Functional Analysis Of AtCIPKL In Ethylene Biosynthesis In Arabidopsis Thaliana

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. The research in Arabidopsis shows that CIPKs are involved in a variety of biological or abiotic stresses and regulate plant growth and morphogenesis. More and more data demonstrate that CIPKs can participate in many kinds of phytohormone signal transduction. In this study,a gene encoding one of CIPK family kinases, AtCIPKL, was identified and cloned. With CIPKL overexpressing transgenic plants andcipkl mutant, the function of AtCIPKL was analyzed and the results showed that AtCIPKL may be involved in the regulation of ethylene synthesis and Brassinosteroids signal transduction.1. The assay of AtCIPKL promoter activityThe promoter sequences of AtCIPKL was isolated from Arabidopsis genome and cloned into pBI101 vector.The construction was introduced into Arabidopsis by floral dip method.Under normal conditions, AtCIPKL promoter acticity was detected mainly in the root(except for root tip).But the activity of AtCIPKLpromoter was changed under different processing.We found that both Jasmonic acid and ethephon can enhance GUS activity indicating the expression of AtCIPKL induced by the two phytohormone signals. Whereas, GUS activitywas reduced under BRtreated conditons.Theresults suggested that AtCIPKL were regulated by multiple hormones.2. AtCIPKL overexpression reduce the sensitivityto ACC and BL signalingAtCIPKL controlled by 35S promoter was introduced into Arabidopsis and transgenic plants were obtained. On the other hand, the salk T-DNA insertion mutant line cipkl was identified. The phenotype analysis of transgenic plants was carried out under different phytohormones treatment.indicated that Compared with wild type and mutant on MS medium,the growth of overexpression transgenic seedling is similar to them and no obvious difference.On MS with luM ACC,the length primary root was significantly longer than WT and the mutant..On MS with ethephon(the active form of ethylene),there is no difference between them. The overexpression transgenic plants shows a tolerability of ACC but ethephon, we suspect AtCIPKL may regulate the synthesis of ethylene to change the content of ethylene.On MS with 10nM BL,the transgenic plants was able to decrease the sensitive of BR.3.Screening and identification of AtCIPKL-interacting proteins fromArabidopsis cDNA libraries.Yeast two-hybrid analysis was performed using the AtCIPKL as bait to screen the two-hybrid library of Arabidopsis cDNAs constructed on the prey vector.There are 28 unique proteins were identified as positive clones.The AtCIPK5-interacting proteins were related to various aspects of plant development..metabolism and signal transduction.4.35S:AtCIPKLreduces the ethylene content in the root and increase the anthocyanin content in leavesIn order to prove the AtCIPKL reduces the production of ethylene,we determine the gaseous ethylene content of root by Meteorological chromatography. There are no obvious difference on the MS medium.The experimental results explain that the ethylene content of transgenic plant is lower than wild type and mutant under ACC processing.Ehhylene can lead to the accumulation of anthocyanin,on the MS with ACC, Mutants have the highest level of anthocyanin accumulation, This result also proves that due to the disfunction of CIPKL result in higher ethylene content, resulting in anthocyanin accumulation.5. AtCIPKLcan interact with AtACO2 proteinYeast two hybrid test results show that the AtCIPKL can interact with AtACO2, and the results of BiFC also shows that they are able to interact in the cytoplasm.The AtACO2 protein was phosphorylated by AtCIPKL protein and lose the ability of the ACC into ethylene.