The cloning of VviMYBF1 from the cultivar (cv.) ‘Shiraz’ (GenBank locus accession: FJ948477) into the vector pART7 (Gleave, 1992) to give the construct pART7MYBF1 was described previously (Czemmel et al., 2009). For generation of HRs overexpressing VviMYBF1, pART7MYBF1 was NotI digested and the insert (VviMYBF1 under control of CaMV35S) was cloned into pART27 (Gleave, 1992) to give pART27MYBF1. The binary vector pART27 contains spectinomycin resistance for bacterial selection and kanamycin resistance for selection in planta.

The collection of the samples for the grapevine developmental series of V. vinifera cv. ‘Pinot Noir’ was described before (Höll et al., 2013). In short, sampling was performed from the start of floral initiation until harvest with early flower and later berry samples collected from a commercial vineyard during the 2007 to 2008 season in Schriesheim near Heidelberg, Germany. The berry samples derived from ∼100 berries from 20 different plants and bunches and were sampled at weekly intervals, as described previously (Downey et al., 2003). All samples were frozen in liquid nitrogen upon collection on the field and stored at -80°C until analyzed.

Before HR transformation, pART27-MYBF1 or pKGWFS7 control plasmid expressing GFP-GUS were transferred by electroporation into the Agrobacterium rhizogenes strain ATCC 15834, carrying the Ri plasmid pRi15834 (neither the chromosome nor the plasmid ATCC 15834 of this A. rhizogenes strain carry an antibiotic marker).

Transformation and induction of transgenic VviMYBF1 or GFP-GUS expressing grapevine HRs derived from leaves from in vitro-grown cv. ‘Chardonnay’ plants was performed exactly as described in Höll et al. (2013). After transformation and induction, transgenic HRs were routinely kept on HR) medium (Höll et al., 2013) and transferred to fresh medium after 4 weeks. HR tissues used for RNA extraction and high performance liquid chromatography (HPLC) analysis were rapidly frozen in liquid nitrogen, ground to a fine powder using a Retchmill (MM20, Retch) and stored at -80°C until further use.

Total RNA was extracted from grinded material from the developmental series of V. vinifera cv. ‘Pinot Noir’ as previously described in Höll et al. (2013). RNA of MYBF1 and GFP transgenic grapevine HRs was isolated following the protocol of the EURx GeneMATRIX Universal RNA purification kit (Roboklon). Prior to RT reactions, RNA samples were heated to 65°C for 10 min and immediately placed on ice to destroy secondary structures. Synthesis of cDNA was performed with SuperScript^TM III First-Strand Biosynthesis System (Invitrogen, catalog no. 18080-051) according to the instructions in the manual for RT using oligo dT primers. Typically, 1–2 μg of RNA were reverse transcribed in a volume of 20 μl for 1 h at 50°C followed by termination of the enzyme reaction at 85°C for 5 min. After digest of the residual RNA strands using RNase H, 30 μl of RNase free H₂O were added. This 50 μl solution served as cDNA stock for further application and was stored at -20°C. PCR reactions were carried out for flower and berry skin samples from the cv. ‘Pinot Noir’ developmental series as well as HR samples by using 0.5 μl of 10 μM forward and 0.5 μl of 10 μM reverse primer, 5 μl cDNA (diluted 1:20), 7.5 μl of 2x ABsolute^TM QPCR SYBR^® Green Fluorescein Mix (ABgene) and 1.5 μl of H₂O in a final volume of 15 μl (for all set of qPCR primers used in this work, see Supplementary Table 1). Thermal cycling conditions were identical for all primer pairs: 96°C for 15 min followed by 96°C for 30 s, 58°C for 30 s, and 72°C for 30 s for 35 cycles, followed by a melt cycle from 50 to 96°C. QPCR was carried out using SYBR green method for detection of double stranded PCR products on an iCyclerTM optical Module real-time cycler (Bio-Rad). UBIQUITIN1 (previous TIGR database id: TC32075, VIT_16s0098g01190) was used for normalization of target gene levels in grapevine HRs and light experiments while the three reference genes GAPDH (VIT_17s0000g10430), UBIQUITIN1 and EF1-alpha (VIT_06s0004g03220) were used to normalize gene expression during cv. ‘Pinot Noir’ berry development as done previously (Höll et al., 2013). The primers MYBintF and MYBintR2 were used to detect the transcript level of VviMYBF1 in grapevine by amplification of a 214 bp PCR-fragment from the 3′ region of the gene. The primers VviFLS1 and VviFLS2 were used to detect a 154 bp amplicon of the VviFLS1/FLS4 gene (for other primer pairs see Supplementary Table 1). The efficiency of the primers was tested in preliminary experiments with dilutions of plasmid or purified PCR products maintaining an r² value ≥ 0.96. With all cDNAs used, the primer set gave a single PCR product, which was verified by agarose gel electrophoresis and by determination of the melt curves for the product at the end of each run.

Fresh cross sections from 3 to 4 weeks old HRs were transferred in a freshly prepared solution of 0.25% (w/v) diphenylboric acid 2-amino-ethyl ester (DBPA, Naturstoffreagenz A, Roth) and 0.00375% (v/v) Triton X-100. Fluorescence was visualized with either an inverted epifluorescence microscope (Leica DM IRB, Leica) or a stereomicroscope (Leica MZ FL III, Leica) using a DAPI fluorescence filter with an excitation wavelength of 340–380 nm and an emission wavelength of 425 nm. Images were captured using the Leica Image Manager (IM) 50 software and handled with Adobe Photoshop version 8.0.1 without changing the color parameters.

Microarray raw data generation was produced at the Centro Nacional de Biotecnologia (CNB, Madrid) by using the Grapegen GeneChip, which is an Affymetrix custom made chip containing 23,096 probe sets corresponding to ∼15,800 different annotated genes (Pontin et al., 2010) and covering ∼52% of the genome. Quality assessment of microarray data and differential expression (DE) analysis were carried out in the statistical language R (version 3.2.1) mainly using the affy (version 1.48.0) and LIMMA packages (version 3.26.9) by the Quantitative Biology Center (QBiC) in Tübingen¹. Based on PCR analysis for the overexpressed TF VviMYBF1 and its target VviFLS1, four VviMYBF1 (lines no. 32, 33, 34, and 62) and four GFP overexpressing lines (lines no. 77, 79, 83, and 97) out of 10 stable transformants were selected. During raw data control analysis on all eight lines (four GFP and four VviMYBF1 lines each) several sensitive measures to assess array quality such as Normalized Unscaled Standard Error (NUSE) and Relative Log Expression (RLE) confirmed low quality of array corresponding to the GFP control line 79. Since the array for line 79 was detected as an outlier, DE analysis has been performed on four VviMYBF1 and three GFP lines excluding GFP line 79. Raw data (CEL files) and meta-information (e.g., experimental design, sample names) of microarray data were deposited to NCBI’s Gene Expression Omnibus (GEO) and can be accessed under the GEO identifier GSE95532.

Four different sunlight or UV-B radiation experiments were conducted in cv. ‘Cabernet Sauvignon’ commercial plants (either treated at field or uprooted and transferred to a greenhouse).

High performance liquid chromatography analysis was performed on methanolic extracts of HR lines using a reverse phase HPLC (Kontron Instruments 322 pump system/360 autosampler/335 HPLC detector; Kontron) with a Symmetry C18 column (3,5 μm, 4,6 mm × 150 mm, catalog no. WAT200632, Waters) protected by a guard column (Czemmel et al., 2009). Liquid nitrogen-frozen and subsequently homogenized sample aliquots of 20–50 mg were used to extract flavonoids by adding 200 μl of 50% (v/v) methanol (HPLC grade) in H₂O. Samples were then sonificated for 20 min in an ice water bath and centrifuged for 10 min at 13000 rpm. 200 μl of the clear supernatant were loaded for HPLC analysis. Separation was carried out with a binary gradient of solvent A [10% formic acid (v/v) in H₂O] to solvent B [100% (v/v) methanol, HPLC grade]. The gradient conditions were 0 min, 17% solvent B; 15 min, 35% solvent B; 40 min, 37% solvent B; 42 min, 100% solvent B; 50 min, 100% solvent B; 51 min, 17% solvent B; 58 min, 17% solvent B. The column was maintained at 40°C and the flow rate was 1 mL/min. Data acquisition and processing was performed by Kroma System 2000 software (Kontron). Data are presented as HPLC chromatogram peak areas over time and expressed as milli-absorbance units (mAU) at 520 nm for anthocyanins and at 353 nm for flavonols. Concentrations were calculated from calibration curves prepared from commercial standards (Phytolab) and expressed as quercetin-3-O-glucoside equivalents for flavonols and malvidin-3-O-glucoside equivalents for anthocyanins. Acidic hydrolysis was performed to identify the compounds as flavonols by cleaving the glycosyl group for a shift to the aglycone form by the addition of a 3 N HCL solution, 50% (v/v) methanol in water and incubation for 3 h at 95°C. All HPLC separation experiments were performed in three independent extractions of from the same biological material.

High performance liquid chromatography quantification of flavonols in berry skins from light experiments was different to flavonol measurements from HRs and berry skin and flower samples from the developmental series of cv. ‘Pinot Noir.’ For light experiments, flavonol measurements were conducted as described in Matus et al. (2009).

For statistical evaluation of microarray data, the LIMMA package was used (Ritchie et al., 2015). First, its function lmFit() fitted linear models on the quality controlled and RMA normalized expression values of each gene across the seven samples with the factor genotype (with the two levels MYBF1 and GFP-GUS). Then the functions eBayes() and topTable() with the argument adjust.method = “BH” were used to compute moderated t-statistics and extract a table of the top-ranked genes from a linear model fit with p-values corrected for multiple testing by applying the false discovery rate (FDR) from Benjamini and Hochberg (1995), respectively. Genes were considered differentially expressed (DE) with a FDR corrected p < = 0.05. No log fold change (logFC) cutoff was applied to assess DE of a gene.

To statistically assess differences in expression of genes and flavonol accumulation between VviMYBF1 and GFP control HR lines, an unpaired two-tailed t-test was performed with assumption of equal standard deviation (SD) between both sample groups. Results were considered statistically different at p < 0.05.

For each of the four UV/light experiments, differential gene expression was assessed using a two-way analysis of variance (ANOVA) with a model including main effects for the two factors time (in WAV) and treatment (+UV-B, -UV-B) and an interaction term between those two factors. To analyze at what time points the treatment effects differed, a post hoc analysis was performed on the model using Tukey’s test. Genes were considered statistically DE at this time point at p < 0.05.

Note that for accumulations of individual flavonol in response to light no statistical test was performed.

Microarray data were submitted in the Gene Expression Omnibus (GEO) under accession number GSE95532.

Previous studies indicate that the grapevine HR system provides a suitable tool to identify novel target genes regulated by an ectopically expressed MYB TF (Cutanda-Perez et al., 2009; Terrier et al., 2009; Khater et al., 2012; Höll et al., 2013). Transgenic HRs were generated by infiltration of V. vinifera cv. ‘Chardonnay’ leaves with A. rhizogenes carrying either VviMYBF1 or GFP (control) cDNA. Presence of VviMYBF1 transgene was verified by PCR, whereas control lines expressing GFP were selected by PCR and fluorescence microscopy. Four VviMYBF1 (lines no. 32, 33, 34, and 62) and four GFP overexpressing lines (lines no. 77, 79, 83, and 97) out of 10 stable transformants were analyzed for transcript amount of VviMYBF1 and its known target VviFLS1 (Czemmel et al., 2009). During microarray quality control analysis, line no. 79 was detected as an outlier (for details, see Materials and Methods) and downstream analysis has been performed on four VviMYBF1 and three GFP lines excluding GFP line 79. Additionally, HR flavonols were stained in vivo with diphenylboric acid-2-aminoethylester (DPBA, Figure 2A) and quantified using HPLC (Figure 2B). By using fluorescence microscopy, we observed orange like bodies (indicative of the accumulation of quercetin-derivatives) exclusively in VviMYBF1 overexpressing HRs (Figure 2A). These bodies were prominent in epidermal cell layers while endodermal cells and parenchymatic cells of the cortex accumulate only trace amounts or did not show any orange staining indicative for flavonol accumulation. Flavonols, mainly quercetin 3-glucoside, quercetin 3-galactoside and a so far unknown flavonol derivative accumulated significantly higher in VviMYBF1 overexpressing lines as compared to GFP control lines when measured quantitatively using HPLC (Figure 2B). To show that MYBF1 induces only the flavonol branch of the flavonoid pathway anthocyanins were measured in the HR lines. Neither in the VviMYBF1 nor in the GFP overexpressing lines anthocyanins were detected.

With the aim to decipher the mechanisms that underlie the phenotypic changes observed in HRs ectopically expressing VviMYBF1, qPCR analysis was deployed. Expression of VviMYBF1 in selected VviMYBF1 lines exceeded significantly its expression compared to GFP control lines (Figure 2C), which resulted in a pronounced induction of its target, VviFLS1 (Figure 2D). Results from this homologous expression experiment clearly support the role of VviMYBF1 in regulation of flavonol biosynthesis by induction of VviFLS1 transcription. These results showed that selected VviMYBF1 and GFP HR lines were suitable candidates for a differential transcriptome analysis approach to identify novel target genes of VviMYBF1 involved in flavonol biosynthesis and its regulation.

To identify novel target genes of VviMYBF1, total RNA was isolated from the four VviMYBF1 and the three GFP overexpressing HR lines to perform a Microarray-based transcriptome analysis. Principal component analysis (PCA) clearly separated both, VviMYBF1 and GFP HR lines in distinct groups (Figure 3B) indicating differences between underlying gene expression pattern in VviMYBF1 and GFP control groups which is also visible using a heatmap (Figure 3A). To test the a priori hypothesis that genes will have significantly different mean expression values between VviMYBF1 and GFP lines, pairwise comparison between the two groups VviMYBF1 and GFP was performed and moderated t-statistics and associated p-values were generated, which were further adjusted for multiple testing using Benjamini and Hochberg’s method to control the FDR (Benjamini and Hochberg, 1995). In total, 548 probesets corresponding to 484 differentially expressed genes (DEGs; that is because several oligonucleotides hybridize to the same gene) were DE based on an adjusted p-value smaller than 0.05 (Table 1 and Supplementary Table 1). These 548 probesets represent ∼2% of the probes on the chip (23,096 probesets in total). 279 probesets corresponding to 230 genes have been called positive DE, while the other 269 probes, corresponding to 254 genes, show significant negative regulation in VviMYBF1 compared to GFP HR lines. Amongst the DEGs, three candidate genes were found which could modify flavonol scaffold structures (Table 1): a putative UDP-sugar flavonoid/flavonol glycosyltransferase [VIT_11s0052g01580, VviGT3 (Ono et al., 2010)], a putative UDP-rhamnose:rhamnosyltransferase (VIT_00s0218g00170, VviRhaT1) and a putative flavonol modifying sulfotransferase (VIT_17s0000g04930, VviST1). An investigation of the positive DEGs for TFs revealed VviHYH (VIT_05s0020g01090) and VviMYB4A (VIT_03s0038g02310), which showed 1.5 and a 1.6 fold inductions in gene expression in VviMYBF1 versus control HRs, respectively (Supplementary Table 1). Amongst the pool of significantly downregulated transcripts several genes of the PA biosynthetic pathway were identified: the PA regulatory TF gene VviMYBPA1 (VIT_15s0046g00170) and genes under genetic control of VviMYBPA1 including two chalcone synthase isoforms (VviCHS3 and VviCHS1, VIT_05s0136g00260, VIT_14s0068g00930) and anthocyanidin reductase (VviANR, VIT_00s0361g00040). Microarrays also indicate that transcript abundance of a yet uncharacterized FLAVONOL SYNTHASE isoform, VviFLS5 [(VIT_18s0001g03430 (Fujita et al., 2006)] was negatively affected by overexpression of VviMYBF1 (Table 1).

With the aim to validate the microarray experiment, expression of the three candidates flavonol-modifying genes VviST1, VviRhaT1, and VviGT3 and the PA regulator VviMYBPA1 were analyzed by qPCR (for primers used see Supplementary Table 1). Quantitative analysis revealed that VviGT3, VviST1 and VviRhaT1 were indeed induced in VviMYBF1 lines compared to GFP controls (Figures 4A–C), whereas transcript abundance of VviMYBPA1 was negatively influenced by ectopic expression of the flavonol regulator VviMYBF1 (Supplementary Figure 1). Expression of the previously characterized flavonol modifying genes VviGT5 and VviGT6 were not checked in the HR samples, as probesets representing these genes were not present on the microarray chip design used here.

For correlative analysis of gene expression during grape berry ripening, the same V. vinifera cv. ‘Pinot Noir’ developmental series was used, in which gene expression related to stilbene biosynthesis was measured previously (Höll et al., 2013). Expression pattern correlation between VviMYBF1 and VviFLS1 transcripts was highest at eleven-to-ten weeks prior to beginning of berry ripening (veraison), correlating with previous results on flavonol accumulation and VviFLS1 and VviMYBF1 expression pattern (Downey et al., 2003; Czemmel et al., 2009). Monitoring expression pattern of the putative VviMYBF1 target genes in developing berries showed that VviST1 and VviGT3 are low expressed during berry ripening (Figures 4D,E) though showing correlation with VviFLS1 and VviMYBF1 expressions at some early developmental stages (Downey et al., 2003; Czemmel et al., 2009). There is correlation of VviRhaT1 expression with flavonol accumulation throughout all stages of development as VviRhaT1 transcript amounts gradually increase from early to late stages of development (Figure 4F), which is similar to flavonol accumulation in grapevine (Downey et al., 2003).

Fruit zone leaf removal is a common viticultural practice that exposes grape berry clusters to sunlight, reducing humidity and improving the accumulation of various secondary metabolites important for fruit quality (Reynolds et al., 1995; Teixeira et al., 2013). In order to evaluate whether gene expression of candidate flavonol-modifying genes is influenced by agronomical practices that alter the exposure of berries to UV-containing light, four experiments were conducted: Experiment 1: sunlight depletion generated by leaf displacement around clusters of field-growing plants (Figures 5A–D), Experiments 2–3: control versus ‘High UV-B’ and ‘Low UV-B’ radiation in clusters of 9 year-old plants growing in a greenhouse (Supplementary Figure 2); and Experiment 4: UV-B filtering in clusters of field-growing plants (Figures 5E–H and Supplementary Figure 3A). For Experiment 4 not only gene expression levels were measured (Figures 5E–H), but also total (Supplementary Figure 3B) and individual flavonol levels determined (Supplementary Figure 4). Furthermore, environmental parameters such as solar UV-B irradiance, temperature, solar radiation at cluster level and total solar radiation were measured (Supplementary Figure 5). For further experimental details for these four light experiments, refer to Section “Light and UV-B Radiation Treatments”.

The MYB TF VviMYBF1 was strongly upregulated in all developmental stages at 100% exposure in the sunlight field experiment compared with 0% exposure (Figure 5A). Its regulation was also higher in the +UV-B condition of the filtering experiment at 0 and +3 WAV (Figure 5E). In the greenhouse experiments, VviMYBF1 expression was more strongly induced by UV-B irradiation (+UV-B) at low intensities but not at high intensities (Supplementary Figures 2A,E). The induction at low intensities was prominent at early stages of development (-3 WAV and time point 0, Supplementary Figure 2B).

Consequently, VviFLS1, the target gene of VviMYBF1, was also upregulated by sunlight and UV-B, most prominently at all time points of 100% exposure in the sunlight experiment compared with 0% exposure (Figure 5B), and at 0 and +3 WAV in the UV-B filtering field experiment (Figure 5F). VviFLS1 was already induced at -3 WAV, although not statistically significant (Figure 5F). Expression of VviFLS1 was also affected in the greenhouse experiments under UV-B irradiance, especially at +3 WAV and +6 WAV (Supplementary Figures 2B,F).

VviRhaT1 expression was induced by +UV-B in the UV filtering experiment around veraison (Figure 5G) but showed no significant response to 25 or 100% sun exposure in the light field experiment except at +7 WAV (Figure 5C) or to low UV-B irradiance conditions in the greenhouse (Supplementary Figure 2G). In contrast to VviMYBF1 and VviFLS1, VviRhaT1 expression profile throughout development was similar in the 25 and 100% exposure treatments in the sunlight field experiment (Figure 5C).

Expression of the three genes MYBF1 and FLS1 and RhaT1 are indicative of a rapid induction to late sunlight exposure (stage +7 WAV in the 25% exposure treatment), as this is the stage when leaves are replaced and clusters are exposed to light (see diagram at the bottom of Figure 5 and see Materials and Methods) which is supported by a similar expression level of the respective gene at the same stage in the 100% exposure treatment (Figures 5A–C).

Regarding VviGT3, there is a consistent two-to-three-fold induction of its expression in response to UV-B at 3 weeks before veraison in the greenhouse UV-B irradiance or filtering experiments (Supplementary Figures 2D,H and Figure 5H). In contrast, VviGT3 gene expression was not detectable in post-veraison samples such as in the light field experiment (Figure 5D), which correlates well with its low expression at late stages of development (Figure 4E).

While VviST1 transcript levels were detectable in the HR lines overexpressing VviMYBF1, in none of the light experiments expression of VviST1 was detectable, also correlating to its low expression found in the cv. ‘Pinot Noir’ developmental series (Figure 4D).

In order to correlate gene expression levels with flavonol accumulation, flavonols were quantified in the UV-B filtering field experiment. Flavonols show a significant increase in response to UV-B at different time points (Supplementary Figure 3B). When analyzing for individual flavonol compounds it was found that many flavonols are 3-O-glycosylated, as reported before (Carbonell-Bejerano et al., 2014) and the mostly affected were 3-O-glucosides of quercetin, kaempferol, and isorhamnetin (Supplementary Figure 4).

Several studies have shown that target gene specificity of TFs may differ when introduced into heterologous hosts (Bovy et al., 2002; Luo et al., 2008). Therefore microarray-based transcriptome analysis was performed on the homologous system V. vinifera cv. ‘Chardonnay’ HRs overexpressing VviMYBF1 and compared to GFP expressing control roots. For microarray analysis, suitable HR lines were pre-selected by qPCR to detect the presence of significant amounts of VviMYBF1 and its known target gene, VviFLS1 (Czemmel et al., 2009). In situ staining of transgenic VviMYBF1 HRs for flavonol compounds clearly supported qPCR analysis and demonstrated that flavonols accumulated in VviMYBF1 transgenic roots but not in GFP (control) lines. Flavonol compounds were visible in yellow vacuolar-like cytoplasmic inclusion bodies but not uniformly located in all HR tissues. Interestingly, similar structures named anthocyanin vacuolar inclusions (AVIs) were observed after overexpression of the anthocyanin regulator VviMYBA1 (Cutanda-Perez et al., 2009). The sectors in VviMYBF1 root cross sections accumulating highest levels of flavonols include skin layers (epidermis, hypodermis), endodermis and vascular bundles, whereas no flavonols could be observed in the cortical parenchyma cells. As flavonol regulation by VviMYBF1 is not dependent on the presence of bHLH proteins (Czemmel et al., 2012), a lack of flavonol accumulation in HR tissues such as cortical parenchyma cells might result from either the presence of inhibitors (e.g., transcriptional repressors) or the lack of WD-repeat proteins or other yet unknown activators in these tissues. Main known flavonols found in VviMYBF1 HR extracts were quercetin-3-O-glucoside and also quercetin-3-O-galactoside. Both of these quercetin-derived compounds have been reported as abundant compounds found in grape leaves and berries (Castillo-Muñoz et al., 2007; Downey and Rochfort, 2008).

It is well-known that flavonols, in a similar way as anthocyanins, mainly exist as glycosylated forms whereas corresponding aglycones rarely exist in planta (Mattivi et al., 2006). Conjugation with glucose leads to increased water solubility, bioavailability and reduced toxicity, as respective reaction products can be subsequently stored in the vacuole. For example, kaempferol-diglucoside was suggested as one substrate for the tonoplast localized MATE protein Flower Flavonoid Transporter (FFT) in Arabidopsis (Thompson et al., 2010). Sugar moieties at the flavonol core skeleton also influence antioxidant potential (Vinson et al., 1999; Burda and Oleszek, 2001; Montoro et al., 2005) and they are able to protect polyphenols from enzymatic oxidation by plant peroxidases (Regev-Shoshani et al., 2003). These flavonol modifications, also in addition to their content and composition, can be remarkably different among grape varieties, during berry development (Downey et al., 2003; Mattivi et al., 2006; Castillo-Muñoz et al., 2007) and in response to UV-containing light (Price et al., 1995; Czemmel et al., 2009; Matus et al., 2009). Still, major knowledge gaps exist in the regulatory network underlying flavonol biosynthesis under developmental and environmental conditions that determine visible and UV-B light qualities.

Microarray analysis of VviMYBF1 overexpressing HR lines was therefore centered toward the identification of novel structural and regulatory genes involved in flavonol biosynthesis. Promising candidates, namely two glycosyltransferases (VviGT3, VviRhaT1) and a putative flavonol-sulfotransferase (VviST1) were identified using high throughput DE profiling. qPCR analysis of transgenic HRs overexpressing VviMYBF1 confirmed the microarray results and showed that VviGT3, VviRhaT1, and VviST1 are significantly upregulated (Figure 4).

Results in this work clearly support the idea that VviGT3 is under the control of VviMYBF1 to produce glycosylated flavonols in young developing berries. Despite the many existences of glycosylated flavonols in planta and their high importance as sunscreen pigments, when comparing grapevine to the model plant Arabidopsis (Yonekura-Sakakibara et al., 2007, 2008), only a few genes involved in flavonol glycosylation have been characterized in vitro (Ono et al., 2010). The same holds true for the identification of only a few structural genes involved in glycosylation of other flavonoids (PAs, anthocyanins) in grapes (Ford et al., 1998; Hugueney et al., 2009; Khater et al., 2012). VviGT3 is located in the same orientation on chromosome 11 in close vicinity to VviGT5 and VviGT6 (Ono et al., 2010). This tandem organization suggests that VviGT3 arose by tandem duplication and might provide another flavonol modifying gene. Sequence similarity between VviGT3 and VviGT5 (64%) or VviGT6 (64%) is lower compared to the sequence identity between VviGT5 and VviGT6 (88%), which implies functional differences. Although the GT3 protein has not been functionally categorized yet, co-expression analysis reveals some spatial and potential functional differences compared to GT5/GT6. VviGT5 and VviGT6 were shown to be co-expressed with VviFLS1 in berries, leaves and petioles (Ono et al., 2010) with higher GT5 expressions after the onset of ripening (Loyola et al., 2016). Results from light experiments and berry development show that VviGT3 transcripts are more profoundly expressed and inducible at early stages of development (Figure 4E), which correlates well with peaks of VviMYBF1 and VviFLS1 expression in grapes (Downey et al., 2003; Czemmel et al., 2009). Furthermore, VviGT3 is light inducible only at early stages of development (Figure 5H) where it is expressed (Figure 4E). The absence of induction in the light field experiment (Figure 5D) can be explained by the fact that all samples used there correspond to post-veraison stages where VviGT3 is low expressed, as it was also demonstrated in the cv. ‘Pinot Noir’ developmental series (Figure 4E). VviGT3 is therefore regulated by light in a similar way as VviGT5 and VviGT6 and the flavonol marker genes VviFLS1 and VviMYBF1 (Carbonell-Bejerano et al., 2014). Neither in the cv. ‘Corvina’ gene atlas (Fasoli et al., 2012) nor in the cv. ‘Pinot Noir’ series VviGT3 expression was observed in skin samples of berries toward ripening (Figure 4E). These results indicate that at very early stages of development VviMYBF1 could regulate a cascade of GT expression profiles including VviGT3, VviGT5 and VviGT6 to attach sugar moieties [mainly of the class of 3-O-galactosides and 3-O-glucosides (Supplementary Figure 4)] to flavonol skeletons produced by the co-induced VviFLS1 gene. Later in berry development, flavonol modifications attached by VviGT3 might not be needed in ripening berry skins, in which the transcriptional network of VviMYBF1 activates the expression of other flavonol structural genes except VviGT3. All UV radiation-increased flavonols found in this study were glycosylated, corroborating the results of Carbonell-Bejerano et al. (2014). The structural similarities between GT5, GT6, and GT3 and the pronounced accumulation of 3-O-galactosides and 3-O-glucosides in the field experiment render the possibility that GT3, besides GT5 and GT6, contribute to the glycosylation of flavonols under UV-light regimes to provide non-toxic UV screens to young berries before veraison.

In conjunction with its albeit modest but present induction by UV-containing light during late stages of development, the VviRhaT1 gene was induced in VviMYBF1 HRs when using microarray analysis and confirmative qPCR analysis. The Grape Gene Atlas data showed that cDNA of VviRhaT1 is present ubiquitously in 50 out of 54 measured samples (Fasoli et al., 2012). It is expressed in all tissues where VviGT3 is expressed but in addition in many berry and fruit related tissues and additionally in leaves where VviRhaT1 expression correlates with VviMYBF1 and VviFLS1 transcript abundances. This correlates well with the uniform expression of this gene across the cv. ‘Pinot Noir’ developmental series (Figure 4F). VviRhaT1 shows an expression pattern very similar to flavonol accumulation on a per berry basis (Downey et al., 2003). This indicates its involvement in flavonol modification throughout rather than at the beginning of berry ripening at which stage VviMYBF1 and VviFLS1 expression peaks (Czemmel et al., 2009). This also supported by a co-induction of quercetin-rhamnoside (+1 WAV) and VviRhaT1 (0 and +3 WAV) gene expression around or post-veraison in the UV-B filtering field experiment (Figure 5G and Supplementary Figure 4H) indicating a putative role of VviRhaT1 as flavonol modifying enzyme. This function is further corroborated by the high sequence similarity to At2g22590 (UGT91A1) from Arabidopsis, which presumably encodes a rhamnosyltransferase under transcriptional control of the flavonol regulators MYB12, MYB11, and MYB111 (Stracke et al., 2007). This regulatory link was identified by transcriptome analysis on a flavonol deficient mutant (myb11-myb12-myb111) showing strong downregulation of the transcripts encoding UGT91A1. Transient expression analysis in Arabidopsis protoplasts confirmed that the promoter of UGT91A1 was responsive to flavonol regulators (Stracke et al., 2007).

Whereas O-glycosylation of flavonoids, which may be influenced by VviGT3 and VviRhaT1, has been at least partially studied in grapes, almost nothing is known regarding flavonoid sulfurylation in developing grape berries. In plants, flavonol sulfotransferases (STs) have been initially described in Flaveria spp. (Varin and Ibrahim, 1989) and sulfurylated flavonols have often been associated with the nucleus (Grandmaison and Ibrahim, 1995; Naoumkina and Dixon, 2008). In addition, another study suggests that sulfurylated flavonols might play a positive role in the regulation of polar auxin transport by acting as antagonist to quercetin (Faulkner and Rubery, 1992). A detailed examination of grapevine chromosome 11 revealed that genes encoding GTs alternate with two putative flavonol sulfotransferase genes with one of them, VviST1, being significantly overexpressed in VviMYBF1 transgenic root lines but not induced by sunlight or UV-B. Expression data regarding flavonol STs in grapes are sparse. The Grape Gene Atlas shows that VviST1 is only present in 13 out of 54 tissue samples and only in berry pericarp and berry flesh when considering fruit samples (Fasoli et al., 2012). VviST1 is expressed in roots but not in flowers, which contrasts the expression of VviMYBF1 in these samples. The low expression, as reported in the Grape Gene Atlas, is supported here when studying expression pattern of ST1 in developing berries (Figure 4D) but thereby shows some correlation with early expression of VviMYBF1 in developing berries (Czemmel et al., 2009).

Taken together, these results confirm a regulative effect of VviMYBF1 on VviFLS1, VviRhaT1, and VviGT3 gene expression in VviMYBF1 HRs, during berry development and at particular time points in response to sunlight and UV-B radiation. In contrast, greenhouse and UV-B field experiments did not gain any insight on a light-dependent regulation of the putative flavonol sulfotransferase gene VviST1. These results extend our knowledge about flavonol glycosylation in response to light and leave open the question whether flavonol sulfurylation can be modified by agronomical practices influencing light.

Microarray analysis on VviMYBF1 HRs identified the grape HY5 homolog (VviHYH) and VviMYBPA1 as target of VviMYBF1 (Table 1 and Supplementary Table 1). While the bZIP factor VviHY5 could not be analyzed in this work because it is not represented on the Grapegen GeneChip, transient expression of VviHY5 in grapevine plantlets resulted in an increase of the expression of VviMYBF1 (Loyola et al., 2016). Furthermore, the presence of HY5 binding elements typical for bZIP TFs in the promoters of VviMYBF1 and VviFLS1 genes was proven by in silico approaches (Czemmel et al., 2009). These results suggest a positive feedback loop of VviMYBF1 and VviHYH/VviHY5 in grapevine. A similar relationship is found between the VviMYBF1 and VviHYH homologes AtMYB12 and AtHY5 in Arabidopsis. Lee et al. (2007) identified HY5 binding sites (ACEs) in the promoters of numerous Arabidopsis genes including AtCHS, AtFLS and most prominently AtMYB12 (Lee et al., 2007). This information was used by Stracke et al. (2010) to demonstrate that in Arabidopsis HY5 is required for the transcriptional activation of the AtMYB12 and AtMYB111 genes under UV-B and visible light. This is consistent with the observation that the hy5 mutant shows flavonoid-deficient roots (Sibout et al., 2006) and downregulation of AtMYB12 transcripts (Stracke et al., 2010). Taken together, these results support the theory that the UV-B response machinery (including the TFs VviHY5 and VviMYBF1) exist in grapes to propel flavonol accumulation through the activation of the regulatory network consisting of both, MYB and bZIP TFs (Malacarne et al., 2016; Matus, 2016).

Our transcriptome study also demonstrated a downregulation of structural and regulatory genes of the proanthocyanidin (PA) pathway by VviMYBF1 and therefore indicated a competition between the flavonol and PA branch of the pathway (Table 1). Indeed, qPCR analysis on VviMYBF1 HRs showed that there is a negative influence of the flavonol regulator VviMYBF1 on the expression of the PA regulatory TF VviMYBPA1 (Table 1 and Supplementary Figure 1). Concomitantly, genes which were shown to be under control of VviMYBPA1 such as two CHALCONE SYNTHASE isoforms, ANR and CHI (Terrier et al., 2009) were also downregulated. These results are in line with previous findings that also proposed a competition between flavonol and PA/anthocyanin biosynthesis (Czemmel et al., 2009). Interestingly, an inverse relationship – a differential regulation of VviMYBF1 expression by VviMYBPA1 – seems not to exist as the expression of the key target of VviMYBF1, VviFLS1 was not altered in VviMYBPA1 HRs (Terrier et al., 2009). In context with the contrasting expression profiles of VviMYBF1 and VviMYBPA1 in developing cv. ‘Shiraz’ berries (Bogs et al., 2007; Czemmel et al., 2009), the data presented here imply that VviMYBF1 is a positive regulator of flavonol biosynthesis at the expense of PA accumulation via downregulation of VviMYBPA1 transcription during initial berry ripening stages.