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Most morphogeneticevents in flowering Embryogenesis: occur in the postembryonicsporo- phyte after (Fig. 1) (2). Vegetativeorgan systems differentiate con- to Seed tinuouslyfrom and meristematic regionsthat are formedinitially during em- Robert B. Goldberg,*Genaro de Paiva, RaminYadegari bryogenesis.The reproductiveorgans of the are differentiated from a repro- Mostdifferentiation events in higherplants occur continuouslyin the postembryonicadult grammedshoot meristemafter the phase of the life cycle. Embryogenesisin plants, therefore, is concerned primarilywith has become a matureplant (Fig. 1) (25). establishing the basic shoot-root body patternof the plant and accumulatingfood re- Thus, a germlineanalogous to that foundin serves that willbe used by the germinatingseedling aftera periodof embryonicdormancy (1) is not set aside during plant withinthe seed. Recent studies inArabidopsis have identifiedgenes that provide embryogenesis. new insightinto how embryos formduring . These studies, and others A matureflowering plant embryocon- using molecularapproaches, are beginningto revealthe underlyingprocesses that control tains two primaryorgan systems-the axis . and (Fig. 1) (2). These organs have distinct developmentalfates and are both composedof threebasic, or primordial, layers-protoderm,procambium, and A majorproblem in plant developmentis lecularapproaches suggest that a plant em- groundmeristem-which will become the to unravelthe mechanismsoperating dur- bryohas a modularstructure and consistsof epidermal,vascular, and parenchymatissues ing embryogenesisthat enable a plant to severalregions that formautonomously dur- of the youngseedling, respectively (2). The specifyits and tissue differentia- ing embryogenesis. axis, or -radicleregion of the em- tion patterns.Although progresswith a va- bryo,contains the shoot and root rietyof animalsystems has been spectacular Embryos Begin the Diploid Phase and will give rise to the matureplant after in this regard(1), a detailedunderstanding of the Higher Plant Life Cycle seed germination(Fig. 1). By contrast,the of the events that govern plant embryo cotyledon is a terminallydifferentiated or- formationhas yet to be realized.One obsta- The life cycle is divided gan that accumulatesfood reservesthat are cle in achievingthis goal is the location of into haploid and diploid generationsthat utilizedby the seedlingfor growthand de- embryoswithin the plant and their relative are dependent on each other (Fig. 1) (2, velopmentbefore it becomesphotosynthet- inaccessibilityto experimental manipula- 16-18). The haploid, or gametophytic, ically active (Fig. 1). The cotyledon func- tion, particularlyat the early stagesof em- generationbegins after meiosis with tions primarilyduring seed germinationand bryogenesis.In floweringplants, reproduc- that undergomitosis and differentiateinto senesces shortly after the seedling emerges tive processesoccur within floral organs either a grain(male )or from the soil. Embryogenesisin higher (Fig. 1) (2). The is present in the an embryo sac (female gametophyte) (3, plants, therefore,serves (i) to specifymeri- , a multicellularstructure that is buried 19-20). The pollen grain contains two stems and the shoot-root plant body pat- beneath severalcell layersof the pistil, the cells, whereasthe embryosac con- tern, (ii) to differentiatethe primaryplant female reproductiveorgan (2-4). Because tains a single egg (Fig. 1). Other accessory tissue types, (iii) to generate a specialized formation,fertilization, and embry- cells within the haploid male and female storageorgan essential for seed germination ogenesisoccur within the pistil, it has been gametophyteshelp facilitatethe and seedling development,and (iv) to en- difficultto dissectthe majorevents that take and fertilizationprocesses (3, 19, 20). The able the sporophyteto lie dormant until placeduring the earlystages of higherplant male and female gametophytesare derived conditions are favorablefor postembryonic development. from specializedspore-forming cells within development. Recently, it has become feasible to iso- the reproductiveorgans of the flower(3, 4, late plant and fertilizethem in vitro in 21). By contrast, the diploid, or sporo- The Shoot-Root Body Plan Is order to investigate the initial events of phytic, generationbegins after fertilization Generated During Early planteribryogenesis (5). In addition,genet- with the zygoteand formsthe matureplant Embryogenesis ic approacheshave been used to identify with vegetative organs (, stem, root) requiredfor variousembryogenic pro- and that contain the reproductive How the embryoacquires its three-dimen- cesses, includingpattern formation(6, 7). organs(anther and pistil) (Fig. 1). sional shape with specializedorgans and Genetic manipulationof Arabidopsisthali- Two fertilizationevents occur in flower- tissues,and what networksorchestrate ana, by both chemical mutagenesis(8-12) ing plants (2, 22). One spermunites with embryonicdevelopment remain majorun- and insertionalmutagenesis (13-15), has the egg cell to producea zygoteand initiate resolvedproblems. From a descriptivepoint identifieda large numberof mutants that embryogenesis.The other uniteswith a spe- of view, plant embryogenesiscan be divided are blocked at differentstages of embryo- cializedcell within the embryosac (central into three generalphases in which distinct genesis.In this reviewwe outline the major cell) to initiate the differentiationof the developmentaland physiologicalevents oc- insightsthat have been derivedfrom studies ,a triploidtissue that is neither cur: (i) postfertilization-,(ii) of Arabidopsisembryo mutants, and we sum- gametophyticnor sporophyticin origin(Fig. globular-hearttransition, and (iii) organex- marize gene transcriptionexperiments in 1) (23). The endospermis present during pansion and maturation(26-28) (Fig. 2 other plants that providenew information seed developmentand providesnutrients for and Table 1). Although there is consider- about the processesregulating higher plant eitherthe developingembryo, the germinat- able variationin how embryosin different embryogenesis.Both the genetic and mo- ing seedling,or both (23). Fertilizationalso plant taxa form (29), the overalltrends are causesthe ovule, containingthe embryoand remarkablysimilar (29). We summarizethe Authors are in the Department of Biology, University of endosperm,to develop into a seed and the Capsella and Arabidopsispattern of embryo California,Los Angeles, CA 90024-1606, USA. to differentiateinto a , which development (29-33) because (i) it is one *To whom correspondence should be addressed. facilitatesseed dispersal(Fig. 1) (24). of the most well-studied forms of plant em-

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This content downloaded from 164.67.109.135 on Fri, 18 Jul 2014 20:08:48 PM All use subject to JSTOR Terms and Conditions Fig. 1. The life cycle of a 1 Pollen flowering plant with em- Egg cell formation, grains phasis and fertilization on egg cell forma- pollination, permcells tion and seed develop- -ube ment. [Adapted from (16, Pistil Surviving Embryo Female Style nucleus 26).J mothercell megaspore sac gametophyte(n) Male (2n) (n) \p ntipodal gametoht Nucellus CCentral cells gametophyte / > n~~~~Meiosis M itosis { cell XO (nr)fri - Me~~~~~~~sis F~~~~~~~~J~~~~ ~ Ovary-fruit Ovule rynergid Flower Flower Funiculus Integuments ~~~~Egg cells ~ 1I ~~~~~~~~~~~~~~~~~~~~~cell - Anther Micropyle // Pistil epal Germinating Dormant Developing Fertilizedendosperm Seedling seed seed seed nucle s (3n) aotyledo tyldo Endosperm LShootmeristemI11 1 ootmeristem Root FFAA Embryo Developing egg (2n) CotyledonsJ embryo

Seed developmentand germination bryogenesis,dating back to the classical the (Fig. 2). In Arabidopsis,the to be passedfrom the maternalsporophyte studiesof Hanstein,Schaffner, and Soueges suspensorcontains only 7 to 10 cells (Fig. into the developing proembryo(Fig. 2) with Capsella(30-32), (ii) it has an invari- 2). The suspensoranchors the embryoprop- (35). The suspensorsenesces after the ant division pattem duringthe earlystages, er to the surroundingembryo sac and ovule stage and is not a functional part of the which allows cell lineagesto be tracedhis- tissue and servesas a conduit for nutrients embryoin the matureseed. Derivativesof tologically (33), and (iii) recent studies with Arabidopsismutants have provided new insightsinto the processesthat control Posffertilization Globular-heart transition embryodevelopment (6, 9). Asymmetriccleavage of dtezygote results in Proembryo Pd G C theformation of an embryowith a suspensor and embryoproper that have distinct develop- MAT ~~~~JEP I:P mentalfates. The zygote in Arabidopsisand Capsellahas an asymmetricdistribution of cellularcomponents-the nucleusand most of the cytoplasmare present in the upper Zygote2ce 2/4-PE 8ce 3 Transition Heart portionof the cell, whereasa largevacuole EP 16-cell embryo embryo dominatesthe middleto lowerportion (Fig. EP Globular 2). This spatialasymmetry is derivedfrom embryo the egg cell (30). The zygotedivides asym- metricallyinto two distinct-sizeddaughter Organ expansion and maturation cells-a small, upper terminal cell and a Torpedo embryo Walking-stick embryo Mature embryo large, lower basal cell-which establish a CE polarizedlongitudinal axis within the em- s ~V)J~ \ SC bryo (Fig. 2) (2, 30-33). Histologicalstud- ies over the course of the past 125 years En SC have indicatedthat the terminaland basal Pd cells give rise to different regions of the matureembryo (29-33). The small termi- nal cell gives rise to the embryoproper that will form most of the matureembryo (Fig. ft RM RM 2). Cell lineagesderived from the terminal cell and embryoproper will specifythe cot- yledons, shoot ,hypocotyl region of the embryonicaxis (29-33), and part of Fig. 2. A generalized overview of plant embryogenesis. Schematic representations of embryonic stages are based on light microscopy studciesof Arabidopsis (33) and Capsella (30-32) embryo development. the ,or embryonicroot (Fig. 2) (34). Torpedo and walking-stick refer to specific stages of embryogenesis in Arabidopsis and Capsella. By contrast, the large basal cell derived Abbreviations:T, terminalcell; B, basal cell; EP, embryo proper; S, suspensor; Bc, suspensor basal cell; from the lower portion of the zygote will Pd, protoderm; u, upper tier; 1, lower tier; Hs, hypophysis; Pc, procambium; Gm, ground meristem; C, divide and forma highly specialized,termi- cotyledon; A, axis; MPE, micropylarend; CE, chalazal end; SC, seed coat; En, endosperm; SM, shoot nally differentiatedembryonic organ called meristem; and RM, root meristem.

606 SCIENCE * VOL.266 * 28 OCTOBER1994

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Table 1. Majorevents of flowering plant embryo- layerof the embryoproper, is producedand Embryogenesis Can Occur genesis. the hypophysisforms at the top of the sus- Without Surrounding pensor(Fig. 2). Subsequentcell differentia- Maternal Tissue Postfertilization-proembryo tion events within the embryoproper result Terminaland basal cell differentiation in the productionof an inner procambium It is unclearwhat influence, if any, mater- Formation of suspensor and embryo proper tissue layer and a middle layer of ground nal tissue or accessorycells of the female Globular-hearttransition Differentiationof major tissue-type primordia meristemcells (Fig. 2). The spatialorgani- gametophytehave on egg cell formation Establishment of radial (tissue-type) axis zation of protoderm,ground meristem, and and subsequent Embryo proper becomes bilaterally procambiumlayers establishes a radialaxis (Fig. 1). Forexample, do either the ovule or symmetrical of differentiatedtissues within the globular cells within the embryosac (for example, Visible appearance of shoot-root embryo. By contrast, the presence of the synergids)produce morphogeneticfactors (apical-basal) axis hypophysisat the basal end of the embryo that contributeto the establishmentof lon- Initiationof cotyledon and axis (hypocotyl-radicle)development proper establishesan apical-basalpolarity gitudinalasymmetry within the egg?Sever- Differentiationof root meristem within this region and indicates that the al arguments suggest that the maternal Organ expansion and maturation globularembryo is not radiallysymmetrical sporophyteprovides only physical support Enlargement of and axis by cell in the formalsense (Fig. 2). structuresand nutrientsfor the embryo(Fig. division and expansion A dramaticchange in the morphologyof 1). First, somatic cells from a variety of Differentiationof shoot meristem the embryo proper occurs just after the vegetativeand reproductivetissues can un- Formationof lipid and protein bodies globular stage. Cotyledons are specified dergo embryogenesisin cultureand lead to Accumulation of storage proteins and lipids Vacuolization of cotyledon and axis cells from two lateraldomains at the apical end the production of fertile plants (37-39). Cessation of RNA and protein synthesis (top), the hypocotylregion of the axis be- Somatic embryos undergo developmental Loss of water (dehydration) gins to elongate, and the root meristem events similar to those that occur within Inhibitionof precocious germination becomesdifferentiated from the hypophysis the embryo-properregion of zygoticembry- regionat the basalend (bottom) (34). The os, except that they do not becomedormant embryoproper is now heart-shaped,has a (Fig. 2 and Table 1). In addition, spatial bilateralsymmetry, and the body plan and and temporalgene expressionprograms ap- the uppermost cell of the suspensor, the tissue layers of the mature embryo (and pear to be similar in somatic and zygotic hypophysis (Fig. 2), contribute to the for- postembryonicplant) have been established embryos (39, 40). Second, embryo-like mation of the root meristem (30-34). Thus, (Fig.2). Morphogeneticchanges during this structuresleading to plantletscan form di- cell lineages derived from the basal cell give period are mediatedby differentialcell di- rectly from the attached of some rise to the suspensor and part of the radicle vision and expansionrates and by asymmet- plants (41). Third, produced by fer- region of the embryonic axis (Fig. 2). ric cleavagesin differentcell planes(2). No tilizing egg cells in vitro undergoembryo- Different gene sets must become active cell migration occurs, in contrast to the genesis in culture and give rise to flower- in the terminal and basal cells after the migrationevents that take place in many producingplants (5). Finally,ultrastructural division of the zygote. Whether the polar- types of embryos(1). studies suggest that there is a barrierbe- ized organization of the egg cell, the zygote, Embryogenesisterminates with a dormancy tween the inner ovule cell layer and the or both control differential period.A majorchange in embryonicdevel- embryo sac that prevents the transferof events early in embryogenesis is not known. opment occursduring the organexpansion material directly between these compart- For example, do prelocalized regulatory fac- and maturationphase (Fig. 2). A switch ments (42). Thus, both zygoticand somatic tors within the egg cell initiate a cascade of occurs during this period from a pattern embryogenesiscan occur in the absenceof events leading to the lineage-dependent formationprogram to a storageproduct ac- surroundingovule tissue (Fig. 1). differentiation of terminal and basal cell cumulationprogram in orderto preparethe The embryosac is necessaryfor zygotic derivatives? Alternatively, after fertilization young sporophytefor embryonicdormancy embryogenesisbecause it contains the egg does the zygotic genome direct the de novo and postembryonicdevelopment (Table 1). and associatedaccessory cells that are re- synthesis of regulatory factors that are dis- The cotyledons and axis increase in size quiredfor fertilizationand endospermde- tributed asymmetrically to the terminal and dramaticallyas a resultof and velopment (Fig. 1). However, the embryo basal cells at first ? In either case, expansion events (33). Ground meristem sac is not essentialfor embryogenesisper se these events would lead to the autonomous cells within both these organsbecome high- because(i) somaticembryos produced from specification of the terminal and basal cells ly specializedand accumulatelarge amounts sporophyticcells develop normally(5, 37- as a consequence of intrinsic factors rather of storage proteins and oils that will be 40) and (ii) embryoscan be inducedto form than extracellular signals (1). utilizedas a food sourceby the seedlingafter from microsporesthat, under normal cir- Embryonicorgans and tissue-typesdifferen- germination(Fig. 2 and Table 1) (33). One cumstances,give rise to pollen grains(43). tiate duringthe globular-hearttransition phase. differentiationevent does occurduring this These resultssuggest that normal embryo- Two critical events must occur after the period,however-the shoot meristemforms genic processesdo not requirefactors pro- embryo proper forms: (i) regions along the from cell layerslocalized in the upperaxis duced by either the female gametophyteor longitudinal apical-basal axis must differen- regionbetween the two cotyledons(Fig. 2) maternalsporophytic tissue. This conclu- tiate from each other and generate embry- (36). Thus, the differentiationof shoot and sion is supportedby the fact that the over- onic organ systems, and (ii) the three pri- root meristemsat oppositepoles of the em- whelming majorityof mutationsthat alter mordial tissue layers of the embryo need to bryonicaxis does not occurat the sametime embryodevelopment appearto be due to be specified. Both of these events take place (34, 36). At the end of the organexpansion defects in zygoticallyacting genes (6-14, during the globular-heart transition phase and maturation period the embryo has 44). It is possiblethat somaticcells have the (Fig. 2 and Table 1). The embryo proper reachedits maximumsize, cells of the em- potential to produceputative maternalor has a spherical shape during the proembryo bryo and surroundingseed layershave be- gametophyticfactors under the propercon- and globular stages (Fig. 2). The first visible come dehydrated, metabolic activities have ditions, or that somatic embryos specify cell differentiation events occur at the 16- ceased, and a period of embryonic dormancy their longitudinal apical-basal and radial cell stage when the protoderm, or outer cell within the seed begins (26-28, 33). tissue-type axes by different mechanisms

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This content downloaded from 164.67.109.135 on Fri, 18 Jul 2014 20:08:48 PM All use subject to JSTOR Terms and Conditions than zygoticembryos. However, most of the bryos containing chimeric ,B-glucuronidasegions (Fig.3G). This resultsuggests that the availabledata suggest that embryomorpho- (GUS) reportergenes driven by soybean preferentiallocalization of Kti3 mRNA at genesis and cell specification events are embryo-specific gene promoters showed the micropylarend of a soybean globular directed primarilyby the zygotic genome that a globularembryo is organizedinto stage embryo(Fig. 3C) is due to transcrip- after fertilizationoccurs. If so, then this distinct, nonoverlappingtranscriptional re- tional regulatoryprocesses. By contrast, woulddiffer significantly from the situation gions, or territories(Fig. 3, F to I) (47, 48). GUS activityis visibleas a uniformblue belt with many animalssuch as Drosophilaand Blue color resultingfrom GUS enzymeac- that surroundsthe equatorregion of a glob- , in which matemally supplied tivity occurs specificallyat the micropylar ular embryo containing a soybean lectin/ factors influence the pattem of early em- end of a globularstage embryo containing a GUS reportergene (Fig. 3H) (48). GUS bryodevelopment (1). Moreextensive stud- KI3/GUS reportergene (Fig. 3, F and G) activityis not visible at either the micropy- ies with developing female , (47). No GUS activity is observedwithin lar or the chalazal(apical) ends of the em- egg cells, and zygotic embryosin the early the suspensoror other embryo-properre- bryo proper (Fig. 3H) (48); nor is there stagesof embryogenesisare needed to clar- ify this issue. A ~~~B A GlobularEmbryo Contains DifferentiallyTranscribed Regions ~CE En. A largenumber of genes are expresseddur- ing embryogenesisin higher plants (26). Although it is not known how manygenes are necessary to programmorphogenetic and tissuedifferentiation processes, approx- imately 15,000 diversegenes are active in the embryosof plants as diverseas soybean and cotton (26). Many of these genes are expressedin specificcell types,regions, and organsof the embryo(26, 40) and provide usefulentry points to unravelthe molecular EP G - mechanismsthat regulatecell- and region- specific differentiationevents duringplant -~~~~~~~~~~~~~T embryogenesis(1). The axis region of the embryodoes not becomevisibly distinct until the heartstage ------%N;:i- G ifil:X ;0 0 (Figs.2 and 3, A and B). Localizationstud- ies with a soybeanKunitz trypsin inhibitor mRNA, designatedas Kti3 (45), indicated, however,that cells destinedto become the axis are already specified at the globular stage (40). Figure 3C shows that Kti3 mRNA accumulatesspecifically at the bas- Fig. 3. Differentialtranscriptional activity dunng early embryogenesis. (A and B)Brght-field photographs al, or micropylar,end of a late-globular of developingsoybean embryos sectioned longitudinally(40). (A) Late globular stage embryo.(B) Heart stage soybean embryo.No detectableKti3 stage embryo.(C to E) Localizationof the majorKunitz trypsin inhibitor mRNA (Kti3) in longitudinal mRNA is present in other regions of the sections of soybean embryos(40, 45). In situ hybridizationdata are similarto those shown in (40). embryoproper or in the suspensor(Fig. 3C) Photographswere takenby dark-fieldmicroscopy. (C) Late globular stage embryo.(D) Late globular to (40). In transitionand heartstage embryos, transitionstage embryo.(E) Heart stage embryo.(F) Bright-field photograph of a tobacco globularstage embryo sectioned longitudinally.(G and H) Localizationof GUS activitywithin transgenic tobacco Kti3 mRNA remainsdistributed asymmetri- globularstage embryos.Photographs of whole-mountembryos were takenby bright-fieldmicroscopy cally at the embryomicropylar end (Fig. 3, (G)Embryo contains a chimericKti3/GUS gene (47). (H)Embryo contains a chimericlectin/GUS gene D and E) and is localizedspecifically within (48). (I)Localization of GUS mRNAwithin a transgenictobacco globularstage embryosimilar to that the groundmeristem cell layer (Fig. 2) of shown in (H)containing a lectin/GUSgene (48).Abbreviations: EP, embryoproper; S, suspensor;C, the emerging embryonic axis (Fig. 3D) cotyledon;A, axis; En,endosperm; CE, chalazalend; MPE,micropylar end; and Pd, protoderm. (40). No detectableKti3 mRNA is present within the newly initiatedcotyledons (Fig. 3E). This result differsfrom that obtained WTseed WTseedling emb 30/gnom monopteros gurke fackel with the carrot EP2 lipid transferprotein mRNA, which is localizeduniformly in the outer protodermcell layer that surrounds H ff~~~~HH the embryoproper at the globularand heart stages (46). Taken together, these results indicate that cells along the longitudinal apical-basalaxis of the embryoproper are Complete Complete Apical 8) Central Apical Central 0* Basal 0 Basal *0 8) already differentiatedfrom each other at the globularstage and that earlyin embry- Fig. 4. Schematic representations of Arabidopsis pattem mutants [adapted from (9)].The green, yellow, ogenesis distinct gene sets are expressedin and orange colors delineate the apical, central, and basal regions, respectively. Strong (upper)and weak differentembryonic regions and cell types. (lower)gnom phenotypes are depicted (9, 15). Abbreviations: WT, wild-type; RM, root menstem; SM, Transformationstudies with tobaccoem- shoot meristem; C, cotyledon; h, hypocotyl; and R, root.

608 SCIENCE * VOL.266 * 28 OCTOBER1994

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detectableGUS activitywithin the suspen- 48). These resultssuggest that a prepattem cotyledonsand shoot meristem,the central sor (Fig. 3H) (48). GUS mRNA localiza- of different transcriptionalregulatory do- region consists of the hypocotyl, or upper tion studieswith longitudinalglobular em- mains has been establishedin the globular axis, and the basalregion includes the lower bryo sections indicatedthat the lectinpro- embryo before the morphogeneticevents axis, or radicle,and the root meristem(Fig. moteractivity occurs specifically within the that lead to differentiationof cotyledonand 4). These regions are derived ultimately groundmeristem cell layer of the equator axis regionsat the heartstage (Figs. 2 and 3, from the terminal and basal cell lineages region (Fig. 31) (48). A and B). Presumably,each transcriptional and are maintainedin the young seedling These experiments indicate that both domain sets in motion a cascadeof events (Figs.2 and 4). Can regionsalong the lon- the longitudinalapical-basal and radialtis- leading to the differentiationof specific gitudinal axis develop independently of sue-type axes of a globularembryo are par- embryoregions later in embryogenesis. each other?If territoriesestablished within titioned into discrete transcriptional terri- the embryoproper of a globularstage em- tories (1). The longitudinalaxis of the em- Hormones Affect Embryo bryo are specified autonomously,then the bryoproper contains at least threenonover- Morphogenesis loss or alterationof cells within a territory lapping transcriptionalterritories: (i) the shouldnot affectthe developmentof a con- chalazalregion, (ii) the equatorregion, and What physiologicalevents cause the em- tiguousregion (Figs. 2 and 3). Several ex- (iii) the micropylarregion (Fig. 3, F to H). bryo properto initiate cotyledonsand be- perimentssuggest that this is actually the The suspensor represents an additional come bilaterallysymmetric during the glob- case-that is, a plant embryoforms from transcriptionaldomain along this axis (Fig. ular-hearttransition phase (Fig. 2)? Several modules that develop independently of 3, G and H). Each tissue layerof the radial experimentsimplicate a class of plant hor- each other (9-12, 54-59). embryo-properaxis also has a distinct tran- mones, the auxins, in this morphogenetic Patternmutations delete specific embryonic scriptionalprogram (Fig. 3, E, G, and I). process(49-52). Auxins, such as indoleace- regions. Genetic studies have uncovered Transcriptionalactivity within these layers, tic acid (IAA), are involved in a numberof Arabidopsismutations that alter the organi- however, appears to be established in a plant activities, includingphoto- and grav- zation of the embryobody plan (Fig. 4 and territory-specificmanner-that is, ground itropism, apical dominance, and vascular Table 2) (9, 10). Embyropattem mutations meristemcells within the equator region cell differentiation(2). Embryosin plantsas were found that delete (i) the apicalregion activatepromoters distinct from those with- diverseas bean and pine synthesizeauxins (gurke),(ii) the centralregion (fackel),(iii) in groundmeristem cells of the micropylar (49) and transportthem in a polarized, the central and basal regions(monopteros), region, and vice versa (Fig. 3, E to I) (47, basipetaldirection from the shoot meristem and (iv) the apical and basal regions to root tip along the embryonicaxis (Fig.2) (emb30/gnom)(Fig. 4) (9). Other embryo (50). The highest auxin levels occur at the pattern-forminggenes probablyexist; how- Table 2. Examples of Arabidopsis mutants that globular stage of embryogenesis (49). ever, they have not been identifiedin the have defects in embryo development. Several Agents that inhibit polarizedauxin trans- genetic screens carried out thus far (6, hundred Arabidopsis embryo-defective mutants either block the transition from the 8-10, 14, 44). Each mutant gene acts zy- have been identifiedby both chemical and T-DNA port mutagenesis. Most of these mutants can be ob- globularto heart stage completely (51) or gotically,indicating that majorspecifiers of tained from the Arabidopsis Biological Resource prevent the bilateral initiation of cotyle- the embryobody plan act afterfertilization Center ([email protected]). dons at the top of the globularembryo (Fig. has occurred(Fig. 2) (9, 10). In addition, 2) (52). Forexample, auxin transport inhib- the loss of a specificregion, or combination Mutant class References itors cause carrot somatic embryosto re- of regions,does not affect the development main sphericallyshaped and develop into of an adjacentneighbor (Fig. 4) (9, 10). For Pattern mutants contrast, example,gurke embryos lack an apical re- emb3O/gnom 8-11, 15 giant globularembryos (51). By monopteros 9, 12 zygotic embryos of the Indian mustard gion, but have normal central and basal gurke 9 (Brassicajuncea), an Arabidopsisrelative, fail regions(Fig. 4) (9). monopterosembryos, by fackel 9 to initiate two laterallypositioned cotyle- contrast, have a normal apical region but Cell-type differentiationmutants dons when treated with auxin transport are missing the central and basal regions keule 9 blockersin culture (52). A cotyledon-like (Fig. 4) (9). knolle 9 organ does form, but as a collar-like ring Embryopattem mutationsalter the di- Suspensor transformation mutants aroundthe entire upper (apical) region of vision planesthat areestablished during the twvin 73 the embryo (52). Treated Indian mustard postfertilization-proembryophase of embry- sus1 67 sus2 67 embryosresemble those of the Arabidopsis ogenesis (Fig. 2) (11, 12). Thus, deletions sus3 67 pini-1 mutant,which has a defect in polar- of mature embryo regions can be traced raspberryl 44 ized auxin transport(52, 53). These results back to histologicaldefects at the proem- raspberry2 44 suggest that auxin asymmetriesare estab- bryo and globular stages (Fig. 2) (1 1, Meristem differentiationand identity mutants lished within the embryo-properregion of 12)-a result that providesfunctional evi- shoot meristemless 36 globularstage embryos (Fig. 3, A and F) and dence for the embryofate mapsproposed by embryonicflower 60 that these asymmetriescontribute to the the developmental botanists of the late short-root 61, 62 bilateral at the 19th and early20th centuries(31, 32). For hobbit 61, 62 establishmentof symmetry heart stage (Figs. 2 and 3, A and B). example,emb30/gnom zygotes do not divide Maturationprogram mutants lec1- 1/lec1-2 44, 76-78 asymmetrically(Fig. 2) ( 11). Two similar- Iec2 77 Plant Embryos Form from sized daughtercells are produced in the fus3 79, 80 Regions That Develop emb30/gnomembryo-proper region instead abi3 84, 85 Autonomously of the unequal-sizedterminal and basalcells Seedling lethalitymutants that are found in wild-typeembryos (11). fus1/cop 1 91, 94 The longitudinal axis of a mature plant Laterdivision events in emb30/gnomembry- fus2/det1 91, 95 embryo is made up of several regions that os are also highly variable and abnormal fus6/copl11 91, 96 the monopterosdivision fus7/cop9 91, 97 are designated as apical, central, and basal (11). By contrast, (Fig. 4) (9). The apical region contains the pattern is normal until the 8-cell embryo-

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This content downloaded from 164.67.109.135 on Fri, 18 Jul 2014 20:08:48 PM All use subject to JSTOR Terms and Conditions proper stage (Fig. 2) (12). During the there are genes which regulatethe specifi- to the cotyledonsand shoot meristemof a monopterosglobular-heart transition phase, cation, organization,and fate of meristems transgenictobacco embryo(Fig. 5D) (54). lower tier cells of the embryoproper and that differentiate during embryogenesis. These data, and those of others (55-59), derivativesof the hypophysisundergo ab- Genes controlling meristem development indicatethat uniquetranscription factors are normal divisions (12) (Fig. 2). monopteros most likely act downstreamof embryo-re- active within each embryonicregion and upper tier embryo-propercells, however, gion specifiers,such as monopterosand gurke that these factorsinteract with specificpro- develop normally-that is, defects at the (Fig. 4) (9, 10, 12), in the regulatoryhier- moter elements.The combinationof these basalend of the embryoproper do not affect archyneeded to form a plant embryo. elements and factorsgives rise to the tran- events that occur at the apicalend (Fig. 2) Meristemmutants characterized to date scriptional pattem of the whole embryo (12). The resultis a mutantembryo that has indicate that the shoot and root meristems (Fig. 5A). Identification of transcription cotyledons and a shoot meristem, but is function autonomously-that is, they do factors that interact with region-specific missingthe hypocotyland root regions(Fig. not affect the differentiationof contiguous DNA elementsshould provide reverse entry 4) (12). These observationsindicate that domains such as the cotyledons or hypo- into the independentregulatory networks genes which are responsible,in part,for the cotyl (36, 60-62). Meristems,therefore, requiredfor specifyingeach autonomousre- establishmentof the embryobody plan di- representindependent submoduleswithin gion of a plant embryo. rect territory-specificcell division patterns the apical and basal regionsof the embryo duringearly embryogenesis. Abnormal divi- (Fig. 4). A majorquestion is what effect, if Cell Differentiation and sions in one embryonic territorydo not any, do cells adjacentto the shoot and root Morphogenesis Can Be affect the division pattern of an adjacent meristems have on their development? Uncoupled in Plant Embryos territory-that is, cell lineagesgiving rise to That is, arecell signalingevents involvedin specific regions of the matureembryo de- specifying the root and shoot meristems What is the relationbetween cell differen- velop autonomously. within a specific embryonicregion, or do tiation and morphogenesisin plant embry- AgrobacteriumT-DNA-tagged emb30/ the meristemsdifferentiate autonomously? os? Are processesrequired for tissue differ- gnomalleles have led to the isolationof the The latememutant provides one answerto entiation along the embryoradial axis cou- EMB30/GNOMgene (15). This gene en- this question (9). laterne lack pled to those that specify autonomousre- codes a protein that is relatedto the yeast cotyledons but produce leaves indicating Sec7 secretoryprotein, is active throughout that a shoot meristemis present (9). Thus, the plant life cycle, and is involved in cell cotyledons are not requiredfor the differ- division, elongation, and adhesion events entiation of the shoot meristem during requiredat many stages of sporophyticde- embryogenesis. velopment, including embryogenesis(15). Promoterelerments interpret embryo region- Thus, EMB30/GNOMdoes not appearto specificregulatory network1s. One consequence establishthe embryoniccell division pat- of the modularorganization of a plant em- tern directly, but most likely facilitates a bryois that geneswhich are active through- patternset by other genes. What these pat- out the embryomust intersectwith several tern-forminggenes are and how they inter- region-specificregulatory networks-that is, act with downstreamgenes that mediate the promotersof embryo-specificgenes are events requiredfor the differentiationof requiredto senseand interpretthe transcrip- autonomousregions along the longitudinal tional regulatorymachinery unique to each axis remainto be determined. autonomousregion (Fig. 4). For example, Mutationsaffect meristernatic zones of the Kti3 mRNA accumulateswithin the axis embryolongitudinal axis. Arabidopsismuta- region early in soybeanembryogenesis, but tions have been identified that affect the does not accumulatewithin the cotyledons differentiationof the shoot and root meri- until much later(Fig. 3, C to E) (40). Thus, stems duringembryogenesis (Table 2) (36, discretepromoter elements should exist that 60-62). These mutationstarget a specific areresponsible for interacting with transcrip- meristemand have no other effect on em- tion factorsproduced by separateregulatory bryonic development. For example, shoot circuits. meristemlessfails to differentiate a shoot A Kti3/GUSgene with 2 kb of 5' flank- meristem during embryogenesisand pro- ing sequenceis transcribedin all regionsof a Fig. 5. DNA elements program transcrption to duces seedlingswithout leaves (36). embry- maturetransgenic tobacco embryo (Fig. 5A) specific embryonic regions. Maturationstage em- onicflower, on the other hand, generatesa (54). Deletion of 0.2 kb from the 5' end bryos were hand-dissected from transgenic to- bacco containing the E coi GUS gene shoot meristemat the top of the embryonic eliminatesKti3/GUS transcriptional activity fused with differentsoybean seed protein gene 5' axis (Figs. 2 and 4) (54). Remarkably,em- within the embryoradicle region (Fig. 5B) regions (54). GUS activity in whole-mount embry- bryonicflower seedlings produce flowers (54). Deletion of another 1 kb eliminates os was localized as outlined in (47), and photo- ratherthan leaves, indicatingthat the fate Kti3/GUStranscription within the cotyle- graphs were taken with the use of dark-field mi- of the shoot meristemis alteredduring em- dons and shoot meristem,but still permits croscopy. (A to C) Kti3 gene upstream regions bryogenesis-a floral meristemis specified transcriptionto occurwithin the hypocotyl fused with the E coli GUS gene. (A) A 2-kb Kti3 rather than a vegetative shoot meristem region(Fig. 5C) (54). These resultsindicate gene 5' fragment. (B) A 1.7-kb Kti3 gene 5' frag- (60). By contrast,short root and hobbitaffect that discretecis-acting domains are required ment. (C) A 0.8-kb Kti3 gene 5' CaM\//GUS frag- root meristemdevelopment (61, These for the activationof the Kti3 ment. (D) A minimal CaMV/GUS gene-promoter 62). transcriptional cassette (59) fused with a 0.36-kb Gyl gene 5' mutations lead to abnormalroot develop- gene within the radicle,hypocotyl, and cot- region(-446 to -84) (63).The whiteembryo re- ment after seed germination, indicating yledon-shoot meristem regions of the em- sults fromthe segregationof a single Gy1/GUS that the root meristemis altered,but not bryo. Promoter analysis of the soybean Gyl gene withinthis transgenictobacco lineand rep- eliminated,during embryogenesis (61, 62). storage protein gene (63) also uncovered a resents a negativecontrol. Abbreviations: C, cot- Taken together,these resultsindicate that regulatory domain that directs transcription yledon;H, hypocotyl;and Rd, radicle.

610 SCIENCE * VOL.266 * 28 OCTOBER1994

This content downloaded from 164.67.109.135 on Fri, 18 Jul 2014 20:08:48 PM All use subject to JSTOR Terms and Conditions ~~ ARTICLES gions of the longitudinalapical-basal axis genesis.For example, EP2 lipid transferpro- (Table 2). Surprisingly,raspberry 1 embryos (Fig. 2)? Studiesof Arabidopsisembryo pat- tein mRNA accumulatesspecifically within accumulateEP2 and 2S2 markermRNAs in tern mutants suggest that these processes the epidermalcell layer (Fig. 6B) (46, 64, theircorrect spatial context alongthe radial are not necessarilyinterconnected. For ex- 65), and 2S2 albuminmRNA accumulates axis of both the embryo-properand suspen- ample, a fackel embryo does not have a within storageparenchyma cells (Fig. 6C) sor regions(Fig. 6, F to I) (64). EP2mRNA hypocotyl,but epidermal,ground meristem, (64, 66). Neither mRNA is detectable accumulatesalong the outer perimeterof and vasculartissues differentiate within cot- within the vascularlayer (Fig. 6, B and C) raspberry1 embryos (Fig. 6, F and G), where- yledon and radicle regions (Fig. 4) (9). (46, 64-66). Collectively,the EP2and 2S2 as 2S2 mnRNAaccumulates within interior Thus, the loss of one embryonicregion does mRNAs can identifyembryo epidermal and cells (Fig.6, H and I) (64). By contrast,EP2 not affect the formation of tissue layers storageparenchyma cell layersand, by de- and 2S2 mRNAsdo not accumulatedetect- within the remainingregions (Fig. 4) (9- fault, the inner vasculartissue as well (Fig. ably within the central core of raspberry) 12). A more direct question, however, is 6, B and C). embryos(Fig. 6, F to I) (64). Similarresults whethermutant embryos that arrestearly in An Arabidopsisembryo mutant, desig- wereobtained with raspberry2embryos (Ta- embryonicdevelopment and remainglobu- nated raspberry1,was identifiedin a screen ble 2) (44, 64). lar-shapeddifferentiate the specializedcell of T-DNA-mutagenizedArabidopsis lines These mRNA localizationstudies indi- and tissue layers that are found in organ (Table2) (44). This mutantfails to undergo cate that specializedtissues can differentiate systemsof a mature,wild-type embryo. the globular-hearttransition (Fig. 2), has an within the embryo-properregion of mutant A maturationstage Arabidopsisembryo embryo-properregion that remainsglobu- embryosthat remain globularshaped, and has specializedepidermal, storage parenchy- lar-shapedthroughout embryogenesis, and that these tissuesform in theircorrect spatial ma, and vascularcell layerswithin both the does not differentiatecotyledons and axis contexts. A similarconclusion was inferred cotyledonand axis regions(Fig. 6A). These (Fig. 6, D and E) (64). raspberrylembryos from histologicalstudies of the sus mutants tissues are derivedfrom the three primary also have an enlargedsuspensor region (Fig. (67). Tissue differentiation,therefore, can cell layersthat arespecified along the radial 6, D and E) (64). raspberry2(44) and sus take place independentlyof morphogenesis axis of a globularembryo (Fig. 2), and ex- (67) embryo-defectivemutants also have in a higherplant embryo, implying that mor- pressspecific markergenes late in embryo- phenotypes similar to that of raspberyl phogeneticcheckpoints do not occurbefore cell differentiationevents can proceed(68). It does not follow, however, that morpho- genesiscan occurwithout proper cell differ- entiation events. Arabidopsisembryo mu- tants that alter tissue-specificationpatterns have abnormalmorphologies (Table 2) (9). For example,knolle embryos lack an epider- mal cell layer and have a round, ball-like shape without defined apical and basal re- gions(Table 2) (9). Similarly,the carrottsl 1 somaticembryo mutant has a defectivepro- todermcell layerand fails to undergomor- phogenesis(38, 39). Addition of either an extracellularchitinase (69) or Rhizobium nodulation factors (lipooligosaccharides) (70) can rescuethe tsl 1 mutant.Lipooligo- saccharide nodulation factors have been 4~4 shownto be signalmolecules involved in the differentiationof Rhizobium-inducedroot nodules (70). This suggeststhat in carrot somatic embryos,the protodermcell layer may provide signals necessaryfor embryo- -~~~~~~~-..--~~ ~ ~ ~ ~ ~ ~~~~.... genesis to occur (69, 70). Taken together, experimentswith mutantembryos that have defectivecell layerssuggest that specification of the radialaxis needs to occurin orderfor Wt@=~~~~~~~~~~~k a normalshoot-root axis to form.An impor- Fig. 6. Localization of cell-specific mRNAs wHthinwild-type and mutant Arabidopsis embryos. (A) tant corollaryis that cells within the radial Brght-field photograph of a late-maturation stage Arabidopsis embryo sectioned longitudinally(64). (B axis probablyinteract with each other (9). and C) In situ hybndization of labeled RNA probes with longitudinalsections of Arabidopsis maturation stage embryos (64). Photographs taken by dark-field microscopy. (B) Localization of Arabidopsis EP2 Suspensor Cells Have the lipid transfer protein mRNA (65). (C) Localization of 2S2 albumin mRNA (66). (D and E) Arabidopsis Potential to Generate an Embryo raspberryl embryos (44). Embryos were harvested at a stage when wild-type embryos within the same silique were in late maturation[as in (A)].(D) Longitudinalsection of a raspbenyl embryo. The photograph One intriguingaspect of the raspberry)em- was taken by bright-fieldmicroscopy. (E)Araspberryl embryo photographed with Nomarski interference bryois its largesuspensor (Fig. 6, D and E). optics. (F to 1).In situ hybridizationof labeled RNA probes with raspberryl embryo longitudinalsections raspberry] are indistinguishable (64). (F and G) Localization of EP2 lipid transfer protein mRNA (65). Localization experiments with raspberry2 embryos, which have larger suspensors than raspberTyl embryos (44), confirmed that EP2 from wild-type during the early stages of mRNA is present only in the suspensor outer cell layer (64). (H and 1)Localization of 2S2 albumin mRNA embryogenesis(Fig. 2) (64). Later in seed (66). In situ hybridizationwith serial sections through an entire raspberryl embryo confirmed that there is development,when neighboringwild-type an inner core of cells with no detectable 2S2 mRNA (64). Abbreviations:A, axis; V, ; Ed, embryosundergo maturation, cell prolifera- ; C, cotyledon; P, storage parenchyma; Ep, embryo proper; S, suspensor; and En, endosperm. tion events cause the raspbery) suspensor

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This content downloaded from 164.67.109.135 on Fri, 18 Jul 2014 20:08:48 PM All use subject to JSTOR Terms and Conditions to enlargeat its basalend (Fig. 6, D and E) Fig. 7. An Arabidopsis leafy cotyle- (64). EP2 and 2S2 mRNAs (46, 65, 66) don seedling. An allele of the lecl accumulatein the raspberry1 suspensor (Fig. gene (76, 77), designated as lec1-2, 6, F to I) with a spatial pattem similarto was identifiedin a screen of T-DNA- that which occursin mature,wild-type em- mutagenized Arabidopsis lines (44, __ |78). (A and B) Bright-field photo- bryos (Fig. 6, B and C) (64). These cell- graphs of Arabidopsis seedlings specific mRNAs do not accumulatedetect- a | (78).Wi (A) Wild-type seedling. (B) ablyin wild-typesuspensors, or in raspberry1 lecl-2 seedling. Abbreviations: C, suspensors early in embryogenesis(64). -| | M- cotyledon; H, hypocotyl; R, root; These results indicate that the raspberry1 and Tr, tnchome. suspensorhas enteredan embryogenicpath- way and that an embryoproper-like, radial tissue axis has been specified. Other Arabidopsisembryo mutants have suspensorabnormalities similar to that of raspberryl,including raspberry2,and sus gram is induced that is responsiblefor (i) (76-78). Other lec mutants, such as lec2 (Table 2) (14, 35, 44, 67, 71). Although synthesizinglarge amounts of storageprod- and fus3, have phenotypic characteristics the extent of suspensorenlargement varies, ucts, (ii) inducingwater loss and a desiccat- that overlapthose of lecl (77-80). For ex- all of these mutants have morphological ed state, (iii) preventingpremature germi- ample, fus3 and lecl embryos are almost defects in the embryoproper (14, 35, 44, nation, and (iv) establishinga state of dor- indistinguishablefrom each other (77). lec2 67, 71). Mutant embryos that resemble mancy (Fig. 2 and Table 1) (2, 26-28). embryos,by contrast,have leaf-likecotyle- wild-type,but arrestat specific embryonic Severalspecialized gene sets, such as those dons, but are desiccation tolerant, do not stages,do not have aberrantsuspensors (44, encoding storageproteins and late embryo germinateprecociously, and have normal 64). Disruptionsin embryo-propermorpho- abundant(lea) proteins,are activatedtran- levels of storagebodies in their axis region genesis, therefore,can induce an embryo scriptionallyduring maturation and then re- (77). This indicates that leafy cotyledons proper-likepathway in terminallydifferen- pressedbefore dormancy (26, 74, 75). These can occur without correspondingdefects in tiatedsuspensor cells, a resultfirst observed gene sets remaintranscriptionally quiescent desiccation and dormancy,and that wild- by the embryo-properablation experiments duringseed germinationwhen germination- type LEC genes are activatedindependent- of Haccius30 yearsago (72). The Arabidop- specific and postembryonicgene sets are ly in each embryonicorgan system (77). A sis mutantrepresents a strikingexam- transcribed(26, 55, 75). Genetic studies corollaryis that gene networksthat func- ple of the embryogenicpotential of the with Arabidopsishave identifiedsome of the tion in desiccationand dormancyare inde- suspensorregion (73). tuin causes subtle genes that regulateprocesses required for pendent of those responsiblefor cotyledon defectsto occurin embryo-propermorphol- embryomaturation and dormancy,includ- cell specialization. ogy, generatesa second embryowithin the ing the expressionof storageprotein and lea Molecularstudies with lec embryoshave seed fromproliferating suspensor cells, and genes (Table 2). indicatedthat the transcriptionof matura- resultsin twin embryosthat are connected Leafycotyledon mutations disrupt embryo tion-specificgenes, such as those encoding by a suspensorcell bridge(73). maturation.A mutantgene class,designated storage proteins, is greatly reduced (80). The natureof mutant genes that affect as leafycotyledon (lec), has been identified Conversely,the transcriptionof germina- suspensordevelopment, such as raspberry1, that causes defects in the cotyledon cell tion-specificgenes, such as isocitratelyase, is sus,and twin, is not known.These genes are differentiationprocess and in maturation- activated(78). These data,coupled with the probablynot involved in suspensorspecifi- specificevents such as storageproduct accu- histologicaldescriptions of lec embryos(Fig. cation events, because a normal suspensor mulation,desiccation tolerance, and main- 7) (76-80), indicatethat LEC genes func- formsbefore induction of the embryo-prop- tenanceof dormancy(Table 2) (76-80). lec tion duringmaturation to activategenes in- er pathwayin mutantembryos (44, 64, 67, mutationstransform cotyledons of embryos volved in cotyledoncell specialization,stor- 71, 73). They reveal, however, that inter- and seedlingsinto leaf-likestructures (76- age product accumulation,induction and actions occur between the suspensorand 80). Wild-type cotyledons do not have maintenanceof embryonicdormancy, and embryo-properregions. One possibility is (Fig. 7A). Trichomesare present desiccationtolerance (76-80). LEC genes that the embryoproper transmitsspecific only on leaf, stem, and surfacesin also simultaneouslysuppress the manifesta- inhibitorysignals to the suspensorthat sup- wild-type plants and are markers for tion of leaf-likecharacteristics in cotyledons pressthe embryonicpathway (35, 67, 71- postembryonicdevelopment (81). By con- during embryogenesis,including 73). Altematively,a balanceof growthreg- trast, lecl cotyledons have trichomes on specification.In the absenceof LEC prod- ulatorsmight be establishedwithin the en- their adaxial surface which differentiate ucts, embryoniccotyledons enter a default tire embryo that maintains the develop- duringembryogenesis from the protoderm state and express leaf-like characteristics mental states of both the embryo-proper cell layer (Fig. 7B) (76-78). lecl cotyle- (76, 77), manyof whichdevelop normally in and suspensorregions. Disruptions of such dons have other leaf-like characteristics postgermination,wild-type cotyledons (82). signalswould cause the suspensorto take on includingstomata, mesophyll cells, and an Abscisicacid maintauns embiryonic donman- an embryoproper-like fate, a result analo- absenceof protein and lipid storagebodies cy. The planthormone abscisic acid (ABA) gous to embryoinduction in differentiated (77). The axis region of lecl embryosalso is involved in several plant processes,in- sporophyticor gametophyticcells (37-39). lacks storage organelles, indicating that cluding , responsesto environ- the wild-type LEC1 gene functions in mental stresses, growth inhibition, and The Embryo Is Reprogrammed both embryonicorgans (77). maintenanceof a dormantstate (83). Ex- Late in Embryogenesis In addition to the leaf-like cotyledon ogenousABA preventsseed germinationas transformation,lecl embryos(i) germinate well as the precociousgermination of em- How does the embryo prepare for dormancy precociouslyabout 5% of the time, (ii) have bryos in culture. In addition, Arabidopsis and postembryonic development (Fig. 1)? leaf primordiaemerging from their shoot mutantsthat either cannot synthesizeABA Late in embryogenesis a maturation pro- apex, and (iii) fail to survive desiccation or fail to respondto ABA germinatepreco-

612 SCIENCE * VOL.266 * 28 OCTOBER1994

This content downloaded from 164.67.109.135 on Fri, 18 Jul 2014 20:08:48 PM All use subject to JSTOR Terms and Conditions _ _ __ RTICLES| _ m A gS11 sol.r.1-111.1". ciously (84, 85). These data indicate that ering plants (10, 90-92). Several fusca Biology of Plants (Worth, New York, 1992). 3. L. Reiser and R. L. Fischer, 5, 1291 (1993). ABA preventsgermination while seeds are genes have been shown to be alleles of 4. C. S. Gasser and K. Robinson-Beers, ibid., p. 1231. still dormantor presentwithin siliques. constitutive photomorphogenic(cop)/deeti- 5. E. Kranzand H. Lorz, ibid., p. 739; J.-E. Faure, H. L. Three mutantArabidopsis loci, designat- olated (det) genes that function in light- Mogensen, C. Dumas, H. Lorz, E. Kranz, ibid., p. 747; C. Dumas and H. L. Mogensen, ibid., p. 1337; ed as abil, abi2, and abi3, have been iden- regulateddevelopment during seed germi- J.-E. Faure, C. Digonnet, C. Dumas, Science 263, tified that result in ABA insensitivityand nation (Table 2) (91-97). The productsof 1598 (1994). allowseed germinationto occurin the pres- COP/DET loci appear to suppresslight- 6. D. W. Meinke, Dev. Genet. 12, 382 (1991); Plant Cell ence of ABA (84). In addition to preco- regulatedgene activities in the dark and 3, 857 (1991). 7. W. F. Sheridan, Annu. Rev. Genet. 22, 353 (1988); J. cious germination,abi3 embryos are also activate these activities in the presenceof K. Clarkand W. F. Sheridan, Plant Cell 3, 935 (1991). desiccationintolerant and defective in the light by way of a light-mediated signal 8. D. W. Meinke and 1. M. Sussex, Dev. Biol. 72, 50 synthesis of maturation-specificmRNAs, transductionpathway (91-97). Becausede- (1979); ibid., p. 62; D. W. Meinke, Theor.Appl. Gen- et. 69, 543 (1985). such as those encodingstorage proteins and fective copldetgenes are detected as fusca 9. U. Mayer, R. A. Torres-Ruiz,T. Berlath, S. Mis6ra,G. lea proteins (85, 86). This indicates that embryomutants, their wild-typeCOP/DET Jurgens, Nature 353, 402 (1991); in Cellular Com- the wild-typeABI3 gene is a positive regu- alleles are active duringmaturation. Thus, munication in Plants, R. M. Amasino, Ed. (Plenum, New York, 1993), p. 93. lator of gene networks leading to storage regulatorygenes expressedat the end of 10. G. JOrgens, U. Mayer, R. A. Torres-Ruiz, T. Berlath, product accumulation, desiccation, and embryogenesisprepare the plant for life af- S. Mis6ra, Development 1 (suppl.), 27 (1991). dormancy(85, 86). The ABI3 gene encodes ter the seed. 11. U. Mayer, G. BOttner,G. Jurgens, Development 117, 149 (1993). a transcriptionfactor (87) related to the 12. T. Berlath and G. Jurgens, ibid. 118, 575 (1993). corn viviparous-Iprotein, which can acti- Conclusion 13. K. A. Feldmann, Plant J. 1, 71 (1991); N. R. vate the transcriptionof chimericGUS re- Forsthoefel, Y. Wu, B. Schulz, M. J. Bennett, K. A. Feldmann, Aust. J. Plant Physiol. 19, 353 (1992). porter genes containing embryo matura- Plant embryogenesisprovides a vital bridge 14. D. Errampalliet al., Plant Cell 3, 149 (1991); L. A. tion-specific gene promoters(88). Thus, between the gametophyticgeneration and Castle et al., Mol. Gen. Genet. 241, 504 (1993). ABI3 mediatesits effect on embryomatu- postembryonicdifferentiation events that 15. D. E. Shevell et al., Cell 77, 1051 (1994). rationat the transcriptionallevel. Because occur continuouslyin the shoot and root 16. R. B. Goldberg, Science 240, 1460 (1988). 17. V. Walbot, Trends Genet. 1, 165 (1985). aba embryos, which fail to synthesize meristems of the sporophytic plant. As 18. R. Chasan and V. Walbot, Plant Cell 5,1139 (1993). ABA, are normal with respect to most such, plant embryosmust establishthe po- 19. S. McCormick, ibid., p. 1265. maturation-specific processes (84-86), larizedsporophytic plant body plan and en- 20. J. P. Mascarenhas, ibid., p. 1303. 21. R. B. Goldberg, T. P. Beals, P. M. Sanders, ibid., p. ABA probablydoes not regulatethe ABI3 able the youngplant to surviveharsh envi- 1217. gene (84, 86). Rather,ABI3 probablyop- ronmentalconditions and a period of be- 22. S. D. Russell, ibid., p. 1349. erates through an ABA-independent low-groundgrowth from seeds. Plant em- 23. M. A. Lopes and B. A. Larkins,ibid., p. 1383. 24. G. Gillaspy, H. Ben-David, W. Gruissen, ibid., p. pathway that is involved in establishing bryos are simpler than their animal 1439. desiccation and dormancy states late in counterparts,yet they must carryout the 25. E. S. Coen and R. Carpenter, ibid., p. 1175; J. K. embryogenesis(85, 86). Failureto achieve same developmentaltasks-that is, form a Okamuro, B. G. W. den Boer, K. D. Jofuku, ibid., p. 1183. these developmentalstates resultsin ABA three-dimensionalorganism with special- 26. R. B. Goldberg, S. Barker, L. Perez-Grau, Cell 56, insensitivity-that is, ABA responsive- ized regions,compartments, and cell-types 149 (1989). ness is a consequenceof ABI3 gene activ- from a single-celled zygote. These events 27. K. Lindsey and J. F. Topping, J. Exp. Bot. 44, 359 ity (85, 86). lec embryosare sensitive to occur early in plant embryogenesisand are (1993). 28. M. A. L. West and J. J. Harada, Plant Cell 5, 1361 ABA and fail to germinate if ABA is poorly understood.Genetic studiesin Ara- (1993). present (77, 78). Thus, LEC and ABI3 bidopsishave begun to revealgenes that are 29. V. Raghavan,Experimental Embryogenesis in Vas- genes are part of independent regulatory necessaryfor embryogenicevents such as cular Plants (Academic Press, New York, 1976); S. Natesh and M. A. Rau, in of Angio- networksthat control embryomaturation- patternformation, cell differentiation,and , B. M. Johri, Ed. (Springer-Verlag, Berlin, specific events. organdevelopment. 1984), chap. 8; B. M. Johri, K. B. Ambegaokar, P. S. In contrastto abi3embryos, abil and abi2 The precise molecularmechanisms re- Srvastava, ComparativeEmbryology of Angio- sperms (Springer-Verlag,Berlin, 1992). embryoscarry out normalmaturation-specif- sponsible for specifyingdifferent cell lin- 30. M. Schaffner, Ohio Nat. 7, 1 (1906); R. Schulz and ic events,including the activationof storage eages early in plant embryogenesisare not W. A. Jensen, J. Ultrastruct. Res. 22, 376 (1968); protein and lea genes (84-86). ABI1 and known.A majorvoid in ourknowledge con- Am. J. Bot. 55,541 (1968); ibid., p. 807; ibid., p. 915. ABI2 loci, therefore,are involved only in cerns the events that occur within the egg 31. J. Hanstein, Bot. Abhadl., Bonn. 1, 1 (1870). 32. E. C. Soueges, Ann. Sci. Nat. 9, Bot. 19, 311 (1914); maintaining embryonic dormancy. The cell and in the early embryoafter fertiliza- Ann.Sci. Nat. 10, Bot. 1,1 (1919). ABI1gene encodesa Ca2+-dependent phos- tion. In this respect it is crucial to obtain 33. S. G. Mansfield, L. G. Briarty,S. Erni,Can. J. Bot. 69, phatasewith similarityto serine-threonine molecularmarkers in order to follow the 447 (1991); S. G. Mansfieldand L. G. Briarty,ibid., p. 461; ibid. 70,151 (1992). phosphatasesinvolved in signal transduc- specificationevents that take place during 34. L. Dolan et al., Development 119, 71 (1993). tion processes(89). In responseto ABA, the earlyembryogenesis and gain entryinto reg- 35. E. C. Yeung and D. W. Meinke, Plant Cell 5, 1371 ABII phosphatasemight counteractphos- ulatorynetworks that are activatedin differ- (1993). phorylationevents requiredfor the initia- ent embryonicregions after fertilization. Al- 36. M. K. Barton and R. S. Poethig, ibid., p. 823. 37. J. L. Zimmerman, ibid., p. 141 1. tion of root meristemcell division,resulting thougha largeamount of progresshas been 38. F. A. Van Engelen and S. C. de Vries, Trends Genet. in a dormantembryonic state (89). madein recentyears in understandinghow a 8, 66 (1992). Regulatoryloci requiredfor postembryonic plant embryoforms, there is still a long way 39. A. J. de Jong, E. D. L. Schmidt, S. C. de Vries, Plant Mol. Biol. 22, 367 (1993). developmentare activelate in embryogenesis. to go. The next few yearsshould be a very 40. L. Perez-Grau and R. B. Goldberg, Plant Cell 1, 1095 A largenumber of Arabidopsisfusca mutants exciting time to studyplant embryos. (1989). have been identified that accumulatean- 41. R. L. Taylor, Can. J. Bot. 45,1553 (1967). or red on 42. M. A. Chamberlin, H. T. Horner, R. G. Palmer, ibid. thocyanins, pigments, their coty- REFERENCESAND NOTES 71, 1153 (1993). ledons late in embryogenesis(10, 90-92). 43. J. P. Nitsch, Phytomorphology 19, 389 (1969); B. With the exception of fus3 (Table 2), em- 1. E. H. Davidson,Development 105, 421 (1989);ibid. Norreel, Bull. Soc. Bot. Fr. 117, 461 (1970); R. S. bryogenesis is normal in fuscamutants (10, 108, 365 (1990);ibid. 113, 1 (1991);ibid. 118, 665 Sangwanand B. S. Sangwan-Norreel,Int. Rev. Cy- (1993). tol.107, 221 (1987). 90-92). After germination, however, fusca 2. K. Esau, Anatomy of Seed Plants (John Wiley, New 44. The Embryo21st CenturyGroup, unpublished re- seedlings fail to develop into mature flow- York, 1977); P. H. Raven, R. F. Evert, S. E. Eichhorn, sults.This group is a collaborativeeffort between the

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This content downloaded from 164.67.109.135 on Fri, 18 Jul 2014 20:08:48 PM All use subject to JSTOR Terms and Conditions laboratories of R. B. Goldberg [Universityof Califor- Shimura, Plant Cell 3, 677 (1991). 77. D. Meinke, L. H. Franzmann, T. C. Nickle, E. C. nia, Los Angeles (UCLA)],J. J. Harada (UC Davis), R. 54. G. de Paiva and R. B. Goldberg, unpublished results. Yeung, Plant Cell 6, 1049 (1994). L. Fischer (UC Berkeley), J. L. Zimmerman (Universi- The chimeric Kti3/GUS genes contained the Kti3 78. The Embryo 21st Century Group (44), unpublished ty of Maryland,Baltimore), and A. Koltunow (Univer- gene TATA-boxregion (-28) (47). The chimeric Gyll data. Research on the lecl-2 gene was carried out sity of Adelaide, Australia).A total of 5822 T-DNA- GUS gene contained the cauliflower virus by M. West and J. J. Harada (UC Davis). mutagenized Arabidopsis lines (13) were screened (CaMV) TATA-box region within the CaMV minimal 79. K. Keith, M. Krami,N. G. Dengler, P. McCourt, Plant for mutants defective in embryogenesis (6, 8, 14). promoter (-42 to +0) (59). Cell 6, 589 (1994). Sixty-six heterozygous lines were identifiedthat seg- 55. T. L. Thomas, Plant Cell 5, 1401 (1993). 80. H.,Baumlein et al., Plant J., in press. regated seeds in a ratio of 3 wild-type to 1 mutant 56. A. N. Nunberg, et al., ibid. 6, 473 (1994). 81. M. Hulskamp, S. Mis6ra, G. Jurgens, Cell 76, 555 within their siliques, indicating that they contained 57. M. A. Bustos, D. Begum, F. A. Kalkan,M. J. Battraw, (1994). embryo-defective alleles that were inherited as sim- T. C. Hall, EMBO J. 10, 1468 (1990). 82. Y. Hou, A. G. van Arnim, X.-W. Deng, Plant Cell 5, ple Mendelian recessive genes. The raspberryl, 58. A. da Silva Conceic,o and E. Krebbers, Plant J. 5, 329 (1993). raspberry2, and lecl-2 genes were uncovered in 493 (1994). 83. L. Taiz and E. Zeiger, (Benjamin, three different heterozygous lines. 59. P. N. Benfey, L. Ren, N.-H. Chua, EMBOJ. 9,1677 New York, 1991). 45. K. D. Jofuku, R. D. Schipper, R. B. Goldberg, Plant (1990). 84. M. Koorneef, M. L. Jorna, D. L. C. Brinkhorstovan Cell 1, 427 (1989); K. D. Jofuku and R. B. Goldberg, 60. Z. R. Sung, A. Belancheur, B. Shunong, R. Bertrand- der Swan, C. M. Karssen, Theor. Appl. Genet. 61, ibid., p. 1079. Garcia, Science 258, 1645 (1992). 385 (1982); M. Koorneef, G. Reuling, C. M. Karssen, 46. P. Sterk, H. Booij, G. A. Schellekens, A. Van Kam- 61. P. N. Benfey et al., Development 119, 57 (1993). Physiol. Plant. 61, 377 (1984); M. Koorneef, C. J. men, S. C. DeVries, ibid. 3, 907 (1991). 62. R. A. Aeschbacher, J. W. Schiefelbein, P. N. Benfey, Hanhart, H. M. W. Hilhorst, C. M. Karssen, Plant 47. G. de Paiva and R. B. Goldberg, unpublished results. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 25 Physiol. 90, 463 (1989). A 2-kb 5' region from the soybean Kti3 gene was (1994). 85. E. Nambara, S. Nito, P. McCourt, Plant J. 2, 435 fused with the Escherichia coli GUS gene [R. A. Jef- 63. N. G. Nielsen et al., Plant Cell 1, 313 (1989). (1992). ferson, T. A. Kavanagh, M. W. Swan, EMBO J. 6, 64. The Embryo 21 st Century Group (44), unpublished 86. R. R. Finkelstein,Mol. Gen. Genet. 238, 401 (1993). 3901 (1987)], and the resulting chimeric Kti3/GUS results. Research on the raspberryl and raspberry2 87. J. Giraudatet al., Plant Cell 4, 1251 (1992). gene was transferredto tobacco plants byAgrobac- genes was carriedout by R. Yadegari, G. de Paiva, T. 88. D. R. McCartyet al., Cell 66, 895 (1991). terium-mediated transformation as described (45). Laux, and R. B. Goldberg (UCLA).Wild-type and 89. J. Leung etal., Science 264, 1448 (1994); K. Meyer, GUS activity in isolated whole-mount embryos was raspberryl seeds were harvested from heterozy- M. P. Leube, E. Grill,ibid., p. 1452. analyzed as outlined in G. N. Drews, T. P. Beals, A. gous +/raspberryl Arabidopsis plants (44), fixed, 90. A. J. Muller,Biol. Zentralbl. 82, 133 (1963). 26 Q. Bui, and R. B. Goldberg [Plant Cell 4, 1383 embedded in paraffin,and hybridizedwith 35S-RNA 91. L. A. Castle and D. W. Meinke, Plant Cell 6, (1992)]. antisense probes according to the procedures out- (1994). G. 48. R. Yadegari and R. B. Goldberg, unpublished re- lined in (40, 48). raspberryl seeds were harvested 92. S. Mis6ra, A. J. Muller, U. Weiland-Heidecker, sults. A 2-kb 5' region from the soybean lectin gene from siliques at a time when neighboring wild-type Jurgens, Mol. Gen. Genet. 244, 242 (1994). [R. B. Goldberg, G. Hoschek, L. 0. Vodkin, Cell 33, seeds were in the maturationstage of development. 93. X.-W. Deng et al., Cell 71, 791 (1992). 487 465 (1983)] was fused with the E coli GUS gene and 65. The Arabidopsis EP2 gene probe (46) was provided 94. T. W. McNellis et al., Plant Cell 6, (1994). T. D. Poole, J. transferredto tobacco plants as outlined in (47). Lo- by S. DeVries. 95. A. Pepper, T. Delaney, Washburn, Cell 109 (1994). calization of GUS activity in staged, whole-mount 66. P. Guerche et al., Plant Cell 2, 469 (1990). The Ara- Chory, 78, 96. N. Wei et al., Plant Cell 6, 629 (1994). embryos was determined by the procedure outlined bidopsis 2S2 gene probe was provided by E. Kreb- 35S- 97. N. Wei, D. A. Chamovitz, X.-W. Deng, Cell 78, 117 in (47). In situ hybridizationof a GUS antisense bers. a globular-stage em- (1994). RNA probe with longitudinal 67. B. W. Schwartz, E. C. Yeung, D. W. Meinke, Devel- out to the 98. We express our gratitude to J. Harada, R. Fischer, bryo section was carried according proce- opment, in press. dures outlined in (40) and in K. H. Cox and R. B. 1. Okamuro, D. Jofuku, L. Zimmerman, A. Kol- 68. R. Losick and L. Shapiro, Science 262,1227 (1993). Goldberg [inPlant MolecularBiology:A PracticalAp- tunow, T. Laux, L. Perez-Grau, and G. Drews for 69. A. J. DeJong et al., Plant Cell 4, 425 (1992). proach, C. H. Shaw, Ed. (IRL,Oxford, 1988), pp. many critical and stimulating discussions of plant A. J. De et ibid. 615 1-35]. 70. Jong al., 5, (1993). embryogenesis. We thank B. Timberlake, R. 49. L. Michalczuk, T. J. Cooke, J. D. Cohen, Phyto- 71. M. P. F. Marsden and D. W. Meinke,Am. J. Bot. 72, Chasan, and E. Davidson for insightful comments chemistry 31, 1097 (1992). 1801 (1985). on this manuscript. We gratefully acknowlodge K. 50. S. C. Fryand E. Wangermann, New Phytol. 77, 313 72. B. Haccius, Phytomorphology 13,107 (1963). Brill,B. Phan, and T. Zeyen for diligent typing of this (1976). 73. D. M. Vernon and D. W. Meinke, Dev. Biol., in press. manuscript and M. Kowalczyk for the colorful art 51. F. M. Schiavone and T. J. Cooke, Cell Differ.21, 53 74. L. Walling,G. N. Drews, R. B. Goldberg, Proc. Nat/. work. We dedicate this article to Professor E. H. (1987). Acad. Sci. U.S.A. 83, 2123 (1986). Davidson, who has provided intellectual leadership 52. C.-M. Liu, Z.-H. Xu, N.-H. Chua, ibid., p. 621. 75. L: Comai and J. J. Harada, ibid. 87, 2671 (1990). and direction to the field of development biology 53. K. Okada, J. Ueda, M. K. Komaki, C. J. Bell, Y. 76. D. Meinke, Science 258,1647 (1992). over the past 25 years.

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