Resveratrol Content and Expression of Phenylalanine Ammonia-lyase and Stilbene Synthase Genes in Cell Cultures of Vitis amurensis Treated with Coumaric Acid

Abstract

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.

Regulation of stilbene biosynthesis in plants

“…Exogenous application of phenylalanine, p-coumaric acid, and cinnamic acid had a positive effect on stilbene production in grapevine callus cell cultures (Shumakova et al 2011;Kiselev et al 2013d;. Application of p-coumaric acid and caffeic acid, which is a precursor for piceatannol and some other stilbenes, slightly increased stilbene production.…”

“…The most pronounced positive effect on stilbene accumulation has been observed for cinnamic acid . The application of phenylalanine induced expression of several PAL and STS genes, while treatment with p-coumaric acid did not lead to essential alterations in PAL and STS expression (Shumakova et al 2011;Kiselev et al 2013d). Both cinnamic and caffeic acids considerably increased expression of several STS genes.…”

Salicylic acid induces alterations in the methylation pattern of the VaSTS1, VaSTS2, and VaSTS10 genes in Vitis amurensis Rupr. cell cultures

“…Analysis was performed in an thermocycler (Bis-N) programmed for an initial denaturation step of 2 min at 95°C, followed by 35 cycles of 15 s at 95°C, 10 s at 52°C, 40 s at 72°C, and a last cycle of 72°C for 20 min, using the fastest available transitions between each temperature. The PCR products were isolated from agarose gels using a Cleanup Mini Kit (Evrogene, Moscow, Russia) and were subcloned into the pTZ57R/T plasmid using the InsT/Aclone PCR Product Cloning Kit (Fermentas, Vilnius, Lithuania) and sequenced using a Big Dye Terminator Cycle Sequencing Kit (Perkin-Elmer Biosystems, Forster City, CA), following the manufacturer's protocol and recommendations (Shumakova et al 2011;Dubrovina et al 2013) with an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA). A total of 15 individual clones were sequenced for each region of used genes.…”

Involvement of the cell-specific pigment genes pks and sult in bacterial defense response of sea urchins Strongylocentrotus intermedius

“…PCR products were purified with ethanol and directly sequenced in the Instrumental Centre of Institute of Biology and Soil Sciences (Far Eastern Branch of Russian Academy of Sciences, FEB RAS) using an ABI 3130 Genetic Analyzer (Applied Biosystems) as described previously (Kiselev & Dubrovina 2010, Shumakova et al 2011.…”