Transcriptomic effect of Zebularine, a DNA methylation inhibitor, in grapevine cells.

Anthocyanins are flavonoid compounds responsible for the color of many flowers and fruits including red grape berries. Their biosynthesis in grape has drawn much attention, because anthocyanins play an important role in red wine color and quality. Their biosynthesis is regulated by many nutritional, developmental and environmental factors including sugars, abscisic acid, temperature and light, and a complex network involving several transcription factors and regulatory proteins is being progressively deciphered. In particular MYB transcription factors have been shown to play a key role through the control of the expression of the UDP glucose: flavonoid-3-O-glucosyltransferase encoding genes (UFGT), which are critical for anthocyanin biosynthesis. Indeed in different species specific MYB transcription factors are expressed in a correlated manner with anthocyanin accumulation. Interestingly in apple and pear fruits the expression of MYB10, as well as anthocyanin accumulation, were correlated with the DNA methylation status in MYB10 promoter region, suggesting that DNA methylation could play a role in the regulation of anthocyanin biosynthesis. To determine the role of DNA methylation in anthocyanin accumulation in grape, zebularine, a DNA methyltransferase inhibitor, was used to treat cell suspensions (vitis vinifera L. cv Gamay teinturier). Anthocyanin accumulation was stimulated by zebularine, the drug inducing an increase in anthocyanin quantities in the light, and eliciting their accumulation in the dark. Accordingly RNA-seq analyses revealed that several genes coding for anthocyanin biosynthetic enzymes were upregulated by zebularine. However, the analysis of DNA methylation by McrBC-PCR revealed that the methylation level was not changed significantly in the promoter region of three anthocyanin-related genes known to be under transcriptional control (UGFT, MYBA1, and MYBA2). This suggests that a decrease in DNA methylation may not be the primary cause for anthocyanin accumulation in response to zebularine. The metabolic and transcriptomic characterization of the Gamay teinturier cells 12 days after sub-culturing revealed that zebularine indeed globally impacts cell physiology. Notably the nutritional status of light grown cells appeared to be deeply modified by zebularine. In addition many stress-related genes were specifically up-regulated by zebularine in the light and in the dark, especially genes linked to genotoxic stress. Therefore the induction of anthocyanin production could be part of a stress response elicited by zebularine.