Differential Proteomic Analysises Of Two Arabidopsis Ecotypes In Response To ER Stress And The Underlying Molecular Mechanisms

Plants are usually exposed to various unfavorable environments during growth and development.During adversity conditions such as high temperature and specific developmental stages such as seed storage material accumulation,the endoplasmic reticulum(ER)of plant cells accumulates large amounts of unfolded and misfolded proteins,thus causing endoplasmic reticulum stress(ER stress).ER stress triggers a conserved process called the unfolded protein response(UPR),which triggers the expression change of downstream genes involved in protein folding,vesicle trafficking,and ER-associated degradation(ERAD).The model plant Arabidopsis thaliana has thousands of ecotypes all over the world,but it has not been reported whether different ecotypes are not equally sensitive to ER stress.Although transcriptome dynamics in response to ER stress in Arabidopsis and some key transcriptional regulators such as bZIP28/bZIP60 have been identified,ER stress-induced proteomic changes have not been reported in Arabidopsis.In the current study,we found that the Arabidopsis Landsberg erecta(Ler)ecotype was more sensitive to ER stress than the Columbia(Col)ecotype.Quantitative mass spectrometry analysis with Tandem Mass Tag(TMT)isobaric labeling showed that,in total,7439 and 7035 proteins were identified from Col and Ler seedlings,with 88 and 113 differentially regulated(FC > 1.3 or <0.77,P < 0.05)proteins by ER stress in Col and Ler,respectively.Among them,40 proteins were commonly upregulated in Col and Ler,among which 10 were not upregulated in bzip28bzip60 double mutant(Col background)plants.Of the 19 specifically upregulated proteins in Col,as compared with that in Ler,components in ERAD,N-glycosylation,vesicle trafficking,and molecular chaperones were represented.Quantitative RT-PCR showed that gene expression corresponding to 8 of the 19 proteins specifically upregulated in the Col ecotype was not up-regulated after ER stress;Whereas in both ecotypes,the expression of genes corresponding to 11 additional proteins was upregulated expression after ER stress(FC >2,P < 0.05).However,the expression of genes corresponding to these 19 proteins did not differ significantly in up-regulated expression fold between Col and Ler.Compared to the Col ecotype,an important feature of the Ler ecotype is the deficiency in the function of the receptor-like kinase ERECTA gene.We found that the ERECTA mutant(er105)in the Col background had an ER stress inducer Tunicamycin(TM)-sensitive phenotype by phenotypic analysis,suggesting that function of the receptor-like kinase ERECTA may contribute to the difference in TM sensitivity between Arabidopsis Col and Ler seedlings.Subsequently,we validated the proteomic data by obtaining overexpressed lines of Col-specific upregulated proteins corresponding genes OS9,EBS7 and TGA6.Immunoblot experiments confirmed that ERAD-related OS9-FLAG protein level in the Col background was upregulated by TM in a much higher fold than those in the Ler and er105(Col background).Subsequent treatment with the proteasome inhibitor MG132 revealed that this difference between ecotypes disappeared.Combined with cell-free protein degradation assay,it can be demonstrated that OS9 protein abundance in both ecotypes is tightly regulated under ER stress conditions and that the OS9 protein in the Col ecotype is more stable.The protein levels of ERAD-related EBS7-FLAG protein did not differ before and after TM treatment,and we suggest that the difference in the abundance of this protein before and after TM treatment was mainly caused by the difference in the expression level of its corresponding gene.We also found 9 amino acid site variants in the EBS7 protein sequence between Col and Ler ecotypes,which could be one of the reasons for the accumulation of protein differences between the two ecotypes.Gene expression of transcription factor TGA6 was not induced up-regulation by TM in either ecotype,but TGA6-Flag protein abundance was induced up-regulation by TM treatment in Col background,while there was no difference in Ler background.Combined with the results of cell-free protein degradation assay,it can be concluded that the TGA6 protein in the Col ecotype had higher stability.The EBS7-FLAG protein level is not changed after TM treatment,and we speculate that the regulation of this protein may exist only on the transcriptional level.We also found 9 amino acid site variants in the EBS7 homolog amino acid sequences between the two ecotypes,which could possibly be one of the possibility for the differential protein accumulation.Furthermore phenotypic analysis revealed that overexpression of OS9,EBS7 and TGA6 genes in the Ler ecotype background all enhanced TM resistance in the Ler ecotype.In summary,this study found differences in ER stress sensitivity between two Arabidopsis ecotypes,Col and Ler.Proteomic analysis further revealed that the two ecotypes have different responses to ER stress at the proteome level,explored the reasons for the accumulation of differences under ER stress between the two ecotypes,and revealed that the receptor-like kinase ERECTA,ERAD-related proteins OS9 and the key regulatory roles of the receptor-like kinase ERECTA,ERAD-related proteins OS9 and EBS7,and the transcription factor TGA6 on the differences in ER stress sensitivity in the two ecotypes.