[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Embryogenesis in Dicotyledonous Plants

1. Embryogenesis in Dicotyledonous Plants

RAMIN YADEGARI* and ROBERT B. GOLDBERGDepartment ofMolecular. Cell, and Developmental Biology, University of California,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-fertilizationlpro embryo

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 embryos

from 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 and

Pelvetia) 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 the

zygote 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 cell

fates 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. M uta tions delete specific em bryo 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

mon opteros

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 shap e 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 ens or 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

emb ryo-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 the

deletion 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 global

effect 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 meristem

formation 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 reversible

as 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 just

after 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 which

alter 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 how

the 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 embryogenesis

can 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 in

the 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 WO ODEN 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 ). SCA RECROW 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 SCA RECROW 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 SHOOT MERlSTEMLESS (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 contains

the 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 embryo

development? 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 role

in 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 highlighted by the transcription of STM and ANT genes(Figure 2). It is not clear how these spatial patterns of transcriptional activ-ity relate to the regions which are altered due to the zygotic mutations inapical-basal patterning mutations such as gurke, monopteros, etc. Howev-er, all of the regional transcription programs apparently occur in cell typesthat are clonally unrelated; that is they arise in cells which are derivativesof distinct precursor cells [ (Elliott et aI., 1996; Long et al., 1996); G.R . dePaiva and R.B. Goldberg , unpubl .; R. Yadegari and R.B. Goldberg, unpubl.].Each tissue layer of the radial embryo-proper axis also has a distinct tran-scriptional program (Figure 1 and 2). Transcriptional activity within theselayers , however, appears to be established in a territory-specific manner; thatis, ground meristem cells within the equator region activate promoters dis-tinct from those within ground meristem cells of the micropylar-region, andvice versa (Figure I and 2). Therefore, a radial pattern during the globular-stage embryogenesis is composed of two elements-three concentric layers ofcells (Figure 1), and a partially-overlapping ring of transcriptional-specificregulatory programs (depending on the position along the apical-basal axis)(Figure 2). These results suggest that a "pre-pattern" of different transcrip-tional regulatory domains has been established in the globular embryo prior tothe morphogenetic events that lead to the differentiation of cotyledon and axisregions at the heart stage (Figure I). How each regional transcription programis activated early during development is not known . Later in embryogenesis,each transcriptional domain presumably sets in motion a cascade of eventsleading to the differentiation of specific embryo regions and cell types.

B. Promoter Elements as Interpreters ofRegion-Specific RegulatoryNetworks

One consequence of the modular organization of a plant embryo (see above)is that genes which are active throughout the embryo must intersect withseveral region-specific regulatory networks. That is, the promoters of embryo-specific genes are required to sense and interpret the transcriptional regulatorymachinery unique to each specified region. For example, the Kti3 gene istranscribed within the axis region early in soybean embryogenesis, but is notactivated within the cotyledons until much later [ (Perez-Grau and Goldberg,1989); G.R. de Paiva and R.B. Goldberg, unpubl.] (Figure 2). Thus, discretepromoter elements should exist which are responsible for interacting withtranscription factors produced by separate regulatory circuits.


Kti3(2kb) Kti3(1.7kb)Let (3kb)

Kti3 (O.8kb)Let (O.8kb)

Gy/(-446bp/-84bp) CaMV35SCaMV35S (-90bp/+8bp)(-343bp/-90bp)

Fig . 3. Examples of transcripti onal domains in mature tobacco embryos. Schematic repre-sentations of mature tobacco embryos and the transcriptional activity of selected marker genepromoter regions, including fragment s of the soybean Kri3 (G.R. de Paiva and R.B. Goldberg,unpub !.), soybean LeI (R. Yadegari and R.B. Goldberg, unpub!.), soybean glycin in Gyl (G.R.de Paiva and R.B. Goldberg, unpubl .), and cauliflower mosaic virus 35S rCaMV35S; (Benfeyet al., 1990)] gene promoters.

The tran scriptional activity of a number of embryo-specific or seed proteingenes has been ana lyzed during dicot embryo development (Go ldberg et aI.,1989; Bewley and Marcus, 1990; Bevan et al., 1993; Thomas, 1993). Manyseed protein genes, includ ing the soybean Kti3 , LeI , and glycinin l (GyI)genes, are tran scribed during embryo development before their transcriptionalactivity is shut down prior to dessication (Goldberg et aI., 1989). For example,a Kti3-GUS gene with 2 kb of 5' flanking sequence is transcribed in allregion s of a mature tran sgenic tobacco embryo (G.R. de Paiva and R.B.Goldberg, unpubl. ) (Figure 3). Deletion of 0.2 kb from the 5' end eliminatesKti3-GUS tran scriptional activity within the embryo radicle region (G.R. dePaiva and R.B. Goldberg, unpubl. ) (Figure 3). A similar result is obtainedfor the entire region of the LeI gene in tobacco embryos (R. Yadegari andR.B . Goldberg, unpubl.) (Figure 3). Deletion of another I kb eliminatesKti3-G US transcription within the cotyledons and shoot meristem, but stillpermits transcription to occur within the hypocotyl region (G.R. de Paivaand R.B . Goldberg, unpubl. ) (Figure 3). These results indicate that discretecis-acting domains are required for the transcriptional activation of the Kti3and Lei genes within the radicle, hypocotyl, and cotyledon/shoot meristemregions of the embryo . Promoter analysis of the soybeanGyl storage proteingene (Nielsen et aI., 1989) also uncovered a regulatory domain that direct stran scription to the cot yledons and shoot meristem of a transgenic tobaccoembryo (G.R. de Paiva and R.B . Goldberg, unpubl. ) (Figure 3). All thesegenes , therefore, con tain at least two 5' regulatory modules corresponding


to two major morphological regions of the mature embryo, the apical region(cotyledons and shoot meristem) and the hypocotyl region.Deletion analysis of the 5' regulatory region of a ,B-phaseolin gene from

Phaseolus vulgaris has uncovered a similar pattern of gene activity in maturetobacco embryos where two sets of upstream activating sequences (UAS)were shown to direct reporter gene expression individually to the apical region(UASI) and the hypocotyl region (UAS2) (Bustos et aI., 1991). The distinc-tion between the two regional-specific gene expression programs has beenestablished among members of the Arabidopsis 2S albumin gene family whichshow differential expression in embryo cotyledons during late embryogene-sis (Guerche et aI., 1990). Analysis of other embryonic regulatory sequencesincluding the cauliflower mosaic virus 355 gene (CaMV355) promoter (Ben-fey et aI., 1990), sunflower helianthinin gene promoters (Jordano et aI., 1989;Nunberg et aI., 1994, 1995), and soybean Kunitz trypsin inhibitor gene fam-ily promoters (G.R. de Paiva, and R.B. Goldberg, unpubl.) has identifiedadditional regulatory components, each directing transcription of a modi-fied promoter-reporter gene to a particular region of the mature embryo. Forexample, a fragment comprising sequences between +8 bp and -90 bp of theCaMV35S promoter, directs expression of a reporter gene to the basal tip,or the radicle, of a mature tobacco embryo (Benfey et aI., 1990) (Figure 3).Therefore, in plant embryos, promoters of genes expressed during embryodevelopment act as interpreters of transcription programs which are mostlikely involved in regionalizing the early embryo. Some of these regionaltranscription programs may be derived from the earliest zygotic divisions,including genes which are regulated presumably by asymmetrically-locatedgene products such as ATMLl (see above). The transcriptional patterns ofthe chimeric marker genes discussed suggest that their respective regulatoryproteins should be part of, or interact with, pathways that establish both theapical-basal and the radial pattern elements. Later, the same marker genes aretranscribed in larger domains of the mature embryo, marking the activity ofperhaps distinct or overlapping gene regulatory networks which are involvedin organ and tissue development within the more elaborate, maturing embryo.The study of genes expressed in particular spatial regions of the early ani-

mal embryos have uncovered a modular organization for their cis-regulatorysequences (Davidson, 1994; Cai et aI., 1996; Kirchhamer et aI., 1996). Insea urchin and Drosophila embryos for example, modules composed of spe-cific DNA sequence elements (target sites for transcription factors) performeither specific and independent regulatory functions or interact with each oth-er to establish novel patterns of gene expression during embryo development(Makabe et aI., 1995; Arnosti et aI., 1996; Kirchhamer and Davidson, 1996;Yuh and Davidson, 1996). Whether the regional pattern of embryo gene tran-scription during the globular and later stages (Figures 2 and 3) is mediated byautonomously-functioning cis-regulatory modules remains to be determined.However, the existing data indicate that unique transcription factors must be


active within each embryonic region, and that these factors interact with spe-cific promoter elements. The combination of these elements and factors givesrise to the transcriptional pattern of the whole embryo (Figure 3). Identifica-tion of transcription factors that interact with region-specific DNA elementsshould provide entry into the independent regulatory networks required forspecifying each particular region of a plant embryo.

VII. Signal Molecules and Hormones Affect Embryo Development

How are the putative cellular or regional interactions that participate in apical-basal and radial axis development in plant embryos implemented? Such inter-actions may involve processes similar to those identified in animal develop-ment, including graded morphogens or sequential signaling cascades (Green-wald and Rubin, 1992; Jessell and Melton, 1992; Kenyon , 1995; Perrimon,1995). The latter, for example, relies in part on the close interaction betweentwo adjacent cells and involves cell surface-localized signaling molecules(Greenwald and Rubin, 1992). Several lines of evidence argue for the exis-tence of signaling molecules involved in dicot embryogenesis; although, othercomponents of the putative inducer and receptor pathways largely remain tobe identified.

A. Auxins

The morphological events which cause the embryo proper to initiate cotyle-dons and become bilaterally symmetric during the globular-heart transitionphase of embryogenesis (Figure I) may involve the activity of signaling mole-cules. Several experiments implicate a class of plant hormones, the auxins,in this morphogenetic process (Fry and Wangermann, 1976; Schiavone andCooke, 1987; Michalczuk et aI., 1992; Liu et aI., 1993). The auxin, indole-3-acetic acid (lAA), is involved in a number of plant activities, includingphoto- and gravitropism, apical dominance, and vascular cell differentiation(Taiz and Zeiger, 1991). High endogenous levels of activity have been detect-ed in zygotic embryos in plants as diverse as bean and pine as well as insomatic carrot embryos (Michalczuk et aI., 1992, and references therein).Microscale transport assays have indicated a polar, basipetal direction forauxin transport along the embryo axis (Greenwood and Goldsmith, 1970; Fryand Wangermann, 1976). Analysis of carrot somatic embryo cultures indicat-ed that the highest auxin level s occur at the globular stage of embryogenesis(Michalczuk et aI., 1992). Application of agents that inhibit polarized auxintransport either blocks the transition from the globular to heart stage com-pletely (Schiavone and Cooke, 1987) or prevents the bilateral initiation ofcotyledons at the top of the globular embryo (Liu et aI., 1993) (Figure I) . Forexample, auxin transport inhibitors cause carrot somatic embryos to remain


spherically-shaped and develop into giant globular embryos (Schiavone andCooke, 1987). By contrast, zygotic embryos of the Indian mustard tBrassi -ca juncea), an Arabidopsis relative, fail to initiate two laterally-positionedcotyledons when treated with auxin transport blockers in culture (Liu et aI.,1993). A cotyledon-like organ does form, but as a collar-like ring aroundthe entire upper (apical) region of the embryo (Liu et aI., 1993) . TreatedIndian mustard embryos resemble those of the Arabidopsis pinformed tpinl-I) mutant which has a defect in polarized auxin transport (Okada et aI.,1991; Liu et aI., 1993). In microamputation experiments with carrot somaticembryos, auxin and its polar transport have also been implicated in apicalcontrol of axis elongation and root regeneration (Schiavone, 1988) . Togetherwith more extensive evidence of position-dependent regenerative capabilitiesof cut pieces of carrot somatic embryos in which all or part of the missingroot or shoot structure is replaced, auxin polar transport has been suggestedas means of maintaining structural polarity in somatic embryos (Schiavoneand Racusen, 1990, 1991; Cooke et aI., 1993). These results suggest that aux-in asymmetries are established within the embryo-proper region of globularstage embryos and contribute to an apical-basal patterning process as well asto the formation of bilateral symmetry at the heart stage (Cooke et aI., 1993)(Figure I). A recent analysis of the influence of auxin on the establishmentof bilateral symmetry in the monocot wheat zygotic embryo supports thisdual mode of auxin function. One locus of auxin synthesis is proposed to belocated in the basal part of the embryo proper and generates two pathways ofpolar transport-a basipetal one towards the scutellum and a lateral pathwaytowards the site of future promeristem (Fischer and Neuhaus, 1995).

B. Arabinogalactan Proteins

A more recent set of experiments suggests that arabinogalactan proteins(AGPs) may act as signal molecules during embryogenesis to alter an endoge-nous balance of phytohormones (Schmidt et aI., 1994). Belonging to adiverse class of plant glycoproteins and proteoglycans, AGPs occur both asmembrane-associated and secreted components of many different cell types(Chasan, 1994;Kreuger and van Holst, 1996) . In fact, each organ and cell typepossesses a specific class of AGPs, although no absolute function has beenestablished for anyone class (Chasan, 1994; Kreuger and van Holst, 1996). Apotential function of AGPs has been demonstrated in carrot somatic embryo-genesis where very low concentrations of the proteins, isolated from either theculture medium of embryogenic carrot lines or from dry carrot seeds, promotethe development of somatic embryos in nonembryogenic cultures (Kreugerand van Holst , 1993). By contrast, nonembryogenic cell lines produce AGPswhich inhibit the formation of proembryogenic masses in culture (Kreugerand van Holst, 1993). The presence of AGPs are highly modulated duringsomatic embryogenesis and they mark transitional cell states in the develop-


mental pathway, indicating that they may perform important developmentalfunctions during somatic embryogenesis (Stacey et aI., 1990; Pennell et al.,1992; Kreuger and van Holst, 1993; Egertsdotter and von Arnold, 1995).What is the potential role of AGPs during zygotic embryogenesis? There

is no data that shows directly a functional role for AGPs in zygotic embryodevelopment. However, the temporal and spatial distribution of two AGPepitopes during plant life cycle indicates that AGP gene expression is highlyregulated in reproductive organs and is very sensitive to developmental transi-tions, including the globular-heart transition stage of embryonic developmentand the progressive differentiation of embryo-proper and suspensor cells dur-ing early stages of embryo development (Pennell and Roberts, 1990; Pennellet aI., 1991). For example, a plasma membrane AGP epitope recognizedby the monoclonal antibody MAC 207 is detected in all cells of vegetativemeristems, primordia, organs, and undeveloped flower buds of Pisum sativumexcept those that give rise to the pollen sac and the embryo sac surround-ed by the nucellus (Pennell and Roberts, 1990). Following fertilization, thezygote and the early embryo remain unreactive to the monoclonal antibodyuntil the heart stage of development (Pennell and Roberts, 1990). Anothermonoclonal antibody which reacts with a Brassica napus plasma membraneAGP, designated JIM8, is present in all cells of an early embryo containinga 2-cell embryo proper and a suspensor of six cells (Pennell et aI., 1991).In a globular-stage embryo, the cells of the embryo proper lose the epitoperecognized by the JIM8 monoclonal antibody, although all the cells of the sus-pensor remain reactive (Pennell et a!., 1991). The precise spatial and temporalregulation of specific AGP accumulation patterns in reproductive structuresas well as root development (Knox et aI., 1989, 1991) implies an importantrole for such proteins during plant development.

C. Lipooligosaccharides

Secreted AGPs per se are not the only molecules identified which affectsomatic embryo development. A carrot temperature-sensitive somatic embryovariant, designated tsl l , arrests at the globular stage of embryo developmentand has a defective protoderm cell layer (Lo Schiavo et aI., 1990; de Jong etal., 1992). ts11 embryos can proceed beyond the globular stage of embryogen-esis and form normal-looking protoderm when the medium is complementedwith either an appropriate level of a 32 kDa extracellular endochitinase (deJong et a!., 1992, 1995), or by the addition of Rhizobium lipooligosaccha-rides, also known as nodulation (Nod) factors (de Jong et aI., 1993a). Thechitin-containing Nod factors act as signal molecules involved in the differ-entiation of Rhizobium-induced root nodules (Fisher and Long, 1992; Vijn etaI., 1993). The data suggest that the endochitinase participates in the releaseof Nod factor-like signal molecules from an unknown endogenous precursorpresent in the cell wall (de Jong et al., 1993a). Alternatively, the endochiti-


nase and the Nod factor may act through different mechanisms (Schmidt eta1., 1994). Nod factors have been shown to influence many developmentalprocesses possibly through interaction, or participation, with auxin and/orcytokinin signaling pathways (Fisher and Long , 1992; Schmidt et al. , 1994).For example, synthetic Nod factors alleviate the need for auxin and cytokininto maintain the growth of cultured tobacco protoplasts, and are able to pro-mote cell division, activate an auxin-responsive promoter, and increase thelevel of mRNA accumulation for a gene implicated in auxin response (Rohriget al., 1995). In an analogous manner, endogenous lipooligosaccharides mayinfluence embryogenesis by affecting phytohormone balance critical for thedevelopment of embryonic structures, including a normal protoderm.

D. Putative Cell-Wall-Associated Signaling Mole cule s

It remains to be determined whether the relea sed lipooligosaccharides orAGPs discussed earlier playa truly inductive role during zygotic embryoge-nesis in a manner analogous to the role played by various diffusible factors inanimal development (Slack, 1991; Jessell and Melton, 1992) . Likewise, anyfunctional role for plasma membrane-bound or cell wall-associated proteinsin regulating higher plant embryo development is also unknown; although,the plasma membrane AGP epitope which interacts with the 11M3 mono-clonal antibody (see above) has been speculated to be a marker of cell-cellinteractive processes involved in flower development (Pennell et a1., 1991). Inanimal development, cell adhesion plays an important role in both embryonicmorphogenesis and the maintenance of tissue integrity and organ function(Klymkowsky and Parr, 1995). A recent laser ablation experiment performedin the Fucus embryo suggests that important spatial determinants may beassociated with the cell wall in a clear contrast to the secreted form s of puta-tive signaling molecules discussed earlier (Berger et al. , 1994). A cell wall isrequired for fixation of the embryonic axis in Fucus zygotes even though theinitial process of axis formation is independent of cell wall synthesis (Kropfet al., 1988). Laser microsurgery dissection of two-celled embryos producedisolated protoplasts which dedifferentiated once removed from the cell wall(Berger et al., 1994). However, isolated cells still remaining within the con-fines of the cell walls switched their restricted developmental fates when theycame in contact with the isolated cell wall of the other cell type (Berger et a1.,1994). The conclusion that the Fucus cell wall maintains the differentiatedstate and may direct an already-established cell fate (Berger et a1., 1994) mayhave broader implication in higher plant embryogenesis where the cell wallmay perform a similar information-storage function (Brownlee and Berger,1995).

Embryoge nesis in dicotyledonous plants 37

VIII. Cell Differentiation and Morphogenesis Can Be Uncoupled inPlant Embryos

What is the relatio nship between ce ll different iation and morphogenesis inplant embryos? Are processes requ ired for tissue differen tiation along theembryo radial axis coupled to those that specify independent regions/tiers ofthe longitudinal apic al-basal axis (Figure I)? Studies of Arabidopsis embryopattern mutants suggest that these processes are regulated independently.For example, a Jackel embryo does not have a hypocotyl , but epidermal,ground meristem , and vasc ular tissues differentiate within cotyledon andrad icle regions (Maye r et aI., 1991). Thus, the loss of one embryonic regiondoes not aff ect the formation of tissue layers within the remaining region s(Mayer et aI., 1991, 1993b; Berleth and Jiirgens, 1993).What would happen, however, if elaboration of the initial apical-basal

pattern within a globular embryo was prevented? That is, would a mutantembryo that arrests early in embryonic development and remains globular-shaped differentiate the spec ialized cell and tissue layers that are found inorgan sys tems of a mature, wild-type embryo? Amaturation stageArabidopsisembryo has specialized epide rma l, storage parench yma, and vascular celllayers within both the cotyledon and axis regions (Figure I). These tissuesare derived from the three primary cell layers that are speci fied along theradi al axis of a globular embryo (Figure 1), and express specific markergenes late in embryogenes is. For example, the Arahidopsis lipid transferprotein (AtLTP I) mRNA mentioned earlier accumulates speci fically withinthe epiderma l ce ll layer (Ste rk et aI., 199 1; Thoma et aI., 1994; Vroemen et a!.,1996) and the At2S2 albumin mRNA accumulates within storage parenchymace lls (Guerc he et a!., 1990; Conceicao and Krebbers, 1994). Neither mRNAis detectable within the vasc ular layer (Guerche et aI., 1990; Sterk et aI.,1991; Vroemen et al., 1996). Collectively, the AtLTP I and At2S2 mRNAsca n identify embryo epiderma l and storage parenchyma ce ll layers, and , bydefault, the inner vascular tissue as well.An Arabidopsis embryo mut ant , designated raspberry I , fails to undergo

the globular to heart transition (Figure I), has an embryo-p roper region thatremains globular-shaped throu ghout embryogenesis, and does not differenti-ate co tyledons and axis (Yadegari et al., 1994) (Table 2). raspberryl embryosalso have an enlarged suspensor region (Yadega ri et al., 1994 ). raspherry2(Yadegari et a!., 1994) and sus (Schwartz et a!., 1994) embryo-defectivemutants also have phenotypes similar to that of raspberry! (Table 2). Surpri s-ingly, raspb erry] embryos acc umulate AtLTPI and At2S2 marker mRNAs intheir correct spatial context along the radial axis of both the embryo proper andsuspensor regions (Yadegari et aI., 1994). AtLTP I mRNA accumulates alongthe outer perimeter of raspb erry 1 embryos , while At2S2 mRNA accumulateswithin interior ce lls (Yadegari et aI., 1994). By contrast, AtLTP1 and At2S2mRNAs do not accumulate detectab ly within the central core of raspberry1


embryos (Yadegari et a\., 1994). Similar results are obtained with raspberry2embryos (Yadegari et a\., 1994) (Table 2). These mRNA localization studiesindicate that specialized tissues can differentiate within the embryo-properregion of mutant embryos that remain globular-shaped, and that these tissuesform in their correct spatial contexts. A similar conclusion was inferred fromhistological studies of the sus mutants (Schwartz et a\., 1994). Tissue dif-ferentiation, therefore , can take place independently of morphogenesis in ahigher plant embryo, implying that morphogenetic checkpoints do not occurbefore cell differentiation events can proceed.It does not follow, however, that morphogenesis can occur without proper

cell differentiation events. Arabidopsis embryo mutants that alter tissue spec-ification patterns have abnormal morphologies (Mayer et aI., 1991, 1993a;Jiirgens, 1994; Scheres et a\., 1995, 1996) (Table 2). For example, as men-tioned earlier, knolle seedlings are rounded and lack a well-formed epidermislayer due to abnormal cell divisions and enlargements during embryogenesis(Mayer et a\., 1991; Lukowitz et aI., 1996). Also, a number of mutationsaffecting radial organization of the Arabidopsis root manifest their defectsfirst in the radial organization of the embryonic axis (Scheres et a\., 1995,1996). Similarly, the carrot tsl I somatic embryo mutant that has a defectiveprotoderm cell layer fails to undergo morphogenesis (Lo Schiavo et al., 1990;de Jong et a\., 1992), suggesting that in carrot somatic embryos, the forma-tion of a normal protoderm cell layer may be a prerequisite for subsequentembryonic development (de Jong et a\., 1992). Together, experiments withmutant embryos that have defective cell layers suggest that differentiation ofthe radial axis needs to occur in order for the structures along the shoot-rootaxis to form normally; and as discussed earlier, an important corollary is thatcells within the radial axis probably interact with each other to effect properdevelopment of the embryo and subsequently the seedling.

IX. Suspensor Cells Have the Potential to Generate an Embryo

One intriguing aspect of the raspberry and sus embryos is their large sus-pensors (Schwartz et al., 1994; Yadegari et a\., 1994). raspberry] suspensorsare indistinguishable from wild-type during the early stages of embryoge-nesis (Yadegari et a\., 1994) (Figure I). Later in seed development, whenneighboring wild-type embryos undergo maturation, cell proliferation eventscause the raspberry] suspensor to enlarge at its basal end (Yadegari et a\.,1994). AtLTP I and At2S2 mRNAs (Guerche et a\., 1990; Thoma et a\., 1994)accumulate in the raspberry] suspensor with a spatial pattern similar to thatwhich occurs in mature, wild-type embryos (Yadegari et a\., 1994). Thesecell-specific mRNAs do not accumulate detectably in wild-type suspensors,or in raspberry] suspensors early in embryogenesis (Yadegari et a\., 1994).These results indicate that the raspberry] suspensor has entered an embryo-genic pathway, and that an embryo-proper-like, radial tissue axis has been


specified. In addition, since the stereotyped cell division patterns seen duringearly development of the wild-type embryo-proper differ from those that takeplace in the raspberry] suspensor, cell interactions should occur between thedifferentiating cell layers to establish the correct order of radial tissues in adeveloping embryo-proper, or a converted suspensor for that matter (Yadegariet aI., 1994).OtherArabidopsis embryo mutants have suspensor abnormalities similar to

that of raspberry] ,including raspberry'I, and sus (Marsden and Meinke , 1985;Yeung and Meinke, 1993; Meinke, 1994; Schwartz et al., 1994; Yadegari etal., 1994) (Table 2). Although the extent of suspensor enlargement varies, allof these mutants have morphological defects in the embryo proper (Marsdenand Meinke, 1985; Yeung and Meinke, 1993; Meinke, 1994; Schwartz et al.,1994; Yadegari et al., 1994) . Mutant embryos that resemble wild-type, butarrest at specific embryonic stages, do not have aberrant suspensors (Yadegariet al., 1994). Disruptions in embryo proper morphogenesis, therefore, caninduce an embryo-proper-like pathway in terminally-differentiated suspensorcells - a result first observed by the embryo-proper ablation experimentsof Haccius more than 30 years ago (Haccius , 1963). The Arabidopsis twinmutant represents a striking example of the embryogenic potential of thesuspensor region (Vernon and Meinke, 1994). twin causes subtle defects tooccur in embryo-proper morphology, generates a second embryo within theseed from proliferating suspensor cells , and results in twin embryos that areconnected by a suspensor cell bridge (Vernon and Meinke, 1994).Genes like SUS2 and RASPBERRY] are probably not involved in suspensor

specification events per se because a normal suspensor forms prior to induc-tion of the embryo-proper pathway in mutant embryos (Marsden and Meinke,1985; Schwartz et al., 1994; Vernon and Meinke, 1994; Yadegari et al., 1994).In support of this notion, cloning of SUS2 and RASPBERRY] genes and theirmutant alleles has revealed that they code for proteins involved in generaland essential cellular processes [ (Meinke, 1995); R. Yadegari, N.R. Apuya,and R.B . Goldberg, unpubl.]. SUS2 is homologous to the yeast PRP8 gene, asplicesome assembly factor (Meinke, 1995), while RASPBERRY] codes forthe S I ribosomal protein located in the chloroplast (R. Yadegari, N.R. Apuya,and R.B. Goldberg, unpubl.). Regardless of their molecular nature, thesemutations reveal that interactions occur between the suspensor and embryo-proper regions. One possibility is that the embryo proper transmits specificinhibitory signals to the suspensor that suppress the embryonic pathway (Hac-cius, 1963; Marsden and Meinke, 1985; Yeung and Meinke, 1993; Schwartzet al., 1994; Vernon and Meinke, 1994). Alternatively, a balance of growthregulators might be established within the entire embryo that maintains thedevelopmental states of both the embryo proper and suspensor regions. Dis-ruptions of such signals due to arrest in cell division and subsequent arrest inmorphogenesis would cause the suspensor to take on an embryo-proper-likefate - a result analogous to embryo induction in differentiated sporophytic or


gametophytic cells (Van Engelen and De Vries, 1992; de long et aI., 1993b;Zimmerman, 1993; Yeung, 1995).Superficially, the flexibility of suspensor differentiation pathway in fol-

lowing an embryo proper-like fate, as indicated by the phenotype of the sus,raspberry, and twin mutations, might suggest that the earliest allocation ofapical-basal pattern attributes at the first zygotic division is reversible. How-ever, developmental analysis of the above mutants indicates that suspensordevelopment is apparently normal during early embryogenesis, and once theglobular stage is reached, the suspensor cells take on the characteristics of anembryo proper (Schwartz et aI., 1994; Vernon and Meinke, 1994; Yadegariet aI., 1994). As discussed earlier, the embryo proper might actively supply asignal to maintain the suspensor in its proper developmental pathway beforeits disintegration (Yeung and Meinke, 1993; Schwartz et aI., 1994; Vernonand Meinke, 1994; Yadegari et aI., 1994). If so, such continuous regulationof suspensor differentiation is similar to a model proposed earlier to accountfor the dynamic and often reversible differentiated states observed in manyinstances of animal development (Blau and Baltimore, 1991). Accordingly,the continuous activity of positive and negative regulators has been shown tobe required to maintain differentiation in the course of normal development insuch diverse processes as sperm production and regulation of male courtshipbehavior inCaenorabditis and Drosophila, respectively (Blau and Baltimore,1991). However, the expression of the whole hierarchy of the regulatorypathways establishing a differentiated state is not necessarily required for itsmaintenance also (Blau and Baltimore, 1991). In this manner, the proposedinhibition of the embryogenic pathway in the suspensor cells does not repre-sent an example of an instructive (or directive) induction in which respondingtissue develops along one developmental pathway in the presence of a signaland another pathway in its absence (Slack, 1991).The early differentiation of a normal-looking suspensor in sus, raspber-

ry, and twin embryos suggests a normal basal cell specification during thefirst zygotic division. Because there have been no reported cases of a diver-gent development of the basal cell among the embryo-defective mutationsidentified to date, the suspensor differentiation pathway may be caused bycell-autonomous processes and provide an entry point for unraveling themechanisms that underlie the asymmetric designation of embryo-proper fateversus suspensor-cell fate. Recent experiments have identified a number ofsuspensor-specific mRNAs which are localized to the giant suspensor of theScarlett Runner Bean, Phaseolus coccineus (N.R. Apuya and R.B. Goldberg,unpubI.). Suspensor-specific genes which are activated early in embryo devel-opment can be used to identify DNA-binding regulatory proteins which areresponsible for their suspensor-specific spatial patterns of expression. Evenearlier programs of basal cell-specific gene regulatory networks can be iden-tified using the same approach. The putative cell-cell interactions that occurwithin the suspensor and between the suspensor and the embryo proper can


then be addressed using targeted cell ablation utilizing suspensor-specificpromoters to drive the production of cell-autonomous cytotoxic proteins(Koltunow et aI., 1990; Mariani et aI., 1990; Goldberg et aI., 1995).

X. Conclusions

Plant embryogenesis provides a vital bridge between the gametophytic gen-eration and post-embryonic differentiation events that occur continuously inthe shoot and root meristems of the sporophytic plant. As such, plant embryosmust establ ish the polarized sporophytic plant body plan and enable the youngplant to survive harsh environmental conditions and a period of below-groundgrowth from seeds. These events occur early in plant embryogenesis and arepoorly understood. Only now have genetic and molecular studies begun toreveal some of the important processes involved in dicot embryogenesis.The ultimate product of embryogenesis in dicots, even all higher plants, is

relatively similar. Assuming that the basic regulatory mechanisms involvedin higher plant embryogenesis are conserved during evolution, a unifying setof mechanisms must function in many species exhibiting diverse patterns ofcellular growth (division and elongation) in early embryogenesis. Therefore,it is very likely that cell-cell communication is involved in coordinatingthe recruitment of individual cells or cell groups into specific developmentalpathways. Genetic studies have indicated that the establishment of radial tissuelayers is probably a position-dependent process (see above). But where wouldthe initial signal come from? It is possible that as the protoderm becomesrestricted from the rest of the embryonic cells, either via stereotyped celldivisions or by the virtue of being partially exposed to an outside environment,an internal radial signal partitions the embryo proper into three gross tissuelayers which are subsequently refined to produce specific cell layers typicalof the mature embryo.Superimposed upon this pattern is an apical-basal developmental program

whose molecular origins are even more obscure. Clearly, an apical-basalpolarity is somehow perpetuated during early embryo development. Howfar back in the developmental history is the regulatory/functional polarity ofthe embryo established remains unknown. However, molecular and genet-ic data suggest that the polarity is well established after the first zygoticdivision, and in fact, disturbance of this polarity in the form of a symmet-rical division of the zygote has profound effects on the development of theembryo. Bearing in mind the difficulties associated with genetic screening forfemaie-gametophytic or maternal-sporophytic mutations, the overwhelmingpredominance of the zygotic mutations in Arabidopsis suggests that embryopolarity per se is specified after fertilization. In such a model, spatial determi-nants that direct the apical and basal cells to follow different pathways wouldbe synthesized de novo in the zygote and then asymmetrically distributed intodaughter cells upon first division.


How could these spatial determinants be allocated and maintained in space?As in Fucus , higher plant cell wall might maintain the differentiated state ofindividual daughter cells following cell division, or even initiate cell specifi-cation processes prior to cytokinesis in an anisotropic manner (in the zygotefor example). Moreover, cell wall-bound determinants may not only occur onthe outermost cell walls of an angiosperm embryo in an analogous manner tothe mechanism envisioned in Fucus, but there might be a more sophisticatedpatchwork of cell and tissue type-specific factors clung to cell walls through-out an angiosperm embryo. These and other questions regarding the precisemolecular mechanisms responsible for determination of the embryonic polar-ity and specifying different cell and organ types early in plant embryogenesisremain to be determined. A major void in our knowledge is the events thatoccur within the egg cell and in the early embryo following fertilization . Inthis respect, in combination with genetic analysis of early embryogenesis, itis critical to obtain molecular markers (such as the ATMLl mRNA) in orderto follow the specification events that take place during early embryogenesisand gain entry into regulatory networks that are activated in different embry-onic regions after fertilization . Although a large amount of progress has beenmade in recent years in understanding how dicot embryos form, there is stilla long way to go.

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[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Embryogenesis in Dicotyledonous Plants