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.

Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

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.

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.

Functional characterization of B class MADS-box transcription factors in Gerbera hybrida.

According to the classical ABC model, B-function genes are involved in determining petal and stamen development. Most core eudicot species have B class genes belonging to three different lineages: the PI, euAP3, and TM6 lineages, although both Arabidopsis and Antirrhinum appear to have lost their TM6-like gene. Functional studies were performed for three gerbera (Gerbera hybrida) B class MADS-box genes--PI/GLO-like GGLO1, euAP3 class GDEF2, and TM6-like GDEF1--and data are shown for a second euAP3-like gene, GDEF3. In phylogenetic analysis, GDEF3 is a closely related paralogue of GDEF2, and apparently stems from a duplication common to all Asteraceae. Expression analysis and transgenic phenotypes confirm that GGLO1 and GDEF2 mediate the classical B-function since they determine petal and stamen identities. However, based on assays in yeast, three B class heterodimer combinations are possible in gerbera. In addition to the interaction of GGLO1 and GDEF2 proteins, GGLO1 also pairs with GDEF1 and GDEF3. This analysis of GDEF1 represents the first functional characterization of a TM6-like gene in a core eudicot species outside Solanaceae. Similarly to its relatives in petunia and tomato, the expression pattern and transgenic phenotypes indicate that GDEF1 is not involved in determination of petal identity, but has a redundant role in regulating stamen development.

Cytokinin signaling regulates cambial development in poplar.

Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula x tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXIDASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development.

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

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.