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.

Characterisation of the R2R3 Myb subgroup 9 family of transcription factors in tomato.

Tomato (Solanum lycopersicum) has many epidermal cell outgrowths including conical petal cells and multiple types of trichomes. These include the anther-specific trichome mesh which holds the anthers connate. The R2R3 Myb Subgroup 9 family of transcription factors is involved in development of epidermal cell outgrowths throughout the angiosperms. No previous study has examined all members of this transcription factor family in a single species. All 7 R2R3 Myb Subgroup 9 genes were isolated from tomato. They were ectopically expressed in tobacco to assess their ability to induce epidermal cell outgrowth. Endogenous expression patterns were examined by semi-quantitative RT-PCR at different stages of floral development relative to the development of anther trichomes. We report variation in the degree of epidermal cell outgrowth produced in transgenic tobacco by each ectopically expressed gene. Based on expression profile and ectopic activity, SlMIXTA-2 is likely involved in the production of leaf trichomes. SlMIXTA-2 is expressed most strongly in the leaves, and not expressed in the floral tissue. SlMYB17-2 is the best candidate for the regulation of the anther trichome mesh. SlMYB17-2 is expressed strongly in the floral tissue and produces a clear phenotype of epidermal cell outgrowths when ectopically expressed in tobacco. Analysis of the phenotypes of transgenic plants ectopically expressing all 7 genes has revealed the different extent to which members of the same transcription factor subfamily can induce cellular outgrowth.

Strawberry varieties differ in pollinator-relevant floral traits.

A rising global population will need more food, increasing demand for insect pollination services. However, general insect declines conflict with this demand. One way to mitigate this conflict is to grow crop flowers that are easier for insects to find and more rewarding to those that visit them. This study quantifies variation in the pollinator-relevant traits of nectar and pollen production, flower size and flower shape in commercial strawberry, finding significant variation between varieties in all traits. Bumblebees could learn to distinguish between the extremes of variation in flower shape, but this learning is very slow, indicating that this variation is at the limit of that which can be detected by bumblebees. Bee preferences for nectar of differing sugar concentrations at field-realistic volumes were consistent with previous observations at larger volumes, suggesting that it is valid to translate lab findings to the field. This study builds on our knowledge of the range of pollinator reward present in a single cultivated species and of the impact of field-realistic levels of variation in floral traits on bumblebee preferences.

Bumblebees negotiate a trade-off between nectar quality and floral biomechanics.

How and why pollinators choose which flowers to visit are fundamental, multifaceted questions in pollination biology, yet most studies of floral traits measure simple relative preferences. Here, we used vertically and horizontally oriented slippery-surfaced artificial flowers to test whether bumblebees could make a trade-off between floral handling difficulty and nectar sucrose concentration. We quantified foraging energetics, thereby resolving the rationale behind the bees' foraging decisions. The bees chose flowers with either a high handling cost or low sucrose concentration, depending on which was the energetically favorable option. Their behavior agreed with the critical currency being the rate of energy return (net energy collected per unit time), not energetic efficiency (net energy collected per unit energy spent). This suggests that bumblebees prioritize immediate carbohydrate flow to the nest rather than energy gain over the working lifespan of each bee. Trade-off paradigms like these are a powerful approach for quantifying pollinator trait preferences.

Bumblebee responses to variation in pollinator-attracting traits of Vicia faba flowers.

Adaptations that attract pollinators to flowers are central to the reproductive success of insect-pollinated plants, including crops. Understanding the influence of these non-rewarding traits on pollinator preference is important for our future food security by maintaining sufficient crop pollination. We have identified substantial variation in flower shape, petal size, corolla-tube length, petal spot size and floral volatile compounds among a panel of 30 genetically distinct lines of Vicia faba. Using this variation, we found that Bombus terrestris was able to distinguish between natural variation in petal spot size, floral volatile emissions and corolla-tube length. Foragers showed some innate preference for spotted flowers over non-spotted flowers and preferred shorter corolla-tube lengths over longer tubes. Our results suggest that some floral traits may have significant potential to enhance pollinator attraction to V. faba crops, particularly if paired with optimised rewards.

How do you build a nectar spur? A transcriptomic comparison of nectar spur development in Linaria vulgaris and gibba development in Antirrhinum majus.

Nectar spurs (tubular outgrowths of floral organs) have long fascinated biologists. However, given that no model species possess nectar spurs, there is still much to learn about their development. In this study we combined morphological analysis with comparative transcriptomics to gain a global insight into the morphological and molecular basis of spur outgrowth in Linaria. Whole transcriptome sequencing was performed on two related species at three key developmental stages (identified by our morphological analysis), one with a spur (Linaria vulgaris), and one without a spur (Antirrhinum majus). A list of spur-specific genes was selected, on which we performed a gene enrichment analysis. Results from our RNA-seq analysis agreed with our morphological observations. We describe gene activity during spur development and provide a catalogue of spur-specific genes. Our list of spur-specific genes was enriched for genes connected to the plant hormones cytokinin, auxin and gibberellin. We present a global view of the genes involved in spur development in L. vulgaris, and define a suite of genes which are specific to spur development. This work provides candidate genes for spur outgrowth and development in L. vulgaris which can be investigated in future studies.

Morphoelastic modelling of pattern development in the petal epidermal cell cuticle.

We use the model system Hibiscus trionum as a vehicle to study the origin and propagation of surface nano-ridges in plant petal epidermal cells by tracking the development of the cell shape and the cuticle. In this system, the cuticle develops two distinct sub-layers, (i) an uppermost layer which increases in thickness and in-plane extension and (ii) a substrate, composed of cuticular and cell wall material. We quantify the pattern formation and geometrical changes and then postulate a mechanical model assuming that the cuticle behaves as a growing bi-layer. The model is a quasi-static morphoelastic system and it is numerically investigated in two- and three-dimensional settings, using different laws of film and substrate expansion and boundary conditions. We recreate several features of the observed developmental trajectories in petals. We establish the respective roles of the layers' stiffness mismatch, the underlying cell-wall curvature, the cell in-plane expansion and the thickness growth rates of the layers in determining the observed pattern features, such as the variance observed in the amplitude and wavelength of the cuticular striations. Our observations provide evidence which justifies the growing bi-layer description, and provide valuable insights into why some systems develop surface patterns and others do not.

The evolution of flower colour.

Flowers are the most commonly seen colourful elements of the natural world, and in this primer we explain the evolution of their spectacular range of colours. To understand flower colour, we first explain what colour is and how a flower can have different colours in the eyes of different observers. We briefly introduce the molecular and biochemical basis of flower colour, which is primarily based on well-characterised pigment synthesis pathways. We then consider the evolution of flower colour over four timescales - its origin and deep evolution, its macroevolution, its microevolution and finally, the recent effects of human behaviour on flower colour and its evolution. Because flower colour is so evolutionarily labile, and at the same time so striking to the human eye, it is an exciting subject for current and future research efforts.

Biased pollen transfer by bumblebees favors the paternity of virus-infected plants in cross-pollination.

We used a green fluorescent protein marker gene for paternity analysis to determine if virus infection affected male reproductive success of tomato in bumblebee-mediated cross-pollination under glasshouse conditions. We found that bumblebees that visited flowers of infected plants showed a strong preference to subsequently visit flowers of non-infected plants. The behavior of the bumblebees to move toward non-infected plants after pollinating virus-infected plants appears to explain the paternity data, which demonstrate a statistically significant ∼10-fold bias for fertilization of non-infected plants with pollen from infected parents. Thus, in the presence of bumblebee pollinators, CMV-infected plants exhibit enhanced male reproductive success.

Multiple gene co-options underlie the rapid evolution of sexually deceptive flowers in Gorteria diffusa.

Gene co-option, the redeployment of an existing gene in an unrelated developmental context, is an important mechanism underlying the evolution of morphological novelty. In most cases described to date, novel traits emerged by co-option of a single gene or genetic network. Here, we show that the integration of multiple co-opted genetic elements facilitated the rapid evolution of complex petal spots that mimic female bee-fly pollinators in the sexually deceptive South African daisy Gorteria diffusa. First, co-option of iron homeostasis genes altered petal spot pigmentation, producing a color similar to that of female pollinators. Second, co-option of the root hair gene GdEXPA7 enabled the formation of enlarged papillate petal epidermal cells, eliciting copulation responses from male flies. Third, co-option of the miR156-GdSPL1 transcription factor module altered petal spot placement, resulting in better mimicry of female flies resting on the flower. The three genetic elements were likely co-opted sequentially, and strength of sexual deception in different G. diffusa floral forms strongly correlates with the presence of the three corresponding morphological alterations. Our findings suggest that gene co-options can combine in a modular fashion, enabling rapid evolution of novel complex traits.

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.

Analysis of flavonol regulator evolution in the Brassicaceae reveals MYB12, MYB111 and MYB21 duplications and MYB11 and MYB24 gene loss.

BACKGROUND: Flavonols are the largest subgroup of flavonoids, possessing multiple functions in plants including protection against ultraviolet radiation, antimicrobial activities, and flower pigmentation together with anthocyanins. They are of agronomical and economical importance because the major off-taste component in rapeseed protein isolates is a flavonol derivative, which limits rapeseed protein use for human consumption. Flavonol production in Arabidopsis thaliana is mainly regulated by the subgroup 7 (SG7) R2R3-MYB transcription factors MYB11, MYB12, and MYB111. Recently, the SG19 MYBs MYB21, MYB24, and MYB57 were shown to regulate flavonol accumulation in pollen and stamens. The members of each subgroup are closely related, showing gene redundancy and tissue-specific expression in A. thaliana. However, the evolution of these flavonol regulators inside the Brassicaceae, especially inside the Brassiceae, which include the rapeseed crop species, is not fully understood. RESULTS: We studied the SG7 and SG19 MYBs in 44 species, including 31 species of the Brassicaceae, by phylogenetic analyses followed by synteny and gene expression analyses. Thereby we identified a deep MYB12 and MYB111 duplication inside the Brassicaceae, which likely occurred before the divergence of Brassiceae and Thelypodieae. These duplications of SG7 members were followed by the loss of MYB11 after the divergence of Eruca vesicaria from the remaining Brassiceae species. Similarly, MYB21 experienced duplication before the emergence of the Brassiceae tribe, where the gene loss of MYB24 is also proposed to have happened. The members of each subgroup revealed frequent overlapping spatio-temporal expression patterns in the Brassiceae member B. napus, which are assumed to compensate for the loss of MYB11 and MYB24 in the analysed tissues. CONCLUSIONS: We identified a duplication of MYB12, MYB111, and MYB21 inside the Brassicaceae and MYB11 and MYB24 gene loss inside the tribe Brassiceae. We propose that polyploidization events have shaped the evolution of the flavonol regulators in the Brassicaceae, especially in the Brassiceae.

Conical petal epidermal cells, regulated by the MYB transcription factor MIXTA, have an ancient origin within the angiosperms.

Conical epidermal cells occur on the tepals (perianth organs, typically petals and/or sepals) of the majority of animal-pollinated angiosperms, where they play both visual and tactile roles in pollinator attraction, providing grip to foraging insects, and enhancing colour, temperature, and hydrophobicity. To explore the evolutionary history of conical epidermal cells in angiosperms, we surveyed the tepal epidermis in representative species of the ANA-grade families, the early-diverging successive sister lineages to all other extant angiosperms, and analysed the function of a candidate regulator of cell outgrowth from Cabomba caroliniana (Nymphaeales). We identified conical cells in at least two genera from different families (Austrobaileya and Cabomba). A single SBG9 MYB gene was isolated from C. caroliniana and found to induce strong differentiation of cellular outgrowth, including conical cells, when ectopically expressed in Nicotiana tabacum. Ontogenetic analysis and quantitative reverse transcription-PCR established that CcSBG9A1 is spatially and temporally expressed in a profile which correlates with a role in conical cell development. We conclude that conical or subconical cells on perianth organs are ancient within the angiosperms and most probably develop using a common genetic programme initiated by a SBG9 MYB transcription factor.

Molecular biology for green recovery-A call for action.

Molecular biology holds a vast potential for tackling climate change and biodiversity loss. Yet, it is largely absent from the current strategies. We call for a community-wide action to bring molecular biology to the forefront of climate change solutions.

Cell wall composition determines handedness reversal in helicoidal cellulose architectures of Pollia condensata fruits.

Chiral asymmetry is important in a wide variety of disciplines and occurs across length scales. While several natural chiral biomolecules exist only with single handedness, they can produce complex hierarchical structures with opposite chiralities. Understanding how the handedness is transferred from molecular to the macroscopic scales is far from trivial. An intriguing example is the transfer of the handedness of helicoidal organizations of cellulose microfibrils in plant cell walls. These cellulose helicoids produce structural colors if their dimension is comparable to the wavelength of visible light. All previously reported examples of a helicoidal structure in plants are left-handed except, remarkably, in the Pollia condensata fruit; both left- and right-handed helicoidal cell walls are found in neighboring cells of the same tissue. By simultaneously studying optical and mechanical responses of cells with different handednesses, we propose that the chirality of helicoids results from differences in cell wall composition. In detail, here we showed statistical substantiation of three different observations: 1) light reflected from right-handed cells is red shifted compared to light reflected from left-handed cells, 2) right-handed cells occur more rarely than left-handed ones, and 3) right-handed cells are located mainly in regions corresponding to interlocular divisions. Finally, 4) right-handed cells have an average lower elastic modulus compared to left-handed cells of the same color. Our findings, combined with mechanical simulation, suggest that the different chiralities of helicoids in the cell wall may result from different chemical composition, which strengthens previous hypotheses that hemicellulose might mediate the rotations of cellulose microfibrils.

Mechanical buckling can pattern the light-diffracting cuticle of Hibiscus trionum.

Many species have cuticular striations that play a range of roles, from pollinator attraction to surface wettability. In Hibiscus trionum, the striations span multiple cells at the base of the petal to form a pattern that produces a type of iridescence. It is postulated, using theoretical models, that the pattern of striations could result from mechanical instabilities. By combining the application of mechanical stress with high-resolution imaging, we demonstrate that the cuticle buckles to create a striated pattern. Through mechanical modeling and cryo-SEM fractures, we show that the cuticle behaves like a bilayer system with a stiff film on a compliant substrate. The pattern of buckling aligns with the direction of the stress to create a larger-scale pattern. Our findings contribute to the understanding of the formation of tissue-wide patterns in living organisms.

Humans Share More Preferences for Floral Phenotypes With Pollinators Than With Pests.

Studies on the selection of floral traits usually consider pollinators and sometimes herbivores. However, humans also exert selection on floral traits of ornamental plants. We compared the preferences of bumblebees (Bombus terrestris), thrips (Frankliniella occidentalis), and humans for flowers of snapdragon. From a cross of two species, Antirrhinum majus and Antirrhinum linkianum, we selected four Recombinant Inbred Lines (RILs). We characterised scent emission from whole flowers and stamens, pollen content and viability, trichome density, floral shape, size and colour of floral parts. We tested the preferences of bumblebees, thrips, and humans for whole flowers, floral scent bouquets, stamen scent, and individual scent compounds. Humans and bumblebees showed preferences for parental species, whereas thrips preferred RILs. Colour and floral scent, in combination with other floral traits, seem relevant phenotypes for all organisms. Remarkably, visual traits override scent cues for bumblebees, although, scent is an important trait when bumblebees cannot see the flowers, and methyl benzoate was identified as a key attractant for them. The evolutionary trajectory of flowers is the result of multiple floral traits interacting with different organisms with different habits and modes of interaction.