Regulation Mechanism Of Arabidopsis CBL10 Regulates The Plasma Membrane H~+-ATPase Activity

Plants are sessile,unlike the animals can move freely,Moreover,the plant's habitat is not always good,they often suffer from a variety of environmental stress from outside,such as:salt damage,high pH,low pH,high temperature,low temperature,drought,pests,radiation,etc.Enhancing the bility of resistance is necessary,in order to adapt the changing external environment,this resistance may be a series of complex regulatory mechanisms in plants naturally evolved to be artificially cultivated.Ca2+ is common in plants as a second messenger,when plants suffer from external stress,the concertration and space of Ca2+ in vivo both change,this is called calcium signal.To react to external stimuli,plant needs to get the perception of calcium signal in vivo.CBL(Calcineurin B-like protein,CBL)is a series of important calcium-binding proteins,it involves in the stress signal transduction pathways of plants,ten kinds of genes that encode CBL have been discovered in Arabidopsis and rice,but CBL itself does not have biological activity,it combines with CIPK(CBL-interacting protein kinase)to form complex to regulate downstream taget genes and then involves in signal transduction,eventrually responds to stress.People have done many research on CBL and CIPK,existing studies show that CBL-CIPK transport system by regulating the activity of sodium ions,potassium ions to improve abiotic stress tolerance,but the relationship with the H+transport is not very clear,and H+ is mainly regulated through the plasma membrane H+-ATPase.Hodges,who has found the hydrolysis vigor of caveolae membrane can bound ATPase in 1972,and then found the plasma membrane H+-ATPase,plasma membrane H+-ATPase plays an important role in physiological process of growth and development and stress resistance pathway of plants,plasma membrane H+-ATPase can hydrolyze ATP to produce energy,so that transport the H+ within the cytoplasm out of the cell,is also known as the proton pump,it can generate a transmembrane pH gradient and the potential gradient through this pathway,further formed protons electrochemical potential ? pH,providing the required driving force to cell growth.It has been found that there are 11 kinds of encoding plasma membrane H+-ATPase gene in Arabidopsis,prior studies have shown CIPK11(PKS5)in Arabidopsis regulate the plasma membrane H+-ATPase2(AHA2)negatively,and AtCIPK11 can form a complex with AtCBL10 in Arabidopsis,so we verify whether AtCBL10 can regulate plasma membrane H+-ATPase activity directiy.Bioinformatics analysis shows,the C terminal of AtAHAs protein containing a long transmembrane domain,in order to reserach relationship between it and AtCBL10 protein,we used PCR method to amplify the C-terminus of 11 kinds AtAHAs protein in the cytoplasm,we have proved AtCBL10 may interact with the C-terminal of AtAHA4 and AtAHAll protein by using the yeast two-hybrid method and bimolecular fluorescence complementation experiments,then the interaction shows that AtCBL10 can regulate the plasma membrane +-ATPase directly.Under the high pH(pH7.7 and pH8.1)treatment,compared with wild-type Arabidopsis thaliana,the seeds of cbl10 mutant can better tolerate high pH,also the plant growth and development period,while under the low pH(pH 4.0)treatment,Arabidopsis wild-type and cbl10 mutant plants are both suppressed,but the effect is little.By using the NMT technology to detecting the flow of H+ under the normal condition(pH 5.8)and high pH(pH 8.1),result shows,the concentration of extral H+ is decrease in an alkaline environment,the activity of plasma membrane H+-ATPase in cbl10 mutant is reduced,the efflux of H+ is decrease or no flow,so the cbl10 mutant grows better than wild-type,it also shows AtCBL10 can adjuste the pH balance by regulating the activity of plant plasma membrane H+-ATPase,and respond to external stimuli,thus maintaining normal growth and development of plants.