Regulation Of Plasma Membrane H~+-ATPase Activity By Endogenous Metabolites In Arabidopsis Thaliana

Plasma membrane H+-ATPase(PM H+-ATPase)in plant is a master enzyme with multiple physiological functions,which is essential for plant growth,development and stress responses.PM H+-ATPase hydrolyzes ATP and pumps protons from the cytoplasm to the apoplast to establish proton gradients and electrochemical gradients across the plasma membrane,which are used to drive secondary transport processes of most transport proteins.Many proteins such as kinase,phosphatase,and chaperone protein are all involved in PM H+-ATPase activity regulation.Plant endogenous metabolites are produced with a wide diversity in chemical structures and biological activities because of the complex living environments.However,whether endogenous organic metabolites play a role in PM H+-ATPase activity regulation has not been studied.Here,we describe a bio-guided isolation method to identify endogenous metabolites that regulate PM H+-ATPase activity.We obtained crude extracts from Arabidopsis seedlings with or without salt treatment and then purified them into fractions based on polarity and molecular weight by repeated column chromatography(silica gel,Sephadex LH-20,pre-HPLC).By evaluating the effect of each fraction on PM H+-ATPase activity and further elucidating the compounds in active fractions using spectroscopic methods and mass spectrometry methods,we found that endogenous free unsaturated fatty acids oleic acid(C18:1),linoleic acid(C18:2),and linolenic acid(C18:3)extracted from salt-treated seedlings stimulated PM H+-ATPase activity,and L1 extracted from none-salt treated seedlings inhibited PM H+-ATPase activity.These results were futher confirmed by the addition of exogenous C18:1,C18:2,C18:3 or L1 in the activity assay.The ssi2 mutant,with reduced levels of C18:1,C18:2,and C18:3,displayed reduced PM H+-ATPase activity,and the mutant1 and mutant2,with reduced levels of L1,displayed enhanced PM H+-ATPase activity and salt-tolerant phenotype.Furthermore,C18:1,C18:2,C18:3 and L1 directly bound to the C-terminus of the PM H+-ATPase AHA2.These results suggest that endogenous metabolites C18:1,C18:2,C18:3 could stimulate PM H+-ATPase activity by directly binding to the C-terminus of PM H+-ATPase,and also,L1 could inhibit PM H+-ATPase activity by directly binding to the C-terminus of PM H+-ATPase.Further quantitative real-time PCR analysis revealed that the expression of Gene-L2,which encodes an enzyme to catalyze the metabolism from L1 to L2,was induced by salt treatment.Content analysis for L1 and L2 by mass spectrometry revealed that the ratio of L2/L1 in plasma membrane increased with salt treatment.Activity evaluation of L2 revealed that L2 can activate plasma membrane Na+/H+ antiporter activity but had no effect on PM H+-ATPase activity.These results suggest that L1 may convert to L2 under salt stress to release PM H+-ATPase activity and activate plasma membrane Na+/H+ antiporter activity to increase the salt tolerace of plant.Taken together,endogenous metabolites C18:1,C18:2,C18:3 could stimulate PM H+-ATPase activity under salt stress;L1 could inhibit PM H+-ATPase activity under none-salt stress condition,however,L1 may convert to L2 under salt stress to improve the salt tolerance of plant.The bio-guided isolation model described in this study could enable the identification of new endogenous metabolites that modulate PM H+-ATPase activity,which facilitates our understanding on the regulation of PM H+-ATPase activity,and also provides method for studying endogenous metabolites in modulating other essential protein functions,as well as signal transduction,in plants.