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

Edited by M. T. Clegg, University of California, Irvine, CA, and approved April 4, 2008
July 1, 2008
105 (26) 9117-9122


Several key processes in plant development are regulated by TCP transcription factors. CYCLOIDEA-like (CYC-like) TCP domain proteins have been shown to control flower symmetry in distantly related plant lineages. Gerbera hybrida, a member of one of the largest clades of angiosperms, the sunflower family (Asteraceae), is an interesting model for developmental studies because its elaborate inflorescence comprises different types of flowers that have specialized structures and functions. The morphological differentiation of flower types involves gradual changes in flower size and symmetry that follow the radial organization of the densely packed inflorescence. Differences in the degree of petal fusion further define the distinct shapes of the Gerbera flower types. To study the role of TCP transcription factors during specification of this complex inflorescence organization, we characterized the CYC-like homolog GhCYC2 from Gerbera. The expression of GhCYC2 follows a gradient along the radial axis of the inflorescence. GhCYC2 is expressed in the marginal, bilaterally symmetrical ray flowers but not in the centermost disk flowers, which are nearly radially symmetrical and have significantly less fused petals. Overexpression of GhCYC2 causes disk flowers to obtain morphologies similar to ray flowers. Both expression patterns and transgenic phenotypes suggest that GhCYC2 is involved in differentiation among Gerbera flower types, providing the first molecular evidence that CYC-like TCP factors take part in defining the complex inflorescence structure of the Asteraceae, a major determinant of the family's evolutionary success.
The enormous variety of flowers makes them fascinating targets for comparative developmental genetic studies. Species of the sunflower family (Asteraceae), such as the cut-flower crop Gerbera hybrida, present a unique and challenging benefit: more than one floral phenotype can be analyzed within the same genotype. A typical Asteraceae inflorescence consists of morphologically and functionally differentiated flowers packed into a condensed inflorescence (the capitulum) that resembles (as a pseudanthium) a single large flower. This inflorescence complexity, not shared by other model species used for flower developmental research such as Arabidopsis, Antirrhinum, or Petunia, has apparently proved to be evolutionarily successful because Asteraceae is one of the largest families of flowering plants with >23,000 species (1).
Gerbera bears three flower types (ray, trans, and disk) that are morphologically similar during their early development. One of the most prominent developmental differences, which becomes established early in floral ontogeny, is a gradual change of flower symmetry that follows the radial organization of the capitulum (2). The marginal ray flowers are bilaterally symmetrical (zygomorphic). Three of the five petals are fused ventrally to form the large, elongate ligule, whereas the remaining two (dorsal) petals remain rudimentary. Disk flowers at the center of the capitulum have shorter and less fused petals, and they eventually become radially symmetrical (actinomorphic) with separate petals at the inflorescence apex. Trans flowers, which occupy an intermediate radial position between ray and disk flowers, are strongly zygomorphic, yet smaller in size in comparison with ray flowers. Stamen development also differs among individual flower types (3). In the female ray and trans flowers, stamen development arrests, resulting in rudimentary staminodia (4), whereas in the disk flowers, anthers develop fully and form a postgenitally fused structure that covers the carpel.
Despite continued efforts, the molecular basis for flower type differentiation in Asteraceae remains unclear. Microarray comparison of developing Gerbera ray and disk flower primordia has identified a number of genes that are differentially expressed in individual flower types (3). We have shown that many MADS box genes encoding known regulators of flower organ development are expressed gradientially along the capitulum radius. While this may suggest that different MADS protein complexes participate in developmental regulation of individual flower types, transgenic experiments show that such complexes are not independently sufficient (4, 5). Indeed, classical genetics has shown that the presence or absence of ray flowers in Asteraceae capitula appears to be under control of one or two major and several modifier genes (reviewed in ref. 6). Berti et al. (7) have characterized two sunflower mutants in which flower type identity (and consequently symmetry) has changed. In the chrysanthemoides (chry) mutant, all flowers in the inflorescences are ray-like (zygomorphic), whereas in the tubular ray flower (turf) mutant, all flower types resemble radially symmetrical disk flowers. In Senecio, capitulum organization is principally controlled by the RAY locus, with radiate inflorescences (ray plus disk) dominant over discoid (disk only) (810). For both Senecio and sunflower, hypotheses have been constructed whereby the RAY (6, 11), CHRY and TURF loci (7) could encode homologs of the Antirrhinum floral symmetry gene CYCLOIDEA (12). However, these intriguing postulates remain unevaluated by molecular evidence.
CYCLOIDEA-like genes have been reported to be involved in flower symmetry regulation in various plant species. CYCLOIDEA (CYC) of Antirrhinum was the first gene isolated, and thereafter the most extensively studied (12, 13). It belongs to the plant-specific gene family encoding TCP transcription factors, which share a conserved basic helix–loop–helix TCP domain. The name TCP derives from the three founding members 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 proliferation (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, -2, and -3, have been identified in core eudicots (16). TB1 has played a major role in the evolution of maize from its wild ancestor teosinte. It acts as a repressor of growth by suppressing axillary branching, and thus promotes apical dominance in 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 Antirrhinum regulate flower symmetry by modulating cell-division related gene expression (1214, 19). The wild-type flowers of Antirrhinum have an axis of dorsoventral asymmetry with distinct dorsal, lateral, and ventral organ types. The expression of CYC and DICH is restricted to the dorsal domain of the flowers to establish bilateral petal symmetry and to arrest the development of the dorsalmost stamen (12, 13). CIN arrests the growth of Antirrhinum leaf margins but also affects differentiation of epidermal cells and growth of petal lobes through effects on cell proliferation (20).
In Gerbera, specification of flower types involves differential development of petals and stamens, probably through distinct cell division and elongation as well as organ fusion events. TCP transcription factors have previously been connected with all three of these processes, making them good candidates for regulators of the distinct morphologies of Gerbera flower types. We have isolated several TCP transcription factor genes as candidates from Gerbera. We show that one of these proteins, GhCYC2, has a symmetry function different from that of classic CYC-like genes. Instead of regulating the dorsoventral symmetry of individual flowers, GhCYC2 participates in the control of the identity and radial extent of flower types in Gerbera inflorescences. Moreover, GhCYC2 plays an important and unique role in organ fusion that further differentiates Gerbera flower types.


Isolation of Gerbera CYC Homologs.

To study the role of TCP domain transcription factors during Gerbera flower development, cDNA clones encoding these proteins were isolated from young developing Gerbera inflorescences. The inferred amino acid sequences of the four cDNAs, GhCYC1, GhCYC2, GhCYC3, and GhCYC4 (Gerbera hybrida CYCLOIDEA-like 1, 2, 3, and 4), exhibited the conserved TCP and R domains typical for the CYC/TB1 subfamily. Low stringency hybridization of Gerbera genomic DNA with a full-length GhCYC2 probe suggested the presence of a small gene family [supporting information (SI) Fig. S1]. Although we cannot exclude possible allelism, this inference was supported by isolation of 4 distinct sequence fragments (data not shown) in addition to the four full-length sequences presented in this study. The alignment of full-length GhCYC cDNAs (Fig. S2) showed that the inferred GhCYC1 amino acid sequence is markedly different from the GhCYC2–4 sequences outside the highly conserved regions. Motif Scan (21) analysis of GhCYC amino acid sequences predicted that GhCYC2, 3 and 4 have putative bipartite nuclear localization signals, whereas GhCYC1 does not. Furthermore, GhCYC1 was predicted by TargetP (22) to be targeted to the chloroplast, suggesting a different role for this Gerbera TCP.

Phylogenetic Analysis of Gerbera TCP Domain Factors.

We performed phylogenetic analysis on a selected set of class II TCP factors from various plant species (Table S1) to explore the evolutionary relationships of the Gerbera TCP genes. Nucleotide sequences encoding the conserved TCP and R domains were aligned, and parsimony and maximum likelihood analyses were performed on these data and the inferred amino acid alignment. Robustness of results was assessed using jacknife and bootstrap resampling, respectively (Fig. S3). In most analyses, CIN-like genes from Antirrhinum and Arabidopsis grouped together with support. CYC-like genes from Lamiales, Fabales, and Asteraceae were divided into separate, well supported groups. The greater genetic similarity within rather than among these groups suggests that many functionally important duplication events in the ECE gene subfamily occurred after the divergence of the Lamiales, Fabales, and Asteraceae lineages, respectively. The results of Howarth and Donoghue (16) are similar in this respect in that groupings within subclades CYC1, CYC2, and CYC3 are largely consistent with organismal phylogeny. Given this concurrence, we tested the phylogenetic distribution of our gene data when combined with many of the less-complete Dipsacales and Asterales sequences published by the former authors, and all well supported relationships were consistent with the results reported here (data not shown). According to our trees and their underlying nucleotide alignment, GhCYC1 is deviant from the other three GhCYC genes, which are quite similar to each other (Fig. S2). In addition to its sister gene HpTCP from sunflower, in the most-parsimonious and maximum likelihood trees based on nucleotide data, GhCYC1 groups (without statistical support) with Arabidopsis AtBRC1 and Lotus LjCYC5 (data not shown). AtBRC1 and AtBRC2 have been shown to be functionally related to maize TB1 (15). However, in our analyses, support for particular gene groupings with TB1 is lacking.

The Expression of GhCYC2 Follows the Zonal Organization of the Capitulum.

RNA-blot analysis showed that GhCYC2 is expressed only in floral tissues. We could not detect expression in vegetative organs such as leaves, roots, or floral stems (scapes). Expression was strongest in young inflorescences, and in petals, whereas weaker expression was observed in carpels (stigma and style), in the ovary and in involucral bracts (the leaf-like structures that tightly surround the reproductive centers of capitula) (Fig. 1). We have recently demonstrated that at floral primordium stage 3, the ray and disk flowers of Gerbera are morphologically similar to each other, whereas at later stages (after stage 4) morphological differentiation of the flower types is visible (Fig. 2 B–E) (3). Therefore, a more detailed analysis of GhCYC2 expression in young developing capitula was performed on separately dissected ray and disk flower primordia using both RNA-blots (Fig. 2A) and quantitative PCR (data not shown). The results affirmed that at early developmental stages 3 and 5, GhCYC2 is expressed only in ray flower primordia and not in disk flower primordia located in the center of the capitulum. In later developmental stages 6 and 7, GhCYC2 expression was still lacking from the centermost disk flowers, although present in outer disk flowers and in ray flowers (Fig. 2A).
Fig. 1.
Expression of GhCYC2 in various Gerbera tissues. GhCYC2 showed strongest expression in young inflorescences, petals, and carpel (stigma and style). Weaker expression was also seen in bracts and ovary. The lower blot shows ethidium bromide-stained ribosomal RNA bands to control for RNA loading.
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 μm.)
In situ hybridizations were performed to localize GhCYC2 expression on developing inflorescences. We used inflorescences of ≈12 mm in diameter for which ray flowers were at stage 5 and centermost disk flowers at stage 3. In ray flowers, GhCYC2 was expressed in petals, in rudimentary stamens, and in carpels (Fig. 3 A–C). Intriguingly, GhCYC2 expression was absent from the dorsal rudimentary petals of ray flowers but was clearly present 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. In this basal tube, GhCYC2 expression was detected ubiquitously, with no distinction between the dorsal and the ventral sides (Fig. 3B). GhCYC2 was not expressed in the centermost disk flowers, in accordance with the data from quantitative PCR and RNA-blots. However, in outer disk flowers from later developmental stages, GhCYC2 expression was detected most clearly in stamens but also in carpels and weakly in petals (Fig. 3D). GhCYC2 expression patterns were similar in larger inflorescences ≈18 mm in diameter (data not shown).
Fig. 3.
In situ analysis of GhCYC2 expression in ray flowers (A–C) and in the outer disk flowers (D) of the Gerbera inflorescence (diameter 12 mm). Sections were bridized with GhCYC2 antisense RNA probes labeled with digoxigenin-UTP. In ray flowers, GhCYC2 was expressed in the ventral ligule (vLi) but not in the rudimentary dorsal petals (dPe), as seen in the cross (A) and longitudinal (C) sections. GhCYC2 expression was detected ubiquitously in the basal tubular part of ray flower petals (tuPe) (B). GhCYC2 expression was also detected as 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 and petals (D). (Scale bars: 100 μm.)

Morphological Effects of Constitutive GhCYC2 Expression in Transgenic Gerbera.

For functional characterization, we produced 11 transgenic lines that expressed GhCYC2 constitutively (data not shown). Ectopic expression of 35S::GhCYC2 resulted in delayed growth; all transgenic lines remained longer in juvenile stage, as 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 development 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 expression of GhCYC2 may disrupt the dorsoventral (adaxial/abaxial) polarity of developing flower scapes.
Fig. 4.
Vegetative phenotype of the transgenic 35S::GhCYC2 Gerbera lines. Overexpression lines (tr) showed delayed growth (A) and smaller, more roundly shaped leaves (B). In wild type (wt), the inflorescence stem is bent during growth (C), but in transgenic lines the developing inflorescence faced upward (D). (Scale bars: 1 cm.)
In concordance with the delayed vegetative growth phenotype, capitula appeared smaller in the overexpression lines. This was, however, entirely due to reduction in ray flower petal length in comparison with wild-type petals (Table S2). Most interestingly, in contrast to the shorter petals in ray flowers, petals of disk flowers were significantly longer in the transgenic lines than in wild type (Table S2). In addition, the shape of the disk flower petals was altered (Fig. 5A). In wild type, disk flower petals show reduced bilateral symmetry, and the dorsal and ventral petals are approximately equal in their length. In the overexpression lines, disk flower petals had a distinct ligular structure that resembled the bilaterally symmetric shape of ray and trans flowers in having a larger ventral ligule and smaller dorsal petals. In one of the overexpression lines, all petals in disk flowers were fused together to form tubular structures (Fig. 5C). Furthermore, stamen development was disrupted in these transgenic lines (Fig. 5A). Stamens were brownish in color and unable to release pollen, although some pollen grains developed. These phenotypic changes imply that constitutive expression of GhCYC2 caused disk flowers to obtain ray-like flower characteristics such as enlarged more markedly fused petals and disrupted stamen development.
Fig. 5.
The effect of GhCYC2 overexpression on Gerbera disk flowers. Transgenic disk flowers (tr df) had a clearly distinct phenotype compared with wild-type disk flowers (wt df). (A) Disk flower petals were longer and petals had a more pronounced ligular structure. Development of disk flower stamens (St) was disrupted. At the same developmental stages, wild-type disk flowers release pollen (B), but no pollen was seen in the 35S::GhCYC2 lines (A and C). In one of the overexpression lines, disk flower petals were fused together to form tubular structures (C). (Scale bar: 0.5 cm.)

Effects of Reduced GhCYC2 Expression in Transgenic Gerbera.

We obtained one transgenic line with cosuppressed GhCYC2 expression. In this line, the expression of GhCYC2 could not be detected in RNA blots (Fig. S4). Transformation of the GhCYC2 cDNA in antisense orientation resulted in an additional line that showed similar phenotypic changes as the cosuppression line. This line accumulated very high amounts of antisense transcripts nearly equal to the size of the endogenous transcript corresponding to GhCYC2. Hence, we could not convincingly detect whether the endogenous GhCYC2 expression was silenced (Fig. S4). However, in both lines, we observed flower-type specific alterations. The length of trans flower petals was shorter than in wild type (Fig. 6 and Table S2). In other flower types the lengths of petals did not differ significantly from wild type. We also detected occasional splitting of the trans flower ligules into five to eight separate petals (data not shown).
Fig. 6.
The effect of suppressed GhCYC2 expression on Gerbera inflorescence phenotype. In nontransformed Terra Regina (A and B), trans flowers (wt tf) are longer than in the transgenic lines (A and C). Only the length of the distal ligule and not the basal petal tube differs between wild-type and transgenic trans flowers (tr tf) (A). (Scale bar: 0.5 cm.)


Gerbera TCP Factors Form a Clade Independent from Antirrhinum CYC/DICH and Functionally Characterized Genes from Legumes and Crucifers.

Patterns of homology apparent in our phylogenetic analyses of TCP genes are important for understanding GhCYC2 function. Similar to what has been observed previously for CYC-like genes in legumes (23), GhCYC2 represents one product of several duplication events that occurred within Asteraceae (or perhaps within the larger clade that contains both Asteraceae and Dipsacales). Floral zygomorphy in Lotus and Antirrhinum appears to have evolved independently, because members of two distinct gene clades perform dorsalizing functions within discrete lineages of plants, the rosids and asterids (respectively), which are very likely to be primitively actinomorphic (see, e.g., ref. 24). Likewise, bilateral symmetry in the Arabidopsis relative Iberis, which is distinguished by expression timing effects in its CYC-like gene IaTCP1, may also represent an independent development in yet another rosid lineage (25). Given this diversity of demonstrated CYC-like protein function in eudicots, it is not surprising that duplication and divergence has led to further novelty in Gerbera, and perhaps also within other members of the Asteraceae CYC-like gene clade.

GhCYC2 Affects Growth in Transgenic Gerbera.

In general, TCP domain regulatory proteins have been shown to affect key developmental processes to produce morphological novelties in plants. They function primarily by modulating cell growth and proliferation. We have shown that Gerbera GhCYC2 also regulates growth. Overexpression of GhCYC2 in transgenic Gerbera resulted in slower vegetative growth and reduced inflorescence size. Importantly, the effect of GhCYC2 overexpression on petal growth varied among the different flower types. In ray flowers, petals were shorter (thus the entire inflorescence appeared smaller), whereas in disk flowers petals were longer compared with wild type. GhCYC2 is therefore able to either reduce or promote growth depending on the site of its expression in capitula. Such opposite effects on growth have been described for other TCP transcription factors but not at the inflorescence level. For example in Antirrhinum, CYC reduces growth in the dorsalmost stamen but promotes the growth of dorsal petals (12). Heterologous expression of CYC in Arabidopsis causes reduced vegetative growth but increased growth of petals. The decreased leaf size in these plants was due to reduction in both cell proliferation and cell expansion, whereas enlarged petals resulted from increased cell expansion (26). Furthermore, the class II TCP factor CIN reduces growth in Antirrhinum leaves but promotes the growth of petals (20).

GhCYC2 Expression Is Primarily Ventral and Correlates with Organ Fusion in Different Flower Types.

We found differences between the expression patterns of GhCYC2 and other CYC/TB1-like genes known to be involved in the regulation of floral symmetry. CYC-like genes are expressed in the dorsal parts of the flower to establish bilateral symmetry in the Lamiales and Fabales species studied so far (1214, 23, 27, 28). In Gerbera ray flowers, the expression of GhCYC2 was specifically excluded from the dorsal rudimentary petals, suggesting functional deviation from Lamiales and Fabaceae. A similarly ventral expression pattern was documented for a CYC3-subclade gene from Lonicera (Dipsacales), whereas a CYC2-subclade gene expressed dorsally (16). However, preliminary phylogenetic analyses including some of these authors' shorter sequences suggest that GhCYC2 belongs to the CYC2 subclade (data not shown). If so, ECE CYC-like genes have undergone functional divergence within subclades as well as within organismal groups.
Intriguingly, GhCYC2 expression correlated with organ fusion in petals. In ray flowers, GhCYC2 expression was uniform in the basal petal tube and in the fused ligule but was lacking from the rudimentary dorsal petals, which remain unfused. At the inflorescence level, GhCYC2 was specifically expressed in the bilaterally symmetrical ray flowers with their more markedly fused petals, whereas the lack of GhCYC2 expression in central disk flowers correlated with radial symmetry and decreased petal fusion. Overexpression phenotypes indicate that GhCYC2 not only promotes the growth of ligules in disk flowers but is also capable of generating fully tubular disk flower corollas. Indeed, all three Gerbera flower types can become fully tubular, as observed when a CYC-like gene from Senecio is overexpressed in Gerbera (P.E. and S.K.B., unpublished results). We also observed frequent splitting of trans flower petals in the transgenic Gerbera line with reduced GhCYC2 expression, which in addition to the expression pattern of GhCYC2, provides further support for the role of Asteraceae TCP factors in promoting organ fusion.
Previous studies have connected TCP domain transcription factors with regulation of organ boundaries. In Arabidopsis, CIN-like proteins negatively regulate boundary-specific NAC domain transcription factors (CUP-SHAPED COTYLEDON (CUC) 1, 2, 3) (29). Overexpression of CIN-like genes caused suppression of CUC expression, which resulted in fusion of cotyledons (29, 30). In Antirrhinum, the PCF-like TCP protein TIC (TCP-Interacting with CUP) has been shown to interact with a NAC domain protein that regulates organ fusion (31). Thus, NAC domain proteins have a clear connection with TCP domain proteins, both as downstream targets and as interaction partners.

GhCYC2 Plays a Major Role in Differentiating Gerbera Flower Types.

The experimental evidence shown here suggests that GhCYC2 is involved in differentiation between Gerbera flower types. GhCYC2 is specifically expressed in the marginal ray flower primordia. Based on transgenic phenotypes, the absence of GhCYC2 expression in the central disk flower primordia appears to be crucial for proper flower type specification. Overexpression of GhCYC2 causes disk flowers to obtain characteristics typical for ray flowers, including enlarged ventral petals and disrupted stamen development. These Gerbera lines resemble the semidominant chry mutant of sunflower (7), in which disk flowers also obtain ray-flower like traits. In chry mutants, the form of disk flower petals has shifted toward bilateral symmetry, and stamens are smaller with disrupted pollen production. The similarities of the two phenotypes in these closely related species makes it tempting to speculate that the semidominant chry mutant could result from overexpression of a GhCYC2 ortholog in sunflower.
In Gerbera, some cultivars resemble the transgenic GhCYC2 lines and the sunflower chry mutants. The “crested” trait is characterized by enlarged trans flowers or enlarged trans and disk flowers that are male-sterile and defined by a single locus containing three alleles with semidominant relationships (crested < CRESTED < CRESTEDD) (32). In our overexpression lines, the increase in petal length and reduction in stamen functionality in disk flowers implies similarity with the crested trait. Moreover, the recessive homozygotes of crested are characterized by short trans flowers resembling the transgenic line with suppressed GhCYC2 expression. However, because Gerbera cultivars showing the crested phenotype do not display GhCYC2 expression differences compared with semicrested cultivars (such as Terra Regina; used here) or cultivars having short trans flowers (S.K.B., unpublished results), we postulate that GhCYC2 is not sufficient for complete transformation of flower types.
The transgenic Gerbera line with suppressed GhCYC2 expression did not show alterations in ray flower identity. This again implies that GhCYC2 is not sufficient for regulating differentiation between the rayed and nonrayed form of Asteraceae inflorescences but that it takes part in this process, most likely as a modifier gene. Genetic analyses performed so far have suggested that in addition to the major loci (RAY/turf/CHRY/CRESTED), an unknown number of modifier genes are also involved. The Gerbera GhCYC3 and GhCYC4 genes show ray-flower specific expression similar to GhCYC2 (S.T. and S.K.B., unpublished results). Their expression was not reduced in the transgenic lines with suppressed GhCYC2 expression. Therefore, the absence of altered ray flower phenotype in GhCYC2 suppressed lines might be due to redundant functions of these closely related genes. Functional analyses of GhCYC3 and GhCYC4 are currently underway.
Our data provides the first molecular evidence that CYC-like TCP factors are involved in defining identities of different flower types within a single genotype. The expression of GhCYC2 shows clear radial specificity along the axis of the Gerbera capitulum. In previous work we showed that several MADS box genes also display radial expression differences among developing flower primordia (3). As such, it seems likely that both TCP and MADS domain proteins participate in regulating the developmental specification of Gerbera flower types through response to early acting signals at the inflorescence level. A gradient of a cell nonautonomously moving substance, such as a phytohormone or a transcription factor, could induce differentiation of the distinct flower morphologies by affecting putative downstream targets such as TCP and MADS domain proteins.


Asteraceae (1), Fabales, and Orchidaceae (33) are the three largest lineages of flowering plants, and each are defined by suites of traits that have lent themselves to extensive evolutionary modification. For Asteraceae, the capitulate inflorescence with its capacity to produce different flower types has been its principal innovation. Through analysis of a CYC-like TCP-encoding gene in Gerbera, we have shown that gene duplication and divergence, a frequent theme in the evolution of organismal novelty (3436), has been one major factor in establishing the uniqueness and success of the sunflower family.

Materials and Methods

Plant Material.

Gerbera hybrida (Asteraceae) variety Terra Regina (Terra Nigra) was grown under standard greenhouse conditions. The developmental stages for inflorescences are described in ref. 37 and for the young developing floral primordia in ref. 3.

Isolation of Gerbera TCP Domain Factors.

Gerbera TCP domain factors were amplified from cDNA prepared from young developing inflorescences of Gerbera by PCR using two different pairs of degenerate primers. The first degenerate primer pair was designed from the conserved TCP domain and corresponded to the amino acid sequences KKDRHSKI and ERTKEK. Poly(A)-RNA (450 ng) was used as a template for cDNA synthesis (Boehringer first-strand cDNA kit). PCR conditions were the following: 94°C for 75 sec, 46°C for 2 min, and 72°C for 3 min for 30 cycles. Two different fragments that showed high similarity with CYC-like genes were obtained by using this primer pair. For the second round of PCR screening, degenerate primers were designed by using the CODEHOP strategy (38) and were optimized for codon usage. Primers corresponded to the amino acid sequences DLQDMLGFDK and ARARARERTKEK. cDNA synthesis was performed from poly(A)-RNA by using SuperScript III reverse transcriptase (Invitrogen). Phusion DNA polymerase was used for PCR reactions (Finnzymes). The full-length cDNAs were amplified by using the SMART RACE cDNA amplification kit (Clontech). The TCP mRNA coding sequences have been deposited in the GenBank database (accession numbers EU429302–EU429305).

Phylogenetic Analyses.

Phylogenetic analyses were performed on corresponding nucleotide and amino acid alignments for the TCP and R domains of selected TCP factors. Two phylogeny reconstruction methods were used: parsimony and maximum likelihood (see SI Text for full details).

RNA Blot, in Situ Hybridizations, and Scanning Electron Microscopy (SEM).

Total RNA was isolated by using TRIzol reagent (Invitrogen). Ten micrograms of total RNA was loaded on RNA blots and hybridized by using 32P-labeled DNA probes. For a gene-specific probe, a 253-bp fragment from the 3′ end of the cDNA was used. The blots were washed with 0.5× SSC and 0.1% (wt/vol) SDS at 58°C. In situ hybridization analysis using full-length GhCYC2 probe was performed as in ref. 39, with the following exceptions: probe concentration was 0.7 μg/ml/kb and detection time was 40–45 h. SEM analysis of the Gerbera flower primordia was performed as described in ref. 5.

Plant Transformation and Analysis of Transgenic Lines.

The full-length GhCYC2 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 between wild-type variety Terra Regina and transgenic lines were tested by using the Mann–Whitney rank-sum test. Flowers were counted, and the length of petals was measured from five inflorescences of each line. On average, for ray flowers n = 63 per line, for trans flowers n = 213 per line, and for disk flowers n = 310 per line.

Data Availability

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. EU429302 (GhCYC1), EU429303 (GhCYC2), EU429304 (GhCYC3), and EU429305 (GhCYC4)].


We thank E. Coen (John Innes Centre, Norwich, UK) for providing the Senecio squalidus cDNA (plasmid pJAM2176); the Electron Microscopy Unit of the Institute of Biotechnology, University of Helsinki, for providing laboratory facilities; Eija Takala and Anu Rokkanen for excellent technical assistance; and Sanna Peltola for taking care of the plants in the greenhouse. This work was supported by Academy of Finland Grant 115849 (to P.E.). S.K.B. is supported by the Viikki Graduate School in Biosciences.

Supporting Information

Supporting Information (PDF)
Supporting Information (PDF)


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Published in

Go to Proceedings of the National Academy of Sciences
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Proceedings of the National Academy of Sciences
Vol. 105 | No. 26
July 1, 2008
PubMed: 18574149


Data Availability

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. EU429302 (GhCYC1), EU429303 (GhCYC2), EU429304 (GhCYC3), and EU429305 (GhCYC4)].

Submission history

Received: February 11, 2008
Published online: July 1, 2008
Published in issue: July 1, 2008


  2. flower development
  3. evo-devo
  4. organ fusion


We thank E. Coen (John Innes Centre, Norwich, UK) for providing the Senecio squalidus cDNA (plasmid pJAM2176); the Electron Microscopy Unit of the Institute of Biotechnology, University of Helsinki, for providing laboratory facilities; Eija Takala and Anu Rokkanen for excellent technical assistance; and Sanna Peltola for taking care of the plants in the greenhouse. This work was supported by Academy of Finland Grant 115849 (to P.E.). S.K.B. is supported by the Viikki Graduate School in Biosciences.


This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0801359105/DCSupplemental.



Suvi K. Broholm
Department of Applied Biology, P.O. Box 27, University of Helsinki, Helsinki FIN-00014, Finland;
Sari Tähtiharju
Department of Applied Biology, P.O. Box 27, University of Helsinki, Helsinki FIN-00014, Finland;
Roosa A. E. Laitinen
Max Planck Institute for Developmental Biology, Spemannstrasse 37-39, D-72076 Tübingen, Germany; and
Victor A. Albert
Department of Biological Sciences, State University of New York, Buffalo, NY 14260
Teemu H. Teeri
Department of Applied Biology, P.O. Box 27, University of Helsinki, Helsinki FIN-00014, Finland;
Department of Applied Biology, P.O. Box 27, University of Helsinki, Helsinki FIN-00014, Finland;


To whom correspondence should be addressed. E-mail: [email protected]
Author contributions: S.K.B., R.A.E.L., T.H.T., and P.E. designed research; S.K.B., S.T., R.A.E.L., V.A.A., and P.E. performed research; S.K.B., V.A.A., and P.E. analyzed data; and S.K.B., S.T., V.A.A., T.H.T., and P.E. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    A TCP domain transcription factor controls flower type specification along the radial axis of the Gerbera (Asteraceae) inflorescence
    Proceedings of the National Academy of Sciences
    • Vol. 105
    • No. 26
    • pp. 8803-9130







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