| Bax inhibitor-1(BI-1)like protein is a kind of conserved anti-apoptotic factor localized in eukaryotic cells,and it can participate in unfolded protein response(UPR)in mammals.GAAP is one of Bax inhibitor-1 subfamily proteins.The function of GAAP involved in endoplasmic reticulum(ER)stress and programmed cell death(PCD)in plant cells is largely unknown.There are five GAAP homologous genes in Arabidopsis thaliana.GAAP1 gene has been shown to have resistant response to ER stress and salt stress.GAAP3 mRNA has been detected mainly in cotyledon and root,and its expression level is enhanced by abiotic stress.To further study the function of GAAP3,we analyzed the tolerance effect of GAAP3 to ER stress induced by TM through the physiological,cellular and molecular level.The possible regulatory mechanism of GAAP3 was also studied.The main results are presented as follows:1.GAAP3 enhanced the resistance of plants to ER stress.The etiolation rate and mortality of gaap3 and gaap1gaap3 were significantly higher than that of wild type and transgenic plants with 35S::GAAP3 when seedlings were treated with TM.The etiolation rate and mortality of gaap1gaap3 seedlings were the highest while 35S::GAAP3 seedlings survived the best.The root length of gaaplgaap3 was inhibited most obviously when treated with TM,while the root length inhibition of seedlings over-expressing GAAP3 was the minimum.To further explore the cellular ROS and cell death level in the above-ground under TM treatment,the DAB staining and trypan-blue staining were performed respectively.The results showed that the staining level was very strong in gaaplgaap3 plants,but significantly faint in transgenic plants with 35S::GAAP3.What's more,the main parts stained by trypan-blue were cells around stomatal complex.Additionally,the cell viability and membrane permeability of root tips under ER stress were determined by Fluorescein diacetate(FDA)and Propidium iodide(PI)staining respectively.The cell viability of gaap3 and gaaplgaap3 were significantly decreased and the cell membrane permeability also changed more obviously after treatment with TM,while the cell viability and cell membrane permeability changed a little in 35S::GAAP3 plants.The transcription level of PCD genes were rapidly induced in gaaplgaap3 seedlings upon TM treatment,which was consistent with the related phenotype.These results showed that GAAP3 enhanced the resistance of plants to ER stress.2.GAAP3 might be involved in the regulation of UPR under continued ER stress.Plants relieve their ER stress generally through UPR pathway,and PCD will be induced under the condition of intense stress.To investigate whether GAAP3 influence the gene expression of IRE1 pathway and bZIP28 pathway,fluorescence quantitative PCR assay was performed.The results showed that UPR genes in gaap1gaap3 were significantly up-regulated.The expression of bZIP60s in gaap3 and gaaplgaap3 plants were higher than that in wild type until 24 h treatment with 0.5 ?g mL-1 TM,while the peak level of bZIP60s induced by TM in plants over-expressing GAAP3 was lower than that in wild type,indicating that GAAP3 might negatively regulate the IRE1 pathway.The target genes of bZIP28(PDIL1,Bip3,qSHP70,CNX1)in gaap3 and gaaplgaap3 mutants increased more significantly than those in wild type plants when treated with TM for 24 h.During the low level of ER stress,the genes downstream of bZIP28 in plants over-expressing GAAP3 were not changed significantly or higher if any than in Col plants.However,when treated with 5 ?g·mL-1 TM for 6 h,the UPR genes were not significantly different in plants over-expressing GAAP3,mutants and Col plants,indicating that GAAP3 itself did not influence the expression of UPR genes directly.The protein level of BIP3 in plants over-expressing GAAP3 was up-regulated under normal conditions when detected by Western Blot assay,which might explain the resistance response of GAAP3 to ER stress.3.GST-Pulldown assay showed that GAAP3 interacted with bZIP28.When the interaction between GAAP and IRE1a,IRE1b,bZIP17 and bZIP28 was explored using DUAL-membrane system,we found that IRE1a,IRE1b,bZIP17 and bZIP28 could induce the reporter gene expression by itself.We further knocked out their transmembrane sequences and generated the constructors of bZIP17?C,bZIP28?C,bZIP60?C.The results of yeast two-hybrid showed that GAAP3 did not interact with bZIP17?C,bZIP28?C,bZIP60?C.GAAP3 did not interact with bZIP28?C but interacted with bZIP28 by in vitro GST-Pulldown assay.4.Multi-mutants of GAAP3 with other genes were constructed.Because GAAP3 has four homologous genes,we constructed the multistage mutants of GAAP3.The gaaplgaap2gaap3 triple mutant exhibited the obvious characteristics of early flowering,indicating that all GAAP family members involved in the regulation of plant flowering.In summary,we found that GAAP3 enhanced the resistance response of plants to ER stress through physiological and cellular levels;GAAP3 might be involved in the regulation of sustained ER stress through fluorescence quantitative polymerase chain reaction(PCR)assay and it negatively regulated the IRE1 pathway.GAAP3 interacted with bZIP28 through in vitro assay by GST-Pulldown.These results showed that GAAP3 might regulate the function of UPR receptor to increase the resistance response of plants under ER stress... |
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