Experimental approaches to Arabidopsis embryogenesis

Plant Physiol. B&hem.,

Experimental

1998, 36 (l-2),

69-82

approaches to Arabidopsis embryogenesis

Thomas Berleth University of Toronto, Department of Botany, 25 Willcocks Street, Toronto, Canada M5S 3B2. (Fax I-416 9785878, E-mail [email protected]) Abstract

Arabidopsis embryo represents a simple cellular pattern comprised of few basic tissues and prototypes of leaf- and root-like organs. These structures are generated in a seriesof highly reproducible stages that imply tight control of orientation and frequency of cell division as well as cell morphology and differentiation. Additiona’l temporal control mechanisms are reflected in the successionof developmental programs that control deposition of storage materials and prepare the embryo for desiccation and dormancy. Arubidopsis embryogenesis has been studied in detail and these analyseshave been refined by the use of molecular markers to trace embryonic structures. Embryo defective mutants and new techniques to study embryos in culture have enabled functional analysesthat eventually provide the perspective for understanding plant embryo development at the molecular level. 0 Elsevier, Paris.

Key words

Plant embryo, embryo culture, somatic embryogenesis, embryo defective mutants, embryonic molecular markers, Arabidopsis.

The

INTRODUCTION The understanding of developmental processes in higher plants bears numerous promises at the academic as well as at the applied levels. The fact that higher plants have acquired multicellularity independent of animals suggests that new mechanisms of cellular interaction could be revealed, and the relatively close evolutionary relationship among seed plants implies that new findings in a model species will be of general relevance. In the Arubidopsis embryo, basic prototype plant organs are produced in an experimentally amenable genetic background, and the system has received increasing attention in recent years. In the following sections experimental approaches to Arubidopsis embryogenesis are discussed, mainly with regard to their potential for resolving developmental control mechanisms. However, it will also become obvious that developmental and cellular aspects are not always easily distinguishable, and wider applications of embryo research, covering biochemical and plant cell biological aspects will not be ignored. Due to this broad spectrum, a comprehensive description of examples is clearly Plant Physiol.

Biochem.,

0981-9428/98/1-2/O

Elsevier,

Paris

beyond the scope of this review. For further details the reader is thus referred to specialized publications on plant gametogenesis (Mansfield et al., 1991) and embryogenesis (Mansfield and Briarty, 1991; Bowman, 1994; Jiirgens et al., 1994; Jiirgens and Mayer, 1994; Meinke et al., 1995), somatic embryogenesis (de Jong et al., 1993 b; Schmidt et al., 1994) and Arabidopsis developmental genetics (Bowman, 1994; Meyerowitz and Somerville, 1994 plus other chapters in the same issue). The use of certain terms, variably defined in the literature, will here be defined as the follows (illustrated infigs. 1 and 2): Morphogenesis, will refer to all processes bringing about the overall shape of the organism, ‘including variations in cell shape or numbers. Pattern formation will more specifically refer to the spatial control of cell specification and differentiation to the actual acquisition of cell-type specific features by cells at certain positions. Finally, the unfortunate double-usage of the term polarity (Sachs, 1991) will be avoided by using axiality for all aspects of morphological directionality in which no qualitative or quantitative differences between the termini of the axialized structure (poles) are apparent (for exam-

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ple, oriented cell expansions). The term polarity, thus remains reserved for asymmetric structures in which certain features are qualitatively different, or at least (quantitatively) more pronounced along one preferred direction (Sachs, 199 1). Descriptive

embryology

Embryogenesis in seed plants encompasses the period from fertilization of the egg cell through the dormant embryo in the mature seed. From the cellular point of view, this period can be subdivided into; an early phase in which cell proliferation is intimately linked to differential specification of cell fates; an intermediate phase of maturation in which organs attain their final sizes, tissues differentiate and storage materials accumulate; and a late phase in which the embryo prepares for desiccation and ultimately attains a state of dormancy. The developmental questions to be asked may differ considerably in the three phases. Descriptive embryology raises these questions, formulates models and provides a reference for the interpretation of results from experimental and genetic approaches. Therefore, before describing strategies of experimental interference, a brief summary of the nearly invariant cell divisions during ArubidopsLs embryogenesis is given (for further details, see the above reviews and references therein). The Arubidopsis embryo develops within the protective maternal tissue of the ovule that will later form the seed coat. The ovule of Arubidopsis consists of a small nucellus (tenuinucellate ovule), and two integuments of unequal size enclosing the embryo sac (for details, see Mansfield et al., 1991). Ovule and embryo sac organization are polar, the egg cell and the synergids located at the micropylar pole. At pollination, the pollen tube enters the ovule at the micropyle and delivers the haploid nuclei, one of which fuses with the egg cell nucleus to produce the zygote, the other with the nuclei of the central cell to initiate the development of the triploid endosperm. Thus, a double fertilization event triggers the parallel development of two intimately linked multicellular structures. Although the endosperm primarily serves auxiliary functions in support of the embryo, its development appears tightly regulated. The two daughter nuclei of the primary endosperm nucleus assume positions at opposite poles in the embryo sac. Subsequently, numerous rounds of nuclear divisions produce a free-nuclear endosperm, conspicuously concentrated in the micropylar region around the embryo. Cellularization starts around Plant

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60 hours after fertilization (correlated to the stage of cotyledon initiation in the embryo), and extends from the micropylar pole towards the embryo, and also peripherally towards the chalazal chamber. The endosperm is thought to be ultimately absorbed by the growing embryo, except for a. single outer layer that persists during desiccation. Cell divisions in Arubidopsis embryogenesis follow the Cupsella variation of the Onagrad type, commencing with an unequal division of the conspicuously elongated zygote producing a smaller apical and a larger basal cell. Most of the mature embryo is derived from the apical cell, while only parts of the root apex originate from the basal cell. The apical and the basal cell descendants differ profoundly in orienting planes of cell divisions. Due to right-angle shifts in three successive divisions, the apical cell gives rise to a ball-shaped structure of eight cells, the octant. By contrast, linear divisions of the basal cell descendants result in a filamentous structure, the suspensor cfig. la). Even the sequence of these early divisions of the apical cell descendants is invariable: two vertical and one horizontal division produce the octant, comprising an upper and a lower tier (~.t. and f.r. in the following). The early divisions of the apical cells have been referred to as pseudo-cleavage divisions. since the diameter of the embryo proper has increased by only 14% (21 yM-24 PM) by the octant-stage. One round of tangential divisions in the cells of the octant separates an outer layer of eight epidermal precursor cells (the protoderm) from eight inner cells (dermatogen embryo, jig. 1b). Due to predominantly anticlinal divisions, the protoderm remains separated from the inner cells throughout further development. The embryo remains globular at subsequent stages, but increases in size and displays further specifications among the inner cells. Initially, all inner cells divide longitudinally, but Lt. descendants undergo parallel elongations in the apical-basal direction V;g. Ic). Further divisions among the lower tier descendants are strictly oriented either parallel or perpendicular to the apical-basal axis, thereby generating continuous cell files. Thus, oriented cellular behavior endows the still globular embryo with an anatomically defined axis @ss. lc and d). Successive radial differentiation becomes discernible among the cell files of this region that will ultimately form the concentric tissue layers of the hypocotyl, and parts of the embryonic root. During globular stages, descendants of the apical tier of the octant behave conspicuously different. Those cells

Arabidopsis

embryogenesis

F

Figure 1. Stqes qf Arabidopsis embryogenesis. a) octant stage; 4-8 cells (darkly stained) are visible. Descendants of these 8 cells and of the uppermost suspensor cell (hypophysis cell) will form all structures of the seedling. Being basally attached to the suspensor, the proembryo is clearly polarized, yet no morphological indication of oriented differentiation (axialization) is visible within the octant. b) dermatogen stage; a tangential division of each of the 8 cells of the “octant” produces inner- and epidermis cells. c, d) globular stages; shortly after the dermatogen stage, the cells of the lower tier elongate in the apical basal direction (c) endowing the embryo with a morphologically recognizable axis. While cell shape differences between cortex and procambial cells mark the elaboration of the radial pattern within the lower tier, cells of the upper tier remain isodiametric and do not display regional distinctions (d). e) early heart-stage; at this stage a pattern of major elements is recognizable (see text): A radial pattern comprises 3 basic tissue types: epidermis, ground tissue and vascular tissue. In the apical-basal dimension, symmetrically positioned cotyledon primordia and an axial region, giving rise to the hypocotyl and most of the embryonic root, is visible. At the basal pole, the hypophysis cell (uppermost suspensor cell) has undergone one horizontal division. Its descendants will ultimately form the centre of the primary root meristem and the columella initials f) late-heart stage; hypophysis derivatives have formed a characteristic assemblage of 12 cells arranged in 3 tiers (6 visible in the section). Hypocotyl and cotyledons are enlarged. g) mid-torpedo stage; further enlargement of cotyledons and hypocotyl and further elaboration of the radial pattern. Beginning of vascular differentiation in the cotyledons and formation of the shoot apical meristem (not visible). b) nearly mature embryo with fully elaborated radial pattern in different organs. In the cotyledons a single adaxial subepidermal layer of elongated cells (palisade) can be distinguished from isodiametric spongy mesophyll cells, The radial pattern of the hypocotyl is comprised of a single cell layer of epidermis, two cortex layers, one endodermis and one pericycle layer enclosing the vascular cylinder. (figure is reproduced from Berleth et al.. 1996).

remain isodiametric and their divisions appear randomly oriented fig. Id). At a late globular stage, however, when the cell number has increased to more than a hundred cells, the embryo gradually assumes a triangular shape, due to increased proliferation at two

opposite positions in the apical region (fig. le). The early heart stage (also referred to as ‘triangular’) embryo represents a remarkable cell arrangement, since here, in a population of roughly 250 cells, primordia of major seedling organs, such as the cotylevol. 36, no 1-2 - 1998

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dons, the hypocotyl and the primary root, as well as the basic tissue types, epidermis, cortex and procambium are already anatomically discernible (see jig. le and Genetic analysis below). The development of the suspensor is less precisely controlled. While the total number of suspensor cells may vary between 7 and 9 cells, the behavior of the two terminal cells of the suspensor is invariable. The uppermost suspensor cell (“hypophysis cell”) undergoes an invariant division pattern to give rise to the quiescent centre and to the central root cap initials of the primary root meristem (Scheres et al., 1994). The basalmost suspensor cell enlarges dramatically and has abundant contact to surrounding tissues of the mother plant, likely supporting the supply of the embryo with nutrients. It has been reported for a number of species that embryonic cells could possibly communicate through numerous plasmodesmata, but no symplastic connection to tissues of the mother plant are observed (Lyndon, 1990). Thus, no maternal high molecular mass molecules are transferred to the embryo. Due to ongoing proliferative activity in the cotyledon primordia and in the axis, the embryo successively assumes a “heart”, “torpedo” and “bent cotyledon” shape cfigs. If, g, h). At the heart- to torpedo-stage transition the shoot meristem becomes discernible as three distinct cell layers that will rapidly acquire a tunicacorpus organization (Barton and Poethig, 1993). In the following stages the body pattern is further refined; organs grow and tissues differentiate. For example, provascular tissue becomes clearly discernible in the center of the axis and at the base of the cotyledons, cells in the adaxial ground tissue layer of the cotyledons elongate, and the radial pattern in the hypocotyl and in the embryonic root becomes fully established cf;g. lh). Deposition of lipid and protein reserves commences at the torpedo and early bent cotyledon stage, respectively. Subsequently, the embryo expands to fill most of the embryo sac and the endosperm (fully cellularized by the torpedo stage) is apparently absorbed, except for a single outer layer. At desiccation, the embryo shrinks slightly and the integuments undergo multiple cellular adaptations to function as seed coat. In conclusion, the course of events in Arabidopsis embryogenesis suggests a hierarchy of partitioning events. Groups of cells appear to embark on specific developmental pathways, recognizable by defining characteristics of cell shape and/or division behavior (fig. 1). The first separation distinguishes the cells of Plant

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the embryo proper from those of the suspensor. Next, the protoderm cells are set aside and after the dermatogen stage, axial orientation distinguishes the Lt. cells from the still randomly dividing u.t. cells. Finally, another partitioning event in the apical region ensures restriction of proliferative activities to two opposed positions to produce the properly spaced cotyledon primordia. Later in embryogenesis, the elements of this early pattern are further refined, recognizable by the appearance of new specialized tissue layers. Though embryonic partitioning events are often associated with specific cell divisions, suggesting firm lineage relationships, it should be emphasized that in an invariant environment, predictability of cell fates is equally consistent with specification by positional cues. Precise determination of the cell-lineage relationships and controlled experimental distortion of normal development are tools to define the relative contributions of lineage and position in specifying cell fates. A very detailed cell lineage analysis has been performed tracing the origin of the primary root (Scheres et al., 1994). Clones were marked by random activation of a B-glucuronidase reporter gene and visualized by histological staining in seedlings. The study focused on the question whether characteristic cell divisions separate the hypocotyl and the root regions. The distribution of the observed sectors corroborates a rather high degree of predictability of cell fates. However, occasional deviating sectors confute the idea of an exclusive role of cell lineages in specifying the investigated regions. In fact, the highly variable cell divisions in the embryos of other angiosperms such as Daucus carota suggest a pivotal role of positional information that, due to rigidity, might simply be concealed in Arabidopsis. This speculation can be tested by controlled experimental or genetic distortion of embryogenesis. Experimental

embryology

The inaccessibility of the angiosperm embryo precludes direct experimental manipulation, a common tool in animal embryology. In vitro culture of either zygotic embryos excised from ovules or embryos generated from somatic cells bypass this difficulty, and enable experimental distortion of embryo development and application of potential signal substances. Both strategies have recently been adopted in Arubidopsis. The growth-requirements of Arabidopsis early zygotic embryos have been determined as a prerequi-

Arabidopsis embryogenesis

site for further in vitro studies (Wu et al., 1992). The potential of an in vitro culture system to analyse zygotic embryos can be illustrated by experiments in Brassica juncacea which aimed at determining the role of polar auxin transport in embryo development (Liu et al., 1993). The exposure of globular-stage embryos to auxin transport inhibitor substances (2chloro-9-hydroxyfluorene-9-carboxylic acid (HFCA), 2,3,5-triiodobenzoic acid (TIBA), truns-cinnamic acid) was shown to result in high frequencies of seedlings with variably fused cotyledons. This observation suggests a role of auxin transport in spatial partitioning of the apical region in the early embryo. Interestingly, Avabidopsis mutants provide an intriguing parallel. Mutations in the Arabidopsis genes, PIN FORMED (PIN) and MONOPTEROS (MP), show a similar correlation of cotyledon fusions and reduced auxin transport capacity, here measured in stem segments (Okada et al., 1991; Przemeck et al., 1996). Generating embryos from somatic cells bears numerous advantages: First, large amounts of staged embryos and embryonic tissue can be obtained, facilitating biochemical studies. Second, alternative strategies for mutant selection are feasible. Third, somatic embryogenesis is highly variable, thus allows for the assessment of developmental flexibility. Appropriate methods for generating Arabidopsis somatic embryos have recently been developed (Pillon et al., 1996; Luo and Koop, in press), but experiments combining this strategy with mutant analysis are still awaited. The carrot somatic embryo system illustrates the power of such combined approaches. A temperature sensitive embryo mutant incapable of undergoing globular to heart-stage transition could be rescued by an acidic endochitinase secreted by wild-type cells (de Jong et al., 1992). Interestingly, application of lipooligosaccharides (pentamers of B-l ,4-linked N-acetylglucosamines; also functional as Rhizobium NOD factors) resulted in similar rescue responses (de Jong et al., 1993 b). Thus, low-molecular-mass signal molecules released from cell wall components by the regulated action of endochitinases might be considered as a possible embryonic control mechanism. In conclusion, Arubidopsis embryo culture is still at an early stage, but highly desirable in view of the advantages of combining mutant analysis with other experimental tools. These studies may eventually also help unravel the extent of overlap among the developmental programmes governing zygotic and somatic embryogenesis (see below).

Genetic analysis of Arabidopsis ment

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The strength of the Arabidopsis system lies in the ease with which mutants can be isolated and genes cloned. It is thus not surprising that these types of activities constitute the most important contribution of Arabidopsis investigations to plant embryo research. Historically, the search for embryo defective mutations was among the earliest large-scale mutant screens in Arabidopsis . The large numbers of embryodefective mutants have made them suitable for assessing the mutational spectrum obtained by different types of mutagens, including T-DNA insertion (Mtiller, 1963; Jtirgens et al., 1991; Castle et al., 1993), for creating high-density maps of morphological markers (Franzmann et al., 1995) and to estimate the genetic complexity of this developmental phase (Jtirgens et al., 1991). These quantifications suggest that mutations in large numbers of permanently required genes constitute the bulk of the embryo defective mutations. Classification of embryo mutants is not an easy task. Mutant phenotypes are often extremely variable, showing a high degree of overlap among non-allelic mutations (Meinke, 1985). The difficulty of inferring the type of primary defect from a simple inspection of the phenotype implies that classification of embryo defective mutants is a long-term process, occurring gradually and, most likely, often by revising previous concepts. This has a bearing on the presentation of this review. Mutants are simply grouped by conspicuous features of their phenotype, while the proposed gene functions in specific embryonic processes are discussed within each section. Insertional mutagenesis, giving direct access to the gene product, is the preferred strategy for overcoming the lack of informative phenotypes among embryolethal mutants (Castle et al., 1993). A particularly attractive strategy combines DNA-tagging with information from enhancer or gene trap-reporter gene expression and the technical advantage of an efficient transposon insertion system (Sundaresan et al., 1995). Mutations in the PROLIFERA (PRL) gene illustrate this strategy (Springer et al., 1995). Insertion of a GUS-gene-trap-Ds element in the PRL locus resulted in a highly variable spectrum of mutant phenotypes, ranging from gametophytic lethality to inconspicuous arrest of homozygous embryos. However, the additional information from the associated reporter-gene expression revealed a localized activity of the gene in voL36,n"

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proliferating tissues throughout the Arubidopsis lifecycle. Selected for further investigation by this feature, the tagged gene finally turned out to encode a product with homology to the yeast MCM2-3-5-family, required for initiation of DNA replication. Thus, the prl insertion illustrates how an associated viable dominant (expression) trait can replace the confounding effects caused by gametophytic lethality, impenetrance and an inconspicuous phenotype. The prl example also illustrates that the Arabidopsis embryo mutant collection is also suitable for the genetic analysis of plant cell biology (see further examples among seedling mutants). Two classes of readily recognizable localized early defects have been described: suspensor mutants and twin mutants. Suspensor mutants comprise a fairly heterogeneous class, unified by dramatic alterations in suspensor size. It has been proposed that growth of the suspensor is limited by a signal from the developing embryo. However, the nature of this signal is elusive and it is not known whether specific types of defects in the embryo proper are associated with abnormal suspensor development. The term twin mutant usually refers to all lines segregating conspicuously enhanced frequencies of multiple embryos within single ovules, a phenomenon observed in a broad range of taxa. Polyembryogeny not necessarily interferes with embryo viability, and two seedlings may ultimately emerge from a single seed. The wild-type functions defined by twin mutations would be expected to act in suppressing unscheduled triggering of the embryonic programme from somatic cells. This is consistent with the observation that embryonic cells deprived of their natural context are prone to initiate somatic embryogenesis (Pillon et al., 1996). In this interpretation, twin mutants could genetically identify the systemic signals necessary to suppress infinite reiteration of the embryonic program by individual cells. Irrespective of the type of this signal, the mere occurrence of certain types of double embryo phenotypes enables one to reassess the role of the zygote in embryo pattern formation. The second embryo in a twin mutant may, for example, arise from a suspensor cell, occasionally in an inverted orientation (Vernon and Meinke, 1994) and can thus be viewed as the development of a somatic embryo in an ovule environment. Morphological peculiarities of somatic embryos from suspension cultures have raised the question of alternative genetic programmes in zygotic versus Plant

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somatic embryogenesis, but - unless analysed in twin mutants - it is difficult to exclude distorting influences of the in vitro growth conditions. Thus, the occurrence of features specific for secondary embryos in twin mutants can be seen as a critical test for the assumption of an exclusive role of the zygote in embryogenesis. As yet, no such peculiarities have been reported. Seedling mutants Numerous embryonic distortions are not associated with early lethality, and can therefore be recognized at the seedling stage. In fact, mutants considered to be involved in the specification of the early embryonic pattern were isolated deliberately by their seedling phenotypes. The rationale of this strategy was based on the assumption that specific genes should exist that are functionally restricted to pattern formation (see definitions), and thus are not required for embryo viability (Jtirgens et al., 1991). Similarly, characteristic alteration in the spatial array of defined pattern elements recognizable at early heart-stage (see$gs. 2e, f, g) as well as in the seedling served as a diagnostic feature. The pattern elements scored were the major seedling organs, cotyledons, hypocotyl and root, in the apicalbasal direction and the basic tissue types, epidermis, ground tissue and vascular tissues, as radial pattern elements. As the strategy obviates the need for labourintensive isolation of mutant embryos, large numbers of mutagenized lines can be screened such that saturation for this class of mutants could be achieved. Interestingly, an unexpected class of mutants was isolated in which severe morphological distortions did not result in pattern defects. Despite a dramatically altered overall shape, essential seedling organs and tissues were found in place, and the abnormal appearance of these mutants was largely due to altered cell dimensions. The most dramatic of these “shape mutants”, the cell-elongation defective ,fi~s.s (fi) mutant, is extremely stout at all stages (fig. 2), yet develops beyond the seedling stage and eventually produces stunted, sterile plants (Torres Ruiz and Jtirgens, 1994). Though not altered in its basic seedling pattern, the$y mutant is highly instructive in interpreting plant embryo pattern formation. Cell divisions inJ-s embryo development turned out to be highly variable pg. 2 c and d) demonstrating that rigidity of intermediate stages is not an instrumental part of the mechanism that determines the seedling pattern. This raises the question of what kind of underlying spatial cues guide normal development, irrespective of the precise

Arabidopsis embryogenesis

Figure 2. Seedlings and embryos of wild-type, “shape” and potential “embryo pattern” mutants. Seedlings of the wild-type (a), the “shape” mutant fass (fs) (b) and the potential “embryo pattern” mutant monopteros (mp) (e) at the same magnification. In fs mutants all seedling organs appear extremely stout and deformed, but are present and properly positioned. In mp mutants all basal organs (hypocotyl and primary root) are missing, while the apical region is remarkably normal. c, d) wild-type (c) and fs mutant embryos (d) at mid heart-stage. Althoughfs mutant embryos display regional specifications (e.g. cotyledon primordia in [d]), these emerge from completely variable cell divisions rather than as part of a rigid division sequence as in wild-type embryos. f, g) wild-type (f) and mp mutant embryos (g) at early heart-stage. Defects in mp mutant embryos are confined to the hypocotyl/root region. All mutant embryos display certain characteristic abnormalities indicating an alternative route of embryogenesis. Cells in the central (incipient hypocotyl, embryonic root) region fail to undergo axial elongations and an abnormal pile of cells is observed in the region normally occupied by hypophyseal derivatives. Abbreviations: ep, epidermis; co, cotyledons; rp, incipient root primordium; vp, vascular primordium. (a-d, reproduced from Tomes Ruiz and Jiirgens, 1994; f, g, reproduced from Berleth and Jiirgens, 1993).

suite of embryonic events (see below). Traas et al. (1995) confirmed the above observation with the phenotypically similar (possibly allelic) tonneau (ton) 1 and ton2 mutants. They also showed that the preprophase band, normally marking the position of the future cell wall in plant cell divisions, is missing in ton mutants. McClinton and Sung (1997) further delimited the cytoskeletal defect, showing thatfs mutants selectively lack capacities for spatial organization of microtubules, while still being capable of forming normal mitotic spindles and phragmoplasts. Thus, although the primary defects are still elusive, f&on mutants shed new light on the spatial cues underlying embryo development and provide genetic access to mechanisms of plant cell cytoskeletal organization. If spatial cues dictate the final seedling pattern irrespective of intermediate cell arrangements, what anatomical features in the embryo should then be considered as indicative of a defect in one of those underlying cues? A number of seedling mutants displayed a truly alternative path of embryogenesis, leading to correspondingly altered seedling phenotypes @g. 2). This is suggestive of a switch in an underlying developmental program, and thereby of a gene product in control of this program. However, it should be kept in mind

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that developmental decisions can occasionally be influenced by rather mundane factors (in some examples even temperature), thus phenotype alone can at best serve as a reliable preselection criterion. Mutations in four genes were described to result in deletions of pattern elements along the apical-basal axis, recognizable from the early embryo to the seedling stage. Mutations in the GNOM/EMB30 gene result in dramatic distortions of the apical basal organization (Meinke, 1985; Mayer et al., 1993). gn Seedling phenotypes range from funnel shaped rootless seedlings with fused cotyledons to perfectly spherical “ballshaped” seedlings. The latter do not display any trace of apical basal polarity, while the radial organization seems unaffected. At heart-stage, gn embryos do not display apical-basal partitioning. Moreover, mutant specific abnormalities could be traced back to the zygote, whose asymmetric division depends on GN gene activity (Mayer et al., 1993). GN gene function turned out not to be restricted to the early embryo, demonstrated by the invariably unorganized growth of gn mutant cells in culture (Mayer et al., 1993). These findings are indicative of a general role of the GN product in initiating or stabilizing apical-basal polarity vol. 36. no 1-2 - 1998

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in plant development. The GN gene has been isolated, and the predicted protein product displays limited similarities to the yeast SEC7 gene encoding a secretory protein, and more extensive similarity to YEC2, a yeast gene of unknown function (Shevell et al., 1994; Busch et al., 1996). It may thus require a more detailed understanding of the cell biological mechanism by which plant cells establish and stabilize polarity to understand the crucial role of the GN gene product in this process. Mutations in three genes - MONOPTEROS (MP), GURKE (GK), and FACKEL (FK) - affect only specific parts of the apical-basal pattern. Mutations in the MP gene result in the deletion of both the hypocotyl and the embryonic root (fig. 2e), while the cotyledons can be variably fused (Berleth and Jtirgens, 1993). In culture, mp mutant cells are capable of producing both types of terminal meristems and thereby adult plants. This enables the study of morphology and anatomy in mutant organs over the entire life-cycle (Przemeck et al., 1996). Anatomical analyses revealed no general cellular defects in mp mutant embryos or plants. Rather, there seems to be a specific requirement for MP gene activity in the differentiation and proper alignment of vascular cells. Interestingly, the vascular defects in mp mutant organs are consistent with the early heart-stage embryo phenotype which is characterized by a lack of elongated cells in the hypocotyl region of the embryo @g. 2g). It has therefore been proposed that mp mutant cells lack orienting information from spatial cues that mark the shoot-to-root axis and that the MP gene could be involved in relaying these signals (Przemeck et al., 1996). The recent identification of the MP product as a transcriptional regulator expressed in developing vascular tissues is in good agreement with this interpretation (Hardtke, C.S. and Berleth, T., unpublished). It is interesting to note that apical-basal polarity is strictly maintained in mp mutants. In contrast to ball-shaped gn mutants, there is unequivocal apical-basal asymmetry. Rather, it is axiality (morphological directionality irrespective of polar asymmetry; see definitions above) that is affected in mp mutants. Mutant, abnormalities are most pronounced where cells are extended in the apical-basal direction (e.g. vascular tissues), while not recognizable in apparently non-oriented tissues (e.g. leaf mesophyll). While this type of vascular defect has not been described for any other Arubidopsis mutant, other features of the post-embryonic mp phenotype resemble Plant

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those of the mutant pin firmed @in) (Okada et ul., 1991). Both mp and pin are defective in polar auxin transport, positioning of the cotyledons and the formation of determinate organs from reproductive meristems. They may therefore be isuitable candidates for genetic dissection of the developmental relationship between auxin transport, vascular patterning and the generation of determinate organs. The GK gene seems to be locally required in the apical domain of the embryo. In strong gk mutants (an allele was previously isolated and named emb20. Patton et al., 1991) the entire apical domain, comprising the shoot meristem, the cotyledons and even parts of the hypocotyl may be deleted, while the root meristem and the radial pattern in hypocotyl and root are normal (Ton-es Ruiz et al., 1996). Mutant embryos develop normally up to the globular stage, but fail to initiate the growth of the cotyledons. Notably, there is some variability even in strong alleles. While the cotyledons appear to be most sensitive to lack of gk gene activity, some abnormal shoot structures can be produced even by strong mutants. These features of the phenotype suggest that the GK product directly of indirectly influences the organization of the apical domain, rather than being required for the production of certain apical structures. Seedlings mutant for the gene FK lack the hypocotyl, while cotyledons and the root are present. The seedling phenotype reflects localized defects in the early heart-stage embryo, recognizable by the lack of provascular cells in the hypocotyl region. Thus, there are overlaps in the defects in ,flz, mp and gk mutants, such that more than one of the genes participates in the specification of each of the embryonic regions. Mutations in two genes, KNOLLE (KN) and KEULE (KEU), have been described as affecting the radial pattern by preventing the formation of a proper epidermis (Mayer et al., 199 1). Subsequent ultrastructural analysis of these mutants, however, revealed features indicative of incomplete cell divisions, such as cell wall stubs and multinucleate cells (Assaad, 1996; Lukowitz et al., 1996). The KN product has been identified as a likely Arabidopsis syntaxin, a class of proteins involved in vesicular trafficking in a large variety of organisms (Lukowitz et al., 1996). From these results it seems conceivable that KN, and possibly also KEU, are involved in directing material for cell plate formation during cytokinesis. The radial pattern distortion and the cellular defect could be related, if stable expression of cell fates depends on cytoplasmic sepa-

Arabidopsis embryogenesis

ration of cell groups. Interestingly, similar conclusions are suggested by marker injection experiments in root meristem cells that show diminishing cytoplasmatic connection between maturating cells in different tissue layers (Duckett et al., 1994). The interpretation is also supported by the observation of typical protoderm specific markers being illegitimately expressed among subepidermal cells in kn mutant embryos (Vroemen et al., 1996). The dramatic phenotypes of the seedling mutants described in this section demonstrate that even gross abnormalities in the early embryo do not per se arrest embryogenesis. It is thus not surprising that mutations affecting more subtle aspects of the embryonic pattern were identified by phenotypes at still later stages. A number of mutations lead to the deletion of the shoot meristem or to its delayed appearance. These mutants are described in detail in reviews on shoot meristem development. Since they have no bearing on the remaining embryo they will not be discussed in this review. By contrast, a number of root mutants turned out to reflect highly specific embryonic defects. The radial pattern of the mature embryo axis, as well as of the post-embryonic root comprises six, rather than just three, discernible tissue types: epidermis, cortex, endodermis, pericycle and phloem and xylem strands in the central cylinder. Analysis of mutants defective in the radial organization of the root (and thereby in root growth) revealed corresponding radial defects in the embryo, suggesting that the primary root meristem merely perpetuates the embryonically established radial pattern (Scheres et al., 1995). This raises the possibility of utilizing defective root growth as a diagnostic feature to screen for this otherwise rather inconspicuous type of embryo pattern defect. Mutations in five genes were found to interfere with the formation of specific root/embryonic tissues. Again, a number of unrelated primary defects could conceivably impinge on the radial pattern and need to be sorted out by additional tests. For example, the lack of a specific tissue layer could simply result from a shortage of cells. An elegant genetic test addressed this question. When the number of tissue layers was increased in a$y mutant background, the defect was no longer visible in the majority of the mutants. However, loss of the endodermis layer by mutations in the gene SHR was not suppressed by$y mutations, suggesting a function of the SHR gene in specifying the endodermis element of the radial pattern.

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Three conclusions may be extracted from the above examples: First, severe distortions of the embryonic pattern do not per se arrest embryonic development. Instead, revealing embryo mutants may be identified by phenotypes at surprisingly late stages. Second, cellular defects that impinge on the embryonic pattern may identify developmental correlations, such as the relevance of cytoplasmic separation. Third, Arabidopsis embryo and seedling mutants also prove suitable for characterizing novel functions in plant cell biology, as illustrated by the functional characterization of a plant syntaxin in the kn mutant or by the surprising lack of a preprophase band in thefi mutant. Pigmentation

mutants

Mutants identified by pigmentation abnormalities at the seedling stage have been broadly classified as albino, yellow-green andfusca mutants, the latter displaying a purplish-green pigmentation already visible through the seed coat. Available data from a few systematically analysed pigmentation mutants also heterogeneous types of primary defects, however, within thefusca class, where the majority of 14 identified loci have been implicated in the negative regulation of light dependent signals (Misera et al., 1994; for review Wei and Deng, 1996). Cotyledon identity and late embryogenesis mutants Except for a dark pigmentation, fus3 mutants have little in common with any of the otherfusca mutants. Instead, mutations in the FUS3 gene primarily interfere with embryo maturation (Baumlein et al., 1994; Keith et al., 1994). Conceivably, seed plants have to express complex genetic programs to bring about dormancy late in embryogenesis, while suppressing germinative programmes until unleashed by appropriate environmental signals perceived by the dormant seed. fus3 Mutant embryos do not establish dormancy and desiccation tolerance, but instead express markers of germinative development already during embryogenesis (Keith et al., 1994). Interestingly, the defect also perturbs cotyledon development in that fus3 mutant cotyledons display both cotyledon as well as vegetative-leaf characteristics @g. 3). Partial cotyledon-toleaf transformations associated with maturation defects are also observed in two other non-allelic mutants, leafy cotyledon (1ec)l (Meinke, 1992; West et al., 1994) and lec2 (Meinke et al., 1994). For let mutants, the earliest stages of abnormality have not vol. 36, no l-2 - 1998

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T. Berleth

Figure 3. Cotyledon identity mutant fusca3 (fus3J. Mutants are recognizable by abnormal pigmentation in immature siliques (a). When excised from ovules prior to desiccation (b: wild type [left] and larger ,,fu93 embryo [right] 12 days after fertthaation), desiccation intolerant fus3 mutants can be rescued. Numerous mutant features are related to unscheduled germinative events in the embryo and the FUS3 gene function has thus been implicated in the temporal control of late embryonic versus germinative programmes (Keith et al.. 1994). The arrows in c mark (reduced) trichomes on mutant cotyledons that are normally only produced by postembryor,ic leaves (d) (c, d; scanning electron micrographs; bars = 63 urn). (figure reproduced from Keith et al.. 1994).

precisely been defined. Formally, this defect is both heterochronic and homeotic, since the expression of a developmental program is shifted temporally and spaPlant

Physiol.

Biochem.

tially. However, the correlation of this type of defect with other unscheduled events in embryogenesis of three non-allelic mutants has been interpreted to sug-

Arabidopsis embryogenesis

gest a primary defect in temporal control (Keith et al., 1994). A fairly large number of molecular probes defining late stages of embryogenesis should enable one to test this hypothesis in the available mutants, as well as to further dissect the underlying regulatory network (for examples see West et al., 1994). In contrast to fus3 and Zec mutants, maturation defects in abscisic acid insensitive abi mutants are associated with generally reduced ABA responses, demonstrating the role of this plant hormone in the establishment and maintenance of seed dormancy. Remarkably, the ABZ3 product encodes a transcriptional regulator related to the maize embryo maturation control gene VP1 (Giraudat et al., 1992), and phenotype analysis of ubi3 mutants suggests complex regulatory functions of the gene in orchestrating maturation and germinative programmes (Nambara et al., 1995).

Molecular

markers

The early progress of descriptive plant embryology was facilitated by the rigid framework of plant cell walls that is readily recognizable even in whole-mount preparations and also precludes confounding morphogenetic movements. However, the experimental strategies described above depends on the availability of markers to identify specific cells, irrespective of shape and arrangement. Suitable molecular markers are of three types: Reporter gene expression, nucleic acid antisense sequences to detect genes by in situ hybridization and specific antisera to localize the respective protein products. Probes of all three types have been compiled in previous reviews (Jiirgens et al., 1994). Recently published highly specific expression profiles include the shoot meristem specific STM gene (Long et al., 1996) the cortical initial and endodermis specific SCR gene (Di Laurenzio et al., 1996) the provascular specific ATHB-8 gene (Baima et al., 1995) and the epidermis specific genes ATLTPI (Vroemen et al., 1996) and ATMLl (Lu et al., 1996). Large numbers of new expression patterns are to be expected from systematic analyses of available enhancer and gene-trap collections (Topping et al., 1994; Sundaresan et al., 1995). Conclusions

and prospects

What are the major contributions of the different fields of Arubidopsis embryo research and is there an emerging view of the mechanisms governing embryo

79

development? Detailed descriptions of wild-type embryogenesis, and a steadily increasing number of reliable embryonic markers have enabled more precise evaluations of the phenotypes of distorted embryos. Experimental manipulation of embryogenesis is still at an early stage, but sufficiently established to be applied in mutant background. Not surprisingly, mutant analysis in combination with molecular characterization of the corresponding gene products has become the broadest area of Arubidopsis embryo related research. Characterization of embryo mutants has led in different directions. Developmental control mechanisms are only part of the appearing spectrum; basic plant cell biology and biochemistry are others. For the latter aspects, early embryo-lethal mutants seem to play an important role. This stresses the need for rapid assessment of primary defects among early lethal mutants which thus far has precluded their use as tools in analysing defined wild-type processes. Fortunately, rapid progress can be expected from ongoing saturating approaches. Large-scale insertional mutagenesis in combination with gene-trap expression patterns (e.g. Sundaresan et al., 1995) and the tools of reverse genetics (e.g. McKinney et al., 1995) may soon assign functional information to a larger fraction of the embryolethal mutants. Identifying genes regulating embryo development is a difficult task: unlike in several animal systems, the key regulatory genes are still elusive and there is no generally accepted model integrating the roles of identified genes. Nevertheless, several developmental control mechanisms have become genetically tractable. Temporal control of dormancy programmes, for example, turned out to be amenable to mutant analysis. Heterochronic/homeotic shifts, indicative of alternative control of developmental programmes, were observed, and diagnostic features have been elaborated. Finally, the question of how the embryo establishes the threedimensional order of specified cell fates receives elucidatory input from several sides. First, there is accumulating evidence for the importance of positional information in the development of plant organs (for reviews see Scheres, 1996; Meyerowitz, 1997). Second, embryo culture techniques provide test systems for developmental flexibility and enable one to screen for genetically inaccessible signal molecules, such as low~molecular-mass substanc&. Third, Arubidopsis mutants have identified genes involved in fundamental aspects of plant architecture, such as polarity, axiality, vol.

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80

radial organization. Apparently, this basic architecture is sufficient for viability, since “shape” mutants like$y that are distorted in virtually all other aspects of morphology are viable up to the reproductive stages. If a viable pattern is that robust, possibly fewer cues than in animals are required for its specification (though still a number of them are likely to be identified). In this interpretation, additional fine-tuning controls would be required solely to dictate morphology in detail. This view has a bearing on mutant screens, since genes involved in such line-tune control mechanisms would more likely be identified by subtle developmental defects rather than by lethal embryo distortions. Acknowledgements. I would like to thank C. Hardtke, P. McCourt, A. Mordhorst, W. Lukowitz, S. Ploense and B. Scheres for helpful suggestions on the manuscript and Kluwer Academic Publishers, the Company of Biologists Ltd. as well as the American Society of Plant Physiology for the permission to reproduce published photographs. (Received July 18, 1997; accepted August 25, 1997)

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