Proteome-Level Investigation of Vitis amurensis Calli Transformed with a Constitutively Active, Ca2+-Independent Form of the Arabidopsis AtCPK1 Gene

Abstract

Calcium-dependent protein kinases (CDPKs) are one of the main Ca2+ decoders in plants. Among them, Arabidopsis thaliana AtCPK1 is one of the most studied CDPK genes as a positive regulator of plant responses to biotic and abiotic stress. The mutated form of AtCPK1, in which the autoinhibitory domain is inactivated (AtCPK1-Ca), provides constitutive kinase activity by mimicking a stress-induced increase in the Ca2+ flux. In the present study, we performed a proteomic analysis of Vitis amurensis calli overexpressing the AtCPK1-Ca form using untransformed calli as a control. In our previous studies, we have shown that the overexpression of this mutant form leads to the activation of secondary metabolism in plant cell cultures, including an increase in resveratrol biosynthesis in V. amurensis cell cultures. We analyzed upregulated and downregulated proteins in control and transgenic callus cultures using two-dimensional gel electrophoresis, and Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF). In calli transformed with AtCPK1-Ca, an increased amounts of pathogenesis-related proteins were found. A quantitative real-time PCR analysis confirmed this result.