Induction of resveratrol biosynthesis in Vitis amurensis cells by heterologous expression of the Arabidopsis constitutively active, Ca-independent form of the AtCPK1 gene Running Title: CDPK effect on resveratrol production

In the present study, we established transgenic calli of Vitis amurensis that expressed a constitutively active, Ca-independent form of the AtCPK1 gene (AtCPK1-Ca) and calli with a non-active form of the gene. High-performance liquid chromatography with UV and highresolution mass-spectrometry revealed that the predominant metabolites synthesized in our transgenic callus cultures were trans-resveratrol di-glucoside, trans-piceid, trans-resveratrol, trans-ε-viniferin and trans-δ-viniferin. The resveratrol content in the AtCPK1-Ca-transformed callus cultures exceeded that in the control cultures up to 90-fold. Furthermore, the expression of the AtCPK1-Ca gene caused cell growth activation, which led to the enhancement of resveratrol production up to 137 times that of the control calli (69.7 mg L vs. 0.51 mg L). Real-time PCR analysis showed that AtCPK1-Ca overexpression caused increasing of the expression of the key enzymes of phenylpropanoid pathway of resveratrol biosynthesis, 4-coumarate-CoA ligases. Thus, heterologous expression of constitutively active CDPK genes can be used to bioengineer plant cell cultures that produce stilbenes. Possible mechanisms for AtCPK1-mediated signal transduction were proposed by the reconstruction of known protein-protein interactions within CPK1-assotiated protein modules.