Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.

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The present study examines the effect of p-coumaric acid (CA), a precursor of stilbenes and isoflavonoids, on biosynthesis of resveratrol in cell cultures of Vitis amurensis. Earlier, we transformed V. amurensis with the rolB gene of Agrobacterium rhizogenes and showed increased level of resveratrol production in the rolB transgenic cell culture. We used control and the rolB-transgenic cell culture of V. amurensis as a model system in this study. CA was capable of increasing resveratrol production in the control and the rolB-transgenic cell cultures in 10.3 and 1.5 times, respectively. The CA-treated control and rolB transgenic calli produced up to 0.06% and 1.1% DW of resveratrol. Using quantitative real-time RT-PCR, we characterized the expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes in the CA-treated control and rolB transgenic cell cultures. The expression of PAL genes remained essentially unchanged under 0.1 mM of CA, while expression of VaPAL1, VaPAL2, VaPAL3, and VaPAL5 genes was considerably decreased under 0.5 and 2 mM CA compared with the untreated cells. In the CA-treated calli, expression of VaSTS2 and VaSTS3 was increased, while expression of VaSTS5, VaSTS8, VaSTS9, and VaSTS10 was significantly decreased. These results indicate that CA increased resveratrol accumulation in V. amurensis calli via selective enhancement of expression of individual STS genes.

KEY MESSAGE : VaCDPK3a is actively expressed in leaves, stems, inflorescences, and berries of Vitis amurensis and may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis. Calcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.

Resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of a Vitis amurensis Rupr. cell suspension with the gene VaCPK20 for a calcium-dependent protein kinase (CDPK) under the control of double CaMV 35S promoter increased resveratrol production in five independently transformed cell lines in 9-68 times compared with control cells. The VaCPK20-transformed calli were capable of producing 0.04-0.42 % dry wt. of resveratrol, while the control calli produced up to 0.008 % dry wt. of resveratrol Also, we characterized expression of stilbene synthase (STS) genes in the five VaCPK20-transgenic cell lines of V. amurensis. In all VaCPK20-transgenic cell lines, expression of VaSTS7 increased; while expression of VaSTS1 decreased. We suggest that transformation of V. amurensis calli with the VaCPK20 gene induced resveratrol accumulation via enhancement of expression of the VaSTS7 gene involved in resveratrol biosynthesis. The obtained data first demonstrate that overexpression of a CDPK gene resulted in increased accumulation of a stilbenoid phytoalexine in transgenic plant cells. We propose that the VaCPK20 gene could play an important role in the regulation of resveratrol biosynthesis in grape cells.