CBL And CIPK Involved In Cold Signal Pathway In Arabidopsis Thaliana

Low temperature is one of the adverse environmental stresses that cause significant reductions in crop yield and quality and the studies on how to enhance the chilling/freezing tolerance of plants is of great importance. Low temperature alters the cytosolic Ca2+ concentration in cells. The initial perception of this calcium signal is via the binding of calcium to various calcium sensors, which transmit the stress signal downstream in the pathway. The family of calcineurin B-like (CBL) proteins is a unique group of Ca2+sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL-CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL-CIPK involved in cold stress responses. In the present study, we take 3-week-old Arabidopsis thaliana as object and analyze the behavior of calcium sensor CBL and its interacted protein kinase CIPK7 under cold stress. The major results are as follows:1. The expression of CIPK7 and CBL1 under cold stress was analyzed by real time quantitative PCR. The results revealed that the expression of both CIPK7 and CBL1 were induced by cold stress in 3-week-old Arabidopsis plants.2. CIPK7 demonstrated to be interacted with CBL1 in vitro in GST pull-down assay. And affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo.3. We compared the wild type and cbll mutant plants, and found that the sensitivity to cold was altered in cbll mutant. Under cold stress, there is no difference in survival rate of wild type and cbll mutant, however, the analyses of electrolyte leakage and proline content indicated that cbll plants was more sensitive than wild type to low temperature. Furthermore, the analyses of some cold-responsive genes expression in wild type and cbll mutant showed that the disruption of CBL1 gene alters the induction kinetics of several cold marker genes under cold stress. These results indicated that CBL1 is responsive to cold stress and that it participates in the cold stress signaling pathway. 4. We subsequently analyzed the expression patterns of CIPK7 in wild type and cbll mutant under cold stress. The results of both real-time quantitative PCR and Western blot analysis showed that the disruption of CBLl resulted in the delayed and diminished induction of CIPK7 by cold stress.Thus, our results demonstrate that expression of CIPK7 is induced by cold, and CIPK7 interacts with CBL1. Expression of CBLl is also cold inducible, and CBLl has a role in cold signal pathway. By comparing expression pattern of CIPK7 between wild-type and cbll mutant plants, we found that CIPK7 may be involved in cold stress response via its interaction with CBLl in Arabidopsis thaliana. Through exploring the function of CBLl and CIPK7 under cold stress, we hope our study might provide some promising clues for revealing how plant sense and response to low temperature.