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Plant Embryogenesis: Plants Occur in the Postembryonic Sporo- Phyte After Seed Germination (Fig

Plant Embryogenesis: Plants Occur in the Postembryonic Sporo- Phyte After Seed Germination (Fig

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Most morphogenetic events in flowering Embryogenesis: occur in the postembryonic sporo- phyte after (Fig. 1) (2). to Seed Vegetative organ systems differentiate con- tinuously from and meristematic regions that are formed initially during em- Robert B. Goldberg,* Genaro de Paiva, Ramin Yadegari bryogenesis. The reproductive organs of the are differentiated from a repro- Most differentiation events in higher plants occur continuously in the postembryonic grammed shoot after the phase of the life cycle. Embryogenesis in plants, therefore, is concerned primarily with has become a mature plant (Fig. 1) (25). establishing the basic shoot-root body pattern of the plant and accumulating food re- Thus, a germline analogous to that found in serves that will be used by the germinating seedling after a period of embryonic (1) is not set aside during plant within the seed. Recent studies in have identified that provide embryogenesis. new insight into how form during . These studies, and others A mature con- using molecular approaches, are beginning to reveal the underlying processes that control tains two primary organ systems-the axis . and (Fig. 1) (2). These organs have distinct developmental fates and are both composed of three basic, or primordial, layers-protoderm, procambium, and A major problem in plant development is lecular approaches suggest that a plant em- ground meristem-which will become the to unravel the mechanisms operating dur- bryo has a modular structure and consists of epidermal, vascular, and parenchyma tissues ing embryogenesis that enable a plant to several regions that form autonomously dur- of the young seedling, respectively (2). The specify its and tissue differentia- ing embryogenesis. axis, or - region of the em- tion patterns. Although progress with a va- bryo, contains the shoot and root riety of systems has been spectacular Embryos Begin the Diploid Phase and will give rise to the mature plant after in this regard (1), a detailed understanding of the Higher Plant Life Cycle seed germination (Fig. 1). By contrast, the of the events that govern plant embryo cotyledon is a terminally differentiated or- formation has yet to be realized. One obsta- The flowering plant life cycle is divided gan that accumulates food reserves that are cle in achieving this goal is the location of into haploid and diploid generations that utilized by the seedling for growth and de- on March 26, 2012 embryos within the plant and their relative are dependent on each other (Fig. 1) (2, velopment before it becomes photosynthet- inaccessibility to experimental manipula- 16-18). The haploid, or gametophytic, ically active (Fig. 1). The cotyledon func- tion, particularly at the early stages of em- generation begins after meiosis with tions primarily during seed germination and bryogenesis. In flowering plants, reproduc- that undergo and differentiate into senesces shortly after the seedling emerges tive processes occur within floral organs either a grain (male ) or from the . Embryogenesis in higher (Fig. 1) (2). The is present in the an embryo sac (female gametophyte) (3, plants, therefore, serves (i) to specify meri- , a multicellular structure that is buried 19-20). The pollen grain contains two stems and the shoot-root plant body pat- beneath several cell layers of the pistil, the cells, whereas the embryo sac con- tern, (ii) to differentiate the primary plant www.sciencemag.org female reproductive organ (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 essential for seed germination ogenesis occur within the pistil, it has been help facilitate the and seedling development, and (iv) to en- difficult to dissect the major events that take and fertilization processes (3, 19, 20). The able the to lie dormant until place during the early stages of higher plant male and female gametophytes are derived conditions are favorable for postembryonic development. from specialized -forming cells within development. Recently, it has become feasible to iso- the reproductive organs of the flower (3, 4, late plant and fertilize them in vitro in 21). By contrast, the diploid, or sporo- The Shoot-Root Body Plan Is Downloaded from order to investigate the initial events of phytic, generation begins after fertilization Generated During Early plant embryogenesis (5). In addition, genet- with the zygote and forms the mature plant Embryogenesis ic approaches have been used to identify with vegetative organs (, stem, root) genes required for various embryogenic pro- and that contain the reproductive How the embryo acquires its three-dimen- cesses, including (6, 7). organs (anther and pistil) (Fig. 1). sional shape with specialized organs and Genetic manipulation of Arabidopsis thali- Two fertilization events occur in flower- tissues, and what networks orchestrate ana, by both chemical mutagenesis (8-12) ing plants (2, 22). One sperm unites with remain major un- and insertional mutagenesis (13-15), has the egg cell to produce a zygote and initiate resolved problems. From a descriptive point identified a large number of mutants that embryogenesis. The other unites with a spe- of view, plant embryogenesis can be divided are blocked at different stages of embryo- cialized cell within the embryo sac (central into three general phases in which distinct genesis. In this review we outline the major cell) to initiate the differentiation of the developmental and physiological events oc- insights that have been derived from studies , a triploid tissue that is neither cur: (i) postfertilization-, (ii) ofArabidopsis embryo mutants, and we sum- gametophytic nor sporophytic in origin (Fig. globular- transition, and (iii) organ ex- marize gene transcription experiments in 1) (23). The endosperm is present during pansion and maturation (26-28) (Fig. 2 other plants that provide new information seed development and provides nutrients for and Table 1). Although there is consider- about the processes regulating higher plant either the developing embryo, the germinat- able variation in how embryos in different embryogenesis. Both the genetic and mo- ing seedling, or both (23). Fertilization also plant taxa form (29), the overall trends are causes the ovule, containing the embryo and remarkably similar (29). We summarize the Authors are in the Department of Biology, University of endosperm, to develop into a seed and the Capsella and Arabidopsis pattern of embryo California, Los Angeles, CA 90024-1606, USA. to differentiate into a , which development (29-33) because (i) it is one *To whom correspondence should be addressed. facilitates (Fig. 1) (24). of the most well-studied forms of plant em-

SCIENCE * VOL. 266 * 28 OCTOBER 1994 605 liiiiiiiiniiinii!iii -- ....-

Fig. 1. The life cycle of a Pollen flowering plant with em- Egg cell formation, grains ?"' phasis on egg cell forma- pollination, and fertilization Sperm cells tion and seed develop- FTube ment. [Adapted from (16, Pistil Surviving Embryo Female Style nucleus 26).] cell megaspore sac gametophyte (n) (2n) (n) Male us I \ Anti.iFipdal gametophyte Nucellu ' - b Central Aceells Meiosis Mitosis cell s l -_ ___ Ovary-fruitvary-ft(n) Ovule 'Synergid Flower cells Funiculus /Integuments Micropyle yW

Germinating Dormant Developing Fertilized endosperm seed seed seed nucleus (3n) Endosperm 0*

Fertilized meristem Root Axis lvEmbryo eeloping egg (2n) j embryo

Seed development and germination

bryogenesis, dating back to the classical the (Fig. 2). In Arabidopsis, the to be passed from the maternal sporophyte studies of Hanstein, Schaffner, and Soueges suspensor contains only 7 to 10 cells (Fig. into the developing proembryo (Fig. 2) with Capsella (30-32), (ii) it has an invari- 2). The suspensor anchors the embryo prop- (35). The suspensor senesces after the heart on March 26, 2012 ant division pattern during the early stages, er to the surrounding embryo sac and ovule stage and is not a functional part of the which allows cell lineages to be traced his- tissue and serves as a conduit for nutrients embryo in the mature seed. Derivatives of tologically (33), and (iii) recent studies mutants have provided with Arabidopsis Postfertilization Globular-heart transition new insights into the processes that control I a 5 A embryo development (6, 9). l II1 A Proembryo C Asymmetric of the zygote results in -cPd ti the formation of an embryo with a suspensor A and embryo proper that have distinct develop- www.sciencemag.org mental fates. The zygote in Arabidopsis and 0 Capsella has an asymmetric distribution of cellular components-the nucleus and most Zygote 2-cell of the are present in the upper 2/4-cell Heart EP 8-cell portion of the cell, whereas a large EP 16-cell embryo dominates the middle to lower portion (Fig. EP Globular embryo

2). This spatial asymmetry is derived from Downloaded from the egg cell (30). The zygote divides asym- metrically into two distinct-sized daughter 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 polarized longitudinal axis within the em- SM bryo (Fig. 2) (2, 30-33). Histological stud- ies over the course of the past 125 years BeEn - SC have indicated that the terminal and basal -Pd A- cells give rise to different regions of the - SM mature embryo (29-33). The small termi- EGm nal cell gives rise to the embryo proper that -Pc will form most of the mature embryo (Fig. RM RM 2). Cell lineages derived from the terminal En cell and embryo proper will specify the cot- W -~ MPE - yledons, shoot meristem, hypocotyl region Fig. 2. A generalized overview of plant embryogenesis. Schematic representations of embryonic stages of the embryonic axis (29-33), and part of are based on light microscopy studies of Arabidopsis (33) and Capsella (30-32) embryo development. the radicle, or embryonic root (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, terminal cell; 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; I, lower tier; Hs, hypophysis; Pc, procambium; Gm, ground meristem; C, divide and form a highly specialized, termi- cotyledon; A, axis; MPE, micropylar end; CE, chalazal end; SC, seed coat; En, endosperm; SM, shoot nally differentiated embryonic organ called meristem; and RM, root meristem. 606 SCIENCE * VOL. 266 * 28 OCTOBER 1994 moum~ lm im iiiiiiii ....

Table 1. Major events of flowering plant embryo- layer of the embryo proper, is produced and Embryogenesis Can Occur genesis. the hypophysis forms at the top of the sus- Without Surrounding pensor (Fig. 2). Subsequent cell differentia- Maternal Tissue Posffertilization-proembryo tion events within Terminal and basal cell differentiation the embryo proper result Formation of suspensor and embryo proper in the production of an inner procambium It is unclear what influence, if any, mater- Globular-heart transition tissue layer and a middle layer of ground nal tissue or accessory cells of the female Differentiation of major tissue-type primordia meristem cells (Fig. 2). The spatial organi- gametophyte have on egg cell formation Establishment of radial (tissue-type) axis zation of protoderm, ground meristem, and and subsequent embryonic development Embryo proper becomes bilaterally procambium layers establishes a radial axis (Fig. 1). For example, do either the ovule or symmetrical of differentiated tissues within the globular cells within the embryo sac (for example, Visible appearance of shoot-root embryo. contrast, the (apical-basal) axis By presence of the synergids) produce morphogenetic factors Initiation of cotyledon and axis hypophysis at the basal end of the embryo that contribute to the establishment of lon- (hypocotyl-radicle) development proper establishes an apical-basal polarity gitudinal asymmetry within the egg? Sever- Differentiation of root meristem within this region and indicates that the al arguments suggest that the maternal Organ expansion and maturation globular embryo is not radially symmetrical sporophyte provides only physical support Enlargement of cotyledons and axis by cell in the formal sense (Fig. 2). structures and nutrients for the embryo (Fig. division and expansion A dramatic change in the morphology of 1). First, somatic cells from a variety of Differentiation of shoot meristem the embryo proper occurs just after the and Formation of lipid and protein bodies vegetative reproductive tissues can un- Accumulation of storage proteins and lipids globular stage. Cotyledons are specified dergo embryogenesis in culture and lead to Vacuolization of cotyledon and axis cells from two lateral domains at the apical end the production of fertile plants (37-39). Cessation of RNA and protein synthesis (top), the hypocotyl region 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 Inhibition of precocious germination becomes differentiated from the hypophysis the embryo-proper region of zygotic embry- Dormancy region at the basal end (bottom) (34). The os, except that they do not become dormant embryo proper is now heart-shaped, has a (Fig. 2 and Table 1). In addition, spatial bilateral symmetry, and the body plan and and temporal programs 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- postembryonic plant) have been established embryos (39, 40). Second, embryo-like mation of the root meristem (30-34). Thus, (Fig. 2). Morphogenetic changes during this structures leading to plantlets can form di- on March 26, 2012 cell lineages derived from the basal cell give period are mediated by differential cell di- rectly from the attached of some rise to the suspensor and part of the radicle vision and expansion rates and by asymmet- plants (41). Third, produced by fer- region of the embryonic axis (Fig. 2). ric cleavages in different cell planes (2). No tilizing egg cells in vitro undergo embryo- 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 migration events that take place in many producing plants (5). Finally, ultrastructural division of the zygote. Whether the polar- types of animal embryos (1). studies suggest that there is a barrier be- ized organization of the egg cell, the zygote, Embryogenesis terminates with a dormancy tween the inner ovule cell layer and the or both control differential gene expression period. A major change in embryonic devel- embryo sac that prevents the transfer of events early in embryogenesis is not known. opment occurs during the organ expansion material directly between these compart- www.sciencemag.org For example, do prelocalized regulatory fac- and maturation phase (Fig. 2). A switch ments (42). Thus, both zygotic and somatic tors within the egg cell initiate a cascade of occurs during this period from a pattern embryogenesis can occur in the absence of events leading to the lineage-dependent formation program to a storage product ac- surrounding ovule tissue (Fig. 1). differentiation of terminal and basal cell cumulation program in order to prepare the The embryo sac is necessary for zygotic derivatives? Alternatively, after fertilization young sporophyte for embryonic dormancy embryogenesis because it contains the egg does the zygotic genome direct the de novo and postembryonic development (Table 1). and associated accessory cells that are re-

synthesis of regulatory factors that are dis- The cotyledons and axis increase in size quired for fertilization and endosperm de- Downloaded from tributed asymmetrically to the terminal and dramatically as a result of and velopment (Fig. 1). However, the embryo basal cells at first cleavage? In either case, expansion events (33). Ground meristem sac is not essential for embryogenesis per se these events would lead to the autonomous cells within both these organs become high- because (i) somatic embryos produced from specification of the terminal and basal cells ly specialized and accumulate large amounts sporophytic cells develop normally (5, 37- as a consequence of intrinsic factors rather of storage proteins and oils that will be 40) and (ii) embryos can be induced to form than extracellular signals (1). utilized as a food source by the seedling after from that, under normal cir- Embryonic organs and tissue-types differen- germination (Fig. 2 and Table 1) (33). One cumstances, give rise to pollen grains (43). tiate during the globular-heart transition phase. differentiation event does occur during this These results suggest that normal embryo- Two critical events must occur after the period, however-the shoot meristem forms genic processes do not require factors pro- embryo proper forms: (i) regions along the from cell layers localized in the upper axis duced by either the female gametophyte or longitudinal apical-basal axis must differen- region between the two cotyledons (Fig. 2) maternal sporophytic tissue. This conclu- tiate from each other and generate embry- (36). Thus, the differentiation of shoot and sion is supported by the fact that the over- onic organ systems, and (ii) the three pri- root meristems at opposite poles of the em- whelming majority of mutations that alter mordial tissue layers of the embryo need to bryonic axis does not occur at the same time embryo development appear to be due to be specified. Both of these events take place (34, 36). At the end of the organ expansion defects in zygotically acting genes (6-14, during the globular-heart transition phase and maturation period the embryo has 44). It is possible that somatic cells have the (Fig. 2 and Table 1). The embryo proper reached its maximum size, cells of the em- potential to produce putative maternal or has a spherical shape during the proembryo bryo and surrounding seed layers have be- gametophytic factors under the proper con- 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 SCIENCE * VOL. 266 * 28 OCTOBER 1994 607 than zygotic embryos. However, most of the bryos containing chimeric 3-glucuronidase gions (Fig. 3G). This result suggests that the available data suggest that embryo morpho- (GUS) reporter genes driven by soybean preferential localization of Kti3 mRNA at genesis and cell specification events are embryo-specific gene promoters showed the micropylar end of a soybean globular directed primarily by the zygotic genome that a globular embryo is organized into stage embryo (Fig. 3C) is due to transcrip- after fertilization occurs. If so, then this distinct, nonoverlapping transcriptional re- tional regulatory processes. By contrast, would differ significantly from the situation gions, or territories (Fig. 3, F to I) (47, 48). GUS activity is visible as a uniform blue belt with many animals such as Drosophila and Blue color resulting from GUS enzyme ac- that surrounds the equator region of a glob- , in which maternally supplied tivity occurs specifically at the micropylar ular embryo containing a soybean lectin/ factors influence the pattern of early em- end of a globular stage embryo containing a GUS reporter gene (Fig. 3H) (48). GUS bryo development (1). More extensive stud- Kii3/GUS reporter gene (Fig. 3, F and G) activity is not visible at either the micropy- ies with developing female gametophytes, (47). No GUS activity is observed within lar or the chalazal (apical) ends of the em- egg cells, and zygotic embryos in the early the suspensor or other embryo-proper re- bryo proper (Fig. 3H) (48); nor is there stages of embryogenesis are needed to clar- ify this issue. A Globular Embryo Contains Differentially Transcribed Regions A large number of genes are expressed dur- ing embryogenesis in higher plants (26). Although it is not known how many genes are necessary to program morphogenetic and tissue differentiation processes, approx- imately 15,000 diverse genes are active in the embryos of plants as diverse as soybean and cotton (26). Many of these genes are expressed in specific cell types, regions, and organs of the embryo (26, 40) and provide useful entry points to unravel the molecular on March 26, 2012 mechanisms that regulate cell- and region- specific differentiation events during plant embryogenesis (1). The axis region of the embryo does not become visibly distinct until the heart stage (Figs. 2 and 3, A and B). Localization stud- ies with a soybean Kunitz trypsin inhibitor mRNA, designated as Kti3 (45), indicated, however, that cells destined to become the www.sciencemag.org axis are already specified at the globular stage (40). Figure 3C shows that Kti3 mRNA accumulates specifically at the bas- Fig. 3. Differential transcriptional activity during early embryogenesis. (A and B) Bright-field photographs al, or micropylar, end of a late-globular of developing soybean embryos sectioned longitudinally (40). (A) Late globular stage embryo. (B) Heart stage soybean embryo. No detectable Kti3 stage embryo. (C to E) Localization of the major Kunitz trypsin inhibitor mRNA (Kti3) in longitudinal mRNA is present in other regions of the sections of soybean embryos (40, 45). In situ hybridization data are similar to those shown in (40). embryo proper or in the suspensor (Fig. 3C) Photographs were taken by dark-field microscopy. (C) Late globular stage embryo. (D) Late globular to transition stage embryo. (E) Heart stage embryo. (F) Bright-field photograph of a tobacco globular stage Downloaded from (40). In transition and heart stage embryos, embryo sectioned longitudinally. (G and H) Localization of GUS activity within transgenic tobacco Kti3 mRNA remains distributed asymmetri- globular stage embryos. Photographs of whole-mount embryos were taken by bright-field microscopy. cally at the embryo micropylar end (Fig. 3, (G) Embryo contains a chimeric Kti3/GUS gene (47). (H) Embryo contains a chimeric lectin/GUS gene D and E) and is localized specifically within (48). (I) Localization of GUS mRNA within a transgenic tobacco globular stage embryo similar to that the ground meristem cell layer (Fig. 2) of shown in (H) containing a lectin/GUS gene (48). Abbreviations: EP, embryo proper; S, suspensor; C, the emerging embryonic axis (Fig. 3D) cotyledon; A, axis; En, endosperm; CE, chalazal end; MPE, micropylar end; and Pd, protoderm. (40). No detectable Kti3 mRNA is present within the newly initiated cotyledons (Fig. 3E). This result differs from that obtained WT seed WT seedling emb 30/gnom monopteros gurke fackel with the carrot EP2 lipid transfer protein H SM SM SM mRNA, which is localized uniformly in the (E)~ SMc outer protoderm cell layer that surrounds .~~~~ the embryo proper at the globular and heart stages (46). Taken together, these results indicate that cells along the longitudinal apical-basal axis of the embryo proper are Complete Complete 0 Basal * Basal already differentiated from each other at the globular stage and that early in embry- Fig. 4. Schematic representations of Arabidopsis pattern mutants [adapted from (9)]. The green, yellow, ogenesis distinct gene sets are expressed in and orange colors delineate the apical, central, and basal regions, respectively. Strong (upper) and weak different embryonic regions and cell types. (lower) gnom phenotypes are depicted (9, 15). Abbreviations: WT, wild-type; RM, root meristem; SM, Transformation studies with tobacco em- shoot meristem; C, cotyledon; h, hypocotyl; and R, root. 608 SCIENCE * VOL. 266 * 28 OCTOBER 1994 detectable GUS activity within the suspen- 48). These results suggest that a prepattern cotyledons and shoot meristem, the central sor (Fig. 3H) (48). GUS mRNA localiza- of different transcriptional regulatory do- region consists of the hypocotyl, or upper tion studies with longitudinal globular em- mains has been established in the globular axis, and the basal region includes the lower bryo sections indicated that the lectin pro- embryo before the morphogenetic events axis, or radicle, and the root meristem (Fig. moter activity occurs specifically within the that lead to differentiation of cotyledon and 4). These regions are derived ultimately ground meristem cell layer of the equator axis regions at the heart stage (Figs. 2 and 3, from the terminal and basal cell lineages region (Fig. 31) (48). A and B). Presumably, each transcriptional and are maintained in the young seedling These experiments indicate that both domain sets in motion a cascade of events (Figs. 2 and 4). Can regions along the lon- the longitudinal apical-basal and radial tis- leading to the differentiation of specific gitudinal axis develop independently of sue-type axes of a globular embryo are par- embryo regions later in embryogenesis. each other? If territories established within titioned into discrete transcriptional terri- the embryo proper of a globular stage em- tories (I). The longitudinal axis of the em- Hormones Affect Embryo bryo are specified autonomously, then the bryo proper contains at least three nonover- loss or alteration of cells within a territory lapping transcriptional territories: (i) the should not affect the development of a con- chalazal region, (ii) the equator region, and What physiological events cause the em- tiguous region (Figs. 2 and 3). Several ex- (iii) the micropylar region (Fig. 3, F to H). bryo proper to initiate cotyledons and be- periments suggest that this is actually the The suspensor represents an additional come bilaterally symmetric during the glob- case that is, a plant embryo forms from transcriptional domain along this axis (Fig. ular-heart transition phase (Fig. 2)? Several modules that develop independently of 3, G and H). Each tissue layer of the radial experiments implicate a class of plant hor- each other (9-12, 54-59). embryo-proper axis also has a distinct tran- mones, the , in this morphogenetic Pattern mutations delete specific embryonic scriptional program (Fig. 3, E, G, and I). process (49-52). Auxins, such as indoleace- regions. Genetic studies have uncovered Transcriptional activity within these layers, tic acid (IAA), are involved in a number of Arabidopsis mutations that alter the organi- however, appears to be established in a plant activities, including photo- and grav- zation of the embryo body plan (Fig. 4 and territory-specific manner-that is, ground itropism, apical dominance, and vascular Table 2) (9, 10). Embyro pattern mutations meristem cells within the equator region cell differentiation (2). Embryos in plants as were found that delete (i) the apical region activate promoters distinct from those with- diverse as bean and pine synthesize auxins (gurke), (ii) the central region (fackel), (iii) in ground meristem cells of the micropylar (49) and transport them in a polarized, the central and basal regions (monopteros), region, and vice versa (Fig. 3, E to I) (47, basipetal direction from the shoot meristem and (iv) the apical and basal regions to root tip along the embryonic axis (Fig. 2) (emb30/gnom) (Fig. 4) (9). Other embryo on March 26, 2012 (50). The highest levels occur at the pattern-forming genes probably exist; how- Table 2. Examples of Arabidopsis mutants that globular stage of embryogenesis (49). ever, they have not been identified in the have defects in embryo development. Several Agents that inhibit polarized auxin trans- genetic screens carried out thus far (6, hundred Arabidopsis embryo-defective mutants have been identified by both chemical and T-DNA port either block the transition from the 8-10, 14, 44). Each mutant gene acts zy- mutagenesis. Most of these mutants can be ob- globular to heart stage completely (51) or gotically, indicating that major specifiers of tained from the Arabidopsis Biological Resource prevent the bilateral initiation of cotyle- the embryo body plan act after fertilization Center (arabidopsis+ @osu.edu). dons at the top of the globular embryo (Fig. has occurred (Fig. 2) (9, 10). In addition, 2) (52). For example, auxin transport inhib- the loss of a specific region, or combination Mutant class References itors cause carrot somatic embryos to re- of regions, does not affect the development www.sciencemag.org main spherically shaped and develop into of an adjacent neighbor (Fig. 4) (9, 10). For Pattern mutants an re- emb3O/gnom 8-11, 15 giant globular embryos (51). By contrast, example, gurke embryos lack apical monopteros 9, 12 zygotic embryos of the Indian mustard gion, but have normal central and basal gurke 9 (Brassicajuncea), an Arabidopsis relative, fail regions (Fig. 4) (9). monopteros embryos, by fackel to initiate two laterally positioned cotyle- contrast, have a normal apical region but Cell-type differentiation mutants dons when treated with auxin transport are missing the central and basal regions keule 9 blockers in culture A (52). cotyledon-like (Fig. 4) (9). Downloaded from knolle 9 organ does form, but as a collar-like ring Embryo pattern mutations alter the di- Suspensor transformation mutants around the entire upper (apical) region of vision planes that are established during the 73 the embryo (52). Treated Indian mustard postfertilization-proembryo phase of embry- sus1 67 sus2 67 embryos resemble those of the Arabidopsis ogenesis (Fig. 2) (11, 12). Thus, deletions sus3 67 pin]-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 histological defects at the proem- raspberry2 44 suggest that auxin asymmetries are estab- bryo and globular stages (Fig. 2) (11, Meristem differentiation and identity mutants lished within the embryo-proper region of 12) a result that provides functional evi- shoot meristemless 36 globular stage embryos (Fig. 3, A and F) and dence for the embryo fate maps proposed by embryonic flower 60 that these asymmetries contribute to the the developmental botanists of the late short-root 61, 62 establishment of bilateral symmetry at the 19th and early 20th centuries (31, 32). For hobbit 61, 62 heart 2 and A and do not divide Maturation program mutants stage (Figs. 3, B). example, emb30/grnom zygotes lecl-/lec1-2 44, 76-78 asymmetrically (Fig. 2) (1 1). Two similar- lec2 77 Plant Embryos Form from sized daughter cells are produced in the fus3 79, 80 Regions That Develop emb30/gnom embryo-proper region instead abi3 84, 85 Autonomously of the unequal-sized terminal and basal cells Seedling lethality mutants that are found in wild-type embryos (11). fus 1/cop1 91, 94 The longitudinal axis of a mature plant Later division events in emb30/gnom embry- fus2/det1 91, 95 embryo is made up of several regions that os are also highly variable and abnormal fus6/cop 1 1 91, 96 are designated as apical, central, and basal (11). By contrast, the monopteros division fus7/cop9 91, 97 (Fig. 4) (9). The apical region contains the pattern is normal until the 8-cell embryo- SCIENCE * VOL. 266 * 28 OCTOBER 1994 609 proper stage (Fig. 2) (12). During the there are genes which regulate the specifi- to the cotyledons and shoot meristem of a monopteros globular-heart transition phase, cation, organization, and fate of meristems transgenic tobacco embryo (Fig. 5D) (54). lower tier cells of the embryo proper and that differentiate during embryogenesis. These data, and those of others (55-59), derivatives of the hypophysis undergo ab- Genes controlling meristem development indicate that unique transcription factors are normal divisions (12) (Fig. 2). monopteros most likely act downstream of embryo-re- active within each embryonic region and upper tier embryo-proper cells, however, gion specifiers, such as monopteros and gurke that these factors interact with specific pro- develop normally that is, defects at the (Fig. 4) (9, 10, 12), in the regulatory hier- moter elements. The combination of these basal end of the embryo proper do not affect archy needed to form a plant embryo. elements and factors gives rise to the tran- events that occur at the apical end (Fig. 2) Meristem mutants characterized to date scriptional pattern of the whole embryo (12). The result is a mutant embryo 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 missing the hypocotyl and root regions (Fig. not affect the differentiation of contiguous DNA elements should provide reverse entry 4) (12). These observations indicate that domains such as the cotyledons or hypo- into the independent regulatory networks genes which are responsible, in part, for the cotyl (36, 60-62). Meristems, therefore, required for specifying each autonomnous re- establishment of the embryo body plan di- represent independent submodules within gion of a plant embryo. rect territory-specific cell division patterns the apical and basal regions of the embryo during early embryogenesis. Abnormal divi- (Fig. 4). A major question is what effect, if Cell Differentiation and sions in one embryonic territory do not any, do cells adjacent to 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 lineages giving rise to That is, are events involved in specific regions of the mature embryo de- specifying the root and shoot meristems What is the relation between cell differen- velop autonomously. within a specific embryonic region, or do tiation and morphogenesis in plant embry- Agrobacterium T-DNA-tagged emb30/ the meristems differentiate autonomously? os? Are processes required for tissue differ- gnom alleles have led to the isolation of the The laterne mutant provides one answer to entiation along the embryo radial axis cou- EMB30/GNOM gene (15). This gene en- this question (9). laterne lack pled to those that specify autonomous re- codes a protein that is related to the yeast cotyledons but produce leaves indicating Sec7 secretory protein, is active throughout that a shoot meristem is present (9). Thus, the plant life cycle, and is involved in cell cotyledons are not required for the differ- division, elongation, and adhesion events entiation of the shoot meristem durino on March 26, 2012 required at many stages of sporophytic de- embryogenesis. velopment, including embryogenesis (15). Promoter elements interpret embryo region- Thus, EMB30/GNOM does not appear to specific regulatory networks. One consequence establish the embryonic cell division pat- of the modular organization of a plant em- tern directly, but most likely facilitates a bryo is that genes which are active through- pattern set by other genes. What these pat- out the embryo must intersect with several tern-forming genes are and how they inter- region-specific regulatory networks that is. act with downstream genes that mediate the promoters of embryo-specific genes are events required for the differentiation of required to sense and interpret the transcrip- www.sciencemag.org autonomous regions along the longitudinal tional regulatory machinery unique to each axis remain to be determined. autonomous region (Fig. 4). For example, Mutations affect meristematic zones of the Kti3 mRNA accumulates within the axis embryo longitudinal axis. Arabidopsis muta- region early in soybean embryogenesis, but tions have been identified that affect the does not accumulate within the cotyledons differentiation of the shoot and root meri- until much later (Fig. 3, C to E) (40). Thus, stems during embryogenesis (Table 2) (36, discrete promoter elements should exist that Downloaded from 60-62). These mutations target a specific are responsible for interacting with transcrip- meristem and have no other effect on em- tion factors produced by separate regulatory bryonic development. For example, shoot circuits. meristemless fails to differentiate a shoot A Kti3/GUS gene with 2 kb of 5' flank- meristem during embryogenesis and pro- ing sequence is transcribed in all regions of a Fig. 5. DNA elements program transcription to duces seedlings without leaves (36). embry- mature transgenic tobacco embryo (Fig. 5A) specific embryonic regions. Maturation stage em- onic flower, on the other hand, generates a (54). Deletion of 0.2 kb from the 5' end bryos were hand-dissected from transgenic to~ shoot meristem at the top of the embryonic eliminates Kti3/GUS transcriptional bacco containing the E. col/ GUS gene activity fused with different soybean seed 5' axis (Figs. 2 and 4) (54). Remarkably, em- within the embryo radicle region (Fig. 5B) protein gene bryonic flower seedlings regions (54). GUS activity in whole-mount embry- produce flowers (54). Deletion of another 1 kb eliminates os was localized as outlined in (47), and photo- rather than leaves, indicating that the fate Kti3/GUS transcription within the cotyle- graphs were taken with the use of dark-field mi- of the shoot meristem is altered during em- dons and shoot meristem, but still permits croscopy. (A to C) Kti3 gene upstream regions bryogenesis a floral meristem is specified transcription to occur within the hypocotyl fused with the E coli GUS gene. (A) A 2-kb KtO3 rather than a vegetative shoot meristem region (Fig. 5C) (54). These results indicate gene 5' fragment. (B) A 1 .7-kb Kfi3 gene 5' frag- (60). By contrast, short root and hobbit affect that discrete cis-acting domains are required ment. (C) A 0.8-kb Kfi3 gene 5' CaMVIGUS frag- root meristem development (61, 62). These for the transcriptional activation of the Kti3 ment. (D) A minimal CaMVIGUS gene-promoter mutations lead to abnormal root develop- gene within the radicle, hypocotyl, and cot- cassette (59) fused with a 0.36-kb Gyl gene 5' ment after seed germination, indicating yledon-shoot meristem regions of the em- region (-446 to -84) (63). The white embryo re- that the root meristem is altered, but not sults from the segregation of a single GylIGUS bryo. Promoter analysis of the soybean Gy 1 gene within this transgenic tobacco line and rep- eliminated, during embryogenesis (61, 62). storage protein gene (63) also uncovered a resents a negative control. Abbreviations: C, cot- Taken together, these results indicate that regulatory domain that directs transcription yledon; H, hypocotyl: and Rd, radicle. 610 SCIENCE * VOL. 266 * 23 OCTOBER 1994 N1. .WB. gions of the longitudinal apical-basal axis genesis. For example, EP2 lipid transfer pro- (Table 2). Surprisingly, raspberry) embryos (Fig. 2)? Studies of Arabidopsis embryo pat- tein mRNA accumulates specifically within accumulate EP2 and 2S2 marker mRNAs in tern mutants suggest that these processes the epidermal cell layer (Fig. 6B) (46, 64, their correct spatial context along the radial are not necessarily interconnected. For ex- 65), and 2S2 albumin mRNA accumulates axis of both the embryo-proper and suspen- ample, a fackel embryo does not have a within storage parenchyma cells (Fig. 6C) sor regions (Fig. 6, F to I) (64). EP2 mRNA hypocotyl, but epidermal, ground meristem, (64, 66). Neither mRNA is detectable accumulates along the outer perimeter of and vascular tissues differentiate within cot- within the vascular layer (Fig. 6, B and C) raspberry] embryos (Fig. 6, F and G), where- yledon and radicle regions (Fig. 4) (9). (46, 64-66). Collectively, the EP2 and 2S2 as 2S2 rnRNA accumulates within interior Thus, the loss of one embryonic region does mRNAs can identify embryo epidermal and cells (Fig. 6, H and I) (64). By contrast, EP2 not affect the formation of tissue layers storage parenchyma cell layers and, by de- and 2S2 mRNAs do not accumulate detect- within the remaining regions (Fig. 4) (9- fault, the inner as well (Fig. ably within the central core of raspberry 1 12). A more direct question, however, is 6, B and C). embryos (Fig. 6, F to I) (64). Similar results whether mutant embryos that arrest early in An Arabidopsis embryo mutant, desig- were obtained with raspberry2 embryos (Ta- embryonic development and remain globu- nated raspberry1, was identified in a screen ble 2) (44, 64). lar-shaped differentiate the specialized cell of T-DNA-mutagenized Arabidopsis lines These mRNA localization studies indi- and tissue layers that are found in organ (Table 2) (44). This mutant fails to undergo cate that specialized tissues can differentiate systems of a mature, wild-type embryo. the globular-heart transition (Fig. 2), has an within the embryo-proper region of mutant A maturation stage Arabidopsis embryo embryo-proper region that remains globu- embryos that remain globular shaped, and has specialized epidermal, storage parenchy- lar-shaped throughout embryogenesis, and that these tissues form in their correct spatial ma, and vascular cell layers within both the does not differentiate cotyledons and axis contexts. A similar conclusion was inferred cotyledon and axis regions (Fig. 6A). These (Fig. 6, D and E) (64). raspberry1 embryos from histological studies of the sus mutants tissues are derived from the three primary also have an enlarged suspensor region (Fig. (67). Tissue differentiation, therefore, can cell layers that are specified along the radial 6, D and E) (64). raspberry2 (44) and sus take place independently of morphogenesis axis of a globular embryo (Fig. 2), and ex- (67) embryo-defective mutants also have in a higher plant embryo, implying that mor- press specific marker genes late in embryo- phenotypes similar to that of raspberry 1 phogenetic checkpoints do not occur before cell differentiation events can proceed (68). It does not follow, however, that morpho- genesis can occur without proper cell differ- entiation events. Arabidopsis embryo mu- on March 26, 2012 tants that alter tissue-specification patterns have abnormal morphologies (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 carrot tsl 1 somatic embryo mutant has a defective pro- toderm cell layer and fails to undergo mor- phogenesis (38, 39). Addition of either an www.sciencemag.org extracellular chitinase (69) or Rhizobium nodulation factors (lipooligosaccharides) (70) can rescue the ts 11 mutant. Lipooligo- saccharide nodulation factors have been shown to be signal molecules involved in the differentiation of Rhizobium-induced root

nodules (70). This suggests that in carrot Downloaded from .1i somatic embryos, the protoderm cell layer may provide signals necessary for embryo- genesis to occur (69, 70). Taken together, 3 experiments with mutant embryos that have defective cell layers suggest that specification of the radial axis needs to occur in order for a normal shoot-root axis to form. An impor- Fig. 6. Localization of cell-specific mRNAs within wild-type and mutant Arabidopsis embryos. (A) tant corollary is that cells within the radial Bright-field photograph of a late-maturation stage Arabidopsis embryo sectioned longitudinally (64). (B axis probably interact with each other (9). and C) In situ hybridization of labeled RNA probes with longitudinal sections 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) Longitudinal section of a raspberryl embryo. The photograph One intriguing aspect of the raspberry1 em- was taken by bright-field microscopy. (E) A raspberryl embryo photographed with Nomarski interference bryo is its large suspensor (Fig. 6, D and E). optics. (F to 1). In situ hybridization of labeled RNA probes with raspberryl embryo longitudinal sections raspberry 1 are indistinguishable (64). (F and G) Localization of EP2 lipid transfer protein mRNA (65). Localization experiments with from wild-type during the early stages of raspberry2 embryos, which have larger suspensors than raspberryl embryos (44), confirmed that EP2 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 hybridization with serial sections through an entire raspberryl embryo confirmed that there is development, when neighboring wild-type an inner core of cells with no detectable 2S2 mRNA (64). Abbreviations: A, axis; V, vascular tissue; Ed, embryos undergo maturation, cell prolifera- ; C, cotyledon; P, storage parenchyma; Ep, embryo proper; S, suspensor; and En, endosperm. tion events cause the raspberry 1 suspensor

SCIENCE * VOL. 266 * 28 OCTOBER 1994 Air to enlarge at its basal end (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 accumulate in the raspberry1 suspensor (Fig. gene (76, 77), designated as lec1-2, 6, F to I) with a spatial pattern similar to was identified in a screen of T-DNA- mutagenized Arabidopsis lines (44, that which occurs in mature, wild-type em- 78). (A and B) Bright-field photo- bryos (Fig. 6, B and C) (64). These cell- graphs of Arabidopsis seedlings specific mRNAs do not accumulate detect- (78). (A) Wild-type seedling. (B) ably in wild-type suspensors, or in raspberry 1 lecl-2 seedling. Abbreviations: C, suspensors early in embryogenesis (64). cotyledon; H, hypocotyl; R, root; These results indicate that the raspberry) and Tr, . suspensor has entered an embryogenic path- way and that an embryo proper-like, radial tissue axis has been specified. Other Arabidopsis embryo mutants have suspensor abnormalities similar to that of raspberry), including raspberry2, and sus gram is induced that is responsible for (i) (76-78). Other lec mutants, such as lec2 (Table 2) (14, 35, 44, 67, 71). Although synthesizing large amounts of storage prod- and fus3, have phenotypic characteristics the extent of suspensor enlargement varies, ucts, (ii) inducing water loss and a desiccat- that overlap those of led (77-80). For ex- all of these mutants have morphological ed state, (iii) preventing premature germi- ample, fus3 and lec embryos are almost defects in the embryo proper (14, 35, 44, nation, and (iv) establishing a state of dor- indistinguishable from each other (77). lec2 67, 71). Mutant embryos that resemble mancy (Fig. 2 and Table 1) (2, 26-28). embryos, by contrast, have leaf-like cotyle- wild-type, but arrest at specific embryonic Several specialized gene sets, such as those dons, but are desiccation tolerant, do not stages, do not have aberrant suspensors (44, encoding storage proteins and late embryo germinate precociously, and have normal 64). Disruptions in embryo-proper morpho- abundant (lea) proteins, are activated tran- levels of storage bodies in their axis region genesis, therefore, can induce an embryo scriptionally during maturation and then re- (77). This indicates that leafy cotyledons proper-like pathway in terminally differen- pressed before dormancy (26, 74, 75). These can occur without corresponding defects in tiated suspensor cells, a result first observed gene sets remain transcriptionally quiescent desiccation and dormancy, and that wild- by the embryo-proper ablation experiments during seed germination when germination- type LEC genes are activated independent- on March 26, 2012 of Haccius 30 years ago (72). The Arabidop- specific and postembryonic gene sets are ly in each embryonic organ system (77). A sis twin mutant represents a striking exam- transcribed (26, 55, 75). Genetic studies corollary is that gene networks that func- ple of the embryogenic potential of the with Arabidopsis have identified some of the tion in desiccation and dormancy are inde- suspensor region (73). twin causes subtle genes that regulate processes required for pendent of those responsible for cotyledon defects to occur in embryo-proper morphol- embryo maturation and dormancy, includ- cell specialization. ogy, generates a second embryo within the ing the expression of storage protein and lea Molecular studies with lec embryos have seed from proliferating suspensor cells, and genes (Table 2). indicated that the transcription of matura- results in twin embryos that are connected Leafy cotyledon mutations disrupt embryo tion-specific genes, such as those encoding by a suspensor cell bridge (73). maturation. A mutant gene class, designated storage proteins, is greatly reduced (80). www.sciencemag.org The nature of mutant genes that affect as leafy cotyledon (lec), has been identified Conversely, the transcription of germina- suspensor development, such as raspberry1, that causes defects in the cotyledon cell tion-specific genes, such as isocitrate lyase, is sus, and twin, is not known. These genes are differentiation process and in maturation- activated (78). These data, coupled with the probably not involved in suspensor specifi- specific events such as storage product accu- histological descriptions of lec embryos (Fig. cation events, because a normal suspensor mulation, desiccation tolerance, and main- 7) (76-80), indicate that LEC genes func- forms before induction of the embryo-prop- tenance of dormancy (Table 2) (76-80). iec tion during maturation to activate genes in- er pathway in mutant embryos (44, 64, 67, mutations transform cotyledons of embryos volved in cotyledon cell specialization, stor-

71, 73). They reveal, however, that inter- and seedlings into leaf-like structures (76- age product accumulation, induction and Downloaded from actions occur between the suspensor and 80). Wild-type cotyledons do not have maintenance of embryonic dormancy, and embryo-proper regions. One possibility is (Fig. 7A). Trichomes are present desiccation tolerance (76-80). LEC genes that the embryo proper transmits specific only on leaf, stem, and surfaces in also simultaneously suppress the manifesta- inhibitory signals to the suspensor that sup- wild-type plants and are markers for tion of leaf-like characteristics in cotyledons press the embryonic pathway (35, 67, 71- postembryonic development (81). By con- during embryogenesis, including trichome 73). Alternatively, a balance of growth reg- trast, led cotyledons have trichomes on specification. In the absence of LEC prod- ulators might be established within the en- their adaxial surface which differentiate ucts, embryonic cotyledons enter a default tire embryo that maintains the develop- during embryogenesis 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), many of which develop normally in and suspensor regions. Disruptions of such dons have other leaf-like characteristics postgermination, wild-type cotyledons (82). signals would cause the suspensor to take on including stomata, mesophyll cells, and an Abscisic acid maintains embryonic dorman- an embryo proper-like fate, a result analo- absence of protein and lipid storage bodies cy. The abscisic acid (ABA) gous to embryo induction in differentiated (77). The axis region of led embryos also is involved in several plant processes, in- sporophytic or gametophytic cells (37-39). lacks storage organelles, indicating that cluding , responses to environ- the wild-type LEC 1 gene functions in mental stresses, growth inhibition, and The Embryo Is Reprogrammed both embryonic organs (77). maintenance of a dormant state (83). Ex- Late in Embryogenesis In addition to the leaf-like cotyledon ogenous ABA prevents seed germination as transformation, lec embryos (i) germinate well as the precocious germination of em- How does the embryo prepare for dormancy precociously about 5% of the time, (ii) have bryos in culture. In addition, Arabidopsis and postembryonic development (Fig. 1)? leaf primordia emerging from their shoot mutants that either cannot synthesize ABA Late in embryogenesis a maturation pro- apex, and (iii) fail to survive desiccation or fail to respond to ABA germinate preco-

612 SCIENCE * VOL. 266 * 28 OCTOBER 1994 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 prevents germination while seeds are genes have been shown to be alleles of 4. C. S. Gasser and K. Robinson-Beers, ibid., p. 1231. still dormant or present within siliques. constitutive photomorphogenic (cop)/deeti- 5. E. Kranz and H. Lbrz, ibid., p.739; J.-E. Faure, H. L. Three mutant Arabidopsis loci, designat- olated (det) genes that function in light- Mogensen, C. Dumas, H. Lbrz, E. Kranz, ibid., p. 747; C. Dumas and H. L. Mogensen, ibid., p. 1337; ed as abil, abi2, and abi3, have been iden- regulated development during seed germi- J.-E. Faure, C. Digonnet, C. Dumas, Science 263, tified that result in ABA insensitivity and nation (Table 2) (91-97). The products of 1598 (1994). allow seed germination to occur in the pres- COP/DET loci appear to suppress light- 6. D. W. Meinke, Dev. Genet. 12, 382 (1991); Plant Cell in the dark 3, 857 (1991). ence of ABA (84). In addition to preco- regulated gene activities and 7. W. F. Sheridan, Annu. Rev. Genet. 22, 353 (1988); J. cious germination, abi3 embryos are also activate these activities in the presence of K. Clark and W. F. Sheridan, Plant Cell 3, 935 (1991). desiccation intolerant 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-specific mRNAs, transduction pathway (91-97). Because de- (1979); ibid., p. 62; D. W. Meinke, Theor. Appl. Gen- et. 69, 543 (1985). such as those encoding storage proteins and fective cop/det genes are detected as fusca 9. U. Mayer, R. A. Torres-Ruiz, T. Berlath, S. Misera, G. lea proteins (85, 86). This indicates that embryo mutants, their wild-type COP/DET Jurgens, Nature 353, 402 (1991); in Cellular Com- the wild-type ABI3 gene is a positive regu- alleles are active during maturation. Thus, munication in Plants, R. M. Amasino, Ed. (Plenum, New York, 1993), p. 93. lator of gene networks leading to storage regulatory genes expressed at the end of 10. G. JOrgens, U. Mayer, R. A. Torres-Ruiz, T. Berlath, product accumulation, desiccation, and embryogenesis prepare the plant for life af- S. Misera, Development 1 (suppl.), 27 (1991). dormancy (85, 86). The ABI3 gene encodes ter the seed. 11. U. Mayer, G. Bbttner, G. Jbrgens, Development 117, 149 (1993). a transcription factor (87) related to the 12. T. Berlath and G. Jdrgens, ibid. 118, 575 (1993). corn viviparous-1 protein, which can acti- Conclusion 13. K. A. Feldmann, Plant J. 1, 71 (1991); N. R. vate the transcription of chimeric GUS 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 embryogenesis provides a vital bridge 14. D. Errampalli et al., Plant Cell 3, 149 (1991); L. A. tion-specific gene promoters (88). Thus, between the gametophytic generation and Castle et al., Mol. Gen. Genet. 241, 504 (1993). ABI3 mediates its effect on embryo matu- postembryonic differentiation events that 15. D. E. Shevell et al., Cell 77, 1051 (1994). in root 16. R. B. Goldberg, Science 240, 1460 (1988). ration at the transcriptional level. Because occur continuously the shoot and 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 embryos must establish the po- 19. S. McCormick, ibid., p. 1265. maturation-specific processes (84 -86), larized sporophytic 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 probably does not regulate the ABI3 able the young plant to survive harsh envi- 1217. on March 26, 2012 gene (84, 86). Rather, ABI3 probably op- ronmental conditions and a period of be- 22. S. D. Russell, ibid., p. 1349. erates through an ABA-independent low-ground growth 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 carry out the 25. E. S. Coen and R. Carpenter, ibid., p. 1175; J. K. embryogenesis (85, 86). Failure to achieve same developmental tasks-that is, form a Okamuro, B. G. W. den Boer, K. D. Jofuku, ibid., p. 1183. these developmental states results in ABA three-dimensional 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 consequence of 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 embryos are sensitive to occur early in plant embryogenesis and are (1993). 28. M. A. L. West and J. J. Harada, Plant Cell 5, 1361 www.sciencemag.org ABA and fail to germinate if ABA is poorly understood. Genetic studies in Ara- (1993). present (77, 78). Thus, LEC and ABI3 bidopsis have begun to reveal genes that are 29. V. Raghavan, Experimental Embryogenesis in Vas- genes are part of independent regulatory necessary for embryogenic events such as cular Plants (Academic Press, New York, 1976); S. Natesh and M. A. Rau, in of Angio- networks that control embryo maturation- pattern formation, cell differentiation, and , B. M. Johri, Ed. (Springer-Verlag, Berlin, specific events. organ development. 1984), chap. 8; B. M. Johri, K. B. Ambegaokar, P. S. In contrast to abi3 embryos, abi 1 and abi2 The precise molecular mechanisms re- Srivastava, Comparative Embryology of Angio- sperms (Springer-Verlag, Berlin, 1992). embryos carry out normal maturation-specif- sponsible for specifying different cell lin- 30. M. Schaffner, Ohio Nat. 7, 1 (1906); R. Schulz and

ic events, including the activation of storage eages early in plant embryogenesis are not W. A. Jensen, J. Ultrastruct. Res. 22, 376 (1968); Downloaded from protein and lea genes (84-86). ABIJ and known. A major void in our knowledge con- Am. J. Bot. 55,541 (1968); ibid., p. 807; ibid., p. 915. ABI2 are involved in events occur the 31. J. Hanstein, Bot. Abhadl., Bonn. 1, 1 (1870). loci, therefore, only cerns the that within egg 32. E. C. Soueges, Ann. Sci. Nat. 9, Bot. 19, 311 (1914); maintaining embryonic dormancy. The cell and in the early embryo after fertiliza- Ann. Sci. Nat. 10, Bot. 1,1 (1919). ABI 1 gene encodes a 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, phatase with similarity to serine-threonine molecular markers in order to follow the 447 (1991); S. G. Mansfield and L. G. Briarty, ibid., p. 461; ibid. 70, 151 (1992). phosphatases involved in signal transduc- specification events that take place during 34. L. Dolan et al., Development 1 1 9, 71 (1 993). tion processes (89). In response to ABA, the early embryogenesis and gain entry into reg- 35. E. C. Yeung and D. W. Meinke, Plant Cell 5, 1371 ABI 1 phosphatase might counteract phos- ulatory networks that are activated in differ- (1993). events for the initia- ent after fertilization. Al- 36. M. K. Barton and R. S. Poethig, ibid., p. 823. phorylation required embryonic regions 37. J. L. Zimmerman, ibid., p. 141 1. tion of root meristem cell division, resulting though a large amount of progress has been 38. F. A. Van Engelen and S. C. de Vries, Trends Genet. in a dormant embryonic state (89). made in recent years in understanding how a 8, 66 (1992). Regulatory loci required for postembryonic plant embryo forms, 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). development are active late in embryogenesis. to go. The next few years should be a very 40. L. Perez-Grau and R. B. Goldberg, Plant Cell 1, 1095 A large number of Arabidopsis fusca mutants exciting time to study plant embryos. (1989). have been identified that accumulate an- 41. R. L. Taylor, Can. J. Bot. 45, 1553 (1967). 42. M. A. Chamberlin, H. T. Homer, R. G. Palmer, ibid. thocyanins, or red pigments, on their coty- REFERENCES AND 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 fusca mutants (10, 108, 365 (1990); ibid. 113, 1 (1991); ibid. 118, 665 Sangwan and 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 Embryo 21st Century Group, 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 collaborative effort between the SCIENCE * VOL. 266 * 28 OCTOBER 1994 613 .Ml MM.l MM.M

laboratories of R. B. Goldberg [University of 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 Kti3IGUS genes contained the Kti3 78. The Embryo 21st Century Group (44), unpublished ty of Maryland, Baltimore), and A. Koltunow (Univer- gene TATA-box region (-28) (47). The chimeric GylI 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 identified that 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. JOrgens, 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 ConceigAo and E. Krebbers, Plant J. 5, 329 (1993). raspberry2, and lecl-2 genes were uncovered in 493 (1994). 83. L. Taiz and E. Zeiger, Plant (Benjamin, three different heterozygous lines. 59. P. N. Benfey, L. Ren, N.-H. Chua, EMBO J. 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 coi 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 21st 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. Giraudat et al., Plant Cell 4, 1251 (1992). gene was transferred to tobacco plants byAgrobac- genes was carried out by R. Yadegari, G. de Paiva, T. 88. D. R. McCarty et al., Cell 66, 895 (1991). terium-mediated transformation as described (45). Laux, and R. B. Goldberg (UCLA). Wild-type and 89. J. Leung et al., 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 +Iraspberryl Arabidopsis plants (44), fixed, 90. A. J. MOller, Biol. Zentralbl. 82, 133 (1963). Q. Bui, and R. B. Goldberg [Plant Cell 4, 1383 embedded in paraffin, and hybridized with 35S-RNA 91. L. A. Castle and D. W. Meinke, Plant Cell 6, 26 (1992)]. antisense probes according to the procedures out- (1994). 48. R. Yadegari and R. B. Goldberg, unpublished re- lined in (40, 48). raspberryl seeds were harvested 92. S. Mis6ra, A. J. MOller, U. Weiland-Heidecker, G. sults. A 2-kb 5' region from the soybean lectin gene from siliques at a time when neighboring wild-type JArgens, Mol. Gen. Genet. 244, 242 (1994). [R. B. Goldberg, G. Hoschek, L. 0. Vodkin, Cell 33, seeds were in the maturation stage of development. 93. X.-W. Deng et al., Cell 71, 791 (1992). 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, 487 (1994). transferred to tobacco plants as outlined in (47). Lo- by S. DeVries. 95. A. Pepper, T. Delaney, T. Washburn, D. Poole, J. calization of GUS activity in staged, whole-mount Chory, Cell 78,109 (1994). was determined the 66. P. Guerche et al., Plant Cell 2, 469 (1990). The Ara- embryos by procedure outlined bidopsis 2S2 gene probe was provided by E. Kreb- 96. N. Wei et al., Plant Cell 6, 629 (1994). on March 26, 2012 in (47). In situ hybridization of a GUS antisense 35S_ 97. N. Wei, D. A. Chamovitz, X.-W. Deng, Cell 78, 117 RNA bers. probe with a longitudinal globular-stage em- 67. B. W. Schwartz, E. C. Yeung, D. W. Meinke, Devel- (1994). bryo section was carried out according to the proce- opment, in press. 98. We express our gratitude to J. Harada, R. Fischer, dures outlined in (40) and in K. H. Cox and R. B. J. Okamuro, D. Jofuku, L. Zimmerman, A. Kol- Goldberg [in Plant Molecular Biology:A PracticalAp- 68. R. Losick and L. Shapiro, Science 262,1227 (1993). J. tunow, T. Laux, L. Perez-Grau, and G. Drews for proach, C. H. Shaw, Ed. (IRL, Oxford, 1988), pp. 69. A. DeJong et al., Plant Cell 4, 425 (1992). many critical and stimulating discussions of plant 1-35]. 70. A. J. De Jong et al., ibid. 5, 615 (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 acknowledge K. 50. S. C. Fry and 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. Natl. work. We dedicate this article to Professor E. H. www.sciencemag.org (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. Downloaded from

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