An evolutionary conserved CBL1-CIPK6 complex of oil persimmon involved in responses to ABA, salt and drought stress

Calcineurin B-like proteins (CBLs) interact with CBL-interacting protein kinases (CIPKs) to form CBL-CIPK complexes, which regulate plant responses to biotic/abiotic stresses. These complexes originated from a common ancestor and subsequently diverged during evolution of land plants. In our previous work, a pair of CBL-CIPK genes from oil persimmon (Diospyros oleifera Cheng) were identified to play different roles in plant stress responses. However, their evolutionary trajectories, biological functions and regulatory networks remain unclear.

In this work, the cloned CBL and CIPK genes from oil persimmon were named as DoCBL1 and DoCIPK6 based on their homology. The phylogenetic analysis and sequence alignment showed that CBL1 and CIPK6 proteins are evolutionarily and structurally conserved across various plant species. The expansion of these proteins in angiosperms might enhance plant adaptation to abiotic stress. Additionally, DoCBL1 and DoCIPK6 proteins were localized at the plasma membrane in tobacco (Nicotiana tabacum L.) leaves, and DoCBL1 interacted with DoCIPK6 in both yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Furthermore, the potential functions of DoCBL1 and DoCIPK6 were explored by mutating and overexpressing them in Arabidopsis thaliana (Columbia), using wild-type plants as controls, under ABA, salt and drought stress. The results revealed that DoCBL1 and DoCIPK6 transgenic plants exhibited enhanced tolerance to salt and drought stress. This tolerance was achieved by altering the expression of ABI1, AKT1, CAX1, SOS1/2/3, PP2CA, DREB1A and RD26, thereby regulating the balance of K⁺, Ca²⁺ and Na⁺, as well as ROS-scavenging activities. Meanwhile, the transgenic Arabidopsis exhibited increased sensitivity to ABA, characterizing by elevated contents of Na⁺ and reactive oxygen species (ROS), including H₂O₂, O₂⁻, and MDA. These plants also showed significant reductions in biomass, K⁺ content, and the activities of SOD, POD and CAT.

Our findings suggested that DoCBL1 and DoCIPK6 might confer enhanced salt and drought tolerance, as well as increased sensitivity to ABA, in Arabidopsis plants. We proposed that DoCBL1-DoCIPK6 complex serves as a conserved bridge between stress signals and downstream transcriptional regulation, and thereby offering a novel perspective on their roles in abiotic stress responses.