Preliminary Function Analysis Of GAAP4 In Arabidopsis

Endoplasmic reticulum(ER)is an important organelle providing protein folding processing,modification,and lipid synthesis in eukaryotes.And various environmental or biological factors caused the acc?mulation of misfolded proteins or unfolded proteins leading to ER stress.ER homeostasis can be maintained through regulating unfolded protein response(UPR)signaling in response to different ER-stress.Under the condition of long-term severe stress,UPRcan also induce programmed cell death.In yeast and mammalian cells,BAX inhibitor-1(BI-1)like proteins are one class of the most intensively characterized cell death suppressors In mammals,BI-1 like proteins play roles in cell protective through regulating UPR signaling.GAAPs(Golgi anti-apoptotic protein)are a novel,evolutionarily conserved sub-group of BI-1 related proteins.In arabidopsis thaliana,there are five GAAP genes.AtGAAP1 and AtGAAP3 have been found to be involved in resistance ER stress by our group.In order to further analyze the function of GAAPs in the plant and clarify the molecular mechanism of programmed cell death in plants,The function of AtGAAP4 was analyzed and main results are as follows:1.The expression of GAAP4 gene is special in different tissues and development stages of plants.By generating transformants with promoter and GUS fusion constructor(pGAAP4::GUS),GUS staining assay and situ hybridization,GAAP4 gene expression pattern was analyzed.The results showed that GAAP4 gene expressed mainly in cotyledon blade tip,leaf primordi?m,hypocotyl and root in vegetative growth period.And it expressed in leaf,flower and fruit in reproductive period.2.GAAP4 was involved in anti-ER stress through regulation of UPR.Col and gaap4 seedlings were treated with tunicamycin(TM),an inducer of ER stress,and the roots of mutant gaap4 were inhibited more significantly than those of the wild type.GAAP4 gene expression was significantly strengthened by ER stress.In addition,The changes of UPR marker genes duing the course of the moderate strength of stress were analyzed.And the result showed that UPR signaling was enhanced in gaap4 mutant in short time stress.With the lasting of ER stress,UPR signaling pathway mediated by IRE1 was shut down gradually first and followed by bZIP28 pathwan The change trend of UPR in gaap4 mutant was similar to wild type,but GAAP4 mutation weakened the increase of UPR gene expression Especially both mutation of GAAP4 and interactor MAPR3 severally affected the increase of UPR genes alonge the stress course.Based on these results we proposed that GAAP4 might regulate UPR or other cellular resitance pathway in the long ER stress conditions.3.GAAP4 negatively regulated the heat resistance.When gaap4,Col,GAAP4-OX treated with heat stress,we found that survival rate of gaap4 mutant seedling was obviously higher than that of Col.While GAAP4 restored the heat sensitivity.In addition,the analysis of UPR genes and heat stress response genes expression upon low strength of heat stress showed thatgaap4 mutation did not affec UPR signaling pathway but enhanced the expression of heat stress marker genes such as HSFA2,HSP90.1,DREB2.GAAP4-OX also restored the expression level to the level in CoL Additionally,upon high strength heat stress,mutation of single GAAP4 or both with MAPR3 severally reduced the increase of UPR genes,but enhanced the heat response genes.These results suggested that GAAP4 negatively regulates the heat resistance and played different roles in various stresses.4.GAAP4 and interaction factor were co-localized in the cell membrane and induced by ER stress.Fluorescent detection with YFP and CFP fusion proteins in tobacco leaf showed that GAAP4 and ATMAPR3 both located in the cell membrane.Under normal conditions,the GAAP4 protein expression is weak in 35::eYFP::GAAP4 Arabidopsis stable transformants,while the protein signal was enhanced in the roots remarkably when treated with TM.The data indicated that GAAP4 was regulated post-transcription.5.GAAP4 was involved in flowering regulation in plant.Under normal conditions,gaap4 mutant exhibited early flowering.And statistics analysis found that the bolting rate and rosette etiolation rate of gaap4 were significantly higher than the wild type in reproductive period,indicating that GAAP4 played an important role in regulating on plant development.6.Double mutants of GAAP4 and GAAPs family genes,and its interaction factor were generated.Homozygous mutans of gaap4gaap5 and gaap4 Atmapr3 were identified throuth genetic cross.These double mutants all showed early flowierng.Further study the function will be conducted.In conclusion,GAAP4 gene expressionwere induced by stress and GAAP4 mutation increased the sensitivity to ER stress and enhanced anti-heat stress.GAAP4 alleviated ER stress might through the regulation ofUPRat least.In addition,GAAP4 negatively regulated heat resistance,which was mediated by heat shock protein response pathway And the molecular mechanism of GAAP4 with anti-ER stress and heat sensitivity will be further addressed in the future.