Reproductive meristem fates in Gerbera

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1 Journal of Experimental Botany, Vol. 57, No. 13, pp , 2006 Major Themes in Flowering Research Special Issue doi: /jxb/erl181 Advance Access publication 5 October, 2006 Reproductive meristem fates in Gerbera Teemu H. Teeri 1,2, *, Anne Uimari 1,y, Mika Kotilainen 1, Roosa Laitinen 1, Hanna Help 1, Paula Elomaa 1 and Victor A. Albert 3 1 Gerbera Laboratory, Department of Applied Biology, PO Box 27, University of Helsinki, Finland 2 Department of Biology, University of Tromsø, N-9037 Tromsø, Norway 3 Natural History Museum, University of Oslo, PO Box 1172, Blindern, NO-0318 Oslo, Norway Received 13 June 2006; Accepted 4 September 2006 Abstract Flowering plants go through several phases between regular stem growth and the actual production of flower parts. The stepwise conversion of vegetative into inflorescence and floral meristems is usually unidirectional, but under certain environmental or genetic conditions, meristems can revert to an earlier developmental identity. Vegetative meristems are typically indeterminate, producing organs continuously, whereas flower meristems are determinate, shutting down their growth after reproductive organs are initiated. Inflorescence meristems can show either pattern. Flower and inflorescence development have been investigated in Gerbera hybrida, an ornamental plant in the sunflower family, Asteraceae. Unlike the common model species used to study flower development, Gerbera inflorescences bear a fixed number of flowers, and the architecture of the flowers differ in that Gerbera ovaries are inferior (borne below the perianth). This architectural difference has been exploited to show that floral meristem determinacy and identity are spatially and genetically distinct in Gerbera, and we have shown that a single SEPALLATA-like MADS domain factor controls both flower and inflorescence meristem fate in the plant. Although these phenomena have not been directly observed in Arabidopsis, the integrative role of the SEPALLATA function in reproductive meristem development may be general for all flowering plants. Key words: Asteraceae, flower, MADS box, meristem, SEPALLATA. Introduction Over the last two decades, flower development has become a major paradigm in plant developmental genetics. Starting from an informative series of floral homeotic mutant lines (Haughn and Somerville, 1988; Komaki et al., 1988; Bowman et al., 1989; Meyerowitz et al., 1989), the wellknown ABC model of flower development was built over 15 years ago (Coen and Meyerowitz, 1991). In this model, the identities of the four floral organs (sepals, petals, stamens, and carpels), arranged in a whorled phyllotaxis in the eudicot flower, are determined by a combinatorial code of three floral functions, each covering two adjacent whorls of the flower. In a well-known fashion, function A alone codes for sepals, A plus B petals, B plus C stamens, and, finally, C alone carpels (Fig. 1). The ABC model was initially developed in the model plants Arabidopsis (Arabidopsis thaliana) and snapdragon (Antirrhinum majus), although the first version of the snapdragon model was described without the A function (Schwarz-Sommer et al., 1990; Meyerowitz et al., 1991). This remarkable merging of Arabidopsis and snapdragon research was accompanied by an equally remarkable identification of homologous genes carrying out the major functions of floral organ identity determination. With a single exception (the APETALA2 gene of Arabidopsis; Jofuku et al., 1994), all genes originally identified as determinants of the A, B, and C functions code for transcription factors of a single eukaryote-wide family of DNA binding proteins, the MADS domain factors. MADS domain proteins, and the MADS box genes that encode them, are found in all major taxa of organisms, including distantly related forms in prokaryotes (Theissen et al., 2000; Entry?ac¼IPR002100). However, in plants they have * To whom correspondence should be addressed. teemu.teeri@helsinki.fi y Present address: A.I. Virtanen Institute, University of Kuopio, PO Box 1627, Kuopio, Finland. ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 3446 Teeri et al. experienced a significant increase in number, and while humans have 16 MADS box genes and yeast four (see Arabidopsis has 107 genes in this family (Parenicova et al., 2003). Further, plants have acquired a second conserved element in their MADS box genes, the K box, which unifies the type II (or MIKC type) MADS box genes of plants (Ma et al., 1991; Münster et al., 1997). Although type I MADS box genes (lacking the K-box) are more Fig. 1. The current ABCDE model of flower development. The classical ABC model was first expanded by adding the ovule specifying the D function, then by including the E function that is necessary for the development of all floral organs. In the upper panel, Arabidopsis genes are marked. The D function is best specified in Petunia and coded for by FBP7 and FBP11 (the cognate homologues of STK in Arabidopsis). In the lower panel, Gerbera ABCDE genes are marked when known. While the E function is redundantly encoded in Arabidopsis, Gerbera harbours specific E function genes that are needed for stamen and carpel development. numerous (67 genes in Arabidopsis; Parenicova et al., 2003), very little is known about their function (Kohler et al., 2003; Nam et al., 2004; Portereiko et al., 2006), and all ABC MADS box genes that determine flower organ identity in the combinatorial way are type II genes. The ABC model of floral organ identity determination can be remarkably well generalized among most flowering plants, especially concerning the C function (Kim et al., 2005), and particularly so among the eudicots, which represent approximately 75% of all angiosperm species (Drinnan et al., 1994). MADS box genes have been isolated from a large number of angiosperm plants, and in phylogenetic analyses, B and C function genes from different species without exception group in conserved clades (Doyle, 1994; Purugganan et al., 1995). Genes related to the Arabidopsis A-function gene APETALA1 also group together in a single clade, although the A-function as such has proved to be difficult to generalize. In a complementary fashion, the phylogenetic positions of newly isolated MADS box genes now provide first-level hypotheses for gene function. Still, out of the 39 type II MADS box genes in Arabidopsis, only four code for the classical ABC functions (two genes are necessary for the B function). The remainder are found scattered within the ABC clades, or form independent branches in the MADS phylogenetic tree, of which the SEPALLATA (SEP) clade (see below) is the largest. Importantly, functional analysis has shown that many of the non-abc MADS box genes relate to flower development, being involved in the induction of flowering, determining reproductive meristem identities, forming coregulators of the ABC genes, and/or participating in stamen (anther), ovule, and seed development (Garcia- Maroto et al., 2003). Still other MADS box genes have vegetative functions (Zhang and Forde, 1998). Fig. 2. (A) The Gerbera inflorescence is composed of three types of flowers. The marginal ray flowers are female and bear showy petals, while the central disc flowers are hermaphrodite and less conspicuous. Trans flowers are female like ray flowers but their petals are shorter. (B) Nearly all Gerbera flowers are bilaterally symmetrical (zygomorphic), but zygomorphy vanishes in the centre of the inflorescence. The central most floret on the capitulum can be seen as a terminal flower. Scale bar ¼ 2 cm.

3 The significant conservation of the ABC model of flower development stands in contrast to the broad phenotypic variation of flowers. Most research in flower development has been done with Arabidopsis and snapdragon, with Petunia (Petunia hybrida) providing important additional input. In addition to developing hypotheses from central model plants, contributions from a variety of additional taxa representing the diversity of angiosperms are crucially important (Fornara et al., 2003; Whipple et al., 2004; Albert et al., 2005). While the whorled, bipartite (sepal petal) design of the eudicot flower is very conserved, and the ABC model is fairly general, what genetic factors are responsible for the morphological variation seen among 75% of flowering plants? A plant species from the sunflower family, Asteraceae, has been chosen as our experimental model. The ornamental plant Gerbera (Gerbera hybrida) displays most typical features of this large family of flowering plants, including a dense inflorescence (the capitulum) bearing individual flowers that play different roles and develop according to different interpretations of the general floral theme. Marginal flowers on the Gerbera capitulum are strongly zygomorphic with an extended fused corolla, having taken up the role of petals of solitary flowers in attracting pollinating insects. These marginal (ray) flowers are also female-only; while stamen development starts normally in these flowers, it progresses slowly and stamens abort before the flowers open. On the other hand, the central (disc) flowers are hermaphroditic, with both stamens and carpels. Their corolla is much reduced and inconspicuous, and their main function is to produce pollen in the centre of the capitulum. In Gerbera, a third flower type can be distinguished between the ray and the disc flowers. These trans flowers are female, like the ray flowers, but their corolla is often (depending on the cultivar) smaller (Fig. 2A). The organization of the dense inflorescence of Asteraceae into flowers with different roles is typical for the family, although some species bear inflorescences with only single flower types, either of the zygomorphic ray or the actinomorphic disc type. What is also typical is the packing of the flowers into a dense flower head, which itself takes the role of attracting pollinators, like single flowers in other insect-pollinated species. This organization forms another hierarchical level with respect to flowering, and although inflorescences as structures are shared among all angiosperms, the sunflower family has patterned the inflorescence in a distinct way that imposes major questions on how this higher-order groundplan is organized in genetic terms. As an introduction to the remainder of this paper, let us imagine two options, which may not be mutually exclusive: (i) that regulatory gene systems present in all angiosperm plants may have been co-opted for new roles in development of the flower-like capitulum, or (ii) that the new roles observable in Reproductive meristem fates in Gerbera 3447 Asteraceae may actually turn out to be present yet hidden in species that bear inflorescences with no apparent higherorder role in reproduction. Gerbera is a fine model for Asteraceae. What makes it a great model is the set of molecular genetics tools that are available. As Gerbera is an out-crossing species, development of inbred lines for mutation screens is not readily within reach. However, efficient genetic transformation of Gerbera permits the use of reverse genetics, where functional analysis of genes is carried out by modifying their expression levels in transgenic plants. Depending on the transferred gene construct, plants with reduced or enhanced (including ectopic and heterochronic) expression of the gene of interest can be generated. Further, a fairly large collection of expressed sequence tags (ESTs) have been developed for Gerbera, along with a cdna microarray representing all 8500 unique sequences in the EST set (Laitinen et al., 2005). With these tools, approximately 20 type II MADS-box genes in Gerbera have been identified as cdna molecules. In phylogenetic analysis, they fall into all of the major clades of A, B, C, and E class (SEP-like) genes, and it has been shown that the B and C functions are well conserved in the plant (Yu et al., 1999). Five of the Gerbera genes are assignable to the SEP clade (Kotilainen et al., 2000; Teeri et al., 2002; TH Teeri, unpublished results). These genes carry the name GERBERA REGULATOR OF CAPITU- LUM DEVELOPMENT (GRCD), based on the pleiotropic function of GRCD2 in control of Gerbera inflorescence architecture (see below). Gene duplication and subsequent subfunctionalization of MADS-box genes may be the key element in developing regulatory systems that control multiple tiers of reproductive structures in the complex inflorescences of Asteraceae (Kotilainen et al., 2000; Uimari et al., 2004). Reprogramming of the apical meristem makes the flower Apical meristems (AMs) at the growing tips of shoots (and roots) contain the undifferentiated stem cells from which all axial organ systems differentiate in the plant. Unlike animal stem cells, plant meristems are not static structures, they can form de novo from differentiated cells. For example, during the normal developmental process of flower formation, floral meristems form from non-meristematic cells on the flanks of the inflorescence meristem (Weigel and Jürgens, 2002). During regeneration (e.g. in vitro), the whole plant with its full meristematic system can grow from a single differentiated cell (Gamborg and Miller, 1973). The shoot apical meristem (SAM) is maintained by an interplay of non-cell autonomous signalling at the shoot tip. In Arabidopsis, the homeodomain-containing transcription factor encoded by WUSCHEL (WUS) is expressed

4 3448 Teeri et al. in cells just below the meristem tip, and induces (by an unknown mechanism) the gene CLAVATA3 (CLV3) in the meristem tip. CLV3 codes for a secreted peptide ligand that is recognized by the CLAVATA1 (CLV1) encoded receptor, which is expressed more widely in the SAM. CLV3 expression is dependent on WUS expression, but WUS expression is repressed by the CLV3 signal, forming a feedback loop that maintains both the condition and size of the SAM. The SAM is not characterized by the meristematic state only; it also has identity. During vegetative growth, the SAM produces leaves and has a vegetative identity. Upon induction to flowering, the SAM is reprogrammed and its identity changes to an inflorescence meristem. Instead of leaves, the inflorescence meristem produces other AMs, ones that have the identity of a floral meristem. Induction to flowering has been best studied in Arabidopsis, and it is known to result from several pathways that can be viewed to act in independent, alternate ways. Arabidopsis, for example, is a long-day plant, meaning that an inductive signal is generated in leaves when night length is shorter than a threshold value (Goto et al., 1991).However,inthe absence of this signal during short days (long nights), the plant will eventually flower anyway, after a delay compared to long-day conditions (Boss et al., 2004). Several genetic factors are known from Arabidopsis that control the switch to flowering, i.e. to the reprogramming of the SAM from a vegetative to an inflorescence meristem. Although details will vary (for example, short-day plants interpret day length in the opposite way than long-day plants; Hayama et al., 2003), Arabidopsis provides a good framework for the genetic mechanisms that control the identity and fate of the SAM. Day length is interpreted (in Arabidopsis) by CONSTANS (CO), which is under transcriptional control by the circadian clock and under a protein turnover control by photoreceptors (Valverde et al., 2004). Consequently, the CO polypeptide accumulates in leaf cells only when transcriptional activity of CO and stability of the protein temporally overlap in the long summer evenings. CO is a putative transcriptional regulator that induces expression of FLOWERING LOCUS T (FT) in leaves, which then travels as mrna to the SAM and causes induction of a number of downstream genes known to be involved in SAM reprogramming (Huang et al., 2005). One of these, SUPPRESSOR OF OVER- EXPRESSION OF CONSTANS 1 (SOC1), is a key regulatory gene needed for the activation of genes that define inflorescence and floral meristem identity. SOC1 is not only under a positive regulation by CO (and FT) but also under negative regulation by FLOWERING LOCUS C (FLC), a repressor that is itself regulated by epigenetic phenomena, the activity state of chromatin at its chromosomal position. Thus, in Arabidopsis, the memory of winter is encoded as the methylation state of histones at the FLC locus (Sung and Amasino, 2004). Self-regulation and genetic switches The Arabidopsis SAM is marked by gene activities that can be interpreted as having meristem identity function. A key player in defining floral meristem identity is LEAFY (LFY), a gene that codes for a plant-specific transcription factor. Together with the MADS-box genes APETALA 1 (AP1)and its redundant homologue CAULIFLOWER (CAL), LFY defines the SAM identity as floral: mutations in these genes cause conversion of floral identity into inflorescence identity. The inflorescence identity of the SAM can be seen as a basal state in relation to floral identity, i.e. in the absence of floral identity factors, the reproductive meristem maintains inflorescence identity. Nevertheless, at least one gene is known to be a possible inflorescence meristem identity gene. It is not known if AGAMOUS LIKE 24 (AGL24) is induced by SOC1 or parallel to SOC1 (Jack, 2004), but AGL24 is needed to turn LFY expression on (Yu et al., 2002).Subsequently,LFY expression shuts down AGL24 expression, making AGL24 necessary for inflorescence function (including the capacity to produce floral meristems), but inhibitory for the floral function: failure to repress AGL24 causes a failure in establishing the floral AM (Michaels et al., 2003;Yuet al., 2004). Expression of LFY is crucial for the SAM to acquire floral meristem identity. In view of this, it is important to keep LFY expression segregated from the inflorescence meristem, so long as the latter identity should be maintained. One of these factors is TERMINAL FLOWER 1 (TFL1), a gene that is expressed at a low level in the vegetative SAM, but induced to higher levels in the inflorescence meristem. Ectopic TFL1 expression down-regulates both LFY and AP1 expression, demonstrating a direct or indirect repression of the floral meristem identity genes (Ratcliffe et al., 1988; Pidkowich et al., 1999). Reciprocally, LFY downregulates TFL1, most probably directly, pointing out that a single regulatory protein can have both activating and repressing roles depending on its targets (Parcy et al., 2002). Mutations in LFY result in the incapacity to define floral identity for the SAM. Conversely, mutations in TFL1 cause loss of the ability to maintain inflorescence identity, and the AM converts to a terminal flower. Just as many of the genes involved in induction and identity of floral structures are MADS-box genes (FLC, SOC1, AGL24, AP1, CAL, and most ABCDE genes), TFL1 (a repressor) is a close homologue of FT (an inducer). MADS-box genes code for transcription factors, but FT and TFL1 code for phosphatidylethanolamine-binding proteins with likely roles in modifying signalling through ligand binding (Pnueli et al., 2001; Abe et al., 2005; Wigge et al., 2005). Intriguingly, a single amino acid change can convert the repressor activity of TFL1 to the activator activity of FT (Hanzawa et al., 2005). Vegetative AMs are indeterminate in the sense that they produce an undetermined number of vegetative organs.

5 Flower meristems, on the other hand, are determinate, and once the fourth floral whorl (the carpel) is generated, the AM is consumed and no further organs develop. The homeotic floral organ identity gene AGAMOUS (AG), encoding the C function in the ABC model, controls floral meristem determinacy in addition to carpel identity. Mutations in AG cause not only perianth-like conversion of the inner floral whorls, but also cause the floral meristem to proliferate and produce an increased number of organs. These two functions are separate: weak ag mutants show that loss of determinacy can be caused without organ identity change (Mizukami and Ma, 1995; Sieburth, 1995). The indeterminate meristematic state of the SAM is maintained through WUS. Although WUS, together with LFY, is necessary to induce AG, expression of AG, together with other flower specific factors, then shuts down expression of WUS (Lenhard et al., 2001; Lohmann et al., 2001). The sequence of SAM reprogramming involves genes that sequentially induce subsequent genes on the pathway. However, certain checkpoints on the induction pathway also act backwards, burning the bridges to genes that were necessary for their own induction (e.g. LFY on AGL24, AG on WUS). After this action, the identity gene must become independent of its original inducer. As first suggested by regulatory network simulations (Espinosa- Soto et al., 2004), AG is self activating once induced (Gómez-Mena et al., 2005). Similarly, induction of the B function floral homeotic genes is first dependent on the genes LFY and UNUSUAL FLOWER ORGANS (UFO), but the proteins then form a self-regulatory loop and gain independence of their inducers (Tröbner et al., 1992; Goto and Meyerowitz, 1994). LFY itself is not known to be self activated, but LFY activates AP1 (Wagner et al., 1999; Saddic et al., 2006) and AP1 in turn up-regulates LFY (Bowman et al., 1993; Liljegren et al., 1999), providing a similar self-regulatory loop. de Folter et al. (2005) have observed that self-regulation is a re-occurring phenomenon among the MADS domain proteins, which are important as both floral organ identity genes and as inflorescence identity and floral induction genes. Backwards repression and self-regulation can both be seen as genetic switches that promote unidirectionality of floral induction. Once the decision to flowering is made, there is, in most cases, no way to turn back (Tooke et al., 2005). Flexibility at the level of the homeotic E function The homeotic ABC function genes are necessary for the determination of flower organ identity, but they are not sufficient for it. A triple mutant that lacks all three functions grows leaves (with a whorled phyllotaxis) instead of flower organs, but ectopic expression of ABC genes in leaves does not convert leaves into flower organs. The missing links are other, redundant MADS-box factors, phylogenetically positioned outside of the A, B, and C Reproductive meristem fates in Gerbera 3449 gene clades in a branch originally referred to as the AGL2 clade, but later as the SEP lineage. At least one of three genes, SEP1, SEP2,orSEP3, is necessary for petal, stamen, and carpel identity. The triple sep1/sep2/sep3 mutant grows sepals on all floral whorls (Pelaz et al., 2000). In fact, sepal identity also requires the SEP function. In sep1/ sep2/sep3/sep4 quadruple mutants all flower organs are replaced by leaves. SEP4 is a fourth redundantly acting gene in the SEP clade, known previously as AGL3 (Ditta et al., 2004). When expressed together with A, B, and C function MADS-box genes, the SEP function promotes the conversion of Arabidopsis leaves into flower organs (Honma and Goto, 2001; Pelaz et al., 2001). In the expanded model for floral organ identity determination, the SEP function was labelled E (Fig. 1). The D function was previously described in Petunia, where the homeotic genes FBP7 and FBP11 are needed for proper identity of the ovules within the carpel (Angenent et al., 1995; Colombo et al., 1995). FBP7 and FBP11 are also MADS-box genes. The current model for action of the floral MADS domain proteins is that they function as pairs of pairs, i.e. as quaternary (or even higher-order) complexes in the activation of their target genes (Egea- Cortines et al., 1999; Honma and Goto, 2001; Theissen, 2001; Theissen and Saedler, 2001). Since many single MADS domain proteins can form complexes with several other MADS domain proteins, the number of possible combinations is very large and may permit the proteins to function in different contexts. The current functional view of, for example, the AG protein in determining stamen identity, carpel identity, and floral determinacy, is that AG interacts with different sets of MADS domain factors to carry out these different functions. A comprehensive analysis of protein protein interactions (in yeast) was recently carried out for all Arabidopsis MADS domain proteins by de Folter et al. (2005). While the (A)BC functions and expression patterns are more rigorously maintained within angiosperms, there is considerable variation of expression and inferred function of the E function genes characterized in different plants (Malcomber and Kellogg, 2005). The genetic redundancy within the SEP encoding genes seen in Arabidopsis is not obvious in many other plants, and there is evidence for subfunctionalization of the SEP gene paralogues in various species. Gerbera is no exception; it appears that one of the Gerbera SEP-like genes, GRCD1, is necessary for stamen development, and another, GRCD2, for carpel development (Kotilainen et al., 2000; Uimari et al., 2004). GRCD2 has, in addition, other pleiotropic functions, being a determinant of inflorescence meristem determinacy and flower meristem identity (Uimari et al., 2004). Gerbera GRCD2 is expressed early in inflorescence and flower development. In flower primordia, the initial expression in flower meristems includes all organ primordia, but later expression concentrates in the centre of the

6 3450 Teeri et al. flowers (Uimari et al., 2004). In transgenic Gerbera lines where GRCD2 expression is down-regulated, a homeotic change occurs in whorl 4. Instead of a stigma and a style, the innermost floral organs show a mixed identity with cell types resembling carpel, petal, and pappus (sepal) cells. This phenotype, including a weaker phenotype in which greenish organs replace the carpel, is similar to the phenotype of transgenic lines where the Gerbera C function (encoded by GAGA1 and GAGA2 MADS-box genes) is down-regulated (Yu et al., 1999). However, unlike the phenotypes of strong anti-gaga2 lines, no repetition of the inner organs occurs, nor is whorl 3 affected. On the other hand, Gerbera lines in which GRCD1 is down-regulated show homeotic conversion of whorl 3 organs from stamens to petals, again phenocopying the change in anti-gaga1 or anti-gaga2 lines, but affecting whorl 3 only, and mainly in the female marginal flowers (Kotilainen et al., 2000). GRCD1 and GRCD2 group phylogenetically with Arabidopsis SEP genes, and like SEP genes, they are needed for stamen and carpel development. However, while the Arabidopsis SEP genes affect all floral whorls in a redundant fashion, GRCD1 is whorl 3-specific and GRCD2 is whorl 4-specific, showing that subfunctionalization has taken place during the evolution of the Gerbera genes. Just as the Arabidopsis SEP proteins have been shown to interact with the AG protein, the Gerbera GRCD1 and GRCD2 proteins interact with both GAGA1 and GAGA2 (Kotilainen et al., 2000; Uimari et al., 2004). Loss of GRCD1 and GRCD2 activity does not affect development of floral organs in whorls 1 and 2. It is tempting to predict that Gerbera harbours as-yet-undiscovered whorl 1- and whorl 2-specific E function genes, which may act either alone or in a redundant fashion like in Arabidopsis (Fig. 1). In Arabidopsis mutants that lack the AG or the SEP function, floral determinacy is lost and extra whorls grow in the centre of the flower. This occurs also in strong anti- GAGA2 transgenic Gerbera lines, but not in anti-grcd2 lines. Instead, the latter show dramatic alterations in the ovary. Nothing less than a full (but compressed) inflorescence, with bracts, floral primordia, and (altered) flowers grow in place of the ovule (Uimari et al., 2004). Loss of GRCD2 function therefore leads to loss of floral meristem identity, with the meristem reverting back to inflorescence identity. Furthermore, anti-grcd2 transgenic lines have an inflorescence phenotype. Inflorescences in the family Asteraceae are traditionally considered to be indeterminate (Harris, 1995; Stevens, 2001), although they usually produce a fixed number of flowers. Grown side-by-side, non-transgenic Gerbera lines (of variety Terra Regina) produce 593 (656) flowers, after which the inflorescence meristem is consumed and replaced by floral meristems (Fig. 5B): in fact, the centre of the inflorescence becomes occupied by a terminal symmetrical flower (Fig. 2B). Anti-GRCD2 lines, on the other hand, produce on average flowers, and the inflorescence meristem remains undifferentiated until production of flowers ceases due to senescence (Uimari et al., 2004). Hence, GRCD2 also plays a role in controlling determinacy of the inflorescence meristem. As described above, GRCD2 displays pleiotropy, its down-regulation affecting phenotypes at at least three different levels (Fig. 3). In Gerbera, homeotic function in floral organ identity determination can be seen as a whorl 4-specific counterpart of the Arabidopsis SEP function. The function in promoting flower meristem identity can be seen as unrelated to the Arabidopsis SEP function, but this may be due to a different and more illuminating structure of the Gerbera flower (it has inferior as opposed to superior ovaries). The third function, promoting determinacy at the inflorescence level, has not been seen in Arabidopsis, snapdragon or Petunia SEP homologue mutants, but all of these plants except Petunia normally bear indeterminate inflorescences. It is important to note, however, that Petunia inflorescences are not highly compressed like those of Gerbera, nor are the flowers borne noticeably heteromorphic. Fig. 3. Pleiotropic phenotypes in anti-grcd2 Gerbera lines. (A) Loss of carpel identity (but not floral determinacy) in whorl 4. (B) Reversion of flowers to inflorescences that occupy the space where ovaries normally develop. (C) Loss of inflorescence determinacy (the fixed number of flowers on the capitulum). Scale bar (A and B) ¼ 1 mm. Scale bar (C) ¼ 2 cm.

7 Common themes for the E function SEP-like genes have been isolated from a large number of flowering plants, and they show more variation in their C-terminal motifs and expression patterns than the other MADS domain determinants of the extended ABCDE model of flower development (Malcomber and Kellogg, 2005; Zahn et al., 2005). Still, one may ask if the pleiotropic phenotypes shown in transgenic Gerbera lines with downregulated GRCD2 are, apart from the homeotic function in whorl 4, specific to Gerbera (or Asteraceae), or whether they may be reflections of general functions of E class MADS-box genes across flowering plants. In Arabidopsis, the sep1/2/3 triple mutant is described as similar to a B and C function double mutant, for example pi ag, phenocopying not only the homeotic conversion of organs in all whorls into sepals, but also the loss of determinacy of the flower meristem (Pelaz et al., 2000). However, in Gerbera, loss of GRCD2 expression does not lead to loss of determinacy, but rather to a change in the meristem identity from floral to non-floral. In Petunia, the SEP function is encoded by several MADS-box genes. These include FBP2 and FBP5 (Ferrario et al., 2003), and probably one or more additional genes that act redundantly (Vandenbussche et al., 2003). Transgenic Petunia lines with 35S-FBP2 show, through cosuppression of several endogenous genes, a phenotype similar to the Arabidopsis sep1/2/3 mutant, including extra organs growing in the centre of the flower. Ferarrio et al. (2003, 2006) refer to both the Petunia and the Arabidopsis structure as a floral shoot, but still maintain the description of the changed meristematic state as loss of determinacy and not reversion. A consistent interpretation of the phenotypes in all three species (Arabidopsis, Petunia, and Gerbera) is that the SEP function is not controlling the determinacy state of the floral meristem, but rather its identity as floral. The important distinction is that SEP is not merely participating in the regulation of determinacy, but that it has a role distinct from the C function MADS-box gene AG. InGerbera, loss of determinacy is seen in the stigma and style, while reversion from floral to inflorescence identity is visible in the ovary. Gerbera ovaries are inferior in that they develop below the whorls of flower organs, making the spatial separation of these phenomena very clear. In Arabidopsis with its superior ovaries, indeterminacy and floral reversion may simply not be set apart (Fig. 4). Gerbera lines in which either the C function (anti- GAGA2 transformants) or the E function (anti-grcd2 transformants) is down-regulated show that indeterminacy and floral reversion are distinct phenomena with spatially separate sites of occurrence (Yu et al., 1999; Uimari et al., 2004). In Arabidopsis, where spatial separation of these changes is not apparent, C function and E function mutant phenotypes can be interpreted similarly. Nevertheless, under certain environmental conditions the Reproductive meristem fates in Gerbera 3451 Fig. 4. Loss of determinacy and floral reversion in inflorescences bearing flowers with inferior versus superior ovaries. The cartoon compares wild type and mutant/transgenic phenotypes between plants that bear flowers with inferior (Gerbera) versus superior (e.g. Arabidopsis or Petunia) ovaries. The cartoon ignores the fact most species bear several flowers on their inflorescence stems (hatched), and that some, such as Arabidopsis and Petunia, bear several ovules in their ovaries. (A, B) Wild-type flowers showing the arrangement of floral organs with respect to the ovary. (C, D) Loss of determinacy of the flower meristem, caused by transgenic or mutant down-regulation of the floral C function. (E, F) Floral reversion, caused by transgenic or mutant down-regulation of the floral E-function. Note the position where reiterative inflorescences emerge. When the ovaries are superior, reversion may resemble loss of floral determinacy. indeterminate ag mutants of Arabidopsis show floral reversion (Okamuro et al., 1996; Mizukami and Ma, 1997), suggesting that AG, together with its companion regulators (which include SEP proteins), may in fact be responsible for both the control of determinacy and meristem identity in Arabidopsis. Therefore, if indeterminacy and floral reversion indeed turn out to be part of a single phenotypic spectrum in Arabidopsis, this may imply that, as compared with Arabidopsis yet another

8 3452 Teeri et al. series of gene duplication and subfunctionalization events has occurred within the Gerbera SEP-like gene family. The third pleiotropic function of GRCD2 in the control of inflorescence determinacy cannot be easily related to Arabidopsis or Antirrhinum since these plants have indeterminate inflorescences to begin with. In Arabidopsis and snapdragon, on the other hand, mutations have been characterized in genes that lead to the acquisition of a determinate state of the inflorescence, i.e. loss of indeterminacy. In both plants these mutations map to homologous (yet not orthologous) genes, TFL1 and CENTRORADIALIS (CEN), respectively (Shannon and Meeks-Wagner, 1991; Bradley et al., 1996). Indeterminacy of the inflorescence has been suggested to be a derived state that has arisen many times in evolution (Coen and Nugent, 1994). Apparently genes that may cause this condition are ancestral in all flowering plants (Coen and Nugent, 1994), but it is not known if evolution of inflorescence indeterminacy always (or often) uses the same mechanism of control. Still, it is intriguing to speculate that the Gerbera counterpart of TFL1 or CEN could be a target for regulation by GRCD2. This hypothesis is not testable yet since corresponding Gerbera genes have not been isolated. This may not be a simple task since TFL1 and CEN are members of gene families that include similar genes with opposing (activating or repressing) biological roles (Mimida et al., 2001; Hanzawa et al., 2005). Another perspective on the regulation of inflorescence determinacy can be borrowed from the regulation of the floral meristem. During the formation of floral organs, the centre of the flower primordium is kept meristematic, and is marked by expression of the meristem organizer gene WUS. WUS, together with LFY, induces AG (see above). Subsequently, AG represses WUS, and failure to do this is reflected by continuous meristematic activity (indeterminacy) in ag mutants. However, for repression of WUS, expression of AG alone is not sufficient. Transgenic Arabidopsis lines ectopically expressing AG (from a 35S- AG construct), show a curly leaf phenotype, but not a loss of WUS activity, and no loss of apical meristems (Mizukami and Ma, 1997). The missing factors were recently identified in Petunia and shown to be the class D and E MADS-box genes FBP11 and FBP2 (Ferrario et al., 2006). D and E function genes acting as cofactors with C function genes provide an explanation for the timing of WUS repression, which occurs later than the onset of C (or E) function genes. The timing of WUS down-regulation is precisely co-ordinated with onset of the Petunia D function genes FBP11 and FBP7 and, furthermore, ectopic expression of C, D, and E function MADS-box genes in transgenic Petunia seedlings causes loss of WUS activity and arrest of seedling growth before the appearance of the first true leaves (Ferrario et al., 2006). As such, regulation of the consumption of the flower meristem is handled by C, D, and E function MADS-domain proteins. For building mechanistic explanations to the involvement of GRCD2 in controlling the fate of the Gerbera inflorescence meristem, if we hypothetically transfer down-regulation of WUS to the inflorescence meristem level, GRCD2 could be a repressor for the still unidentified Gerbera WUS orthologue instead of a TFL1 homologue repressor. At least in Arabidopsis, WUS is a general meristem organizer gene, expressed in all SAMs, including the inflorescence meristem (Mayer et al., 1998). Again, this repression could be a general feature of the E class MADS-domain proteins. It is interesting to note, in this context, that ectopic expression of the E class gene SEP3 (from a 35S-SEP3 construct) leads to a terminal flower, i.e. determinacy at the inflorescence level in Arabidopsis (Honma and Goto, 2001). Fractal nature of reproductive structures As described above, the inflorescences of Asteraceae are false flowers (pseudanthia), structures that resemble single flowers. From the point of view of a pollinator, the capitulum might appear to have showy petals at the perimeter and sex organs in the centre. The adaptive nature of the pseudanthium is apparent, but its molecular developmental evolution still requires explanation at a mechanistic level. Indeed, it is possible to view flowers themselves as composite structures descended from branching systems that are specialized in protective (sepals), showy (petals), male (stamens), or female (carpels) functions. Carpels provide a good example of a further nested structure; they appear to be laminate leaf-like structures that contain the ovules, terminal organs (containing female sporangia) that become seeds when pollinated. Like Goethe s 18th century view (von Goethe, 1790), modern research (Honma and Goto, 2001) backs up the homology of sepals, petals, stamens, and carpels. Clearly then, the hierarchical level at which sexuality is functionally operable varies among plants, and may best be characterized as like a babushka doll one branching level internested within another (Albert, 1999). Conclusion and future prospects Functional analysis of the Gerbera E class MADS-box gene GRCD2 shows that this gene is involved in several processes that can together be seen as representing different steps in the fate of the reproductive meristem. Apart from a homeotic function in defining carpel identity, the pleiotropic functions in controlling floral reversion (flower meristem identity) and number of flowers (inflorescence meristem determinacy) at first appear to be Gerbera (or Asteraceae) specific. However, as described above, all functions of GRCD2 could be considered common functions of E class genes, at least when reflected

9 Reproductive meristem fates in Gerbera 3453 Fig. 5. Phenotypes of wild-type and transgenic Gerbera inflorescences demonstrate different determinacy states. (A) Wild-type inflorescence centre at an early stage showing undifferentiated cells. Inflorescences of anti-grcd2 lines look identical to the wild type at this stage. (B) Wild-type inflorescence centre at a later stage, when the terminal flower is already initiated. (C) Inflorescence of anti-grcd2 line at the stage corresponding to the wild type in (B). Note that the inflorescence apex stays undifferentiated. by data accumulating from Arabidopsis and Petunia. This demonstrates that comparative studies of flower development outside the most common model species are valuable not only for uncovering variations on floral developmental themes, but also from the point of view of clarifying certain functions in the central model systems themselves. Common themes made visible through Gerbera versus truly Gerbera (or Asteraceae) specific modifications in flower development require further hypothesis-driven experimentation. For example, is the role of GRCD2 in controlling inflorescence determination mediated through regulation of TFL1 or WUS homologues in Gerbera? A new microarray resource (Laitinen et al., 2005) will be available to test such hypotheses. Microarray experiments have been used to compare gene expression in inflorescence apical tissue during the transition from the undifferentiated early stage of capitulum development to the stage when the inflorescence meristem is consumed in wild-type versus anti-grcd2 lines (Fig. 5). Transcripts can be distinguished that are up- or down-regulated during this process and those that are dependent on GRCD2 expression in the capitulum can be separated. Many of these transcripts represent genes with proposed regulatory functions, including DNA binding proteins and protein kinases. It is not known which of these are direct targets for GRCD2 and which function further downstream, but there are high hopes that this snap-shot of the inflorescence meristem transcriptome will guide us toward new discoveries. References Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T FD, a bzip protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309, Albert VA Shoot apical meristems and floral patterning: an evolutionary perspective. Trends in Plant Science 4, Albert VA, Soltis DE, Carlson JE, et al Floral gene resources from basal angiosperms for comparative genomics research. BMC Plant Biology 5, 5. Angenent GC, Franken J, Busscher M, van Dijken A, van Went JL, Dons HJ, van Tunen AJ A novel class of MADS box genes is involved in ovule development in petunia. The Plant Cell 7, Boss PK, Bastow RM, Mylne JS, Dean C Multiple pathways in the decision to flower: enabling, promoting, and resetting. The Plant Cell 16, S18 S31. Bowman JL, Alvarez J, Weigel D, Meyerowitz EM, Smyth DR Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes. Development 119, Bowman JL, Smyth DR, Meyerowitz EM Genes directing flower development in Arabidopsis. The Plant Cell 1, Bradley D, Carpenter R, Copsey L, Vincent C, Rothstein S, Coen E Control of inflorescence architecture in Antirrhinum. Nature 379, Coen ES, Meyerowitz EM The war of the whorls: genetic interactions controlling flower development. Nature 353, Coen ESJ, Nugent JM Evolution of flowers and inflorescences. Development (Supplement) 120, Colombo L, Franken J, Koetje E, van Went J, Dons HJ, Angenent GC, van Tunen AJ The petunia MADS box gene FBP11 determines ovule identity. The Plant Cell 7, de Folter S, Immink RG, Kieffer M, et al Comprehensive interaction map of the Arabidopsis MADS box transcription factors. The Plant Cell 17, Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky MF The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology 14, Doyle JJ Evolution of a plant homeotic multigene family: toward connecting molecular systematics and molecular developmental genetics. Systematic Biology 43, Drinnan AN, Crane PR, Hoot SB Patterns of floral evolution in the early diversification of non-magnoliid dicotyledons (eudicots). Plant Systematics and Evolution (Supplement) 8, Egea-Cortines M, Saedler H, Sommer H Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. EMBO Journal 18, Espinosa-Soto C, Padilla-Longoria P, Alvarez-Buylla ER A gene regulatory network model for cell-fate determination during Arabidopsis thaliana flower development that is robust and

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