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CELLULAR AND MOLECULAR BIOLOGYOF PLANT SEED DEVELOPMENT

Advances in Cellular and Molecular Biology of Plants

VOLUME 4

Editor-in-Chief

Indra K. Vasil, Laboratory ofPlant Cell and Mole cular Biology,University ofFlorida, Gainesville, Florida , USA

Editorial Advisory Board

Robert T. Fraley, St. Louis, Missouri. USARobert B. Goldberg, Los Angeles. California. USACharle s S. Levings, III, Raleigh, North Carolina. USARonald L. Phillips, St. Paul. Minnesota, USAJeff Schell , Cologne. Germany

The titles published in this series are listed at the end of this volume.

Cellular andMolecular Biology of

Plant Seed Development

Edited by

BRIAN A. LARKINS

Department ofPlant Sciences. University ofArizona, Tucson, Arizona, USA

and

INDRA K. VASIL

Laboratory ofPlant Cell and Molecular Biology, University of Florida, Gainesville,Florida , USA

Springer-Science+Business Media, B.V.

A C.I.P. catalogue record for this book is available from the Library of Congress

ISBN 978-90-481-4878-3 ISBN 978-94-015-8909-3 (eBook)DOl 10.1007/978-94-015-8909-3

Printed on acid-free paper

All rights reserved

1997 Springer Science+Business Media DordrechtOriginally published by Kluwer Academic Publishers in 1997.Softcover reprint of the hardcover Ist edition 1997

No part of the material protected by this copyright notice may be reproduced orutilized in any form or by any means, electronic or mechanical,including photocopying, recording or by any information storage and retrieval system,without written permission from the copyright owner.

Table of Contents

PrefaceB. Larkins and I.K. Vasil

Section A - Control of Seed Development

VII-Vlll

I . Embryogenesis in Dicotyledonous PlantsR. Yadegari and R. Goldberg 3

2. Development of the Suspensor: Differentiation, Communication,and Programmed Cell Death During Plant EmbryogenesisB.W. Schwartz, D.M. Vernon, D. Meinke 53

3. Endosperm Structure and DevelopmentD.A. DeMason 73

4. Hormonal Regulation of Seed DevelopmentR. Morris 117

Section B - The Synthesis and Accumulation of Stored Metabolites

5. The Biochemistry and Cell Biology of Embryo Storage ProteinsN.C. Nielsen, R. Bassiiner, T. Beaman 151

6. The Prolamin Storage Proteins of Wheat and its RelativesG.Galili 221

7. The Prolamin Proteins of Maize, Sorghum and CoixC.E . Coleman, J. M. Dannenhoffer, B.A. Larkins 257

8. The Storage Proteins of Rice and OatD.G. Muench, T. W. Okita 289

9. The Protease Inhibitors of SeedsK.A . Wilson 331

VI Table a/ Contents

10. Starch Synthesis in the Mai ze Seedi.c. Hannah 375

II . Synthesis and Storage of Fatty AcidsJ. Browse 407

12. Accumulation and Storage of Phosphate and MineralsV.Raboy 441

13. Genetic Regulation of Carbohydrate and Protein Accumulationin SeedsM. Motto , R. Thompson , F. Salamini 479

Section C - Control of Seed Maturation and Germination

14. Lea Proteins and the Desiccation Tolerance of Seed sL. Dure

15. Seed Maturation and Control of DormancyJ. Harada

Section D - Manipulation of Seeds Through Biotechnology

525

545

16. Biotechnological Approaches to Altering Seed CompositionE. Krebbers, R. Brogli e, B. Hitz, T. Jones , N. Hubbard 595

Index 635

Preface

The beginnings of human civili zation can be traced back to the time, near-ly 12,000 years ago , when the early humans gradually changed from a lifeof hunting and gathering food , to producing food. This beginning of primi-tive agriculture ensured a dependable supply of food, and fostered the livingtogether of people in groups and the development of society. During thistime, plant seeds were recognized as a valuable source of food and nutrition ,and began to be used for growing plants for food. Ever since, plant seedshave played an important role in the development of the human civilization.Even today, seeds of a few crop species, such as the cereals and legumes, arethe primary source of most human food , and the predominant commodity ininternational agriculture.

Owing to their great importance as food for human s and in internationaltrade , seeds have been a favorite object of study by developmental biologistsand physiologists , nutritionists and chemists. A wealth of useful informationis available on the biology of seeds. However, studies on the molecular biol-ogy of plant seed development are rather recent, and have begun to providecritical new information about the control of seed development, dormancy,germination and storage reserves.

The age of plant molecular biology began twenty years ago with the isola-tion and characterization ofmRNAs encoding seed storage proteins, followedby the cloning of related genes. The success of these early studie s encour-aged many plant biologists to focus their research on seeds. This was alsobecause seeds are vitally important agricultural products providing much ofthe starch, protein and oil for human and livestock diets, and are good modelsystems to study the biochemical and genetic mechanisms regulating protein ,starch and oil biosynthesis. In addition, seed development embodies severalunique biological processes, such as embryogenesis, dormancy, germination,etc .

During the past twenty years we have learned a great deal about the bio-chemical and genetic regulation of the processes involved in seed develop-ment. Nevertheless, much remains to be discovered about the physiologicalregulation of embryo development, and the mechanisms leading to seed des-iccation and dormancy, and germination. It is hoped that the rapid evolution

B.A. Larkins and IX. Vasil (eds.), Cellular and Molecular Bio!ogy ofPlant Seed Development. vi i-vi ii. 1997 Kluwer Academic Publishers,

viii Preface

of genome projects, and the isolation and characterization of mutants affect-ing seed development, will soon unravel these complex processes.

This volume presents a compilation of chapters describing fundamentalaspects of seed development and maturation by some of the world's leadingexperts. Emerging concepts of embryogenesis, endosperm development andseed maturation and desiccation are discussed in light of recently isolatednovel mutants. The in-depth and up-to-date reviews provide insights into thegenetics, biochemistry and cell biology of metabolic reserve (starch, protein,oil, mineral) synthesis and accumulation. The concluding chapter describesthe practical applications of this knowledge to the manipulation of storagereserve content of seeds by molecular genetic manipulation.

The human population is projected to double in the next thirty years. Foodproduction, primarily in the form of seeds, must be doubled too during thisperiod to provide food security for the increased population. It is vitallyimportant, therefore, to find ways to further enhance the productivity, qualityand utility of seeds. It is our hope that the information and ideas presented inthese pages will provide the insight and inspiration needed to achieve theseobjectives.

We thank each of the authors for providing state-of-the-art accounts offascinating and important advances in the cellular and molecular biology ofseed development.

Brian A. LarkinsIndra K. Vasil

Part A

CONTROL OF SEED DEVELOPMENT

1. Embryogenesis in Dicotyledonous Plants

RAMIN YADEGARI* and ROBERT B. GOLDBERGDepartment ofMolecular. Cell, and Developmental Biology, University ofCalifornia,Los Angeles , CA 90095-1606, USA

ABSTRACT, Embryogenesis in higher plants establishes the basic shoot-root body pattern,the primary tissue layers, and the meristematic zones of the plant. Continuous differentiationof the meristems is the basis of postembryonic development, the adult phase of the lifecycle . Critical to this process is not only the pattern forming or morphogenetic events takingplace mainly during early embryogenesis, but also a series of cellular and physiologicalprocesses which prepare the maturing embryo for dormancy and germination. Recent geneticand molecular studies in Arabidopsis and other model plants have begun to identify criticalprocesses involved in higher plant embryogenesis. Likewise, Arabidopsis mutations defectivein embryo structure or seedling viability are providing the tools for an analysis of molecularmechanisms responsible for dicot embryogenesis. One critical question is whether cellularinteractions play a role in the formation of embryo pattern, or whether the nearly regularpatterns of cell division observed in many species, including Arabidopsis, are a reflectionof a lineage-dependent mode of cell specification . Analysis of mutations altering cellularpatterns in Arabidopsis embryo indicate that cell-cell interactions most likely take place toestablish cell and tissue layers . Further, there is evidence for inter-regional interactions tocoordinate the overall development of the dicot embryo. However, differentiation processesbased on the activity of cell-autonomous determinants may also operate particularly duringthe earliest zygotic divisions which establish the principal embryonic elements. A secondmajor question concerns the specific gene regulatory mechanisms involved in initiating andmaintain ing differentiation programs within the developing embryo. These and other questionsregarding the underlying processes that control dicot embryogenesis are only beginning to beanswered using a combination of molecular and genetic tools.

I. Introduction

New genetic and molecular tools have been used in recent years to dis-sect the mechanisms that control plant embryogenesis. Many genes requiredfor various embryogenic processes in both monocotyledons and dicotyle-dons have been identified using genetic approaches (Meinke, 1985; Clarkand Sheridan, 1991; Mayer et aI., 1991; Johnson et aI., 1994; Hong et aI.,1995). Genetic manipulation of Arabidopsis thaliana by irradiation muta-genesis (Muller, 1963; Usmanov and Muller, 1970), chemical mutagenesis

* Present Address: Department of Plant and Microbial Biology, University of California,Berkeley, California 94720-3102

B.A. Larkins and IX. Vasil (eds .). Cellular and Molecular Biology ofPlant Sad Development, 3-5 2. 1997 Kluwer Academic Publishers,

4 Ramin Yadegari and Robert B . Goldberg

(Meinke and Sussex, 1979a,b; Meinke, 1985; Jurgens et a!., 1991; Mayeret aI., 1991, 1993a) and insertional mutagenesis (Errampalli et aI., 1991;Feldmann, 1991; Forsthoefe1 et a!., 1992; Castle et a!., 1993), has identi-fied a large number of zygotic mutants that are defective at different stagesof embryogenesis. These mutants have provided insights into the processesthat perform essential functions during embryogenesis, regulatory as wellas general housekeeping functions. Furthermore, some of these mutationscan be traced back to specific defects during early stages of embryogenesisrevealing the importance of specific cell division patterns and tissue organiza-tions in normal embryo development processes. Both genetic and molecularapproaches have identified genes which are transcribed in specific regionsof the dicot embryo suggesting an underlying prepattem of gene regulatoryprograms involved in embryo tissue and organ development. The correspond-ing regulatory sequences responsible for the region-specific transcription ofthese genes are beginning to be deciphered allowing an entry into the generegulatory pathways involved in embryo pattern specification and develop-ment (see below). In this review we outline the major conceptual insightsthat have been gained from studies ofArabidopsis embryo mutants and geneexpression experiments in other plants that provide new information about theprocesses regulating dicotyledon embryogenesis especially during the earlydevelopmental stages. Recent experimental evidence suggests that a plantembryo has a modular structure and consists of regions which are distinct atthe molecular levels.

II. General Features of Embryogenesis Are Similar in Higher Plants

In flowering plants (angiosperms), double fertilization of the egg cell and thepolar nuclei (within the central cell) by sperm nuclei produces a diploid zygoteand a triploid endosperm, respectively (Esau, 1977; Raven et al., 1992) . Asa differentiated organ , the endosperm is present during seed developmentand provides nutrients for either the developing embryo, the germinatingseedling, or both (Lopes and Larkins, 1993). The zygote, on the other hand ,develops into an embryo and will give rise to the body plan of the matureplant (sporophyte) after seed germination. Angiosperm embryos contain twoprimary organ systems- the axis and the cotyledon (Raven et a!., 1992) (Fig-ure I). These organs have distinct developmental fates and are composed ofthree basic, or primordial , tissue layers - protoderm, procambium, and groundmeristem - which will become the epidermal, vascular, and parenchyma tis-sues of the young seedling, respectively (Esau, 1977; Raven et a!., 1992).The axis, or hypocotyl-radicle region of the embryo, contains the shoot androot meristems, and will give rise to the mature plant after seed germina-tion (Figure 1). The root meristem will give rise to only one organ, the root,while the shoot meristem will produce, directly or indirectly, all the vegeta-tive and reproductive organs of the mature plant. By contrast, the cotyledon

POST-FERTILIZATION

Embryogenesis in dicotyledonous plants 5

GLOBULAR/HEART TRANSITION

HEARTEMBRYO

TRANSITIONEMBRYO

IHs

uPd

tG-CELL GL08ULAREP EMBRYO

2/4-CELL 8-CELLEP EP

PROEMBRYO

~ ~: .,~j:P ~ZYGOTE HELL

.....--------------,1'I ------- - - - - - - - - --,

ORGAN EXPANSION AND MATURATION

Gm

Pc

SC

PdA

RM

TORPEDO EMBRYO WALKING-STICK EMBRYO MATURE EMBRYO

-CE

SC

En

Fig. I . A generalized overview of dicot embryogenesis. Schematic representations of embry-onic stages are based on light microscopy studies of Arabidopsis (Mansfield and Briarty, 1991,1992; Mayer et al., 1991) and Capsella (Schulz and Jensen, 1968a, b) embryo development.For a comprehens ive description of the stages of Arabidopsi s embryo development refer toJUrgens and Mayer (1994). Abbreviations: T, terminal (apical) cell; B, embryo basal cell; EP,embryo proper ; S, suspensor; Be, suspensor basal cell; Pd, protoderm; u, upper tier; I, lowertier; Hs, hypophy sis; Pc, procambium; Gm, ground meristem; C, cotyledon; A, axis; MPE,micropylar end; CE, chalazal end; SC, seed coat; En, endosperm; SM, shoot meristem; RM,root meristem.

functions primarily in accumulation of food reserve s that are utilized by theseedling for growth and development after germination, becomes photos yn-thet ically acti ve during the seedling stage, and senesces shortly after theseedling emerge s from the so il (Figure I). That is, during embryogenesis, theco tyledon mobil izes food reserves and then switches roles during seedlingdevelopment to break down these reserves prior to the emergence of leaves,allowing the plant to become photosynthetically active. In many higher plants,incl uding Arabidopsis, the embryo might be photosynthetically active prior

6 Ramin Yadegariand Robert B. Goldberg

TABLE 1

Major events of flowering plant embryogenesis.

Post-fertilizationlproembryo

Apical and basal cell differentiation

Formation of suspensor and embryo proper

Globular-heart transition

Differentiation of major tissue-type primordia

Establishment of radial (tissue-type) axis

Embryo proper becomes bilaterally symmetrical

Visible appearance of shoot/root (apical-basal) axis

Initiation of cotyledon and axis (hypocotyl/radicle) development

Differentiation of root meristem

Organ expansion and maturation

Enlargement of cotyledons and axis by cell division and expansion

Differentiation of shoot meristem

Formation of lipid and protein bodies

Accumulat ion of storage proteins and lipids

Vacuolization of cotyledon and axis cell s

Cessation of RNA and protein synthesis

Loss of water/dehydration

Inhibition of precocious germ ination

Dormancy

to dessication as indicated by the presence of chlorophyll and distinctly-differentiated plastids at particular stages of embryo development (Yakovlevand Zhukova, 1980; Mansfield and Briarty, 1991, 1992). Embryogenesis inhigher plants, therefore, serves to (a) specify the shoot/root plant body patternand the meristematic zones, (b) differentiate the primary plant tissue types,(c) generate a specialized storage organ essential for seed germination andseedling development, and (d) enable the sporophyte to lie dormant untilconditions are favorable for post-embryonic development.

III. The Basic Body Plan of the Dicot Plant Is Established during EarlyEmbryogenesis

How the embryo acquires its three-dimensional shape with specializedorgans and tissues, and what gene networks orchestrate embryonic devel-opment remain major unresolved problems. From a descriptive point of


view, plant embryogenesis can be divided into three conceptual phasesin which distinct developmental and physiological events occur: (a) post-fertilization/proembryo, (b) globular-heart transition, and (c) organ expan-sion/maturation (Goldberg et aI., 1989; Lindsey and Topping , 1993; West andHarada, 1993) (Figure 1 and Table 1).

Although there is considerable variation in how angiosperm embryosfrom different species form, the overall trends in developmental patternsare remarkably similar (Natesh and Rau, 1984; Johri et aI., 1992). Amongmany, but not all, dicot and monocot species, the early zygotic divisions arerelatively regular, a feature which has prompted many workers to devise var-ious classification systems based on early embryonic development (Nateshand Rau, 1984). In this review, we summarize the early patterns of embryo-genesis in Arabidopsis and the closely-related plant Capsella bursa-pastoristo illustrate major aspects of dicot embryogenesis (Figure I). Both plantspossess two of the best-studied forms of plant embryogenesis, and display anearly regular cell division pattern during the very early stages of embryoge-nesis (Hanstein, 1870; Schaffner, 1906; Soueges, 1919, 1948; Misra, 1962;Schulz and Jensen, 1968a,b; Yakovlev and Alimova, 1976; Mansfield andBriarty, 1991; Mansfield et al., 1991). The first few cell divisions followa pattern known as the Onagrad (Crucifer) type of embryogeny (Johansen,1950; Maheshwari, 1950) . After the initial cleavage of the zygote, the apical(terminal) cell , which contributes exclu sively to the embryo proper, divideslongitudinally while the basal cell divides transversely to produce the cellsthat will give rise to the suspensor (Schaffner, 1906; Soueges, 1919; Misra ,1962; Schulz and Jensen, 1968b; Yakovlev and Alimova , 1976;Mansfield andBriarty, 1991) (Figure 1). Other type s of embryogeny are distinguished by therelative contribution of the zygote to the formation of the embryo proper andthe suspensor, where the first cell wall formation takes place within the apicalcell, and whether it is longitudinal or transverse (Esau, 1977; Natesh and Rau,1984; Raghavan and Sharma, 1995). The regularity of early embryogenesis inArabidopsis has allowed cell lineages to be traced histologically with relativeconfidence (Misra, 1962; Yakovlev and Alimova, 1976; Mansfield and Bri-arty, 1991; Mansfield et al., 1991). Recent studie s with embryonic mutantsof Arabidopsis have provided new insights into the processes that controlembryo development by tracing back the defects to specific aberrations in theearly stages of embryogenesis (Jiirgens , 1994, 1995).It is important to point out that although we use the relatively regular

Capsella/Arabidopsis pattern of embryogenesis to highlight the general fea-tures of dicot embryogenesis, there are some plants which display irregularpatterns of cell division during early embryogenesis (Natesh and Rau, 1984).For example, the grasses, a major group of monocots which include maize,generally do not show regular embryonic cell divisions (Randolph, 1936;Natesh and Rau, 1984; Sheridan and Clark, 1994). Even among the plantswhich do display similar types of early embryonic divisions, the subsequent


divisions are rather diverse and do not conform to any given pattern (Wardlaw,1955; Steeves and Sussex, 1989). These diverse embryonic patterns ultimate-ly result in a mature body plan which is remarkably similar across manyangiosperm plant familie s (Wardlaw, 1955), undermining the importance ofstrict cell lineage pattern s in regulating embryogenesis, at least after the firstfew embryonic divisions. Such obse rva tions may suggest an important mech-anistic aspect of embryo development ; that is, evolutionarily-conservativeinteractions among embryonic ce lls or regions may play an important role inthe development of the mature embryo.

A. Formation of the Apical-Basal Embryo Pattern

1. Asymmetric cleavage of the zygote initiates the apical -basal patterningprocesses .In angiosperms, the embryo sac (female gametophyte) has an inherent polar-ity, the egg cell is attached to the micropylar (basal ) half of the embryo sacwith its chalazal end pointing towards the central ce ll (Esau, 1977 ; Willemseand van Went, 1984; Raven et aI., 1992). The egg cell itself can also displaymorphological polarity along the micropylar-chalazal (apical-basal) ax is ofthe embryo sac (Willemse and van Went , 1984). InArabidops is, for example,the egg cell has a large micropylar vacuole whereas the nucl eus is localizedto the chalazal end (Mansfield et aI., 1991) . After fertili za tion , the zygote inArabidopsis and Capsella, similar to that of most angiosperms (Natesh andRau, 1984), maintains the asymmetric distribution of ce llular componentsobserved in the egg cell (Misra, 1962; Schulz and Jen sen , 1968b; Yakovlevand Alimova, 1976; Mansfield and Briarty, 1991 ; Mansfield et aI., 199 1)(Figure I). Other angiosperms however, might exhibit a switch in egg cellpolarity immediately after fertilization as the cellular components markingthe orientation of the egg cell invert to establish a polarity in the oppos itedirection (Natesh and Rau , 1984). Despite these diverse cellular appearances ,the embryonic apical-basal polarity is identi cal in all angiosperm s-the futureapical end of the embryo (e.g. the cotyledons) points toward s the chalazawhereas the basal end (e.g. the radicl e) points towards the micropyle end ofthe female gametophyte. How zygotic polarity is achieved and whether it is adirect result of an asymmetric distribution of regulatory components withinthe embryo sac and/or the egg are major unresolved questions. Recent analy-sis of several chromosomal deficiencie s in maize sugges ts that the polarityof embryo sac itself is conferred to some extent by the activity of female-gametophytic genes, and it is not exclusively a function of the developmentalprocesses that shape the maternal ovule in which the embryo sac develops(Vollbrecht and Hake, 1995).

Prior to the first division , the Arabidopsis zyg ote undergoes ce ll e lon-gation along the apical-basal axis (Webb and Gunning, 1991). By contrast,the fertilized egg in other species may undergo shrinkage in size before the


first division (Natesh and Rau , 1984). The elongation of the Arabidopsiszygote is accompanied by a gradual reorganization of the randomly-arrangedmicrotubules found throughout its cytoplasm at the chalazal tip of the zygote(Webb and Gunning, 1991). These microtubules, found primarily in the cellcortex, are oriented perpendicular to the direction of zygote elongation and,in addition , to the zygote's internal cellular organization (i.e., asymmetriclocalization of the nucleus and the vacuole), provide a marker for embry-onic polarity prior to the first cleavage (Webb and Gunning, 1991). Later,the microtubules become evenly spaced along the elongating zygote whilemaintaining their transverse orientation (Webb and Gunning, 1991). Finally,during the initial stages of zygotic division, a broad preprophase band ofmicrotubules marks the future site of the new cell wall separating the api-cal and the basal cells (Webb and Gunning, 1991), as has been shown inother plant cells undergoing cytokinesis (Cyr, 1994; Staehelin and Hepler,1996). The microtubule cytoskeleton within the zygote and the early embryois, therefore, highly dynamic and rearranges to prepare for the morphologi-cal changes accompanying early embryogenesis. Although there is no directevidence, microtubules in conjunction with other cytoskeletal elements, suchas microfilaments, may be involved directly in establishing and maintainingzygotic and embryonic polarity. For instance , the polarity established in theegg cell may be transmitted to the zygote via cytoskeletal architecture perse. Alternatively, the polar appearance of the egg may have no bearing onthe zygotic polarity; once fertilization occurs , cytoskeletal elements couldrearrange to participate in establishing a functional asymmetry that will giverise to the distinct fates of the apical and basal cells.

Studies of zygotic polarization in the lower plants Fucales (Fucus andPelvetia) have indicated that the establishment of polarity is associated withthe actin cytoskeleton (Kropf, 1994). It is thought that later in develop-ment, the zygote's axis is fixed irreversibly by the formation of transmem-brane complexes composed of cortical F-actin and cell wall proteins at oneend of the cell (Goodner and Quatrano, 1993; Kropf, 1994). Whether thecytoskeleton is involved in localizing anisotropic cell fate determinants dur-ing plant embryogenesis is unknown. There is some evidence that at leastin the Fucus zygote asymmetric localization of actin mRNA requires intactmicrofilaments (Bouget et al., 1996). In many animal systems, the asym-metric localization of cell fate determinants has been shown to be mediatedby the cytoskeletal elements (Rhyu and Knoblich, 1995; Doe, 1996; Dru-bin and Nelson, 1996), including the intracellular localization of mRNAswhich encode critical polarizing proteins (St John ston, 1996). For example,in Drosophila , signaling between the germ line and the somatic componentsof the egg chamber polarizes the cytoskeletal network within the oocyte,thereby initiating both anterior-posterior and dorsal-ventral polarity of theoocyte and subsequently the embryo (Lehmann, 1995). Axis formation inthe Drosophila oocyte (and ultimately the embryo) is mediated by the local-

10 Ramin Yadegari and Robert B. Goldberg

ization of critical RNA molecules such as bicoid and oskar via polarizedorientation of the microtubule network (Lehmann, 1995; St Johnston, 1996).It remains to be seen whether similar subcellular mechanisms operate in thehigher plant egg cell or zygote to effect embryonic polarity. It is importantto note, however, that there has been no genetic evidence thus far indicatinga direct influence of either the embryo sac or the egg on embryo polarity. Inaddition, there have been no documented maternally-acting mutations identi-fied to date affecting embryo pattern in plants. In Drosophila, such mutationshave helped to uncover the molecular processes involved in early embryoge-nesis (see above). Maternally-acting mutations affect axis formation duringearly Drosophila embryogenesis, causing embryonic lethality regardless ofthe zygotic genotype (St Johnston and Nusslein-Volhard, 1992) .

In nearly all angiosperm species surveyed to date, the first cleavage of thezygote occurs in a transverse plane relative to the chalazal-micropylar axisof the embryo sac (Natesh and Rau, 1984). In Arabidopsis and Capsella,the zygote divides asymmetrically into two distinct-sized daughter cells - asmall, upper terminal cell (also known as the apical cell) and a large, lowerbasal cell - which establish a polarized longitudinal axis within the embryo(Schaffner, 1906; Soueges, 19 I9; Misra, 1962; Schulz and Jensen, 1968b;Yakovlev and Alimova, 1976; Mansfield and Briarty, 1991) (Figure 1). His-tological studies have indicated that the apical and basal cells give rise todifferent regions of the mature embryo (JUrgens, 1994). The small apical(terminal) cell gives rise to the embryo proper that will form most of themature embryo (Figure 1). Cell lineages derived from the apical cell will con-tribute to the development of cotyledons, shoot meristem, hypocotyl regionof the embryonic axis (Mansfield and Briarty, 1991; Mayer et aI., 1991),and part of the radicle, or embryonic root (Dolan et aI., 1993; Scheres et aI.,1994) (Figure I). The large basal cell derived from the lower portion of thezygote will contribute to the development of the hypophysis and the highlyspecialized, terminally differentiated embryonic organ called the suspensor(Schulz and Jensen, 1968a; Scheres et aI., 1994) (Figure I). In Arabidopsis,the hypophysis will contribute to the cells that comprise the quiescent centreof the root meristem and the central portion of the root cap (Scheres et aI.,1994). The Arabidopsis suspensor contains only 7-10 cells and anchors theembryo proper to the surrounding embryo sac and ovule tissue, and servesas a conduit for nutrients to be passed from the maternal sporophyte into thedeveloping proembryo (Yeung and Meinke, 1993) (Figure I). The suspensorsenesces after the heart stage and is not a functional part of the embryo in themature seed (Yeung and Meinke, 1993).

What are the mechanisms which underlie the asymmetric allocation of cellfates during the first zygotic division? Are there mechanisms similar to thosethat operate in animal cells to produce the divergent fates of the embryoniclineages? These questions have just begun to be addressed using genetic andmolecular tools. Genetic analysis of Arabidopsis embryonic mutations have


uncovered aberrations of the earliest embryonic divisions (see below). Inaddition, molecular markers to trace the development of the plant zygote andea rly embryo have become available ju st recently. For example, an mRNAencoding a homeodomain pro tein, designated as ATML I, is first detected inthe apic al cell of a 2-ce ll embryo in Arabidops is (Lu et al., 1996). ATMLlmRNA accumulates in all embryo-proper cells until the eight-ce ll stage, afterwhich it becomes restricted to the protoderm layer and maintains a protoderm-spec ific pattern of acc umulation durin g globular and heart stages of embryodevelopment. After disappearing during the torpedo stage of embryo devel-opment, the ATML 1 mRNA reappears in the L1 layer of the shoot apicalmeristem (SAM) of the mature embryo. ATMLl mRNA is also detectable indeveloping endosperm, post-germinative SAM Lllayer, and in the epidermisof leafprimord ia (Lu et aI., 1996 ). The accumulation of ATML1mRNA marksthe earlies t partitioning of the apical versus basal gene product s. Its accumu-lation could be due to the activity of pre-localized determin ants derived fromthe egg cell in a lineage-dependent manner. Alternatively, the positioning ofthe apica l cell following zygotic division might result in de novo synthesis ofregulatory products which in part activate ATMLl gene. Isolation and char-acter ization of regulatory gene products which are responsible for the apicalce ll-spec ific transcription of ATMLl and other apical cell-spec ific genes willbegin to unravel the developmental processes involved in apical-basal cellfate determination. A complementary approach using the gene products thatmark the basal fate, the suspenso r development , will converge on the samekey processes (see below).

2. Muta tions delete specific embryo nic regions .Analysis of a large number of zygotica lly-acting seedling-lethal mutations inArabidopsis has ind icated that deletions of spec ific seedling structures can betraced back to abnormaliti es in early embryos (Mayer et aI., 1991). Four ofthese mutations, designatedfackel , gurke, monopteros, and zwi//e, alter theapical-basal organi zation of the seedling (Mayer et aI., 1991 ; Jiirgens, 1994)(Table 2). The defect infacke! seedlings causes the absence of the hypocotylorgan which is traced to the abnormal cell divisions in the central region ofthe globular stage of embryo development-the vascular precursor cells of theprospective hypocotyl do not divide properly (Mayer et aI., 1991; JUrgens etaI., 1994). gurke seedlings are deficient in the most apical region, missingcotyledons and shoot meristem (Mayer et al., 1991). The first sign of thegurke phenotype is evident at the early-heart stage of embryogenesis. Insteadof producing cotyledonary primordia, gurke embryos remain triangular inshape (J iirgens et aI., 1994). On the other hand, monopteros seedlings have acomplementary phenotype to gurke and lack the basal structures including thehypocotyl , root meristem and root cap (Mayer et al., 1991). The embryonicabe rrations caused by monopteros mutation are due to random cell-divisionpattern s of the lower tier of the embryo proper and the hypopheseal deriv-


TABLE 2

Examples of Arabidopsis mutants that have defects in embryo development

Mutant class References

fackel

monopteros

gurke

Apical-basal development mutants

emb30/gnom (Mayer et al., 1991, 1993b; Shevell et al., 1994; Busch et al., 1996; Vroemen et al.,1996)

(Mayer et al., 1991; Ber1eth and JUrgens , 1993)

(Mayer et al., 1991)

(Mayer et al., 1991)

Ce ll-type differentiation and embryo shape mutants

keule (Mayer et al., 1991; Vroemen et al., 1996)

knolle (Mayer et al., 1991; Lukowitz et aI., 1996; Vroemen et aI., 1996)

fa ss (Mayer et al., 1991; Torres-Ruiz and JUrgens, 1994)

Susp ensor transformation mutants

twin (Vernon and Meinke, 1994)

sus I (Schwartz et al., 1994)

sus2 (Schwartz et al., 1994; Meinke , 1995)

sus3 (Schwartz et al., 1994)

raspb erry I (Yadegari et al., 1994)

raspberry2 (Yadegari et al., 1994)

Late embryo -def ective mutants

embryo-defective c1ass(Vemon and Meinke, 1995)

schleppe rless (N.R. Apuya and R.B. Goldbe rg, unpubl.)

Meristem differentiation/identity mutan ts

shoot meristemless (Barton and Poethig, 1993; Endrizzi et al., 1996; Long et al., 1996)

wuschel (Endrizzi et al., 1996; Laux et al., 1996)

zwille (Jurgens et al., 1994; Endrizzi et al., 1996)

pinh ead (McConnell and Barton, 1995)

embryonic flo wer (Sung et al., 1992; Bai and Sung, 1995; Yang et al., 1995)

shortroot (Bcnfey et al., 1993; Schere s et al., 1995)

hobbit (Aeschbacher et al., 1994)

scarecrow (Scheres et al., 1995)

wooden leg (Scheres et al., 1995)

pinocchio (Scheres et al., 1995)

gollum (Scheres et al., 1995)

Maturation program mutants

lecl- IIle cl-2 (Meinke, 1992; Meinke et al., 1994; West et al., 1994)

lec2 (Meinke et al., 1994)

fus3 (Baumlein et aI., 1994; Keith et al., 1994; Misera et al., 1994)

abi3 (Koorneef et al., 1982, 1984, 1989; Giraudat et al., 1992; Nambara et al., 1992,1995)

Emb ryogenesis in dicotyledonous plants 13

TABLE 2

(Continued)

Mutant class References

Seed ling lethality mutan tslconstitutive pho tomo rp hogenic

fus l tcopl tembl os (Deng et al., 1992; Ang and Deng, 1994; McNellis el al., 1994; Misera et al., 1994)

f l/s2/de/ / (Misera et al., 1994; Pepper et al., 1994)

fl/s6 /cop / l /emb78 (Castle and Meinke. 1994; Misera et al., 1994; Wei et al., 1994b)

fl/s7/cop9 (Misera et al., 1994; Wei et al., 1994a)

f l/s4 (Misera et al., 1994)

f l/s5 (Misera et al., 1994)

f l/s8/cop8/emh/3 4 (Misera et al., 1994; Wei el al., 1994b)

ji ls9/cop JO/emb / 44 (Misera et al., 1994; Wei el al., 1994b)

[usII (Misera el al., 1994).fi1S12 (Misera et al., 1994 )

* Severa l hundred Arabidopsis embryo-defective mutants have been identified using bothchemical and T-DNAmutagenesis, Most of these mutant s can be obtained from the ArabidopsisBiological Resource Center (http;//a ims,cps.msu.edu/aimsl) or the Nottingham ArabidopsisStock Centre (http;//nasc,nott.ac,ukl) ,

atives resulting in an embryo proper with more cells than normal (Berlethand JUrgens, 1993). Finally, the zwille mutation causes the most restrictedabnormality in the seedling by only deleting the shoot meristem (JUrgens etal., 1994). These mut ations ind icate that the loss of a specific region, or com-bination of regions, does not affec t the development of an adjacent neighboras manifested by the phenotype apparent in the seedlings (Mayer et al., 1991).

Does the loss ofa struc ture within the seedling necessarily correspond to thedeletion of a discrete population , or lineage , of cells derived from the earliestembryonic precursor s? A nearly invariant pattern of cell division has made itpossible to follow the development of specific cells of the early Arabidopsisembryo to specific seedling structures with some certainty (JUrgens, 1994).For example, as mentioned earlier, the two products of the zygotic divisionfollow completely separate paths-the apical (terminal) cell form s the embryoproper, while the basal cell give s rise to the suspensor and the hypophysis(Figure I ), By the 8-cell embryo-proper (octant) stage, three tiers of cellscan be recognized in an apical-basal direction: an upper tier, a lower tier,and the suspensor/hy pophys is tier (Figure 1), The three tiers remain fairlydistinct through the heart stage of development and are distinguished by thetypes of cell division pattern s they undergo (JUrgens, 1994). Cells of theupper tier divide nearl y in random planes durin g the globular stage, while thelower tier ce lls divide to form files of cells (JUrgens, 1994). A typical patternof division also characterizes the derivatives of the basal cell (Figure I).The upper tier will form the shoot meristem and most of the cotyledons


(Jurgens , 1994); the lower tier will generate hypocotyl, radicle, and rootmeristem initials as well as contributing to portions of cotyledon (Jurgens,1994, 1995; Scheres et a\., 1994). As mentioned earlier, the hypophysis,from the lowest tier, will contribute to the remainder of the root meristem,the quiescent center and the central portion of the root cap (Scheres et a\.,1994). Because the derivatives of more than one tier contribute to any givenseedling structure, the three embryonic cell tiers do not correspond directlyto the presumptive primordia of the seedling (Jurgens, 1995). The simplestexplanation is that interactions exist between the derivatives of each lineageto effect a simultaneous development. For example, the root meristem isderived from cells contributed by the lower tier and the hypophysis, twolineages that are separated as early as the first zygotic division (Dolan eta\., 1994; Scheres et a\., 1994). The MONOPTEROS gene product may beactive in derivatives of the lower tier and the hypophysis, or may act onlyin one lineage with the other receiving signals to coordinate development(Berleth and Jurgens, 1993). A similar argument has been made to explainthe development of cotyledons as a result of interactions between the upperand the lower tier derivatives (Jurgens, 1995). Deletion of distinct seedlingstructures, therefore, may include the derivatives of more than one earlyembryonic region, whose developmental history may involve inter-regionaland/or inter-cellular processes.

A zygotically-acting, seedling-lethal mutation which has the most globaleffect on the apical-basal pattern is gnom (Mayer et al., 1991, 1993b) (Table 2).gnom seedlings are highly abnormal, and possess reduced shoots and com-pletely lack roots. The strongest phenotype, represented by 'ball-shaped'seedlings, lacks any sign of apical-basal polarity. The gnom phenotype hasbeen traced back to the first zygotic division in which two similar-sizeddaughter cells are produced instead of the unequal-sized apical and basalcells that are found in wild-type embryos. The gnom apical daughter celldivides obliquely or perpendicular to the apical-basal axis to produce an octantembryo containing twice the normal number of cells while the presumptivehypophysis fails to develop (Mayer et a\., 1993b). TheGNOM gene most like-ly acts upstream ofMONOPTEROS since gnom has been shown to be epistaticto monopteros, that is, gnom monopteros double mutants have a gnom pheno-type (Mayer et a\., 1993b). GURKE, FACKEL, and MONOPTEROS probablyrepresent genes that play a role in region-specific development, workingdownstream of genes which in part partition the early embryo into the threemajor tiers along the apical-basal axis through an unknown mechanism. Theprecise role of these genes is still unknown. The early partitioning of the dicotembryo may be a result of zygotic polarity, and based on the limited dataon epistatic relationships, it likely requires the activity of the GNOM gene(Jilrgens, 1994).

T-DNA tagging and positional cloning ofgnom alleles (also called emb30)have led to the isolation of the GNOMIEMB30 gene (Shevell et al., 1994;


Busch et aI., 1996). GNOMIEMB30-encoded protein has an overall similarityto a yeast protein which is encoded by the non-essential gene YEC2 (Buschet aI., 1996). It also includes a conserved domain similar to one found inthe yeast Sec? secretory protein (Shevell et aI., 1994). The GNOM/EMB30mRNA is prevalent in all organs of the adult plant studied so far, and ispresent in seedlings at roughly equivalent levels to that found in mature organs(Shevell et aI., 1994). Microscopic analysis of mutant seedlings has indicatedthat the gene mutation affects cell division, elongation and adhesion duringdevelopment (Shevell et aI., 1994). Intragenic complementation of gnomalleles has suggested that an active GNOM protein may consist of identicalsubunits (Busch et aI., 1996). GNOMIEMB30 is not essential for cell viabilityas demonstrated by the fact that bisected gnom seedlings produce greencallus in culture (Mayer et aI., 1993b), and it is also not required for normalgametophytic development as indicated by its zygotic activity (Mayer et aI.,1991 , 1993b). The specific function ofGNOM during embryogenesis might bemediated post-transcriptionally, via physical association of GNOM subunits(Busch et aI., 1996). Alternatively, rather than establishing the embryoniccell division pattern directly, GNOM expression may facilitate a pattern setby other genes. If the GNOM gene product only plays a secretory function ,as Sec? does in yeast, then a structural prerequisite might be required inorder to localize apical-basal determinants in the zygote or the 2-cell embryo,a process which is prevented by the gnom mutation. How GNOM mightperform its specific function and the nature of the upstream apical-basalpatterning (or partitioning) genes and their interaction with downstream genesthat mediate events required for the differentiation of independent regionsalong the longitudinal axis, remain to be determined.

3. Allocation ofapical-basal pattern characteristics in the embryo propermay be reversible.Different gene sets must become active in the apical and basal cells after thedivision of the zygote. As stated earlier, whether the polarized organizationof the egg cell, the zygote, or both control differential gene expression eventsearly in embryogenesis is not known. For example, do pre-localized regulatoryfactors within the egg cell initiate a cascade of events leading to the lineage-dependent differentiation of apical and basal cell derivatives? Alternatively,after fertilization, does the zygotic genome direct the de novo synthesis ofregulatory factors that are distributed asymmetrically to the apical and basalcells at first cleavage? The initial processes which polarize the young embryowill set into motion the subsequent batteries of genes which are responsible forthe establishment of embryonic pattern (cellular organization), cell divisionand growth.

Recent evidence, such as a clonal analysis of Arabidopsis root meristemformation using transposon excision has undermined the importance of alineage-dependent mode of cell specification in establishing the apical-basal

16 Ramin Yadegari and Rohert B . Goldherg

Kti3 La1 STM ANT EP2IAtLTP1

Fig. 2. A summary of transcriptional domain s in dicot globular embryo . Schematic repre-sentations of globular stage embryos and the accumulation of embryonic mRNAs and/or thetranscriptional activity of selected embryonic marker genes, including the soybean Kfi3 (GKde Paiva and R.B. Goldberg, unpubl.), soybean LeI (R. Yadegar i and R.B. Goldberg, unpubl.),Arabidopsis STM (Long et al., 1996), Arabidopsis ANT (Elliott et aI., 1996), and carrot EP2(Sterk et aI., 1991) or Arabidopsis AfLT?1 (Thoma et aI., 1994; Vroemen et al., 1996) genes.

polarity. For example, in a few instances the boundaries of clones (sectors)generated by random excision of the transposable element were variable andinfringed into the neighboring apical-basal compartments of the seedling(Scheres et al., 1994). Laser ablation experiments have shown that the clonalboundary established by the first zygotic division which separates the futurelineages of root initials from the hypophyseal lineage does not restrict thederivatives of the two lineages in a rigid developmental pathway duringArahidopsis root development (van den Berg et al., 1995). Upon ablation of allof the quiescent center cells (basal and later, hypophysis cell derivatives), cellsfrom the proximal vascular bundle (apical and later, the central tier derivative)take up the previous position of the ablated cells and rather than expressing avascular marker gene, they express a root cap marker gene (van den Berg etal., 1995). Thus, the position-dependent mode of cell differentiation and cellreplacement during post-embryonic root development may indicate a similarlack of rigidity in cell differentiation pathways during embryo development.

The apical-basal polarity of the embryo proper appears to be reversibleas well according to a recent set of experiments. As discussed earlier, themost severe gnom phenotype, a ball-shaped embryo, completely lacks anyapical-basal polarity (Mayer et al., 1991, 1993b) . Localization experimentswith a position-specific lipid transfer protein gene, designated as AtLTP I, hasshed some light on the nature of embryonic polarity (Vroemen et al., 1996).AtLTP1 is transcribed in the protoderm layer during early globular stage ofArahidopsis embryo development (Vroemen et al. , 1996) (Figure 2). Later,AtLTPl transcription becomes restricted to the apical end of the embryos,within the epidermis of the developing cotyledons and upper regions of thehypocotyl. Therefore, AtLTPI is a marker of apical-epidermal differentiation(Vroemen et al., 1996). In maturation-stage ball-shaped gnom embryos, threediscrete patterns of AtLTP1 expression are observed in nearly equivalentnumbers . Among mutant embryos whose polarity was already determined


using the suspensor (basal direction) as a criterion, the first group displayeda normal polarity; that is an apical AtLTP1 gene expression pattern; thesecond group had a basal pattern ofAtLTP1 gene activity; and the third groupshowed an uniform gene activity pattern (Vroemen et al., 1996). Consideringthat CNOM is a zygotic gene (Mayer et al. , 1993b), these results implythat the apical-basal polarity of the embryo proper is not fixed very early inembryogenesis and is susceptible to reversion (Vroemen et al., 1996). Thechalazal positioning of the suspensor in all of the embryos (in fact, used asa marker of embryo orientation) which show aberrant AtLTP1 expres sionsuggests that the basal cell fate has not been reversed, but rather a reversionin the embryo-proper (apical cell) fate has occurred. Suspensor fate mightbe tied to the development of the embryo sac chalaza. Alternatively, basalcell-fate specification may be invariant during early development.

B. Organ and Tissue Differentiation Patterns

1. Embryonic organs and tissue-types differentiate during theglohular-heart transition period.Two critical events must occur after the embryo proper forms - (a) particularregions along the apical-basal axis must differentiate from each other andcontribute to the development of particular embryonic organs, and (b) thethree primordial tissue layers of the embryo need to differentiate (Table 1).The embryo proper has a spherical shape during the proembryo and globularstages (Figure I) . The first visible cell differentiation events occur at the 16-cell stage when the protoderm, or outer cell layer of the embryo proper, isproduced and the hypophysis forms at the top of the suspensor (Figure 1).Subsequent cell differentiation events within the embryo proper result in theproduction of an inner procambium tissue layer and a middle layer of groundmeristem cells (Figure 1). The spatial organ ization of protoderm, groundmeristem, and procambium layers establishes a radial axis of differentiatedtissues within the globular embryo.

A dramatic change in the morphology of the embryo proper occurs justafter the globular stage (Table 1). Cotyledons are specified from two lateraldomains at the apical end (top), the hypocotyl region of the axis begins toelongate, and the embryonic root primordium differentiates from the apicalcell and hypophyseal derivatives at the micropylar end (bottom) (Dolan etal., 1993; Scheres et al., 1994). The embryo proper is now heart-shaped, hasa bilateral symmetry, and the body plan and the main tissue layers of themature embryo (and post-embryonic plant) have been established (Figure 1).Morphogenetic changes during this period are mediated by differential celldivision and expansion rates, and by asymmetric cleavages in different cellplanes (Esau, 1977; Lyndon, 1990) . No cell migration occurs, in contrast tothe migration events that take place in many types of animal embryos (Slack ,1991). Further elaboration of the radial tissue organization occurs through


addition of new tissue layers during the heart and early torpedo stages ofembryo development-periclinal divisions of the procambium layer producethe precursors of the vasculature, the pericycle, while ground tissue splitsinto the cortex (outer) and the endodennis (inner) layers (Jiirgens and Mayer,1994).

2. Cellular interactions may playa role in the formation and maintenanceof embryonic tissue characteristics.What are the mechanisms involved in the establishment of the specific celltypes along the radial axis? Demarcation of the first tissue layer, the pro-toderrn, occurs through synchronous, periclinal divisions of all the cells inan 8-cell embryo proper (Mansfield and Briarty, 1991) (Figure 1). However,protoderm initiation does not occur at the same stage of embryo developmentin all plants, as indicated by the delayed occurrence of the protoderm inCitrusand Gossypium (Pollock and Jensen, 1964; Bruck and Walker, 1985a). Con-sidering that embryo development in both plants is highly irregular (Pollockand Jensen, 1964; Bruck and Walker, 1985a) , a highly ordered , lineage-dependent mode of protodermal specification does not seem to operate inhigher plants. The protoderm, and later the epidermis, are maintained duringdevelopment by a restriction of cell division to the anticlinal plane. Excisionof epidermis in globular and heart-stage embryos ofCitrus does not induce theunderlying tissues to replace it, suggesting that protodenn/epidennis specifi-cation is a one-time event during embryogenesis (Bruck and Walker, I985b) .A number of different morphological markers, including cuticle synthesis anddeposition, have been used to argue that the zygote and all subsequent sur-face derivatives are in fact epidermal in nature, and that during development,internal cells diverge from an epidermal fate to form the ground tissue andprocambium (Bruck and Walker, 1985a). Interestingly, the ATMLI mRNAaccumulation parallels this presumed sequence of epidermal differentiationvery closely (see above). As mentioned earlier, ATML I mRNA is first detect -ed in the apical cell following the first zygotic division, and it remains presentwithin all of the cells of the embryo proper until the 16-cell stage when itbecomes restricted to the newly-formed protoderm (Lu et al., 1996). There-fore, the early zygotic derivatives possessing epidermal attributes may becomedetermined as epidermal cells after prolonged contact with the outside envi-ronment. The protoderm differentiation pathway could represent an early, anda necessary, spatial cue to demarcate an outer boundary in the early embryo.

Some insight regarding the importance of cellular organization in embryo-genesis has been gained from the study of a class of seedling-lethal mutationsin Arabidopsis.fass belongs to a group of zygotic mutations which producegrossly misshapen seedlings (Mayer et al., 1991) (Table 2). fass embryoslack the correct temporal and spatial patterns of cell divisions seen in earlywild-type embryos, resulting in the absence of distinctive radial organiza-tion of tissue types (Torres-Ruiz and Jurgens, 1994).fass seedlings are wide


around the hypocotyl and compressed in the apical-basal axis; however, theydisplay all the functi onal tissues found in the wild-type seedlings (Mayer etal., 1991; Torres -Rui z and Jurgens, 1994). In fact,fass embryos do not lackradi al ce ll laye rs, rather they exhibit additional cell layers during embryoge-nesis and later during seedling root development (Torres-Ruiz and Jurgens,1994; Scheres et al., 1995). The fact that the f ass seedlings contain all the pat-tern elements found in the wild-type seedlings has prompted the suggestionthat the regularity of cell division is not critical in pattern formation in theArabidops is embryo (Torres -Ruiz and Jurgens, 1994). That is, positioning ofthe cell s through regular division pattern s is not a prerequ isite for normal tis-sue di fferenti ation, and furth ermore, cellular interaction s may be the primarydeterminant s of gen erating and maintaining the radial pattern (Mayer et aI.,1991 ; Jurgens, 1995). Analysis of a similar (possibly allelic) class of muta-tion s, design ated as ton , has shown that the mutant seedlings are defectivein cytoskeletal architectures, lacking transverse arrays of interphase micro -tubules and preprophase band s in meri stematic zones; even though , all celltypes and organs differentiate in correct relative positions (Traa s et al., 1995).Becausefass seedlings appear to have a nearly normal apical-basal structure,the regular cell division patterns are unnece ssary for the corre ct developmentof the entire embryo proper in Arabidopsis (Torres-Ruiz and Jurgens , 1994).

Another cla ss of seedling-lethal mutants include knolle and keule whichalter the radial pattern ofArabidopsis embryo (Mayer et al., 1991) (Table 2).kno lle seedlings are round ed with a rough and abnormal-looking epider-mis. At the globular stage of embryo development no distinctly-separatedprotoderm is evident within a structure composed of enlarged, irregularl y-pos itioned cell s (Maye r et aI., 1991 ). keule seedlings also have a rough epi-dermis and are usually elongated. The defect in keule can be traced backto globular-stage embryos with abnormal-looking protoderm which remainbloated during development regardl ess of apparent, normal morphology ofthe inner tissue layers (Mayer et aI., 1991). A similar experiment as thatdescribed for the anal ysis of the gnom mutant embryos, using the AtLTP1marker gene (see abov e) has been performed with knolle and keule embryos(Vroemen et aI., 1996). knolle embryos show an uniform localization of theepidermis-specific mark er in all ce lls during early development. Later, as theembryos mature, no AtLTP1 mRNA is detected in the center of the mutantembryos (Vroemen et aI., 1996), concomitant with the appearance of vas-cular tissue (Mayer et aI., 1991). Thi s pattern suggests that knolle embryosinitially contain only ce lls that are epidermal in charac ter and that a sub-population most internal differentiates into vascular cells (Vroemen et aI.,1996 ). keule embryos, on the other hand , display a normal, outer-cell patternof AtLTPI express ion, even though they lack a strictly-protodermal cell mor-pholo gy (Vroemen et aI., 1996 ). Therefore , radial tissue pattern formationcan be viewed as a multi step proc ess of epidermal differentiation followedby added steps, including vasc ular different iation , which are not necessarily


dependent upon the regular cellular divisions in the early embryo or the finalcellular morphology (Vroemen et al., 1996). The first step in establishing thetissue layers could be fulfilled by the invariable positioning of the protoderm,represented during the transition from the octant to the 16-cell (dermatogen)embryo proper stage of Arabidopsis embryogenesis (Figure I). Obviously,this pattern of cell division is not required to obtain a protoderm (epidermis)as shown in the case of the Citrus embryo (Bruck and Walker, 1985a), theGossypium embryo (Pollock and Jen sen , 1964), or the fass mutant embryo(Torres-Ruiz and JUrgens, 1994). In one model of radial tissue specification,a protoderm layer could be specified by the simple fact that the cells whichhave come to reside on the surface of the embryo are not enclosed by otherembryonic cells . After initiating a protodermal differentiation pathway, thesecells could then participate in signaling the inner cell s to take up the otherfates-ground tissue and procambium.

An important question regarding the differentiation of tissue layers is howthe individual fates are fixed and maintained during embryogenesis. An uniqueaspect of cellular organization in plants is the presence of plasmodesmatawhich connect the protoplasts of neighboring cells together to form a largecommunity of cells known as the symplast. The mature root and hypocotylepidermis of Arabidopsis are symplastically isolated from the underlying tis-sue, presumably through inhibiting the plasmodesmatal exchange (Duckett etal., 1994) . A similar mechanism might be involved to effect symplastic iso-lation of the embryonic protoderm/epidermis, thus blocking in and fixing theepidermal fate to a particular cell layer. Positional cloning and characteriza-tion of the mutation responsible for the knolle phenotype have provided someinsight into the possible mechanisms by which differentiation of individualcell layers is implemented (Lukowitz et al. , 1996) . As discussed earlier, theknolle embryo lacks correctly-oriented cell division patterns that are typicalof wild-type embryos including the tangential cell divisions required to createthe protoderm (Mayer et al., 1991) . Mutant embryos contain both small andenlarged cells with incomplete cross wall s and polyploid nuclei (Lukowitzet al., 1996). This phenotype is reminiscent of the cytokinesis-defective (cyd)mutation in pea which primarily affects cotyledon cell morphology where cellplates form partially, resulting in cell wall stubs and a multinucleate character(Liu et al., 1995).Based on homology, the predicted KNOLLE protein belongsto a phylogenetically-diverse family of syntaxin-related proteins involved invesicular trafficking. KNOLLE mRNA can be detected in single cells or smallclusters of cells at varying intensities in all stages of embryo developmentbefore declining at embryo maturation (Lukowitz et al ., 1996). The tissueand cellular morphology of knolle embryos, and the presumed function of themutated gene, have suggested that the failure of the mutant embryos to initi-ate and maintain a normal radial tissue pattern might be due to a protractedassociation between protodermal and internal layers through groups of inter-connected cells (Lukowitz et al., 1996). The uniform localization of AtLTP1


expression in early knolle embryos (see above) supports the notion that theinternal cells are developmentally similar to the epidermal cells, at least earlyduring development (Vroemen et aI., 1996). Therefore, although KNOLLEgene product is not involved in the specification of radial patterning per se,it may segregate the inner cells from the outer cell layer (Lukowitz et aI.,1996; Vroemen et aI., 1996) in a morphological/physical manner reminiscentof the potential function performed by the GNOMIEMB30 gene product inapical-basal patterning.

The study of mutations that manifest a phenotype late in embryogenesiscan be instructive of the processes that take place early during development asseen with seedling-lethal mutations like gurke,fackel, andfass. In the case ofthe fass mutant for example, additional cells generated during embryogenesisapparently do not interfere with apical-basal polarity or tissue differentiation(see above). What would be the result of decreasing the available number ofcells, or cell layers, on tissue differentiation? Analysis of mutations affectingthe radial organization of the Arabidopsis root, such as shortroot, scarecrow,and wooden leg, has shown that the absence of layer-specific cell divisionsduring embryonic axis development causes the absence of specific cell typesin the developing mutant roots (Scheres et aI., 1995) (Table 2). The defectsin scarecrow, and shortroot can be recognized as early as the heart stagewhen the periclinal division contributing to the doubling of the ground tissueis absent within the axis (Scheres et aI., 1995). During late embryogenesisand post-embryonic development, both mutants exhibit only one cell layer inplace of the ground tissue derivatives, the endodermis and cortex (Benfey etaI., 1993; Scheres et aI., 1995). After germination, shortroot and scarecrowinterfere with the asymmetric divisions of the cortex/endodermis initial withinthe root meristem (Benfey et aI., 1993; Scheres et aI., 1995; Di Laurenzioet aI., 1996) . The resulting cell layer in shortroot lacks endodermis-specificmarkers, suggesting that the endodermallayer is missing (Benfey et aI., 1993;Scheres et aI., 1995). The mutant layer in scarecrow, on the other hand, hasdifferentiated attributes of both cortex and endodermis (Di Laurenzio et aI.,1996). In wooden leg embryos, all tissue layers are present prior to the heartstage of development. Later, the last division to achieve the wild-type numberof cells in the pericycle of a mature embryo (a procambium derivative) isabsent, so that instead of 18 cells around the pericycle layer, only ten cellsappear. The roots ofwooden leg seedlings have fewer cells around the vascularbundle, all of which differentiate into xylem elements in exclusion of thephloem (Scheres et aI., 1995).

Double mutation combinations have provided insight into how reduction inthe number of cells within radial layers cause pattern deletions. For example,fass , in combination with wooden leg, scarecrow, or shortroot, relieves blockson cell division in the ground tissue (shortroot, scarecrow) and vascular cells[wooden leg; (Scheres et aI., 1995)]. fass is epistatic to wooden leg andscarecrow, but does not suppress shortroot phenotype. That is, althoughfass


shortroot embryos produce extra ground tissue layers, the main defect inendode rmis development cannot be overcome, suggesting that SHORTROOTmight be directl y responsible for the endoderm is layer development (Schereset aI., 1995). The function of WOODEN LEG seems to be spec ific to theprocambium as a whol e. shortroot wooden leg double mut ant seedlings showan additive pheno type, sugges ting that at least two independent mechanismsprodu ce the defects in the ground tissue and the vascular ce lls (Scheres et aI.,1995). Therefore, SCARECROW and WOODEN LEG appear to be involvedin the organization of cells within the ground tissue and vasc ular bundle,respectively. A reduction in the availability of ce lls within radial layers (asin the ca se of scarecrow and wooden leg embryos) would cause cell layerdeletions. However, if enou gh cell s are avail able, they can be recruited, in aposition-dependent mann er, first into tissue compartments (vascular or groundtissue ), and then they become spec ified as cell types such as peri cycle, cortex,etc . (Scheres et aI., 1995).

The SCARECROW gene has recently been cloned, and its sequence sug-gests that it may encode a put ative tran scription factor (Di Laurenzio etal., 1996). This gene is transcribed in the cortex/endodermal initial , and inthe endodermis, con sistent with a role in regulating the asymmetric divi-sion of the initial du ring root development. During embryo development, theSCARECROW gene is transcribed in the ground tissue of the late heart-stageembryos, and after the division of the ground tissue, it is transcribed in theendode rmis only (Di Laurenzio et aI., 1996 ). SCARECROW may functionin both phases of development , or it may function primaril y during embryodevelopment. According to the latter model , the pattern of ground tissue dif-ferentiation mediated by SCARECROW would be transmitted and maintainedpostembryonically in the form of the asymmetric division pattern seen incortex/end odermis-initial differenti ation (Di Laurenzio et aI., 1996). Th e pat-tern of expression for the SCARECROW gene and the phenotype of the mutantallele support the notion that it plays an important function in establishing theradial pattern of cell types during embryo development (Sche res et aI., 1995;Oi Laurenzio et al., 1996).

Taken together, the evidence from a number of different experiments sug-gests a multi -step model of ce ll specification during embryogenes is. A pro-toderm is specified, perhaps through positioning of cells on the surface ofan embryo. This may be follow ed by a recruitment of cell s into radial com-partment s which are then further differentiated into individu al cell types.This model raises a number of important questions. For example, how arethe individual cell/tissue fates perp etuated during embryo development andeven germination? One way of accomplishing thi s would be through clonaltransmi ssion of the cell fates, as has been proposed for the tran smission ofcell-specific phenotype s of knol!e, keule (epiderm is) , and shortroot (endoder-mis) embryos (JUrgens, 1995). Other developmental processes may impingeupon clonally-transmitted cell fates later during development and germ ina-


tion. For example, dye-coupling experiments have shown that the hypocotylepidermis cell s are symplastically connected to one another, even though,they remain distinctly isolated from the root epidermis (Duckett et aI., 1994).Therefore, although all epidermal cells are clonally related , regionally-actingmechanisms may intervene to effect the differentiation of individual segmentsof the tissue layer-a point of intersection between the apical-basal pattern-ing processes and tissue layer differentiation. An alternative mechanism tothe clonal transmission of cell fates may involve a model similar to the oneproposed for the establishment of the radial pattern of tissues in the Arabidop-sis root (Scheres et aI., 1995; van den Berg et aI., 1995). Information fromthe more mature cells may be required for the correct division pattern andspecification of developing regions of the embryo, or the seedling, along theindividual tissue/celllayers.

IV. The Organ Expansion and Maturation Phase

A. Preparation of the Embryo f or a Dorman cy Period

A major change in embryonic development occurs during the organ expansionand maturation phase (Figure I ). A switch occurs during this period from aregional and cell-specification program to a storage product accumulationprogram in order to prepare the young sporophyte for embryonic dormancyand post-embryonic development (Table I ). The axis and cotyledons increasein size dramatically due to cell divi sion and expansion events (Mansfieldand Briarty, 1991, 1992). Ground meristem cell s within both these organsbecome highl y specialized and accumulate large amounts of storage protein sand oils that will be utilized as a food source by the seedling after germination(Mansfield and Briarty, 1992 ) (Figure I , Table 1). One differentiation eventdoe s occur during this period, however - the characteristic organization of theshoot apical meristem (SAM) becomes evident in the intercotyledonary regionat the bending-cotyledon stage of Arabidopsis embryo development (Bartonand Poethig, 1993) (Figure I). By contrast, the distinct cellular organizationof the embryonic root is visible as early as the heart stage of embryogenesis(Dolan et aI., 1993; Scheres et aI., 1994). At the end of the organ expansionand maturation period the embryo has reached its maximum size, cells ofthe embryo and surrounding seed layers have become dehydrated, metabolicactivities have ceased, and a period of embryonic dormancy within the seedhas begun (Mansfield and Briarty, 1992; Lindsey and Topping, 1993; Westand Harada, 1993).

B. Shoot Apical Meristem Development

At what stage of embryo development is the shoot meristem specified? His-tological differentiation of the shoot apical meri stem (SAM) is defined by

24 Ramin Yadegari and Rohert B. Goldberg

the specific patterns of cell division occurring in the vegetative meristern.In the angiosperms, a peripheral region of one or two layers (tunica) showanticlinal cell divisions, while the interior cells (corpus) divide in variousplanes (Esau, 1977). In Arabidopsis, the SAM is visible first after the torpedostage of embryogenesis (Barton and Poethig, 1993). Yet, the SAM lineagecan be traced to a subset of cells in the apical half of the globular embryo(Barton and Poethig, 1993). The morphological appearance and the analysisof shoot meristem mutants (see below) suggest that shoot meristem initiationoccurs relatively late in embryogenesis and that an organized shoot meris-tern is not required to form cotyledons or leaves (Barton and Poethig, 1993).Mutations in the SHOOTMERlSTEMLESS (STM) gene prevent the formationof a normal SAM during Arabidopsis embryogenesis (Barton and Poethig,1993) (Table 2). SAM development is essentially blocked at or just after thetorpedo stage of embryo development in these mutants. stm explants produceabnormal shoot structures in culture, suggesting that STM is also requiredfor postembryonic SAM development. The defect in stm plants is specificto the shoot meristem and does not affect other developmental processesincluding root meristem development (Barton and Poethig, 1993). This, andother observations, suggest that root and shoot meristem initiation processesare based on different mechanisms (Barton and Poethig, 1993; McConnelland Barton , 1995; Endrizzi et aI., 1996; Laux et aI., 1996). As mentionedearlier, the zwille mutation also disrupts shoot meristem development duringembryogenesis, although its cellular phenotype does not appear until aftergermination (Jurgens et aI., 1994). In contrast to stm plants, zwille does notinterfere with post-embryonic shoot formation (Jurgens et aI., 1994). There-fore, the development of a proper SAM relies on the activity of a group ofgenes, including STM and ZWlLLE, whose function is either to demarcatethe shoot meristem region early during embryogenesis, or bring about theproper development of the subsequent subdivision during the torpedo stageof embryo development, or both (Jurgens et aI., 1994; Long et aI., 1996).

The STM gene has been cloned and shown to be a member of the KNOT-TED clas s of homeodomain protein genes found in maize and soybean(Long et aI., 1996). STM mRNA is detected in early to mid-globular stagesof Arabidopsis embryo development within one or two cells positioned inbetween the presumptive cotyledonary initials. As embryogenesis proceeds,the domain of STM mRNA accumulation expands with the differentiation ofthe presumptive SAM. During seedling germination and adult plant develop-ment , STM mRNA is detectable in all types of SAM, including vegetative,inflorescence, floral, and axillary apices (Long et aI., 1996). Therefore, STMgene activity marks the initiation of the SAM early during embryogenesis, andpersists with the development of the SAM during embryogenesis and matureplant development. The expression ofKNOTTED l , the STM homologue frommaize (Vollbrecht et aI., 1991), has also been localized to the apical regionof the maize embryo (Smith et aI., 1995). However, in contrast to STM, the


start of KNOTTED I mRNA accumulation appears to be concomitant withthe onset of histological changes associated with meristem formation (Ran-dolph, 1936; Smith et aI., 1995) . Further analysis of the STM function duringArabidopsis development has indicated that STM plays an important role inmaintaining shoot and floral meristem activity by preventing the direct incor-poration of cells in the meristem center (central zone) into differentiatingorgan primordia (Endrizzi et aI., 1996). The undifferentiated cells within thecentral zone lie at the summit of the shoot apex and replenish the cells usedup for the initiation of primordia during vegetative development (Steeves andSussex, 1989) . Therefore, STM is involved in the formation of SAM duringembryogenesis, and it is also required for SAM 's proper development bymaintaining the undifferentiated state of the cells in the central zone (Bartonand Poethig, 1993; Endrizzi et aI., 1996).

How does the STM gene product affect SAM development? In maize,KNOTTED1 encodes a protein which is present in Ll layer cells of thevegetative shoot where KNOTTED I mRNA is not detected (Lucas et aI.,1995). In fact, microinjection experiments have shown that the KNOTTED Iprotein is able to mediate cell-to-cell transport of its own sense RNA intobacco mesophyll cells (Lucas et aI., 1995). Whether similar cell-to-celltrafficking ofSTM gene products takes place in Arabidopsis embryos remainsto be seen. Interestingly, although STM mRNA is detectable in the cellspredicted to form the embryonic SAM, the first indication of a stm phenotypeis not seen until the bending-cotyledon stage (Barton and Poethig, 1993;Long et aI., 1996). Unless the synthesis and accumulation of the STM proteinproduct is much delayed relative to the accumulation of the STM mRNA, theSTM gene product appears to function in the derivatives of cells that have beenspecified earlier by an STM-independent process. Alternatively, STM may beinvolved in specification of SAM precursor cells early in embryogenesis;whereas, in an stm background, another gene product might complement theSTM activity within these precursors.

STM's role in the development of shoot meristem (Barton and Poethig,1993; Endrizzi et aI., 1996) is accompanied by the action of other gene s whichfunction downstream to or in concert with STM (McConnell and Barton, 1995;Endrizzi et aI., 1996; Laux et aI., 1996). For example, wuschel mutants donot properly organize a shoot meristem in the embryo while postembryonicshoots are defective and exhibit ectopic primordia initiation (Laux et aI., 1996)(Table 2). Along with ZWILLE (JUrgens et aI., 1994),WUSCHEL is thought tobe involved in the maintenance and proper functioning of the shoot meristem(JUrgens et al ., 1994; Endrizzi et aI., 1996; Laux et aI., 1996). Genetic studiessuggest that STM functions upstream of WUSCHEL and ZWILLE in shootmeristem development (Endrizzi et aI., 1996). The vegetative character ofSAM can also be modified by mutations in the EMBRYONIC FLOWER genewhich cause seedlings to produce flowers rather than leaves, indicating thatthe fate of the shoot meristem is altered during embryogenesis - a floral


meristem is specified rather than a vegetative shoot meristem (Sung et al.,1992) (Table 2). embryonic flower embryos display abnormal patterns of celldivisions in the intercotyledonary region as early as the heart -stage of embryodevelopment (Bai and Sung , 1995). Therefore, proper development of theSAM requires multiple steps in differentiation of a subset of cells in theapical half of the globular-stage embryo. An indication of this process is theaccumulation of STM mRNA in the presumptive SAM precursor cells beforeSAM initiation. It remains to be seen whether STM is involved directly inSAM initiation, or whether it is only responsible for the maintenance of SAMalong with other gene products after the SAM is initiated.

V. Maternal and Female Gametophytic Contribution to Embryogenesis

As discussed earlier, it is unclear what influence, if any, maternal tissuesand/or accessory cells of the female gametophyte have on egg cell formationand subsequent embryonic development. For example, does either the ovule orcells within the embryo sac (e.g., synergids) produce morphogenetic factorsthat contribute to the establishment of longitudinal asymmetry within theegg? Identification of mutations and chromosomal deficiencies in Arabidopsisand maize has indicated that fernale-gametophytic genes are required forthe correct development of the embryo sac (Redei, 1965; Kermicle, 1969;Castle et al., 1993; Springer et al., 1995; Vollbrecht and Hake, 1995; Ohadet al., 1996). That is, development of the embryo sac and its componentsis not controlled exclusively by the processes that establish the structureof the ovule per se. Even after fertilization, development of the endospermis, in part, under the influence of female-gametophytic genes (Ohad et al.,1996). In fact, in the few instances where female-gametophyte mutationshave indicated embryonic lethality or arrest, an abnormal endosperm hasbeen implicated as the source of the defect (Castle et al., 1993; Ohad et al.,1996). To date, there has been no strong evidence for the direct contributionof the female gametophyte to embryonic development. Therefore, the geneticevidence suggests that female gametophyte-specific genes are required forthe development of the embryo sac, and that the female gametophyte in partaffects the development of the endosperm, and indirectly, the embryo.

A. Sporophytic and Gametophytic Contributions to Embryogenesis

Based on cytoskeletal morphology, the surrounding tissue is considered to behighly important in the determination of the embryo sac structure (Webb andGunning, I994a,b ). Furthermore, in Arabidopsis, analysis of female-sterilemutations which are defective in ovule and embryo sac morphogenesis hassuggested that either the embryo sac and ovule interact with each other, orthat a normal ovule tissue is required for embryo sac development (Reiser


and Fischer, 1993). On the other hand , the contribution of the embryo sac tothe functional (biochemical/molecular) polarity of one of its components, theegg ce ll, remains unknown. Furth ermore, the lack of information about thecontribution of the egg ce ll, or the embryo sac as a whole, to the developmentof the embryo most likely reflect s the techn ical complexity of distinguishingthe mutations which affect the female gametophyte development and thosethat affec t other aspec ts of reprodu ctive development. Therefore, female-gametophytic mutations are thought to be underscored frequently in mutantsc reens (Re iser and Fischer, 1993). A similar reason could explain the lack ofevidence for 'materna lly-acting' mutation s which would affect embryo devel-opment (see above). In contrast to the female-gametophytic mutations whichare determined by the hapl oid genotype of the embryo sac, maternal muta-tions would knock out genes expressed in the parent sporophyte, causing allembryos within embryo sacs of a homozygous plant to show the defect (Reis-er and Fischer, 1993) . Although maternal inheritance of embryonic defect shas not been documented , there are few examples of the maternal inheri-tance of ovule mutations (Reiser and Fischer, 1993; Angenent and Colombo,1996) and testa mutations (Leo n-Kloosterziel et aI., 1994), as well as muta-tions which affect meio sis in both male and female gametophytes (Reiser andFischer, 1993).

B. Somatic Embryogenesis

The capac ity to form somatic embryos spontaneously and in culture sugges tsthat except for contributing physical support structures and nutrients for theembryo, the matern al sporophyte prov ides very little, if any, developmen-tal signaling. Emb ryo-like structures leading to plantlets can form directl yfrom the attached leaves of some plants (Raghavan, 1976), including thefoliar embryos ofMalaxis which are very similar to zygotic embryos (Taylor,1967). Non-zygoti c embryos may also form via asexual embryogenesis orapomictic processes (Koltunow, 1993; Sharma and Thorpe, 1995). Somat-ic ce lls from a variety of vegetative and reproductive tissues can undergoembryogenesis in culture and lead to the production of fertile plant s (Thorpe,1995). Somatic embryos undergo morphological events similar to those thatoccur within the embryo-proper region of zygotic embryos (e.g., progres-sive development of globular, heart, and torpedo stages), except that they donot become dormant (Table I ), and have altered cotyledon morphology andstorage product deposition (Zimmerman, 1993; Yeung, 1995). The similari-ties between early zygotic and somatic embryo morphologies are especiallysignificant since the immediate physical environments are so divergent (Zim-merman, 1993). Among the species studied to date, a few show similar cellularpatterns of development during ea rly embryogenesis when somatic embryoswere compared to the zygotic counterparts (Yeung, 1995). Somatic embryossynthes ize and acc umulate storage proteins, starch, and lipids even though


they may exhibit altered spatial and temporal patterns of particular storageproduct(s) depo sition, an aspect most likel y due to the dissimilar embryonicenvironments (Yeung, 1995) Thi s is probably due to the physiological anddevelopmental state of the particul ar somatic embryo culture, as shown inthe case of soybean somatic and zyg otic embryos for example (Dahmer etal., 1992). Remarkably, the spatial and temporal gene transcription program sappear to be highly conserved in somatic and zygotic embryos, as indicated byequivalent patterns of cell or region-specific embryonic mRNA accumulationpattern for the soybean Kunit z trypsin inhibitor (Kti) ge nes (Pe rez-Grau andGoldberg , 1989), carrot lipid transfer protein gene EP2 (Sterk et al ., 1991),and the soybean seed lectin gene Lei (R. Yadegari , L. Perez-Grau , and RB.Goldberg , unpubl.) . In maize, maternal sporophyte-independent zygotes caneven be produced by fertili zing egg cell s in vitro which undergo embryogene-sis in culture and give rise to flower-producing plants (Dumas and Mogensen,1993; Kranz and Lorz, 1993; Faure et al ., 1994; Kranz et al ., 1995). Thus, bothzygotic and somatic embryogenes is can occur in the ab sence of surroundingovule tissue.

The embryo sac is nece ssary for zygotic embryogenesis because it containsthe egg and associ ated accessory ce lls that are required for fertilization andendosperm development (Figure I). However, the embryo sac is not essentialfor embryoge nes is per se because (a) somatic embryos produ ced from sporo-phytic cells develop normally (see abo ve), and (b) embryos can be induced toform from microspores that , under normal circumstances, give rise to pollengra ins (Raghavan, 1976). Taken together, these results suggest that normalembryogenic processes do not require factors produced by either the fema legametophyte or maternal sporophytic tissue. Thi s concl usion is supported toa large extent by the fact that the overwhelming majority of mutations thatalter embryo development appear to be due to defects in zygotically-actinggenes (Meinke, 1985, 1994; Jurgen s et al ., 1991 ; Ca stle et al., 1993; Yadegariet al., 1994). It is possible that soma tic cell s have the potential to produceputative maternal or gametophytic factors under the proper condition s, or thatsomatic embryos specify their longitudinal apical-basal and radial tissue-typeaxes via different mechanisms than zygotic embryos. Therefore, most of theavailable data suggest that embryo axis determination and cell specificationevent s are directed primarily by the zygotic genome after fertilization occurs.

VI. Transcriptional Networks Mark Processes Involved in EmbryoDevelopment

A. Differentially-Transcribed Regions within a Globular-Stage Embryo

A large number of genes are expressed during embryogenesis in high er plants(Goldberg et al. , 1989). Although it is not known how many genes are neces-


sary to program morphogenetic and tissue differentiation processes, approx-imately 15,000 diverse genes are active in the embryos of plants as diverseas soybean and cotton (Goldberg et aI., 1989). Many of these genes areexpressed in specific cell types, regions, and organs of the embryo (Goldberget al., 1989; Perez-Grau and Goldberg, 1989), and provide useful entry pointsto unravel the molecular mechanisms that regulate cell- and region-specificdifferentiation events during plant embryogenesis (Davidson, 1994).

What are the spatial domains of gene expression during early embryodevelopment? Recent studies have uncovered four transcriptionally-activeregions within the globular stage of dicot embryo development. Localizationstudies with a soybean Kunitz trypsin inhibitor mRNA, designated as Kti3(Jofuku and Goldberg, 1989), and GUS enzyme localization conferred bya Kti3 5' region indicated, however, that cells destined to form the axisregion of soybean and tobacco embryos are already specified at the globularstage [(Perez-Grau and Goldberg, 1989); G.R. de Paiva and R.B. Goldberg ,unpubl.] (Figure 2). The region of Kti3 differential expression overlaps thepresumptive embryonic root initials of the tobacco embryo (Soueges, 1920).This result differs from that obtained with the carrot EP2lipid transfer proteinmRNA or the Arabidopsis AtLTPl lipid transfer protein mRNA which arelocalized uniformly in the outer protoderm cell layer that surrounds the entireembryo proper at the globular and heart stages (Sterk et aI., 1991; Vroemen etaI., 1996) (Figure 2). Another soybean seed protein gene, encoding the seedlectin [Lei; (Goldberg et aI., 1983; Okamuro et aI., 1986)], is transcribedin a doughnut-shaped cluster of cell s located medially (along the equator)within a tobacco globular-stage embryo (R. Yadegari and R.B. Goldberg,unpubl.) (Figure 2). Lei-transcribing cells overlap the ground meristem andan adjacent equatorial band of the protoderm (R. Yadegari and R.B. Goldberg,unpubI.) . A third program ofdifferential gene expression in the globular-stageembryos has been described recently in Arabidops is by virtue of the specificexpression of the STM gene in a discrete domain at the chalazal end of aglobular-stage embryo that is destined to become the shoot meristematiczone later in embryogenesis (Barton and Poethig, 1993; Long et aI., 1996)(Figure 2). Although it is not known whether the spatial pattern of STMmRNA is principally regulated at the transcriptional level, the activity of theSTM gene marks a regional specification process that precedes downstreamevents that lead to embryo shoot meristem initiation-events that might beregulated, in part , by the STM gene (Long et aI., 1996). Finally, a fourthprogram of differential gene expression can be attributed to the transcriptionof another Arabidopsis gene, AINTEGUMENTA (ANT), which is involvedin ovule and floral organ development (Elliott et aI., 1996). ANT mRNA islocalized within two bilaterally-symmetrical clusters of cells which would bepresumably involved in cotyledon initiation (Elliott et aI., 1996) (Figure 2).

The activity of these four genes, regardless of whether they playa rolein regulating the ultimate differentiation of the embryonic regions in which


they are expressed, nevertheless indicates that both the longitudinal, apical-basal axis and the radial, tissue-type axis of a globular embryo are partitionedinto discrete transcriptional territories (Davidson, 1990, 1994) . The longi-tudinal axis of the embryo proper contains at least three non-overlappingtranscriptional territories - (a) chalazaI region, (b) equator region, and (c)micropylar region (Figure 2). The chalazal region is partitioned into at leasttwo sub-regions hi