Quantitative Proteomic Analysis of ER Stress Response Reveals both Common and Specific Features in Two Contrasting Ecotypes of Arabidopsis thaliana

Abstract

Accumulation of unfolded and misfolded proteins in endoplasmic reticulum (ER) elicits a well-conserved response called the unfolded protein response (UPR), which triggers the upregulation of downstream genes involved in protein folding, vesicle trafficking, and ER-associated degradation (ERAD). Although dynamic transcriptomic responses and the underlying major transcriptional regulators in ER stress response in Arabidopsis have been well established, the proteome changes induced by ER stress 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 <0.7, 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 bzip28 bzip60 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 transcripts of eight out of 19 proteins were not upregulated (FC > 1.3 or <0.7, p < 0.05) by ER stress in Col ecotype, while transcripts of 11 out of 19 proteins were upregulated by ER stress in both ecotypes with no obvious differences in fold change between Col and Ler. Our results experimentally demonstrated the robust ER stress response at the proteome level in plants and revealed differentially regulated proteins that may contribute to the differed ER stress sensitivity between Col and Ler ecotypes in Arabidopsis.

Keywords: Arabidopsis thaliana; ER stress; TMT; UPR; ecotype; proteomics.

Figure 1

Endoplasmic reticulum (ER) stress sensitivity assays in Columbia (Col), Landsberg erecta (Ler), and double mutant (DM) plants. (A) Wild-type Col and Ler plants were grown on ½ MS plates without or with various concentrations of tunicamycin (TM) for 9 days, and then photographed; (B) The percentage of green-big (G–B), green-small (G-S) and yellow-small (Y-S) plants was calculated after photo-taking. Bars depict SE (n = 3). The difference of each group between Ler and Col under ER stress conditions is significant (p < 0.05). The bzip28 bzip60 double mutant (DM) plants in the Col background was used as a control. Bar = 5 mm.

Figure 2

Gene expression analysis of ER stress marker genes in Col and Ler plants. Nine-day-old Col and Ler seedlings grown on ½ MS plates were treated without or with 5 μg/mL tunicamycin (TM) for 8 h or 12 h, and total RNA was extracted for quantitative RT-PCR (RT-qPCR). Fold change is the expression level of genes in TM-treated plants relative to that in non-treated (CK) plants, both of which were normalized to that of the control ACTIN. Error bars represent SE (n = 3). Asterisks indicate significance levels as compared with two ecotypes in t-test. (*, p < 0.05; **, p < 0.01).

Figure 3

An overview of protein identifications in Col, Ler, and DM plants. Wild-type Col and Ler plants were grown on ½ MS plates for 9 days, and then seedlings were treated without or with 5 μg/mL tunicamycin (TM) for 12 h. Total proteins were extracted from the whole seedlings for TMT-based quantitative proteomics analysis. The bzip28 bzip60 double mutant (DM) plants in the Col background was used as a control.

Figure 4

Differentially regulated proteins by ER stress among Col, Ler, and DM plants. (A–C) Volcano plots of differential abundance of ER stress-regulated proteins among Col, Ler, and DM plants. (D–F) Number of differentially regulated proteins by ER stress in Col, Ler, or DM plants. Parameters were set as fold change >1.3 (upregulation) or <0.7 (downregulation), p < 0.05.

Figure 5

Commonly and specifically regulated proteins at the protein level by ER stress among Col, Ler, and DM plants. (A,B) Venn diagrams showing the number of overlapping and non-overlapping proteins that were differentially regulated among Col, Ler, and DM plants. Criteria for differential regulation were set as fold change >1.3 for upregulation or <0.7 for downregulation, p < 0.05; (C) Functional enrichment of cellular components for the proteins that are commonly upregulated by ER stress in both Col and Ler plants.

Figure 6

Expression analysis of genes encoding the differentially regulated proteins by ER stress between Col and Ler plants. (A,B) Ten-day-old Col and Ler seedlings grown on ½ MS plates were treated without or with 5 μg/mL tunicamycin (TM) for 8 h (A) or 12 h (B), and total RNA was extracted for quantitative RT-PCR (RT-qPCR). Fold change is the expression level of genes in TM-treated plants divided by that in control (CK) plants, both of which were normalized to the expression of ACTIN. Error bars represent SE (n = 3). Asterisks indicate significance levels when comparing two ecotypes in t-test. (* p < 0.05; ** p < 0.01).