Expression of calcium-dependent protein kinase (CDPK) genes in Vitis amurensis under abiotic stress conditions

Abiotic stresses, such as extreme temperatures, soil salinity, or water deficit, are one of the major limiting factors of crop productivity worldwide. Examination of molecular and genetic mechanisms of abiotic stress tolerance in plants is of great interest to plant biologists. Calcium-dependent protein kinases (CDPKs), which are the most important Ca sensors in plants, are known to play one of the key roles in plant adaptation to abiotic stress. CDPK is a multigene family of enzymes. Analysis of CDPK gene expression under various abiotic stress conditions would help identify those CDPKs that might play important roles in plant adaptation to abiotic stress. We focused on studying CDPK gene expression under osmotic, water deficit, and temperature stress conditions in a wild-growing grapevine Vitis amurensis Rurp., which is native to the Russian Far East and is known to possess high adaptive potential and high level of resistance against adverse environmental conditions. Healthy V. amurensis cuttings (excised young stems with one healthy leaf) were used for the treatments. For the non-stress treatment, we placed the cuttings in distilled water for 12 h at room temperature. For the water-deficit stress, detached cuttings were laid on a paper towel for 12 h at room temperature. For osmotic stress treatments, the cuttings were placed in 0.4 М NaCl and 0.4 М mannitol solutions for 12 h at room temperature. To examine temperature stress tolerance, the V. amurensis cuttings were placed in a growth chamber at +10C and +37C for 12 h. The total expression of VaCDPK genes was examined by semiquantitative RT-PCR with degenerate primers designed to the CDPK kinase domain. The total level of CDPK gene expression increased under salt and decreased under low temperature stress conditions. We sequenced 300 clones of the amplified part of different CDPK transcripts obtained from the analyzed cDNA probes. Analysis of the cDNA sequences identified 8 different CDPK genes (VaCDPK1a, 1e, 1d, 2a, 3a, 3b, 3c, 3d). We sequenced full cDNA sequences of the genes and analyzed their expression levels by real-time PCR and FAPP method, which has been recently developed by our research group. The prevalent CDPK transcript was VaCDPK3a under both non-stress and abiotic stress conditions. Under high-salt conditions, VaCDPK1d, 1e, 3b, and 3d transcripts were up-regulated. Under high mannitol conditions, expression of VaCPK1e and 3b was up-regulated, while expression of VaCDPK1d, 3c, and 3d was only slightly induced. Under water-deficit, expression of only VaCDPK3b and 3c genes was induced. Cold stress induced expression of VaCDPK2a and 3d genes; while hot stress induced expression of VaCDPK1a, 1d, 1e, 2a, 3a, and 3c genes. Taken together, the data show that the VaCDPK genes are transcriptionally regulated by osmotic, water-deficit, and temperature stresses. The differential expression of the VaCDPK genes during osmotic, water-deficit, and temperature stresses is suggestive of their involvement in the underlying signal transduction pathways.