Inactivation of the auto-inhibitory domain in Arabidopsis AtCPK1 leads to increased salt, cold and heat tolerance in the AtCPK1-transformed Rubia cordifolia L cell cultures.

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.