NADPH oxidase activity is required for ER stress survival in plants

Abstract

In all eukaryotes, the unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress, which is a potentially lethal condition caused by the accumulation of misfolded proteins in the ER. In mammalian and yeast cells, reactive oxygen species (ROS) generated during ER stress attenuate the UPR, negatively impacting cell survival. In plants, the relationship between the UPR and ROS is less clear. Although ROS develop during ER stress, the sources of ROS linked to ER stress responses and the physiological impact of ROS generation on the survival from proteotoxic stress are yet unknown. Here we show that in Arabidopsis thaliana the respiratory burst oxidase homologs, RBOHD and RBOHF, contribute to the production of ROS during ER stress. We also demonstrate that during ER stress RBOHD and RBOHF are necessary to properly mount the adaptive UPR and overcome temporary and chronic ER stress situations. These results ascribe a cytoprotective role to RBOH-generated ROS in the defense from proteotoxic stress in an essential organelle, and support a plant-specific feature of the UPR management among eukaryotes.

Keywords: Arabidopsis thaliana; RBOHD; RBOHF; ER stress; Unfolded protein response; reactive oxygen species.

Figure 1.. NADPH oxidase dependent O2- is generated during ER Stress partially through RBOHD and RBOHF activity

In situ detection and semi-quantification of superoxide in root tissues by staining with nitro-tetrazolium blue (NBT) A. WT or rbohd rbohf seedlings treated with 1.0 μg/ml Tm, 1.0 μg/ml TM + 2.5 μM DPI, or DMSO were stained with NBT after 24 or 48 hours. †: mature zone; ‡: maturation zone; Δ: root tip. B. Tissue-specific differences in superoxide are shown by relative pixel intensities of NBT stain from the root tip ± SE.

Figure 2.. ER Stress induced H₂O₂ are controlled by RBOHD and RBOHF as well as intact UPR signalling

Development and application of an Amplex Ultra Red protocol for H₂O₂ quantification from seedling tissues subjected to ER stress. A. Comparison of H₂O₂ extraction methods using potassium phosphate buffer (PHOS) or 10% TCA neutralized with NaHCO₃ (NTCA) incubated in the presence or absence of horseradish peroxidase (HRP). B. Evaluation of tissue loading during PHOS extraction and catalase treatment. C. Evaluation of tissue loading during NTCA extraction and catalase treatment. D. WT, rbohd rbohf, ire1a ire1b, and bzip60 bzip28 seedlings were treated in a plate system with Tm or DMSO for the indicated time and H₂O₂ quantified; see materials and methods. Data represent the mean concentration ± SE. E. From the values in (D) average fold change ± SE (Tm/DMSO) was determined from biological replicates in the order that they were measured. Statistical significance compared to equivalent WT value, unless a bracket is used to indicate comparison. Statistical significance determined using Student’s unpaired t-test and indicated by: *=p<0.05, **=p<0.005 ***=p<0.0005, NS=not significant.

Figure 3.. Intact UPR signaling is required to maintain homeostasis of RBOH transcript levels and ROS signaling, while RBOH activity affects UPR homeostasis.

Time course gene expression analysis of WT, rbohd rbohf, ire1a ire1b, and bzip60 bzip28 seedlings subjected to ER stress. qRT-PCR analyses were performed using primers specific for either RBOHF (A), RBOHD (B), ZAT12 (C), spliced bZIP60 (sbZIP60; D), BIP3 (E), or ERdj3B (E). Data represent the mean ratio ± SE (biological replicates=3); Statistical significance compared to equivalent WT value, unless a bracket is used to indicate comparison. Statistical significance determined using Student’s unpaired t-test and indicated by: *=p<0.05, **=p<0.005 ***=p<0.005, NS=not significant.

Figure 4.. RBOHD and RBOHF are required in the recovery from temporary ER stress.

Phenotypic analyses of the various genetic backgrounds subjected to temporary ER stress. A. WT, rbohd rbohf, and ire1a ire1b seedlings were subjected to ER stress and were imaged after a 3 day recovery. From the seedlings grown in (A) shoot fresh weight (B), average total chlorophyll (C), and root length (D), were recorded as described in the materials and methods. Data represent the mean ± SE. (at least 10 biological replicates). Letters above each data point indicate statistically significant groups using Student’s unpaired t-test (p<0.05).

Figure 5.. RBOHD and RBOHF are required for adaptation to chronic ER stress.

Phenotypic analysis of different seedling genetic backgrounds subjected to chronic ER stress. A. WT, rbohd rbohf, ire1a ire1b, and bzip60 bzip28 were germinated on media containing the indicated concentration of Tm or DMSO grown for two weeks. Shoots fresh weight (B) and total chlorophyll content (C) were measured. Data represent the mean ± SE (at least 10 biological replicates). Letters above each data point indicate statistically significant groups using Student’s unpaired t-test (p<0.05).

Figure 6.. RBOHD and RBOHF act to prevent ER stress induced cell death

Determining the extent of cell death in seedlings of different genetic background subjected to prolonged ER stress. A. WT, rbohd rbohf, ire1a ire1b, and bzip60 bzip28 seedlings treated with Tm in a plate system for 48 and 144 hours, and then photographed at 144 hours. B. The extent of cell death was determined by quantification of percent electrolyte leakage. Data represent the mean ± SE (4 biological replicates). Letters above each data point indicate statistically significant groups using Student’s unpaired t-test (p<0.05).