No abstract

DNA methylation is known to be involved in the regulation of plant development and defense mechanisms. However, there is a general lack of data on the role of methylation in plant secondary metabolism. We have investigated the effect of a cytidine analog, 5-azacytidine (azaC), which is known to block DNA methylation, on resveratrol biosynthesis and stilbene synthase (STS) gene expression in Vitis amurensis cultured cells. Resveratrol is a naturally occurring polyphenol that has been reported to exhibit a wide range of important biological and pharmacological properties. We previously obtained a control cell line of V. amurensis (VV) as well as a rolB-transgenic cell line of V. amurensis (VB2) that has a higher level of resveratrol accumulation. In our experimental setup, the azaCtreated VV and VB2 calli produced 0.092% and 0.455% dry weight (DW) resveratrol, respectively. We found that treatment with 200 lM of azaC resulted in 1.9-and 2.0-fold increases in resveratrol production in VV and VB2 calli, respectively. A quantitative real-time PCR assay for STS gene expression in the azaC-treated VV and VB2 cells revealed that there were statistically increased expression levels of VaSTS10 in VV calli and of VaSTS5, VaSTS6, and VaSTS10 in VB2 calli. These results demonstrate that azaC is able to increase resveratrol production in V. amurensis calli through a mechanism that involves the induction of STS gene expression.

Salicylic acid (SA) treatment selectively reduced the cytosine DNA methylation of stilbene synthase ( STS ) genes and stimulated resveratrol production in cell cultures of Vitis amurensis. The effect of salicylic acid (SA) on plant growth, flowering time, and fruit number is known to correlate with the level of DNA methylation, while the potential correlation between SA-induced changes in DNA methylation and biosynthesis of secondary metabolites has not been studied. Trans-resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In this study, cell cultures of Vitis amurensis capable of producing t-resveratrol were used as a model system to study whether the SA-induced increase in t-resveratrol production is associated with changes in DNA methylation of stilbene synthase (STS) genes. T-resveratrol is synthesized via the phenylpropanoid pathway, in which STS genes are the key enzymes. Treatment of V. amurensis callus cultures with SA significantly increased t-resveratrol production and the expression of certain STS genes (e.g., VaSTS2 and VaSTS10). A marked decrease in the methylation of the VaSTS2 and VaSTS10 genes in response to SA was demonstrated using bisulfite sequencing, while no considerable changes were detected in the methylation of VaSTS1, a constitutively and highly expressed STS gene. The obtained results show that SA treatment selectively reduced cytosine methylation of VaSTS genes. The data suggest that selective DNA demethylation of particular STS genes could be necessary for the activation of t-resveratrol biosynthesis in response to SA. This finding provides an insight into the mechanism of SA action and biosynthesis of secondary metabolites in plant cells.

DNA methylation is known to play an important role in various developmental processes and defense mechanisms in plants and other organisms. However, it is not known whether DNA methylation is implicated in the genetic regulation of plant secondary metabolism, including resveratrol biosynthesis. Resveratrol is a naturally occurring polyphenol that is present in grapes, peanuts, and other plant sources, and it exhibits a wide range of valuable biologically active properties. The transformation of the wild-growing grape Vitis amurensis with the oncogene rolB from Agrobacterium rhizogenes has been demonstrated to considerably increase resveratrol production. To investigate whether DNA methylation regulates resveratrol biosynthesis, we treated both rolB transgenic and empty vector control V. amurensis cell cultures with the DNA demethylation agent 5-azacytosine (azaC). The azaC treatment significantly increased stilbene synthase 10 gene (VaSTS10) expression and resveratrol content in the V. amurensis cell cultures. Using bisulfite sequencing, we examined the methylation status of VaSTS10 in cell cultures under normal conditions and after azaC treatment. Both the promoter and 3'-end of the protein coding region of the VaSTS10 gene were hypermethylated (54-67 %) in the control cell culture. The rolB transgenic cell culture had high levels of resveratrol and lower hypermethylation levels of the VaSTS10 gene (20-47 %). The azaC treatment resulted in reduction in the DNA methylation levels in the promoter and coding regions of the VaSTS10 gene in both cell cultures. These data suggest that the DNA methylation may be involved in the control of resveratrol biosynthesis via the regulation of STS genes expression.