Regulation Of Plasma Membrane H~+-ATPase Activity By SNARE Protein VAMP711 Under Drought Stress In Arabidopsis

Drought stress is one of the major abiotic stresses that negatively affect plant growth and development,and crop yield.The plant hormone abscisic acid(ABA)plays an important role in plant drought stress response.Previous studies showed that,under drought stress,ABA inhibits plasma membrane(PM)H+-ATPase activity,leading to the promotion of stomatal closure,thereby reducing water loss.The ost2-2D mutant which contains two mutations in AHA1(the point mutation of H+-ATPase family member AHA1)result in constitutive higher PM H+-ATPase activity,and it is hypersensitive to drought stress,indicating that PM H+-ATPase plays a very important role in drought stress response.However,the underlying molecular mechanisms are not well understood.To identify components involved in ABA mediated PM H+-ATPase regulation progress during stomatal closure,ABA-treated AHA1 transgenetic materials were used to screen for the putative interaction protein of AHA1 by co-immunoprecipitation(Co-IP)and liquid chromatography-tandem mass spectrometry(LC-MS/MS)assays.The SNARE(Soluble NSF-Attachment Protein Receptor)protein VAMP711(Vesicle-Associated Membrane Protein711)was identified as an interacting protein of AHA1.VAMP711 plays an essential role in vesicle trafficking by facilitating the fusion of vesicles and the target membrane.The interaction between VAMP711 and AHA1 was found to be induced by ABA treatment by firefly luciferase complementation(LCI)assay.Bimolecular fluorescence complementation(BiFC)assay indicated that VAMP711 interacted with AHAI on plasma membrane partially.Yeast two hybrid assay showed that 11 amino acid residues between 887 to 897 of AHA1/AHA2 were required for the interaction between VAMP711 and AHA1/AHA2.Subsequently,the PM H+-ATPase activity assay showed that the PM H+-ATPase activity in vamp711 mutant was higher than that of wild type;the recombinant protein of VAMP711 inhibited the PM H+-ATPase activity directly in vitro;VAMP711 inhibited yeast growth in which the growth is dependent on exogenous PM H+-ATPase activity.These results indicated that VAMP711 was a negative regulator of the PM H+-ATPase in Arabidopsis.In order to determine the molecular mechanism how VAMP711 regulates PM H+-ATPase in response to drought stress in Arabidopsis,we detected whether VAMP711 regulates the amount of PM H+-ATPase proteins on plasma membrane.The immunoblot analysis showed that VAMP711 had no effect on the amount of PM H+-ATPase on plasma membrane.The location of GFP-VAMP711 got close to plasma membrane under ABA treatment.After ABA treatment,the PM H+-ATPase activity decreased in both the wild type and the vamp711 mutant;however,greater reduction was detected in the wild type than that in the vamp711 mutant using the non-invasive micro-test technique(NMT)and PM H+-ATPase activity assays.These results suggest that ABA inhibits PM H+-ATPase activity at least partially through VAMP711.In Arabidopsis,overexpression of VAMP711 in ost2-2D mutants partially rescued the drought stress sensitive phenotype of ost2-2D,indicating that VAMP711 regulates drought stress by inhibiting the activity of PM H+-ATPase in Arabidopsis.Taken together,this study finds that the SNARE protein VAMP711 is involved in ABA mediated inhibition of PM H+-ATPase activity to promote stomatal closure in response drought stress.Under drought stress response,the accumulation of ABA enhances the interaction between VAMP711 and AHA1/AHA2 on the plasma membrane,leading to the reduction of PM H+-ATPase activity,which therefore promotes stomatal closure,reduces water loss.