The Molecular Mechanism Of AtRSA1 Regulation Of Lateral Root Growth And Stomatal Movement

As a critical plant hormone,abscisic acid?ABA?participates in regulating the whole processes of plant growth and development.Based on guard cell researches,the discovery of ABA receptors and its sig-nal pathway obtained unprecedented development.However,the molecular mechanism of ABA regulating plant root growth and development and its complex physiological process are still unclear.To understand-ing the molecular mechanism,we identified a lateral root growth and stomatal movement related mutant,rsa1-1?root growth and stomatal aperture regulated by ABA 1,RSA1?,in the forward genetic screen for ABA insensitive mutants.By map-based cloning,we found AtRSA1 encodes an acetyl-CoA synthetase pro-tein,ACN1?acetate non-utilizing 1?.The lateral root?LR?development of the two allelic mutants of AtRSA1,atrsa1-1 and atrsa1-2,was sensitive to ABA.The lateral root length,number and density of the mutants were inhibited by ABA,com-pared to those of wild type?WT?.In addition,the complement lines recovered the ABA insensitive pheno-type in the loss function of AtRSA1 mutant.Except for the lateral root growth responsed to ABA,both of atrsa1-1 and atrsa1-2 showed smaller stomatal aperture,lower water loss,and higher leaf temperature than WT.Moreover,the classic response of ABA induced stomatal closure showed that atrsa1-1 was less sensi-tive to exogenous ABA-induced stomatal closure,indicating that AtRSA1 also involved in the ABA-regulated stomatal movement.Protein subcellular localization showed that AtRSA1 located in peroxisome.According to the results of histochemical staining and real-time quantitative PCR,AtRSA1 expresses in various tissues,including root and guard cell,and can be induced by ABA.Based on the bioinformatics data,AtRSA1 promoter con-tains ABRE?ABA-responsive elements?conserved sequence,which responses to ABA signaling.Using yeast one-hybrid and transient expression system,we found ABREs from AtRSA1 combined with ABFs transcription factors,which will regulate the expression of AtRSA1 to ABA.AtRSA1 was implicated in regulating plant metabolism,when it was mutation,the contents of meta-bolic intermediates measured?including malate,citrate,?-ketoglutarate,et al.?are decreased.And the de-crease also happened after ABA treatment in the WT plants,while no significant or slight changes occur in the mutant plants in the presence of ABA,suggesting that AtRSA1 is involved in regulating metabolism in ABA responses.Furthermore,the smaller stomatal aperture of atrsa1-1 could be restored to the level of WT by exogenous malate,which further evidenced the function of AtRSA1 in regulating plant metabolism.We have tested the internal K⁺channel activity of guard cell plasma membrane,and found that[K+]_in current of atrsa1-1 was smaller than that of WT,and exogenous malate could recover K⁺channel activity up to the WT level.Moreover,the guard cell intracellular pH of atrsa1-1 was higher than WT,and it can be restored to the WT level by exogenous malate.These results suggested that AtRSA1 regulates stomatal aperture,the activity of plasma membrane K⁺channels and intracellular pH by regulating the level of malate.In conclusion,our results suggest that,AtRSA1 recycles free acetate into acetyl-CoA in peroxisome,and into the TCA cycle eventually,which providing carbon skeleton for malate synthesis in the ABA response.The malate(Malate^2-)can balance intracellular cation?K⁺?,regulates cell turgor pressure,and promotes stomatal opening and lateral root formation.The researches of AtRSA1 revealed the ABA regulation of root development and stomatal movement mechanism,which coordinating the shoot and root.Moreover,the discovery that AtRSA1 mediates stomatal movement by regulating the intracellular metabolites malate,provides a powerful evidence for the malate production theory in traditional stomatal movement mechanism.