Effect of spruce PjSTS1a, PjSTS2, or PjSTS3 gene overexpression on stilbene biosynthesis in callus cultures of Vitis amurensis Rupr.

Stilbenes are natural compounds protecting plants against microbial pathogens and known to possess valuable biologically active properties. In the present study, we established transgenic grapevine callus cell cultures overexpressing three stilbene synthase (STS) genes of spruce Picea jezoensis PjSTS1a, PjSTS2, and PjSTS3. Transformation of Vitis amurensis calli with the PjSTS1a, PjSTS2, and PjSTS3 genes significantly increased total content of stilbenes in 3.6-6, 2.5-2.9, and 4.1-16.1 times, respectively, in comparison with the control calli. The most pronounced positive effect on the accumulation of stilbenes was observed for the PjSTS3-overexpressing calli where the total content of stilbenes was increased up to 3.1 mg/g DW, and the stilbene production reached 25.4 mg/L. These values were higher than those achieved for the grapevine callus cell cultures overexpressing three STS genes from V. amurensis. Thus, transformation of grapevine cell cultures with spruce STS genes with a relatively low degree of homology to the endogenous VaSTSs is a more effective strategy for induction of plant secondary metabolite biosynthesis than using the grapevine genes for the overexpression experiments.

The effect of ultraviolet-C and precursor feeding on stilbene biosynthesis in spruce Picea jezoensis.

This study examines the effects of p-coumaric (CoA) and caffeic (CaA) acids on stilbene biosynthesis in one-year-old needles of Picea jezoensis (Lindl. et Gord.) Fisch ex Carr. under control conditions and after ultraviolet (UV-C) irradiation. HPLC analysis revealed that while UV-C irradiation slightly affected the total stilbene content, CoA and CaA feeding increased the total content of stilbenes by 1.2-1.3-fold. UV-C treatment combined with CoA-feeding of the P. jezoensis cuttings exerted the most pronounced stimulatory effect on stilbene accumulation leading to the total stilbene content of 9.18 mg/g of dry weight or DW. This increase correlated with the elevated transcription of the stilbene synthase PjSTS1a and PjSTS1b genes. UV-C treatment in combination with CaA feeding of P. jezoensis did not considerably influence stilbene content. These results revealed a positive effect of UV-C radiation and phenolic precursors on the content of stilbenes in spruce needles.

Stilbene biosynthesis in the needles of spruce Picea jezoensis.

Stilbenes are valuable phenolic compounds that are synthesized in plants via the phenylpropanoid pathway where stilbene synthase (STS) directly catalyzes resveratrol or pinosylvin formation. Currently, there is a lack of information about the stilbene biosynthetic pathway in spruce (Picea). Resveratrol and piceatannol derivatives have been detected in the spruce bark, needles, and roots. We analyzed seasonal variation in stilbene spectrum and content in the needles of different ages of one tree of spruce Picea jezoensis. HPLC analysis revealed the presence of nine stilbenes: t- and cis-astringin, t- and cis-piceid, t- and cis-isorhapontin, and t-piceatannol were present in amounts of 0.01-6.07 mg/g of dry weight (DW), while t-isorhapontigenin and t-resveratrol were present in traces (0.001-0.312 µg/g DW). T-astringin prevailed over other stilbenoid compounds (66-86% of all stilbenes). The highest total stilbene content was detected in one-year-old needles collected in the autumn and spring (5.4-7.77 mg/g DW). We previously cloned and sequenced full-length cDNAs of the four STS transcripts (PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3) of P. jezoensis. This study presents a detailed analysis of seasonal variations in PjSTS1a, 1b, 2, and 3 transcript levels in the needles of P. jezoensis of different ages using qRT-PCR. PjSTS1a and PjSTS1b transcription was higher in the needles collected in the autumn, spring, or summer than in the winter. PjSTS2 was actively transcribed in the needles of all ages collected in the winter, spring, and summer. PjSTS3 expression did not significantly change during the year and did not depend on the age of the needles. Therefore, the data show that high levels of the stilbene glucosides and PjSTS expression are present in the needles of P. jezoensis.

[Expression of the stilbene synthase genes in the needles of spruce Picea jezoensis].

Stilbenes are valuable plant phytoalexins, the biosynthesis of which is characteristic of different groups of phylogenetically unrelated plants. It is believed that all the stilbenes are the derivatives of resveratrol (3,5,4'-trihydroxy-trans-stilbene) or compounds close to it (pinosylvin or piceatannol). The last stage of the resveratrol biosynthesis takes place with the involvement of stilbene synthase or resveratrol synthase (STS). The family Pinaceae is characterized by the presence of the derivatives of pinosylvin (genus Pinus) and piceatannol (genus Picea), the biosynthetic pathways of which are scarcely examined. Previously, in different species of the genus Picea, only two stilbene synthase genes were described. On the basis of RNA isolated from the needles of spruce Picea jezoensis, the full-length cDNAs of the four stilbene synthase genes, PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3, were obtained. Then, using the clone frequency analysis and real-time PCR, expression of the PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3 genes was examined in the needles of P. jezoensis accessions of different age and sampled in different seasons (spring, summer, autumn, winter). Among the analyzed transcripts, the PjSTS1a and PjSTS1b genes were the most frequent, indicating their higher level of expression compared to other STS genes. The highest level of PjSTS1a and PjSTS1b expression was observed in autumn, while the level of PjSTS2 and PjSTS3 expression was the highest in spring and winter. Moreover, the highest PjSTS expression was detected in the young tissues of P. jezoensis in autumn, which may indicate a higher level of stilbene biosynthesis in these tissues.