UV-C Promotes the Accumulation of Flavane-3-ols in Juvenile Fruit of Grape through Positive Regulating VvMYBPA1.

Flavane-3-ol monomers are the precursors of proanthocyanidins (PAs), which play a crucial role in grape resistance. Previous studies showed that UV-C positively regulated leucoanthocyanidin reductase (LAR) enzyme activity to promote the accumulation of total flavane-3-ols in juvenile grape fruit, but its molecular mechanism was still unclear. In this paper, we found that the contents of flavane-3-ol monomers increased dramatically at the early development stage grape fruit after UV-C treatment, and the expression of its related transcription factor VvMYBPA1 was also enhanced significantly. The contents of (-)-epicatechin and (+)-catechin, the expression level of VvLAR1 and VvANR, and the activities of LAR and anthocyanidin reductase (ANR) were improved significantly in the VvMYBPA1 overexpressed grape leaves compared to the empty vector. Both VvMYBPA1 and VvMYC2 could interact with VvWDR1 using bimolecular fluorescence complementation (BiFC) and yeast two hybrid (Y2H). Finally, VvMYBPA1 was proven to bind with the promoters of VvLAR1 and VvANR by yeast one hybrid (Y1H). To sum up, we found that the expression of VvMYBPA1 increased in the young stage of grape fruit after UV-C treatment. VvMYBPA1 formed a trimer complex with VvMYC2 and VvWDR1 to regulate the expression of VvLAR1 and VvANR, thus positively promoting the activities of LAR and ANR enzyme, and eventually improved the accumulation of flavane-3-ols in grape fruit.

VvANR silencing promotes expression of VvANS and accumulation of anthocyanin in grape berries.

Virus-induced gene silencing (VIGS) technology was applied to silence VvANR in cv. Zaoheibao grape berries, and the effects of VvANR silencing on berries phenotype; gene expression level of ANS, LAR1, LAR2, and UFGT; enzyme activity of ANS; and accumulations of anthocyanin and flavan-3-ol were investigated. At the third day after treatment, the VvANR silenced grape berries began to turn red slightly, which was 2 days earlier than that of the control group. And the flavan-3-ol content in VvANR-silenced grape berries had been remarkable within 1 to 5 days, the ANR enzyme activity in VvANR-silenced grapes extremely significantly decreased in 3 days, and LAR enzyme activity also decreased, but the difference was not striking. The ANS enzyme activity of the transformed berries was significantly higher than that of the control after 3 days of infection, and it was exceedingly significantly higher than that of the control after 5 to 10 days. The content of anthocyanin in transformed berries increased of a very marked difference within 3 to 15 days. pTRV2-ANR infection resulted in an extremely significant decrease in the expression of VvANR gene, and the expression of VvLAR1, VvLAR2, VvMYBPA1, VvMYBPA2, and VvDFR were also down-regulated. However, the expression of VvANS and VvUFGT was up-regulated significantly. After VvANR silencing via VIGS, VvANR expression in grape berries was extremely significantly decreased, resulting in decreased ANR enzyme activity and flavan-3-ol content; berries turned red and deeper in advance. In addition, VvANR silencing can induce up-regulation of VvANS and VvUFGT expression, significantly increase ANS enzyme activity, and increase of anthocyanin accumulation.