Unfolded protein-independent IRE1 activation contributes to multifaceted developmental processes in Arabidopsis

Abstract

As an initial step for the unfolded protein response (UPR) pathway, the luminal domain of inositol requiring enzyme 1 (IRE1) senses unfolded proteins in the endoplasmic reticulum (ER). Recent findings in yeast and metazoans suggest alternative IRE1 activation without the sensor domain, although its mechanism and physiological significance remain to be elucidated. In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay (RIDD) under ER stress, while the mutant does not exhibit severe developmental defects and is fertile under non-stress conditions. In this study, we focused on a third Arabidopsis IRE1 gene, designated as IRE1C, whose product lacks a sensor domain. We found that even though ire1c and ire1a/c mutants did not exhibit defective bZIP60 splicing and RIDD under ER stress, the ire1a/b/c triple mutant is lethal. Heterozygous IRE1C (ire1c/+) mutation in the ire1a/b mutants resulted in growth defects and reduction of the number of pollen grains. Genetic analysis revealed that IRE1C is required for male gametophyte development in the ire1a/b mutant background. Expression of a mutant form of IRE1B that lacks the luminal sensor domain (ΔLD) in the ire1a/b mutant did not complement defects in ER stress-dependent bZIP60 splicing and RIDD. Nevertheless, expression of Δ LD complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the Δ LD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate RIDD but not bZIP60 splicing. Phenotypes of IRE1B mutants lacking the sensor domain produced by CRISPR/Cas9-mediated gene editing in the ire1a/c mutant background were essentially same as those of Δ LD-expressing ire1a/b mutant. These observations suggest that IRE1 contributes to plant development, especially male gametogenesis, using an alternative activation mechanism that bypasses the unfolded protein-sensing luminal domain. KiM directed the project. KiM and NK design the experiments. KiM, YI, RH and NN produced transgenic plants. TM and KiM performed histological analysis. AM performed fatty acid analysis.KiM performed all other experiments. KiM and YI provided the materials and reagents. KiM, YI, and NK wrote the manuscript. All authors have read and approved the manuscript. Conflict of interestThe authors declare that they have no conflict of interest. ReferencesChen Y, Brandizzi F (2012) AtIRE1A/AtIRE1B and AGB1 independently control two essential unfolded protein response pathways in Arabidopsis. Plant J 69: 266-277 Clough SJ, Bent AF (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 Deng Y, Humbert S, Liu JX, Srivastava R, Rothstein SJ, Howell SH (2011) Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis.

Evolution of the unfolded protein response in plants

“…A. thaliana has three IRE1 genes, two full length genes, AtIRE1a and ‐b , discovered by Koizumi et al (2001), and a shorter version called AtIRE1c (Figure 4). Arabidopsis lyrata has four IRE1 genes, AlIRE1a , ‐b , ‐c and c‐like (Mishiba et al, 2019). Surprisingly, there are three ZmIRE1 genes in maize ( Zea mays B73) – two full‐length genes, which cannot be classified as either IRE1a or ‐b ‐like and a shorter one, which by virtue of its size suggests that it is IRE1c ‐like.…”

“…IRE1c is largely limited to the Brassicas, is a smaller form that lacks the N‐terminal lumenal or sensory domain and is expressed primarily in flowers (Winter et al, 2007; Figure 4). Reporter constructs involving the IRE1c promoter demonstrated that the gene is expressed in the anther and embryo (Mishiba et al, 2019). The function of IRE1c remained elusive for a long time, because single knockout mutations in IRE1c had no demonstrable phenotype.…”

“…The function of IRE1c remained elusive for a long time, because single knockout mutations in IRE1c had no demonstrable phenotype. However, a role of IRE1c came to light when two groups created a triple ire1a/b/c mutant and reported that it was lethal, and when a heterozygous IRE1c mutation was created in conjunction with ire1a/ire1b ( ire1a/ire1b/ire1c/+ ) it was found to be defective in male gametogenesis (Mishiba et al, 2019; Pu et al, 2019).…”

“…A very interesting result was obtained, when Mishiba et al (2019) created an IRE1c mimic construct by deleting the lumenal domain of IRE1b ( ire1bΔLD ). They first showed that this construct was ineffective in its support of the UPR during vegetative development but that it or the full‐length IRE1b could rescue the defect in gametogenesis and support the recovery of the mutant ire1c allele through pollen.…”

AtIRE1C, an unconventional isoform of the UPR master regulator AtIRE1, is functionally associated with AtIRE1B in Arabidopsis gametogenesis

“…While this manuscript was under revision, complementary work on the characterization of IRE1C was published (Mishiba et al, ). The authors indicate that they were not able to isolate an ire1b ire1c homozygous knockout allele using the same combination of parental alleles used in our work.…”