Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway

Abstract

Grapevine (Vitis vinifera) proanthocyanidins contribute to plant defense mechanisms against biotic stress and also play a critical role in organoleptic properties of wine. In grapevine berry, these compounds are mainly accumulated in exocarps and seeds in the very early stages of development. A previous study has already identified VvMybPA1 as the first transcription factor involved in the regulation of the proanthocyanidin pathway during seed development in grapevine. A novel Myb factor, VvMybPA2, which is described in this study, is in contrast mainly expressed in the exocarp of young berries and in the leaves. This transcription factor shows very high protein sequence homology with other plant Myb factors, which regulate flavonoid biosynthesis. Ectopic expression of either VvMybPA1 or VvMybPA2 in grapevine hairy roots induced qualitative and quantitative changes of the proanthocyanidin profiles. High-throughput transcriptomic analyses of transformed grapevine organs identified a large set of putative targets of the VvMybPA1 and VvMybPA2 transcription factors. Both genes significantly activated enzymes of the flavonoid pathway, including anthocyanidin reductase and leucoanthocyanidin reductase 1, the specific terminal steps in the biosynthesis of epicatechin and catechin, respectively, but not leucoanthocyanidin reductase 2. The functional annotation of the genes whose expression was modified revealed putative new actors of the proanthocyanidin pathway, such as glucosyltransferases and transporters.

Figure 1.

Multialignment of VvMybPA2 with related MYB proteins by ClustalW: Arabidopsis AtMyb123 (TT2; Q9FJA2), rice (Oryza sativa) OsMYB3 (BAA23339), and grapevine VvMYB5b (AAX51291) and VvMybPA1 (CAJ90831). Identical amino acids are indicated in black, and similar amino acids are indicated in gray. The R2 and R3 repeats of the MYB domain, the motif participating in the interaction with bHLH proteins (motif 1), and the C-terminal conserved motif (motif 2) are indicated.

Figure 2.

Phylogenetic tree showing selected plant MYB transcription factors retrieved from public databases. The phylogenic tree was constructed from the Clustal alignment using the neighbor-joining method in the MEGA4 package. The scale bar represents 0.05 substitutions per site. The GenBank accession numbers of the MYB proteins are as follows: AtMYB12 (NP_182268), AtMYB11 (NP_191820), AtMYB111 (NP_199744), ZmP (P27898), PhPH4 (AAY52377), VvMyb5b (AAX51291), VvMYb5a (AAS68190), VvMybPA1 (CAJ90831), PmMBF1 (AAA82943), ZmC1 (AAA33482), ZmPl (AAA19821), VvMybPA2 (EU919682), OsMYB3 (BAA23339), AtTT2 (Q9FJA2), BnTT2-1(ABI13038), BnTT2-2 (ABI13039), BnTT2-3 (ABI13040), FaMYB1 (AAK84064), AmMIXTA (CAA55725), AtMyb4 (BAA21619), PhODO1 (AAV98200), AtGL1 (NP_189430), AtWER (AAF18939), MdMYB1 (ABK58136) MdMYB10 (ABB84753) AtPAP2 (NP_176813), AtPAP1 (NP_176057), LeANT1 (AAQ55181), VvMYBA1 (BAD18977), and VvMYBA2 (BAD18978).

Figure 3.

Transcript levels of VvMybPA2 during berry pericarp development (A), in different berry tissues at three developmental stages (B), and in different organs of vine plants (C). Véraison (V) is marked with the arrows. Gene expression was determined by real-time PCR and normalized with the expression of EF1α. All data are means of three replicates, with error bars indicating sd.

Figure 4.

PA contents and mean characteristics (mDP, percentage galloylation, percentage trihydroxylation) in grapevine organs overexpressing VvMybPA1 (lines PA1-2A and PA1-7A) or VvMybPA2 (lines PA2-1A and PA2-7A) versus the wild type (lines control-1 and control-2). Data are means ± sd of triplicate extractions. Stars indicate that values are significantly different between control and transformed hairy roots (P < 0.01). FW, Fresh weight.

Figure 5.

Phenotype of grapevine hairy roots stained with DMACA. A, Wild-type hairy roots showing the distribution of blue dye formed with PA on main and secondary organs. Bar = 2 mm. B, Wild-type hairy root section. Bar = 250 μm. C, VvMybPA1-expressing hairy root. Bar = 250 μm. D, VvMybPA2-expressing hairy root. Bar = 250 μm. DZ, Division zone; EZ, elongation zone; SR, secondary roots.

Figure 6.

Transcript levels of putative targets (A) and validation of microarray results (B) in grapevine organs overexpressing VvMybPA1 (lines PA1-2A and PA1-7A) or VvMybPA2 (lines PA2-1A and PA2-7A) versus the wild type (lines control-1 and control-2). Gene expression was determined by real-time PCR and normalized with the expression of EF1α. All data are means ± sd of three replicates. Stars indicate that expression levels are significantly different between wild-type and transformed hairy roots (P < 0.01).