Functional Analysis Of ZmCIPK9 And ZmCBL8 From Maize In Response To Abiotic Stress

Concentrations of calcium increase in plant cells rapidly and dramatically under environmental stresses. This kind of change in calcium concentrations is sufficient to trigger various physiological adjustment respond to these stimuli. CBL/CIPK signaling pathway in plant plays an important role in calcium signature transduction. After sensing the calcium signature, CBLs activate the kinase activity of the downstream component CIPK subsequently to relay the calcium signal. Although a numbers of CIPK and CBL family members were found to be involved in plant responding to abiotic stress, detailed researches are still needed to uncover the mechanism of CBL/CIPK signaling pathway in maize. Previous studies in our lab have shown that there were more than 40 CIPK and 10 CBL members in maize genome, respectively, some of which were upregulated in various abiotic stresses. This suggested that these genes play an crucial part in response to abiotic stress in maize.Firstly, we previously found that ZmCIPK9, as a CIPK gene in maize, was upregulated significantly in drought stress and its overexpression rescued the low potassium sensitivity of Atcipk23 mutant. In this study, Real-time PCR showed that ZmCIPK9 mRNA expressed in various organ of maize, and ears expressed the highest expression level of ZmCIPK9. Abiotic stresses, including low potassium, ABA, NaCl, dehydration, cold and heat, strongly induced expression of ZmCIPK9. ZmCIPK9 was proved to interact with ZmCBL1, ZmCBL4 and ZmCBL9 in yeast two-hybrid system and BiFC assay. ZmCIPK9/ZmCBLs complexes located on plasma membrane after their interaction. Atcipk23 mutant transgenic lines harboring the 35S:ZmCIPK9 showed elevated potassium content and uptake, respectively. For screening the ZmCIPK9’s target, we used the yeast two-hybrid, and found that ZmCIPK9 could interact ZORK, a Shaker potassium channel in maize. These findings indicated that ZmCIPK9 is involved in the CBL/CIPK signaling network, which is related to potassium signal transduction.In addition, we cloned ZmCBL8, a homolog of AtCBL8, via phylogenic method. Sequence analysis showed that ZmCBL8 have a high sequence identity with AtCBLS and AtCBL4. Expression level of ZmCBL8 was significantly suppressed under ABA, NaCl, cold and heat treatment.3 ZmCIPKs were found to interact with ZmCBL8 in yeast two-hybrid system. ZmCBL8::GFP fusion protein expressed in cytoplasm and nucleus. Then the ZmCBL8 was over-expressed in Arabidopsis Col-0. Transgenic lines showed ABA insensitive phenotype compared with wild type, of which the green cotyledon rate, fresh weight and root length were less inhibited under ABA treatment. Real-time PCR showed that ZmCBL8’s overexpression elevated the ability of ABA degeneration and influenced ABA biosynthesis related genes; no obvious changes were found in genes involved in ABA signaling pathway. These results suggested that ZmCBL8 may regulate ABA biosynthesis and metabolism, and function negatively in maize responding to abiotic stresses.