Effect of Calmodulin-like Gene (CML) Overexpression on Stilbene Biosynthesis in Cell Cultures of Vitis amurensis Rupr.

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.