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An IRE1-proteasome system signalling cohort controls cell fate determination in unresolved proteotoxic stress of the plant endoplasmic reticulum

Nature Plants volume 9, pages 1333–1346 (2023)Cite this article

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Abstract

Excessive accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, which is an underlying cause of major crop losses and devastating human conditions. ER proteostasis surveillance is mediated by the conserved master regulator of the unfolded protein response (UPR), Inositol Requiring Enzyme 1 (IRE1), which determines cell fate by controlling pro-life and pro-death outcomes through as yet largely unknown mechanisms. Here we report that Arabidopsis IRE1 determines cell fate in ER stress by balancing the ubiquitin–proteasome system (UPS) and UPR through the plant-unique E3 ligase, PHOSPHATASE TYPE 2CA (PP2CA)-INTERACTING RING FINGER PROTEIN 1 (PIR1). Indeed, PIR1 loss leads to suppression of pro-death UPS and the lethal phenotype of an IRE1 loss-of-function mutant in unresolved ER stress in addition to activating pro-survival UPR. Specifically, in ER stress, PIR1 loss stabilizes ABI5, a basic leucine zipper (bZIP) transcription factor, that directly activates expression of the critical UPR regulator gene, bZIP60, triggering transcriptional cascades enhancing pro-survival UPR. Collectively, our results identify new cell fate effectors in plant ER stress by showing that IRE1’s coordination of cell death and survival hinges on PIR1, a key pro-death component of the UPS, which controls ABI5, a pro-survival transcriptional activator of bZIP60.

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Fig. 1: ism93 is a partial suppressor of the ER stress sensitivity of ire1a/b.
Fig. 2: Identification of the causal mutation in ism93.
Fig. 3: ER stress activates pro-death UPS, which is suppressed by IRE1.
Fig. 4: Transcriptomic reprogramming in pir1-1 under ER stress and identification of ABI5 downstream of PIR1.
Fig. 5: ABI5 activates the expression of UPR genes by directly regulating bZIP60.

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Data availability

All data supporting the findings of this study are available within this paper and its Supplementary Materials files. The raw data of WGS and RNA-seq have been deposited to the National Center for Biotechnological Information Sequence Read Archive and are accessible via BioProject accession codes PRJNA813856 and PRJNA880604. The Col-0 reference genome (TAIR10) was used for sequence analyses. The UPR eY1H dataset and DAP-seq dataset were downloaded from the corresponding database (https://brandizzilab.natsci.msu.edu/resources/upromics.aspx and http://neomorph.salk.edu/dev/pages/shhuang/dap_web/pages/index.php, respectively). The full results of WGS analyses are available in Supplementary Data 1. The processed data of RNA-seq analyses are available in Supplementary Data 2 and 3. Source data are provided with this paper.

Code availability

The scripts used in this study are available in GitHub (https://github.com/DaeKwan-Ko/ism93).

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Acknowledgements

This study was supported primarily by the National Institutes of Health (R35GM136637) with contributing support from the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC0018409), Chemical Sciences, Geoscience and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (DE-FG02-91ER20021) and MSU AgBioResearch (MICL02598). We thank the Research Technology Support Facility Genomics Core and Mass Spectrometry and Metabolomics Core facilities at Michigan State University for the next-generation sequencing and quantitative LC–MS/MS, respectively.

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Authors and Affiliations

  1. MSU-DOE Plant Research Lab, Michigan State University, East Lansing, MI, USA

    Dae Kwan Ko, Joo Yong Kim, Ethan A. Thibault & Federica Brandizzi

  2. Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI, USA

    Dae Kwan Ko & Federica Brandizzi

  3. Department of Plant Biology, Michigan State University, East Lansing, MI, USA

    Ethan A. Thibault & Federica Brandizzi

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  1. Dae Kwan Ko
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Contributions

D.K.K. and F.B. conceived the project, and designed the experiments and research plan. D.K.K., J.Y.K. and E.A.T. performed the experiments and data analysis. F.B. supervised the project. D.K.K. and F.B. interpreted the data and wrote the paper.

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Correspondence to Federica Brandizzi.

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Extended data

Extended Data Fig. 1 Characterization of ism93 and the experimental design for BSA.

a, Semi-quantitative RT-PCR of IRE1a and IRE1b in Col-0, ire1a/b and ism93. Total RNA was isolated from 5-day-old seedlings showing that, similar to ire1a/b, neither IRE1a nor IRE1b transcripts were amplified in ism93, in support that ism93 is isogenic to ire1a/b. ACT7 gene was used as an internal control. b, Relative values of the primary root growth in unresolved ER stress trigged by DTT. The values were measured at 12-day of growth except for fresh weight which was measured at 10-day of growth. Means ± SEM; n = 5 biological replicates (5 seedlings per replicate). c, Relative growth values (Tm/DMSO) of primary root length at 3-, 5-, 7-, and 10-day of unresolved ER stress. Means ± SEM; n = 5 biological replicates (30 seedlings per replicate). d, ism93 shows no suppression of growth defects in ire1a/b during recovery from ER stress. Five-day-old seedlings were treated with either 500 ng l−1 tunicamycin (Tm) or DMSO for 6 h and then transferred to drug-free growth media to allow ER stress to subside and growth to resume. The relative length (Tm/DMSO) of primary roots was measured at 7-day of recovery from ER stress. Means ± SEM; n = 5 biological replicates (6 seedlings per replicate). e, Schematic illustration of BSA to identify the causal mutation in ism93. WGS was performed in the following samples: ire1a/b, ism93 M3, BC1F2 with the suppressor phenotype, and BC1F2 without the suppressor phenotype (underlined). Green triangles indicate SNPs not linked to the phenotype. Red stars indicate the causal mutation. f, A representative picture of BC1F2 screening plates (9-day-old). In unresolved ER stress conditions, the BC1F2 progeny segregated with a 3:1 ratio for ER stress resistance (1,022 Tm-susceptible: 375 Tm-resistant; two-tailed Chi-square test, P = 0.1116). Scale bar (white line) = 1.8 cm. b–d, One-way ANOVA followed by Duncan’s multiple range test was used to analyze the significance of differences (P < 0.05) among the multiple samples. Means with the same letter are not significantly different. The experiments (a-d) were independently repeated three times with similar results.

Source data

Extended Data Fig. 2 Molecular features of PIR1 and its product.

a, PIR1 protein structure predicted by AlphaFold, showing the position of the R44 affected by the ism93 mutation. The color indicates the level of prediction confidence. b, Multiple Sequence Alignment of PIR1 homologs from 11 plant species including Arabidopsis. The causal mutation site of ism93 is indicated by red color. Solyc08g006980, Solanum lycopersicum. Brara.E00909, Brassica rapa L. Thhalv10006944m, Eutrema salsugineum. Lesat.0031s0210, Lepidium sativum L. Bostr.23794s0569, Boechera stricta. Alyli.0025s0183, Meniocus linifolius. Orange1.1g008232m, Citrus sinensis. EL10Ac5g12173, Beta vulgaris. Glyma.17G154400, Glycine max. Medtr4g098560, Medicago truncatula. c, A snapshot of the eFP browser for PIR1. The diagrams indicate the expression profiles of PIR1 in various tissues of Arabidopsis plants, which were obtained from the Arabidopsis eFP browser website (http://bar.utoronto.ca/eplant/).

Extended Data Fig. 3 PIR1 homologs are present broadly in eudicot species.

PIR1 plant homologs were identified by blasting the PIR1 amino acid sequence against the Phytozome database (v13) with an E-value threshold of 1.0 × 10−20, a minimum of 50% coverage, and 50% identity. The phylogenetic tree was constructed by the neighbor-joining tree method. The size of tip circles increases proportionally to the percentage identity as indicated. Tip labels (protein names annotated to corresponding species) and circles are colored by taxonomy. The black star indicates PIR1 (Arabidopsis thaliana). PIR1 homologs were found in all eudicot clades and its ancestor (Cinnamomum kanehirae where CKAN_01633100 (green) exists), but not monocot, non-Angiosperm or non-plant species (Mus musculus, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens).

Extended Data Fig. 4 The functional roles of PIR1 in the UPR are not associated with the ABA signaling.

a, LC-MS/MS analyses for quantification of ABA content in Col-0, ire1a/b, and ism93 at 24 h of adaptive ER stress. Means ± SEM; n = 5 biological replicates (>50 seedlings per replicate). ns, not significant (two-tailed Student’s t-test). FW, fresh weight. b, Representative blot images of cell-free degradation assay for PP2CA in Col-0 and pir1-1. The glutathione S-transferase-tagged PP2CA (GST–PP2CA) protein was incubated for 0, 15, 30, 60, 90 and 120 min with crude extracts prepared from Tm-treated 5-day-old Col-0 or pir1-1 plants in the presence or absence of MG132. Relative signal intensity vales (GST/ACTIN) are indicated. c, Unresolved ER stress assay of pir1-1 pp2ca double mutant. The relative growth values of the primary root were measured at 14-day of growth. Means ± SEM; n = 5 biological replicates (5 seedlings per replicate). a,c, One-way ANOVA followed by Duncan’s multiple range test was used to analyze the significance of differences (P < 0.05) among the multiple samples. Means with the same letter are not significantly different. The experiments were independently repeated three times with similar results.

Source data

Extended Data Fig. 5 qRT-PCR analyses of UPR biomarker genes in Col-0, pir1-1 and ire1a/b under ER stress condition.

Relative expression levels of BiP3, ERdj3B, and sbZIP60 (Log2(Tm/DMSO)) in Col-0, pir1-1 and ire1a/b at 24 h of adaptive ER stress. Expression values were calculated relative to UBQ10. Means ± SEM; n = 3 biological replicates (12 seedlings per replicate). The significance (P-value) measured by two-tailed Student’s t-tests is shown. The experiments were independently repeated three times with similar results.

Extended Data Fig. 6 Screenshot of genome browser with DAP-seq peaks for bZIP and bHLH TFs in the promoters of BiP3, bZIP60, and bZIP28.

DAP-seq profiles of bZIP and bHLH TFs are visualized in the genome browser. The binding motif corresponding to each selected TF is shown at the right. The ACGT motif is underlined by red lines. The scale of the y-axis (0–30) is equal across the DAP-seq of all TFs and target genes analyzed. Arrows indicate the gene orientation.

Extended Data Fig. 7 In vitro ubiquitination assay of His-T7-ABI5 and MBP-PIR1 Δ1-367 (MBP-PIR1ΔN).

The MBP-PIR1ΔN protein was assayed for ubiquitination activity on the His-T7-ABI5 protein in the presence or absence of E1, E2, ubiquitin and ATP. While PIR1 can self-ubiquitinate, it does not ubiquitinate ABI5. Western blot analyses were performed with α-Ub and α-T7 sera. The experiments were independently repeated three times with similar results.

Source data

Extended Data Fig. 8 Global ubiquitination levels between Col-0 and pir1-1.

Western blot for ubiquitin conjugations showing induction of ubiquitination at 24 h of Tm treatment in Col-0 and pir1-1 compared to the corresponding mock controls. Ponceau S staining of the small subunit of ribulose bisphosphate carboxylase was used as the loading control. The experiments were independently repeated two times with similar results.

Source data

Extended Data Table 1 An ire1a/b pir1-1 mutation is lethal
Full size table

Supplementary information

Reporting Summary

Supplementary Tables 1 and 2 and Data 1–5

Supplementary Table 1. Genome coverage of WGS data. Supplementary Table 2. Primers used in this study. F, forward; R, reverse; Ori, orientation. Restriction enzyme sites are indicated by red colour. Supplementary Data 1. A full list of SNP markers obtained through the BSA. Chr, chromosome; CDS, coding sequence; Nonsyn, non-synonymous; Syn, synonymous. Supplementary Data 2. The normalized read values (FPKM) for gene expression in all samples analyzed. Sample name consists of genotype, treatment and biological replicate with hyphen. C, Col-0; P, pir1-1. Supplementary Data 3. A full list of DEGs identified in Col-0 and pir1-1 in ER stress conditions. Dw, downregulated; Up, upregulated. Supplementary Data 4. log2-transformed fold change (Tm/DMSO) of the 3,477 DEGs in both genotypes and cluster membership. Clusters are colour-coded as shown in Fig. 4d. Supplementary Data 5. A full list of GO terms for 3,477 DEGs K-means clustered. Top-ranked GO terms are visualized in Fig. 4e. P, biological process; F, molecular function; C, cellular component

Source data

Source Data Fig. 1

Statistical source data for Figs. 1–5 and Extended Data Figs. 1, 4, 5, 7 and 8.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 1

Unprocessed DNA gel blots.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 8

Unprocessed western blots.

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Ko, D.K., Kim, J.Y., Thibault, E.A. et al. An IRE1-proteasome system signalling cohort controls cell fate determination in unresolved proteotoxic stress of the plant endoplasmic reticulum. Nat. Plants 9, 1333–1346 (2023). https://doi.org/10.1038/s41477-023-01480-3

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