A Dynamic Proteomic Atlas Of A Drought Tolerance Arabidopsis VAM3 Mutant

With the intensification of the global greenhouse effect and the emergence of the global extreme environment,drought stress has become one of the main environmental factors limiting crop productivity around the world.Improving drought resistance in crops is of great importance in enhancing crop productivity and food security.Arabidopsisthaliana was used in our research.A drought tolerant mutant(vam3)of Arabidopsis thaliana was successfully identified by using phenotypic analysis and related physiological parameters.VAM3 belongs to SNARE(soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors)protein family,which is located on vacuole.Based on previously studies,VAM3 is involved in vacuole fusion and can affect guard cell movement.In order to further elucidate the drought tolerance mechanism of the vam3,Top-down Label-free proteomics was used to compare vam3 and wild type Arabidopsis,with and without drought treatments.By comparing proteomic data from different genotypes and different treatment,we provided a strong foundation for analyzing the vam3 phenotypes and their drought tolerance mechanisms.(1)Dynamic phenotypes under drought treatment were successfully obtained by withholding watering for 2-week.According to the different time points between these two genotypes(WT and vam3),the relative water content,anthocyanin and proline content of the two groups were determined.By comparing the phenotypic and physiological data,the drought resistance of vam3 was determined.(2)By using exogenous ABA treatment,we successfully found that the stomatal movement of vam3 is more sensitive to ABA than that of wild type Arabidopsis.Seed germination experiments were carried out onvam3 and wild type Arabidopsis thaliana seeds were carried out by using different concentrations of ABA treatment.We found that during seed germinationvam3 were more sensitive to ABA treatment than wild type.(3)By using high performance liquid chromatography(HPLC),we found that the metabolism of indole glucosinolates and the aliphatic glucosinolates in vam3 showed strong responses to drought stress whereas,in the WT,indole glucosinolates metabolism showed no response to drought stress and aliphatic glucosinolate metabolism showed a little bit response to drought.(4)By using Top-down Label-free proteomics approach,we compared vam3 and wild type proteomes at 4 drought treatment time points(day-8,day-10,day-12,day-14).On comparing the proteomics data between vam3 and wild type under normal growth condition,we identified 765 and 383 increased and decreased proteins in vam3,respectively.There are 70 different expressed proteins in vam3 related to Arabidopsis morphogenesis.Furthermore,ACC1 was highly expressed in vam3 compared with WT and it's a key protein in the network analysis.ACC1,an acetyl-CoA carboxylase from Arabidopsis thaliana,can promote acetyl coenzyme A content in Arabidopsis thaliana which is related to histone H3 lysine 27(H3K27)acetylation.Furthermore,the acetylation level of histone H3 can affect the drought tolerance of Arabidopsis.By using String to analyze these differential proteins,we have obtained a series of candidate proteins that may cause the obvious phenotypes in vam3.The function of some key proteins is directly related to the drought resistance of plants,and this further illustrates the credibility of our proteomics data.(5)By comparing the overlap of vam3 and wild type drought stress response proteins,we isolated vam3 specifice differentially expressed proteins.There are 529 and 943 increased and decreased proteins specificly response to drought stress in vam3,respectively.Through functional and network analysis of these proteins,we obtained several key node proteins,such as PP2 C protein(AT4G16580),CDPK protein(AT4G37490)and chromatin remodeling protein(AT3G12810).Based on transcription factor analysis,there were 21 increased and 27 decreased transcription factor proteins specificly showed up in vam3.The use of comparable proteomics to identify the key proteins and transcription factors in vam3 will provide a better understanding of the mechanism responsible for the vam3 drought tolerance.