J. Plant Res. 111: 295-298, 1998 Journal of Plant Research @ by The Botanical Society of Japan 1998

JPR Symposium

Genetic Pathways Controlling Carpel Development in Arabidopsis thaliana

John A l va rez and Dav id R. Smy th

Department of Biological Sciences, Monash University, Clayton, Melbourne, Victoria 3168, Australia

Carpel development in Arabidopsis is known to be controlled by the organ identity gene AGAMOUS. How- ever, even in the absence of AGAMOUS function, many carpel properties can arise suggesting that other genes are also involved. Two new carpel genes, CRABS CLAW and SPATULA, have been recognised by their specific disrup- tions to carpel development in mutant plants. These dis- ruptions suggest that CRABS CLAW normally plays a role in promoting the growth of specific regions of the carpel wall, whereas SPATULA apparently has a primary function in promoting development of the transmitting tract. When the function of these genes is also compromised along with that of AGAMOUS in multiply mutant plants, carpelloid properties vanish. Thus AGAMOUS, CRABS CLAW and SPATULA act together in specifying carpel development, although none can do this alone. Because SPATULA mutants are epistatic to mutants of another carpel development gene, E-ITIN, the latter may normally act by suppressing the action of SPATULA in specific regions of the developing gynoecium. There is indirect evidence that ETTIN, and another morphegenetic gene, PINOID, act through regulat- ing auxin-induced growth in specific regions of the develop- ing flower, but it is not yet known how this could result in the suppression of SPATULA function.

Key words: AGAMOUS - - Arab idopsis - Carpel devel- opment - - CRABS C L A W - - ETT IN - - Gynoecium - - PINOID - - SPA TULA

A central question in developmental biology is how cells know their position within the developing organism and grow and differentiate appropriately. This information comes ultimately from genes and environmental signals. In plants, studies over the last decade have thrown light on how gene products control the morphogenesis of embryos, leaves, roots and flowers. In flowers, a series of organ identity genes has been identified and their molecular structure and function are now being deduced (Weigel and Meyerowitz 1994). Also, recent studies have characterised genes that set up boundaries within the flower (Sakai et al. 1995), and

The extended abstract of a paper presented at the 13th Inter- national Symposium in Conjugation with Award of the Inter- national Prize for Biology "Frontier of Plant Biology"

genes that control the rate of proliferation of regions within the floral meristem (Clark et al. 1997). Less well known are genes that control the growth and differentiation of individual floral organs, the most complex of which are the carpels.

Flowers of all Angiosperms contain carpels. These are the female reproductive organs that enclose and protect the ovules, gamete-bearing structures that ultimately develop into seeds. Indeed, it is the carpel that defines this suc- cessful plant group (Cronquist 1988). They may occur as individual organs or as fused aggregates, and together make up the gynoecium.

Much of the recent progress in understanding floral morphogenesis has come from studies of model plant species including Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida (Weigel and Meyerowitz 1994). In Arabidopsis, the gynoecium is composed of two congenitally fused carpels (Hill and Lord 1989, Okada et al. 1989, Smyth et al. 1990, Sessions and Zambryski 1995). These arise from the central dome of the floral meristem. First a rim of cells begins to extend as a hollow tube that eventually fuses post- genitally near the apex to form a solid style capped with a dry stigma made up of many papillae. Inside the tube, a ridge of tissue grows outward from each point of fusion of the two carpels, and eventually these outgrowths fuse post-genitally to form a false septum that divides the gynoecium into two Iocules. Placental tissue arises on each side of the growing septum and about 40-50 ovules arise per flower. Just before maturity, specialised cells within the style and septum secrete an extracellular matrix that provides a pathway (transmitting tract) for pollen tube growth.

Genes involved in the carpel development program are likely to disrupt the program in characteristic ways when they are inactive or dysfunctional. Several such genes are already known from Arabidopsis. Firstly, mutants of three clavata genes usually result in the formation of a gynoecium with four fused carpels instead of two. However, this is apparently the consequence of an earlier disruption to signalling that controls the flower meristem's growth pattern rather than reflecting a specific involvement of CLAVATA genes in carpel morphogenesis (Clark et al. 1997). Second- ly, mutants of the ETTIN gene have gynoecia that contain bizarre disruptions (Sessions and Zambryski 1995). In this case there are reversals of normal tissue distributions, in both inner-outer and apical-basal orientations. It has been proposed that ETTIN allows the setting up or perception of

296 J. Alvarez and D.R. Smyth

two radial boundaries early in gynoecium development that distinguish subsequent tissue differentiation pathways (Ses- sions 1997). The ETTIN gene encodes a protein related to transcription factors that respond rapidly to the presence of the plant growth substance auxin (Sessions et al. 1997). Thus ETTIN may be having an effect on carpel development by specifying where auxin-stimulated growth will occur.

The best known gene controlling carpel development in Arabidopsis is AGAMOUS. This is one of the so-called ABC organ identity genes, and is the only known member

encoding C function (Bowman et al. 1991). When C function is active in the absence of B function, organogenesis follows the carpel developmental program. This has been deduced from the phenotype of A function mutants in which AGAMOUS is ectopically active in first whorl organs. Instead of sepals, these organs now develop with all the features of carpels. AGAMOUS encodes a transcription factor and is a founding member of a large class of such regulatory products containing a conserved MADS box (Rounsley et al. 1995, Riechmann and Meyerowitz 1997).

Carpel Development Genes in Arabidopsis 29"7

However, AGAMOUS C function is insufficient to specify all carpel properties (Bowman et al. 1991). When AGAMOUS function is also removed from A class apetala2 mutants, as in AC double mutants, the medial first whorl organs that result are not devoid of carpel features. The only tissue type that is absent is the specialised ovary wall. The stigma, septum and ovules are still present.

We have identified two further carpel development genes, CRABS CLAW (CRC) and SPATULA (SPT), that, when com- bined with A and C mutants in a quadruply mutant plant, result in the almost complete loss of these remaining carpel properties. Thus we believe that CRABS CLAW and SPAT- ULA act in parallel with AGAMOUS to specify the full carpel developmental program.

Results

The CRABS CLAW gene has a mutant phenotype in which the carpels are wider and shorter than normal, and unfused at the top (Fig. 1B c.f. Fig. 1A). The two unfused regions bend inwards in the mature fruit and resemble the pincers of a crab's claw. No other floral organs or tissues (except nectaries) are affected. Developmental studies of mutants suggest that the CRABS CLAW protein controls growth of the gynoecium, suppressing lateral growth of the early primordial dome but enhancing vertical growth of the elon- gating cylinder (Alvarez and Smyth 1993a). The CRABS CLAW gene has been cloned by chromosome walking, and it encodes a protein likely to represent a new class of transcription factors (J.L. Bowman and D.R. Smyth, unpub- lished results). Studies of the expression of CRABS CLAW are consistent with its proposed role in gynoecial growth.

The SPATULA gene, on the other hand, plays an important role in septum and transmitting tract development. In spat- ula mutants, the septum and stigma are greatly reduced, and cells that secrete the extracellular matrix in the style and septum are absent (Fig. 1C). There is also some effect on gynoecium growth so that the fruits are often flattened

laterally at the top and thus appear spatula-l ike. No other floral organs are affected (AIvarez and Smyth 1993b). Genetic evidence indicates that SPATULA and CRABS CLAW play an overlapping role in carpel development. In double mutants, growth of the gynoecium is much more severely disrupted than in either single mutant, suggesting that this role involves specifying growth in defined regions and at defined times (Alvarez and Smyth 1997).

Ectopic carpels develop in the first whorl medial positions of the A function mutant apetala2 (Fig. 1D) as a consequence of deregulation of C function genes. When the C function encoded by AGAMOUS is also removed in apetala2 agamous double mutants, the wall of these ectopic organs becomes leaf- and sepal-l ike, but other carpel properties remain (Fig. 1E). We have tested whether these residual carpel features are specified by CRABS CLAW and SPATULA by generating the agamous apetala2 crabs claw spatula quadruple mutant (Alvarez and Smyth 1997, J. Alvarez and D.R. Smyth, unpub- lished results). Clearly, almost all carpel properties are now lost (Fig. 1F). Only an occasional marginal outgrowth arises that may represent an ovule precursor. Thus AGAMOUS, CRABS CLAW and SPATULA are all required simultaneously for complete carpel morphogenesis.

The ETTIN gene specifies pattern formation within the developing gynoecium rather than being necessary for the presence or differentiation of any specific tissue type. In ettin mutants, a thin stalk-l ike structure (gynophore) arises in place of much of the gynoecium (Sessions and Zambryski 1995). This displays two switches of the usual tissue polar- ity, the normally internal septum and transmitting tract are now external, and the normally apical style now extends for much of the organ's length. The phenotype of an interme- diate mutant allele, ettin-3, is shown in Fig. lG. When double mutants are made between ettin and spatula, a remarkable restoration of gynoecium structure is seen (Fig. 1H). The carpel walls are mostly restored, and there is no longer any external transmitting tract. This epistasis of spatula mutants over ettin mutants indicates that the ettin

Fig. 1. Morphology of the gynoecium in mature flowers of wild type and a range of carpel development mutants of Arabidopsis. (A) Medial view of wild type flower. The gynoecium consists of a stigma (stg), style (sty) and ovary. The ovary has two walls (ovw) joined by a thin strip of replum tissue. A septum arises internal to the replum that divides the ovary into two Iocules, and ovules arise from each side of the septum. Overall, the gynoecium arises as two carpels congenitally fused in the medial plane of the flower. (A medial sepal, two petals and two medial stamens have been removed.) (B) Medial view of a crabs claw-I mutant flower. Note that the carpels are wider but shorter than normal, and at the apex they are unfused and bend inwards. (C) Medial view of a spatula-2 mutant flower. The carpels are unfused at the apex, and transmitting tract is absent in the hollow style and reduced septum. Later the gynoecium will be flattened laterally and widened medially as it develops into the fruit. (D) Lateral view of an apetala2-2 mutant flower. The two medial first whorl organs resemble solitary unfused carpels, with stigmatic papillae (stg) and stylar tissues (sty) extending around the edges of the ovary wall (ovw). A flange of septal tissue (sep) and a row of ovules (ovu) arise along the edge internal to the style. (Lateral first whorl organs, second whorl and most third whorl organs are absent.) (E) Lateral view of an apetala2-2 agamous-1 double mutant flower. The ovary wall (ovw) now appears leaf- and sepal-like, the style is reduced/absent, but other carpelloid tissues remain (stigma (stg), septum (sep) and ovules (ovu)). (F) Medial view of an apetala2-2 agamous-1 crabs claw-1 spatula-2 quadruple mutant. All carpelloid tissues are now absent from the margins of the medial first whorl organs (arrows). (G) Medial view of the intermediate ettin- 3 mutant flower. The lower half of the gynoecium appears as a stem-like gynophore (gph), while stigma (stg) and stylar (sty) tissues have expanded in the upper half. A small patch of ovary wall (ovw) remains. (A medial sepal, two petals and several stamens have been removed.) (H) Medial view of an ettin-3 spatula-2 double mutant flower, spatula-2 is largely epistatic to ettin-3 in that the gynophore (gph) is now greatly reduced, the ovary wall (ovw) extended, and the style (sty) and stigma (stg) occupy close to normal apical positions. (I) Side view of pinoid-I mutant flower. The gyncecium develops as a long, thin gynophore (gph) capped with a large stigma (stg). Other floral organs arise in variable numbers, with sepals and stamens usually reduced, but petals increased. (Several of each organ type have been removed.)

298 J. Alvarez and D.R. Smyth

mutant phenotype may arise through ectopic SPATULA expression. E'I-I-IN is known to encode a product related to auxin responsive transcription factors (Sessions et al. 1997), and one of its roles may lie in negatively regulating SPATULA expression in the carpel wall. These proposals can be tested once SPATULA has been cloned (M.G.B. Heisler and D.R. Smyth, in progress).

It is interesting that mutants of another gene, PINOID, result in stem-like gynoecial growth (Fig. 11; Bennett et al. 1995). Similar disruptions may occur in flowers of weaker mutants of a further gene, PIN-FORMED (Bennett et al. 1995). While pinoid and pin-formed mutants resemble ettin mutants in having stem-like gynoecia, the gynoecia do not show the same ettin-like disruptions to tissue polarity. There is indirect evidence that PINOID and PIN-FORMED also involve an auxin-related process, because phenocopies of pinoid and pin-formed mutant disruptions can be induced by applying specific inhibitors of polar auxin transport (Okada et al. 1991). Even so, polar auxin transport is not abolished in mutants of either gene (Bennett et al. 1995), so specifica- tion of transport is unlikely to represent their primary function.

Conclusion

The intricate network of developmental decisions (Meyer- owitz 1997) underlying carpel morphogenesis is beginning to be unravelled. Several transcription factors that regulate expression of "realistor" genes have been identifed, although specific realisator targets are as yet unknown. Also we are in the dark about signals that specifically control carpel development (although more general signals are beginning to be uncovered (Roe et al. 1997)). It is expected that such signals exist in developmental time and space, but informa- tion about their generation, perception, transmission and response is lacking. Continued study of genes whose mutants specifically disrupt carpel morphogenesis, and genes that are specifically expressed in sub-regions of developing carpels (Rounsley et al. 1995), should soon rem- edy this situation.

Carpel evolution is intimately linked with Angiosperm evolution. The enclosure of ovule-bearing structures, and the generation of a specialised transmitting tract for pollen tube growth, are the two properties that define carpels. It is interesting to speculate that ancestors of genes such as CRABS CLAW (controlling carpel wall growth) and SPA TULA (necessary for development of the transmitting tract) may have played an early role in carpel evolution.

This work has been generously supported by the Aust- ralian Research Council.

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(Received March 5, 1998: Accepted March 30, 1998)