Painted flowers: Eluta generates pigment patterning in Antirrhinum.

In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum/Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face. We identified the gene underlying the Eluta locus by transposon-tagging, using an Antirrhinum line that spontaneously lost the nonsuppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and promoter activation/repression assays. Eluta competes with MYB activators for promoter cis-elements, and also by titrating essential cofactors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3-MYB factors, Rosea and Venosa, with the greatest repression on those parts of the petals where Eluta is most highly expressed. Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties.

Vaccinium as a comparative system for understanding of complex flavonoid accumulation profiles and regulation in fruit.

The genus Vaccinium L. (Ericaceae) contains premium berryfruit crops, including blueberry, cranberry, bilberry, and lingonberry. Consumption of Vaccinium berries is strongly associated with various potential health benefits, many of which are attributed to the relatively high concentrations of flavonoids, including the anthocyanins that provide the attractive red and blue berry colors. Because these phytochemicals are increasingly appealing to consumers, they have become a crop breeding target. There has been substantial recent progress in Vaccinium genomics and genetics together with new functional data on the transcriptional regulation of flavonoids. This is helping to unravel the developmental control of flavonoids and identify genetic regions and genes that can be selected for to further improve Vaccinium crops and advance our understanding of flavonoid regulation and biosynthesis across a broader range of fruit crops. In this update we consider the recent progress in understanding flavonoid regulation in fruit crops, using Vaccinium as an example and highlighting the significant gains in both genomic tools and functional analysis.

Stress, senescence, and specialized metabolites in bryophytes.

Life on land exposes plants to varied abiotic and biotic environmental stresses. These environmental drivers contributed to a large expansion of metabolic capabilities during land plant evolution and species diversification. In this review we summarize knowledge on how the specialized metabolite pathways of bryophytes may contribute to stress tolerance capabilities. Bryophytes are the non-tracheophyte land plant group (comprising the hornworts, liverworts, and mosses) and rapidly diversified following the colonization of land. Mosses and liverworts have as wide a distribution as flowering plants with regard to available environments, able to grow in polar regions through to hot desert landscapes. Yet in contrast to flowering plants, for which the biosynthetic pathways, transcriptional regulation, and compound function of stress tolerance-related metabolite pathways have been extensively characterized, it is only recently that similar data have become available for bryophytes. The bryophyte data are compared with those available for angiosperms, including examining how the differing plant forms of bryophytes and angiosperms may influence specialized metabolite diversity and function. The involvement of stress-induced specialized metabolites in senescence and nutrient response pathways is also discussed.

Hierarchical regulation of MYBPA1 by anthocyanin- and proanthocyanidin-related MYB proteins is conserved in Vaccinium species.

Members of the Vaccinium genus bear fruits rich in anthocyanins, a class of red-purple flavonoid pigments that provide human health benefits, although the localization and concentrations of anthocyanins differ between species: blueberry (V. corymbosum) has white flesh, while bilberry (V. myrtillus) has red flesh. Comparative transcriptomics between blueberry and bilberry revealed that MYBPA1.1 and MYBA1 strongly correlated with the presence of anthocyanins, but were absent or weakly expressed in blueberry flesh. MYBPA1.1 had a biphasic expression profile, correlating with both proanthocyanidin biosynthesis early during fruit development and anthocyanin biosynthesis during berry ripening. MYBPA1.1 was unable to induce anthocyanin or proanthocyanidin accumulation in Nicotiana benthamiana, but activated promoters of flavonoid biosynthesis genes. The MYBPA1.1 promoter is directly activated by MYBA1 and MYBPA2 proteins, which regulate anthocyanins and proanthocyanidins, respectively. Our findings suggest that the lack of VcMYBA1 expression in blueberry flesh results in an absence of VcMYBPA1.1 expression, which are both required for anthocyanin regulation. In contrast, VmMYBA1 is well expressed in bilberry flesh, up-regulating VmMYBPA1.1, allowing coordinated regulation of flavonoid biosynthesis genes and anthocyanin accumulation. The hierarchal model described here for Vaccinium may also occur in a wider group of plants as a means to co-regulate different branches of the flavonoid pathway.

Evolution and function of red pigmentation in land plants.

BACKGROUND: Land plants commonly produce red pigmentation as a response to environmental stressors, both abiotic and biotic. The type of pigment produced varies among different land plant lineages. In the majority of species they are flavonoids, a large branch of the phenylpropanoid pathway. Flavonoids that can confer red colours include 3-hydroxyanthocyanins, 3-deoxyanthocyanins, sphagnorubins and auronidins, which are the predominant red pigments in flowering plants, ferns, mosses and liverworts, respectively. However, some flowering plants have lost the capacity for anthocyanin biosynthesis and produce nitrogen-containing betalain pigments instead. Some terrestrial algal species also produce red pigmentation as an abiotic stress response, and these include both carotenoid and phenolic pigments. SCOPE: In this review, we examine: which environmental triggers induce red pigmentation in non-reproductive tissues; theories on the functions of stress-induced pigmentation; the evolution of the biosynthetic pathways; and structure-function aspects of different pigment types. We also compare data on stress-induced pigmentation in land plants with those for terrestrial algae, and discuss possible explanations for the lack of red pigmentation in the hornwort lineage of land plants. CONCLUSIONS: The evidence suggests that pigment biosynthetic pathways have evolved numerous times in land plants to provide compounds that have red colour to screen damaging photosynthetically active radiation but that also have secondary functions that provide specific benefits to the particular land plant lineage.

Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants.

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name "auronidin," with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants.

Discrete bHLH transcription factors play functionally overlapping roles in pigmentation patterning in flowers of Antirrhinum majus.

Floral pigmentation patterning is important for pollinator attraction as well as aesthetic appeal. Patterning of anthocyanin accumulation is frequently associated with variation in activity of the Myb, bHLH and WDR transcription factor complex (MBW) that regulates anthocyanin biosynthesis. Investigation of two classic mutants in Antirrhinum majus, mutabilis and incolorata I, showed they affect a gene encoding a bHLH protein belonging to subclade bHLH-2. The previously characterised gene, Delila, which encodes a bHLH-1 protein, has a bicoloured mutant phenotype, with residual lobe-specific pigmentation conferred by Incolorata I. Both Incolorata I and Delila induce expression of the anthocyanin biosynthetic gene DFR. Rosea 1 (Myb) and WDR1 proteins compete for interaction with Delila, but interact positively to promote Incolorata I activity. Delila positively regulates Incolorata I and WDR1 expression. Hierarchical regulation can explain the bicoloured patterning of delila mutants, through effects on both regulatory gene expression and the activity of promoters of biosynthetic genes like DFR that mediate MBW regulation. bHLH-1 and bHLH-2 proteins contribute to establishing patterns of pigment distribution in A. majus flowers in two ways: through functional redundancy in regulating anthocyanin biosynthetic gene expression, and through differences between the proteins in their ability to regulate genes encoding transcription factors.

MYBA and MYBPA transcription factors co-regulate anthocyanin biosynthesis in blue-coloured berries.

The regulatory network of R2R3 MYB transcription factors in anthocyanin biosynthesis is not fully understood in blue-coloured berries containing delphinidin compounds. We used blue berries of bilberry (Vaccinium myrtillus) to comprehensively characterise flavonoid-regulating R2R3 MYBs, which revealed a new type of co-regulation in anthocyanin biosynthesis between members of MYBA-, MYBPA1- and MYBPA2-subgroups. VmMYBA1, VmMYBPA1.1 and VmMYBPA2.2 expression was elevated at berry ripening and by abscisic acid treatment. Additionally, VmMYBA1 and VmMYBPA1.1 expression was strongly downregulated in a white berry mutant. Complementation and transient overexpression assays confirmed VmMYBA1 and VmMYBA2 to induce anthocyanin accumulation. Promoter activation assays showed that VmMYBA1, VmMYBPA1.1 and VmMYBPA2.2 had similar activity towards dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS), but differential regulation activity for UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) and flavonoid 3'5'-hydroxylase (F3'5'H) promoters. Silencing of VmMYBPA1.1 in berries led to the downregulation of key anthocyanin and delphinidin biosynthesis genes. Functional analyses of other MYBPA regulators, and a member of novel MYBPA3 subgroup, associated them with proanthocyanidin biosynthesis and F3'5'H expression. The existence of 18 flavonoid-regulating MYBs indicated gene duplication, which may have enabled functional diversification among MYBA, MYBPA1 and MYBPA2 subgroups. Our results provide new insights into the intricate regulation of the complex anthocyanin profile found in blue-coloured berries involving regulation of both cyanidin and delphinidin branches.

UVR8-mediated induction of flavonoid biosynthesis for UVB tolerance is conserved between the liverwort Marchantia polymorpha and flowering plants.

Damaging UVB radiation is a major abiotic stress facing land plants. In angiosperms the UV RESISTANCE LOCUS8 (UVR8) photoreceptor coordinates UVB responses, including inducing biosynthesis of protective flavonoids. We characterised the UVB responses of Marchantia polymorpha (marchantia), the model species for the liverwort group of basal plants. Physiological, chemical and transcriptomic analyses were conducted on wild-type marchantia exposed to three different UVB regimes. CRISPR/Cas9 was used to obtain plant lines with mutations for components of the UVB signal pathway or the flavonoid biosynthetic pathway, and transgenics overexpressing the marchantia UVR8 sequence were generated. The mutant and transgenic lines were analysed for changes in flavonoid content, their response to UVB exposure, and transcript abundance of a set of 48 genes that included components of the UVB response pathway characterised for angiosperms. The marchantia UVB response included many components in common with Arabidopsis, including production of UVB-absorbing flavonoids, the central activator role of ELONGATED HYPOCOTYL5 (HY5), and negative feedback regulation by REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1). Notable differences included the greater importance of CHALCONE ISOMERASE-LIKE (CHIL). Mutants disrupted in the response pathway (hy5) or flavonoid production (chalcone isomerase, chil) were more easily damaged by UVB. Mutants (rup1) or transgenics (35S:MpMYB14) with increased flavonoid content had increased UVB tolerance. The results suggest that UVR8-mediated flavonoid induction is a UVB tolerance character conserved across land plants and may have been an early adaptation to life on land.

Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants.

The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light- and nutrient-deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress-induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization.

A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots.

Plants require sophisticated regulatory mechanisms to ensure the degree of anthocyanin pigmentation is appropriate to myriad developmental and environmental signals. Central to this process are the activity of MYB-bHLH-WD repeat (MBW) complexes that regulate the transcription of anthocyanin genes. In this study, the gene regulatory network that regulates anthocyanin synthesis in petunia (Petunia hybrida) has been characterized. Genetic and molecular evidence show that the R2R3-MYB, MYB27, is an anthocyanin repressor that functions as part of the MBW complex and represses transcription through its C-terminal EAR motif. MYB27 targets both the anthocyanin pathway genes and basic-helix-loop-helix (bHLH) ANTHOCYANIN1 (AN1), itself an essential component of the MBW activation complex for pigmentation. Other features of the regulatory network identified include inhibition of AN1 activity by the competitive R3-MYB repressor MYBx and the activation of AN1, MYB27, and MYBx by the MBW activation complex, providing for both reinforcement and feedback regulation. We also demonstrate the intercellular movement of the WDR protein (AN11) and R3-repressor (MYBx), which may facilitate anthocyanin pigment pattern formation. The fundamental features of this regulatory network in the Asterid model of petunia are similar to those in the Rosid model of Arabidopsis thaliana and are thus likely to be widespread in the Eudicots.

Anthocyanin leaf markings are regulated by a family of R2R3-MYB genes in the genus Trifolium.

Anthocyanin pigments accumulate to form spatially restricted patterns in plants, particularly in flowers, but also occur in vegetative tissues. Spatially restricted anthocyanin leaf markings are poorly characterised in plants, but are common in forage legumes. We hypothesised that the molecular basis for anthocyanin leaf markings in Trifolium spp. is due to the activity of a family of R2R3-MYB genes. R2R3-MYB genes were identified that are associated with the two classic pigmentation loci in T. repens. The R locus patterns 'red leaf', 'red midrib' and 'red fleck' are conditioned by a single MYB gene, RED LEAF. The 'diffuse red leaf' trait is regulated by the RED LEAF DIFFUSE MYB gene. The V locus was identified through mapping two V-linked traits, 'V-broken yellow' (Vby) and 'red leaflet' (Vrl). Two highly similar R2R3-MYB genes, RED V-a and RED V-b, mapped to the V locus and co-segregated with the RED V pigmentation pattern. Functional characterisation of RED LEAF and RED V was performed, confirming their function as anthocyanin regulators and identifying a C-terminal region necessary for transactivation. The mechanisms responsible for generating anthocyanin leaf markings in T. repens provide a valuable system to compare with mechanisms that regulate complex floral pigmentation.

Failure to launch: the self-regulating Md-MYB10 R6 gene from apple is active in flowers but not leaves of Petunia.

KEY MESSAGE: The Md - MYB10 R6 gene from apple is capable of self-regulating in heterologous host species and enhancing anthocyanin pigmentation, but the activity of MYB10 is dependent on endogenous protein partners. Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples (Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md-MYB10 R6 , are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md-MYB10 R6 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md-MYB10 R6 transgene (MYB10-R6 pro :MYB10:MYB10 term ) activated anthocyanin synthesis when transiently expressed in Antirrhinum rosea (dorsea) petals and petunia leaf discs. Stable transgenic petunias containing Md-MYB10 R6 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of 'Mitchell' petunia. The absence of foliar pigmentation was due to the failure of the Md-MYB10 R6 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md-MYB10 R6 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals.

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in Cymbidium orchid.

MAIN CONCLUSION: This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. ß-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3' hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3'H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

Two genes, ANS and UFGT2, from Vaccinium spp. are key steps for modulating anthocyanin production.

Anthocyanins are a major group of red to blue spectrum plant pigments with many consumer health benefits. Anthocyanins are derived from the flavonoid pathway and diversified by glycosylation and methylation, involving the concerted action of specific enzymes. Blueberry and bilberry (Vaccinium spp.) are regarded as 'superfruits' owing to their high content of flavonoids, especially anthocyanins. While ripening-related anthocyanin production in bilberry (V. myrtillus) and blueberry (V. corymbosum) is regulated by the transcriptional activator MYBA1, the role of specific structural genes in determining the concentration and composition of anthocyanins has not been functionally elucidated. We isolated three candidate genes, CHALCONE SYNTHASE (VmCHS1), ANTHOCYANIDIN SYNTHASE (VmANS) and UDP-GLUCOSE : FLAVONOID-3-O-GLYCOSYLTRANSFERASE (VcUFGT2), from Vaccinium, which were predominantly expressed in pigmented fruit skin tissue and showed high homology between bilberry and blueberry. Agrobacterium-mediated transient expression of Nicotiana benthamiana showed that overexpression of VcMYBA1 in combination with VmANS significantly increased anthocyanin concentration (3-fold). Overexpression of VmCHS1 showed no effect above that induced by VcMYBA1, while VcUFGT2 modulated anthocyanin composition to produce delphinidin-3-galactosylrhamnoside, not naturally produced in tobacco. In strawberry (Fragaria × ananassa), combined transient overexpression of VcUFGT2 with a FLAVONOID 3´,5´-HYDROXYLASE from kiwifruit (Actinidia melanandra) modulated the anthocyanin profile to include galactosides and arabinosides of delphinidin and cyanidin, major anthocyanins in blueberry and bilberry. These findings provide insight into the role of the final steps of biosynthesis in modulating anthocyanin production in Vaccinium and may contribute to the targeted breeding of new cultivars with improved nutritional properties.

Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures) as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium - a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures), as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium-a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning.

We present an investigation of anthocyanin regulation over the entire petunia plant, determining the mechanisms governing complex floral pigmentation patterning and environmentally induced vegetative anthocyanin synthesis. DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) encode members of the R2R3-MYB transcription factor family that regulate anthocyanin synthesis in petunia, and control anthocyanin production in vegetative tissues and contribute to floral pigmentation. In addition to these two MYB factors, the basic helix-loop-helix (bHLH) factor ANTHOCYANIN1 (AN1) and WD-repeat protein AN11, are also essential for vegetative pigmentation. The induction of anthocyanins in vegetative tissues by high light was tightly correlated to the induction of transcripts for PHZ and AN1. Interestingly, transcripts for PhMYB27, a putative R2R3-MYB active repressor, were highly expressed during non-inductive shade conditions and repressed during high light. The competitive inhibitor PhMYBx (R3-MYB) was expressed under high light, which may provide feedback repression. In floral tissues DPL regulates vein-associated anthocyanin pigmentation in the flower tube, while PHZ determines light-induced anthocyanin accumulation on exposed petal surfaces (bud-blush). A model is presented suggesting how complex floral and vegetative pigmentation patterns are derived in petunia in terms of MYB, bHLH and WDR co-regulators.

The Coordinated Action of MYB Activators and Repressors Controls Proanthocyanidin and Anthocyanin Biosynthesis in Vaccinium.

Vaccinium berries are regarded as "superfoods" owing to their high concentrations of anthocyanins, flavonoid metabolites that provide pigmentation and positively affect human health. Anthocyanin localization differs between the fruit of cultivated highbush blueberry (V. corymbosum) and wild bilberry (V. myrtillus), with the latter having deep red flesh coloration. Analysis of comparative transcriptomics across a developmental series of blueberry and bilberry fruit skin and flesh identified candidate anthocyanin regulators responsible for this distinction. This included multiple activator and repressor transcription factors (TFs) that correlated strongly with anthocyanin production and had minimal expression in blueberry (non-pigmented) flesh. R2R3 MYB TFs appeared key to the presence and absence of anthocyanin-based pigmentation; MYBA1 and MYBPA1.1 co-activated the pathway while MYBC2.1 repressed it. Transient overexpression of MYBA1 in Nicotiana benthamiana strongly induced anthocyanins, but this was substantially reduced when co-infiltrated with MYBC2.1. Co-infiltration of MYBC2.1 with MYBA1 also reduced activation of DFR and UFGT, key anthocyanin biosynthesis genes, in promoter activation studies. We demonstrated that these TFs operate within a regulatory hierarchy where MYBA1 activated the promoters of MYBC2.1 and bHLH2. Stable overexpression of VcMYBA1 in blueberry elevated anthocyanin content in transgenic plants, indicating that MYBA1 is sufficient to upregulate the TF module and activate the pathway. Our findings identify TF activators and repressors that are hierarchically regulated by SG6 MYBA1, and fine-tune anthocyanin production in Vaccinium. The lack of this TF module in blueberry flesh results in an absence of anthocyanins.

A chromosome-scale assembly of the bilberry genome identifies a complex locus controlling berry anthocyanin composition.

Bilberry (Vaccinium myrtillus L.) belongs to the Vaccinium genus, which includes blueberries (Vaccinium spp.) and cranberry (V. macrocarpon). Unlike its cultivated relatives, bilberry remains largely undomesticated, with berry harvesting almost entirely from the wild. As such, it represents an ideal target for genomic analysis, providing comparisons with the domesticated Vaccinium species. Bilberry is prized for its taste and health properties and has provided essential nutrition for Northern European indigenous populations. It contains high concentrations of phytonutrients, with perhaps the most important being the purple colored anthocyanins, found in both skin and flesh. Here, we present the first bilberry genome assembly, comprising 12 pseudochromosomes assembled using Oxford Nanopore (ONT) and Hi-C Technologies. The pseudochromosomes represent 96.6% complete BUSCO genes with an assessed LAI score of 16.3, showing a high conservation of synteny against the blueberry genome. Kmer analysis showed an unusual third peak, indicating the sequenced samples may have been from two individuals. The alternate alleles were purged so that the final assembly represents only one haplotype. A total of 36,404 genes were annotated after nearly 48% of the assembly was masked to remove repeats. To illustrate the genome quality, we describe the complex MYBA locus, and identify the key regulating MYB genes that determine anthocyanin production. The new bilberry genome builds on the genomic resources and knowledge of Vaccinium species, to help understand the genetics underpinning some of the quality attributes that breeding programs aspire to improve. The high conservation of synteny between bilberry and blueberry genomes means that comparative genome mapping can be applied to transfer knowledge about marker-trait association between these two species, as the loci involved in key characters are orthologous.