The Regulatory Effect Of GAAP1 To Endoplasmic Reticulum Stress In Arabidopsis

Protein is the bearer of life, whether it is higher or lower organisms are, proteins are necessary. The endoplasmic reticulum (ER) is one of the main organelles of protein processing. When the plant suffering from the Various environmental stress conditions, it may will lead to the accumulating of the unfolder unfolded or misfolded proteins in endoplasmic reticulum, which was called the endoplasmic reticulum stress (ER Stress). Sin the early moment of ER stress, the plant cells will start someeveral protrction pathways including unfolded protein response (UPR) pathway will be initiated to relieve the ER stress, the unfolded protein response(UPR)pathway is included. But when the stress is soover-serious, that itUPR will occur induce the programmed cell death (PCD). Bax-inhibitor (BI-1) like protein is one family of ocell death suppressor and they chave been found involved in mmodulating tUPR. GAAPs are subfamily of BI-1 like factors and the function in plant chave not been reported. There are five GAAPs homologous genes in Arabidopsis. Previous studies in our laboratory showed that GAAP1 can resiste salt stress and ER stress. However, the mechanism is not known. Here Ithe role of GAAP1 in UPR and ER stress was studied and the main results are as following:1. GAAP1 is induced by ER stress. pGAAP1::GUS reporter assay showed that GAAP1 mainly expressed in roots during the vegetative growth period, and ER stress enhanced its expression. During the reproductive groth stages, GAAP1mainly expressed in pistil and stigma,filaments, immature seed beads stalk.2. GAAP1 relieve the ER stress which induced by TM. We found that GAAP1 reduced the generation of H2O2 and autopHagy induced by ER stress through 3,3'-diaminobenzidine (DAB) staining and mondansylcadverin (MDC) staining assay.3. GAAP1 participates in the ER stress by negative regulating UPR.Measuring the overexpression and mutation through QRT-PCR, results in that in acute stress the lack and mutation of GAAP1 have no influence in the expression of UPR passway;in the early period and lasting time,the overexpression of GAAPI can inhibit and decrease the up-regulation of UPR;however,the mutation of GAAP1 do not influent the UPR in the early period,and it can weaken the up-regulation of UPR in the lasting ER stress, which concluses that GAAP1 may be related with the influence of UPR in the lasting ER stress.By analysizing the expression of UPR in the different time after the acute stress,wild type,gaap1gaap3-the double mutation and GAAP1-OX further identified that GAAP1 can inhibit the expression of UPR.To explore the molecular mechanism that GAAP1 inhibits UPR,the protein expression of gaapl,Col,gaap1gaap3 and GAAP1-OX are tested.It turned out that GAAP1 may be weakening UPR pathway by up-regulating the expression of BIP protein.4. GAAP1 and its interacting factor were co-localized in the plasma membrane, and its cellular location was change upon ER stress. Confocal fluorescence microscopic assay showed that GAAP1 was located in the cell membrane, and had the common location in the cell membrane.The change of GAAP1's location was analysed in 35S::GAAP1-eYFP stable transformants in Arabidopsis. The result showed that GAAP1 located in the cell membrane, membrane and cytoskeleton of stomata guard cell, chloroplast envelope under normal growth conditions. And its cellular localization were changed after TM treatment.5. The interaction between GAAP1 and some factors in plant were verified. They were fused with FLAG and TAP expression vectors respectively, and their interaction were conducted by Co-Immunoprecipitation (CO-IP) assay.6. Conducted the double mutants of gaap1 and its interaction factors. The double mutant of GAAP1 and its interaction factors were generated to explore the relationship between them.And the double mutants were screened and identificated by PCR.In summary, GAAPI reduced reactive oxygen and autopHagy bodies induced by ER stress. GAAPI suppressed UPR during the early and late stage of ER stress. GAAPI also upregulated the protein level of BIP. Obviouly, GAAP1 regulated ER stress in various levels. The further study the function of the interaction of GAAP1 and its interactors will provide more detail information.