Abstract
Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.
Highlights
Stilbenes are part of a vast group of plant polyphenolic compounds with antimicrobial activities synthesized via the phenylpropanoid pathway by a broad range of unrelated plant families [1,2,3]
To establish Vitis amurensis Rupr. cell cultures overexpressing the full-length VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes and the control cell line, the V7 suspension culture of V. amurensis was incubated with A. tumefaciens strains to bear the pZP-RCS2VaCML-nptII construct for VaCML-transgenic cells or the pZP-RCS2-nptII for the control KA0 cell line
We investigated the effect of overexpression of five grapevine calmodulin-like protein (CML) genes, which were highly up-regulated in wild-growing grapevine V. amurensis in response to stilbene-inducing conditions, and were suggested as promising candidates playing important roles in stilbene biosynthesis [43]
Summary
Stilbenes are part of a vast group of plant polyphenolic compounds with antimicrobial activities synthesized via the phenylpropanoid pathway by a broad range of unrelated plant families [1,2,3]. Plant stilbenes are well-known to confer a variety of health benefits and possess promising pharmacological applications [4,5,6]. Stilbenes are classified as phytoalexins that rapidly accumulate in plants in response to injury or microbial pathogens and contribute to plant environmental stress resistance [3,7]. Trans-resveratrol (t-resveratrol) is the most prominent stilbene with a variety of valuable biologically active properties and was widely used in the food and medicine industries [8]. Stilbenes are known to be involved in plant constitutive and inducible defense reactions against plant fungal and bacterial pathogens, nematodes, and herbivores [7,9]. Biosynthesis of natural stilbenoids is activated in response to a number of other environmental stresses, such as ultraviolet (UV) irradiation, wounding, drought, or unfavorable temperatures [3,9]
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