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

Abstract

The unfolded protein response (UPR), a highly conserved set of eukaryotic intracellular signaling cascades, controls the homeostasis of the endoplasmic reticulum (ER) in normal physiological growth and situations causing accumulation of potentially toxic levels of misfolded proteins in the ER, a condition known as ER stress. During evolution, eukaryotic lineages have acquired multiple UPR effectors, which have increased the pliability of cytoprotective responses to physiological and environmental stresses. The ER‐associated protein kinase and ribonuclease IRE1 is a UPR effector that is conserved from yeast to metazoans and plants. IRE1 assumes dispensable roles in growth in yeast but it is essential in mammals and plants. The Arabidopsis genome encodes two isoforms of IRE1, IRE1A and IRE1B, whose protein functional domains are conserved across eukaryotes. Here, we describe the identification of a third Arabidopsis IRE1 isoform, IRE1C. This protein lacks the ER lumenal domain that has been implicated in sensing ER stress in the IRE1 isoforms known to date. Through functional analyses, we demonstrate that IRE1C is not essential in growth and stress responses when deleted from the genome singularly or in combination with an IRE1A knockout allele. However, we found that IRE1C exerts an essential role in gametogenesis when IRE1B is also depleted. Our results identify a novel, plant‐specific IRE1 isoform and highlight that at least the control of gametogenesis in Arabidopsis requires an unexpected functional coordination of IRE1C and IRE1B. More broadly, our findings support the existence of a functional form of IRE1 that is required for development despite the remarkable absence of a protein domain that is critical for the function of other known IRE1 isoforms.

Evolution of the unfolded protein response in plants

“…Reporter constructs involving the IRE1c promoter demonstrated that the gene is expressed in the anther and embryo (Mishiba et al, 2019) Moreno et al (2012) [Colour figure can be viewed at wileyonlinelibrary.com] 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).…”

“…Lu and Christopher were the first to report that ire1b‐2 homozygotes could not be recovered upon selfing heterozygotes (Lu & Christopher, 2008). Pu, Ruberti, Angelos, and Brandizzi (2019) argued that ire1b‐4 , which maintains viability as a homozygote is a ‘functional knockdown allele’ and that complete loss‐of‐function alleles of IRE1b are not viable as homozygotes. Nagashima et al (2011) tried but failed to complement an ire1b‐2 mutant with the wild type copy of the IRE1b gene.…”

A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

“…In animals, plants, and yeast, the most conserved axis of UPR is Inositol-Requiring Enzyme 1 (IRE1), a hybrid kinase equipped with an N-terminal ER lumen sensor domain and a C-terminal endonuclease (RNase) domain ( Chen and Brandizzi, 2013b ; Ruberti and Brandizzi, 2014 ; Ruberti et al, 2015 ; Srivastava et al, 2018 ; Stefano and Brandizzi, 2018 ; Bao et al, 2019 ). The Arabidopsis genome encodes three isoforms of IRE1: AtIRE1a, AtIRE1b, and AtIRE1c ( Koizumi et al, 2001 ; Mishiba et al, 2019 ; Pu et al, 2019 ). Although AtIRE1a and AtIRE1b have partly overlapping functions in UPR ( Moreno et al, 2012 ; Chen and Brandizzi, 2013b ; Afrin et al, 2020a ; Verchot and Pajerowska-Mukhtar, 2021 ), the two isoforms show functional divergence and play distinct roles in response to various forms of stress.…”

“…AtIRE1a is preferentially involved in pathogen-induced UPR whereas AtIRE1b shows higher activation when exposed to abiotic and chemical stress treatments, such as the N -glycosylation inhibitor tunicamycin (Tm) ( Moreno et al, 2012 ). AtIRE1c lacks the N-terminal domain and was shown to be primarily involved in reproductive development ( Mishiba et al, 2019 ; Pu et al, 2019 ).…”

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

“…Recently, the endonuclease-independent role of IRE1 has been revealed in Arabidopsis. The protein kinase domain of IRE1 is essential for its role in RIDP to regulate plant growth and development, although the phosphorylation substrates of IRE1 have not yet been identified [ 21 , 22 , 23 ].…”