The Function And Mechanism Of Calcium-dependent Protein Kinases CPK28 In Tomato Response To Heat Stress

Under the background of global warming,the temperature keeps rising and accompanied by frequent hot weather.Our country is the country with the largest area of protected vegetable cultivation,it is easier to form high temperature environment in semi-closed facility environment.Tomato is an important horticultural crop and the main cultivated crop in China.Heat stress seriously harms the quality of tomato and restricts the annual production of tomato industry in China.Therefore,it is of great significance for the healthy and stable development of vegetable industry in China to explore the internal regulation mechanism of tomato under heat stress and explore the molecular mechanism of plant response to heat,so as to innovate resistance technology based on the heat characteristics of plants.Ca²⁺as a ubiquitous secondary messenger,regulates plant stress signaling and plays an important role in plant response to heat stress.Calcium-dependent protein kinase（CPK）is an important calcium sensing protein that transmits calcium signals and performs its kinase function through regulating the activity of target proteins.In recent years,more and more studies have confirmed the role of CPK in abiotic stress,however,the role of CPK in plant response to thermotolerance and its molecular regulatory mechanism remain unclear.In this study,tomato was used as the research material,and plant physiology,genetics,molecular biology and other techniques were used to study the role of CPK28 in response to heat stress in tomato and the effect of CPK28 mutation on plant antioxidant system,and its target protein APX2 was identified to further clarify the internal molecular mechanism of CPK28 regulation of APX2.The main results are as follows:1.We identified the positive regulatory role of CPK28 in tomato response to heat stress.The thermotolerance of cpk28#36 and cpk28#44 mutants with loss of function of CPK28 gene was significantly reduced,which showed that the leaves were wilting after heat treatment,the electrolyte permeability of leaves increased,and the content of MDA increased.2.We found that CPK28 enhanced the antioxidant capacity of tomato in response to heat stress.We found that CPK28 also participated in and positively regulated the antioxidant system of plants.We found the accumulation of intracellular oxidative substances O₂.^-and H₂O₂ was significantly increased in cpk28,the content of ROS-scavenging protein 2-CP monomer was significantly decreased,APX expression was decreased,the activities of CAT,APX,DHAR,GR and other antioxidant enzymes were inhibited,and the ratios of GSH/GSSG and As A/DHA were significantly decreased.Secondly,we found that APX activity and As A/DHA ratio in CPK28 were also inhibited at normal temperature.We effectively improved CPK28’s thermotolerance and As A/DHA ratio at heat by exogenous treatment of As A.These results suggested that CPK28was involved in the regulation of plant redox system by affecting antioxidant enzyme activities and As A/DHA levels,and thus affected the accumulation of plant oxides and protein deformation under heat stress.3.We identified APX2,the interaction target of CPK28.APX2 positively regulates tomato thermotolerance.The interaction between CPK28 and APX2 was found by lucilucase complementation assay and co-immunoprecipitation Co-IP assay,and the interaction between CPK28 and APX2 was enhanced by Ca²⁺induction,while the interaction between CPK28 and APX2 was inhibited by calcium ion chelator EGTA.Furthermore,we constructed apx2,and found that APX2 also positively regulates tomato high temperature resistance.Under heat stress,The thermotolerance,photochemical efficiency and APX activity of the apx2 mutant decreased.4.We identified that CPK28 affected APX activity and thermotolerance by phosphorylating APX2’s Thr-59 and Thr-169.We found that CPK28 phosphorylates APX2 and further studied the function of the phosphorylation site in plant response to heat stress.CPK28 can phosphorylate and activate APX2 activity,and phosphorylation is positively regulated by Ca²⁺.The inactivated phosphorylation site mutation blocked the phosphorylation of CPK28.In tobacco and tomato,the inactivated phosphorylation site not only weakened thermotolerance,but also reduced the activity of APX.These results suggested that Ca²⁺mediated the phosphorylation of APX2 by CPK28,and CPK28 may regulate the activity of APX2 through phosphorylation of two residues of APX2:Thr-59 and Thr-164,and then participate in the regulation of redox homeostasis of tomato and improve the thermotolerance of tomato.