Functional Analysis Of Endoplasmic Reticulum-plasma Membrane Tether VAP27-1 During Endoplasmic Reticulum Stress In Arabidopsis

The efficient life process in eukaryotic cells depends on the complex network of organelle interactions.Endoplasmic reticulum(ER)is closely related to the plasma membrane(PM),forming ER-PM contact sites with a distance of less than 30 nm,which allow the material transport and information exchange between ER and PM.When the protein folding process is damaged due to adversity stress,ER stress can be triggered and the cell can initiate unfolded protein response(UPR)to resist the stress.In yeast and animal cells,cortical ER and ER-PM contact sites are involved in the response to ER stress.However,the roles of ER-PM contact sites and their functions in ER stress remain largely unknown in plant cells.In this study,multiple approaches including cell biology,genetics,molecular and biochemical assays were used to investigate the functions and regulatory mechanisms of ER-PM tethering protein VAP27-1 in ER stress in Arabidopsis.The main results are as follows:(1)Observations of transgenic seedlings expressing VAP27-1-EGFP using confocal microscopy revealed that VAP27-1-EGFP was widely distributed in various tissues of Arabidopsis seedlings.The subcellular localization of VAP27-1-EGFP is mainly concentrated at PM,which is unevenly distributed and enriched at specific sites.(2)Single-molecule analysis of VAP27-1-EGFP was performed with variable angle-total internal reflection fluorescence microscopy(VA-TIRFM)to track the trajectories of VAP27-1-EGFP particles over a period of time at PM.Single particle tracking was employed to analyze the velocity and diffusion coefficient of VAP27-1-EGFP.The results showed that the lateral motion of VAP27-1-EGFP at PM was stable.(3)By CRISPR/Cas9 system,vap27-1 vap27-3 vap27-4 triple mutants were obtained.The gene editing efficiency of Cas9 was different for different target sites,and gene editing for the same target site also resulted in multiple mutation types.Homozygous mutation mediated by CRISPR/Cas9 can be stably inherited.CRISPR/Cas9 bears an extremely low risk of off-target effects.(4)Observation of the growth state of vap27-1 vap27-3 vap27-4 and wild-type(WT)Arabidopsis roots under normal growth conditions and ER stress conditions showed that vap27-1 vap27-3 vap27-4had shorter root length and higher root tip cell mortality than that of WT under ER stress.After the rescue of VAP27-1-EGFP in vap27-1 vap27-3 vap27-4,the phenotype of root shortening under ER stress was alleviated to a certain extent.(5)Through quantitative real-time PCR(q RT-PCR)and western blot assay,it was found that the transcription level of VAP27-1 increased slightly under ER stress,while the expression level of VAP27-1-EGFP fusion protein did not change significantly.Single-molecule analysis showed that the velocity and diffusion coefficient of VAP27-1-EGFP decreased,and its motion mode was more stable at PM under ER stress.The distribution of VAP27-1-EGFP at PM was also inclined to have more cisternaes.(6)Using transcriptome sequencing technology and q RT-PCR,it was detected that higher constitutive unfolded protein response signal in vap27-1 vap27-3 vap27-4 than in WT under normal growth condition.The expressions of UPR relative genes in vap27-1 vap27-3 vap27-4 were also higher than that of WT under ER stress.(7)The ultrastructures of vap27-1 vap27-3 vap27-4 and WT Arabidopsis roots were investigated by use of high-pressure freeze fixation and transmission electron microscopy.The results revealed that there were smaller ER membrane area and weaker ER-PM connectivity in vap27-1 vap27-3 vap27-4compared with that of WT under normal growth condition or ER stress.(8)The blue-native polyacrylamide gel-electrophoresis(BN-PAGE)assay unveiled that VAP27-1was distributed in the same complex with AGB1 which mediated an unforded protein response pathway.Moreover,vap27-1 vap27-3 vap27-4 agb1 quadruple mutant was more sensitive to ER stress than vap27-1 vap27-3 vap27-4 triple mutant and agb1 single mutant.Taken together,this study deepened the molecular and cytological mechanisms of ER stress and unfolded protein response signaling pathways in plants.The results provided new clues for improving broad-spectrum resistance to different stress in plants,which may also have certain application value in plant stress tolerance.