Impaired PSII proteostasis triggers a UPR-like response in the var2 mutant of Arabidopsis

Abstract

Cellular protein homeostasis (proteostasis) is maintained through the balance between de novo synthesis and proteolysis. The unfolded/misfolded protein response (UPR) that is triggered by stressed endoplasmic reticulum (ER) also plays an important role in proteostasis in both plants and animals. Although ER-triggered UPR has been extensively studied in plants, the molecular mechanisms underlying mitochondrial and chloroplastic UPRs are largely uncharacterized despite the fact that these organelles are sites of production of harmful reactive oxygen species (ROS), which damage proteins. In this study, we demonstrate that chloroplasts of the Arabidopsis yellow leaf variegation 2 (var2) mutant, which lacks the metalloprotease FtsH2, accumulate damaged chloroplast proteins and trigger a UPR-like response, namely the accumulation of a suite of chloroplast proteins involved in protein quality control (PQC). These PQC proteins include heat-shock proteins, chaperones, proteases, and ROS detoxifiers. Given that FtsH2 functions primarily in photosystem II proteostasis, the accumulation of PQC-related proteins may balance the FtsH2 deficiency. Moreover, the apparent up-regulation of the cognate transcripts indicates that the accumulation of PQC-related proteins in var2 is probably mediated by retrograde signaling, indicating the occurrence of a UPR-like response in var2.

Keywords: PSII repair; Photosystem II; damaged protein response; proteostasis; retrograde signaling; singlet oxygen; unfolded protein response.

Fig. 1.

Impaired PSII proteostasis alters the chloroplast proteome in the Arabidopsis var2 mutant. (A) Schematic representation of MS-based analysis of the total chloroplast proteins isolated from 3-week-old plants of var2 and the WT grown under continuous light (80 µmol m^–2 s^–1) at 20±2 °C. (B) Distribution of 1474 proteins detected in either var2 or the wild-type (WT) (see Supplementary Table S2). (C) Label-free quantitation identified a total of 603 differentially accumulated proteins (Log₂ FC ≥ 1; P<0.05, Student’s t-test), among which, 317 were increased and 286 were reduced in var2 as compared to the WT (Supplementary Tables S3, S5).

Fig. 2.

Proteins related to protein quality control (PQC) are highly accumulated whereas photosynthesis-related proteins are significantly reduced in the Arabidopsis var2 mutant. (A) Heat maps showing the expression of PQC-related proteins in var2 as compared with the wild-type (WT). GO analysis of the proteins highly accumulated in var2 compared with the WT revealed a significant enrichment in protein folding, proteolysis, detoxification, and chloroplast organization processes (Supplementary Fig. S2A). A complete GO analysis of proteins accumulated in var2 is shown in Supplementary Table S4. (B) Heat maps showing the expression of photosynthesis-related proteins in var2 as compared with the WT. GO analysis of the proteins reduced in var2 revealed a significant enrichment of proteins involved in photosynthetic protein import, PSI and PSII assembly, PSII repair, photosynthetic electron transport chain (PETC), and chlorophyll biosynthesis (Supplementary Fig. S2B). A complete GO analysis of down-regulated proteins in var2 is shown in Supplementary Table S6.

Fig. 3.

Validation of the label-free protein quantitation. Proteins including Hsp70, Cpn60A, and Cpn60B exhibited higher steady-state levels in the Arabidopsis var2 mutant compared with the wild-type (WT) in both western blots (A) and label-free quantitation data (B). (A) Total chloroplast proteins were extracted from intact chloroplasts isolated from 3-week-old plants grown under continuous light and were assessed by western blotting using protein-specific antibodies. (B) Label-free quantitation-based steady-state levels (expressed as relative abundance) of Hsp70 (Hsc70-1, Hsc70-2), Cpn60A, and Cpn60B (Cpn60B1 and Cpn60B2) in var2 as compared to the WT. Relative abundance was calculated using mean intensity values. Data shown are means (±SD) of n=3 replicates.

Fig. 4.

The accumulation of proteins related to proteostasis in the Arabidopsis var2 mutant is transcriptionally regulated. (A) Relative abundance of proteins exhibiting a higher accumulation in var2 compared with the wild-type (WT). The ClpD protein is shown as the control in which the steady-state levels remained unchanged. (B) Relative transcript levels of the proteins accumulated in var2 were examined using qRT-PCR. (C) The expression of -shock transcription factor A2 (HSFA2) and its downstream target genes Hsp21 and ClpB3 are up-regulated in var2. The relative abundance of the proteins was calculated using mean protein intensities. The relative transcript levels were calculated by qRT-PCR using PPA2 as a control. The data are means (±SD) of n=3 replicates. Significant differences between mean values were determined using Student’s t-test (*P<0.05).

Fig. 5.

The cpUPR-like response contributes to the refolding of enzymes involved in the MEP pathway in the Arabidopsis var2 mutant. (A, B) DXS is the first enzyme in the MEP pathway and is a prone-to-aggregate protein under oxidative stress conditions. Interaction of Hsp70 with ClpC1/ClpC2 results in the unfolding and degradation of DXS (A). In contrast, Hsp70–ClpB3 interaction assists the refolding and reactivation of DXS (B). (C) Label-free quantitation indicating the steady-state levels of ClpB3, Hsp70 (Hsc70-1 and Hsc70-2), ClpC1, ClpC2, and DXS in var2 as compared to the wild-type (WT). The data are means (±SD) of n=3 replicates.

Fig. 6.

Trp-oxidized photosynthetic proteins are significantly accumulated in the Arabidopsis var2 mutant. (A) Steady-state levels photosynthetic proteins and GAPA-2 in var2 and the wild-type (WT). All proteins except HCF136 and PsaH exhibited at least 1.8-fold higher levels in var2. The data represent mean intensity values of the related peptides (n=3 replicates) (Supplementary Table S2). (B) Oxidation levels of the proteins. These proteins exhibited at least 1.6-fold higher oxidation levels in var2 as compared with the WT. The levels of oxidation were calculated using total intensities of peptides carrying oxidized Trp residues (Supplementary Table S8). The data represent the means of n=3 replicates. GAPA-2, a non-photosynthetic protein, was also found to undergo Trp-oxidation but showed no change in expression or in oxidation levels in var2 as compared with the WT.

Fig. 7.

A model depicting the cpUPR-like damaged protein response (DPR) triggered by impaired proteostasis in the Arabidopsis var2 mutant. Inactivation of FtsH2 results in the accumulation of damaged proteins in the chloroplasts of var2. Simultaneously, it also leads to accumulation of reactive oxygen species (ROS), as demonstrated previously (Kato et al., 2009), which may subsequently alter the redox status in the chloroplasts, exaggerating the impairment of proteostasis. This then triggers chloroplast-to-nucleus retrograde signaling (RS) to activate the DPR, which is comprised of increased expression of heat-shock proteins (HSPs), proteases, and detoxifiers in order to restore the protein and redox homeostasis. In addition, this retrograde response also includes the repression of the photosynthetic apparatus to reduce the generation of ROS. Impaired Clp protease also exhibits a similar phenotype and activates cpUPR. In wild-type (WT) plants, stress conditions cause a burst of ROS in chloroplasts, which may also lead to the accumulation of damaged/misfolded/unfolded chloroplast proteins. As an adaptive mechanism, chloroplasts of WT plants may also initiate a similar retrograde cpUPR/DPR to reinstate chloroplastic homeostasis.