Development and anatomy of petals with specialized nectar holder and pollen container in Fumarioideae (Papaveraceae).

MAIN CONCLUSION: Petal developmental characteristics in Fumarioideae were similar at early stages, and the specialized nectar holder/pollen container formed by the outer/inner petals. The micro-morphology of these two structures, however, shows diversity in seven species. Elaborate petals have been modified to form different types, including petal lobes, ridges, protuberances, and spurs, each with specialized functions. Nectar holder and pollen container presumably have a function in plant-pollinator interactions. In Fumarioideae, four elaborate petals of the disymmetric/zygomorphic flower present architecture forming the "nectar holder" and "pollen container" structure at the bottom and top separately. In the present study, the petals of seven species in Fumarioideae were investigated by scanning electron microscopy, light microscope, and transmission electron microscopes. The results show that petal development could divided into six stages: initiation, enlargement, adaxial/abaxial differentiation, elaborate specializations (sacs, spurs, and lobes formed), extension, and maturation, while the specialized "nectar holder" and "pollen container" structures mainly formed in stage 4. "Nectar holder" is developed from the shallow sac/spur differentiated at the base of the outer petal, eventually forming a multi-organized complex structure, together with staminal nectaries (1-2) with individual sizes. A semi-closed ellipsoidal "pollen container" is developed from the apical part of the 3-lobed inner petals fused by middle lobes and attain different sizes. The adaxial epidermis cells are specialized, with more distinct punctate/dense columnar protrusions or wavy cuticles presented on obviously thickening cell walls. In addition, a large and well-developed cavity appears between the inner and outer epidermis of the petals. As an exception, Hypecoum erectum middle lobes present stamen mimicry. Elaborate petal structure is crucial for comprehending the petal diversity in Fumarioideae and provides more evidence for further exploration of the reproductive study in Papaveraceae.

Chrysanthemum lavandulifolium homolog ClCYCA2;1 modulates cell division in ray florets.

Morphology of ray florets in chrysanthemums is tightly associated with cell division and cell expansion, both of which require proper cell cycle progression. Here we identified a Chrysanthemum lavandulifolium homolog ClCYCA2;1, whose expression in ray florets is negatively correlated with petal width in C. lavandulifolium. Two TCP transcription factors in CYCLOIDEA2 (CYC2) family, ClCYC2a interacts with and stabilizes ClCYC2b and the latter can bind to the promoter of ClCYCA2;1 to activate its transcription. Overexpression of ClCYCA2;1 in C. lavandulifolium reduces the size of capitula and ray florets. Cytological analysis reveals that ClCYCA2;1 overexpression inhibits both cell division and cell expansion via repressing mitotic cell cycle in ray florets whose latitudinal development was more negatively influenced leading to increased ratios of petal length to width at later developmental stages. Yeast two hybrid library screening reveals multiple ClCYCA2;1 interacting proteins including ARP7, and silencing ClARP7 inhibits the development of ray florets. Co-immunoprecipitation assays confirm that ClCYCA2;1 can induce the degradation of ClARP7 to inhibit the development of ray florets. Taken together, our study constitutes a regulatory network containing ClCYC2b-ClCYCA2;1-ClARP7 in ray floret development via governing mitosis, which may facilitate breeding efforts targeted for novel ornamental traits of chrysanthemums.

POPOVICH, encoding a C2H2 zinc-finger transcription factor, plays a central role in the development of a key innovation, floral nectar spurs, in Aquilegia.

The evolution of novel features, such as eyes or wings, that allow organisms to exploit their environment in new ways can lead to increased diversification rates. Therefore, understanding the genetic and developmental mechanisms involved in the origin of these key innovations has long been of interest to evolutionary biologists. In flowering plants, floral nectar spurs are a prime example of a key innovation, with the independent evolution of spurs associated with increased diversification rates in multiple angiosperm lineages due to their ability to promote reproductive isolation via pollinator specialization. As none of the traditional plant model taxa have nectar spurs, little is known about the genetic and developmental basis of this trait. Nectar spurs are a defining feature of the columbine genus Aquilegia (Ranunculaceae), a lineage that has experienced a relatively recent and rapid radiation. We use a combination of genetic mapping, gene expression analyses, and functional assays to identify a gene crucial for nectar spur development, POPOVICH (POP), which encodes a C2H2 zinc-finger transcription factor. POP plays a central role in regulating cell proliferation in the Aquilegia petal during the early phase (phase I) of spur development and also appears to be necessary for the subsequent development of nectaries. The identification of POP opens up numerous avenues for continued scientific exploration, including further elucidating of the genetic pathway of which it is a part, determining its role in the initial evolution of the Aquilegia nectar spur, and examining its potential role in the subsequent evolution of diverse spur morphologies across the genus.

Brassinosteroids regulate petal spur length in Aquilegia by controlling cell elongation.

BACKGROUND AND AIMS: Aquilegia produce elongated, three-dimensional petal spurs that fill with nectar to attract pollinators. Previous studies have shown that the diversity of spur length across the Aquilegia genus is a key innovation that is tightly linked with its recent and rapid diversification into new ranges, and that evolution of increased spur lengths is achieved via anisotropic cell elongation. Previous work identified a brassinosteroid response transcription factor as being enriched in the early developing spur cup. Brassinosteroids are known to be important for cell elongation, suggesting that brassinosteroid-mediated response may be an important regulator of spur elongation and potentially a driver of spur length diversity in Aquilegia. In this study, we investigated the role of brassinosteroids in the development of the Aquilegia coerulea petal spur. METHODS: We exogenously applied the biologically active brassinosteroid brassinolide to developing petal spurs to investigate spur growth under high hormone conditions. We used virus-induced gene silencing and gene expression experiments to understand the function of brassinosteroid-related transcription factors in A. coerulea petal spurs. KEY RESULTS: We identified a total of three Aquilegia homologues of the BES1/BZR1 protein family and found that these genes are ubiquitously expressed in all floral tissues during development, yet, consistent with the previous RNAseq study, we found that two of these paralogues are enriched in early developing petals. Exogenously applied brassinosteroid increased petal spur length due to increased anisotropic cell elongation as well as cell division. We found that targeting of the AqBEH genes with virus-induced gene silencing resulted in shortened petals, a phenotype caused in part by a loss of cell anisotropy. CONCLUSIONS: Collectively, our results support a role for brassinosteroids in anisotropic cell expansion in Aquilegia petal spurs and highlight the brassinosteroid pathway as a potential player in the diversification of petal spur length in Aquilegia.

Genome-Wide Identification, Characterization and Expression Analysis of TCP Transcription Factors in Petunia.

The plant-specific TCP transcription factors are well-characterized in both monocots and dicots, which have been implicated in multiple aspects of plant biological processes such as leaf morphogenesis and senescence, lateral branching, flower development and hormone crosstalk. However, no systematic analysis of the petunia TCP gene family has been described. In this work, a total of 66 petunia TCP genes (32 PaTCP genes in P. axillaris and 34 PiTCP genes in P. inflata) were identified. Subsequently, a systematic analysis of 32 PaTCP genes was performed. The phylogenetic analysis combined with structural analysis clearly distinguished the 32 PaTCP proteins into two classes-class Ι and class Ⅱ. Class Ⅱ was further divided into two subclades, namely, the CIN-TCP subclade and the CYC/TB1 subclade. Plenty of cis-acting elements responsible for plant growth and development, phytohormone and/or stress responses were identified in the promoter of PaTCPs. Distinct spatial expression patterns were determined among PaTCP genes, suggesting that these genes may have diverse regulatory roles in plant growth development. Furthermore, differential temporal expression patterns were observed between the large- and small-flowered petunia lines for most PaTCP genes, suggesting that these genes are likely to be related to petal development and/or petal size in petunia. The spatiotemporal expression profiles and promoter analysis of PaTCPs indicated that these genes play important roles in petunia diverse developmental processes that may work via multiple hormone pathways. Moreover, three PaTCP-YFP fusion proteins were detected in nuclei through subcellular localization analysis. This is the first comprehensive analysis of the petunia TCP gene family on a genome-wide scale, which provides the basis for further functional characterization of this gene family in petunia.

Comparative transcriptomics of early petal development across four diverse species of Aquilegia reveal few genes consistently associated with nectar spur development.

BACKGROUND: Petal nectar spurs, which facilitate pollination through animal attraction and pollen placement, represent a key innovation promoting diversification in the genus Aquilegia (Ranunculaceae). Identifying the genetic components that contribute to the development of these three-dimensional structures will inform our understanding of the number and types of genetic changes that are involved in the evolution of novel traits. In a prior study, gene expression between two regions of developing petals, the laminar blade and the spur cup, was compared at two developmental stages in the horticultural variety A. coerulea 'Origami'. Several hundred genes were differentially expressed (DE) between the blade and spur at both developmental stages. In order to narrow in on a set of genes crucial to early spur formation, the current study uses RNA sequencing (RNAseq) to conduct comparative expression analyses of petals from five developmental stages between four Aquilegia species, three with morphologically variable nectar spurs, A. sibirica, A. formosa, and A. chrysantha, and one that lacks nectar spurs, A. ecalcarata. RESULTS: Petal morphology differed increasingly between taxa across the developmental stages assessed, with petals from all four taxa being indistinguishable pre-spur formation at developmental stage 1 (DS1) and highly differentiated by developmental stage 5 (DS5). In all four taxa, genes involved in mitosis were down-regulated over the course of the assessed developmental stages, however, many genes involved in mitotic processes remained expressed at higher levels later in development in the spurred taxa. A total of 690 genes were identified that were consistently DE between the spurred taxa and A. ecalcarata at all five developmental stages. By comparing these genes with those identified as DE between spur and blade tissue in A. coerulea 'Origami', a set of only 35 genes was identified that shows consistent DE between petal samples containing spur tissue versus those without spur tissue. CONCLUSIONS: The results of this study suggest that expression differences in very few loci are associated with the presence and absence of spurs. In general, it appears that the spurless petals of A. ecalcarata cease cell divisions and enter the cell differentiation phase at an earlier developmental time point than those that produce spurs. This much more tractable list of 35 candidates genes will greatly facilitate targeted functional studies to assess the genetic control and evolution of petal spurs in Aquilegia.

RABBIT EARS regulates the transcription of TCP4 during petal development in Arabidopsis.

Plant organ growth requires the proper transition from cell proliferation to cell expansion and differentiation. The CIN-TCP transcription factor gene TCP4 and its post-transcriptional regulator microRNA319 play a pivotal role in this process. In this study, we identified a pathway in which the product of the C2H2 zinc finger gene RABBIT EARS (RBE) regulates the transcription of TCP4 during Arabidopsis (Arabidopsis thaliana) petal development. RBE directly represses TCP4 during the early stages of petal development; this contributes to the role of RBE in controlling the growth of petal primordia. We also found that the rbe-1 mutant strongly enhanced the petal phenotypes of tcp4soj6 and mir319a, two mutants with compromised miR319 regulation of TCP4 Our results show that transcriptional and post-transcriptional regulation function together to pattern the spatial and temporal expression of TCP4 This in turn controls petal size and shape in Arabidopsis.

Evolution of petaloidy in the zingiberales: An assessment of the relationship between ultrastructure and gene expression patterns.

BACKGROUND: The development of petal-like organs has occurred repetitively throughout angiosperm evolution. Despite homoplasy, it is possible that common underlying molecular mechanisms are repeatedly recruited to drive the development of petaloid organs. In Zingiberales, infertile, petal-like structures replace fertile stamens, resulting in petaloidy in androecial whorls. Assuming that androecial petaloidy is a shared derived characteristic, we expect to find common ultrastructure and molecular mechanisms underlying androecial petaloidy across Zingiberales. RESULTS: We show that petaloidy in Zingiberales is associated with tightly packed, protruding epidermal cells. Expression patterns for candidate genes involved in petal identity differ between the petaloid organs of Costaceae v. Cannaceae, despite similar macro- and microscopic organization. For all candidate gene families analyzed, our data suggest at least one Zingiberales-specific duplication event. CONCLUSIONS: Our data suggest that the patterns of B-class gene expression across the Zingiberales do not correlate with the occurrence of petaloidy, indicating that androecial petaloidy might have evolved independently of B-class gene expression in some lineages. It is possible that gene duplication may play a role in the diversity of petaloid structures found throughout the Zingiberales. It is likely that Zingiberales petaloidy may also result from the deployment of genes other than those involved in specification of petal identity. Developmental Dynamics 244:1121-1132, 2015. © 2015 Wiley Periodicals, Inc.

A flat petal as ancestral state for Ranunculaceae.

Ranunculaceae comprise ca. 2,500 species (ca. 55 genera) that display a broad range of floral diversity, particularly at the level of the perianth. Petals, when present, are often referred to as "elaborate" because they have a complex morphology. In addition, the petals usually produce and store nectar, which gives them a crucial functional role in the interaction with pollinators. Its morphological diversity and species richness make this family a particularly suitable model group for studying the evolution of complex morphologies. Our aims are (1) to reconstruct the ancestral form of the petal and evolutionary stages at the scale of Ranunculaceae, (2) to test the hypothesis that there are morphogenetic regions on the petal that are common to all species and that interspecific morphological diversity may be due to differences in the relative proportions of these regions during development. We scored and analyzed traits (descriptors) that characterize in detail the complexity of mature petal morphology in 32 genera. Furthermore, we described petal development using high resolution X-Ray computed tomography (HRX-CT) in six species with contrasting petal forms (Ficaria verna, Helleborus orientalis, Staphisagria picta, Aconitum napellus, Nigella damascena, Aquilegia vulgaris). Ancestral state reconstruction was performed using a robust and dated phylogeny of the family, allowing us to produce new hypotheses for petal evolution in Ranunculaceae. Our results suggest a flat ancestral petal with a short claw for the entire family and for the ancestors of all tribes except Adonideae. The elaborate petals that are present in different lineages have evolved independently, and similar morphologies are the result of convergent evolution.

Cuticle chemistry drives the development of diffraction gratings on the surface of Hibiscus trionum petals.

Plants combine both chemical and structural means to appear colorful. We now have an extensive understanding of the metabolic pathways used by flowering plants to synthesize pigments, but the mechanisms remain obscure whereby cells produce microscopic structures sufficiently regular to interfere with light and create an optical effect. Here, we combine transgenic approaches in a novel model system, Hibiscus trionum, with chemical analyses of the cuticle, both in transgenic lines and in different species of Hibiscus, to investigate the formation of a semi-ordered diffraction grating on the petal surface. We show that regulating both cuticle production and epidermal cell growth is insufficient to determine the type of cuticular pattern produced. Instead, the chemical composition of the cuticle plays a crucial role in restricting the formation of diffraction gratings to the pigmented region of the petal. This suggests that buckling, driven by spatiotemporal regulation of cuticle chemistry, could pattern the petal surface at the nanoscale.

The Petunia CHANEL Gene is a ZEITLUPE Ortholog Coordinating Growth and Scent Profiles.

The floral perianth, comprising sepals and petals, conceals the sexual organs and attracts pollinators. The coordination of growth and scent emission is not fully understood. We have analyzed the effect of knocking down CHANEL (PhCHL), the ZEITLUPE ortholog in petunia (PhCHL) by hairpin RNAs. Plants with low PhCHL mRNA had overall decreased size. Growth evaluation using time lapse image analysis showed that early leaf movement was not affected by RNAi:PhCHL, but flower angle movement was modified, moving earlier during the day in knockdown plants than in wild types. Despite differences in stem length, growth rate was not significantly affected by loss of PhCHL. In contrast, petal growth displayed lower growth rate in RNAi:PhCHL. Decreased levels of PhCHL caused strongly modified scent profiles, including changes in composition and timing of emission resulting in volatile profiles highly divergent from the wild type. Our results show a role of PhCHL in controlling growth and development of vegetative and reproductive organs in petunia. The different effects of PhCHL on organ development indicate an organ-specific interpretation of the down regulation of PhCHL. Through the control of both timing and quantitative volatile emissions, PhCHL appears to be a major coordinator of scent profiles.

CsPI from the perianthless early-diverging Chloranthus spicatus show function on petal development in Arabidopsis thaliana.

BACKGROUND: In the floral ABC model, B-class genes comprised of DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI) had been proposed to involve in second and third whorl floral organ development. However, less is known about the function of B-class genes from early-diverging angiosperms. Chloranthaceae is one of the early-diverging angiosperm families. In this study, we characterized the role of the PI-like gene CsPI cloned from Chloranthus spicatus which have the simplest perianthless bisexual flowers. RESULTS: The expression profile analysis reveals high levels of CsPI mRNA in stamens in Chloranthus spicatus, with weak distribution in leaves and other floral organs. Nevertheless, CsPI rescued both stamen and petal development in Arabidopsis thaliana pi-1 mutants and caused partially conversion of sepals into petaloid organs in wild-type Arabidopsis thaliana plants. Yeast two-hybrid analysis showed that CsPI can form not only homodimers but also heterodimers with proteins encoded by Arabidopsis thaliana and Chloranthus spicatus AP3-like genes. CONCLUSIONS: These results suggested that CsPI has an ancestral function on stamen development and that CsPI has capability to specify petal development in Arabidopsis thaliana. The finding indicates that the activity of the encoded PI-like proteins is highly conserved between the early-diverging Chloranthus and Arabidopsis. Moreover, our results appear to suggest that B-function genes may not play a role in perianth development in Chloranthus spicatus.

Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis.

RNA splicing is crucial to the production of mature mRNAs (mRNA). In Arabidopsis thaliana, the protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. SR45 is alternatively spliced into 2 isoforms. Isoform 1 (SR45.1) plays an important role in the flower petal development whereas isoform 2 (SR45.2) is important for root growth. In this study, we used immunoprecipitation to isolate an SR45.1-GFP fusion protein from transgenic plants complementing a null mutant, sr45-1. Mass spectrometry suggested a single phosphorylation event in a peptide from the alternatively spliced region unique to SR45.1. Substituting alanine for threonine 218, a candidate site for phosphorylation, did not complement the sr45-1 mutant with narrow flower petals whereas substituting aspartic acid or glutamic acid for threonine 218 did complement the sr45-1 mutant. Mass spectrometry also revealed that other proteins involved in the spliceosome co-precipitated with SR45.1, and RT-qPCR revealed that phosphorylation of threonine 218 promotes the function of SR45.1 in promoting the constitutive splicing of SR30 mRNA. This is the first demonstration of a specific phosphorylation site that differentially regulates the function of a plant splicing activator in physiologically and morphologically distinct plant tissues.