Plant Embryogenesis: Zygote to Seed Robert B. Goldberg ... · animals (1) is not set aside during plant embryogenesis. Amature flowering plant embryo con-tains two primary organ systems-the

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Plant Embryogenesis:Zygote to Seed

Robert B. Goldberg,* Genaro de Paiva, Ramin Yadegari

Most differentiation events in higher plants occur continuously in the postembryonic adultphase of the life cycle. Embryogenesis in plants, therefore, is concerned primarily withestablishing the basic shoot-root body pattern of the plant and accumulating food re-serves that will be used by the germinating seedling after a period of embryonic dormancywithin the seed. Recent genetics studies in Arabidopsis have identified genes that providenew insight into how embryos form during plant development. These studies, and othersusing molecular approaches, are beginning to reveal the underlying processes that controlplant embryogenesis.

A major problem in plant development isto unravel the mechanisms operating dur-ing embryogenesis that enable a plant tospecify its body plan and tissue differentia-tion patterns. Although progress with a va-

riety of animal systems has been spectacularin this regard (1), a detailed understandingof the events that govern plant embryoformation has yet to be realized. One obsta-cle in achieving this goal is the location ofembryos within the plant and their relativeinaccessibility to experimental manipula-tion, particularly at the early stages of em-bryogenesis. In flowering plants, reproduc-tive processes occur within floral organs

(Fig. 1) (2). The egg cell is present in theovule, a multicellular structure that is buriedbeneath several cell layers of the pistil, thefemale reproductive organ (2-4). Becauseegg cell formation, fertilization, and embry-ogenesis occur within the pistil, it has beendifficult to dissect the major events that takeplace during the early stages of higher plantdevelopment.

Recently, it has become feasible to iso-late plant eggs and fertilize them in vitro inorder to investigate the initial events ofplant embryogenesis (5). In addition, genet-ic approaches have been used to identifygenes required for various embryogenic pro-

cesses, including pattern formation (6, 7).Genetic manipulation of Arabidopsis thali-ana, by both chemical mutagenesis (8-12)and insertional mutagenesis (13-15), hasidentified a large number of mutants thatare blocked at different stages of embryo-genesis. In this review we outline the majorinsights that have been derived from studiesof Arabidopsis embryo mutants, and we sum-

marize gene transcription experiments inother plants that provide new informationabout the processes regulating higher plantembryogenesis. Both the genetic and mo-

lecular approaches suggest that a plant em-

bryo has a modular structure and consists ofseveral regions that form autonomously dur-ing embryogenesis.

Embryos Begin the Diploid Phaseof the Higher Plant Life Cycle

The flowering plant life cycle is dividedinto haploid and diploid generations thatare dependent on each other (Fig. 1) (2,16-18). The haploid, or gametophytic,generation begins after meiosis with spores

that undergo mitosis and differentiate intoeither a pollen grain (male gametophyte) or

an embryo sac (female gametophyte) (3,19-20). The pollen grain contains twosperm cells, whereas the embryo sac con-

tains a single egg (Fig. 1). Other accessorycells within the haploid male and femalegametophytes help facilitate the pollinationand fertilization processes (3, 19, 20). Themale and female gametophytes are derivedfrom specialized spore-forming cells withinthe reproductive organs of the flower (3, 4,21). By contrast, the diploid, or sporo-

phytic, generation begins after fertilizationwith the zygote and forms the mature plantwith vegetative organs (leaf, stem, root)and flowers that contain the reproductiveorgans (anther and pistil) (Fig. 1).

Two fertilization events occur in flower-ing plants (2, 22). One sperm unites withthe egg cell to produce a zygote and initiateembryogenesis. The other unites with a spe-

cialized cell within the embryo sac (centralcell) to initiate the differentiation of theendosperm, a triploid tissue that is neithergametophytic nor sporophytic in origin (Fig.1) (23). The endosperm is present duringseed development and provides nutrients foreither the developing embryo, the germinat-ing seedling, or both (23). Fertilization alsocauses the ovule, containing the embryo andendosperm, to develop into a seed and theovary to differentiate into a fruit, whichfacilitates seed dispersal (Fig. 1) (24).

SCIENCE * VOL. 266 * 28 OCTOBER 1994

Most morphogenetic events in floweringplants occur in the postembryonic sporo-

phyte after seed germination (Fig. 1) (2).Vegetative organ systems differentiate con-

tinuously from root and shoot meristematicregions that are formed initially during em-

bryogenesis. The reproductive organs of theflower are differentiated from a repro-

grammed shoot meristem after the seedlinghas become a mature plant (Fig. 1) (25).Thus, a germline analogous to that found inanimals (1) is not set aside during plantembryogenesis.A mature flowering plant embryo con-

tains two primary organ systems-the axisand cotyledon (Fig. 1) (2). These organs

have distinct developmental fates and are

both composed of three basic, or primordial,tissue layers-protoderm, procambium, andground meristem-which will become theepidermal, vascular, and parenchyma tissuesof the young seedling, respectively (2). Theaxis, or hypocotyl-radicle region of the em-

bryo, contains the shoot and root meristemsand will give rise to the mature plant afterseed germination (Fig. 1). By contrast, thecotyledon is a terminally differentiated or-

gan that accumulates food reserves that are

utilized by the seedling for growth and de-velopment before it becomes photosynthet-ically active (Fig. 1). The cotyledon func-tions primarily during seed germination andsenesces shortly after the seedling emergesfrom the soil. Embryogenesis in higherplants, therefore, serves (i) to specify meri-stems and the shoot-root plant body pat-tern, (ii) to differentiate the primary planttissue types, (iii) to generate a specializedstorage organ essential for seed germinationand seedling development, and (iv) to en-

able the sporophyte to lie dormant untilconditions are favorable for postembryonicdevelopment.

The Shoot-Root Body Plan IsGenerated During Early

Embryogenesis

How the embryo acquires its three-dimen-sional shape with specialized organs andtissues, and what gene networks orchestrateembryonic development remain major un-

resolved problems. From a descriptive pointof view, plant embryogenesis can be dividedinto three general phases in which distinctdevelopmental and physiological events oc-

cur: (i) postfertilization-proembryo, (ii)globular-heart transition, and (iii) organ ex-

pansion and maturation (26-28) (Fig. 2and Table 1). Although there is consider-able variation in how embryos in differentplant taxa form (29), the overall trends are

remarkably similar (29). We summarize theCapsella and Arabidopsis pattern of embryodevelopment (29-33) because (i) it is one

of the most well-studied forms of plant em-

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Authors are in the Department of Biology, University ofCalifornia, Los Angeles, CA 90024-1606, USA.

*To whom correspondence should be addressed.

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Fig. 1. The life cycle of aflowering plant with em-phasis on egg cell forma-tion and seed develop-ment. [Adapted from (16,26).]

Flower

Egg cell formation,pollination, and fertilization

Pistil

Nucellu

Ovule

Megasporemother cell

(2n)us ' -

meristem

Survivingmegaspore

(n)

Meiosisl -_

Funiculus /IntegumentsMicropyle

Germinatingseed

0*

Root

Embryo Femalesac gametophyte (n)

I \ Anti

Mitosis___

b Centralcell s

Ace

Pollengrains ?"'

Stigma Sperm cellsFTube

Style nucleus

ipdal Male

ells gametophyte

vary-ft(n)Ovary-fruit

'Synergidcells

yW

Dormant Developing Fertilized endospermseed seed nucleus (3n)

Endosperm

Fertilized

Axis lvEmbryo eeloping egg (2n)

Cotyledons j embryo

Seed development and germination

bryogenesis, dating back to the classicalstudies of Hanstein, Schaffner, and Souegeswith Capsella (30-32), (ii) it has an invari-ant division pattern during the early stages,which allows cell lineages to be traced his-tologically (33), and (iii) recent studieswith Arabidopsis mutants have providednew insights into the processes that controlembryo development (6, 9).

Asymmetric cleavage of the zygote results inthe formation of an embryo with a suspensorand embryo proper that have distinct develop-mental fates. The zygote in Arabidopsis andCapsella has an asymmetric distribution ofcellular components-the nucleus and mostof the cytoplasm are present in the upperportion of the cell, whereas a large vacuoledominates the middle to lower portion (Fig.2). This spatial asymmetry is derived fromthe egg cell (30). The zygote divides asym-metrically into two distinct-sized daughtercells a small, upper terminal cell and alarge, lower basal cell-which establish apolarized longitudinal axis within the em-bryo (Fig. 2) (2, 30-33). Histological stud-ies over the course of the past 125 yearshave indicated that the terminal and basalcells give rise to different regions of themature embryo (29-33). The small termi-nal cell gives rise to the embryo proper thatwill form most of the mature embryo (Fig.2). Cell lineages derived from the terminalcell and embryo proper will specify the cot-yledons, shoot meristem, hypocotyl regionof the embryonic axis (29-33), and part ofthe radicle, or embryonic root (Fig. 2) (34).By contrast, the large basal cell derivedfrom the lower portion of the zygote willdivide and form a highly specialized, termi-nally differentiated embryonic organ called

606

the suspensor (Fig. 2). In Arabidopsis, thesuspensor contains only 7 to 10 cells (Fig.2). The suspensor anchors the embryo prop-er to the surrounding embryo sac and ovuletissue and serves as a conduit for nutrients

Postfertilization

to be passed from the maternal sporophyteinto the developing proembryo (Fig. 2)(35). The suspensor senesces after the heartstage and is not a functional part of theembryo in the mature seed. Derivatives of

Globular-heart transitionII1

Proembryo

0Zygote 2-cell 2/4-cell

EP

-cPd ti

8-cellEP 16-cell

EP Globularembryo

C

A

Heartembryo

Organ expansion and maturation

Torpedo embryo Walking-stick embryo Mature embryo

CE

BeEn

A-- SM

RM

SM

- SC

-Pd

EGm-Pc

EnRM

W -~ MPE -

Fig. 2. A generalized overview of plant embryogenesis. Schematic representations of embryonic stagesare based on light microscopy studies of Arabidopsis (33) and Capsella (30-32) embryo development.Torpedo and walking-stick refer to specific stages of embryogenesis in Arabidopsis and Capsella.Abbreviations: T, terminal cell; B, basal cell; EP, embryo proper; S, suspensor; Bc, suspensor basal cell;Pd, protoderm; u, upper tier; I, lower tier; Hs, hypophysis; Pc, procambium; Gm, ground meristem; C,cotyledon; A, axis; MPE, micropylar end; CE, chalazal end; SC, seed coat; En, endosperm; SM, shootmeristem; and RM, root meristem.


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Table 1. Major events of flowering plant embryo-genesis.

Posffertilization-proembryoTerminal and basal cell differentiationFormation of suspensor and embryo proper

Globular-heart transitionDifferentiation of major tissue-type primordiaEstablishment of radial (tissue-type) axisEmbryo proper becomes bilaterally

symmetricalVisible appearance of shoot-root

(apical-basal) axisInitiation of cotyledon and axis

(hypocotyl-radicle) developmentDifferentiation of root meristem

Organ expansion and maturationEnlargement of cotyledons and axis by cell

division and expansionDifferentiation of shoot meristemFormation of lipid and protein bodiesAccumulation of storage proteins and lipidsVacuolization of cotyledon and axis cellsCessation of RNA and protein synthesisLoss of water (dehydration)Inhibition of precocious germinationDormancy

the uppermost cell of the suspensor, thehypophysis (Fig. 2), contribute to the for-mation of the root meristem (30-34). Thus,cell lineages derived from the basal cell giverise to the suspensor and part of the radicleregion of the embryonic axis (Fig. 2).

Different gene sets must become activein the terminal and basal cells after thedivision of the zygote. Whether the polar-ized organization of the egg cell, the zygote,or both control differential gene expressionevents early in embryogenesis is not known.For example, do prelocalized regulatory fac-tors within the egg cell initiate a cascade ofevents leading to the lineage-dependentdifferentiation of terminal and basal cellderivatives? Alternatively, after fertilizationdoes the zygotic genome direct the de novosynthesis of regulatory factors that are dis-tributed asymmetrically to the terminal andbasal cells at first cleavage? In either case,these events would lead to the autonomousspecification of the terminal and basal cellsas a consequence of intrinsic factors ratherthan extracellular signals (1).

Embryonic organs and tissue-types differen-tiate during the globular-heart transition phase.Two critical events must occur after theembryo proper forms: (i) regions along thelongitudinal apical-basal axis must differen-tiate from each other and generate embry-onic organ systems, and (ii) the three pri-mordial tissue layers of the embryo need tobe specified. Both of these events take placeduring the globular-heart transition phase(Fig. 2 and Table 1). The embryo properhas a spherical shape during the proembryoand globular stages (Fig. 2). The first visiblecell differentiation events occur at the 16-cell stage when the protoderm, or outer cell

layer of the embryo proper, is produced andthe hypophysis forms at the top of the sus-pensor (Fig. 2). Subsequent cell differentia-tion events within the embryo proper resultin the production of an inner procambiumtissue layer and a middle layer of groundmeristem cells (Fig. 2). The spatial organi-zation of protoderm, ground meristem, andprocambium layers establishes a radial axisof differentiated tissues within the globularembryo. By contrast, the presence of thehypophysis at the basal end of the embryoproper establishes an apical-basal polaritywithin this region and indicates that theglobular embryo is not radially symmetricalin the formal sense (Fig. 2).A dramatic change in the morphology of

the embryo proper occurs just after theglobular stage. Cotyledons are specifiedfrom two lateral domains at the apical end(top), the hypocotyl region of the axis be-gins to elongate, and the root meristembecomes differentiated from the hypophysisregion at the basal end (bottom) (34). Theembryo proper is now heart-shaped, has abilateral symmetry, and the body plan andtissue layers of the mature embryo (andpostembryonic plant) have been established(Fig. 2). Morphogenetic changes during thisperiod are mediated by differential cell di-vision and expansion rates and by asymmet-ric cleavages in different cell planes (2). Nocell migration occurs, in contrast to themigration events that take place in manytypes of animal embryos (1).

Embryogenesis terminates with a dormancyperiod. A major change in embryonic devel-opment occurs during the organ expansionand maturation phase (Fig. 2). A switchoccurs during this period from a patternformation program to a storage product ac-cumulation program in order to prepare theyoung sporophyte for embryonic dormancyand postembryonic development (Table 1).The cotyledons and axis increase in sizedramatically as a result of cell division andexpansion events (33). Ground meristemcells within both these organs become high-ly specialized and accumulate large amountsof storage proteins and oils that will beutilized as a food source by the seedling aftergermination (Fig. 2 and Table 1) (33). Onedifferentiation event does occur during thisperiod, however-the shoot meristem formsfrom cell layers localized in the upper axisregion between the two cotyledons (Fig. 2)(36). Thus, the differentiation of shoot androot meristems at opposite poles of the em-bryonic axis does not occur at the same time(34, 36). At the end of the organ expansionand maturation period the embryo hasreached its maximum size, cells of the em-bryo and surrounding seed layers have be-come dehydrated, metabolic activities haveceased, and a period of embryonic dormancywithin the seed begins (26-28, 33).


Embryogenesis Can OccurWithout Surrounding

Maternal Tissue

It is unclear what influence, if any, mater-nal tissue or accessory cells of the femalegametophyte have on egg cell formationand subsequent embryonic development(Fig. 1). For example, do either the ovule orcells within the embryo sac (for example,synergids) produce morphogenetic factorsthat contribute to the establishment of lon-gitudinal asymmetry within the egg? Sever-al arguments suggest that the maternalsporophyte provides only physical supportstructures and nutrients for the embryo (Fig.1). First, somatic cells from a variety ofvegetative and reproductive tissues can un-dergo embryogenesis in culture and lead tothe production of fertile plants (37-39).Somatic embryos undergo developmentalevents similar to those that occur withinthe embryo-proper region of zygotic embry-os, except that they do not become dormant(Fig. 2 and Table 1). In addition, spatialand temporal gene expression programs ap-pear to be similar in somatic and zygoticembryos (39, 40). Second, embryo-likestructures leading to plantlets can form di-rectly from the attached leaves of someplants (41). Third, zygotes produced by fer-tilizing egg cells in vitro undergo embryo-genesis in culture and give rise to flower-producing plants (5). Finally, ultrastructuralstudies suggest that there is a barrier be-tween the inner ovule cell layer and theembryo sac that prevents the transfer ofmaterial directly between these compart-ments (42). Thus, both zygotic and somaticembryogenesis can occur in the absence ofsurrounding ovule tissue (Fig. 1).

The embryo sac is necessary for zygoticembryogenesis because it contains the eggand associated accessory cells that are re-quired for fertilization and endosperm de-velopment (Fig. 1). However, the embryosac is not essential for embryogenesis per sebecause (i) somatic embryos produced fromsporophytic cells develop normally (5, 37-40) and (ii) embryos can be induced to formfrom microspores that, under normal cir-cumstances, give rise to pollen grains (43).These results suggest that normal embryo-genic processes do not require factors pro-duced by either the female gametophyte ormaternal sporophytic tissue. This conclu-sion is supported by the fact that the over-whelming majority of mutations that alterembryo development appear to be due todefects in zygotically acting genes (6-14,44). It is possible that somatic cells have thepotential to produce putative maternal orgametophytic factors under the proper con-ditions, or that somatic embryos specifytheir longitudinal apical-basal and radialtissue-type axes by different mechanisms

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than zygotic embryos. However, most of theavailable data suggest that embryo morpho-genesis and cell specification events aredirected primarily by the zygotic genomeafter fertilization occurs. If so, then thiswould differ significantly from the situationwith many animals such as Drosophila andsea urchin, in which maternally suppliedfactors influence the pattern of early em-bryo development (1). More extensive stud-ies with developing female gametophytes,egg cells, and zygotic embryos in the earlystages of embryogenesis are needed to clar-ify this issue.

A Globular Embryo ContainsDifferentially Transcribed Regions

A large number of genes are expressed dur-ing embryogenesis in higher plants (26).Although it is not known how many genesare necessary to program morphogeneticand tissue differentiation processes, approx-imately 15,000 diverse genes are active inthe embryos of plants as diverse as soybeanand cotton (26). Many of these genes areexpressed in specific cell types, regions, andorgans of the embryo (26, 40) and provideuseful entry points to unravel the molecularmechanisms that regulate cell- and region-specific differentiation events during plantembryogenesis (1).

The axis region of the embryo does notbecome visibly distinct until the heart stage(Figs. 2 and 3, A and B). Localization stud-ies with a soybean Kunitz trypsin inhibitormRNA, designated as Kti3 (45), indicated,however, that cells destined to become theaxis are already specified at the globularstage (40). Figure 3C shows that Kti3mRNA accumulates specifically at the bas-al, or micropylar, end of a late-globularstage soybean embryo. No detectable Kti3mRNA is present in other regions of theembryo proper or in the suspensor (Fig. 3C)(40). In transition and heart stage embryos,Kti3 mRNA remains distributed asymmetri-cally at the embryo micropylar end (Fig. 3,D and E) and is localized specifically withinthe ground meristem cell layer (Fig. 2) ofthe emerging embryonic axis (Fig. 3D)(40). No detectable Kti3 mRNA is presentwithin the newly initiated cotyledons (Fig.3E). This result differs from that obtainedwith the carrot EP2 lipid transfer proteinmRNA, which is localized uniformly in theouter protoderm cell layer that surroundsthe embryo proper at the globular and heartstages (46). Taken together, these resultsindicate that cells along the longitudinalapical-basal axis of the embryo proper arealready differentiated from each other atthe globular stage and that early in embry-ogenesis distinct gene sets are expressed indifferent embryonic regions and cell types.

Transformation studies with tobacco em-

bryos containing chimeric 3-glucuronidase(GUS) reporter genes driven by soybeanembryo-specific gene promoters showedthat a globular embryo is organized intodistinct, nonoverlapping transcriptional re-gions, or territories (Fig. 3, F to I) (47, 48).Blue color resulting from GUS enzyme ac-tivity occurs specifically at the micropylarend of a globular stage embryo containing aKii3/GUS reporter gene (Fig. 3, F and G)(47). No GUS activity is observed withinthe suspensor or other embryo-proper re-

gions (Fig. 3G). This result suggests that thepreferential localization of Kti3 mRNA atthe micropylar end of a soybean globularstage embryo (Fig. 3C) is due to transcrip-tional regulatory processes. By contrast,GUS activity is visible as a uniform blue beltthat surrounds the equator region of a glob-ular embryo containing a soybean lectin/GUS reporter gene (Fig. 3H) (48). GUSactivity is not visible at either the micropy-lar or the chalazal (apical) ends of the em-bryo proper (Fig. 3H) (48); nor is there

Fig. 3. Differential transcriptional activity during early embryogenesis. (A and B) Bright-field photographsof developing soybean embryos sectioned longitudinally (40). (A) Late globular stage embryo. (B) Heartstage embryo. (C to E) Localization of the major Kunitz trypsin inhibitor mRNA (Kti3) in longitudinalsections of soybean embryos (40, 45). In situ hybridization data are similar to those shown in (40).Photographs were taken by dark-field microscopy. (C) Late globular stage embryo. (D) Late globular totransition stage embryo. (E) Heart stage embryo. (F) Bright-field photograph of a tobacco globular stageembryo sectioned longitudinally. (G and H) Localization of GUS activity within transgenic tobaccoglobular stage embryos. Photographs of whole-mount embryos were taken by bright-field microscopy.(G) Embryo contains a chimeric Kti3/GUS gene (47). (H) Embryo contains a chimeric lectin/GUS gene(48). (I) Localization of GUS mRNA within a transgenic tobacco globular stage embryo similar to thatshown in (H) containing a lectin/GUS gene (48). Abbreviations: EP, embryo proper; S, suspensor; C,cotyledon; A, axis; En, endosperm; CE, chalazal end; MPE, micropylar end; and Pd, protoderm.

WT seed WT seedling emb 30/gnom monopteros gurke fackel

H SM SM SM

(E)~ SMc.~~~~Complete Complete 0 Basal * Basal

Fig. 4. Schematic representations of Arabidopsis pattern mutants [adapted from (9)]. The green, yellow,and orange colors delineate the apical, central, and basal regions, respectively. Strong (upper) and weak(lower) gnom phenotypes are depicted (9, 15). Abbreviations: WT, wild-type; RM, root meristem; SM,shoot meristem; C, cotyledon; h, hypocotyl; and R, root.


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detectable GUS activity within the suspen-sor (Fig. 3H) (48). GUS mRNA localiza-tion studies with longitudinal globular em-bryo sections indicated that the lectin pro-moter activity occurs specifically within theground meristem cell layer of the equatorregion (Fig. 31) (48).

These experiments indicate that boththe longitudinal apical-basal and radial tis-sue-type axes of a globular embryo are par-titioned into discrete transcriptional terri-tories (I). The longitudinal axis of the em-bryo proper contains at least three nonover-lapping transcriptional territories: (i) thechalazal region, (ii) the equator region, and(iii) the micropylar region (Fig. 3, F to H).The suspensor represents an additionaltranscriptional domain along this axis (Fig.3, G and H). Each tissue layer of the radialembryo-proper axis also has a distinct tran-scriptional program (Fig. 3, E, G, and I).Transcriptional activity within these layers,however, appears to be established in aterritory-specific manner-that is, groundmeristem cells within the equator regionactivate promoters distinct from those with-in ground meristem cells of the micropylarregion, and vice versa (Fig. 3, E to I) (47,

Table 2. Examples of Arabidopsis mutants thathave defects in embryo development. Severalhundred Arabidopsis embryo-defective mutantshave been identified by both chemical and T-DNAmutagenesis. Most of these mutants can be ob-tained from the Arabidopsis Biological ResourceCenter (arabidopsis+ @osu.edu).

Mutant class References

Pattern mutantsemb3O/gnommonopterosgurkefackel

8-11, 159, 129

Cell-type differentiation mutantskeule 9knolle 9

Suspensor transformation mutantstwin 73sus1 67sus2 67sus3 67raspberryl 44raspberry2 44

Meristem differentiation and identity mutantsshoot meristemless 36embryonic flower 60short-root 61, 62hobbit 61, 62

Maturation program mutantslecl-/lec1-2 44, 76-78lec2 77fus3 79, 80abi3 84, 85

Seedling lethality mutantsfus 1/cop 1fus2/det1fus6/cop 1 1fus7/cop9

91, 9491, 9591, 9691, 97

48). These results suggest that a prepatternof different transcriptional regulatory do-mains has been established in the globularembryo before the morphogenetic eventsthat lead to differentiation of cotyledon andaxis regions at the heart stage (Figs. 2 and 3,A and B). Presumably, each transcriptionaldomain sets in motion a cascade of eventsleading to the differentiation of specificembryo regions later in embryogenesis.

Hormones Affect EmbryoMorphogenesis

What physiological events cause the em-bryo proper to initiate cotyledons and be-come bilaterally symmetric during the glob-ular-heart transition phase (Fig. 2)? Severalexperiments implicate a class of plant hor-mones, the auxins, in this morphogeneticprocess (49-52). Auxins, such as indoleace-tic acid (IAA), are involved in a number ofplant activities, including photo- and grav-itropism, apical dominance, and vascularcell differentiation (2). Embryos in plants asdiverse as bean and pine synthesize auxins(49) and transport them in a polarized,basipetal direction from the shoot meristemto root tip along the embryonic axis (Fig. 2)(50). The highest auxin levels occur at theglobular stage of embryogenesis (49).Agents that inhibit polarized auxin trans-port either block the transition from theglobular to heart stage completely (51) orprevent the bilateral initiation of cotyle-dons at the top of the globular embryo (Fig.2) (52). For example, auxin transport inhib-itors cause carrot somatic embryos to re-main spherically shaped and develop intogiant globular embryos (51). By contrast,zygotic embryos of the Indian mustard(Brassicajuncea), an Arabidopsis relative, failto initiate two laterally positioned cotyle-dons when treated with auxin transportblockers in culture (52). A cotyledon-likeorgan does form, but as a collar-like ringaround the entire upper (apical) region ofthe embryo (52). Treated Indian mustardembryos resemble those of the Arabidopsispin] -1 mutant, which has a defect in polar-ized auxin transport (52, 53). These resultssuggest that auxin asymmetries are estab-lished within the embryo-proper region ofglobular stage embryos (Fig. 3, A and F) andthat these asymmetries contribute to theestablishment of bilateral symmetry at theheart stage (Figs. 2 and 3, A and B).

Plant Embryos Form fromRegions That Develop

Autonomously

The longitudinal axis of a mature plantembryo is made up of several regions thatare designated as apical, central, and basal(Fig. 4) (9). The apical region contains the


cotyledons and shoot meristem, the centralregion consists of the hypocotyl, or upperaxis, and the basal region includes the loweraxis, or radicle, and the root meristem (Fig.4). These regions are derived ultimatelyfrom the terminal and basal cell lineagesand are maintained in the young seedling(Figs. 2 and 4). Can regions along the lon-gitudinal axis develop independently ofeach other? If territories established withinthe embryo proper of a globular stage em-bryo are specified autonomously, then theloss or alteration of cells within a territoryshould not affect the development of a con-tiguous region (Figs. 2 and 3). Several ex-periments suggest that this is actually thecase that is, a plant embryo forms frommodules that develop independently ofeach other (9-12, 54-59).

Pattern mutations delete specific embryonicregions. Genetic studies have uncoveredArabidopsis mutations that alter the organi-zation of the embryo body plan (Fig. 4 andTable 2) (9, 10). Embyro pattern mutationswere found that delete (i) the apical region(gurke), (ii) the central region (fackel), (iii)the central and basal regions (monopteros),and (iv) the apical and basal regions(emb30/gnom) (Fig. 4) (9). Other embryopattern-forming genes probably exist; how-ever, they have not been identified in thegenetic screens carried out thus far (6,8-10, 14, 44). Each mutant gene acts zy-gotically, indicating that major specifiers ofthe embryo body plan act after fertilizationhas occurred (Fig. 2) (9, 10). In addition,the loss of a specific region, or combinationof regions, does not affect the developmentof an adjacent neighbor (Fig. 4) (9, 10). Forexample, gurke embryos lack an apical re-gion, but have normal central and basalregions (Fig. 4) (9). monopteros embryos, bycontrast, have a normal apical region butare missing the central and basal regions(Fig. 4) (9).

Embryo pattern mutations alter the di-vision planes that are established during thepostfertilization-proembryo phase of embry-ogenesis (Fig. 2) (11, 12). Thus, deletionsof mature embryo regions can be tracedback to histological defects at the proem-bryo and globular stages (Fig. 2) (11,12) a result that provides functional evi-dence for the embryo fate maps proposed bythe developmental botanists of the late19th and early 20th centuries (31, 32). Forexample, emb30/grnom zygotes do not divideasymmetrically (Fig. 2) (1 1). Two similar-sized daughter cells are produced in theemb30/gnom embryo-proper region insteadof the unequal-sized terminal and basal cellsthat are found in wild-type embryos (11).Later division events in emb30/gnom embry-os are also highly variable and abnormal(11). By contrast, the monopteros divisionpattern is normal until the 8-cell embryo-

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proper stage (Fig. 2) (12). During themonopteros globular-heart transition phase,lower tier cells of the embryo proper andderivatives of the hypophysis undergo ab-normal divisions (12) (Fig. 2). monopterosupper tier embryo-proper cells, however,develop normally that is, defects at thebasal end of the embryo proper do not affectevents that occur at the apical end (Fig. 2)(12). The result is a mutant embryo that hascotyledons and a shoot meristem, but ismissing the hypocotyl and root regions (Fig.4) (12). These observations indicate thatgenes which are responsible, in part, for theestablishment of the embryo body plan di-rect territory-specific cell division patternsduring early embryogenesis. Abnormal divi-sions in one embryonic territory do notaffect the division pattern of an adjacentterritory that is, cell lineages giving rise tospecific regions of the mature embryo de-velop autonomously.

Agrobacterium T-DNA-tagged emb30/gnom alleles have led to the isolation of theEMB30/GNOM gene (15). This gene en-codes a protein that is related to the yeastSec7 secretory protein, is active throughoutthe plant life cycle, and is involved in celldivision, elongation, and adhesion eventsrequired at many stages of sporophytic de-velopment, including embryogenesis (15).Thus, EMB30/GNOM does not appear toestablish the embryonic cell division pat-tern directly, but most likely facilitates apattern set by other genes. What these pat-tern-forming genes are and how they inter-act with downstream genes that mediateevents required for the differentiation ofautonomous regions along the longitudinalaxis remain to be determined.

Mutations affect meristematic zones of theembryo longitudinal axis. Arabidopsis muta-tions have been identified that affect thedifferentiation of the shoot and root meri-stems during embryogenesis (Table 2) (36,60-62). These mutations target a specificmeristem and have no other effect on em-bryonic development. For example, shootmeristemless fails to differentiate a shootmeristem during embryogenesis and pro-duces seedlings without leaves (36). embry-onic flower, on the other hand, generates ashoot meristem at the top of the embryonicaxis (Figs. 2 and 4) (54). Remarkably, em-bryonic flower seedlings produce flowersrather than leaves, indicating that the fateof the shoot meristem is altered during em-bryogenesis a floral meristem is specifiedrather than a vegetative shoot meristem(60). By contrast, short root and hobbit affectroot meristem development (61, 62). Thesemutations lead to abnormal root develop-ment after seed germination, indicatingthat the root meristem is altered, but noteliminated, during embryogenesis (61, 62 ).Taken together, these results indicate that

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there are genes which regulate the specifi-cation, organization, and fate of meristemsthat differentiate during embryogenesis.Genes controlling meristem developmentmost likely act downstream of embryo-re-gion specifiers, such as monopteros and gurke(Fig. 4) (9, 10, 12), in the regulatory hier-archy needed to form a plant embryo.

Meristem mutants characterized to dateindicate that the shoot and root meristemsfunction autonomously that is, they donot affect the differentiation of contiguousdomains such as the cotyledons or hypo-cotyl (36, 60-62). Meristems, therefore,represent independent submodules withinthe apical and basal regions of the embryo(Fig. 4). A major question is what effect, ifany, do cells adjacent to the shoot and rootmeristems have on their development?That is, are cell signaling events involved inspecifying the root and shoot meristemswithin a specific embryonic region, or dothe meristems differentiate autonomously?The laterne mutant provides one answer tothis question (9). laterne seedlings lackcotyledons but produce leaves indicatingthat a shoot meristem is present (9). Thus,cotyledons are not required for the differ-entiation of the shoot meristem durinoembryogenesis.

Promoter elements interpret embryo region-specific regulatory networks. One consequenceof the modular organization of a plant em-bryo is that genes which are active through-out the embryo must intersect with severalregion-specific regulatory networks that is.the promoters of embryo-specific genes arerequired to sense and interpret the transcrip-tional regulatory machinery unique to eachautonomous region (Fig. 4). For example,Kti3 mRNA accumulates within the axisregion early in soybean embryogenesis, butdoes not accumulate within the cotyledonsuntil much later (Fig. 3, C to E) (40). Thus,discrete promoter elements should exist thatare responsible for interacting with transcrip-tion factors produced by separate regulatorycircuits.A Kti3/GUS gene with 2 kb of 5' flank-

ing sequence is transcribed in all regions of amature transgenic tobacco embryo (Fig. 5A)(54). Deletion of 0.2 kb from the 5' endeliminates Kti3/GUS transcriptional activitywithin the embryo radicle region (Fig. 5B)(54). Deletion of another 1 kb eliminatesKti3/GUS transcription within the cotyle-dons and shoot meristem, but still permitstranscription to occur within the hypocotylregion (Fig. 5C) (54). These results indicatethat discrete cis-acting domains are requiredfor the transcriptional activation of the Kti3gene within the radicle, hypocotyl, and cot-yledon-shoot meristem regions of the em-bryo. Promoter analysis of the soybean Gy 1storage protein gene (63) also uncovered aregulatory domain that directs transcription


to the cotyledons and shoot meristem of a

transgenic tobacco embryo (Fig. 5D) (54).These data, and those of others (55-59),indicate that unique transcription factors are

active within each embryonic region andthat these factors interact with specific pro-moter elements. The combination of theseelements and factors gives rise to the tran-

scriptional pattern of the whole embryo(Fig. 5A). Identification of transcriptionfactors that interact with region-specificDNA elements should provide reverse entryinto the independent regulatory networksrequired for specifying each autonomnous re-

gion of a plant embryo.

Cell Differentiation andMorphogenesis Can Be

Uncoupled in Plant EmbryosWhat is the relation between cell differen-tiation and morphogenesis in plant embry-os? Are processes required for tissue differ-entiation along the embryo radial axis cou-pled to those that specify autonomous re-

Fig. 5. DNA elements program transcription tospecific embryonic regions. Maturation stage em-bryos were hand-dissected from transgenic to~bacco seeds containing the E. col/ GUS genefused with different soybean seed protein gene 5'regions (54). GUS activity in whole-mount embry-os was localized as outlined in (47), and photo-graphs were taken with the use of dark-field mi-croscopy. (A to C) Kti3 gene upstream regionsfused with the E coli GUS gene. (A) A 2-kb KtO3gene 5' fragment. (B) A 1 .7-kb Kfi3 gene 5' frag-ment. (C) A 0.8-kb Kfi3 gene 5' CaMVIGUS frag-ment. (D) A minimal CaMVIGUS gene-promotercassette (59) fused with a 0.36-kb Gyl gene 5'region (-446 to -84) (63). The white embryo re-sults from the segregation of a single GylIGUSgene within this transgenic tobacco line and rep-resents a negative control. Abbreviations: C, cot-yledon; H, hypocotyl: and Rd, radicle.

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gions of the longitudinal apical-basal axis(Fig. 2)? Studies of Arabidopsis embryo pat-tern mutants suggest that these processesare not necessarily interconnected. For ex-ample, a fackel embryo does not have ahypocotyl, but epidermal, ground meristem,and vascular tissues differentiate within cot-yledon and radicle regions (Fig. 4) (9).Thus, the loss of one embryonic region doesnot affect the formation of tissue layerswithin the remaining regions (Fig. 4) (9-12). A more direct question, however, iswhether mutant embryos that arrest early inembryonic development and remain globu-lar-shaped differentiate the specialized celland tissue layers that are found in organsystems of a mature, wild-type embryo.A maturation stage Arabidopsis embryo

has specialized epidermal, storage parenchy-ma, and vascular cell layers within both thecotyledon and axis regions (Fig. 6A). Thesetissues are derived from the three primarycell layers that are specified along the radialaxis of a globular embryo (Fig. 2), and ex-press specific marker genes late in embryo-

genesis. For example, EP2 lipid transfer pro-tein mRNA accumulates specifically withinthe epidermal cell layer (Fig. 6B) (46, 64,65), and 2S2 albumin mRNA accumulateswithin storage parenchyma cells (Fig. 6C)(64, 66). Neither mRNA is detectablewithin the vascular layer (Fig. 6, B and C)(46, 64-66). Collectively, the EP2 and 2S2mRNAs can identify embryo epidermal andstorage parenchyma cell layers and, by de-fault, the inner vascular tissue as well (Fig.6, B and C).An Arabidopsis embryo mutant, desig-

nated raspberry1, was identified in a screenof T-DNA-mutagenized Arabidopsis lines(Table 2) (44). This mutant fails to undergothe globular-heart transition (Fig. 2), has anembryo-proper region that remains globu-lar-shaped throughout embryogenesis, anddoes not differentiate cotyledons and axis(Fig. 6, D and E) (64). raspberry1 embryosalso have an enlarged suspensor region (Fig.6, D and E) (64). raspberry2 (44) and sus(67) embryo-defective mutants also havephenotypes similar to that of raspberry 1

i.1

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Fig. 6. Localization of cell-specific mRNAs within wild-type and mutant Arabidopsis embryos. (A)Bright-field photograph of a late-maturation stage Arabidopsis embryo sectioned longitudinally (64). (Band C) In situ hybridization of labeled RNA probes with longitudinal sections of Arabidopsis maturationstage embryos (64). Photographs taken by dark-field microscopy. (B) Localization of Arabidopsis EP2lipid transfer protein mRNA (65). (C) Localization of 2S2 albumin mRNA (66). (D and E) Arabidopsisraspberryl embryos (44). Embryos were harvested at a stage when wild-type embryos within the samesilique were in late maturation [as in (A)]. (D) Longitudinal section of a raspberryl embryo. The photographwas taken by bright-field microscopy. (E) A raspberryl embryo photographed with Nomarski interferenceoptics. (F to 1). In situ hybridization of labeled RNA probes with raspberryl embryo longitudinal sections(64). (F and G) Localization of EP2 lipid transfer protein mRNA (65). Localization experiments withraspberry2 embryos, which have larger suspensors than raspberryl embryos (44), confirmed that EP2mRNA is present only in the suspensor outer cell layer (64). (H and 1) Localization of 2S2 albumin mRNA(66). In situ hybridization with serial sections through an entire raspberryl embryo confirmed that there isan inner core of cells with no detectable 2S2 mRNA (64). Abbreviations: A, axis; V, vascular tissue; Ed,epidermis; C, cotyledon; P, storage parenchyma; Ep, embryo proper; S, suspensor; and En, endosperm.


N1. .WB.

(Table 2). Surprisingly, raspberry) embryosaccumulate EP2 and 2S2 marker mRNAs intheir correct spatial context along the radialaxis of both the embryo-proper and suspen-sor regions (Fig. 6, F to I) (64). EP2 mRNAaccumulates along the outer perimeter ofraspberry] embryos (Fig. 6, F and G), where-as 2S2 rnRNA accumulates within interiorcells (Fig. 6, H and I) (64). By contrast, EP2and 2S2 mRNAs do not accumulate detect-ably within the central core of raspberry 1embryos (Fig. 6, F to I) (64). Similar resultswere obtained with raspberry2 embryos (Ta-ble 2) (44, 64).

These mRNA localization studies indi-cate that specialized tissues can differentiatewithin the embryo-proper region of mutantembryos that remain globular shaped, andthat these tissues form in their correct spatialcontexts. A similar conclusion was inferredfrom histological studies of the sus mutants(67). Tissue differentiation, therefore, cantake place independently of morphogenesisin a higher plant embryo, implying that mor-phogenetic checkpoints do not occur beforecell differentiation events can proceed (68).It does not follow, however, that morpho-genesis can occur without proper cell differ-entiation events. Arabidopsis embryo mu-tants that alter tissue-specification patternshave abnormal morphologies (Table 2) (9).For example, knolle embryos lack an epider-mal cell layer and have a round, ball-likeshape without defined apical and basal re-gions (Table 2) (9). Similarly, the carrot tsl 1somatic embryo mutant has a defective pro-toderm cell layer and fails to undergo mor-phogenesis (38, 39). Addition of either anextracellular chitinase (69) or Rhizobiumnodulation factors (lipooligosaccharides)(70) can rescue the ts 11 mutant. Lipooligo-saccharide nodulation factors have beenshown to be signal molecules involved in thedifferentiation of Rhizobium-induced rootnodules (70). This suggests that in carrotsomatic embryos, the protoderm cell layermay provide signals necessary for embryo-genesis to occur (69, 70). Taken together,experiments with mutant embryos that havedefective cell layers suggest that specificationof the radial axis needs to occur in order fora normal shoot-root axis to form. An impor-tant corollary is that cells within the radialaxis probably interact with each other (9).

Suspensor Cells Have thePotential to Generate an Embryo

One intriguing aspect of the raspberry1 em-bryo is its large suspensor (Fig. 6, D and E).raspberry 1 suspensors are indistinguishablefrom wild-type during the early stages ofembryogenesis (Fig. 2) (64). Later in seeddevelopment, when neighboring wild-typeembryos undergo maturation, cell prolifera-tion events cause the raspberry 1 suspensor

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to enlarge at its basal end (Fig. 6, D and E)(64). EP2 and 2S2 mRNAs (46, 65, 66)accumulate in the raspberry1 suspensor (Fig.6, F to I) with a spatial pattern similar tothat which occurs in mature, wild-type em-bryos (Fig. 6, B and C) (64). These cell-specific mRNAs do not accumulate detect-ably in wild-type suspensors, or in raspberry 1suspensors early in embryogenesis (64).These results indicate that the raspberry)suspensor has entered an embryogenic path-way and that an embryo proper-like, radialtissue axis has been specified.

Other Arabidopsis embryo mutants havesuspensor abnormalities similar to that ofraspberry ), including raspberry2, and sus(Table 2) (14, 35, 44, 67, 71). Althoughthe extent of suspensor enlargement varies,all of these mutants have morphologicaldefects in the embryo proper (14, 35, 44,67, 71). Mutant embryos that resemblewild-type, but arrest at specific embryonicstages, do not have aberrant suspensors (44,64). Disruptions in embryo-proper morpho-genesis, therefore, can induce an embryoproper-like pathway in terminally differen-tiated suspensor cells, a result first observedby the embryo-proper ablation experimentsof Haccius 30 years ago (72). The Arabidop-sis twin mutant represents a striking exam-ple of the embryogenic potential of thesuspensor region (73). twin causes subtledefects to occur in embryo-proper morphol-ogy, generates a second embryo within theseed from proliferating suspensor cells, andresults in twin embryos that are connectedby a suspensor cell bridge (73).

The nature of mutant genes that affectsuspensor development, such as raspberry1,sus, and twin, is not known. These genes areprobably not involved in suspensor specifi-cation events, because a normal suspensorforms before induction of the embryo-prop-er pathway in mutant embryos (44, 64, 67,71, 73). They reveal, however, that inter-actions occur between the suspensor andembryo-proper regions. One possibility isthat the embryo proper transmits specificinhibitory signals to the suspensor that sup-press the embryonic pathway (35, 67, 71-73). Alternatively, a balance of growth reg-ulators might be established within the en-tire embryo that maintains the develop-mental states of both the embryo-properand suspensor regions. Disruptions of suchsignals would cause the suspensor to take onan embryo proper-like fate, a result analo-gous to embryo induction in differentiatedsporophytic or gametophytic cells (37-39).

The Embryo Is ReprogrammedLate in Embryogenesis

How does the embryo prepare for dormancyand postembryonic development (Fig. 1)?Late in embryogenesis a maturation pro-

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gram is induced that is responsible for (i)synthesizing large amounts of storage prod-ucts, (ii) inducing water loss and a desiccat-ed state, (iii) preventing premature germi-nation, and (iv) establishing a state of dor-mancy (Fig. 2 and Table 1) (2, 26-28).Several specialized gene sets, such as thoseencoding storage proteins and late embryoabundant (lea) proteins, are activated tran-scriptionally during maturation and then re-

pressed before dormancy (26, 74, 75). Thesegene sets remain transcriptionally quiescentduring seed germination when germination-specific and postembryonic gene sets are

transcribed (26, 55, 75). Genetic studieswith Arabidopsis have identified some of thegenes that regulate processes required forembryo maturation and dormancy, includ-ing the expression of storage protein and leagenes (Table 2).

Leafy cotyledon mutations disrupt embryomaturation. A mutant gene class, designatedas leafy cotyledon (lec), has been identifiedthat causes defects in the cotyledon celldifferentiation process and in maturation-specific events such as storage product accu-

mulation, desiccation tolerance, and main-tenance of dormancy (Table 2) (76-80). iecmutations transform cotyledons of embryosand seedlings into leaf-like structures (76-80). Wild-type cotyledons do not havetrichomes (Fig. 7A). Trichomes are presentonly on leaf, stem, and sepal surfaces inwild-type plants and are markers forpostembryonic development (81). By con-

trast, led cotyledons have trichomes on

their adaxial surface which differentiateduring embryogenesis from the protodermcell layer (Fig. 7B) (76-78). lecl cotyle-dons have other leaf-like characteristicsincluding stomata, mesophyll cells, and an

absence of protein and lipid storage bodies(77). The axis region of led embryos alsolacks storage organelles, indicating thatthe wild-type LEC 1 gene functions inboth embryonic organs (77).

In addition to the leaf-like cotyledontransformation, lec embryos (i) germinateprecociously about 5% of the time, (ii) haveleaf primordia emerging from their shootapex, and (iii) fail to survive desiccation


Fig. 7. An Arabidopsis leafy cotyle-don seedling. An allele of the leclgene (76, 77), designated as lec1-2,was identified in a screen ofT-DNA-mutagenized Arabidopsis lines (44,78). (A and B) Bright-field photo-graphs of Arabidopsis seedlings(78). (A) Wild-type seedling. (B)lecl-2 seedling. Abbreviations: C,cotyledon; H, hypocotyl; R, root;and Tr, trichome.

(76-78). Other lec mutants, such as lec2and fus3, have phenotypic characteristicsthat overlap those of led (77-80). For ex-ample, fus3 and lec embryos are almostindistinguishable from each other (77). lec2embryos, by contrast, have leaf-like cotyle-dons, but are desiccation tolerant, do notgerminate precociously, and have normallevels of storage bodies in their axis region(77). This indicates that leafy cotyledonscan occur without corresponding defects indesiccation and dormancy, and that wild-type LEC genes are activated independent-ly in each embryonic organ system (77). Acorollary is that gene networks that func-tion in desiccation and dormancy are inde-pendent of those responsible for cotyledoncell specialization.

Molecular studies with lec embryos haveindicated that the transcription of matura-tion-specific genes, such as those encodingstorage proteins, is greatly reduced (80).Conversely, the transcription of germina-tion-specific genes, such as isocitrate lyase, isactivated (78). These data, coupled with thehistological descriptions of lec embryos (Fig.7) (76-80), indicate that LEC genes func-tion during maturation to activate genes in-volved in cotyledon cell specialization, stor-age product accumulation, induction andmaintenance of embryonic dormancy, anddesiccation tolerance (76-80). LEC genesalso simultaneously suppress the manifesta-tion of leaf-like characteristics in cotyledonsduring embryogenesis, including trichomespecification. In the absence of LEC prod-ucts, embryonic cotyledons enter a defaultstate and express leaf-like characteristics(76, 77), many of which develop normally inpostgermination, wild-type cotyledons (82).

Abscisic acid maintains embryonic dorman-cy. The plant hormone abscisic acid (ABA)is involved in several plant processes, in-cluding senescence, responses to environ-mental stresses, growth inhibition, andmaintenance of a dormant state (83). Ex-ogenous ABA prevents seed germination aswell as the precocious germination of em-bryos in culture. In addition, Arabidopsismutants that either cannot synthesize ABAor fail to respond to ABA germinate preco-

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ciously (84, 85). These data indicate thatABA prevents germination while seeds arestill dormant or present within siliques.

Three mutant Arabidopsis loci, designat-ed as abil, abi2, and abi3, have been iden-tified that result in ABA insensitivity andallow seed germination to occur in the pres-ence of ABA (84). In addition to preco-cious germination, abi3 embryos are alsodesiccation intolerant and defective in thesynthesis of maturation-specific mRNAs,such as those encoding storage proteins andlea proteins (85, 86). This indicates thatthe wild-type ABI3 gene is a positive regu-lator of gene networks leading to storageproduct accumulation, desiccation, anddormancy (85, 86). The ABI3 gene encodesa transcription factor (87) related to thecorn viviparous-1 protein, which can acti-vate the transcription of chimeric GUS re-porter genes containing embryo matura-tion-specific gene promoters (88). Thus,ABI3 mediates its effect on embryo matu-ration at the transcriptional level. Becauseaba embryos, which fail to synthesizeABA, are normal with respect to mostmaturation-specific processes (84 -86),ABA probably does not regulate the ABI3gene (84, 86). Rather, ABI3 probably op-erates through an ABA-independentpathway that is involved in establishingdesiccation and dormancy states late inembryogenesis (85, 86). Failure to achievethese developmental states results in ABAinsensitivity-that is, ABA responsive-ness is a consequence of ABI3 gene activ-ity (85, 86). lec embryos are sensitive toABA and fail to germinate if ABA ispresent (77, 78). Thus, LEC and ABI3genes are part of independent regulatorynetworks that control embryo maturation-specific events.

In contrast to abi3 embryos, abi 1 and abi2embryos carry out normal maturation-specif-ic events, including the activation of storageprotein and lea genes (84-86). ABIJ andABI2 loci, therefore, are involved only inmaintaining embryonic dormancy. TheABI 1 gene encodes a Ca2+_-dependent phos-phatase with similarity to serine-threoninephosphatases involved in signal transduc-tion processes (89). In response to ABA, theABI 1 phosphatase might counteract phos-phorylation events required for the initia-tion of root meristem cell division, resultingin a dormant embryonic state (89).

Regulatory loci required for postembryonicdevelopment are active late in embryogenesis.A large number of Arabidopsis fusca mutantshave been identified that accumulate an-thocyanins, or red pigments, on their coty-ledons late in embryogenesis (10, 90-92).With the exception of fus3 (Table 2), em-bryogenesis is normal in fusca mutants (10,90-92). After germination, however, fuscaseedlings fail to develop into mature flow-

ering plants (10, 90-92). Several fuscagenes have been shown to be alleles ofconstitutive photomorphogenic (cop)/deeti-olated (det) genes that function in light-regulated development during seed germi-nation (Table 2) (91-97). The products ofCOP/DET loci appear to suppress light-regulated gene activities in the dark andactivate these activities in the presence oflight by way of a light-mediated signaltransduction pathway (91-97). Because de-fective cop/det genes are detected as fuscaembryo mutants, their wild-type COP/DETalleles are active during maturation. Thus,regulatory genes expressed at the end ofembryogenesis prepare the plant for life af-ter the seed.

Conclusion

Plant embryogenesis provides a vital bridgebetween the gametophytic generation andpostembryonic differentiation events thatoccur continuously in the shoot and rootmeristems of the sporophytic plant. Assuch, plant embryos must establish the po-larized sporophytic plant body plan and en-able the young plant to survive harsh envi-ronmental conditions and a period of be-low-ground growth from seeds. Plant em-bryos are simpler than their animalcounterparts, yet they must carry out thesame developmental tasks-that is, form athree-dimensional organism with special-ized regions, compartments, and cell-typesfrom a single-celled zygote. These eventsoccur early in plant embryogenesis and arepoorly understood. Genetic studies in Ara-bidopsis have begun to reveal genes that arenecessary for embryogenic events such aspattern formation, cell differentiation, andorgan development.

The precise molecular mechanisms re-sponsible for specifying different cell lin-eages early in plant embryogenesis are notknown. A major void in our knowledge con-cerns the events that occur within the eggcell and in the early embryo after fertiliza-tion. In this respect it is crucial to obtainmolecular markers in order to follow thespecification events that take place duringearly embryogenesis and gain entry into reg-ulatory networks that are activated in differ-ent embryonic regions after fertilization. Al-though a large amount of progress has beenmade in recent years in understanding how aplant embryo forms, there is still a long wayto go. The next few years should be a veryexciting time to study plant embryos.

REFERENCES AND NOTES

1. E. H. Davidson, Development 105, 421 (1989); ibid.108, 365 (1990); ibid. 113, 1 (1991); ibid. 118, 665(1993).

2. K. Esau, Anatomy of Seed Plants (John Wiley, NewYork, 1977); P. H. Raven, R. F. Evert, S. E. Eichhorn,


Biology of Plants (Worth, New York, 1992).3. L. Reiser and R. L. Fischer, Plant Cell 5, 1291 (1993).4. C. S. Gasser and K. Robinson-Beers, ibid., p. 1231.5. E. Kranz and H. Lbrz, ibid., p.739; J.-E. Faure, H. L.

Mogensen, C. Dumas, H. Lbrz, E. Kranz, ibid., p.747; C. Dumas and H. L. Mogensen, ibid., p. 1337;J.-E. Faure, C. Digonnet, C. Dumas, Science 263,1598 (1994).

6. D. W. Meinke, Dev. Genet. 12, 382 (1991); Plant Cell3, 857 (1991).

7. W. F. Sheridan, Annu. Rev. Genet. 22, 353 (1988); J.K. Clark and W. F. Sheridan, Plant Cell 3, 935 (1991).

8. D. W. Meinke and 1. M. Sussex, Dev. Biol. 72, 50(1979); ibid., p. 62; D. W. Meinke, Theor. Appl. Gen-et. 69, 543 (1985).

9. U. Mayer, R. A. Torres-Ruiz, T. Berlath, S. Misera, G.Jurgens, Nature 353, 402 (1991); in Cellular Com-munication in Plants, R. M. Amasino, Ed. (Plenum,New York, 1993), p. 93.

10. G. JOrgens, U. Mayer, R. A. Torres-Ruiz, T. Berlath,S. Misera, Development 1 (suppl.), 27 (1991).

11. U. Mayer, G. Bbttner, G. Jbrgens, Development 117,149 (1993).

12. T. Berlath and G. Jdrgens, ibid. 118, 575 (1993).13. K. A. Feldmann, Plant J. 1, 71 (1991); N. R.

Forsthoefel, Y. Wu, B. Schulz, M. J. Bennett, K. A.Feldmann, Aust. J. Plant Physiol. 19, 353 (1992).

14. D. Errampalli et al., Plant Cell 3, 149 (1991); L. A.Castle et al., Mol. Gen. Genet. 241, 504 (1993).

15. D. E. Shevell et al., Cell 77, 1051 (1994).16. R. B. Goldberg, Science 240, 1460 (1988).17. V. Walbot, Trends Genet. 1, 165 (1985).18. R. Chasan and V. Walbot, Plant Cell 5,1139 (1993).19. S. McCormick, ibid., p. 1265.20. J. P. Mascarenhas, ibid., p. 1303.21. R. B. Goldberg, T. P. Beals, P. M. Sanders, ibid., p.

1217.22. S. D. Russell, ibid., p. 1349.23. M. A. Lopes and B. A. Larkins, ibid., p. 1383.24. G. Gillaspy, H. Ben-David, W. Gruissen, ibid., p.

1439.25. E. S. Coen and R. Carpenter, ibid., p. 1175; J. K.

Okamuro, B. G. W. den Boer, K. D. Jofuku, ibid., p.1183.

26. R. B. Goldberg, S. Barker, L. Perez-Grau, Cell 56,149 (1989).

27. K. Lindsey and J. F. Topping, J. Exp. Bot. 44, 359(1993).

28. M. A. L. West and J. J. Harada, Plant Cell 5, 1361(1993).

29. V. Raghavan, Experimental Embryogenesis in Vas-cular Plants (Academic Press, New York, 1976); S.Natesh and M. A. Rau, in Embryology of Angio-sperms, B. M. Johri, Ed. (Springer-Verlag, Berlin,1984), chap. 8; B. M. Johri, K. B. Ambegaokar, P. S.Srivastava, Comparative Embryology of Angio-sperms (Springer-Verlag, Berlin, 1992).

30. M. Schaffner, Ohio Nat. 7, 1 (1906); R. Schulz andW. A. Jensen, J. Ultrastruct. Res. 22, 376 (1968);Am. J. Bot. 55,541 (1968); ibid., p. 807; ibid., p. 915.

31. J. Hanstein, Bot. Abhadl., Bonn. 1, 1 (1870).32. E. C. Soueges, Ann. Sci. Nat. 9, Bot. 19, 311 (1914);

Ann. Sci. Nat. 10, Bot. 1,1 (1919).33. S. G. Mansfield, L. G. Briarty, S. Erni, Can. J. Bot. 69,

447 (1991); S. G. Mansfield and L. G. Briarty, ibid., p.461; ibid. 70, 151 (1992).

34. L. Dolan et al., Development 1 1 9, 71 (1 993).35. E. C. Yeung and D. W. Meinke, Plant Cell 5, 1371

(1993).36. M. K. Barton and R. S. Poethig, ibid., p. 823.37. J. L. Zimmerman, ibid., p. 141 1.38. F. A. Van Engelen and S. C. de Vries, Trends Genet.

8, 66 (1992).39. A. J. de Jong, E. D. L. Schmidt, S. C. de Vries, Plant

Mol. Biol. 22, 367 (1993).40. L. Perez-Grau and R. B. Goldberg, Plant Cell 1, 1095

(1989).41. R. L. Taylor, Can. J. Bot. 45, 1553 (1967).42. M. A. Chamberlin, H. T. Homer, R. G. Palmer, ibid.

71, 1153 (1993).43. J. P. Nitsch, Phytomorphology 19, 389 (1969); B.

Norreel, Bull. Soc. Bot. Fr. 117, 461 (1970); R. S.Sangwan and B. S. Sangwan-Norreel, Int. Rev. Cy-tol. 107, 221 (1987).

44. The Embryo 21st Century Group, unpublished re-

sults. This group is a collaborative effort between the

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laboratories of R. B. Goldberg [University of Califor-nia, Los Angeles (UCLA)], J. J. Harada (UC Davis), R.L. Fischer (UC Berkeley), J. L. Zimmerman (Universi-ty of Maryland, Baltimore), and A. Koltunow (Univer-sity of Adelaide, Australia). A total of 5822 T-DNA-mutagenized Arabidopsis lines (13) were screenedfor mutants defective in embryogenesis (6, 8, 14).Sixty-six heterozygous lines were identified that seg-regated seeds in a ratio of 3 wild-type to 1 mutantwithin their siliques, indicating that they containedembryo-defective alleles that were inherited as sim-ple Mendelian recessive genes. The raspberryl,raspberry2, and lecl-2 genes were uncovered inthree different heterozygous lines.

45. K. D. Jofuku, R. D. Schipper, R. B. Goldberg, PlantCell 1, 427 (1989); K. D. Jofuku and R. B. Goldberg,ibid., p. 1079.

46. P. Sterk, H. Booij, G. A. Schellekens, A. Van Kam-men, S. C. DeVries, ibid. 3, 907 (1991).

47. G. de Paiva and R. B. Goldberg, unpublished results.A 2-kb 5' region from the soybean Kti3 gene wasfused with the Escherichia coi GUS gene [R. A. Jef-ferson, T. A. Kavanagh, M. W. Swan, EMBO J. 6,3901 (1987)], and the resulting chimeric Kti3/GUSgene was transferred to tobacco plants byAgrobac-terium-mediated transformation as described (45).GUS activity in isolated whole-mount embryos wasanalyzed as outlined in G. N. Drews, T. P. Beals, A.Q. Bui, and R. B. Goldberg [Plant Cell 4, 1383(1992)].

48. R. Yadegari and R. B. Goldberg, unpublished re-sults. A 2-kb 5' region from the soybean lectin gene[R. B. Goldberg, G. Hoschek, L. 0. Vodkin, Cell 33,465 (1983)] was fused with the E. coli GUS gene andtransferred to tobacco plants as outlined in (47). Lo-calization of GUS activity in staged, whole-mountembryos was determined by the procedure outlinedin (47). In situ hybridization of a GUS antisense 35S_RNA probe with a longitudinal globular-stage em-bryo section was carried out according to the proce-dures outlined in (40) and in K. H. Cox and R. B.Goldberg [in Plant Molecular Biology:A PracticalAp-proach, C. H. Shaw, Ed. (IRL, Oxford, 1988), pp.1-35].

49. L. Michalczuk, T. J. Cooke, J. D. Cohen, Phyto-chemistry 31, 1097 (1992).

50. S. C. Fry and E. Wangermann, New Phytol. 77, 313(1976).

.51. F. M. Schiavone and T. J. Cooke, Cell Differ. 21, 53(1987).

52. C.-M. Liu, Z.-H. Xu, N.-H. Chua, ibid., p. 621.53. K. Okada, J. Ueda, M. K. Komaki, C. J. Bell, Y.

Shimura, Plant Cell 3, 677 (1991).54. G. de Paiva and R. B. Goldberg, unpublished results.

The chimeric Kti3IGUS genes contained the Kti3gene TATA-box region (- 28) (47). The chimeric GylIGUS gene contained the cauliflower mosaic virus(CaMV) TATA-box region within the CaMV minimalpromoter (-42 to +0) (59).

55. T. L. Thomas, Plant Cell 5,1401 (1993).56. A. N. Nunberg, et al., ibid. 6, 473 (1994).57. M. A. Bustos, D. Begum, F. A. Kalkan, M. J. Battraw,

T. C. Hall, EMBO J. 10, 1468 (1990).58. A. da Silva ConceigAo and E. Krebbers, Plant J. 5,

493 (1994).59. P. N. Benfey, L. Ren, N.-H. Chua, EMBO J. 9, 1677

(1990).60. Z. R. Sung, A. Belancheur, B. Shunong, R. Bertrand-

Garcia, Science 258,1645 (1992).61. P. N. Benfey et al., Development 119, 57 (1993).62. R. A. Aeschbacher, J. W. Schiefelbein, P. N. Benfey,

Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 25(1994).

63. N. G. Nielsen et al., Plant Cell 1, 313 (1989).64. The Embryo 21st Century Group (44), unpublished

results. Research on the raspberryl and raspberry2genes was carried out by R. Yadegari, G. de Paiva, T.Laux, and R. B. Goldberg (UCLA). Wild-type andraspberryl seeds were harvested from heterozy-gous +Iraspberryl Arabidopsis plants (44), fixed,embedded in paraffin, and hybridized with 35S-RNAantisense probes according to the procedures out-lined in (40, 48). raspberryl seeds were harvestedfrom siliques at a time when neighboring wild-typeseeds were in the maturation stage of development.

65. The Arabidopsis EP2 gene probe (46) was providedby S. DeVries.

66. P. Guerche et al., Plant Cell 2, 469 (1990). The Ara-bidopsis 2S2 gene probe was provided by E. Kreb-bers.

67. B. W. Schwartz, E. C. Yeung, D. W. Meinke, Devel-opment, in press.

68. R. Losick and L. Shapiro, Science 262,1227 (1993).69. A. J. DeJong et al., Plant Cell 4, 425 (1992).70. A. J. De Jong et al., ibid. 5, 615 (1993).71. M. P. F. Marsden and D. W. Meinke, Am. J. Bot. 72,

1801 (1985).72. B. Haccius, Phytomorphology 13,107 (1963).73. D. M. Vernon and D. W. Meinke, Dev. Biol., in press.74. L. Walling, G. N. Drews, R. B. Goldberg, Proc. Natl.

Acad. Sci. U.S.A. 83, 2123 (1986).75. L. Comai and J. J. Harada, ibid. 87, 2671 (1990).76. D. Meinke, Science 258,1647 (1992).

77. D. Meinke, L. H. Franzmann, T. C. Nickle, E. C.Yeung, Plant Cell 6, 1049 (1994).

78. The Embryo 21st Century Group (44), unpublisheddata. Research on the lecl-2 gene was carried outby M. West and J. J. Harada (UC Davis).

79. K. Keith, M. Krami, N. G. Dengler, P. McCourt, PlantCell 6, 589(1994).

80. H. Baumlein et al., Plant J., in press.81. M. Hulskamp, S. Mis6ra, G. JOrgens, Cell 76, 555

(1994).82. Y. Hou, A. G. van Arnim, X.-W. Deng, Plant Cell 5,

329 (1993).83. L. Taiz and E. Zeiger, Plant Physiology (Benjamin,

New York, 1991).84. M. Koorneef, M. L. Jorna, D. L. C. Brinkhorstovan

der Swan, C. M. Karssen, Theor. Appl. Genet. 61,385 (1982); M. Koorneef, G. Reuling, C. M. Karssen,Physiol. Plant. 61, 377 (1984); M. Koorneef, C. J.Hanhart, H. M. W. Hilhorst, C. M. Karssen, PlantPhysiol. 90, 463 (1989).

85. E. Nambara, S. Nito, P. McCourt, Plant J. 2, 435(1992).

86. R. R. Finkelstein, Mol. Gen. Genet. 238, 401 (1993).87. J. Giraudat et al., Plant Cell 4, 1251 (1992).88. D. R. McCarty et al., Cell 66, 895 (1991).89. J. Leung et al., Science 264, 1448 (1994); K. Meyer,

M. P. Leube, E. Grill, ibid., p. 1452.90. A. J. MOller, Biol. Zentralbl. 82, 133 (1963).91. L. A. Castle and D. W. Meinke, Plant Cell 6, 26

(1994).92. S. Mis6ra, A. J. MOller, U. Weiland-Heidecker, G.

JArgens, Mol. Gen. Genet. 244, 242 (1994).93. X.-W. Deng et al., Cell 71, 791 (1992).94. T. W. McNellis et al., Plant Cell 6, 487 (1994).95. A. Pepper, T. Delaney, T. Washburn, D. Poole, J.

Chory, Cell 78,109 (1994).96. N. Wei et al., Plant Cell 6, 629 (1994).97. N. Wei, D. A. Chamovitz, X.-W. Deng, Cell 78, 117

(1994).98. We express our gratitude to J. Harada, R. Fischer,

J. Okamuro, D. Jofuku, L. Zimmerman, A. Kol-tunow, T. Laux, L. Perez-Grau, and G. Drews formany critical and stimulating discussions of plantembryogenesis. We thank B. Timberlake, R.Chasan, and E. Davidson for insightful commentson this manuscript. We gratefully acknowledge K.Brill, B. Phan, and T. Zeyen for diligent typing of thismanuscript and M. Kowalczyk for the colorful artwork. We dedicate this article to Professor E. H.Davidson, who has provided intellectual leadershipand direction to the field of development biologyover the past 25 years.


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Plant Embryogenesis: Zygote to Seed Robert B. Goldberg ... · animals (1) is not set aside during plant embryogenesis. Amature flowering plant embryo con-tains two primary organ systems-the