Influence of overexpression of the true and false alternative transcripts of calcium-dependent protein kinase CPK9 and CPK3a genes on the growth, stress tolerance, and resveratrol content in Vitis amurensis cell cultures

Abstract

Calcium-dependent protein kinases (CDPKs or CPKs) are important Ca2+ sensors in plants implicated in plant abiotic and biotic stress responses, e.g. in drought-induced stomata closure or pathogen-induced stilbene production. We previously found that the VaCPK9 gene of Vitis amurensis was subjected to unusual post-transcriptional processing mediated by canonical splice sites leading to production of three mRNA isoforms that lacked important catalytic kinase subdomains. We also detected numerous unusual VaCPK9 and VaCPK3a cDNAs possessing short direct repeated sequences instead of canonical splice sites and suggested that these cDNAs were false alternative transcripts generated by reverse transcriptase template switching in vitro. The present study has been attempted to determine if the full-length and spliced CDPKs play a role in the grapevine growth regulation, stilbene biosynthesis, and abiotic stress adaptation. We generated transgenic V. amurensis callus cultures overexpressing different true and false VaCPK9 and VaCPK3a transcripts under control of the CaMV 35S promoter. Overexpression of the full-length VaCPK9 and VaCPK3a transcripts slightly increased biomass accumulation but did not affect resistance to abiotic stresses and biosynthesis of resveratrol (3,5,4′-trihydroxy-trans-stilbene) in the calli. Overexpression of the true alternative VaCPK9SF1, VaCPK9SF2, and VaCPK9SF3 transcripts considerably improved growth of the transgenic calli, while overexpression of the false alternative VaCPK3aSF1, VaCPK3aSF2, and VaCPK3aSF3 transcripts did not considerably influence it. The results show that the VaCPK9 and VaCPK3a genes function in growth regulation of V. amurensis but not in stilbene biosynthesis and abiotic stress tolerance. Alternative splicing of VaCPK9 may serve to reinforce the positive effect of VaCPK9 expression on biomass accumulation.