Resistance breeding offers invaluable perspectives for environment-friendly crop protection, but its success may be limited by the breakdown of plant resistance by pathogen strains. With the breeding and use of varieties carrying multiple genetic resistances, grapevine (Vitis spp.) represents a distinctive model for perennials to investigate the agreement that pyramiding broadens and enhances the efficacy and durability of resistance. To this end, grapevine progenies segregating for four major resistance loci against Plasmopara viticola (Rpvs) were used to evaluate single and pyramided loci when confronted with naive and Rpv-breaking pathogen strains. In pyramiding, undefeated and defeated Rpvs provided either beneficial, neutral, or detrimental quantitative effects, depending on the loci combination and pathogen strain. In particular, the fact that defeated loci may compromise resistance highlighted important implications for the breeding of perennials. Thorough phenotypic investigations of pyramiding schemes emerge as a critical step for the effective and durable management of genetic resistances.

Grape varieties carrying “Resistance to Plasmopara viticola” (Rpv) loci represent an effective solution to mitigate the environmental impact of fungicide application in viticulture. However, P. viticola strains able to overcome major Rpv have become a main threat to their cultivation. Pyramiding resistance loci in the same variety enhances plant resistance, but interactions involving stacked and defeated Rpv and different P. viticola strains are poorly documented. Investigation of these interactions may uncover information for the efficient deployment of Rpv and durable resistances. In the present study, a grapevine offspring carrying single and pyramided Rpv1, Rpv3.1 and Rpv10 was phenotyped for the resistance to P. viticola using a naive strain and a strain virulent towards Rpv10. By using high-resolution phenotyping applied to grapevine phytoalexins and P. viticola metabolic biomarkers, we demonstrated that the efficacy of Rpv combinations and aggressiveness of P. viticola strains are differentiated in the early phase of infection. Quantitative early and late infection outcomes based on P. viticola lipid biomarkers were closely related and provided complementary information. In particular, they evidenced the residual effect of a defeated Rpv and its importance in pyramiding, thereby providing keys to streamline resistance research and utilization in grapevine. In contrast, the accumulation of grapevine stilbenoids depended on infection severity, Rpv combination and genotype, highlighting the need for caution in their use as resistance biomarkers. The implementation of pathogen metabolic biomarkers-based phenotyping in resistance breeding represents an innovation that can be adapted to other plant pathosystems.

Resistance breeding offers invaluable perspectives for environment-friendly crop protection, but its success may be limited by the breakdown of plant resistance by pathogen strains. This threat is particularly acute for perennial crops, which may be cultivated for several decades. With the increasing use of new varieties carrying multiple major resistance loci, grapevine ( Vitis spp.) represents a distinctive model to investigate the broad agreement that combining several resistance genes (pyramiding) enhances both resistance efficacy and durability. To this end, grapevine progenies segregating for four resistance loci against Plasmopara viticola ( Rpvs ) were used to evaluate the efficiency of single and pyramided major loci when confronted to naive and Rpv -breaking pathogen strains. In the context of polygenic resistance, both undefeated and defeated Rpvs provided significant quantitative effects. However, interactions between pyramided Rpvs were either beneficial, neutral or detrimental to the level of resistance, depending on the loci combination and pathogen strain. In particular, the fact that the presence of defeated resistance loci may compromise the resistance provided by functional major loci has important implications for crops resistance breeding. Thorough phenotypic investigations of pyramiding breeding schemes emerge as a critical step for the effective and durable management of genetic resistances and plant diseases. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la RechercheAgence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-22-CE92-0005 Deutsche ForschungsgemeinschaftDeutsche Forschungsgemeinschaft, , 504993256

Sunburn on grapes has emerged as an economic concern in recent decades. The phenomenon can be attributed to climatic changes, characterised by elevated air temperatures and intense solar radiation, leading to increased crop failures as well as quality losses of wines. This study aimed to evaluate comprehensively selected preventive strategies for the sunburn-sensitive, white cultivar ‘Riesling’. To this end, the impact of the timing of bunch zone defoliation on sunburn necrosis within three wine-growing regions in Central Europe, as well as the application of sunscreens onto grapes and shading by protective nets at one of these sites, were visually assessed in the contrasting growing seasons 2021 and 2022, respectively. Early defoliation between the end of flowering and fruit set decreased sunburn necrosis severity by down to 59.6 % in comparison to late defoliation at bunch closure across the two years. Furthermore, rot severity was reduced by down to 85.3 % in comparison to no defoliation across both growing seasons. The application of lime onto grapes following late defoliation diminished sunburn damage by 41.9 % across both experimental years, while kaolin showed no effect. Combining early defoliation with application of lime revealed an increased effectiveness of 79.2 % in comparison to late defoliation within one growing season. Different protective nets were able to lower sunburn damage within each growing season, but the black, small-meshed shading net with the highest shading factor investigated revealed the highest effectiveness of 63.7 % across both experimental years. However, the effectiveness of defoliation, sunscreens and protective nets depended on the meteorological conditions during each growing season. Total yield, including grapes with different levels of damage, as well as the vigour of vines, were not impacted by the viticultural measures. Malic acid and total acidity concentrations in the must were decreased by early defoliation compared to no defoliation within each year, while must weight was not influenced by any measure. In conclusion, this study compared the potential of different prevention strategies and highlighted a combined positive effect of early defoliation at two experimental sites and within each growing season: Both sunburn necrosis and rot severity were reduced in comparison to the respective maximum extent.

The high susceptibility of European grapevine cultivars to downy mildew (DM) and powdery mildew (PM) causes the intensive use of fungicides. Fungus-resistant cultivars (FRCs) with different resistance (R) loci have been bred and could play an important role in reducing plant protection treatments (PPTs). However, little information is available about the extent to which PPTs can be reduced in the field through the use of FRCs and the associated economic advantages. In this study, different strategies with reduced PPTs on FRCs were tested in field experiments. The results demonstrated that the number of PPTs can be reduced by 60 to 90%, resulting in reductions in applied copper and sulfur by 52 to 79% through the use of FRCs compared with susceptible cultivars, without affecting grape or plant health. The saving potential varied among years, depending on the type of R loci and climatic conditions. Furthermore, this study highlights that completely omitting PPTs in the cultivation of FRCs can result in PM or DM infections and possible loss of yield and fruit quality. In addition to the field experiments, a two-year observation of the performance of FRCs in commercial vineyards was undertaken, which highlighted not only the significant reduction in PPTs but also the financial savings that can be achieved through the use of FRCs.

Climate change is an environmental challenge that impacts the wine industry due to frequent sunburn damage triggered by heat waves, sun radiation and severe water deficits. This leads to severe yield losses and sensory changes in the resulting wines, presumably caused by climate -induced off-flavours. This study aims to develop viticultural and oenological strategies to mitigate sunburn damage in the highly sensitive variety Riesling and its detrimental sensory properties. In 2020, we combined the timing and intensity of defoliation measures with the application of kaolin and calcium hydroxide suspensions, reflecting a portion of the solar radiation. Seven treatments were replicated in three field experiments. Grapes from each field replication were maintained as a fermentation replication and, therefore, separately vinified following a standardised protocol. Replicates, including field and fermentation replicates, were further used as sensory replications. Descriptive analysis (DA) and temporal dominance of sensations (TDS) revealed a significant sensory impact of experimental trials in eight of twelve attributes. Additionally, wines were analysed by gas chromatography-mass spectrometry. The early sun exposition treatment with partial defoliation of the grape zone after flowering, followed by a second defoliation at berry closure, reduced fruity aroma but increased smoky notes due to enhanced 4-vinylguajacol formation as well as the intensity of the atypical ageing note (ATA) reminiscent of acacia blossom, naphthalene and fusel alcohols. Applying kaolin or calcium hydroxide particles on the berry skin slightly mitigated these unpleasant effects and even increased fruitiness and sweetness. Late defoliation at berry closure diminished the green note, which was most prominent in the non-defoliated control. Further smokiness and ATA intensities were lowered, in contrast to their rise due to early defoliation. However, late defoliation increased 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), causing the petrol off -flavour. TDS analysis revealed a higher fruity and sweet dominance during the first 10 seconds due to early defoliation, while late defoliation fostered dominant and lingering sourness. In conclusion, early defoliation, which lowers sunburn incidence due to an early adaptation towards sun exposure, increases smoky and ATA flavours and diminishes fruitiness. These detrimental sensory effects could be significantly mitigated by applying reflecting particles of kaolin and calcium hydroxide on berry skins.

The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis vinifera L.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we demonstrate how DNA affinity purification sequencing (DAP-Seq) allows for genome-wide TF binding site interrogation in grape. We obtained 5,190 and 4,443 binding events assigned to 4,041 and 3,626 genes for MYB14 and MYB15, respectively (around 40% of peaks located within -10kb of transcription start sites). DAP-Seq of MYB14/MYB15 was combined with aggregate gene co-expression networks (GCNs) built from more than 1,400 transcriptomic datasets from leaves, fruits and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15 and MYB13, a third uncharacterised member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 out of 47 STS family genes. Moreover, all three MYBs bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene candidates amongst which ROMT2-3 were validated enzymatically. A high proportion of DAP-Seq bound genes was induced in the activated transcriptomes of transient MYB15-overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3-MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP-Seq and reciprocal GCNs offers a rapid framework for gene function characterisation using genome-wide approaches in the context of non-model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialised metabolism.

The stilbenoid pathway is responsible for the production of resveratrol and its derivatives in grapevine. A few transcription factors (TFs) have been previously identified as regulators of this pathway but the extent of this control is yet to be fully understood. Here we demonstrate how DNA affinity purification sequencing (DAP-Seq) allows for genome-wide TF binding site interrogation in a non-model species. We obtained 5,190 and 4,443 binding events assigned to 4,041 and 3,626 genes for MYB14 and MYB15, respectively (around 40% of peaks being located within -10kb of transcription start sites). DAP-Seq of MYB14 and MYB15 was combined with aggregate gene centred co-expression networks built from more than 1,400 transcriptomic datasets from leaves, fruits and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15 and MYB13, a third uncharacterised member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS ) targets, these three regulators bind to 30 out of 47 STS family genes. Moreover all three MYBs bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and novel resveratrol-modifying gene candidates amongst which ROMT2 -3 were validated enzymatically. A high proportion of DAP-Seq bound genes was induced in the activated transcriptomes of transient MYB15 -overexpressing stilbenoid-producing grapevine leaves, validating our methodological approach for identifying gene regulatory networks of specialised metabolism. Overall, MYB genes from Subgroup 2 appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production.

Background and Aims The flavonoid composition of grapes and wine was determined in transgenic grapevines with altered expression of the tannin synthesis genes anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). Methods and Results Shiraz grapevines were transformed with constructs aimed at decreasing expression of genes encoding VvANR and VvLAR1. Many of the ANRsi (silenced) transgenic lines had an altered appearance and a low concentration of leaf tannin but produced no fruit. Those that did produce fruit had a higher concentration of flavonol and a lower concentration of tannin in the skin but no change in seed or overall berry tannin. The ANRsi wines had a higher concentration of anthocyanin, flavonol, tannin and phenolic substances and higher colour. The LARsi lines had a lower concentration of tannin in the leaves and seeds but not in berry skins and produced wines with a lower concentration of anthocyanin, tannin and phenolic substances and lower colour. Conclusions Manipulation of tannin genes altered flavonoid composition in grapevines and suggests that ANR is more important for tannin synthesis in leaves and berry skins whereas LAR1 is involved more in tannin production in seeds. Significance of the Study There is potential to alter wine colour and tannin properties by manipulating the flavonoid pathway in grapevines.

The control of plant specialised metabolism is exerted by transcription factors and co-regulators acting on cis -regulatory DNA sequences of pathway-structural genes, determining when, where, and how metabolites are accumulated. A particularly interesting case for studying the transcriptional control of metabolism is represented by stilbenoids, produced within the phenylpropanoid pathway, as their ability to inhibit infection by coronaviruses MERS-CoV and SARS-CoV has been recently demonstrated in vitro . Integrative omic studies in grapevine ( Vitis vinifera L.), including gene coexpression networks, have previously highlighted several transcription factors (TFs) from different gene families as potential modulators of stilbenoid accumulation, offering an ideal framework for gene function characterisation using genome-wide approaches. In the context of non-model plant species, DNA affinity purification sequencing (DAP-Seq) results a novel and potentially powerful tool for the analysis of novel uncharacterised regulators, however, it has not yet been applied in fruit crops. Accordingly, we tested as a proof-of-concept the binding of two previously characterised R2R3-MYB TFs to their known targets of the stilbene pathway, MYB14 and MYB15, obtaining 5,222 and 4,502 binding events assigned to 4,038 and 3,645 genes for each TF, respectively. Bound genes (putative targets) were overlapped with aggregated gene centred co-expression networks resulting in shared and exclusive High Confidence Targets (HCTs) suggesting a high, but not complete, redundancy. Our results show that in addition to the previously known but few STS targets, these regulators bind to almost half of the complete STS family in addition to other phenylpropanoid-and stilbenoid-related genes. We also suggest they are potentially involved in other processes such as the circadian rhythm or the synthesis of biotin. We searched the activated transcriptomes of transiently MYB15 -overexpressing grapevine plants and observed a large activation of its high confidence targets, validating our methodological approach. Our results also show that MYB15 seems to play a role in regulating other stilbenoid-related TFs such as WRKY03. were sequenced on an Illumina NextSeq 500 (sequencing of libraries was set at 30 million and 1x75bp single-end reads). As a negative control, we performed DAP-Seq with the pIX-HALO expression vector without any ORF inserted, accounting for possible non-specific DNA binding, as well as copy number variations at specific genomic loci. All experiments included two biological replicates. Grapevine Genome Characterization. The grapevine genome sequence suggests ancestral hexaploidization angiosperm

A wild grape haplotype (Rpv3-1) confers resistance to Plasmopara viticola. We mapped the causal factor for resistance to an interval containing a TIR-NB-LRR (TNL) gene pair that originated 1.6-2.6 million years ago by a tandem segmental duplication. Transient coexpression of the TNL pair in Vitis vinifera leaves activated pathogen-induced necrosis and reduced sporulation compared with control leaves. Even though transcripts of the TNL pair from the wild haplotype appear to be partially subject to nonsense-mediated mRNA decay, mature mRNA levels in a homozygous resistant genotype were individually higher than the mRNA trace levels observed for the orthologous single-copy TNL in sensitive genotypes. Allelic expression imbalance in a resistant heterozygote confirmed that cis-acting regulatory variation promotes expression in the wild haplotype. The movement of transposable elements had a major impact on the generation of haplotype diversity, altering the DNA context around similar TNL coding sequences and the GC-content in their proximal 5 '-intergenic regions. The wild and domesticated haplotypes also diverged in conserved single-copy intergenic DNA, but the highest divergence was observed in intraspecific and not in interspecific comparisons. In this case, introgression breeding did not transgress the genetic boundaries of the domesticated species, because haplotypes present in modern varieties sometimes predate speciation events between wild and cultivated species.

List of 2042 differentially expressed genes identified using interaction term analysis and displayed in Venn diagram (Fig. 7). xInteraction term analyses to identify DEG characteristic for a successful defence by comparing the pairwise contrasts of Rpv3–1 samples with susceptible samples. (adjusted P ≤ 0.1). FC, fold change. (XLSX 305 kb)

Background European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance toP.viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance. Results In this study, Rpv3-mediated defense responses were investigated in Rpv3(+) and Rpv3 grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3(+) and avrRpv3, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-1-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-1-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses. Conclusion This study used histochemical, transcriptomic and metabolomic analyses of Rpv3(+) and susceptible cultivars inoculated with avirulent and virulent P. viticola isolates to investigate mechanism underlying the Rpv3-1-mediated resistance response. We demonstrated a strong correlation between the expressions of stilbene biosynthesis related genes, the accumulation of fungi-toxic stilbenes, pathogen growth inhibition and PCD.

Stilbene synthase (STS) is the key enzyme leading to the biosynthesis of resveratrol. Recently we reported two R2R3-MYB transcription factor (TF) genes that regulate the stilbene biosynthetic pathway in grapevine: VviMYB14 and VviMYB15. These genes are strongly co-expressed with STS genes under a range of stress and developmental conditions, in agreement with the specific activation of STS promoters by these TFs. Genome-wide gene co-expression analysis using two separate transcriptome compendia based on microarray and RNA sequencing data revealed that WRKY TFs were the top TF family correlated with STS genes. On the basis of correlation frequency, four WRKY genes, namely VviWRKY03, VviWRKY24, VviWRKY43 and VviWRKY53, were further shortlisted and functionally validated. Expression analyses under both unstressed and stressed conditions, together with promoter-luciferase reporter assays, suggested different hierarchies for these TFs in the regulation of the stilbene biosynthetic pathway. In particular, VviWRKY24 seems to act as a singular effector in the activation of the VviSTS29 promoter, while VviWRKY03 acts through a combinatorial effect with VviMYB14, suggesting that these two regulators may interact at the protein level as previously reported in other species.

Upon continuous stress exposure, plants display attenuated metabolic stress responses due to regulatory feedback loops. Here, we have tested the hypothesis that pulsed stress exposure with intervening recovery periods should affect these feedback loops, thereby causing increased accumulation of stress-induced metabolites. The response of Arabidopsis plantlets to continuous UV-B exposure (Cuv ) was compared with that of pulsed UV-B exposure (Puv ). The differential responses to Puv versus Cuv were monitored at the level of gene expression and metabolite accumulation, using wild type (WT) and different mutant lines. In comparison with Cuv , Puv increased sinapyl and flavonol (S + F) content, whereas adaptive growth attenuation was reduced. Furthermore, in a myb4 mutant (AtMYB4, repressor-type R2R3-MYB transcription factor), the S + F content was increased only for Cuv , but not beyond the level for Puv observed in WT. These observations and the ability of AtMYB4 to repress AtMYB12/AtMYB111-mediated activation of target gene promoters (pCHS and pFLS) indicate that the increase of S + F content after Puv observed in WT plants results from reduced feedback inhibition by AtMYB4. The results support the notion that stress-induced metabolic changes not necessarily cause a growth penalty. Furthermore, the observed Puv -induced increase in flavonol accumulation may stimulate reevaluation of commercial plant production practices.

Flavonols constitute a group of flavonoids with important photoprotective roles in plants. In addition, flavonol content and composition greatly influences fruit quality. We previously demonstrated that the grapevine R2R3-MYB transcription factor (TF) VviMYBF1 promotes flavonol accumulation by inducing the expression of flavonol synthase (VviFLS1/VviFLS4), a key step of the initial flavonol pathway. Despite this, gene networks underlying flavonol modification in grapevine including both structural and regulatory genes remain poorly understood. In order to identify flavonol modifying genes and TFs acting downstream of VviMYBF1 a microarray-based transcriptome analysis was performed on grapevine hairy roots ectopically expressing VviMYBF1 or a Green Fluorescent Protein as control. VviFLS1 was induced in VviMYBF1 transgenic roots and glycosylated flavonols accumulated significantly compared with control lines. Among the differentially expressed genes, potential flavonol-modifying enzymes with predicted rhamnosyltransferase (e.g., RhaT1) or glycosyltransferase (e.g., GT3) activities were identified. In addition, important TFs of the MYB and bZIP families such as the proanthocyanidin regulator VviMYBPA1 and the UV-B light responsive HY5 homolog VviHYH were significantly altered in their expression pattern by overexpression of VviMYBF1. Co-temporal expression analysis demonstrated positive correlation of VviMYBF1 with VviFLS1, VviGT3, and VviRhaT1 during berry development and in fruits ripened with different light and UV-B radiation conditions at field. These results show that VviMYBF1 overexpression led to the identification of novel genes of the flavonol pathway and that the flavonol modifying machinery can be influenced by agricultural practices to optimize flavonol composition in grapes.

Grapevine organs accumulate anthocyanins in a cultivar-specific and environmentally induced manner. The MYBA1-A2 genes within the berry color locus in chromosome 2 represent the major genetic determinants of fruit color. The simultaneous occurrence of transposon insertions and point mutations in these genes is responsible for most white-skinned phenotypes; however, the red pigmentation found in vegetative organs suggests the presence of additional regulators. This work describes a genomic region of chromosome 14 containing three closely related R2R3-MYB genes, named MYBA5, MYBA6 and MYBA7. Ectopic expression of the latter two genes in grapevine hairy roots promoted anthocyanin accumulation without affecting other phenylpropanoids. Transcriptomic profiling of hairy roots expressing MYBA1, MYBA6 and MYBA7 showed that these regulators share the activation of late biosynthetic and modification/transport-related genes, but differ in the activation of the FLAVONOID-3'5'-HYDROXYLASE (F3'5'H) family. An alternatively spliced MYBA6 variant was incapable of activating anthocyanin synthesis, however, because of the lack of an MYC1 interaction domain. MYBA1, MYBA6.1 and MYBA7 activated the promoters of UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UFGT) and ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (3AT), but only MYBA1 induced F3'5'H in concordance with the low proportion of tri-hydroxylated anthocyanins found in MYBA6-A7 hairy roots. This putative new color locus is related to the red/cyanidic pigmentation of vegetative organs in black- and white-skinned cultivars, and forms part of the UV-B radiation response pathway orchestrated by ELONGATED HYPOCOTYL 5 (HY5). These results demonstrate the involvement of additional anthocyanin regulators in grapevine and suggest an evolutionary divergence between the two grape color loci for controlling additional targets of the flavonoid pathway.

Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures.

In grapevine, flavonoids constitute one of the most abundant subgroups of secondary metabolites, influencing the quality, health value, and typicity of wines. Their synthesis in many plant species is mainly regulated at the transcriptional level by modulation of flavonoid pathway genes either by single regulators or by complexes of different regulators. In particular, bZIP and MYB factors interact synergistically in the recognition of light response units present in the promoter of some genes of the pathway, thus mediating light-dependent flavonoid biosynthesis. We recently identified VvibZIPC22, a member of clade C of the grapevine bZIP family, in a quantitative trait locus (QTL) specifically associated with kaemperol content in mature berries. Here, to validate the involvement of this candidate gene in the fine regulation of flavonol biosynthesis, we characterized its function by in vitro and in vivo experiments. A role for this gene in the control of flavonol biosynthesis was indeed confirmed by its highest expression at flowering and during UV light-mediated induction, paralleled by accumulation of the flavonol synthase 1 transcript and flavonol compounds. The overexpression of VvibZIPC22 in tobacco caused a significant increase in several flavonoids in the flower, via induction of general and specific genes of the pathway. In agreement with this evidence, VvibZIPC22 was able to activate the promoters of specific genes of the flavonoid pathway, alone or together with other factors, as revealed by transient reporter assays. These findings, supported by in silico indications, allowed us to propose VvibZIPC22 as a new regulator of flavonoid biosynthesis in grapevine.

R2R3-MYB transcription factors (TFs) belong to a large and functionally diverse protein superfamily in plants. In this study, we explore the evolution and function of this family in grapevine (Vitis vinifera L.), a high-value fruit crop. We identified and manually curated 134 genes using RNA-Seq data, and named them systematically according to the Super-Nomenclature Committee. We identified novel genes, splicing variants and grapevine/woody-specific duplicated subgroups, suggesting possible neo- and sub-functionalization events. Regulatory network analysis ascribed biological functions to uncharacterized genes and validated those of known genes (e.g. secondary cell wall biogenesis and flavonoid biosynthesis). A comprehensive analysis of different MYB binding motifs in the promoters of co-expressed genes predicted grape R2R3-MYB binding preferences and supported evidence for putative downstream targets. Enrichment of cis-regulatory motifs for diverse TFs reinforced the notion of transcriptional coordination and interaction between MYBs and other regulators. Analysis of the network of Subgroup 2 showed that the resveratrol-related VviMYB14 and VviMYB15 share common co-expressed STILBENE SYNTHASE genes with the uncharacterized VviMYB13. These regulators have distinct expression patterns within organs and in response to biotic and abiotic stresses, suggesting a pivotal role of VviMYB13 in regulating stilbene accumulation in vegetative tissues and under biotic stress conditions.