The Roles Of GAAP1 And PAS2 Under Endoplasmic Reticulum Stress In Arabidopsis

Endoplasmic reticulum is an important organelle in eukaryotes,regulating the folding or processing of secreted proteins and most membrane proteins.When the accumulation of unfolded or misfolded proteins more than the folding mechanism,which is called ER stress,unfolded protein response(UPR)can be actived to relieve stress.When stress is severe or persistent for a long time,programmed cell death(PCD)will be induced.BAX-inhibitor 1(BI-1)is a conservative apoptosis inhibiting factor in plants and mammals,which can regulate the survival and death signals from ER stress.Golgi anti-apoptotic proteins(GAAPs)are sub-group of BI-1 proteins.Arabidopsis thaliana GAAP1 has been shown to play an important role in resisting ER stress and salt stress.In addition,PAS2(PASTICCIN02)was an interaction factor of GAAP1.PAS2 is an indispensable enzyme involved in VLCFA(Very-long-chain fatty acids)synthesis.It's known that VLCFAs not only are related to plant resistance,but also participate in the PCD signal transduction through impacting the synthesis of sphingolipid.In order to clarify the function of GAAP1 and PAS2 in ER stress and the molecular mechanism,we explored the roles of GAAP1 and PAS2 in UPR and PCD.The main results are as follows:1.GAAP1 enhanced the ER stress resistance on the whole and cellular levels.Under ER stress condition,the healthy plant percentages of GAAP1 mutants and 35S::eYFP-GAAP1 were significantly lower than that of wild type,and mortalities and root inhibition rates were significantly higher than those of wild type,but 35S::GAAP1 was opposite.On the cellular level,reactive oxygen species and dead cells in gaaplgaap3 cotyledons were more than in wild type,but the lowest in 35S::GAAPl.Autophagosomes in gaaplgaap3 root cell induced by ER stress were more than in wild type assayed by MDC staining.Cells in root transition zone were most sensitive to ER stress aasyed by the DAB,PI and trypan-blue staining.And H2O2 level,cell membrane permeability and dead cells in gaap1-1 and gaaplgaap3 roots were higher than in wild type after treated with TM,but the least in 35S::GAAP1.These results suggested that GAAP1 enhanced the ER stress resistance on the whole and cellular levels,while 35S::e YFP-GAAP1 was sensitive.2.GAAP1 might play a role in weakening UPR and retarding the initiation of PCD under chronic ER stress.When detected UPR downstream genes expression,we found that 35S::GAAP1 weakened the expression of protective genes of IRE1 pathway in advance,but had a little effect on bZIP28 pathway under chronic ER stress condition.In addition,35S::GAAP1 postponed to start PCD,which was consistent with its resistance to ER stress.Recovered for different time after TM treatment,35S::GAAP1 promoted to reduce the expression of genes in IRE1 and bZIP28 pathways.Additionally,PCD related gene PR-1 and RIDD related gene PR-4 in 35S::GAAP1 were lower than in wild type,showing GAAP1 inhibiting on the transcription level of some genes encoded secretion proteins.These results suggested that GAAP1 promoted to weaken IRE1 and bZIP28 pathways in the process of recovery after ER stress and negatively regulated PCD.3.GAAP1 and PAS2 double mutation enhanced ER stress sensitivity on the whole and cellular levels.GAAP1 and PAS2 double mutation could affect germination.The healthy plant percentages of gaap1-1,pas2-5 and gaap1pas2 were significantly lower than that of wild type,but mortalities were significantly higher than that of wild type under ER stress condition.The average fresh weight,the average water content and net photosynthetic rate of gaaplpas2 were significantly lower than those of wild type,but relative conductivity and the root inhibition rate of gaaplpas2 were significantly higher than those of wild type under ER stress condition.These results showed that PAS2 single mutation,GAAP1 and PAS2 double mutation enhanced ER stress sensitivity.Consistent with these,reactive oxygen species and dead cells of gaap1pas2 cotyledons were more than in wild type assayed by DAB and trypan-blue staining.Through FDA and PI staining,root cell viability of gaaplpas2 was lower than that of wild type,whereas cell membrane permeability of gaaplpas2 roots was higher after treated with TM.In a word,GAAP1 and PAS2 double mutation enhanced ER stress sensitivity on the whole and cellular levels.4.GAAP1 and PAS2 double mutation might delay turning off UPR and promoted PCD.After treated with TM,UPR signaling pathway was induced strongly in pas2-5 and gaap1pas2.IRE1 pathway of gaap1pas2 could be reactivated under chronic ER stress.bZIP28 pathway of gaaplpas2 could be maintained,but single gene mutation of GAAP1 or PAS2 had a little effect on bZIP28 pathway.Detected the expression of cytoplasm stress marker gene and metabolic activity gene,we found that pas2-5 showed cytoplasmic stress earlier and metabolic activity was seriously inhibited in gaaplpas2,which might be the result of more energy allocation in UPR.In addition,PCD related genes in gaaplpas2 were higher than in wild type.These results suggested that GAAP1 and PAS2 double mutation could maintain UPR protective response,and earlier start PCD pathway under chronic ER stress.5.PAS2 might regulate UPR through the interaction with BIP.Western blot assay showed that pas2-5 mutant upregulate BIP expression earlier to launch stress response under ER stress.Through tobacco instantaneous transformation system and CO-IP detection means,we found the interaction between PAS2 and BIP in vivo.We proposed that PAS2 might participate in the regulation of UPR at least through the interaction w th BIP.6.GAAP1 and PAS2 double mutation might reduce JA level in normal growth condition,and upregulated JA and SA pathways activity under chronic ER stress.In order to explore the regulatory mechanism of GAAP1 and PAS2 double mutation to ER stress,RNA sequence assay was adopted.The result showed that JA metabolism,JA pathway and SA pathway in gaaplpas2 were mainly affected under ER stress.The further qPCR assay showed that the expressions of JA synthesis pathway genes(LOX2 and LOX3)in gaaplpas2 were lower than in wild type under control condition,signifying GAAP1 and PAS2 double mutation might affect the synthesis of JA.Consistent with these,the expressions of JA response genes PDF1.3,PDF1.2A,PR-4 and ORA59 in gaaplpas2 were lower under control condition.But JA response genes were highly upregulated in gaaplpas2 after TM treatment for 2 d,showing part genes of JA pathway could be upregulated and gaaplpas2 promoted JA pathway activity under ER stress.In addition,gaaplpas2 could specifically activate PCD ralated gene PR-1,which is SA response gene,under chronic ER stress.Based on these results we proposed that GAAP1 and PAS2 double mutation could reduce JA level,and upregulated JA and SA pathways activity under chronic ER stress.In conclusion,through the overall phenotype observation and staining of cellular levels,we found GAAP1 enhanced ER stress resistance on the whole and cellular levels.On transcription level,GAAP1 might participate in weakening UPR and retarded to start PCD under ER stress.Additionally,we found GAAP1 and PAS2 double mutation enhanced ER stress sensitivity.GAAP1 and PAS2 double mutation could maintain UPR protective response,and promoted to start PCD pathway under ER stress.PAS2 might participate in the regulation of UPR through the interaction with BIP.In addition,GAAP1 and PAS2 double mutation could reduce JA level,and upregulated JA and SA pathways activity under ER stress.These showed that GAAP1 and PAS2 double mutation could allocate more energy for resistance leading to extremely sensitive under ER stress.