Holden, M.A. (1997) Strawberry flavour: analysis and biosynthesis. Journal of the Science of Food and Agriculture 74, 421–434. Zuzunaga, M., Serrano, M., Martinez-Romero, D., Valero, D. and Riquelme, F. (2001) Comparative study of two plum (Prunus salicina Lindl.) cultivars during growth and ripening. Food Science and Technology International 7, 123–130.

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Annual Plant Reviews (2009) 38, 326–345 doi: 10.1002/9781444314557.ch9

www.interscience.wiley.com

Chapter 9

PARTHENOCARPY IN CROP PLANTS Tiziana Pandolfini,1 Barbara Molesini2 and Angelo Spena2 1

Department of Science, Technology and Market of the Vine and Wine, University of Verona, San Floriano (VR), Italy 2 Department of Biotechnology, University of Verona, Verona, Italy

Abstract: Fruit set and growth in the absence of fertilization (parthenocarpy) is a useful trait in plants grown for the value of their fruit. Auxins and gibberellins are widely used to spray flowers to chemically confer parthenocarpy. In recent years, genetic modifications of either auxin or gibberellin biology have been used to confer parthenocarpy to tomato and other crops. Present knowledge indicates that parthenocarpy can be achieved by genetic modification of either auxin synthesis (iaaM), auxin sensitivity (rolB), auxin content (Aucsia) or auxin signal transduction (IAA9 or ARF8). Genetic modification of gibberellin signal transduction (DELLA) has also been shown to confer parthenocarpy. Available data, obtained under both open field and protected cultivation, show that genetic parthenocarpy can be used to improve fruit production and/or fruit quality. The mechanisms, genetically modified to confer parthenocarpy, are active also in other plant organs. Observations consistent with the Euanthial theory that envisages the fruit as a modified leaf predict that the mechanisms underlying fruit initiation have been recruited from molecular machineries present and controlling other plant developmental processes. The flower/fruit represents the last evolutionary innovation of the green plant lineage, and yet genes (i.e. Aucsia) controlling fruit initiation are most likely older than 1 billion years being present in Prasinophytes (i.e. probable ancestors of Charophytes, which themselves are considered ancestors of all land plants). Keywords: auxin; gibberellin; fruit initiation; fruit production; fruit quality; parthenocarpy

9.1

Introduction

A crop is any plant that is grown to be harvested as food, livestock fodder, wood or for any other economic purpose. Some crop plants are cultivated 326

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for the economic value of their fruit. Fruits usually develop from the ovary, the basal part of the pistil. However, extracarpellary structures can contribute to the fruit. Fruit development usually takes place only after pollination/fertilization. Fruit set and development in the absence of pollination and fertilization is called parthenocarpy and parthenocarpic fruits are either seedless or contain a much reduced number of seeds. Parthenocarpic fruits have been known since IV century B.C. and by the middle of XX century, plant breeders had already appreciated that ‘virginfruiting’ could be a useful trait in many horticultural species cultivated for the value of their fruit (Ewert, 1929). Parthenocarpy has two main advantages. First, in parthenocarpic plants fruit set and production are less affected by environmental factors adverse for pollination and fertilization. The second main advantage of parthenocarpy is fruit seedlessness. In several fruit crops, seedlessness is advantageous either for fruit processing (e.g. tomato paste) or because it improves fruit quality (e.g. eggplant) or simply because seedlessness is a feature appreciated by consumers. Moreover, seedless fruit has often a shelf life longer than seeded fruits. Lastly, in parthenocarpic plants fruit set and growth often start before anthesis. Consequently, parthenocarpy might allow early fruit production and harvest. Thus, parthenocarpy is a valuable trait to be used to improve both fruit quality and productivity in horticultural species grown for the value of their fruit. Parthenocarpy has heuristic interest too. The pistil, and consequently the fruit, is an evolutionary innovation of plant development typical of Angiosperms. The fruit is a specialized structure that provides an appropriate environment for the development of the seeds and consequently of the embryos. Seed and embryo development within sporophytic organs takes place in Gymnosperms and in all Angiosperms, the most evolved plants. Another evolutionary function of the fruit is to assist seed dispersal. Thus, seedless fruit represents a biological paradox. However, wild parsnip (Pastinaca sativa), a crop plant having a quite high percentage (up to 20%) of parthenocarpic fruits, is an exception to this paradox. Webworms (Depressaria pastinacella) prefer to eat parsnip parthenocarpic fruits (Zangerl et al., 1991) because parthenocarpic fruits contain less furanocoumarin toxin and less octyl-butyrate, a volatile deterrent for webworms (Cianfrogna et al., 2002). Thus, the seedless fruits lure the predators and consequently preserve seeded fruits and the progeny contained within their seeds. A somewhat similar role for empty seeds in reducing seed predation by birds has been proposed in a Gymnosperm, Juniperus osteosperma (Fuentes and Schupp, 1998). Seedless fruits are often smaller than seeded fruits indicating that the development of the fruit is coordinated with the development of the seed/embryo. This cross-talk relies on signals produced by pollinated embryos/seeds. Nitsch was the first to show that auxin can replace fertilized ovules and seeds to set and further sustain fruit development (Nitsch, 1950). Consequently, auxin was considered an early signal coordinating embryo/seed and fruit development (Nitsch, 1970). More recently, the identification of molecular

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Fruit Development and Seed Dispersal mechanisms underlying parthenocarpy has shed light on the control of fruit set in Angiosperms (for review see, Pandolfini et al., 2007) and it might contribute to our understanding of plant development and evolution too. The methods commonly used in agricultural practice to elicit artificial parthenocarpy rely on phytohormones and related substances (Schwabe and Mills, 1981). Conceptually, all methods based on the exogenous application of phytohormones and/or related substances stem from the work of Gustafson showing that exogenous application of auxin to tomato flowers triggers parthenocarpic fruit development in the absence of pollination/fertilization (Gustafson, 1936). Recent knowledge derived from studies on genetic parthenocarpy also highlights the role of phytohormones, that is auxin and gibberellin, in the control of fruit set (for review see, Gorguet et al., 2005). The main form of natural auxin is indole-3-acetic acid (IAA). The discovery of IAA was accomplished via its biological (Went, 1926) and chemical identification (Koegl and Haagen Smit, 1931), and yet its search had been triggered by previous studies on gravitropism (Ciesielski, 1872) and phototropism (Darwin and Darwin, 1880). Tropisms are crucial for the adaptation of plants to their environment and consequently for plant evolution. Nowadays, auxin has been shown to control many plant processes including tropic responses (Woodward and Bartel, 2005). From embryo and leaf development to fruit development, auxin is crucial to sporophyte (Woodward and Bartel, 2005) and probably gametophyte development too. IAA is widespread from prokaryotes to higher eukaryotes, and yet only in plants IAA function, at submicromolar concentrations, as a phytohormone. In fact, at much higher concentrations, IAA is toxic for eukaryotic cells (de Melo et al., 2004). IAA is usually derived from tryptophan, an ancient molecule in life history. Thus, it has been argued that Darwin’s hypothetical substance now called auxin might have played a crucial role in the evolution of novel body plans during the late Silurian–early Devonian radiation of land plants (Cooke et al., 2003).

9.2

Parthenocarpy

Parthenocarpy can be either elicited by artificial means (artificial parthenocarpy) or via genetic modifications (genetic parthenocarpy). Genetic parthenocarpy can be either facultative or obligatory. In facultative parthenocarpy, the fruits are seedless only when grown under conditions adverse for pollination and/or fertilization or when the flowers are emasculated. Parthenocarpy is obligated when the fruit is always seedless. In the past, obligate and facultative genetic parthenocarpy was either obtained by alterations of ploidy or caused by gene(s) mutations. In recent years, parthenocarpy has been achieved via transgenesis too. Pollination either with dead pollen (Massart, 1902) and/or with pollen extracts (Fitting, 1909; Yasuda, 1934) were the first methods used to trigger

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artificial parthenocarpy. The chemical identification of auxin (Koegl and Haagen-Smit, 1931) was followed by the demonstration that pollen extracts contain auxin and that exogenous auxin triggers parthenocarpy in several plant species (Gustafson, 1936, 1937, 1942). Auxin transport inhibitors can also elicit parthenocarpy (Robinson et al., 1971) most likely by blocking auxin flow from the ovules/ovary and consequently causing an increase in auxin content. The discovery of other types of phytohormones allowed to evaluate their effects on parthenocarpic fruit set (Schwabe and Mills, 1981). Gibberellins, also present in pollen extracts (Tsao, 1980), emerged as another class of phytohormones able to induce fruit set in the absence of pollination/fertilization. Nowadays, it is quite a common agricultural practice in some horticultural crop plants such as tomato and eggplant, to spray flowers with auxin and/or gibberellin to trigger parthenocarpic fruit development. Other phytohormones are known to affect fruit set and development. In this regard, cytokinins have also been used in some species (Schwabe and Mills, 1981). Although delivery of phytohormones via pollen tube to ovules has not been experimentally proven, all previous experiments are usually interpreted as indicating that pollen might provide phytohormones, such as auxin and gibberellin, to the ovules (Nitsch, 1970). However, fertilized ovules can also accumulate auxin (Nitsch, 1952), probably either via de novo auxin synthesis and/or by reducing auxin transport. Each one of these three mechanisms – pollen-derived auxin, de novo auxin synthesis and inhibition of auxin transport – might by itself lead to an increased auxin content within fertilized ovules/ovaries. It is however likely that, during fruit initiation, all three mechanisms might concur to increase auxin content within the fertilized ovules/ovary. The increased auxin content within the ovules and ovary will then stimulate gibberellin (GA) synthesis (Ross and O’Neill, 2001). In several species and tissues, auxin stimulates GA content acting both on GA biosynthesis and inactivation (Weiss and Ori, 2007). The increase in both auxin and GA content would then activate auxin and GA signalling pathways based on degradation of specific target proteins causing fruit set and growth (Fu and Harberd, 2003; Goetz et al., 2006). Auxin and GA are the most common phytohormones used to trigger artificial parthenocarpy in crop plants. Recent studies on genetic parthenocarpy have contributed to the mechanisms underlying the biological effects triggered by auxin and GA.

9.3 Auxin-synthesis parthenocarpy Auxin synthesis within unpollinated ovary has been achieved by expressing the iaaM gene from Pseudomonas syringae under the control of the DefH9 (Deficiens Homologue 9) promoter from Antirrhinum majus (Rotino et al., 1997). The DefH9 promoter confers placenta–ovule specific expression to the tryptophan 2-monoxigenase encoded by the iaaM gene. Tryptophan 2-monoxigenase

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Fruit Development and Seed Dispersal converts trytophan to indole-acetamide (Kosuge et al., 1966). Within plant cells, indole-acetamide is converted by either spontaneous or enzymatic hydrolysis to IAA and ammonia. Consistently, IAA content of DefH9-iaaM flower buds is higher than wild-type flowers (Pandolfini et al., 2002; Costantini et al., 2007). The DefH9-iaaM gene has been used to confer parthenocarpy to several crop plant species (eggplant, tobacco: Rotino et al., 1997; tomato: Ficcadenti et al., 1999; raspberry, strawberry: Mezzetti et al., 2004; cucumber: Yin et al., 2006). When cultivated in vitro, wild-type pre-anthesis tomato flowers need exogenous auxin to develop fruits (Nitsch, 1951), whilst DefH9-iaaM pre-anthesis flower buds develop fruits in medium not supplemented with auxin (Fig. 9.1a). Altogether, the data prove that an increased auxin content, in this case achieved via endogenous synthesis within the placenta/ovules, triggers parthenocarpic fruit development in species belonging to distinct botanical families (e.g. Solanaceae, Rosaceae, Cucurbitaceae) and having different fruit types (e.g. tomato, raspberry, tobacco). Auxin-synthesis parthenocarpy is facultative. Under conditions prohibitive for fertilization/pollination, fruits are seedless. Under environmental conditions favourable for pollination, fruits contain seeds. In DefH9-iaaM auxin-synthesis flower buds, ovary growth starts before anthesis, whereas in wild-type pre-anthesis flower buds ovary growth is restricted (Spena and Rotino, 2001; Fig. 9.1b). Transcript profiles from pre-anthesis DefH9iaaM tomato flower buds are consistent with the interpretation that fruit-set mechanisms are already active before anthesis in auxin-synthesis parthenocarpic flowers (Fig. 9.2). Polyamines are known to be involved in tomato fruit set and growth (Alabad´ı et al., 1996). The ornithine decarboxylase (ODC) gene of tomato, which encodes the enzyme that catalyzes the first step of polyamine biosynthesis, is up-regulated in fruits induced to grow either by exogenous auxin (Alabad´ı and Carbonell, 1998) or in unpollinated ovaries of the parthenocarpic tomato mutant pat-2 (Fos et al., 2003) or in pre-anthesis DefH9-iaaM flower buds (Fig. 9.2). A tomato gene homologous to the ArgE protein of Escherichia coli, a putative N-acetylornithine deacetylase that catalyzes the hydrolysis of N-acetylornithine to ornithine (LeArgE) is also upregulated in DefH9-iaaM pre-anthesis flower buds (Fig. 9.2). Interestingly, in auxin-synthesis parthenocarpy tomato flower buds Aucsia gene expression is already down-regulated at pre-anthesis (see Section 9.6 ). Altogether, these observations suggest that the fruit developmental programme is already active in pre-anthesis auxin-synthesis parthenocarpic flower buds. In conclusion, auxin synthesis within the placenta–ovules triggers parthenocarpic fruit development.

9.4

Parthenocarpy via auxin signal transduction

In plant cells, changes in auxin content triggers auxin signal transduction and response. Auxin-dependent transcriptional regulation is mediated by

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Figure 9.1 In vitro development of parthenocarpic DefH9-iaaM tomato fruit. (a) (Left panel) Flower buds from auxin-synthesis parthenocarpic plants collected at pre-anthesis and cultivated in medium not supplemented with auxin. (Middle panel) Ovaries growth after 10 days of in vitro cultivation. (Right panel) Mature fruits after approximately 30 days of in vitro cultivation. (b) (Left panel) Ovaries present in pre-anthesis (stage a) wild-type (wt) flower buds as compared with the ovaries present in DefH9-iaaM flower buds. (Right panel) DefH9-iaaM pre-anthesis flower bud showing enlarged ovary. (For a colour version of this figure, please see Plate 4 of the colour plate section.)

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Figure 9.2 Transcript profiles from pre-anthesis auxin-synthesis parthenocarpic flower buds. Northern blot analysis showing steady state mRNAs levels of ArgE, ODC and actin genes in different developmental stages of wt flower buds (a: 0.5 cm-long flower bud at 6–7 days before anthesis; b: 0.7–0.9 cm-long flower bud at 4–5 days before anthesis; c: 1.0–1.1 cm-long flower bud at 1–3 days before anthesis; d: flower bud at anthesis; e: flower bud at 4–5 days after anthesis; f: ovary, 0.5–1 cm-long, at 6 days after anthesis; g: fruit at 11 days after anthesis). (Bottom panel) ArgE, ODC and actin mRNAs levels in pre-anthesis flower buds (stages a, b and c) from DefH9-iaaM and wt tomatoes.

regulatory proteins belonging to auxin/indole-3-acetic acid (AUX/IAA) and auxin response factor (ARF) families of transcription factors (Dharmasiri et al., 2005; Leyser, 2006). A crucial role in auxin perception is played by SCF-type ubiquitin-ligase complexes (Tan et al., 2007). The SCF-TIR1 complex is activated by auxin binding (Tan et al., 2007). SCF-TIR1 and probably related auxin-regulated SCF-AFB complexes poly-ubiquitinilate target proteins, such as AUX/IAA transcriptional regulators, triggering their degradation (Dharmasiri et al., 2005). Degradation of AUX/IAA proteins usually derepress transcription of auxin-regulated genes (Woodward and Bartel, 2005; Leyser, 2006). In tomato, silencing IAA9 gene expression confers parthenocarpy

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333 Box 9.1 Phenotype is the result of the interactions between the introgressed gene, the genetic background and the environment. The available data obtained with parthenocarpic plants engineered via novel genetic tools confirm this well-known plant breeding principle. In the case of the DefH9-iaaM auxin-synthesis gene all the available data can be summarized as follows: 1. DefH9-iaaM confers facultative parthenocarpy to several plant species (Rotino et al., 1997; Ficcadenti et al., 1999; Mezzetti et al., 2004; Yin et al., 2006). 2. Within a plant species (e.g. tomato. eggplant), the DefH9-iaaM gene or its derivative DefH9-RI-iaaM confers parthenocarpy to several lines or varieties with rather different fruit size and form (Rotino et al., 1997, 2008; Acciarri et al., 2000, 2002; Donzella et al., 2000; Pandolfini et al., 2002). 3. Unpollinated DefH9-iaaM tomato ovaries produce seedless fruit. Pollinated DefH9-iaaM ovaries produce seeds, although their number is drastically reduced (Rotino et al., 2005). 4. Fruit set is improved in all tomato genetic backgrounds tested, and yet the increase in fruit set is more pronounced in cultivars low-productive due to a low fruit-set efficiency (Acciarri et al., 2000). 5. Increased tomato and eggplant fruit production under greenhouse cultivation conditions (Acciarri et al., 2000; Donzella et al., 2000). 6. Fruit quality of parthenocarpic fruits, besides seedlessness, is either equivalent or better than that of seeded fruits (Maestrelli et al., 2003; Rotino et al., 2005; Costantini et al., 2007). 7. Under open field conditions, fruit production (total fruit weight per plant) was either equivalent or increased compared to wild-type control (Acciarri et al., 2002; Rotino et al., 2005). 8. Auxin synthesis within placenta–ovules often causes fruit set before the development of stamens and anthers. Consequently, under open field cultivation conditions most DefH9-iaaM fruits are seedless although the environmental conditions did not hamper pollination/fertilization (Acciarri et al., 2002; Rotino et al., 2005).

and other auxin-related phenotypes (Wang et al., 2005). The down-regulation of IAA9 mRNAs most likely mimics the degradation of IAA9 protein caused by the increased auxin content of the ovules/ovary that follows pollination/fertilization. In tomato and Arabidopsis, parthenocarpy has also been obtained by genetic alterations of ARF8 function (Goetz et al., 2006, 2007). ARF8 is an ovule-specific transcription factor that negatively regulates fruit set (Goetz et al., 2006, 2007). ARF8 gene expression is switched off after pollination/fertilization. ARF family proteins are transcriptional regulators acting either as repressors or activators of transcription (Woodward and Bartel, 2005). ARFs form heterodimers with AUX/IAA proteins that bind to promoters of auxin-responsive genes and also ARF8 action is most likely regulated via heterodimerization with specific AUX/IAA proteins (Goetz et al., 2007). At the simplest, the data can be interpreted as indicating that before anthesis ARF8/IAA9 heterodimers repress tomato ovary growth. The increased auxin content in the fertilized ovules would activate the degradation of IAA9

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Fruit Development and Seed Dispersal protein via the ubiquitin-proteasome pathway releasing the inhibitory effect of ARF8/IAA9 heterodimers on target gene expression and fruit growth. A genetic mutation of ARF8 would have a rather similar effect on ARF8/IAA9 heterodimers releasing their inhibitory action. However, ARF8 might also have other molecular functions during fruit growth as suggested by the observation that parthenocarpic fruits obtained via ARF8 genetic ablation are smaller than seeded fruits (Goetz et al., 2007).

9.5

Parthenocarpy via gibberellin signal transduction

Derepression of a repressed state is considered also the key step in GA signal transduction pathways (Jiang and Fu, 2007). DELLA proteins are a subfamily of the GRAS protein family of putative transcription factors characterized by the conserved amino acid motif DELLA (Thomas and Sun, 2004). DELLA proteins repress GA signal transduction and GA targets DELLA proteins for degradation via ubiquitin-proteasome-mediated proteolysis releasing their repressive action (Fleet and Sun, 2005). Silencing DELLA gene expression in tomato causes parthenocarpic fruit development (Mart´ı et al., 2007). DELLA gene silencing should mimic the degradation of DELLA protein stimulated by the increased GA content of pollinated ovaries. In several plant species and tissues, auxin stimulates transcription of GA biosynthetic genes (Ross and O’Neill, 2001; Weiss and Ori, 2007) and auxin-induced GA synthesis might lead to an increased GA content. Thus, the increased GA content of the ovary could be caused either by pollen-derived GAs (Nitsch, 1970; Tsao, 1980) and/or by stimulating endogenous GA synthesis and/or inhibiting GA inactivation within fertilized ovules. Moreover, in Arabidopsis roots, it has been shown that GA-dependent DELLA protein degradation is stimulated by auxin (Fu and Harberd, 2003). Tomato parthenocarpic fruits obtained via DELLA silencing are smaller in size and elongated in shape compared with either wild type or DELLAsilenced pollinated fruits (Mart´ı et al., 2007). This observation might indicate that to achieve optimal fruit set and growth both auxin and GA signal transduction pathways have to be active.

9.6 Aucsia-silencing parthenocarpy We have identified two tomato genes, called Aucsia genes, and shown that RNAi of both Aucsia genes causes parthenocarpic fruit development in tomato (Molesini et al., 2009). In wild-type tomato ovaries, both Aucsia genes are drastically down-regulated at fruit set (Molesini et al., 2009). Thus, a reduced expression of both Aucsia genes at fruit set is part of the molecular mechanisms controlling fruit initiation. Interestingly, in auxin-synthesis

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parthenocarpy down-regulation of Aucsia genes already takes place in preanthesis (0.5 cm long) flower buds (Molesini et al., 2009). The Aucsia genes have been functionally identified in tomato. However, Aucsia homologous genes and transcripts are widespread in green plants from Chlorophytes to Streptophytes. Aucsia genes encode small peptides 44–54 aa long (Molesini et al., 2009). AUCSIA peptides have a conserved 16 amino acidlong AUCSIA motif, a conserved tyrosine-based sorting motif involved in endocytosis and a lysine-rich carboxyterminal region. The first serine residue of the AUCSIA motif is a likely candidate for alternate phosphorylation/ON-acetylglucosamine glycosylation (www.cbs.dtu.dk/services/YinOYang/). Consequently, AUCSIA peptides could be present in alternative modified forms, either phosphorylated or glycosylated. The Arabidopsis genome contains two O-GlcNAc transferases, SPINDLY and SECRET AGENT, that catalyze the transfer of GlcNAc (N-acetylglucosamine) to serine or threonine of proteins (Hartweck et al., 2002). Arabidopsis spindly mutants show parthenocarpic fruit development and other alterations of growth (Jacobsen and Olszewski, 1993). The minimal molecular mass of AUCSIA peptides and their conserved features suggest that AUCSIA peptides are probably regulatory subunits of large protein complexes involved in auxin biology. Endocytic recycling of auxin transporters is required for auxin distribution and action (Geldner et al., 2003). Auxin polar transport, both at long and short distance, is crucial for auxin responses (Leyser, 2006). Inhibitors of auxin transport cause parthenocarpy (Robinson et al., 1971) indicating that a block of auxin transport within the ovules/ovary might trigger fruit set and development likely by increasing auxin content within the ovules/ovary. Several genes involved in auxin transport are known (Leyser, 2006) and Arabidopsis mutants altered in auxin transport have been extensively analyzed (Vieten et al., 2007). However, so far there has been no genetic evidence linking auxin transport to parthenocarpy.

9.7 Auxin sensitivity and parthenocarpy In tomato, parthenocarpy has been conferred by the auxin-like action of the rolB gene from Agrobacterium rhizogenes expressed under the control of a fruit-specific promoter (Carmi et al., 2003). The mechanism of action of RolB is not known. It has been proposed that RolB expression increases auxin sensitivity either by its phosphatase activity (Carmi et al., 2003) or via its ␤-glucosidase activity (Spena et al., 1996). Phosphorylation– dephosphorylation regulates many biological processes including auxin signal transduction and auxin transport (Zegzouti et al., 2006). Thus, a phosphatase activity might alter auxin response by affecting either auxin signalling and/or transport. A ␤-glucosidase activity might release indolic compounds, such as indolethanol from indolethanol-glucoside, and consequently it might affect IAA homeostasis (Spena et al., 1996). However, phosphatases and glucosidases are rather

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9.8

Apetalous parthenocarpy and the role of other floral organs

Apetalous parthenocarpy has often been observed in apple cultivars (Tobutt, 1994). The flowers lack petals and stamens, have two whorls of sepals and up to 15 styles (Tobutt, 1994). Yao et al. (2001) have shown that apple apetalous parthenocarpy is due to a mutation in Pistillata, a MADS-box transcription factor involved in the control of floral organ identity. In tomato, parthenocarpic development has been caused by mutations in TM8 (Lifschitz et al., 1993) and by genetic alterations of TM5 (Pnueli et al., 1994) and TM29 (Ampomah-Dwamena et al., 2002). TM5, TM8 and TM29 are tomato SEPALLATA homologous genes. SEPALLATA genes are involved in the control of floral organ identity (Pelaz et al., 2000). The demonstration that different genes controlling floral organ identity cause parthenocarpy suggests that mutations in genes affecting floral organ identity might either display parthenocarpy or facilitate parthenocarpic fruit development. Other natural parthenocarpic mutants display developmental alterations affecting either outer floral organs and/or ovule development (e.g. pat gene of tomato; Pecaut and Philouze, 1978). Concerning the influence of outer floral organs on fruit set and growth, it is worthwhile mentioning that a successful fertilization triggers not only fruit growth (i.e. ovary cell division and expansion), but also the senescence of the outer floral organs (stamens, petals, sepals). In this regard, the mechanical deletion of outer floral organs enhances the parthenocarpy caused by ARF8 genetic ablation in Arabidopsis (Vivian-Smith et al., 2001). In conclusion, outer floral organs influence fruit initiation and growth although the underlying mechanisms are unknown.

9.9

Stenospermocarpy

Stenospermocarpy is the production of seedless fruits having only seed traces. In watermelon, a method to breed triploid plants that develop seedless fruits with only residual integuments has been widely used (Kihara, 1951). The triploid plants are not self-fertile and their fruit is stenospermocarpic because ovule/embryo development is blocked quite early on. The molecular mechanisms conferring seedlessness in triploid watermelons are unknown. In Saccharomyces cerevisiae, ploidy is known to affect the expression of several genes (Galitski et al., 1999).

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Thompson seedless grape cultivars are widely cultivated. Thompson seedless cultivars are stenospermocarpic and their berries have only seed traces. GA treatment of Thompson seedless cultivars is commonly used to increase berry size, to elongate the grape cluster and to reduce seed traces. The same effects have been obtained by introducing the DefH9-iaaM auxin-synthesizing gene in the genome of a Thompson seedless cultivar (Costantini et al., 2007). GA content has not been assayed in DefH9-iaaM Thompson seedless fruit. Thus, the observed effects can be only interpreted as probably due to auxininduced GA synthesis within the berry.

9.10

Parthenocarpy in perennial crop plants

In perennial crop plants, data on novel genetic tools to confer parthenocarpy are quite limited. The DefH9-iaaM gene has been shown to cause parthenocarpic fruit development in raspberry (Mezzetti et al., 2004) and to improve seedlessness in table grape (Costantini et al., 2007). The effect of the DefH9-iaaM gene on fruit production has been tested under standard cultivation conditions in three Rosaceae species, Fragaria vesca, Fragaria x ananassa and Rubus idaeus (Mezzetti et al., 2004) and in two table grape cultivars (Costantini et al., 2007). DefH9-iaaM plants of all three Rosaceae species tested have an increased number of flowers per inflorescence and an increased number of inflorescences per plant resulting in an increased number of fruits per plant. Moreover, the weight and size of DefH9-iaaM transgenic Rosaceae fruits was also increased. The increase in fruit yield was approximately 180% in cultivated strawberry, 140% in wild strawberry and 100% in raspberry. In table grape, the DefH9-iaaM gene caused a more than 100% increased fruit production in the Thompson seedless genetic background and an 8% increase in the Silcora genetic background (Costantini et al., 2007). The increase in fruit productivity observed in all these perennial species is due to the cumulative effects on fruit size, flower/fruit number and inflorescence number per plant.

9.11

Parthenocarpy and fruit crop breeding

Parthenocarpy is an efficient way to improve fruit production under cultivation conditions negative for fruit set (Gorguet et al., 2005). Moreover, parthenocarpy can also be beneficial under conditions favourable for fruit set and growth. This because parthenocarpic plants often display an early fruit set, taking place before anthesis (Fig. 9.1b; Spena and Rotino, 2001), that allows early fruit production. In some fruit crops (e.g. eggplant, cucumber, grape, industrial tomato), parthenocarpy improves fruit quality too. Genetic

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Fruit Development and Seed Dispersal parthenocarpy might therefore offer the possibility of improving fruit quality, fruit productivity and to rationalize fruit production in horticulture. Genetic (natural) parthenocarpy has been reported in many horticultural species, however, parthenocarpy has been so far widely used only in few fruit crops (e.g. banana, cucumber). Musa spp. (e.g. banana) cultivated as crop plants are usually triploid sterile plants that produce parthenocarpic fruits. The parthenocarpic trait in Musa spp. is polygenic (Ortiz and Vuylsteke, 1995). In cucumber, parthenocarpy is controlled by a single dominant gene and by other modifier genes (Pike and Peterson, 1969). The limited use of natural parthenocarpic lines and cultivars in horticulture is quite often due to the smaller size of parthenocarpic fruits in comparison to seeded ones. Thus, a more widespread use of parthenocarpic plants in horticulture requires methods valid for most fruit crop plant species/varieties and reliable under different environmental conditions. Furthermore, and most importantly, the methods of choice should preferably improve both fruit quality and productivity. Alternatively, they should either improve fruit quality without curtailing productivity, or increase fruit productivity without affecting fruit quality. Tomato is one of the most important fruit crop plants and provides sufficient data to evaluate novel genetic strategies for breeding parthenocarpy in annual species. In tomato, there is a huge genetic diversity evident also in the different sizes and forms of tomato fruits. Natural sources of parthenocarpy are mainly ‘Severianin’ and line RP75/79 (for review, see Gorguet et al., 2005). ‘Severianin’ parthenocarpy is due to a single gene (pat-2), while RP75/79 parthenocarpy is due to two genes (pat-3/pat-4). Both types of parthenocarpy have an incomplete penetration and expression of the trait, and the quality of their fruit is curtailed by several negative ‘collateral effects’ such as reduced firmness, malformation, inefficient fruit set (Philouze et al., 1988). Thus, novel genetic methods for parthenocarpy in tomato, and in other horticultural crops showing high phenotypical variability (e.g. eggplant), should be capable of improving fruit set and productivity in most, if not all available cultivars and lines. This aim has to be accomplished either by preserving or improving the quality of the seedless fruit. In tomato and related crop plants (e.g. eggplant), parthenocarpy is valuable both to produce tomato seedless fruit for the processing industry (e.g. tomato paste and peeled tomatoes production) and to improve productivity in tomato and eggplant under cultivation conditions adverse for fruit set (e.g. winter production of fresh market fruit in unheated greenhouses). Thus, new tomatoes for the processing industry will have to be tested under open field cultivation conditions, while tomato and eggplant lines cultivated for fresh fruit consumption have to be tested under both winter and spring cultivation conditions. Seedless tomato fruit for the processing industry provide an example of the relevance of the genetic background for transgenic/cisgenic methods. The importance of testing parthenocarpy in different genetic backgrounds can be well appreciated by UC82 tomato plants transgenic for either the DefH9-iaaM

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or the TRPF1-rolB genes. The DefH9-iaaM and TRPF1-rolB have conferred facultative parthenocarpy in several genetic tomato backgrounds producing seedless fruits of normal shape (Ficcadenti et al., 1999; Carmi et al., 2003). However, the TRPF1-rolB transgene in the UC82 background, a tomato grown for the processing industry, causes under summer cultivation conditions, the production of fruits containing more seeds than wild-type fruits (Shabtai et al., 2007). In the UC82 genetic background, the DefH9-iaaM gene causes the production of seedless parthenocarpic fruits, and yet the fruits are usually morphologically altered due to an excess of auxin. UC82 parthenocarpic fruits of normal shape and size have been obtained by using the DefH9-RI-iaaM gene, a DefH9-iaaM gene version curtailed in its expression at the post-transcriptional level (Pandolfini et al., 2002). Under open field cultivation conditions permissive for pollination/fertilization, DefH9-RI-iaaM fruits were either seedless or contained few seeds (Rotino et al., 2005). Consequently, the average seed number per fruit was 60–80% decreased in comparison to wild type. The UC82 example shows the importance of testing the gene(s) of interest in different genetic backgrounds and the importance, in modern plant breeding, of knowledge of the molecular function of the gene(s) used. Knowledge of the molecular function of the chosen gene can assist the rational development of modifications necessary to optimize gene action in a preferred genetic background. For example, a reduced action of the DefH9-iaaM gene has been achieved by curtailing gene expression (e.g. DefH9-RI-iaaM; Pandolfini et al., 2002), however, a similar result could probably have been achieved by using a tryptophan 2-monoxigenase variant (C511S) obtained by single amino acid substitution and curtailed in its enzymatic activity (Sobrado and Fitzpatrick, 2002). Seedless tomato fruit for the fresh market provide an example of the relevance of the environment. Parthenocarpy is particularly advantageous under cultivation conditions adverse for pollination and fertilization. During winter cultivation in unheated greenhouses, suboptimal temperatures and low light intensity inhibit fruit set and growth curtailing fruit production. Auxinsynthesis parthenocarpy (DefH9-iaaM transgene) has been tested for fruit production during spring and winter cultivation conditions in tomato and eggplant (Acciarri et al., 2000; Donzella et al., 2000). Under spring cultivation conditions, with temperatures suboptimal for tomato fruit set and growth, auxin-synthesis parthenocarpy has improved both fruit set and fruit production in tomato hybrid lines with round-shaped fruits (e.g. Giasone, 95–514, 95–516) and cherry tomatoes (L.CMxL.4). Fruit set in DefH9-iaaM tomato lines was usually higher than 90%, whilst in control plants ranged from 42% for cherry tomato to a maximum of 70% in the hybrid tomato line 95–514 (Acciarri et al., 2000). Fruit yield per plant was increased by 61% for tomato line 95–516, 123% for line 95–514 and 150% for line Giasone (tomato with round-shaped fruits) compared to controls not treated with exogenous phytohormones (Acciarri et al., 2000). In cherry tomato, the increase in fruit yield was higher than 250% compared to untreated controls (Acciarri et al., 2000).

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Fruit Development and Seed Dispersal During spring cultivation DefH9-iaaM tomato fruits were seedless. Similar results were obtained by testing fruit set and growth in DefH9-iaaM parthenocarpic eggplants during winter cultivation (Donzella et al., 2000). Total eggplant fruit production from late January to the 1st of June was 45% and 27% increased in the parthenocarpic hybrid lines compared to controls treated with exogenous phytohormones (Donzella et al., 2000). Without phytohormone treatment of the flowers, the increment in total yield was 157% and 78% in the two hybrid lines tested (Donzella et al., 2000). Under cultivation conditions not limiting for fruit set and growth, auxin-synthesis tomatoes (Rotino et al., 2005) and eggplants (Acciarri et al., 2002) showed the parthenocarpic trait and displayed an increased ability to set fruits. Due to the higher fruit number and weight, summer yield of parthenocarpic auxin-synthesis eggplants was increased by 36–76% compared to control. Tomato auxin-synthesis parthenocarpic lines grown under cultivation conditions not limiting for pollination/fertilization showed a summer total fruit production per plant not different from control (Rotino et al., 2005). This because in auxin-synthesis UC82 tomato lines, fruit number per plant was increased, but the average fruit weight was decreased (Rotino et al., 2005). The reduced average fruit weight observed in UC82 parthenocarpic fruit grown under open field conditions might be due to the increased number of fruits per plant caused by the improved fruit set. In such cases, appropriate agronomical practices (i.e. fertilizers, watering) could be adopted to increase fruit weight and consequently fruit production. Besides the absence of seeds, auxin-synthesis parthenocarpic eggplant and tomato fruits analyzed for fruit quality parameters were found either substantially equivalent or better than seeded fruits (Maestrelli et al., 2003; Rotino et al., 2005).

9.12

From green plants to fruit crop plants

More than 1 billion years ago, green plants split into two clades: Chlorophytes and Streptophytes (Bowman et al., 2007). For quite a long time, green plants were exclusively aquatic organisms. The first land plants probably appeared 470 Mya and slightly later, land plants radiated in bryophytes and vascular plants. Vascular plants evolved, approximately 300 Mya, a seed-plant lineage that gave rise to two sister groups: the Gymnosperms and the Angiosperms or flowering plants. The evolution of the flower is typical of Angiosperms and the development of fertile organs, the carpel(s) and stamens of the flower, was crucial for the evolution of innovative systems for pollination/fertilization, seed production and dispersal. The Euanthial theory of Angiosperm evolution envisions that carpels and other floral organs are modified leaves (Arber and Parkin, 1907; Stebbins, 1974). According to this theory, Angiosperms might have developed the carpel/pistil via cooptation and successive modifications of pre-existing mechanisms controlling leaf development. Leaf vascular development and

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phillotaxy are controlled by auxin (Scarpella et al., 2006), and young leaves are one of the main sites of auxin synthesis. The identification of molecular mechanisms controlling fruit initiation in crop plants has highlighted a crucial role for auxin in the cross-talk between fertilized ovules, which develop seeds and embryos, and the surrounding sporophytic tissues that develop in fruit. As far as regards auxin, it appears that the control of fruit set and development in both crop and model plants has been achieved by recruiting signal-transduction and signal-controlling mechanisms similar to those already acting in other organs and structures of Angiosperms. It has been argued that phyllotaxis and leaf vascularization could be controlled by the same mechanisms (Berleth et al., 2007). If so, then also fruit initiation might be based on a similar molecular mechanics. Seedless parthenocarpic crops have shown that it is feasible to reduce the number of seeds producing seedless fruit. Knowledge of the molecular mechanisms controlling fruit initiation and development is currently driving the development of novel transgenic and cisgenic tools to confer parthenocarpy to crop plants.

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Spena, A. and Rotino, G.L. (2001) Parthenocarpy: state of the art. In: Current Trends in the Embryology of Angiosperms (eds D.S. Bhojwani and W.Y. Soh). Kluwer Academic Publishers, Dordrecht, pp 435–450. Spena, A., Saedler, H., Sommer, H. and Rotino, G.L. (1996) Phytowelt Greentechnologies/Istituto Sperimentale Orticultura/Spena Angelo. Methods for producing parthenocarpic or female sterile transgenic plants and methods for enhancing fruit setting and development. US Patent 6483012, 19 November 2002, WO98/28430. Stebbins, G.L. (1974) Flowering Plants: Evolution above the Species Level. Harvard University Press, Cambridge. Tan, X., Calderon-Villalobos, L.I.A., Sharon, M., Zheng, C., Robinson, C.V., Estelle, M. and Zheng, N. (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645. Thomas, S.G. and Sun, T. (2004) Update on gibberellin signaling. A tale of the tall and the short. Plant Physiology 135, 668–676. Tobutt, K.R. (1994) Combining apetalous parthenocarpy with columnar growth habit in apple. Euphytica 77, 51–54. Tsao, T. (1980) Growth substances: role in fertilization and sex expression. In: Plant Growth Substances (ed. F. Skoog). Springer-Verlag, New York, pp 345–348. Vieten, A., Sauer, M., Brewer, P.B. and Friml, J. (2007) Molecular and cellular aspects of auxin-transport-mediated development. Trends in Plant Sciences 12, 160–168. Vivian-Smith, A., Luo, M., Chaudhury, A. and Koltunow, A. (2001) Fruit development is actively restricted in the absence of fertilization in Arabidopsis. Development 128, 2321–2331. Wang, H., Jones, B., Li, Z., Frasse, P., Delalande, C., Regad, F., Chaabouni, S., Latch´e, A., Pech, J.C. and Bouzayen, M. (2005) The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell 17, 2676–2692. Weiss, D. and Ori, N. (2007) Mechanisms of cross talk between gibberellin and other hormones. Plant Physiology 144, 1240–1246. Went, F.W. (1926) On growth-accelerating substances in the coleoptile of Avena sativa. Proceedings of the Koniklijke Nederlandse Akademie van Wetenschappen. Series C, Biological and Medical Sciences 30, 10–19. Woodward, A.W. and Bartel, B. (2005) Auxin: regulation, action and interaction. Annals of Botany 95, 707–735. Yao, J., Dong, Y. and Morris, B.A. (2001) Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. Proceedings of the National Academy of Sciences of the United States of America 98, 1306–1311. Yasuda, S. (1934) Parthenocarpy caused by the stimulus of pollination in some plants of Solanaceae. Agriculture and Horticulture 9, 647–656. ´ Yin, Z., Malinowski, R., Ziołkowska, A., Sommer, H., Plcader, W. and Malepszy, S. (2006) The DefH9-iaaM-containing construct efficiently induces parthenocarpy in cucumber. Cellular & Molecular Biology Letters 11, 279–290. Zangerl, A.R., Nitao, J.K. and Berenbaum, M.R. (1991) Parthenocarpic fruits in wild parsnip: decoy defence against a specialist herbivore. Evolutionary Ecology 5, 136–145. ¨ Zegzouti, H., Anthony, R.G., Jahchan, N., Bogre, L. and Christensen, S.K. (2006) Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 103, 6404–6409.

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INDEX

(1→5)-α-L-arabinan, 300 β-1,3-glucans (callose), 261 γ-aminobutryic acid (GABA), 118 abaxial, 15, 38, 72, 182 abaxial–adaxial polarity, 48, 74, 77, 182 ABC model, 12, 44 ABCE model, 12 ABERRANT TESTA SHAPE (ATS), 78 abominable mystery, 72 abscissic acid (ABA), 257, 260 Abu Hureyra, 243 aca9, 121 ACC oxidase, 304 ACC synthase, 303 ACS2, 140 ACS6, 140 adaxial, 38, 74, 182 A-function, 13, 44 AGAMOUS (AG), 13, 44, 79, 177 AGAMOUS-LIKE6 (AGL6), 17 AGL23, 85 AGL24, 277 AGL80, 85 Agrobacterium rhizogenes, 335 AINTEGUMENTA/aintegumenta (ANT/ant), 25, 40, 75 ALCATRAZ (ALC), 49, 178, 301 allogeny, 278 Amaranthaceae, 261 Amborellales, 7 Amborella trichopoda, 7, 72 ampisporangiate strobilius, 3 amplified fragment length polymorphism (AFLP), 251 ANA grade, 7 anatropous placentation, 12 androecium, 3 angiosperm, 1, 36, 71, 108, 173, 340 ANGUSTIFOLIA3 (AN3), 40

346

anthesis, 42, 111, 174, 330 anthocyanin, 306 antipodal cells, 82, 117 Antirrhinum, 12, 53 APETALA1 (AP1), 23, 179, 301 APETALA2 (AP2), 22, 44, 251 apetalous parthenocarpy, 336 apical meristem, 3, 182 apocarpic, 10 Apocynaceae, 24 Arabidopsis thaliana, 5, 36, 70, 115, 174 ARABINOGALACTAN PROTEIN18, 85 Araucariaceae, 7 archaeobotany, 239 ard tillage, 267 ARGONAUTE (AGO), 141 ARGOS, 40 Asarum canadense, 209 asterids, 2 ASYMMETRIC LEAVES1 and 2 (AS1/2), 37, 185 ASYNAPTIC (ASY1), 84 A. tauschii, 251 Atlantic meridional overturning circulation, 229 AtMPK3, 140 AtMPK6, 140 Aucsia, 330 Austrobaileya scandens, 10 Austrobaileyaceae, 7 Austrobaileyales, 7 Aux/IAA, 124 auxin, 124, 192, 305 biosynthesis, 54, 129 gradient, 193 in parthenocarpy, 327, 329–30 response, 120 signalling, 55, 124, 194 transport, 40, 48, 55, 129, 192

Fruit Development and Seed Dispersal Edited by Lars Østergaard © 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-18946-0

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Index
347

auxin response factor (ARF), 16, 305, 332 ARF1, 141 ARF2, 139 ARF3/ETT, 39, 193 ARF4, 39, 193 ARF5/MP, 55, 128 ARF6, 54 ARF8, 54, 114, 333 AuxRE, 125 Avena fatua, 260 B (barrel type), 251 basic helix-loop-helix (bHLH), 16, 45, 177 BELL1 (BEL1), 90, 182 BELLRINGER, 47 B-function, 12 benzyl adenine (BA), 136 B&H hypothesis, 6 bicarpelate, 174 biodiversity, 2 bitegmic, 72 BLADE-ON-PETIOLE1 and 2 (BOP1/2), 41 BODENLOS (BDL)/IAA12, 128 Botrytis cinerea, 309 bract, 3 Brassica napus, 136 Brassicaceae, 82, 114 brassinazole, 304 brassinosteroids, 139, 304 BREVIPEDICELLUS (BP), 47, 184 bristles, 248 Bronze Age Gujarat, India, 260 bryophytes, 340 btr1, 251 btr2, 251 Cabombaceae, 7 capsaicin, 297 Capsella, 82 Capsicum, 113 CArG box, 17 Carolus Linneaus, 35 CAROTENOID CLEAVAGE DIOXYGENASE 1, 308 carotenoids, 308 carpel, 1, 36, 73, 172, 340 Caryophyllales, 2 cassowaries (Casuarius casuarius), 218

Catharanthus roseus (The Madagascar Periwinkle), 24 CAULIFLOWER (CAL), 179 cdka;1, 87 Cenozoic extinctions, 72 central cell, 73, 82, 111, 117 central cell guidance (ccg), 88 Cephalotaxaceae, 7 Cerratophyllum, 8 C-function, 12, 44 chalaza, 72 Chalcolithic Kosak Shimali, 264 CHALCONE SYNTHASE 1 and 2 (CHS1/CHS2), 112 Chenopodiaceae, 261 Chenopodium album, 258 Chenopodium berlanderii, 260 chickpea (Cicer arietinum), 259 chitinases, 301 Chloranthanceae, 8 Chlorophytes, 340 CHROMATIN REMODELLING PROTEIN11, 85 CLAVATA3 (CLV3), 79 climacteric fruits, 302 CO, 277 coat dormancy, 257 coatlique (coa), 87 coenocytic embryo sac, 82 Colorless non-ripening (Cnr), 298 combination model, 251 compitum, 25 cones, 4 congenital, 24 conifers, 7 CONSTITUTIVE PHOTOMORPHOGENESIS 10 (COP10), 307 core eudicots, 2 Corystospermales, 5 CRABS CLAW (CRC), 15, 45, 76 crinkly4, 272 cross-pollination, 246, 269 Cucurbitaceae, 303, 330 Cupressaceae, 7 CUP-SHAPED COTYLEDON1, 2 and 3 (CUC1/2/3), 26, 38, 75, 188 cupule, 4–5, 249 cuticle, 26

ind

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Char Count=

348
Index Cuticular water permeability 1 (Cwp1), 299, 310 c-value, 16 Cycadales, 7 Cycas revoluta, 16 CYP78A9, 118 cytokinin, 37, 115, 136, 329 Darwin, 9, 72, 262, 278, 328 DE-ETIOLATED 1 (DET1), 307 DefH9, 329 DefH9-iaaM, 330 DefH9-RI-iaaM, 338 dehiscence zone, 43, 176 delayed fruit deterioration (dfd), 311 DELLA proteins, 134, 333 DEMETER, 85 determinant infertile1 (dif1), 86 Devil’s claw (Proboscidea parviflora), 255 D-function, 13 diaspore units, 252 DICER-LIKE1 (DCL1), 143 dieocy, 10 dispersal distance kernel, 206, 213 dispersal kernel, 206 dispersal vector, 206 distal-proximal, 38, 40–41, 77 domestication, 238 dormancy, 261 dorsoventral, 39 Dpo1, 256 Dpo2, 256 DR12, 305 DR5rev::eGFP, 123 DROOPING LEAF (DL), 19, 46 dwarf (dx ), 305 Early Cretaceous, 9 eelgrass (Zostera marina), 222 effective pollination period (EPP), 113 E-function, 12 egg cell, 73, 82, 111, 117 Ehd1, 277 EIN3, 140 elaiosome, 208 embryogenesis, 71 embryo sac, 72

embryo sac defective mutants (emd), 91 endocarp a (ena), 175 endocarp b (enb), 175 endosperm, 12, 82 endozoochorous dispersal, 216–17, 219–20 ent-copalyl diphosphate synthase (CPS), 132 ent-kaurene oxidase (KO), 132 ent-kaurene synthase (KS), 132 ent-kaurenoic acid oxidase (KAO), 132 eostre, 89 epigenetic mutations, 301 epizoochorous dispersal, 219–20 ethylene, 136, 298, 302 ETTIN/ettin (ETT/ett), 25, 39, 128, 193 Euanthial theory of Angiosperm evolution, 340 eudicots, 2 eudicotyldons, 72 eumagnolids, 8, 72 evolutionary convergence, 250 EXCESS MICROSPOROCYTES1 (EMS1), 84 exocarp, 175 EXTRASPOROGENOUS CELLS (EXS), 84 Fabaceae, 255 facultative parthenocarpy, 135, 328 FARINELLI (FAR), 20 farnesylsation site, 23 fat-tailed dispersal kernel, 206 F-box protein, 6, 305 FERONIA (FER), 88, 122 Fertile Crescent, 222 fertilization, 2, 12, 36, 82, 173 FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), 85 FERTILIZATION-INDEPENDENT SEED2 (FIS2), 85 fiddlehead (fdh), 26 FILAMENTOUS FLOWER (FIL), 45, 75, 182 flavonoid, 112, 306 flax (Linum usitatissimum), 256 FLC, 277

ind

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Index
349

fleshy fruits, 296 FLORAL BINDING PROTEIN (FBP) FBP2, 96 FBP5, 96 FBP6, 22 FBP7, 13, 96 FBP11, 96 floral determinacy, 22, 46 floral meristem, 179 Fragaria ananassa, 308 Fragaria vesca, 308 French flag model, 185 frugivores, 297, 307 fruit, 2, 36, 107, 172, 296, 327 FRUITFULL (FUL), 23, 49, 179, 301 fruit weight locus (fw2.2), 147 FT, 276 funiculus, 22, 72, 111 fwf/arf8, 115 GA20-OXIDASE (GA20OX), 37, 132 GA2ox2, 113 GA3ox, 55, 132 gai dominant (gai-1d), 138 gametophyte, 72, 117, 173, 328 gametophytic factor2 (gfa2), 88 GARGOYLE, 196 GEX3, 118 GFA2, 85 GH3 gene family, 131 Ghd7, 277 GI, 276 gibberellin (GA), 109, 132, 257 biosynthesis, 37, 55, 132 in parthenocarpy, 329, 333 signalling, 55, 134 Ginkgoales, 7 Ginkgo biloba, 7 Glume, 271 glume tenacity (Tg), 251 Gnetales, 7 Gnetum ula, 16 Goethe, 3, 36 Gossypium, 255 Gossypium barbadense, 255 Gossypium hirsutum, 255 Gramineae, 82 Green-ripe (Gr), 303

GROWTH-REGULATING-FACTOR5 (GRF5), 40 GS3, 272 Guitarerro Cave, 256 GW2, 271 gymnosperm, 1, 71, 141, 340 gynoecium, 3, 36, 73, 172 gynophore, 42, 74, 173 haplochory, 206, 209 Hd1, 277 Hd3a, 277 Hd6, 277 HD-ZIPII, 39 HECATE (HEC), 54 Hemudu, 253 high pigment 1 and 2 (hp-1 and hp-2), 307 Holocene, 246 homogalacturonan, 300 homoplasy, 278 Hordeum pussilum, 254, 271 Hordeum spontaneum/vulgare, 243 HUA ENHANCER1 (HEN1), 143 HUELLENLOS (HLL), 76 Hydatella, 12 Hydatellaceae, 7 HYPONASTIC LEAVES1 (HYL1), 143 IAA1/AXR5, 128 IAA9, 305, 333 IAA18/CRANE, 128 IAA19/MSG2, 128 IAA28, 128 iaaM, 129, 329 Illiciaceae, 7 Illicium, 12 inbreeding, 2 INDEHISCENT (IND), 49, 177, 301 indeterminate gametophyte1 (ig1), 89 Indian Vigna spp., 259 indica rice, 250 indole-3-acetic acid (IAA), 328 initiation phase, 212 inner integument, 72 INNER NO OUTER (INO), 15, 78 integument, 15, 71, 111 inter-specific incompatibility, 2 introgression lines, 308 involucrum, 3, 248

ind

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Char Count=

350
Index Iridaceae, 223 Iron Age Europe, 260 irregular dispersal vector, 209, 224 ISOPENTENYL TRANSFERASE7 (IPT7), 37 JAGGED (JAG), 40, 183 JAGGED LATERAL ORGANS (JLO), 38 japonica rice, 250 jasmonic acid, 140 Javanica rice, 270 Jerf el Ahmar, 264 Juniperus osteosperma, 327 KANADI (KAN), 39, 78, 189 Karkarichinkat, 248 kelp (Pterygophora californica), 221 KNAT2, 185 KNAT6, 185 KNOTTED1-like homeobox (KNOX), 37, 181 KNUCKLES (KNU), 56, 117 Lablab purpureus, 259 lachesis (lis), 89 Late Jurassic, 9 leaf lamina, 39 LEAFY (LFY), 3, 44 LEAFY HULL STERILE1, 19 LEAFY PETIOLE (LEP), 40 LeCOP1-like, 307 Leguminoseae, 82 LeHY5, 307 lemma, 20 Lens, 259 lentil, 261 leptokurtic distribution, 214 LeSPL-CNR, 301 LEUNIG/leunig (LUG/lug), 25, 75 lignification, 43, 177 long-distance dispersal of seeds (LDD), 204 Lower Cretaceous, 9 Lower Yangtze, 253, 270 LPAT2, 85 macrocalyx (mc), 298 MADS-box gene, 5, 77, 177, 250 maize (Zea mays), 246

Malus, 27 Malvaceae, 82 marginal tissue, 43, 74, 175 marsheldar (Iva annua), 271 Mauretania, 265 MdPI, 116 MEDEA (MEA), 85 megagametogenesis, 71, 81 megagametophyte, 82 megasporangium, 71 megaspore, 71 megaspore mother cell (MMC), 73 megasporogenesis, 73, 81 megasporogenesis defective (msd), 91 megasporophylls, 6 Mehrgarh, 254 meristem, 3, 185 meristem determinacy, 46, 97 mesocarp, 175 mesokurtic distribution, 214 methodical selection, 262 micropyle, 72, 111, 118 microsporangia, 3 microsporocytes, 84 microsporophylls, 3 middle lamellae, 309 MIR164, 26 miRNA165/166, 39 mn1, 272 Monimiaceae (Laurales), 25 monocarpelate, 172 monocots, 2 monocotyledons, 72 monophyletic, 7 MONOPTEROS (MP), 128 morphogen, 55 morphotype, 242 Mostly Male Theory (MMT), 3 mulicage, 25 multiple archesporial cells (mac1), 84 MULTIPLE SPOROCYTES1 (MSP1), 84 Mureybit, 243 MYB98, 85 Nabta Playa, 246 N-acetylornithine deacetylase, 330 Narhan, 267 NEEDLY (NLY), 4 neo-functionalization, 22

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Index
351

neoteny, 279 NEROLIDOL SYNTHASE 1 (NES1), 308 Nevasa, 267 New Caledonia, 7 New World Chenopodium, 260 NGATHA (NGA), 54 NO TRANSMITTING TRACT (NTT), 54 NOMEGA, 85 non-climacteric fruits, 304 non-ripening (nor), 298 non-standard dispersal vector, 206 NOZZLE, 76 NUBBIN (NUB), 46 nucellus, 72 Nuphar, 12 Nuphar/Schisandra-type gametophyte, 117 Nymphaea alba, 15 Nymphaeaceae, 7 Nymphaeales, 7 obligate parthenocarpy, 328 Old World Chenopodium album, 260 ornithine decarboxylase (ODC), 330 orthologue, 5 Oryza nivara, 250 Oryza rufipogon, 250 OsMADS3, 18 OsMADS5, 20 OsMADS7, 20 OsMADS8, 20 OsMADS13, 18, 45, 96 OsMADS21, 18 OsMADS24, 96 OsMADS45, 96 OsMADS58, 18 Out-of-Female (OOF) hypothesis, 5 Out-of-Male (OOM) hypothesis, 5 outer integument, 72 ovary, 42, 74, 173, 327 ovule, 1, 36, 70, 108, 173, 327 paedomorphosis, 279 palea, 20 panicoid group, 274 pappus, 228 parenchyma, 24, 130, 176

parsnip (Pastinaca sativa), 327 parthenocarpy, 107, 326, 328 pat-2 tomato mutant, 113, 330 PEAPOD (PPD), 40 pearl millet (Pennisetum glaucum), 246 Pedaliaceae/Martyniaceae (Sesamum, Proboscidea), 261 PENNYWISE, 47 peramorphosis, 279 pericarp, 300 perisperm, 12 petals, 17, 44, 92, 335 Petunia, 96 Petunia hybrida, 8 PHABULOSA (PHB), 39, 78 Phaeseolus, 256 Phaeseolus lunatus, 256 Phaeseolus vulgaris (common bean), 256 Phalaenopsis orchids, 110 PHAVOLUTA (PHV), 39 photoperiod, 273 Phyllocladaceae, 7 phylogenetic, 7 phylogeography, 250 phytoliths, 249 PINFORMED (PIN), 37 PIN1, 130 PIN2, 130 PIN3, 130 PIN6, 130 Pinaceae, 7 PINENE SYNTHASE (PINS), 308 PINOID/pinoid (PID/pid), 48, 130, 131 Pinus ayacahuite, 16 pistil, 2, 91, 111, 173, 327 PISTILLATA, 116 Pisum abyssinicum (the Ethiopian pea), 259 Pisum sativum subsp. elatius, 259 Pisum sativum subsp. sativum, 259 placenta, 24, 72, 146, 175, 330 plasmodesmata, 24, 261 pleisiomorphic, 15 PLENA (PLE), 20 pluricarpelate, 172 PMADS3, 21 Poaceae, 18 Podocarpaceae, 7

ind

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Char Count=

352
Index pollen, 1, 86, 110 pollen capture, 1 pollen germination, 2, 173 pollen tube, 1, 10, 11, 25, 36, 43, 73, 173 pollen-stigma recognition, 110 pollination, 110, 172, 327 polyamines (PAs), 305 polychorous, 211 polychory, 206, 209 Polygonum divaricatum, 82 Polygonum type, 12, 72, 117 polyploids, 9 pop1/cer6-1, 115 POP2, 118 postgenital, 24 ppd-H1, 276 ppd1, 272 pre-cultivation domestication, 259 Pre-Pottery Neolithic Jericho, 256 Pre-Pottery Neolithic A (PPNA), 264 Pre-Pottery Neolithic B (PPNB), 245 prickle pollination, 112 protogynous, 10 Pseudomonas syringae, 129, 329 PsMAPK3, 140 putrescine, 305 Pyrus, 27 Q gene, 251 qSH1, 250 qSW5, 272 quantile of dispersal distance, 205 quantitative trait loci (QTL), 124, 308 rachilla, 240 rachis, 240 ragwort (Senecio inaequidens), 223 Ranunculales, 9 receptacle, 173 regular dispersal vector, 224 replum, 42, 43, 74, 131, 173 REPLUMLESS (RPL), 47, 181, 250, 301 RETINOBLASTOMARELATED1, 84 REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1), 303 REVOLUTA (REV), 39 ribosomal protein L24/SHORT VALVE1 (RPL24/STV1), 128 rice, 8, 96, 195

rice (Oryza sativa), 246 ripening inhibitor (rin), 298 RMADS217, 20 Rosaceae, 312, 330 rosids, 2 RP75/79, 337 S-adenosylmethionine, 305 SBP-box (SQUAMOSA Promoter Binding Protein-like), 301 SCFTIR1, 305, 332 Schissandraceae, 7 Sciadopityaceae, 7 scylla (syl), 122 SECRET AGENT, 334 seed, 206, 297 seed abscission, 22, 77 seed dispersal, 2, 109, 204, 206, 297, 327 seed passage time (P), 206, 213 SEEDSTICK (STK), 22, 93 self-incompatibility (SI), 10, 110 semi-domesticated, 240 SEPALLATA (SEP), 13, 36, 94, 116, 298, 335 sepals, 17, 44, 93, 298, 335 septum, 42, 74, 173 SERRATE (SE), 143 sesame (Sesamum indicum), 256 Setaria spp., 252 SEUSS (SEU), 52, 75 Severianin, 337 sh4, 250 SHATTERPROOF1 and 2 (SHP1/2), 20, 45, 93, 177, 250, 301 shoot apical meristem (SAM), 37, 79 SHOOT MERISTEMLESS (STM), 47, 92, 114, 184 SHORT INTEGUMENTS1 and 2 (SIN2), 76 Sht1, 252 Sht2, 252 silique, 43, 173 single nucleotide polymorphism (SNP), 250 sirene (srn), 88, 122 SlARF7, 125 SlARF8, 125 SlARF9, 125 SlDELLA, 134

ind

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Index
353

SlIAA2, 125 SlIAA9, 125 SlIAA14, 125 SLOW WALKER1, 85 Small Auxin Upregulated RNA (SAUR), 114 snowball model, 251 Solanaceae, 82, 297, 330 Solanum chmiliewskii, 310 Solanum habrochaites, 310 Solanum lycopersicum, 308 Solanum pennellii, 308 Solanum peruvianum, 310 Sorghum bicolour, 246 SPATULA/spatula (SPT/spt), 25, 45, 193 spider monkeys (Ateles paniscus), 218 spikelet, 240 SPINDLY (SPY), 134, 334 sporocyteless (spl), 87 SPOROCYTELESS/NOZZLE (SPL/NZZ), 116 sporophylls, 3 sporophytic and megagametogenesis defective mutants (smd), 91 stamens, 3, 4, 14, 17, 44, 115, 336 standard dispersal vector, 206 stenospermocarpic, 113, 336 stigma, 42, 74, 110, 173 stigmatic, 2, 42 stilbene synthases (STSs), 313 Streptophytes, 340 strobilus, 3 style, 42, 74, 112, 173 STYLISH1 and 2 (STY1/2), 53, 114 STYLOSA, 53 sub-functionalization, 22 sunflower (Helianthus annus), 270 SUPERMAN (SUP), 80, 114 Surkotada, Gujarat, 267 syncarpic, 2 synergid cells, 82, 117 synteny, 250 Tambourissa, 25 tapetum, 84 TAPETUM DETERMINANT1 (TPD1), 84 TAS3, 39, 193 Taxaceae, 7

Taxodiaceae, 7 Taxus baccata, 26 TCP proteins (TEOSINTE BRANCHED1, CYCLOIDEA, PCF), 40 TDR4, 301 Tell Ramad, 256 telomic theory, 71 teosinte (Zea mexicana), 249 tepals, 17 tepitzin1, 118 termination phase, 212 TERMINATOR (TER), 97 terpene synthase (TPS), 313 Thompson seedless grape, 337 TM29, 116 TOMLOX-C, 308 total dispersal kernel (TDK), 206, 209 TOUSLED/tousled (TSL/tsl), 25, 54 transmitting tract, 43, 74, 173 transport phase, 212 trichomes, 253 Trillium grandiflorum, 226 Trimenia moorei, 10 Trimeniaceae, 7 Triticum boeitucm/monococcum, 243 TRPF1-rolB, 339 UC82 tomato, 338 unitegmetic ovule, 27 unitegmic, 72 UNUSUAL FLORAL ORGANS (UFO), 6 UV-DAMAGE DNA BINDING PROTEIN 1 (DDB1), 307 VAAMANA, 47 Vaccinium, 27 valve, 42, 74, 173 valve margin, 42, 173, 301 VARICOSE (VCS), 143 vascular plants, 340 vector displacement velocity (V), 206, 212 vector seed load (Q), 206, 212 vernalization, 240 Vigna angularis (azuki bean), 256 Vigna unguiculata, 269 VIN3, 277 vivipary (preharvest sprouting), 261

ind

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7:26

Char Count=

354
Index VP1, 261 VRN1, 274 VRN2, 274 VRN3, 274 Vrs-1, 272 W (wedge type), 251 webworms (Depressaria pastinacella), 327 Welwitschia, 27 white-tailed deer (Odocoileus virginianus), 211 WOX5, 118 WUSCHEL (WUS), 44, 79

YABBY transcription factors, 15, 38-9, 78, 182 YABBY3 (YAB3), 45 Yangtze valley, 242 YUCCA (YUC), 48 YUC2, 116, 194 YUC6, 116, 194 ZAG1, 18 ZmEA1, 88 ZMM2, 18 ZMM23, 18 zygote, 111, 119