The Mechanism Of Calcium-associated Protein Kinase Genes In Regulating The Response To Abiotic Stress In Canola And Arabidopsis

The yield and quality of canola (Brassica napus L.) and other plants are frequentlythreatened by environmental stresses including drought, cold and high salinity. Calcium is aubiquitous intracellular secondary messenger in plants. Proteins that decode Calciumsignaling in plants are divided into four categories. Calcineurin B-like proteins (CBLs) areCa2+sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting proteinkinases (CIPKs). Calcium-dependent protein kinases (CPKs) are also Ser/Thr protein kinasesfound only in plants and some protozoans. There are quite some studies on CBL, CIPK, CPKgene families in model plants Arabidopsis and rice, however, little is known about these genesin canola (Brassica napus L.), which is one of the most important oil-producing crops inChina and worldwide, and belongs to the genus of Brassica with Arabidopsis. As the yield andquality of canola are frequently threatened by stresses, it will be of theoretical significanceand practical value to study calcium signaling related genes and clarify their signalingnetworks in response to stresses at the molecular level, and to compare with the relevantconclusions from Arabidopsis.In the present study, we further studied a number of CBL, CIPK and CPK genesidentified and cloned from canola previously, using a combination of plant physiological,biochemical, molecular biological and genetic approaches. The subcellular localization ofthree BnaCBLs, two BnaCIPKs and five BnaCIPKs were determined using greenfluorescence protein (GFP) as the reporter. Interactions between six BnaCBLs and17BnaCIPKs were examined using yeast two-hybrid assay (Y2H), and a subset of interactionswere further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore,the expression levels of six BnaCBL,12BnaCIPK and21BnaCPK genes in response to salt,drought, cold, heat, ABA, methyl viologen (MV) and low potassium (LK) were examined byquantitative RT-PCR and these genes were found to respond to multiple stimuli. Morover, thefunction of BnaCIPK24gene was dissected in Arabidopsis. This study suggests that thecanola CBL-CIPK and CPK network may be a point of convergence for several differentsignaling pathways. We also characterized an Arabidopsis Ser/Thr protein kinase PKSA5gene and found out that it is involved in the ABA and drought signaling pathway, possibly throughphosphorylating ABF transcription factors. Besides, the promoter activity of PKSA5wasanalyzed through using GUS reporter gene with multiple interaction proteins identifiedthrough yeast two-hybrid and BiFC.Our findings indicate that CBL and CIPK family members may form a dynamic complexto respond to different abiotic or hormone signaling. Our comparative analyses of theCBL-CIPK network between canola and Arabidopsis highlight functional differences and thenecessity to study CBL-CIPK gene functions in canola. Besides, the study on CPK genefamily in canola provides a foundation for further functional studies and signal transductionmechanism in plants. The identified novel Arabidopsis PKSA5gene modulating ABA anddrought response, and the primary elucidation of its regulatory mechanism, will make it agood candidate gene for molecular breeding of stress-tolerant crop plants and also lays atheoretical foundation.