A TCP domain transcription factor controls flower type specification along the radial axis of the Gerbera () inflorescence

Suvi K. Broholm*, Sari Ta¨ htiharju*, Roosa A. E. Laitinen†, Victor A. Albert‡, Teemu H. Teeri*, and Paula Elomaa*§

*Department of Applied Biology, P.O. Box 27, University of Helsinki, Helsinki FIN-00014, Finland; †Max Planck Institute for Developmental Biology, Spemannstrasse 37-39, D-72076 Tu¨bingen, Germany; and ‡Department of Biological Sciences, State University of New York, Buffalo, NY 14260

Edited by M. T. Clegg, University of California, Irvine, CA, and approved April 4, 2008 (received for review February 11, 2008) Several key processes in development are regulated by TCP inflorescence apex. Trans flowers, which occupy an intermediate transcription factors. CYCLOIDEA-like (CYC-like) TCP domain pro- radial position between ray and disk flowers, are strongly teins have been shown to control flower symmetry in distantly zygomorphic, yet smaller in size in comparison with ray flowers. related plant lineages. Gerbera hybrida, a member of one of the Stamen development also differs among individual flower types largest clades of angiosperms, the sunflower family (Asteraceae), (3). In the female ray and trans flowers, stamen development is an interesting model for developmental studies because its arrests, resulting in rudimentary staminodia (4), whereas in the elaborate inflorescence comprises different types of flowers that disk flowers, anthers develop fully and form a postgenitally fused have specialized structures and functions. The morphological dif- structure that covers the carpel. ferentiation of flower types involves gradual changes in flower Despite continued efforts, the molecular basis for flower type size and symmetry that follow the radial organization of the differentiation in Asteraceae remains unclear. Microarray com- densely packed inflorescence. Differences in the degree of petal parison of developing Gerbera ray and disk flower primordia has fusion further define the distinct shapes of the Gerbera flower identified a number of genes that are differentially expressed in types. To study the role of TCP transcription factors during speci- individual flower types (3). We have shown that many MADS fication of this complex inflorescence organization, we character- box genes encoding known regulators of flower organ develop- ized the CYC-like homolog GhCYC2 from Gerbera. The expression ment are expressed gradientially along the capitulum radius. of GhCYC2 follows a gradient along the radial axis of the inflo- While this may suggest that different MADS protein complexes rescence. GhCYC2 is expressed in the marginal, bilaterally symmet- participate in developmental regulation of individual flower rical ray flowers but not in the centermost disk flowers, which are types, transgenic experiments show that such complexes are not nearly radially symmetrical and have significantly less fused petals. independently sufficient (4, 5). Indeed, classical genetics has Overexpression of GhCYC2 causes disk flowers to obtain morphol- shown that the presence or absence of ray flowers in Asteraceae ogies similar to ray flowers. Both expression patterns and trans- capitula appears to be under control of one or two major and genic phenotypes suggest that GhCYC2 is involved in differentia- several modifier genes (reviewed in ref. 6). Berti et al. (7) have PLANT BIOLOGY tion among Gerbera flower types, providing the first molecular characterized two sunflower mutants in which flower type evidence that CYC-like TCP factors take part in defining the com- identity (and consequently symmetry) has changed. In the plex inflorescence structure of the Asteraceae, a major determinant chrysanthemoides (chry) mutant, all flowers in the inflorescences of the family’s evolutionary success. are ray-like (zygomorphic), whereas in the tubular ray flower (turf) mutant, all flower types resemble radially symmetrical disk CYCLOIDEA ͉ flower development ͉ evo-devo ͉ organ fusion flowers. In Senecio, capitulum organization is principally con- trolled by the RAY locus, with radiate inflorescences (ray plus he enormous variety of flowers makes them fascinating disk) dominant over discoid (disk only) (8–10). For both Senecio Ttargets for comparative developmental genetic studies. Spe- and sunflower, hypotheses have been constructed whereby the cies of the sunflower family (Asteraceae), such as the cut-flower RAY (6, 11), CHRY and TURF loci (7) could encode homologs crop Gerbera hybrida, present a unique and challenging benefit: of the Antirrhinum floral symmetry gene CYCLOIDEA (12). more than one floral phenotype can be analyzed within the same However, these intriguing postulates remain unevaluated by genotype. A typical Asteraceae inflorescence consists of mor- molecular evidence. phologically and functionally differentiated flowers packed into CYCLOIDEA-like genes have been reported to be involved a condensed inflorescence (the capitulum) that resembles (as a in flower symmetry regulation in various plant species. pseudanthium) a single large flower. This inflorescence com- CYCLOIDEA (CYC)ofAntirrhinum was the first gene isolated, plexity, not shared by other model species used for flower and thereafter the most extensively studied (12, 13). It belongs developmental research such as Arabidopsis, Antirrhinum,or to the plant-specific gene family encoding TCP transcription Petunia, has apparently proved to be evolutionarily successful factors, which share a conserved basic helix–loop–helix TCP because Asteraceae is one of the largest families of flowering with Ͼ23,000 species (1). Author contributions: S.K.B., R.A.E.L., T.H.T., and P.E. designed research; S.K.B., S.T., Gerbera bears three flower types (ray, trans, and disk) that are R.A.E.L., V.A.A., and P.E. performed research; S.K.B., V.A.A., and P.E. analyzed data; and morphologically similar during their early development. One of S.K.B., S.T., V.A.A., T.H.T., and P.E. wrote the paper. the most prominent developmental differences, which becomes The authors declare no conflict of interest. established early in floral ontogeny, is a gradual change of flower This article is a PNAS Direct Submission. symmetry that follows the radial organization of the capitulum Data deposition: The sequences reported in this paper have been deposited in the GenBank (2). The marginal ray flowers are bilaterally symmetrical (zygo- database [accession nos. EU429302 (GhCYC1), EU429303 (GhCYC2), EU429304 (GhCYC3), morphic). Three of the five petals are fused ventrally to form the and EU429305 (GhCYC4)]. large, elongate ligule, whereas the remaining two (dorsal) petals §To whom correspondence should be addressed. E-mail: paula.elomaa@helsinki.fi. remain rudimentary. Disk flowers at the center of the capitulum This article contains supporting information online at www.pnas.org/cgi/content/full/ have shorter and less fused petals, and they eventually become 0801359105/DCSupplemental. radially symmetrical (actinomorphic) with separate petals at the © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801359105 PNAS ͉ July 1, 2008 ͉ vol. 105 ͉ no. 26 ͉ 9117–9122 Downloaded by guest on September 28, 2021 domain. The name TCP derives from the three founding mem- bers of the family, TEOSINTE BRANCHED1 (TB1) of maize, CYC, and PROLIFERATING CELL FACTOR (PCF) of rice, all of which control meristem growth by affecting cell prolifer- ation (14). Phylogenetic analysis based on the TCP domain has uncovered two subfamilies; PCF proteins form one clade (class I) whereas CYC/TB1 and CINCINNATA (CIN) group together to form the class II TCP proteins (15). The CYC/TB1 group is also called the ECE clade, within which three subclades, CYC1, Fig. 1. Expression of GhCYC2 in various Gerbera tissues. GhCYC2 showed -2, and -3, have been identified in core (16). TB1 has strongest expression in young inflorescences, petals, and carpel (stigma and played a major role in the evolution of maize from its wild style). Weaker expression was also seen in bracts and ovary. The lower blot ancestor teosinte. It acts as a repressor of growth by suppressing shows ethidium bromide-stained ribosomal RNA bands to control for RNA axillary branching, and thus promotes apical dominance in loading. domesticated maize (17). TB1 homologs have also been shown to prevent the growth of axillary buds in rice and Arabidopsis (15, 18). The two TCP genes CYC and DICHOTOMA (DICH)in evolutionary relationships of the Gerbera TCP genes. Nucleotide Antirrhinum regulate flower symmetry by modulating cell- sequences encoding the conserved TCP and R domains were division related gene expression (12–14, 19). The wild-type aligned, and parsimony and maximum likelihood analyses were flowers of Antirrhinum have an axis of dorsoventral asymmetry performed on these data and the inferred amino acid alignment. with distinct dorsal, lateral, and ventral organ types. The expres- Robustness of results was assessed using jacknife and bootstrap sion of CYC and DICH is restricted to the dorsal domain of the resampling, respectively (Fig. S3). In most analyses, CIN-like flowers to establish bilateral petal symmetry and to arrest the genes from Antirrhinum and Arabidopsis grouped together with development of the dorsalmost stamen (12, 13). CIN arrests support. CYC-like genes from Lamiales, , and Asteraceae the growth of Antirrhinum leaf margins but also affects differ- were divided into separate, well supported groups. The greater entiation of epidermal cells and growth of petal lobes through genetic similarity within rather than among these groups sug- effects on cell proliferation (20). gests that many functionally important duplication events in the In Gerbera, specification of flower types involves differential ECE gene subfamily occurred after the divergence of the Lamia- development of petals and stamens, probably through distinct les, Fabales, and Asteraceae lineages, respectively. The results of cell division and elongation as well as organ fusion events. TCP Howarth and Donoghue (16) are similar in this respect in that transcription factors have previously been connected with all groupings within subclades CYC1, CYC2, and CYC3 are largely three of these processes, making them good candidates for consistent with organismal phylogeny. Given this concurrence, regulators of the distinct morphologies of Gerbera flower types. we tested the phylogenetic distribution of our gene data when We have isolated several TCP transcription factor genes as combined with many of the less-complete Dipsacales and Aste- candidates from Gerbera. We show that one of these proteins, rales sequences published by the former authors, and all well GhCYC2, has a symmetry function different from that of classic supported relationships were consistent with the results reported CYC-like genes. Instead of regulating the dorsoventral symmetry here (data not shown). According to our trees and their under- of individual flowers, GhCYC2 participates in the control of the lying nucleotide alignment, GhCYC1 is deviant from the other identity and radial extent of flower types in Gerbera inflores- three GhCYC genes, which are quite similar to each other (Fig. cences. Moreover, GhCYC2 plays an important and unique role S2). In addition to its sister gene HpTCP from sunflower, in the in organ fusion that further differentiates Gerbera flower types. most-parsimonious and maximum likelihood trees based on nucleotide data, GhCYC1 groups (without statistical support) Results with Arabidopsis AtBRC1 and Lotus LjCYC5 (data not shown). Isolation of Gerbera CYC Homologs. To study the role of TCP AtBRC1 and AtBRC2 have been shown to be functionally domain transcription factors during Gerbera flower develop- related to maize TB1 (15). However, in our analyses, support for ment, cDNA clones encoding these proteins were isolated from particular gene groupings with TB1 is lacking. young developing Gerbera inflorescences. The inferred amino acid sequences of the four cDNAs, GhCYC1, GhCYC2, Gh- The Expression of GhCYC2 Follows the Zonal Organization of the CYC3, and GhCYC4 (Gerbera hybrida CYCLOIDEA-like 1, 2, 3, Capitulum. RNA-blot analysis showed that GhCYC2 is expressed and 4), exhibited the conserved TCP and R domains typical for only in floral tissues. We could not detect expression in vege- the CYC/TB1 subfamily. Low stringency hybridization of Ger- tative organs such as leaves, roots, or floral stems (scapes). bera genomic DNA with a full-length GhCYC2 probe suggested Expression was strongest in young inflorescences, and in petals, the presence of a small gene family [supporting information (SI) whereas weaker expression was observed in carpels (stigma and Fig. S1]. Although we cannot exclude possible allelism, this style), in the ovary and in involucral bracts (the leaf-like struc- inference was supported by isolation of 4 distinct sequence tures that tightly surround the reproductive centers of capitula) fragments (data not shown) in addition to the four full-length (Fig. 1). We have recently demonstrated that at floral primor- sequences presented in this study. The alignment of full-length dium stage 3, the ray and disk flowers of Gerbera are morpho- GhCYC cDNAs (Fig. S2) showed that the inferred GhCYC1 logically similar to each other, whereas at later stages (after stage amino acid sequence is markedly different from the GhCYC2–4 4) morphological differentiation of the flower types is visible sequences outside the highly conserved regions. Motif Scan (21) (Fig. 2 B–E) (3). Therefore, a more detailed analysis of GhCYC2 analysis of GhCYC amino acid sequences predicted that Gh- expression in young developing capitula was performed on CYC2, 3 and 4 have putative bipartite nuclear localization separately dissected ray and disk flower primordia using both signals, whereas GhCYC1 does not. Furthermore, GhCYC1 was RNA-blots (Fig. 2A) and quantitative PCR (data not shown). predicted by TargetP (22) to be targeted to the chloroplast, The results affirmed that at early developmental stages 3 and 5, suggesting a different role for this Gerbera TCP. GhCYC2 is expressed only in ray flower primordia and not in disk flower primordia located in the center of the capitulum. In Phylogenetic Analysis of Gerbera TCP Domain Factors. We per- later developmental stages 6 and 7, GhCYC2 expression was still formed phylogenetic analysis on a selected set of class II TCP lacking from the centermost disk flowers, although present in factors from various plant species (Table S1) to explore the outer disk flowers and in ray flowers (Fig. 2A).

9118 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801359105 Broholm et al. Downloaded by guest on September 28, 2021 Fig. 2. GhCYC2 is expressed in the outermost ray flower primordia but not in the centermost disk flower primordia. RNA-blot analysis (A) was done for ray (RF) and disk flower (DF) primordia in developmental stages 3, 5, 6, and 7. Outer (DF-o) and centermost (DF-c) disk flowers were dissected separately at stages 3 and 5, whereas at stages 6 and 7, outer, inner (DF-i), and centermost disk flowers were separated. At stages 3 and 5, GhCYC2 expression was detected only in ray flowers and not in outer or centermost disk flowers. At later stages 6 and 7, in addition to ray flowers, GhCYC2 expression was detected in the outer and inner disk flowers (DF-i), but GhCYC2 expression was still excluded from the centermost disk flowers. SEM pictures (B–E) show that at stage 3 the Gerbera ray and disk flower primordia have similar petal (pe) and stamen (st) morphology but that at stage 5 the two flower types have distinct characteristics, such as altered petal symmetry. (Scale bars: 100 ␮m.) Fig. 3. In situ analysis of GhCYC2 expression in ray flowers (A–C) and in the outer disk flowers (D)oftheGerbera inflorescence (diameter 12 mm). Sections In situ hybridizations were performed to localize GhCYC2 were bridized with GhCYC2 antisense RNA probes labeled with digoxigenin- expression on developing inflorescences. We used inflorescences UTP. In ray flowers, GhCYC2 was expressed in the ventral ligule (vLi) but not of Ϸ12 mm in diameter for which ray flowers were at stage 5 and in the rudimentary dorsal petals (dPe), as seen in the cross (A) and longitudinal centermost disk flowers at stage 3. In ray flowers, GhCYC2 was (C) sections. GhCYC2 expression was detected ubiquitously in the basal tubular expressed in petals, in rudimentary stamens, and in carpels (Fig. part of ray flower petals (tuPe) (B). GhCYC2 expression was also detected as 3 A–C). Intriguingly, GhCYC2 expression was absent from the well in rudimentary stamens (ruSt) and in carpels (Ca) (B and C). In the outer disk flowers, GhCYC2 was expressed most clearly in stamens but also in carpel dorsal rudimentary petals of ray flowers but was clearly present ␮

and petals (D). (Scale bars: 100 m.) PLANT BIOLOGY in the large ventral ligule that is formed via fusion of three (sometimes four) petals (Fig. 3A). The basalmost parts of all five Gerbera petals fuse together to form a tube in all flower types. was, however, entirely due to reduction in ray flower petal length In this basal tube, GhCYC2 expression was detected ubiqui- in comparison with wild-type petals (Table S2). Most interest- tously, with no distinction between the dorsal and the ventral ingly, in contrast to the shorter petals in ray flowers, petals of sides (Fig. 3B). GhCYC2 was not expressed in the centermost disk flowers were significantly longer in the transgenic lines than disk flowers, in accordance with the data from quantitative PCR in wild type (Table S2). In addition, the shape of the disk flower and RNA-blots. However, in outer disk flowers from later petals was altered (Fig. 5A). In wild type, disk flower petals show developmental stages, GhCYC2 expression was detected most reduced bilateral symmetry, and the dorsal and ventral petals are clearly in stamens but also in carpels and weakly in petals (Fig. approximately equal in their length. In the overexpression lines, 3D). GhCYC2 expression patterns were similar in larger inflo- disk flower petals had a distinct ligular structure that resembled rescences Ϸ18 mm in diameter (data not shown). the bilaterally symmetric shape of ray and trans flowers in having a larger ventral ligule and smaller dorsal petals. In one of the Morphological Effects of Constitutive GhCYC2 Expression in Trans- overexpression lines, all petals in disk flowers were fused to- genic Gerbera. For functional characterization, we produced 11 gether to form tubular structures (Fig. 5C). Furthermore, sta- transgenic lines that expressed GhCYC2 constitutively (data not men development was disrupted in these transgenic lines (Fig. shown). Ectopic expression of 35S::GhCYC2 resulted in delayed 5A). Stamens were brownish in color and unable to release growth; all transgenic lines remained longer in juvenile stage, as pollen, although some pollen grains developed. These pheno- defined by smaller and more roundly shaped leaves (Fig. 4 A and B). One of the lines was never able to transfer from vegetative into reproductive phase. Eventually, most of the lines produced inflorescences. In wild-type Gerbera the scape is bent so that the inflorescence faces downwards during the growth and develop- ment of the capitulum (Fig. 4C). The scape straightens during flower opening when the petals reach their final shape and size. In contrast, scapes in the overexpression lines were not bent, and inflorescences faced straight upward from the beginning of their development (Fig. 4D). This suggests that the constitutive ex- Fig. 4. Vegetative phenotype of the transgenic 35S::GhCYC2 Gerbera lines. pression of GhCYC2 may disrupt the dorsoventral (adaxial/ Overexpression lines (tr) showed delayed growth (A) and smaller, more abaxial) polarity of developing flower scapes. roundly shaped leaves (B). In wild type (wt), the inflorescence stem is bent In concordance with the delayed vegetative growth pheno- during growth (C), but in transgenic lines the developing inflorescence faced type, capitula appeared smaller in the overexpression lines. This upward (D). (Scale bars: 1 cm.)

Broholm et al. PNAS ͉ July 1, 2008 ͉ vol. 105 ͉ no. 26 ͉ 9119 Downloaded by guest on September 28, 2021 Fig. 5. The effect of GhCYC2 overexpression on Gerbera disk flowers. Transgenic disk flowers (tr df) had a clearly distinct phenotype compared with Fig. 6. The effect of suppressed GhCYC2 expression on Gerbera inflores- wild-type disk flowers (wt df). (A) Disk flower petals were longer and petals cence phenotype. In nontransformed Terra Regina (A and B), trans flowers (wt had a more pronounced ligular structure. Development of disk flower stamens tf) are longer than in the transgenic lines (A and C). Only the length of the (St) was disrupted. At the same developmental stages, wild-type disk flowers distal ligule and not the basal petal tube differs between wild-type and release pollen (B), but no pollen was seen in the 35S::GhCYC2 lines (A and C). transgenic trans flowers (tr tf) (A). (Scale bar: 0.5 cm.) In one of the overexpression lines, disk flower petals were fused together to form tubular structures (C). (Scale bar: 0.5 cm.) in yet another rosid lineage (25). Given this diversity of dem- onstrated CYC-like protein function in eudicots, it is not sur- typic changes imply that constitutive expression of GhCYC2 prising that duplication and divergence has led to further novelty caused disk flowers to obtain ray-like flower characteristics such in Gerbera, and perhaps also within other members of the as enlarged more markedly fused petals and disrupted stamen Asteraceae CYC-like gene clade. development. GhCYC2 Affects Growth in Transgenic Gerbera. In general, TCP Effects of Reduced GhCYC2 Expression in Transgenic Gerbera. We domain regulatory proteins have been shown to affect key obtained one transgenic line with cosuppressed GhCYC2 ex- developmental processes to produce morphological novelties in pression. In this line, the expression of GhCYC2 could not be plants. They function primarily by modulating cell growth and detected in RNA blots (Fig. S4). Transformation of the GhCYC2 proliferation. We have shown that Gerbera GhCYC2 also reg- cDNA in antisense orientation resulted in an additional line that ulates growth. Overexpression of GhCYC2 in transgenic Gerbera showed similar phenotypic changes as the cosuppression line. resulted in slower vegetative growth and reduced inflorescence This line accumulated very high amounts of antisense transcripts size. Importantly, the effect of GhCYC2 overexpression on petal nearly equal to the size of the endogenous transcript correspond- growth varied among the different flower types. In ray flowers, ing to GhCYC2. Hence, we could not convincingly detect petals were shorter (thus the entire inflorescence appeared whether the endogenous GhCYC2 expression was silenced (Fig. smaller), whereas in disk flowers petals were longer compared S4). However, in both lines, we observed flower-type specific with wild type. GhCYC2 is therefore able to either reduce or alterations. The length of trans flower petals was shorter than in promote growth depending on the site of its expression in wild type (Fig. 6 and Table S2). In other flower types the lengths capitula. Such opposite effects on growth have been described of petals did not differ significantly from wild type. We also for other TCP transcription factors but not at the inflorescence detected occasional splitting of the trans flower ligules into five level. For example in Antirrhinum, CYC reduces growth in the to eight separate petals (data not shown). dorsalmost stamen but promotes the growth of dorsal petals (12). Heterologous expression of CYC in Arabidopsis causes Discussion reduced vegetative growth but increased growth of petals. The Gerbera TCP Factors Form a Clade Independent from Antirrhinum decreased leaf size in these plants was due to reduction in both CYC/DICH and Functionally Characterized Genes from Legumes and cell proliferation and cell expansion, whereas enlarged petals Crucifers. Patterns of homology apparent in our phylogenetic resulted from increased cell expansion (26). Furthermore, the analyses of TCP genes are important for understanding GhCYC2 class II TCP factor CIN reduces growth in Antirrhinum leaves but function. Similar to what has been observed previously for promotes the growth of petals (20). CYC-like genes in legumes (23), GhCYC2 represents one prod- uct of several duplication events that occurred within Asteraceae GhCYC2 Expression Is Primarily Ventral and Correlates with Organ (or perhaps within the larger clade that contains both Asteraceae Fusion in Different Flower Types. We found differences between the and Dipsacales). Floral zygomorphy in Lotus and Antirrhinum expression patterns of GhCYC2 and other CYC/TB1-like genes appears to have evolved independently, because members of two known to be involved in the regulation of floral symmetry. distinct gene clades perform dorsalizing functions within discrete CYC-like genes are expressed in the dorsal parts of the flower to lineages of plants, the and (respectively), which establish bilateral symmetry in the Lamiales and Fabales species are very likely to be primitively actinomorphic (see, e.g., ref. 24). studied so far (12–14, 23, 27, 28). In Gerbera ray flowers, the Likewise, bilateral symmetry in the Arabidopsis relative Iberis, expression of GhCYC2 was specifically excluded from the dorsal which is distinguished by expression timing effects in its CYC-like rudimentary petals, suggesting functional deviation from Lamia- gene IaTCP1, may also represent an independent development les and . A similarly ventral expression pattern was

9120 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801359105 Broholm et al. Downloaded by guest on September 28, 2021 documented for a CYC3-subclade gene from Lonicera (Dip- cultivars showing the crested phenotype do not display GhCYC2 sacales), whereas a CYC2-subclade gene expressed dorsally (16). expression differences compared with semicrested cultivars However, preliminary phylogenetic analyses including some of (such as Terra Regina; used here) or cultivars having short trans these authors’ shorter sequences suggest that GhCYC2 belongs flowers (S.K.B., unpublished results), we postulate that GhCYC2 to the CYC2 subclade (data not shown). If so, ECE CYC-like is not sufficient for complete transformation of flower types. genes have undergone functional divergence within subclades as The transgenic Gerbera line with suppressed GhCYC2 expres- well as within organismal groups. sion did not show alterations in ray flower identity. This again Intriguingly, GhCYC2 expression correlated with organ fusion implies that GhCYC2 is not sufficient for regulating differenti- in petals. In ray flowers, GhCYC2 expression was uniform in the ation between the rayed and nonrayed form of Asteraceae basal petal tube and in the fused ligule but was lacking from the inflorescences but that it takes part in this process, most likely as rudimentary dorsal petals, which remain unfused. At the inflo- a modifier gene. Genetic analyses performed so far have sug- rescence level, GhCYC2 was specifically expressed in the bilat- gested that in addition to the major loci (RAY/turf/CHRY/ erally symmetrical ray flowers with their more markedly fused CRESTED), an unknown number of modifier genes are also petals, whereas the lack of GhCYC2 expression in central disk involved. The Gerbera GhCYC3 and GhCYC4 genes show ray- flowers correlated with radial symmetry and decreased petal flower specific expression similar to GhCYC2 (S.T. and S.K.B., fusion. Overexpression phenotypes indicate that GhCYC2 not unpublished results). Their expression was not reduced in the only promotes the growth of ligules in disk flowers but is also transgenic lines with suppressed GhCYC2 expression. Therefore, capable of generating fully tubular disk flower corollas. Indeed, the absence of altered ray flower phenotype in GhCYC2 sup- all three Gerbera flower types can become fully tubular, as pressed lines might be due to redundant functions of these observed when a CYC-like gene from Senecio is overexpressed closely related genes. Functional analyses of GhCYC3 and in Gerbera (P.E. and S.K.B., unpublished results). We also GhCYC4 are currently underway. observed frequent splitting of trans flower petals in the trans- Our data provides the first molecular evidence that CYC-like genic Gerbera line with reduced GhCYC2 expression, which in TCP factors are involved in defining identities of different flower addition to the expression pattern of GhCYC2, provides further types within a single genotype. The expression of GhCYC2 shows support for the role of Asteraceae TCP factors in promoting clear radial specificity along the axis of the Gerbera capitulum. organ fusion. In previous work we showed that several MADS box genes also Previous studies have connected TCP domain transcription display radial expression differences among developing flower factors with regulation of organ boundaries. In Arabidopsis, primordia (3). As such, it seems likely that both TCP and MADS CIN-like proteins negatively regulate boundary-specific NAC domain proteins participate in regulating the developmental domain transcription factors (CUP-SHAPED COTYLEDON specification of Gerbera flower types through response to early (CUC) 1, 2, 3) (29). Overexpression of CIN-like genes caused acting signals at the inflorescence level. A gradient of a cell suppression of CUC expression, which resulted in fusion of nonautonomously moving substance, such as a phytohormone or cotyledons (29, 30). In Antirrhinum, the PCF-like TCP protein a transcription factor, could induce differentiation of the distinct TIC (TCP-Interacting with CUP) has been shown to interact flower morphologies by affecting putative downstream targets with a NAC domain protein that regulates organ fusion (31). such as TCP and MADS domain proteins.

Thus, NAC domain proteins have a clear connection with TCP PLANT BIOLOGY domain proteins, both as downstream targets and as interaction Conclusions partners. Asteraceae (1), Fabales, and Orchidaceae (33) are the three largest lineages of flowering plants, and each are defined by GhCYC2 Plays a Major Role in Differentiating Gerbera Flower Types. suites of traits that have lent themselves to extensive evolutionary The experimental evidence shown here suggests that GhCYC2 modification. For Asteraceae, the capitulate inflorescence with is involved in differentiation between Gerbera flower types. its capacity to produce different flower types has been its GhCYC2 is specifically expressed in the marginal ray flower principal innovation. Through analysis of a CYC-like TCP- primordia. Based on transgenic phenotypes, the absence of encoding gene in Gerbera, we have shown that gene duplication GhCYC2 expression in the central disk flower primordia appears and divergence, a frequent theme in the evolution of organismal to be crucial for proper flower type specification. Overexpres- novelty (34–36), has been one major factor in establishing the sion of GhCYC2 causes disk flowers to obtain characteristics uniqueness and success of the sunflower family. typical for ray flowers, including enlarged ventral petals and disrupted stamen development. These Gerbera lines resemble Materials and Methods the semidominant chry mutant of sunflower (7), in which disk Plant Material. Gerbera hybrida (Asteraceae) variety Terra Regina (Terra flowers also obtain ray-flower like traits. In chry mutants, the Nigra) was grown under standard greenhouse conditions. The developmental form of disk flower petals has shifted toward bilateral symmetry, stages for inflorescences are described in ref. 37 and for the young developing and stamens are smaller with disrupted pollen production. The floral primordia in ref. 3. similarities of the two phenotypes in these closely related species Isolation of Gerbera TCP Domain Factors. Gerbera TCP domain factors were makes it tempting to speculate that the semidominant chry amplified from cDNA prepared from young developing inflorescences of mutant could result from overexpression of a GhCYC2 ortholog Gerbera by PCR using two different pairs of degenerate primers. The first in sunflower. degenerate primer pair was designed from the conserved TCP domain and In Gerbera, some cultivars resemble the transgenic GhCYC2 corresponded to the amino acid sequences KKDRHSKI and ERTKEK. Poly(A)- lines and the sunflower chry mutants. The ‘‘crested’’ trait is RNA (450 ng) was used as a template for cDNA synthesis (Boehringer first- characterized by enlarged trans flowers or enlarged trans and strand cDNA kit). PCR conditions were the following: 94°C for 75 sec, 46°C for disk flowers that are male-sterile and defined by a single locus 2 min, and 72°C for 3 min for 30 cycles. Two different fragments that showed containing three alleles with semidominant relationships high similarity with CYC-like genes were obtained by using this primer pair. (crested Ͻ CRESTED Ͻ CRESTEDD) (32). In our overexpres- For the second round of PCR screening, degenerate primers were designed by using the CODEHOP strategy (38) and were optimized for codon usage. sion lines, the increase in petal length and reduction in stamen Primers corresponded to the amino acid sequences DLQDMLGFDK and functionality in disk flowers implies similarity with the crested ARARARERTKEK. cDNA synthesis was performed from poly(A)-RNA by using trait. Moreover, the recessive homozygotes of crested are char- SuperScript III reverse transcriptase (Invitrogen). Phusion DNA polymerase was acterized by short trans flowers resembling the transgenic line used for PCR reactions (Finnzymes). The full-length cDNAs were amplified by with suppressed GhCYC2 expression. However, because Gerbera using the SMART RACE cDNA amplification kit (Clontech). The TCP mRNA

Broholm et al. PNAS ͉ July 1, 2008 ͉ vol. 105 ͉ no. 26 ͉ 9121 Downloaded by guest on September 28, 2021 coding sequences have been deposited in the GenBank database (accession Plant Transformation and Analysis of Transgenic Lines. The full-length GhCYC2 numbers EU429302–EU429305). cDNA was transformed under the CaMV 35S promoter into Gerbera variety Terra Regina as reported in refs. 39 and 40. Differences in the length of petals Phylogenetic Analyses. Phylogenetic analyses were performed on correspond- between wild-type variety Terra Regina and transgenic lines were tested by ing nucleotide and amino acid alignments for the TCP and R domains of using the Mann–Whitney rank-sum test. Flowers were counted, and the selected TCP factors. Two phylogeny reconstruction methods were used: length of petals was measured from five inflorescences of each line. On parsimony and maximum likelihood (see SI Text for full details). average, for ray flowers n ϭ 63 per line, for trans flowers n ϭ 213 per line, and for disk flowers n ϭ 310 per line. RNA Blot, in Situ Hybridizations, and Scanning Electron Microscopy (SEM). Total RNA was isolated by using TRIzol reagent (Invitrogen). Ten micrograms of ACKNOWLEDGMENTS. We thank E. Coen (John Innes Centre, Norwich, UK) 32 total RNA was loaded on RNA blots and hybridized by using P-labeled DNA for providing the Senecio squalidus cDNA (plasmid pJAM2176); the Elec- probes. For a gene-specific probe, a 253-bp fragment from the 3Ј end of the tron Microscopy Unit of the Institute of Biotechnology, University of cDNA was used. The blots were washed with 0.5ϫ SSC and 0.1% (wt/vol) SDS Helsinki, for providing laboratory facilities; Eija Takala and Anu Rokkanen at 58°C. In situ hybridization analysis using full-length GhCYC2 probe was for excellent technical assistance; and Sanna Peltola for taking care of the performed as in ref. 39, with the following exceptions: probe concentration plants in the greenhouse. This work was supported by Academy of Finland was 0.7 ␮g/ml/kb and detection time was 40–45 h. SEM analysis of the Gerbera Grant 115849 (to P.E.). S.K.B. is supported by the Viikki Graduate School in flower primordia was performed as described in ref. 5. Biosciences.

1. Bremer K (1994) Asteraceae: Cladistics and Classification (Timber Press, Portland, OR). 21. Pagni M, et al. (2004) MyHits: A new interactive resource for protein annotation and 2. Teeri TH, et al. (2006) Floral developmental genetics of Gerbera (Asteraceae). Devel- domain identification. Nucleic Acids Res 32:W332–W335. opmental Genetics of the Flower, Advances in Botanical Research, eds Soltis DE, 22. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular Leebens-Mack JH Soltis PS (Elsevier, New York), pp 324–351. localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 3. Laitinen RA, Broholm S, Albert VA, Teeri TH, Elomaa P (2006) Patterns of MADS-box 300:1005–1016. gene expression mark flower-type development in Gerbera hybrida (Asteraceae). BMC 23. Feng X, et al. (2006) Control of petal shape and floral zygomorphy in Lotus japonicus. Plant Biol 6:11. Proc Natl Acad Sci USA 103:4970–4975. 4. Kotilainen M, et al. (2000) GRCD1, an AGL2-like MADS box gene, participates in the C 24. Donoghue MJ, Ree RH, Baum DA (1998) Phylogeny and the evolution of flower function during stamen development in Gerbera hybrida. Plant Cell 12:1893–1902. symmetry in the Asteridae. Trends Plants Sci 3:311–317. 5. Uimari A, et al. (2004) Integration of reproductive meristem fates by a SEPALLATA-like 25. Busch A, Zachgo S (2007) Control of corolla monosymmetry in the Brassicaceae Iberis MADS-box gene. Proc Natl Acad Sci USA 101:15817–15822. amara. Proc Natl Acad Sci USA 104:16714–16719. 6. Gillies ACM, Cubas P, Coen ES, Abbott RJ (2002) Making rays in the Asteraceae: Genetics 26. Costa MM, Fox S, Hanna AI, Baxter C, Coen E (2005) Evolution of regulatory interactions and evolution of radiate versus discoid flower heads. Developmental Genetics and controlling floral asymmetry. Development 132:5093–5101. Plant Evolution, eds Cronk QCB, Bateman RM Hawkins JA (Taylor & Francis, London), 27. Hileman LC, Kramer EM, Baum DA (2003) Differential regulation of symmetry genes pp 233–246. 7. Berti F, Fambrini M, Turi M, Bertini D, Pugliesi C (2005) Mutations of corolla symmetry and the evolution of floral morphologies. Proc Natl Acad Sci USA 100:12814–12819. affect carpel and stamen development in Helianthus annuus. Can J Bot 83:1065–1072. 28. Citerne HL, Pennington RT, Cronk QC (2006) An apparent reversal in floral symmetry 8. Abbott RJ, Ashton PA, Forbes DG (1992) Introgressive origin of the radiate groundsel in the legume Cadia is a homeotic transformation. Proc Natl Acad Sci USA 103:12017– Senecio vulgaris var hibernicus Syme: Aat-3 evidence. Heredity 68:425–435. 12020. 9. Comes H (1998) Major gene effects during weed evolution: Phenotypic characters 29. Koyama T, Furutani M, Tasaka M, Ohme-Takagi M (2007) TCP transcription factors cosegregate with alleles at the ray floret locus in Senecio vulgaris L. (Asteraceae). control the morphology of shoot lateral organs via negative regulation of the expres- J Hered 89:54–61. sion of boundary-specific genes in Arabidopsis. Plant Cell 19:473–484. 10. Andersson S (2001) The genetic basis of floral variation in Senecio jacobaea (Aster- 30. Palatnik JF, et al. (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257– aceae). J Hered 92:409–414. 263. 11. Coen ES, Nugent JM (1994) Evolution of flowers and inflorescences. Development 31. Weir I, et al. (2004) CUPULIFORMIS establishes lateral organ boundaries in Antirrhi- Suppl:107–116. num. Development 131:915–922. 12. Luo D, Carpenter R, Vincent C, Copsey L, Coen E (1996) Origin of floral asymmetry in 32. Kloos WE, George CG, Sorge LK (2004) Inheritance of the flower types of Gerbera Antirrhinum. Nature 383:794–799. hybrida. J Am Soc Hort Sci 129:802–810. 13. Luo D, et al. (1999) Control of organ asymmetry in flowers of Antirrhinum. Cell 33. Endress PK (1994) Diversity and Evolutionary Biology of Flowers (Cambridge Univ Press, 99:367–376. Cambridge, UK). 14. Cubas P, Lauter N, Doebley J, Coen E (1999) The TCP domain: A motif found in proteins 34. Hoekstra HE, Coyne JA (2007) The locus of evolution: evo devo and the genetics of regulating plant growth and development. Plant J 18:215–222. adaptation. Evolution 61:995–1016. 15. Aguilar-Martinez JA, Poza-Carrion C, Cubas P (2007) Arabidopsis BRANCHED1 acts as 35. Roth C, et al. (2007) Evolution after gene duplication: Models, mechanisms, sequences, an integrator of branching signals within axillary buds. Plant Cell 19:458–472. systems, and organisms. J Exp Zool B Mol Dev Evol 308:58–73. 16. Howarth DG, Donoghue MJ (2006) Phylogenetic analysis of the ‘‘ECE’’ (CYC/TB1) clade 36. Baxter CE, Costa MM, Coen ES (2007) Diversification and co-option of RAD-like genes reveals duplications predating the core eudicots. Proc Natl Acad Sci USA 103:9101– in the evolution of floral asymmetry. Plant J 52:105–113. 9106. 37. Helariutta Y, Elomaa P, Kotilainen M, Seppanen P, Teeri TH (1993) Cloning of cDNA 17. Doebley J, Stec A, Hubbard L (1997) The evolution of apical dominance in maize. Nature coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in 386:485–488. 18. Takeda T, et al. (2003) The OsTB1 gene negatively regulates lateral branching in rice. the corollas of Gerbera hybrida var. Regina (Compositae). Plant Mol Biol 22:183–193. Plant J 33:513–520. 38. Morant M, Hehn A, Werck-Reichhart D (2002) Conservation and diversity of gene 19. Gaudin V, et al. (2000) The expression of D-cyclin genes defines distinct developmental families explored using the CODEHOP strategy in higher plants. BMC Plant Biol 2:7. zones in snapdragon apical meristems and is locally regulated by the CYCLOIDEA gene. 39. Elomaa P, et al. (2003) Activation of anthocyanin biosynthesis in Gerbera hybrida Plant Physiol 122:1137–1148. (Asteraceae) suggests conserved protein–protein and protein–promoter interactions 20. Crawford BC, Nath U, Carpenter R, Coen ES (2004) CINCINNATA controls both cell between the anciently diverged monocots and eudicots. Plant Physiol 133:1831–1842. differentiation and growth in petal lobes and leaves of Antirrhinum. Plant Physiol 40. Elomaa P, Teeri TH (2001) Transgenic Gerbera. Biotechnology in Agriculture and 135:244–253. Forestry, Transgenic Crops III, ed YPS Bajaj (Springer, Berlin), pp 139–154.

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