Complex petal spot formation in the Beetle Daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paralogous GdMYBSG6 transcription factors.

Gorteria diffusa has elaborate petal spots that attract pollinators through sexual deception, but how G. diffusa controls spot development is largely unknown. Here, we investigate how pigmentation is regulated during spot formation. We determined the anthocyanin composition of G. diffusa petals and combined gene expression analysis with protein interaction assays to characterise R2R3-MYBs that likely regulate pigment production in G. diffusa petal spots. We found that cyanidin 3-glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three subgroup 6 R2R3-MYB transcription factors (GdMYBSG6-1,2,3) that likely activate the production of spot pigmentation. These genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. Interaction assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes. We demonstrate that the elaboration of complex spots in G. diffusa begins with the accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three paralogous R2R3-MYB transcription factors. Our results indicate that the functional diversification of these GdMYBSG6s involved changes in the spatial control of their transcription, and modification of the duration of GdMYBSG6 gene expression contributes towards floral variation within the species.

Reflections on the ABC model of flower development.

The formulation of the ABC model by a handful of pioneer plant developmental geneticists was a seminal event in the quest to answer a seemingly simple question: how are flowers formed? Fast forward 30 years and this elegant model has generated a vibrant and diverse community, capturing the imagination of developmental and evolutionary biologists, structuralists, biochemists and molecular biologists alike. Together they have managed to solve many floral mysteries, uncovering the regulatory processes that generate the characteristic spatio-temporal expression patterns of floral homeotic genes, elucidating some of the mechanisms allowing ABC genes to specify distinct organ identities, revealing how evolution tinkers with the ABC to generate morphological diversity, and even shining a light on the origins of the floral gene regulatory network itself. Here we retrace the history of the ABC model, from its genesis to its current form, highlighting specific milestones along the way before drawing attention to some of the unsolved riddles still hidden in the floral alphabet.

Multiple origins of lipid-based structural colors contribute to a gradient of fruit colors in Viburnum (Adoxaceae).

Structural color is poorly known in plants relative to animals. In fruits, only a handful of cases have been described, including in Viburnum tinus where the blue color results from a disordered multilayered reflector made of lipid droplets. Here, we examine the broader evolutionary context of fruit structural color across the genus Viburnum. We obtained fresh and herbarium fruit material from 30 Viburnum species spanning the phylogeny and used transmission electron microscopy, optical simulations, and ancestral state reconstruction to identify the presence/absence of photonic structures in each species, understand the mechanism producing structural color in newly identified species, relate the development of cell wall structure to reflectance in Viburnum dentatum, and describe the evolution of cell wall architecture across Viburnum. We identify at least two (possibly three) origins of blue fruit color in Viburnum in species which produce large photonic structures made of lipid droplets embedded in the cell wall and which reflect blue light. Examining the full spectrum of mechanisms producing color in pl, including structural color as well as pigments, will yield further insights into the diversity, ecology, and evolution of fruit color.

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.

Eco-Evo-Devo of petal pigmentation patterning.

Colourful spots, stripes and rings decorate the corolla of most flowering plants and fulfil important biotic and abiotic functions. Spatial differences in the pigmentation of epidermal cells can create these patterns. The last few years have yielded new data that have started to illuminate the mechanisms controlling the function, formation and evolution of petal patterns. These advances have broad impacts beyond the immediate field as pigmentation patterns are wonderful systems to explore multiscale biological problems: from understanding how cells make decisions at the microscale to examining the roots of biodiversity at the macroscale. These new results also reveal there is more to petal patterning than meets the eye, opening up a brand new area of investigation. In this mini-review, we summarise our current knowledge on the Eco-Evo-Devo of petal pigmentation patterns and discuss some of the most exciting yet unanswered questions that represent avenues for future research.

Transcription Factors Evolve Faster Than Their Structural Gene Targets in the Flavonoid Pigment Pathway.

Dissecting the relationship between gene function and substitution rates is key to understanding genome-wide patterns of molecular evolution. Biochemical pathways provide powerful systems for investigating this relationship because the functional role of each gene is often well characterized. Here, we investigate the evolution of the flavonoid pigment pathway in the colorful Petunieae clade of the tomato family (Solanaceae). This pathway is broadly conserved in plants, both in terms of its structural elements and its MYB, basic helix-loop-helix, and WD40 transcriptional regulators, and its function has been extensively studied, particularly in model species of petunia. We built a phylotranscriptomic data set for 69 species of Petunieae to infer patterns of molecular evolution across pathway genes and across lineages. We found that transcription factors exhibit faster rates of molecular evolution (dN/dS) than their targets, with the highly specialized MYB genes evolving fastest. Using the largest comparative data set to date, we recovered little support for the hypothesis that upstream enzymes evolve slower than those occupying more downstream positions, although expression levels do predict molecular evolutionary rates. Although shifts in floral pigmentation were only weakly related to changes affecting coding regions, we found a strong relationship with the presence/absence patterns of MYB transcripts. Intensely pigmented species express all three main MYB anthocyanin activators in petals, whereas pale or white species express few or none. Our findings reinforce the notion that pathway regulators have a dynamic history, involving higher rates of molecular evolution than structural components, along with frequent changes in expression during color transitions.

Concordance-Based Approaches for the Inference of Relationships and Molecular Rates with Phylogenomic Data Sets.

Gene tree conflict is common and finding methods to analyze and alleviate the negative effects that conflict has on species tree analysis is a crucial part of phylogenomics. This study aims to expand the discussion of inferring species trees and molecular branch lengths when conflict is present. Conflict is typically examined in two ways: inferring its prevalence and inferring the influence of the individual genes (how strongly one gene supports any given topology compared to an alternative topology). Here, we examine a procedure for incorporating both conflict and the influence of genes in order to infer evolutionary relationships. All supported relationships in the gene trees are analyzed and the likelihood of the genes constrained to these relationships is summed to provide a likelihood for the relationship. Consensus tree assembly is conducted based on the sum of likelihoods for a given relationship and choosing relationships based on the most likely relationship assuming it does not conflict with a relationship that has a higher likelihood score. If it is not possible for all most likely relationships to be combined into a single bifurcating tree then multiple trees are produced and a consensus tree with a polytomy is created. This procedure allows for more influential genes to have a greater influence on an inferred relationship, does not assume conflict has arisen from any one source and does not force the data set to produce a single bifurcating tree. Using this approach, on three empirical data sets, we examine and discuss the relationship between influence and prevalence of gene tree conflict. We find that in one of the data sets, assembling a bifurcating consensus tree solely composed of the most likely relationships is impossible. To account for conflict in molecular rate analysis we also introduce a concordance-based approach to the summary and estimation of branch lengths suitable for downstream comparative analyses. We demonstrate through simulation that even under high levels of stochastic conflict, the mean and median of the concordant rates recapitulate the true molecular rate better than using a supermatrix approach. Using a large phylogenomic data set, we examine rate heterogeneity across concordant genes with a focus on the branch subtending crown angiosperms. Notably, we find highly variable rates of evolution along the branch subtending crown angiosperms. The approaches outlined here have several limitations, but they also represent some alternative methods for harnessing the complexity of phylogenomic data sets and enrich our inferences of both species relationships and evolutionary processes.[Branch length estimation; consensus tree; gene tree conflict; gene tree filtering; phylogenetics; phylogenomics.].

Sculpting the surface: Structural patterning of plant epidermis.

Plant epidermis are multifunctional surfaces that directly affect how plants interact with animals or microorganisms and influence their ability to harvest or protect from abiotic factors. To do this, plants rely on minuscule structures that confer remarkable properties to their outer layer. These microscopic features emerge from the hierarchical organization of epidermal cells with various shapes and dimensions combined with different elaborations of the cuticle, a protective film that covers plant surfaces. Understanding the properties and functions of those tridimensional elements as well as disentangling the mechanisms that control their formation and spatial distribution warrant a multidisciplinary approach. Here we show how interdisciplinary efforts of coupling modern tools of experimental biology, physics, and chemistry with advanced computational modeling and state-of-the art microscopy are yielding broad new insights into the seemingly arcane patterning processes that sculpt the outer layer of plants.

Using structural colour to track length scale of cell-wall layers in developing Pollia japonica fruits.

Helicoidally arranged layers of cellulose microfibrils in plant cell walls can produce strong and vivid coloration in a wide range of species. Despite its significance, the morphogenesis of cell walls, whether reflective or not, is not fully understood. Here we show that by optically monitoring the reflectance of Pollia japonica fruits during development we can directly map structural changes of the cell wall on a scale of tens of nanometres. Visible-light reflectance spectra from individual living cells were measured throughout the fruit maturation process and compared with numerical models. Our analysis reveals that periodic spacing of the helicoidal architecture remains unchanged throughout fruit development, suggesting that interactions in the cell-wall polysaccharides lead to a fixed twisting angle of cellulose helicoids in the cell wall. By contrast with conventional electron microscopy, which requires analysis of different fixed specimens at different stages of development, the noninvasive optical technique we present allowed us to directly monitor live structural changes in biological photonic systems as they develop. This method therefore is applicable to investigations of photonic tissues in other organisms.

Viburnum tinus Fruits Use Lipids to Produce Metallic Blue Structural Color.

Viburnum tinus is an evergreen shrub that is native to the Mediterranean region but cultivated widely in Europe and around the world. It produces ripe metallic blue fruits throughout winter [1]. Despite its limited fleshy pulp [2], its high lipid content [3] makes it a valuable resource to the small birds [4] that act as its seed-dispersers [5]. Here, we find that the metallic blue appearance of the fruits is produced by globular lipid inclusions arranged in a disordered multilayer structure. This structure is embedded in the cell walls of the epicarp and underlaid with a dark layer of anthocyanin pigments. The presence of such large, organized lipid aggregates in plant cell walls represents a new mechanism for structural coloration and may serve as an honest signal of nutritional content.

Direct Depolymerization Coupled to Liquid Extraction Surface Analysis-High-Resolution Mass Spectrometry for the Characterization of the Surface of Plant Tissues.

The cuticle, the outermost layer covering the epidermis of most aerial organs of land plants, can have a heterogeneous composition even on the surface of the same organ. The main cuticle component is the polymer cutin which, depending on its chemical composition and structure, can have different biophysical properties. In this study, we introduce a new on-surface depolymerization method coupled to liquid extraction surface analysis (LESA) high-resolution mass spectrometry (HRMS) for a fast and spatially resolved chemical characterization of the cuticle of plant tissues. The method is composed of an on-surface saponification, followed by extraction with LESA using a chloroform-acetonitrile-water (49:49:2) mixture and direct HRMS detection. The method is also compared with LESA-HRMS without prior depolymerization for the analysis of the surface of the petals of Hibiscus richardsonii flowers, which have a ridged cuticle in the proximal region and a smooth cuticle in the distal region. We found that on-surface saponification is effective enough to depolymerize the cutin into its monomeric constituents thus allowing detection of compounds that were not otherwise accessible without a depolymerization step. The effect of the depolymerization procedure was more pronounced for the ridged/proximal cuticle, which is thicker and richer in epicuticular waxes compared with the cuticle in the smooth/distal region of the petal.

Ultrastructure and optics of the prism-like petal epidermal cells of Eschscholzia californica (California poppy).

The petals of Eschscholzia californica (California poppy) are robust, pliable and typically coloured intensely orange or yellow owing to the presence of carotenoid pigments; they are also highly reflective at certain angles, producing a silky effect. To understand the mechanisms behind colour enhancement and reflectivity in California poppy, which represents a model species among early-divergent eudicots, we explored the development, ultrastructure, pigment composition and optical properties of the petals using light microscopy and electron microscopy combined with both spectrophotometry and goniometry. The elongated petal epidermal cells each possess a densely thickened prism-like ridge that is composed primarily of cell wall. The surface ridges strongly focus incident light onto the pigments, which are located in plastids at the cell base. Our results indicate that this highly unusual, deeply ridged surface structure not only enhances the deep colour response in this desert species, but also results in strongly angle-dependent 'silky' reflectivity that is anisotropic and mostly directional.

Disorder in convergent floral nanostructures enhances signalling to bees.

Diverse forms of nanoscale architecture generate structural colour and perform signalling functions within and between species. Structural colour is the result of the interference of light from approximately regular periodic structures; some structural disorder is, however, inevitable in biological organisms. Is this disorder functional and subject to evolutionary selection, or is it simply an unavoidable outcome of biological developmental processes? Here we show that disordered nanostructures enable flowers to produce visual signals that are salient to bees. These disordered nanostructures (identified in most major lineages of angiosperms) have distinct anatomies but convergent optical properties; they all produce angle-dependent scattered light, predominantly at short wavelengths (ultraviolet and blue). We manufactured artificial flowers with nanoscale structures that possessed tailored levels of disorder in order to investigate how foraging bumblebees respond to this optical effect. We conclude that floral nanostructures have evolved, on multiple independent occasions, an effective degree of relative spatial disorder that generates a photonic signature that is highly salient to insect pollinators.

The Evolution of Diverse Floral Morphologies.

The angiosperm flower develops through a modular programme which, although ancient and conserved, provides the flexibility that has allowed an almost infinite variety of floral forms to emerge. In this review, we explore the evolution of floral diversity, focusing on our recent understanding of the mechanistic basis of evolutionary change. We discuss the various ways in which flower size and floral organ size can be modified, the means by which flower shape and symmetry can change, and the ways in which floral organ position can be varied. We conclude that many challenges remain before we fully understand the ecological and molecular processes that facilitate the diversification of flower structure.

A link between LEAFY and B-gene homologues in Welwitschia mirabilis sheds light on ancestral mechanisms prefiguring floral development.

Flowering plants evolved from an unidentified gymnosperm ancestor. Comparison of the mechanisms controlling development in angiosperm flowers and gymnosperm cones may help to elucidate the mysterious origin of the flower. We combined gene expression studies with protein behaviour characterization in Welwitschia mirabilis to test whether the known regulatory links between LEAFY and its MADS-box gene targets, central to flower development, might also contribute to gymnosperm reproductive development. We found that WelLFY, one of two LEAFY-like genes in Welwitschia, could be an upstream regulator of the MADS-box genes APETALA3/PISTILLATA-like (B-genes). We demonstrated that, even though their DNA-binding domains are extremely similar, WelLFY and its paralogue WelNDLY exhibit distinct DNA-binding specificities, and that, unlike WelNDLY, WelLFY shares with its angiosperm orthologue the capacity to bind promoters of Welwitschia B-genes. Finally, we identified several cis-elements mediating these interactions in Welwitschia and obtained evidence that the link between LFY homologues and B-genes is also conserved in two other gymnosperms, Pinus and Picea. Although functional approaches to investigate cone development in gymnosperms are limited, our state-of-the-art biophysical techniques, coupled with expression studies, provide evidence that crucial links, central to the control of floral development, may already have existed before the appearance of flowers.

The physics of pollinator attraction.

Contents 350 I. 350 II. 350 III. 352 IV. 353 V. 353 353 References 354 SUMMARY: This Tansley Insight focuses on recent advances in our understanding of how flowers manipulate physical forces to attract animal pollinators and ensure reproductive success. Research has traditionally explored the role of chemical pigments and volatile organic compounds as cues for pollinators, but recent reports have demonstrated the importance of physical and structural means of pollinator attraction. Here we explore the role of petal microstructure in influencing floral light capture and optics, analysing colour, gloss and polarization effects. We discuss the interaction between flower, pollinator and gravity, and how petal surface structure can influence that interaction. Finally, we consider the role of electrostatic forces in pollen transfer and pollinator attraction. We conclude that this new interdisciplinary field is evolving rapidly.

Structural colour from helicoidal cell-wall architecture in fruits of Margaritaria nobilis.

The bright and intense blue-green coloration of the fruits of Margaritaria nobilis (Phyllanthaceae) was investigated using polarization-resolved spectroscopy and transmission electron microscopy. Optical measurements of freshly collected fruits revealed a strong circularly polarized reflection of the fruit that originates from a cellulose helicoidal cell wall structure in the pericarp cells. Hyperspectral microscopy was used to capture the iridescent effect at the single-cell level.

Direct surface analysis coupled to high-resolution mass spectrometry reveals heterogeneous composition of the cuticle of Hibiscus trionum petals.

Plant cuticle, which is the outermost layer covering the aerial parts of all plants including petals and leaves, can present a wide range of patterns that, combined with cell shape, can generate unique physical, mechanical, or optical properties. For example, arrays of regularly spaced nanoridges have been found on the dark (anthocyanin-rich) portion at the base of the petals of Hibiscus trionum. Those ridges act as a diffraction grating, producing an iridescent effect. Because the surface of the distal white region of the petals is smooth and noniridescent, a selective chemical characterization of the surface of the petals on different portions (i.e., ridged vs smooth) is needed to understand whether distinct cuticular patterns correlate with distinct chemical compositions of the cuticle. In the present study, a rapid screening method has been developed for the direct surface analysis of Hibiscus trionum petals using liquid extraction surface analysis (LESA) coupled with high-resolution mass spectrometry. The optimized method was used to characterize a wide range of plant metabolites and cuticle monomers on the upper (adaxial) surface of the petals on both the white/smooth and anthocyanic/ridged regions, and on the lower (abaxial) surface, which is entirely smooth. The main components detected on the surface of the petals are low-molecular-weight organic acids, sugars, and flavonoids. The ridged portion on the upper surface of the petal is enriched in long-chain fatty acids, which are constituents of the wax fraction of the cuticle. These compounds were not detected on the white/smooth region of the upper petal surface or on the smooth lower surface.