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

Abstract

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.

Figure 1.

Simplified Representation of the Grapevine Phe/Polymalonate Pathway, Which Leads to the Biosynthesis of Flavonoids and Stilbenes. Transcriptional regulation of the general enzymes is conducted by the R2R3 MYB TFs MYBPA1, MYBA, MYBF1, MYB14, and MYB15. 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; ROMT, resveratrol-O-methyltransferase.

Figure 2.

Expression of MYB14, MYB15, and Selected STS Group B-Type Genes (STS25/27/29 and STS41/45) in Response to Different Abiotic and Biotic Stresses. (A) V. vinifera cv Shiraz leaf discs were subjected to wounding, (B) UV-C light irradiation. (C) Downy mildew infection. Rapidly expanding leaves collected from nodes 5 to 8 of new shoots harvested at different time points following treatments (hours after treatment). Transcript levels were normalized to the expression of elongation factor EF1-α and plotted as fold change. Fold change for wounded discs was calculated relative to the untreated sample (0 h), whereas fold change for UV-C–treated and downy mildew infected discs was obtained by calculating the ratio between treated (UV-C or downy infected) and untreated (i.e., wounded discs) samples at the same time point. The experiment was repeated twice with the same results. Data show the results of one of these experiments. Bars indicate se of three technical replicates.

Figure 3.

Expression of MYB14, MYB15, STSs, and Stilbene Accumulation during Grape Berry Development of V. vinifera cv Pinot Noir. (A) Columns represent MYB and STS transcript profiles in flower/skins. (B) Stilbene accumulation in flower/skins. (C) MYB and STS transcript profiles in seeds. (D) Stilbene accumulation in seeds. Data points are given as weeks from the onset of ripening (véraison; labeled with an arrow), with expression values as means of three replicate PCRs (n = 6) and error bars indicating se. Note that from 6 weeks before véraison (-6), berry skin has been separated from seeds. Flowering occurred 8 weeks before véraison. Accumulation of stilbenes was measured as methanol extracts of developing grape tissues using reverse-phase HPLC analysis. (B) and (D) show relative stilbene contents of trans-piceid in developing berries and in seeds as the mean of three different experiments, with error bars indicating se. Note that each time point during developmental series is a pool of >100 berries collected from ∼20 plants growing as described in Methods. FW, fresh weight.

Figure 4.

MYB14 and MYB15 Specifically Induce the Promoter Activity of STS41 and STS29, Which Are Involved in the Biosynthesis of Stilbenes. (A) to (C) Fold induction of promoter activity in the presence of a MYB TF relative to promoter activity in the absence of the MYB TF. Control columns show a fold induction of 1.0, indicating no effect on promoter activity. (D) Fold induction of STS promoter activity by MYB14 and MYB15 with and without (w/o) bHLH and WD40 cofactors Vv-MYC1 and At-TTG1. (E) Basal activity of V. vinifera promoters in suspension cell culture in the absence of added MYB TFs. Transient expression in V. vinifera cv Chardonnay suspension cell culture following particle bombardment. Specific promoters linked to a firefly luciferase gene were cobombarded into cells with pART7-MYB TF constructs or with a pART7 (empty vector) control. Each transfection contained the Renilla luciferase plasmid pRluc as an internal control (Horstmann et al., 2004) and the cofactors Vv-MYC1 (bHLH type) and At-TTG1 (WDR type) (except for the experiment shown in [D]). The columns represent relative LUC activity (Firefly/Renilla) of the corresponding promoter plus MYB factor relative to the respective control (without the MYB factor) of six independent experiments with error bars indicating se.

Figure 5.

Expression of STSs and PAL and Accumulation of trans-Piceid in Grapevine Hairy Roots Ectopically Expressing MYB15. (A) Transcript levels of MYB15, STS, and PAL were determined by quantitative PCR in V. vinifera cv Chardonnay hairy root lines transformed with pART27-MYB15 compared with control lines transformed with GFP:GUS (pKGWFS7; Karimi et al., 2002). Each column in shows transcript levels corrected to Ubiquitin1, EF1-α, and GAPDH (Pfaffl et al., 2002), expressed as mean values of three replicate PCRs (n = 6) and error bars indicating se. Note that expression levels can only be compared between MYB15 and GFP:GUS control lines and not between the individual genes using this method. (B) Accumulation of trans-piceid MYB15-overexpressing hairy root lines compared with GFP transformed controls expressed as relative content in nmol/g fresh weight. Trans-piceid levels shown represent the mean of three different experiments with error bars indicating se. FW, fresh weight.