Biosynthesis and regulation of flavonoids in buckwheat.

Buckwheat contains an abundance of antioxidants such as polyphenols and is considered a functional food. Among polyphenols, flavonoids have multiple functions in various aspects of plant growth and in flower and leaf colors. Flavonoids have antioxidant properties, and are thought to prevent cancer and cardiovascular disease. Here, we summarize the flavonoids present in various organs and their synthesis in buckwheat. We discuss the use of this information to breed highly functional and high value cultivars.

The grapevine NaE sodium exclusion locus encodes sodium transporters with diverse transport properties and localisation.

Grapevine (Vitis vinifera L.) is a valuable crop for human consumption and wine production, and is prone to suffering from salinity stress in arid regions or when exposed to low quality irrigation water. A previous study identified a quantitative trait locus (QTL) NaE, containing six High-affinity Potassium Transporter 1 genes, that was associated with shoot Na+ exclusion in grapevine. While HKT1;1 was predicted to be the most likely gene within this QTL to encode for this important salinity tolerance sub-trait, four other HKTs within the QTL remained uncharacterised; VviHKT1;2 encodes a truncated transcript unlikely to form a functional transporter. In this study, two allelic variants for each of VviHKT1;6, VviHKT1;7 and VviHKT1;8 from the heterozygous grapevine variety Cabernet Sauvignon were functionally characterised. Using the Xenopus laevis oocyte heterologous expression system, as well as transient expression in tobacco leaves, we found that the VviHKT1;6 and VviHKT1;7 alleles encoded plasma membrane localised proteins that facilitated significant non-rectifying Na+ transport. Conversely, proteins encoded by the VviHKT1;8 alleles were inwardly-rectifying, weak Na+ transporters that localised to intracellular organelles. Mining of previous RNA-seq gene expression data suggested that VviHKT1;6-8 are weakly expressed in grapevine roots, flower buds, and seeds under normal conditions and different nutrient regimes. We propose that VviHKT1;6 and VviHKT1;7 are likely to have a less significant role in grapevine leaf Na+ exclusion than VviHKT1;1, and that VviHKT1;8 is involved in endomembrane Na+ transport.

A whole canopy gas exchange system for the targeted manipulation of grapevine source-sink relations using sub-ambient CO2.

BACKGROUND: Elucidating the effect of source-sink relations on berry composition is of interest for wine grape production as it represents a mechanistic link between yield, photosynthetic capacity and wine quality. However, the specific effects of carbohydrate supply on berry composition are difficult to study in isolation as leaf area or crop adjustments can also change fruit exposure, or lead to compensatory growth or photosynthetic responses. A new experimental system was therefore devised to slow berry sugar accumulation without changing canopy structure or yield. This consisted of six transparent 1.2 m3 chambers to enclose large pot-grown grapevines, and large soda-lime filled scrubbers that reduced carbon dioxide (CO2) concentration of day-time supply air by approximately 200 ppm below ambient. RESULTS: In the first full scale test of the system, the chambers were installed on mature Shiraz grapevines for 14 days from the onset of berry sugar accumulation. Three chambers were run at sub-ambient CO2 for 10 days before returning to ambient. Canopy gas exchange, and juice hexose concentrations were determined. Net CO2 exchange was reduced from 65.2 to 30 g vine- 1 day- 1, or 54%, by the sub-ambient treatment. At the end of the 10 day period, total sugar concentration was reduced from 95 to 77 g L- 1 from an average starting value of 23 g L- 1, representing a 25% reduction. Scaling to a per vine basis, it was estimated that 223 g of berry sugars accumulated under ambient supply compared to 166 g under sub-ambient, an amount equivalent to 50 and 72% of total C assimilated. CONCLUSIONS: Through supply of sub-ambient CO2 using whole canopy gas exchange chambers system, an effective method was developed for reducing photosynthesis and slowing the rate of berry sugar accumulation without modifying yield or leaf area. While in this case developed for further investigations of grape and wine composition, the system has broader applications for the manipulation and of study of grapevine source-sink relations.

The MYB5-driven MBW complex recruits a WRKY factor to enhance the expression of targets involved in vacuolar hyper-acidification and trafficking in grapevine.

The accumulation of secondary metabolites and the regulation of tissue acidity contribute to the important traits of grape berry and influence plant performance in response to abiotic and biotic factors. In several plant species a highly conserved MYB-bHLH-WD (MBW) transcriptional regulatory complex controls flavonoid pigment synthesis and transport, and vacuolar acidification in epidermal cells. An additional component, represented by a WRKY-type transcription factor, physically interacts with the complex increasing the expression of some target genes and adding specificity for other targets. Here we investigated the function of MBW(W) complexes involving two MYBs (VvMYB5a and VvMYB5b) and the WRKY factor VvWRKY26 in grapevine (Vitis vinifera L.). Using transgenic grapevine plants we showed that these complexes affected different aspects of morphology, plant development, pH regulation, and pigment accumulation. Transcriptomic analysis identified a core set of putative target genes controlled by VvMYB5a, VvMYB5b, and VvWRKY26 in different tissues. Our data indicated that VvWRKY26 enhances the expression of selected target genes induced by VvMYB5a/b. Among these targets are genes involved in vacuolar hyper-acidification, such as the P-type ATPases VvPH5 and VvPH1, and trafficking, and genes involved in the biosynthesis of flavonoids. In addition, VvWRKY26 is recruited specifically by VvMYB5a, reflecting the functional diversification of VvMYB5a and VvMYB5b. The expression of MBWW complexes in vegetative organs, such as leaves, indicates a possible function of vacuolar hyper-acidification in the repulsion of herbivores and/or in developmental processes, as shown by defects in transgenic grape plants where the complex is inactivated.

Grape berry flavonoids: a review of their biochemical responses to high and extreme high temperatures.

Climate change scenarios predict an increase in average temperatures and in the frequency, intensity, and length of extreme temperature events in many wine regions around the world. In already warm and hot regions, such changes may compromise grape growing and the production of high quality wine as high temperature has been found to affect berry composition critically. Most recent studies focusing on the sole effect of temperature, separated from light and water, on grape berry composition found that high temperature affects a wide range of metabolites, and in particular flavonoids-key compounds for berry and wine quality. A decrease in total anthocyanins is reported in most cases, and appears to be directly associated with high temperature. Changes in anthocyanin composition, and flavonol and proanthocyanidin responses are however less consistent, and reflect the complexity of the underlying biosynthetic pathways and diversity of experimental treatments that have been used in these studies. This review examines the impact of high temperature on the biosynthesis, accumulation, and degradation of flavonoids, and attempts to reconcile the diversity of responses in relation to the latest understanding of flavonoid chemistry and molecular regulation.

Buckwheat R2R3 MYB transcription factor FeMYBF1 regulates flavonol biosynthesis.

Buckwheat (Fagopyrum esculentum) contains high amounts of flavonoids, especially flavonols (e.g., rutin), which are thought to be highly beneficial for human health. Little is known, however, about the regulation of flavonol synthesis in buckwheat. We identified a buckwheat gene encoding an R2R3 MYB transcription factor, and named this gene FeMYBF1. Analysis of the deduced amino acid sequence and phylogenetic analysis suggested that FeMYBF1 encodes an ortholog of the Arabidopsis flavonol regulators AtMYB11, AtMYB12 and AtMYB111. Expression of FeMYBF1 in a flavonol-deficient Arabidopsis triple mutant (myb11 myb12 myb111) restored flavonol synthesis. Constitutive expression of FeMYBF1 driven by the CaMV 35S promoter in Arabidopsis resulted in over-accumulation of flavonol glycosides and upregulation of the expression of AtFLS1. Transient expression assays showed that FeMYBF1 activated the promoter of the Arabidopsis gene encoding AtFLS1, and the promoters of buckwheat genes related to anthocyanin and proanthocyanidin synthesis such as dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) in addition to genes encoding FLS. The results indicate that FeMYBF1 regulates flavonol synthesis and may have a role in synthesis of other flavonoid compounds, and also that buckwheat may have alternative pathway of flavonol synthesis through DFR and LDOX.

Measuring Dipole Inversion in Self-Assembled Nano-Dielectric Molecular Layers.

A self-assembled nanodielectric (SAND) is an ultrathin film, typically with periodic layer pairs of high-k oxide and phosphonic-acid-based π-electron (PAE) molecular layers. IPAE, having a molecular structure similar to that of PAE but with an inverted dipole direction, has recently been developed for use in thin-film transistors. Here we report that replacing PAE with IPAE in SAND-based thin-film transistors induces sizable threshold and turn-on voltage shifts, indicating the flipping of the built-in SAND polarity. The bromide counteranion (Br-) associated with the cationic stilbazolium portion of PAE or IPAE is of great importance, because its relative position strongly affects the electric dipole moment of the organic layer. Hence, a set of X-ray synchrotron measurements were designed and performed to directly measure and compare the Br- distributions within the PAE and IPAE SANDs. Two trilayer SANDs, consisting of a PAE or IPAE layer sandwiched between an HfOx and a ZrOx layer, were deposited on the SiOx surface of Si substrates or periodic Si/Mo multilayer substrates for X-ray reflectivity and X-ray standing wave measurements, respectively. Along with complementary DFT simulations, the spacings, elemental (Hf, Br, and Zr) distributions, molecular orientations, and Mulliken charge distributions of the PAE and IPAE molecules within each of the SAND trilayers were determined and correlated with the dipole inversion.

Functional differences in transport properties of natural HKT1;1 variants influence shoot Na+ exclusion in grapevine rootstocks.

Under salinity, Vitis spp. rootstocks can mediate salt (NaCl) exclusion from grafted V. vinifera scions enabling higher grapevine yields and production of superior wines with lower salt content. Until now, the genetic and mechanistic elements controlling sodium (Na+ ) exclusion in grapevine were unknown. Using a cross between two Vitis interspecific hybrid rootstocks, we mapped a dominant quantitative trait locus (QTL) associated with leaf Na+ exclusion (NaE) under salinity stress. The NaE locus encodes six high-affinity potassium transporters (HKT). Transcript profiling and functional characterization in heterologous systems identified VisHKT1;1 as the best candidate gene for controlling leaf Na+ exclusion. We characterized four proteins encoded by unique VisHKT1;1 alleles from the parents, and revealed that the dominant HKT variants exhibit greater Na+ conductance with less rectification than the recessive variants. Mutagenesis of VisHKT1;1 and TaHKT1.5-D from bread wheat, demonstrated that charged amino acid residues in the eighth predicted transmembrane domain of HKT proteins reduces inward Na+ conductance, and causes inward rectification of Na+ transport. The origin of the recessive VisHKT1;1 alleles was traced to V. champinii and V. rupestris. We propose that the genetic and functional data presented here will assist with breeding Na+ -tolerant grapevine rootstocks.

Genotyping by Sequencing in Almond: SNP Discovery, Linkage Mapping, and Marker Design.

In crop plant genetics, linkage maps provide the basis for the mapping of loci that affect important traits and for the selection of markers to be applied in crop improvement. In outcrossing species such as almond (Prunus dulcis Mill. D. A. Webb), application of a double pseudotestcross mapping approach to the F1 progeny of a biparental cross leads to the construction of a linkage map for each parent. Here, we report on the application of genotyping by sequencing to discover and map single nucleotide polymorphisms in the almond cultivars "Nonpareil" and "Lauranne." Allele-specific marker assays were developed for 309 tag pairs. Application of these assays to 231 Nonpareil × Lauranne F1 progeny provided robust linkage maps for each parent. Analysis of phenotypic data for shell hardness demonstrated the utility of these maps for quantitative trait locus mapping. Comparison of these maps to the peach genome assembly confirmed high synteny and collinearity between the peach and almond genomes. The marker assays were applied to progeny from several other Nonpareil crosses, providing the basis for a composite linkage map of Nonpareil. Applications of the assays to a panel of almond clones and a panel of rootstocks used for almond production demonstrated the broad applicability of the markers and provide subsets of markers that could be used to discriminate among accessions. The sequence-based linkage maps and single nucleotide polymorphism assays presented here could be useful resources for the genetic analysis and genetic improvement of almond.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

BACKGROUND: Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. RESULTS: By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. CONCLUSIONS: We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum).

Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat.

Solution-Processed Self-Assembled Nanodielectrics on Template-Stripped Metal Substrates.

The coupling of hybrid organic-inorganic gate dielectrics with emergent unconventional semiconductors has yielded transistor devices exhibiting record-setting transport properties. However, extensive electronic transport measurements on these high-capacitance systems are often convoluted with the electronic response of the semiconducting silicon substrate. In this report, we demonstrate the growth of solution-processed zirconia self-assembled nanodielectrics (Zr-SAND) on template-stripped aluminum substrates. The resulting Zr-SAND on Al structures leverage the ultrasmooth (r.m.s. roughness <0.4 nm), chemically uniform nature of template-stripped metal substrates to demonstrate the same exceptional electronic uniformity (capacitance ∼700 nF cm(-2), leakage current <1 µA cm(-2) at -2 MV cm(-1)) and multilayer growth of Zr-SAND on Si, while exhibiting superior temperature and voltage capacitance responses. These results are important to conduct detailed transport measurements in emergent transistor technologies featuring SAND as well as for future applications in integrated circuits or flexible electronics.

A Grapevine Anthocyanin Acyltransferase, Transcriptionally Regulated by VvMYBA, Can Produce Most Acylated Anthocyanins Present in Grape Skins.

Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species. They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilate N-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro.

Grapevine and Arabidopsis Cation-Chloride Cotransporters Localize to the Golgi and Trans-Golgi Network and Indirectly Influence Long-Distance Ion Transport and Plant Salt Tolerance.

Plant cation-chloride cotransporters (CCCs) have been implicated in conferring salt tolerance. They are predicted to improve shoot salt exclusion by directly catalyzing the retrieval of sodium (Na(+)) and chloride (Cl(-)) ions from the root xylem. We investigated whether grapevine (Vitis vinifera [Vvi]) CCC has a role in salt tolerance by cloning and functionally characterizing the gene from the cultivar Cabernet Sauvignon. Amino acid sequence analysis revealed that VviCCC shares a high degree of similarity with other plant CCCs. A VviCCC-yellow fluorescent protein translational fusion protein localized to the Golgi and the trans-Golgi network and not the plasma membrane when expressed transiently in tobacco (Nicotiana benthamiana) leaves and Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. AtCCC-green fluorescent protein from Arabidopsis also localized to the Golgi and the trans-Golgi network. In Xenopus laevis oocytes, VviCCC targeted to the plasma membrane, where it catalyzed bumetanide-sensitive (36)Cl(-), (22)Na(+), and (86)Rb(+) uptake, suggesting that VviCCC (like AtCCC) belongs to the Na(+)-K(+)-2Cl(-) cotransporter class of CCCs. Expression of VviCCC in an Arabidopsis ccc knockout mutant abolished the mutant's stunted growth phenotypes and reduced shoot Cl(-) and Na(+) content to wild-type levels after growing plants in 50 mm NaCl. In grapevine roots, VviCCC transcript abundance was not regulated by Cl(-) treatment and was present at similar levels in both the root stele and cortex of three Vitis spp. genotypes that exhibit differential shoot salt exclusion. Our findings indicate that CCC function is conserved between grapevine and Arabidopsis, but neither protein is likely to directly mediate ion transfer with the xylem or have a direct role in salt tolerance.

Shoot chloride exclusion and salt tolerance in grapevine is associated with differential ion transporter expression in roots.

BACKGROUND: Salt tolerance in grapevine is associated with chloride (Cl-) exclusion from shoots; the rate-limiting step being the passage of Cl- between the root symplast and xylem apoplast. Despite an understanding of the physiological mechanism of Cl- exclusion in grapevine, the molecular identity of membrane proteins that control this process have remained elusive. To elucidate candidate genes likely to control Cl- exclusion, we compared the root transcriptomes of three Vitis spp. with contrasting shoot Cl- exclusion capacities using a custom microarray. RESULTS: When challenged with 50 mM Cl-, transcriptional changes of genotypes 140 Ruggeri (shoot Cl- excluding rootstock), K51-40 (shoot Cl- including rootstock) and Cabernet Sauvignon (intermediate shoot Cl- excluder) differed. The magnitude of salt-induced transcriptional changes in roots correlated with the amount of Cl- accumulated in shoots. Abiotic-stress responsive transcripts (e.g. heat shock proteins) were induced in 140 Ruggeri, respiratory transcripts were repressed in Cabernet Sauvignon, and the expression of hypersensitive response and ROS scavenging transcripts was altered in K51-40. Despite these differences, no obvious Cl- transporters were identified. However, under control conditions where differences in shoot Cl- exclusion between rootstocks were still significant, genes encoding putative ion channels SLAH3, ALMT1 and putative kinases SnRK2.6 and CPKs were differentially expressed between rootstocks, as were members of the NRT1 (NAXT1 and NRT1.4), and CLC families. CONCLUSIONS: These results suggest that transcriptional events contributing to the Cl- exclusion mechanism in grapevine are not stress-inducible, but constitutively different between contrasting varieties. We have identified individual genes from large families known to have members with roles in anion transport in other plants, as likely candidates for controlling anion homeostasis and Cl- exclusion in Vitis species. We propose these genes as priority candidates for functional characterisation to determine their role in chloride transport in grapevine and other plants.

The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera.

Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB-type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine.

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR-NB-LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated resistance to Uncinula necator (MrRUN1) and resistance to Plasmopara viticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south-eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1-mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR-NB-LRR genes at this locus share a common ancestor.

Identification of key amino acids for the evolution of promoter target specificity of anthocyanin and proanthocyanidin regulating MYB factors.

A complex of R2R3-MYB and bHLH transcription factors, stabilized by WD40 repeat proteins, regulates gene transcription for plant cell pigmentation and epidermal cell morphology. It is the MYB component of this complex which specifies promoter target activation. The Arabidopsis MYB TT2 regulates proanthocyanidin (PA) biosynthesis by activating the expression of ANR (anthocyanidin reductase), the gene product of which catalyzes the first committed step of this pathway. Conversely the closely related MYB PAP4 (AtMYB114) regulates the anthocyanin pathway and specifically activates UFGT (UDP-glucose:flavonoid-3-O-glucosyltransferase), encoding the first enzyme of the anthocyanin pathway. Both at the level of structural and regulatory genes, evolution of PA biosynthesis proceeded anthocyanin biosynthesis and we have identified key residues in these MYB transcription factors for the evolution of target promoter specificity. Using chimeric and point mutated variants of TT2 and PAP4 we found that exchange of a single amino acid, Gly/Arg(39) in the R2 domain combined with an exchange of a four amino acid motif in the R3 domain, could swap the pathway selection of TT2 and PAP4, thereby converting in planta specificity of the PA towards the anthocyanin pathway and vice versa. The general importance of these amino acids for target specificity was also shown for the grapevine transcription factors VvMYBPA2 and VvMYBA2 which regulate PAs and anthocyanins, respectively. These results provide an insight into the evolution of the different flavonoid regulators from a common ancestral gene.

The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries.

Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and health-promoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera 'Shiraz') R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.