Diversity of woodland strawberry inflorescences arises from heterochrony regulated by TERMINAL FLOWER 1 and FLOWERING LOCUS T.

A vast variety of inflorescence architectures have evolved in angiosperms. Here, we analyze the diversity and development of the woodland strawberry (Fragaria vesca) inflorescence. Contrary to historical classifications, we show that it is a closed thyrse: a compound inflorescence with determinate primary monopodial axis and lateral sympodial branches, thus combining features of racemes and cymes. We demonstrate that this architecture is generated by 2 types of inflorescence meristems differing in their geometry. We further show that woodland strawberry homologs of TERMINAL FLOWER 1 (FvTFL1) and FLOWERING LOCUS T (FvFT1) regulate the development of both the racemose and cymose components of the thyrse. Loss of functional FvTFL1 reduces the number of lateral branches of the main axis and iterations in the lateral branches but does not affect their cymose pattern. These changes can be enhanced or compensated by altering FvFT1 expression. We complement our experimental findings with a computational model that captures inflorescence development using a small set of rules. The model highlights the distinct regulation of the fate of the primary and higher-order meristems, and explains the phenotypic diversity among inflorescences in terms of heterochrony resulting from the opposite action of FvTFL1 and FvFT1 within the thyrse framework. Our results represent a detailed analysis of thyrse architecture development at the meristematic and molecular levels.

Development and evolution of the Asteraceae capitulum.

Asteraceae represent one of the largest and most diverse families of plants. The evolutionary success of this family has largely been contributed to their unique inflorescences, capitula that mimic solitary flowers but are typically aggregates of multiple florets. Here, we summarize the recent molecular and genetic level studies that have promoted our understanding of the development and evolution of capitula. We focus on new results on patterning of the enlarged meristem resulting in the iconic phyllotactic arrangement of florets in Fibonacci numbers of spirals. We also summarize the current understanding of the genetic networks regulating the characteristic reproductive traits in the family such as floral dimorphism and differentiation of highly specialized floral organs. So far, developmental studies in Asteraceae are still limited to a very narrow selection of model species. Along with the recent advancements in genomics and phylogenomics, Asteraceae and its relatives provide an outstanding model clade for extended evo-devo studies to exploit the morphological diversity and the underlying molecular networks and to translate this knowledge to the breeding of the key crops in the family.

The hidden diversity of vascular patterns in flower heads.

Vascular systems are intimately related to the shape and spatial arrangement of the plant organs they support. We investigate the largely unexplored association between spiral phyllotaxis and the vascular system in Asteraceae flower heads. We imaged heads of eight species using synchrotron-based X-ray micro-computed tomography and applied original virtual reality and haptic software to explore head vasculature in three dimensions. We then constructed a computational model to infer a plausible patterning mechanism. The vascular system in the head of the model plant Gerbera hybrida is qualitatively different from those of Bellis perennis and Helianthus annuus, characterized previously. Cirsium vulgare, Craspedia globosa, Echinacea purpurea, Echinops bannaticus, and Tanacetum vulgare represent variants of the Bellis and Helianthus systems. In each species, the layout of the main strands is stereotypical, but details vary. The observed vascular patterns can be generated by a common computational model with different parameter values. In spite of the observed differences of vascular systems in heads, they may be produced by a conserved mechanism. The diversity and irregularities of vasculature stand in contrast with the relative uniformity and regularity of phyllotactic patterns, confirming that phyllotaxis in heads is not driven by the vasculature.

Phyllotactic patterning of gerbera flower heads.

Phyllotaxis, the distribution of organs such as leaves and flowers on their support, is a key attribute of plant architecture. The geometric regularity of phyllotaxis has attracted multidisciplinary interest for centuries, resulting in an understanding of the patterns in the model plants Arabidopsis and tomato down to the molecular level. Nevertheless, the iconic example of phyllotaxis, the arrangement of individual florets into spirals in the heads of the daisy family of plants (Asteraceae), has not been fully explained. We integrate experimental data and computational models to explain phyllotaxis in Gerbera hybrida We show that phyllotactic patterning in gerbera is governed by changes in the size of the morphogenetically active zone coordinated with the growth of the head. The dynamics of these changes divides the patterning process into three phases: the development of an approximately circular pattern with a Fibonacci number of primordia near the head rim, its gradual transition to a zigzag pattern, and the development of a spiral pattern that fills the head on the template of this zigzag pattern. Fibonacci spiral numbers arise due to the intercalary insertion and lateral displacement of incipient primordia in the first phase. Our results demonstrate the essential role of the growth and active zone dynamics in the patterning of flower heads.

Polyketide reductases in defense-related parasorboside biosynthesis in Gerbera hybrida share processing strategies with microbial polyketide synthase systems.

Plant polyketides are well-known for their crucial functions in plants and their importance in the context of human health. They are synthesized by type III polyketide synthases (PKSs) and their final functional diversity is determined by post-PKS tailoring enzymes. Gerbera hybrida is rich in two defense-related polyketides: gerberin and parasorboside. Their synthesis is known to be initiated by GERBERA 2-PYRONE SYNTHASE 1 (G2PS1), but the polyketide reductases (PKRs) that determine their final structure have not yet been identified. We identified two PKR candidates in the pathway, GERBERA REDUCTASE 1 (GRED1) and GRED2. Gene expression and metabolite analysis of different gerbera tissues, cultivars, and transgenic gerbera plants, and in vitro enzyme assays, were performed for functional characterization of the enzymes. GRED1 and GRED2 catalyze the second reduction step in parasorboside biosynthesis. They reduce the proximal keto domain of the linear CoA bound intermediate before lactonization. We identified a crucial tailoring step in an important gerbera PKS pathway and show that plant polyketide biosynthesis shares processing strategies with fungi and bacteria. The two tailoring enzymes are recruited from the ancient sporopollenin biosynthetic pathway to a defense-related PKS pathway in gerbera. Our data provide an example of how plants recruit conserved genes to new functions in secondary metabolism that are important for environmental adaptation.

Phyllotaxis without symmetry: what can we learn from flower heads?

Phyllotaxis is commonly considered in the context of circular meristems or receptacles, yet non-circular (fasciated) structures also give rise to new primordia and organs. Here we investigate phyllotactic patterns in fasciated flower heads in the Asteraceae plant family. We begin by surveying the phenomenon of fasciation. We then show that phyllotactic patterns in fasciated heads can be generated by removing the inessential assumption of circularity from the previously published model of gerbera heads. To characterize these patterns, we revisit the conceptual framework in which phyllotactic patterns are commonly described. We note that some notions, in particular parastichies and parastichy numbers, maintain their significance in non-circular phyllotaxis, whereas others, in particular the divergence angle, need to be extended or lose their role. These observations highlight a number of open problems related to phyllotaxis in general, which may be elucidated by studies of fasciated heads.

TCP and MADS-Box Transcription Factor Networks Regulate Heteromorphic Flower Type Identity in Gerbera hybrida.

The large sunflower family, Asteraceae, is characterized by compressed, flower-like inflorescences that may bear phenotypically distinct flower types. The CYCLOIDEA (CYC)/TEOSINTE BRANCHED1-like transcription factors (TFs) belonging to the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) protein family are known to regulate bilateral symmetry in single flowers. In Asteraceae, they function at the inflorescence level, and were recruited to define differential flower type identities. Here, we identified upstream regulators of GhCYC3, a gene that specifies ray flower identity at the flower head margin in the model plant Gerbera hybrida We discovered a previously unidentified expression domain and functional role for the paralogous CINCINNATA-like TCP proteins. They function upstream of GhCYC3 and affect the developmental delay of marginal ray primordia during their early ontogeny. At the level of single flowers, the Asteraceae CYC genes show a unique function in regulating the elongation of showy ventral ligules that play a major role in pollinator attraction. We discovered that during ligule development, the E class MADS-box TF GRCD5 activates GhCYC3 expression. We propose that the C class MADS-box TF GAGA1 contributes to stamen development upstream of GhCYC3 Our data demonstrate how interactions among and between the conserved floral regulators, TCP and MADS-box TFs, contribute to the evolution of the elaborate inflorescence architecture of Asteraceae.

Repatterning of the inflorescence meristem in Gerbera hybrida after wounding.

The Asteraceae plant family is characterized by inflorescences, called flower heads or capitula that may combine hundreds of individual florets into a single flower-like structure. The florets are arranged in a regular phyllotactic pattern with Fibonacci numbers of left- and right-winding spirals. Such a pattern may be disrupted due to physical constraints or by wounding occurring during the early meristem development. Recovery from wounding re-establishes patterning although the mechanisms have remained elusive. In this study, we applied Gerbera hybrida as a model system and established methods to conduct wounding experiments either with syringe needles or using laser ablation combined with live imaging of head meristems. By revisiting the historical experiments in sunflower, we conducted wounding to transgenic auxin reporter lines of gerbera and followed the recovery of cellular growth and meristem patterning. We show that wounding disrupted the expression of the gerbera CLAVATA3 (GhCLV3) gene that marks the undifferentiated meristematic region and led to de novo re-initiation of patterning at the wound margin. During the recovery growth, three to five layers of elongated cells showing periclinal cell division planes and lacking auxin signal were formed at the wound rim. DR5 auxin signal was shown to localize and form regularly spaced maxima in a distance from the wound rim. Consequently, spiral pattern of contact parastichies was re-established by stacking of new auxin maxima on top of the previous ones. The developed methods facilitate future studies on understanding the molecular mechanisms of de novo patterning of meristems.

Evolutionary diversification of CYC/TB1-like TCP homologs and their recruitment for the control of branching and floral morphology in Papaveraceae (basal eudicots).

Angiosperms possess enormous morphological variation in plant architectures and floral forms. Previous studies in Pentapetalae and monocots have demonstrated the involvement of TCP domain CYCLOIDEA/TEOSINTE BRANCHED1-like (CYC/TB1) genes in the control of floral symmetry and shoot branching. However, how TCP/CYC-like (CYL) genes originated, evolved and functionally diversified remain unclear. We conducted a comparative functional study in Ranunculales, the sister lineage to all other eudicots, between Eschscholzia californica and Cysticapnos vesicaria, two species of Papaveraceae with actinomorphic and zygomorphic flowers, respectively. Phylogenetic analysis indicates that CYL genes in Papaveraceae form two paralogous lineages, PapaCYL1 and PapaCYL2. Papaveraceae CYL genes show highly diversified expression patterns as well as functions. Enhanced branching by silencing of EscaCYL1 suggests that the role of CYC/TB1-like genes in branching control is conserved in Papaveraceae. In contrast to the arrest of stamen development in Pentapetalae, PapaCYL genes promote stamen initiation and growth. In addition, we demonstrate that CyveCYLs are involved in perianth development, specifying sepal and petal identity in Cysticapnos by regulating the B-class floral organ identity genes. Our data also suggest the involvement of CyveCYL genes in the regulation of flower symmetry in Cysticapnos. Our work provides evidence of the importance of TCP/CYC-like genes in the promotion of morphological diversity across angiosperms.

Altered regulation of TERMINAL FLOWER 1 causes the unique vernalisation response in an arctic woodland strawberry accession.

Vernalisation requirement is an agriculturally important trait that postpones the development of cold-sensitive floral organs until the spring. The family Rosaceae includes many agriculturally important fruit and berry crops that suffer from crop losses caused by frost injury to overwintering flower buds. Recently, a vernalisation-requiring accession of the Rosaceae model woodland strawberry (Fragaria vesca) has been identified in northern Norway. Understanding the molecular basis of the vernalisation requirement in this accession would advance the development of strawberry cultivars better adapted to temperate climate. We use gene silencing, gene expression analysis, genetic mapping and population genomics to study the genetic basis of the vernalisation requirement in woodland strawberry. Our results indicate that the woodland strawberry vernalisation requirement is endemic to northern Norwegian population, and mapping data suggest the orthologue of TERMINAL FLOWER1 (FvTFL1) as the causal floral repressor. We demonstrate that exceptionally low temperatures are needed to downregulate FvTFL1 and to make these plants competent to induce flowering at low postvernalisation temperatures in the spring. We show that altered regulation of FvTFL1 in the northern Norwegian woodland strawberry accession postpones flower induction until the spring, allowing plants to avoid winter injuries of flower buds that commonly occur in temperate regions.

Dissecting functions of SEPALLATA-like MADS box genes in patterning of the pseudanthial inflorescence of Gerbera hybrida.

The pseudanthial inflorescences of the sunflower family, Asteraceae, mimic a solitary flower but are composed of multiple flowers. Our studies in Gerbera hybrida indicate functional diversification for SEPALLATA (SEP)-like MADS box genes that often function redundantly in other core eudicots. We conducted phylogenetic and expression analysis for eight SEP-like GERBERA REGULATOR OF CAPITULUM DEVELOPMENT (GRCD) genes, including previously unstudied gene family members. Transgenic gerbera plants were used to infer gene functions. Adding to the previously identified stamen and carpel functions for GRCD1 and GRCD2, two partially redundant genes, GRCD4 and GRCD5, were found to be indispensable for petal development. Stepwise conversion of floral organs into leaves in the most severe RNA interference lines suggest redundant and additive GRCD activities in organ identity regulation. We show conserved and redundant functions for several GRCD genes in regulation of flower meristem maintenance, while functional diversification for three SEP1/2/4 clade genes in regulation of inflorescence meristem patterning was observed. GRCD genes show both specialized and pleiotropic functions contributing to organ differentiation and flower meristem fate, and uniquely, to patterning of the inflorescence meristem. Altogether, we provide an example of how plant reproductive evolution has used conserved genetic modules for regulating the elaborate inflorescence architecture in Asteraceae.

Evolutionary Co-Option of Floral Meristem Identity Genes for Patterning of the Flower-Like Asteraceae Inflorescence.

The evolutionary success of Asteraceae, the largest family of flowering plants, has been attributed to the unique inflorescence architecture of the family, which superficially resembles an individual flower. Here, we show that Asteraceae inflorescences (flower heads, or capitula) resemble solitary flowers not only morphologically but also at the molecular level. By conducting functional analyses for orthologs of the flower meristem identity genes LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) in Gerbera hybrida, we show that GhUFO is the master regulator of flower meristem identity, while GhLFY has evolved a novel, homeotic function during the evolution of head-like inflorescences. Resembling LFY expression in a single flower meristem, uniform expression of GhLFY in the inflorescence meristem defines the capitulum as a determinate structure that can assume floral fate upon ectopic GhUFO expression. We also show that GhLFY uniquely regulates the ontogeny of outer, expanded ray flowers but not inner, compact disc flowers, indicating that the distinction of different flower types in Asteraceae is connected with their independent evolutionary origins from separate branching systems.

TERMINAL FLOWER1 is a breeding target for a novel everbearing trait and tailored flowering responses in cultivated strawberry (Fragaria × ananassa Duch.).

The effects of daylength and temperature on flowering of the cultivated octoploid strawberry (Fragaria × ananassa Duch.) have been studied extensively at the physiological level, but information on the molecular pathways controlling flowering in the species is scarce. The flowering pathway has been studied at the molecular level in the diploid short-day woodland strawberry (F. vesca L.), in which the FLOWERING LOCUS T1 (FvFT1)-SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (FvSOC1)-TERMINAL FLOWER1 (FvTFL1) pathway is essential for the correct timing of flowering. In this work, we show by transgenic approach that the silencing of the floral repressor FaTFL1 in the octoploid short-day cultivar 'Elsanta' is sufficient to induce perpetual flowering under long days without direct changes in vegetative reproduction. We also demonstrate that although the genes FaFT1 and FaSOC1 show similar expression patterns in different cultivars, the regulation of FaTFL1 varies widely from cultivar to cultivar and is correlated with floral induction, indicating that the transcription of FaTFL1 occurs at least partially independently of the FaFT1-FaSOC1 module. Our results indicate that changing the expression patterns of FaTFL1 through biotechnological or conventional breeding approaches could result in strawberries with specific flowering and runnering characteristics including new types of everbearing cultivars.

The Fragaria vesca homolog of suppressor of overexpression of constans1 represses flowering and promotes vegetative growth.

In the annual long-day plant Arabidopsis thaliana, suppressor of overexpression of constans1 (SOC1) integrates endogenous and environmental signals to promote flowering. We analyzed the function and regulation of the SOC1 homolog (Fragaria vesca [Fv] SOC1) in the perennial short-day plant woodland strawberry (Fragaria vesca). We found that Fv SOC1 overexpression represses flower initiation under inductive short days, whereas its silencing causes continuous flowering in both short days and noninductive long days, similar to mutants in the floral repressor Fv terminal flower1 (Fv TFL1). Molecular analysis of these transgenic lines revealed that Fv SOC1 activates Fv TFL1 in the shoot apex, leading to the repression of flowering in strawberry. In parallel, Fv SOC1 regulates the differentiation of axillary buds to runners or axillary leaf rosettes, probably through the activation of gibberellin biosynthetic genes. We also demonstrated that Fv SOC1 is regulated by photoperiod and Fv flowering locus T1, suggesting that it plays a central role in the photoperiodic control of both generative and vegetative growth in strawberry. In conclusion, we propose that Fv SOC1 is a signaling hub that regulates yearly cycles of vegetative and generative development through separate genetic pathways.

Functional diversification of duplicated CYC2 clade genes in regulation of inflorescence development in Gerbera hybrida (Asteraceae).

The complex inflorescences (capitula) of Asteraceae consist of different types of flowers. In Gerbera hybrida (gerbera), the peripheral ray flowers are bilaterally symmetrical and lack functional stamens while the central disc flowers are more radially symmetrical and hermaphroditic. Proteins of the CYC2 subclade of the CYC/TB1-like TCP domain transcription factors have been recruited several times independently for parallel evolution of bilaterally symmetrical flowers in various angiosperm plant lineages, and have also been shown to regulate flower-type identity in Asteraceae. The CYC2 subclade genes in gerbera show largely overlapping gene expression patterns. At the level of single flowers, their expression domain in petals shows a spatial shift from the dorsal pattern known so far in species with bilaterally symmetrical flowers, suggesting that this change in expression may have evolved after the origin of Asteraceae. Functional analysis indicates that GhCYC2, GhCYC3 and GhCYC4 mediate positional information at the proximal-distal axis of the inflorescence, leading to differentiation of ray flowers, but that they also regulate ray flower petal growth by affecting cell proliferation until the final size and shape of the petals is reached. Moreover, our data show functional diversification for the GhCYC5 gene. Ectopic activation of GhCYC5 increases flower density in the inflorescence, suggesting that GhCYC5 may promote the flower initiation rate during expansion of the capitulum. Our data thus indicate that modification of the ancestral network of TCP factors has, through gene duplications, led to the establishment of new expression domains and to functional diversification.

Anthocyanin biosynthesis in gerbera cultivar 'Estelle' and its acyanic sport 'Ivory'.

MAIN CONCLUSION: Identification of distinct allelic versions for dihydroflavonol 4-reductase in gerbera cultivars reveals that gerbera DFR enzymes have strong substrate preference in vivo that is not reflected to the activity in vitro. Flavonoids in the model ornamental plant Gerbera hybrida consist of flavones, flavonols and anthocyanins. Anthocyanins accumulate in the adaxial epidermis of petals and give the different cultivars their characteristic red and violet colour. Both pelargonidin and cyanidin derivatives are found in gerbera, but none of the cultivars contain delphinidin. 'Ivory', a cultivar with white petals, is a sport of the pelargonidin-containing pink cultivar 'Estelle', i.e. it originates from an acyanic branch of 'Estelle'. In this work, four different alleles encoding dihydroflavonol 4-reductase (DFR) were identified in gerbera cultivars. We found that, in contrast to 'Estelle' with the functional allele GDFR1-2, 'Ivory' carries a mutation in this gene that results in an inactive enzyme. Interestingly, 'Ivory' also expresses a second, nonmutated allele (GDFR1-3) in petal epidermi, leading to extractable DFR activity but not to anthocyanin biosynthesis. The second allele encodes a protein identical in amino acid sequence to the DFR of the cyanidin-containing variety 'President'. Pelargonidin-containing cultivars do not react to the flavonoid 3'-hydroxylase inhibitor tetcyclacis, but cyanidin-containing cultivars lose their colour, instead of starting to synthesise pelargonidins, indicating the specificity of GDFR1-3 for the cyanidin pathway. This explains why petals of 'Ivory' are white, even when it has lost only one of the two enzymatically functional DFR forms, and shows that anthocyanin biosynthesis in gerbera is under more complex regulation than earlier thought.

Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida.

• Chalcone synthase (CHS) is the key enzyme in the first committed step of the flavonoid biosynthetic pathway and catalyzes the stepwise condensation of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone. In plants, CHS is often encoded by a small family of genes that are temporally and spatially regulated. Our earlier studies have shown that GCHS4 is highly activated by ectopic expression of an MYB-type regulator GMYB10 in gerbera (Gerbera hybrida). • The tissue- and development-specific expression patterns of three gerbera CHS genes were examined. Virus-induced gene silencing (VIGS) was used to knock down GCHS1 and GCHS4 separately in gerbera inflorescences. • Our data show that GCHS4 is the only CHS encoding gene that is expressed in the cyanidin-pigmented vegetative tissues of gerbera cv Terraregina. GCHS3 expression is pronounced in the pappus bristles of the flowers. Expression of both GCHS1 and GCHS4 is high in the epidermal cells of gerbera petals, but only GCHS1 is contributing to flavonoid biosynthesis. • Gerbera contains a family of three CHS encoding genes showing different spatial and temporal regulation. GCHS4 expression in gerbera petals is regulated post-transcriptionally, at the level of either translation elongation or protein stability.

Modification of Tobacco rattle virus RNA1 to serve as a VIGS vector reveals that the 29K movement protein is an RNA silencing suppressor of the virus.

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.

Evolution and diversification of the CYC/TB1 gene family in Asteraceae--a comparative study in Gerbera (Mutisieae) and sunflower (Heliantheae).

Plant-specific TCP domain transcription factors have been shown to regulate morphological novelties during plant evolution, including the complex architecture of the Asteraceae inflorescence that involves different types of flowers. We conducted comparative analysis of the CYCLOIDEA/TEOSINTE BRANCHED1 (CYC/TB1) gene family in Gerbera hybrida (gerbera) and Helianthus annuus (sunflower), two species that represent distant tribes within Asteraceae. Our data confirm that the CYC/TB1 gene family has expanded in Asteraceae, a condition that appears to be connected with the increased developmental complexity and evolutionary success of this large plant family. Phylogenetic analysis of the CYC/TB1 gene family revealed both shared and lineage-specific duplications in gerbera and sunflower, corresponding to the three gene lineages previously identified as specific to core eudicots: CYC1, CYC2, and CYC3. Expression analyses of early stages of flower primordia development indicated that especially within the CYC2 clade, with the greatest number of secondary gene duplications, gene expression patterns are conserved between the species and associated with flower and inflorescence development. All sunflower and gerbera CYC2 clade genes showed differential expression between developing flower types, being upregulated in marginal ray (and trans) flowers. One gene in gerbera (GhCYC3) and two in sunflower (HaCYC2d and HaCYC2c) were indicated to be strong candidates as regulators of ray flower identity, a function that is specific for Asteraceae. Our data further showed that other CYC2 clade genes are likely to have more specialized functions at the level of single flowers, including the late functions in floral reproductive organs that may be more conserved across plant families. The expression patterns of CYC1 and CYC3 clade genes showed more differences between the two species but still pointed to possible conserved functions during vegetative plant development. Pairwise protein-protein interaction assays gave the first molecular evidence that CYC/TB1-like proteins function in complexes. Compared with sunflower, the gerbera proteins showed higher capacity for dimerization, between as well as within CYC clades. Our data from two distant species within the Asteraceae suggest that the expansion and the apparent conservation of especially the CYC2 clade CYC/TB1-like genes are associated with the evolution of the increased complexity of the Asteraceae inflorescence architecture.