Cell layer-specific expression of the homeotic MADS-box transcription factor PhDEF contributes to modular petal morphogenesis in petunia.

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from distinct meristematic layers. How cells from these distinct layers acquire their respective identities and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we studied petunia (Petunia × hybrida) petals that form a limb and tube through congenital fusion. We identified petunia mutants (periclinal chimeras) expressing the B-class MADS-box gene DEFICIENS in the petal epidermis or in the petal mesophyll, called wico and star, respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but greatly reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. These mutants highlight the layer-specific roles of PhDEF during petal development. We explored the link between PhDEF and petal pigmentation, a well-characterized limb epidermal trait. The anthocyanin biosynthesis pathway was strongly downregulated in star petals, including its major regulator ANTHOCYANIN2 (AN2). We established that PhDEF directly binds to the AN2 terminator in vitro and in vivo, suggesting that PhDEF might regulate AN2 expression and therefore petal epidermis pigmentation. Altogether, we show that cell layer-specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

Analyses of Cullin1 homologs reveal functional redundancy in S-RNase-based self-incompatibility and evolutionary relationships in eudicots.

In Petunia (Solanaceae family), self-incompatibility (SI) is regulated by the polymorphic S-locus, which contains the pistil-specific S-RNase and multiple pollen-specific S-Locus F-box (SLF) genes. SLFs assemble into E3 ubiquitin ligase complexes known as Skp1-Cullin1-F-box complexes (SCFSLF). In pollen tubes, these complexes collectively mediate ubiquitination and degradation of all nonself S-RNases, but not self S-RNase, resulting in cross-compatible, but self-incompatible, pollination. Using Petunia inflata, we show that two pollen-expressed Cullin1 (CUL1) proteins, PiCUL1-P and PiCUL1-B, function redundantly in SI. This redundancy is lost in Petunia hybrida, not because of the inability of PhCUL1-B to interact with SSK1, but due to a reduction in the PhCUL1-B transcript level. This is possibly caused by the presence of a DNA transposon in the PhCUL1-B promoter region, which was inherited from Petunia axillaris, one of the parental species of Pe. hybrida. Phylogenetic and syntenic analyses of Cullin genes in various eudicots show that three Solanaceae-specific CUL1 genes share a common origin, with CUL1-P dedicated to S-RNase-related reproductive processes. However, CUL1-B is a dispersed duplicate of CUL1-P present only in Petunia, and not in the other species of the Solanaceae family examined. We suggest that the CUL1s involved (or potentially involved) in the SI response in eudicots share a common origin.

The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

CRISPR/Cas9-mediated mutagenesis of FT/TFL1 in petunia improves plant architecture and early flowering.

Petunias are renowned ornamental species widely cultivated as pot plants for their aesthetic appeal both indoors and outdoors. The preference for pot plants depends on their compact growth habit and abundant flowering. While genome editing has gained significant popularity in many crop plants in addressing growth and development and abiotic and biotic stress factors, relatively less emphasis has been placed on its application in ornamental plant species. Genome editing in ornamental plants opens up possibilities for enhancing their aesthetic qualities, offering innovative opportunities for manipulating plant architecture and visual appeal through precise genetic modifications. In this study, we aimed to optimize the procedure for an efficient genome editing system in petunia plants using the highly efficient multiplexed CRISPR/Cas9 system. Specifically, we targeted a total of six genes in Petunia which are associated with plant architecture traits, two paralogous of FLOWERING LOCUS T (PhFT) and four TERMINAL FLOWER-LIKE1 (PhTFL1) paralogous genes separately in two constructs. We successfully induced homogeneous and heterogeneous indels in the targeted genes through precise genome editing, resulting in significant phenotypic alterations in petunia. Notably, the plants harboring edited PhTFL1 and PhFT exhibited a conspicuously early flowering time in comparison to the wild-type counterparts. Furthermore, mutants with alterations in the PhTFL1 demonstrated shorter internodes than wild-type, likely by downregulating the gibberellic acid pathway genes PhGAI, creating a more compact and aesthetically appealing phenotype. This study represents the first successful endeavor to produce compact petunia plants with increased flower abundance through genome editing. Our approach holds immense promise to improve economically important potting plants like petunia and serve as a potential foundation for further improvements in similar ornamental plant species.

Petunia PHYTOCHROME INTERACTING FACTOR 4/5 transcriptionally activates key regulators of floral scent.

Floral scent emission of petunia flowers is regulated by light conditions, circadian rhythms, ambient temperature and the phytohormones GA and ethylene, but the mechanisms underlying sensitivity to these factors remain obscure. PHYTOCHROME INTERACTING FACTORs (PIFs) have been well studied as components of the regulatory machinery for numerous physiological processes. Acting redundantly, they serve as transmitters of light, circadian, metabolic, thermal and hormonal signals. Here we identified and characterized the phylogenetics of petunia PIF family members (PhPIFs). PhPIF4/5 was revealed as a positive regulator of floral scent: TRV-based transient suppression of PhPIF4/5 in petunia petals reduced emission of volatiles, whereas transient overexpression increased scent emission. The mechanism of PhPIF4/5-mediated regulation of volatile production includes activation of the expression of genes encoding biosynthetic enzymes and a key positive regulator of the pathway, EMISSION OF BENZENOIDS II (EOBII). The PIF-binding motif on the EOBII promoter (G-box) was shown to be needed for this activation. As PhPIF4/5 homologues are sensors of dawn and expression of EOBII also peaks at dawn, the prior is proposed to be part of the diurnal control of the volatile biosynthetic machinery. PhPIF4/5 was also found to transcriptionally activate PhDELLAs; a similar positive effect of PIFs on DELLA expression was further confirmed in Arabidopsis seedlings. The PhPIF4/5-PhDELLAs feedback is proposed to fine-tune GA signaling for regulation of floral scent production.

Rhythmic histone acetylation acts in concert with day-night oscillation of the floral volatile metabolic network.

The biosynthesis of specialized metabolites is strictly regulated by environmental inputs such as the day-night cycle, but the underlying mechanisms remain elusive. In Petunia hybrida cv. Mitchell flowers, the biosynthesis and emission of volatile compounds display a diurnal pattern with a peak in the evening to attract nocturnal pollinators. Using petunia flowers as a model system, we found that chromatin level regulation, especially histone acetylation, plays an essential role in mediating the day-night oscillation of the biosynthetic gene network of specialized metabolites. By performing time-course chromatin immunoprecipitation assays for histone modifications, we uncovered that a specific group of genes involved in the regulation, biosynthesis, and emission of floral volatile compounds, which displays the greatest magnitude in day-night oscillating gene expression, is associated with highly dynamic histone acetylation marks H3K9ac and H3K27ac. Specifically, the strongest oscillating genes featured a drastic removal of histone acetylation marks at night, potentially to shut down the biosynthesis of floral volatile compounds during the morning when they are not needed. Inhibiting daytime histone acetylation led to a compromised evening induction of these genes. Overall, our study suggested an active role of chromatin modification in the diurnal oscillation of specialized metabolic network.

SCARECROW-like GRAS protein PES positively regulates petunia floral scent production.

Emission of scent volatiles by flowers is important for successful pollination and consequently, reproduction. Petunia (Petunia hybrida) floral scent is formed mainly by volatile products of the phenylpropanoid pathway. We identified and characterized a regulator of petunia scent production: the GRAS protein PHENYLPROPANOID EMISSION-REGULATING SCARECROW-LIKE (PES). Its expression increased in petals during bud development and was highest in open flowers. Overexpression of PES increased the production of floral volatiles, while its suppression resulted in scent reduction. We showed that PES upregulates the expression of genes encoding enzymes of the phenylpropanoid and shikimate pathways in petals, and of the core regulator of volatile biosynthesis ODORANT1 by activating its promoter. PES is an ortholog of Arabidopsis (Arabidopsis thaliana) PHYTOCHROME A SIGNAL TRANSDUCTION 1, involved in physiological responses to far-red (FR) light. Analyses of the effect of nonphotosynthetic irradiation (low-intensity FR light) on petunia floral volatiles revealed FR light as a scent-activating factor. While PHYTOCHROME A regulated scent-related gene expression and floral scent production under FR light, the influence of PES on volatile production was not limited by FR light conditions.

Incomplete lineage sorting and hybridization underlie tree discordance in Petunia and related genera (Petunieae, Solanaceae).

Despite the overarching history of species divergence, phylogenetic studies often reveal distinct topologies across regions of the genome. The sources of these gene tree discordances are variable, but incomplete lineage sorting (ILS) and hybridization are among those with the most biological importance. Petunia serves as a classic system for studying hybridization in the wild. While field studies suggest that hybridization is frequent, the extent of reticulation within Petunia and its closely related genera has never been examined from a phylogenetic perspective. In this study, we used transcriptomic data from 11 Petunia, 16 Calibrachoa, and 10 Fabiana species to illuminate the relationships between these species and investigate whether hybridization played a significant role in the diversification of the clade. We inferred that gene tree discordance within genera is linked to hybridization events along with high levels of ILS due to their rapid diversification. Moreover, network analyses estimated deeper hybridization events between Petunia and Calibrachoa, genera that have different chromosome numbers. Although these genera cannot hybridize at the present time, ancestral hybridization could have played a role in their parallel radiations, as they share the same habitat and life history.

Dorsoventrally asymmetric expression of miR319/TCP generates dorsal-specific venation patterning in petunia corolla tube.

Vein-associated pigmentation (venation) is a type of floral coloration adopted by plants to attract pollinators. Several petunia (Petunia hybrida) lines generate dorsoventrally asymmetric venation patterning of the corolla tube, in which venation is only present in the dorsal tube. The molecular mechanism underlying this trait is unknown. Here, we demonstrate that miR319 is preferentially expressed in the dorsal corolla tube, leading to dorsoventrally asymmetric expression of its target genes. Transgenic lines overexpressing phy-miR319a generated uniform venation patterning of the corolla tube. Knockout of TCP genes targeted by miR319 promoted venation patterning in the ventral and dorsal tube, while overexpression of the miR319 target gene, PhTCP6, completely inhibited corolla tube venation patterning. In addition, miR319-targeted TCPs negatively regulated venation patterning, probably by repressing the regulator of venation patterning, AN4. Together, our data demonstrate that asymmetric expression of miR319 promotes venation patterning in the petunia dorsal tube alone by repressing the expression of its target TCP genes, which negatively regulate corolla tube venation patterning. These findings provide novel insights into how the dorsoventrally asymmetric distribution of venation patterning is established in zygomorphic flowers.

Complex evolution of novel red floral color in Petunia.

Red flower color has arisen multiple times and is generally associated with hummingbird pollination. The majority of evolutionary transitions to red color proceeded from purple lineages and tend to be genetically simple, almost always involving a few loss-of-function mutations of major phenotypic effect. Here we report on the complex evolution of a novel red floral color in the hummingbird-pollinated Petunia exserta (Solanaceae) from a colorless ancestor. The presence of a red color is remarkable because the genus cannot synthesize red anthocyanins and P. exserta retains a nonfunctional copy of the key MYB transcription factor AN2. We show that moderate upregulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE, restores anthocyanin biosynthesis in P. exserta. An essential shift in anthocyanin hydroxylation occurred through rebalancing the expression of three hydroxylating genes. Furthermore, the downregulation of an acyltransferase promotes reddish hues in typically purple pigments by preventing acyl group decoration of anthocyanins. This study presents a rare case of a genetically complex evolutionary transition toward the gain of a novel red color.

Enhancing cryopreservation survival in Petunia × Calibrachoa 'Light Yellow' callus: Insights into material characteristics and genetic integrity.

Petunia × Calibrachoa 'Light Yellow' (× Petchoa 'Light Yellow') is a kind of perennial herbaceous flower obtained through intergeneric hybridization of Petunia and Calibrachoa with high ornamental value and wide application, facing challenges in seed acquisition. Expanding propagation through tissue culture is an economically efficient means. Hence, establishing an effective procedure for the storage of callus is essential for × Petchoa 'Light Yellow'. Cryopreservation is an effective method for the in vitro propagation and long-term preservation of × Petchoa 'Light Yellow' germplasms. For formulating the optimization of the vitrification procedure, first, an orthogonal experimental design was employed to pinpoint critical steps in the vitrification protocol (pre-culture, osmoprotection, dehydration, and dilution) for Petunia × Calibrachoa callus tissues and then five additional factors (pre-culture, osmoprotection I and II, dehydration, and dilution) were optimized to further reduce the sample water content and enhance cell viability levels. The vitrification procedure was described as follows: callus tissues were precultured in MS solid medium with 0.3 M sucrose for 5 d, incubated with osmoprotection solution I and II for 15 min at 25 °C, respectively, cryoprotected with PVS2 for 30 min at 0 °C, and rapidly immersed in liquid nitrogen. Cryopreserved callus tissues were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25 °C and recovered on MS solid medium with 0.5 mg/L 6-BA and 0.1 mg/L NAA, and sucrose. The cell viability measured by TTC staining was approximately 16 %-18 % after 72 h-recovery. Following 45 days, the relative survival of callus reached up to 49.48 %. Furthermore, EST-SSR analysis showed no significant difference in the genetic stability of cryopreserved callus compared to the control. Based on the cryopreservation of × Petchoa 'Light Yellow' callus, we further evaluated the response of callus water contents to the osmotic stress in the optimized and original protocols (CK) for a higher cryopreservation survival. A comparative analysis of water content demonstrated that the procedure of gradual and gentle dehydration significantly improved water content and cell survival. Ultrastructural changes between cryopreserved and non-cryopreserved callus were examined and high vacuolation emerged as a key determinant, indicating its substantial impact on the low survival of cryopreserved cells, which should help us to understand the effectiveness of osmotic protectants in dehydration.

Genetic architecture of a pollinator shift and its fate in secondary hybrid zones of two Petunia species.

BACKGROUND: Theory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta. RESULTS: Using population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones. CONCLUSIONS: Among the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype-phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection.

ODORANT1 targets multiple metabolic networks in petunia flowers.

Scent bouquets produced by the flowers of Petunia spp. (petunia) are composed of a complex mixture of floral volatile benzenoid and phenylpropanoid compounds (FVBPs), which are specialized metabolites derived from phenylalanine (Phe) through an interconnected network of enzymes. The biosynthesis and emission of high levels of these volatiles requires coordinated transcriptional activation of both primary and specialized metabolic networks. The petunia R2R3-MYB transcription factor ODORANT 1 (ODO1) was identified as a master regulator of FVBP production and emission; however, our knowledge of the direct regulatory targets of ODO1 has remained limited. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq) in petunia flowers, we identify genome-wide ODO1-bound genes that are enriched not only in genes involved in the biosynthesis of the Phe precursor, as previously reported, but also genes associated with the specialized metabolic pathways involved in generating phenylpropanoid intermediates for FVBPs. ODO1-bound genes are also involved in methionine and S-adenosylmethionine metabolism, which could modulate methyl group supplies for certain FVBPs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and RNA-seq analysis in an ODO1 RNAi knockdown line revealed that ODO1-bound targets are expressed at lower levels when ODO1 is suppressed. A cis-regulatory motif, CACCAACCCC, was identified as a potential binding site for ODO1 in the promoters of genes that are both bound and activated by ODO1, which was validated by in planta promoter reporter assays with wild-type and mutated promoters. Overall, our work presents a mechanistic model for ODO1 controlling an extensive gene regulatory network that contributes to FVBP production to give rise to floral scent.

Diffusion of volatile organics and water in the epicuticular waxes of petunia petal epidermal cells.

Plant cuticles are a mixture of crystalline and amorphous waxes that restrict the exchange of molecules between the plant and the atmosphere. The multicomponent nature of cuticular waxes complicates the study of the relationship between the physical and transport properties. Here, a model cuticle based on the epicuticular waxes of Petunia hybrida flower petals was formulated to test the effect of wax composition on diffusion of water and volatile organic compounds (VOCs). The model cuticle was composed of an n-tetracosane (C24 H50 ), 1-docosanol (C22 H45 OH), and 3-methylbutyl dodecanoate (C17 H34 O2 ), reflecting the relative chain length, functional groups, molecular arrangements, and crystallinity of the natural waxes. Molecular dynamics simulations were performed to obtain diffusion coefficients for compounds moving through waxes of varying composition. Simulated VOC diffusivities of the model system were found to highly correlate with in vitro measurements in isolated petunia cuticles. VOC diffusivity increased up to 30-fold in completely amorphous waxes, indicating a significant effect of crystallinity on cuticular permeability. The crystallinity of the waxes was highly dependent on the elongation of the lattice length and decrease in gap width between crystalline unit cells. Diffusion of water and higher molecular weight VOCs were significantly affected by alterations in crystalline spacing and lengths, whereas the low molecular weight VOCs were less affected. Comparison of measured diffusion coefficients from atomistic simulations and emissions from petunia flowers indicates that the role of the plant cuticle in the VOC emission network is attributed to the differential control on mass transfer of individual VOCs by controlling the composition, amount, and dynamics of scent emission.

Altered profile of floral volatiles and lignin content by down-regulation of Caffeoyl Shikimate Esterase in Petunia.

BACKGROUND: The floral volatile profile of Petunia x hybrida 'Mitchell diploid' (MD) is dominated by phenylpropanoids, many of which are derived from p-coumaric acid. However, the downstream processes involved in the production of caffeoyl-CoA and feruloyl-CoA from p-coumaric acid are complex, as the genes and biosynthesis steps are associated with flavonoids and lignin synthesis as well as floral volatiles benzenoid/phenylpropanoid (FVBP). Caffeoyl shikimate esterase (CSE) converts caffeoyl shikimate to caffeic acid and is considered one of the essential regulators in lignin production. Moreover, CSE in involved in phenylpropanoid production. To investigate the roles of CSE in FVBP biosynthesis, we used RNAi-mediated CSE down-regulated (ir-PhCSE) petunias. RESULTS: Lowered CSE transcript accumulation in ir-PhCSE plants resulted in reduced lignin layers in the stems and stunted growth, suggesting a positive correlation between lignin layers and lignin content. The altered CSE level influenced the expression of many FVBP genes, including elevated transcripts of p-coumarate-3-hydroxylase (C3H), hydroxycinnamoyl transferase (HCT), and 4-coumaric acid: CoA ligase (4CL). In particular, the expression of C4H in ir-PhCSE plants was more than twice the expression in MD plants. Moreover, the production of volatile compounds was alterend in ir-PhCSE plants. Most floral volatiles decreased, and the amounts of phenylalanine and caffeic acid were significantly lower. CONCLUSIONS: Reduced lignin layers in the stems and stunted growth in ir-PhCSE plants suggest that PhCSE is essential for lignin production and plant growth in petunia. The decreased CSE level influenced the expression of many FVBP genes, and interference of shikimate derivates altered volatile compound production. Significantly decreased caffeic acid, but not ferulic acid, in ir-PhCSE plants suggest that CSE is primarily involved in the reaction of caffeoyl shikimate. Higher C3H and C4H transcripts seem to alleviate accumulated p-coumaric acid resulting from altered CSE. Finally, alteration in C3H, HCT, and 4CL in CSE down-regulated plants suggests an interaction of the FVBP genes, leading to the regulation of floral volatiles of petunia.

Transcriptomic analysis implicates ABA signaling and carbon supply in the differential outgrowth of petunia axillary buds.

BACKGROUND: Shoot branching of flowering plants exhibits phenotypic plasticity and variability. This plasticity is determined by the activity of axillary meristems, which in turn is influenced by endogenous and exogenous cues such as nutrients and light. In many species, not all buds on the main shoot develop into branches despite favorable growing conditions. In petunia, basal axillary buds (buds 1-3) typically do not grow out to form branches, while more apical axillary buds (buds 6 and 7) are competent to grow. RESULTS: The genetic regulation of buds was explored using transcriptome analyses of petunia axillary buds at different positions on the main stem. To suppress or promote bud outgrowth, we grew the plants in media with differing phosphate (P) levels. Using RNA-seq, we found many (> 5000) differentially expressed genes between bud 6 or 7, and bud 2. In addition, more genes were differentially expressed when we transferred the plants from low P to high P medium, compared with shifting from high P to low P medium. Buds 6 and 7 had increased transcript abundance of cytokinin and auxin-related genes, whereas the basal non-growing buds (bud 2 and to a lesser extent bud 3) had higher expression of strigolactone, abscisic acid, and dormancy-related genes, suggesting the outgrowth of these basal buds was actively suppressed. Consistent with this, the expression of ABA associated genes decreased significantly in apical buds after stimulating growth by switching the medium from low P to high P. Furthermore, comparisons between our data and transcriptome data from other species suggest that the suppression of outgrowth of bud 2 was correlated with a limited supply of carbon to these axillary buds. Candidate genes that might repress bud outgrowth were identified by co-expression analysis. CONCLUSIONS: Plants need to balance growth of axillary buds into branches to fit with available resources while allowing some buds to remain dormant to grow after the loss of plant parts or in response to a change in environmental conditions. Here we demonstrate that different buds on the same plant with different developmental potentials have quite different transcriptome profiles.

Evolutionary walks through flower colour space driven by gene expression in Petunia and allies (Petunieae).

The structure and function of biochemical and developmental pathways determine the range of accessible phenotypes, which are the substrate for evolutionary change. Accordingly, we expect that observed phenotypic variation across species is strongly influenced by pathway structure, with different phenotypes arising due to changes in activity along pathway branches. Here, we use flower colour as a model to investigate how the structure of pigment pathways shapes the evolution of phenotypic diversity. We focus on the phenotypically diverse Petunieae clade in the nightshade family, which contains ca 180 species of Petunia and related genera, as a model to understand how flavonoid pathway gene expression maps onto pigment production. We use multivariate comparative methods to estimate co-expression relationships between pathway enzymes and transcriptional regulators, and then assess how expression of these genes relates to the major axes of variation in floral pigmentation. Our results indicate that coordinated shifts in gene expression predict transitions in both total anthocyanin levels and pigment type, which, in turn, incur trade-offs with the production of UV-absorbing flavonol compounds. These findings demonstrate that the intrinsic structure of the flavonoid pathway and its regulatory architecture underlies the accessibility of pigment phenotypes and shapes evolutionary outcomes for floral pigment production.

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.