Abstract
In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay under ER stress, whereas the mutant does not exhibit severe developmental defects under normal conditions. In this study, we focused on the Arabidopsis IRE1C gene, whose product lacks a sensor domain. We found that the ire1a/b/c triple mutant is lethal, and 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) complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the ΔLD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate regulated IRE1-dependent decay but not bZIP60 splicing. 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.
Highlights
The ER in eukaryotes copes with an accumulation of unfolded proteins by activating the unfolded protein response (UPR), which increases protein folding capacity and attenuates protein synthesis in the ER (Walter & Ron, 2011)
The sensor domain– lacking Inositol-requiring enzyme 1 (IRE1) was found in some other Brassicaceae species, such as Camelina sativa, and phylogenetic analysis showed that the IRE1C forms an independent cluster from IRE1A and IRE1B groups in dicotyledonous plants (Fig 1B)
To detect bZIP60 splicing and regulated IRE1-dependent decay (RIDD) in the IRE1 mutants under ER stress, expressions of BiP3 and PR-4 mRNA, which are the typical targets of bZIP60 (Iwata & Koizumi, 2005) and RIDD (Mishiba et al, 2013), respectively, and the spliced form of bZIP60 mRNA were analyzed by qPCR
Summary
The ER in eukaryotes copes with an accumulation of unfolded proteins by activating the unfolded protein response (UPR), which increases protein folding capacity and attenuates protein synthesis in the ER (Walter & Ron, 2011). Targets of the cytoplasmic splicing are mRNAs encoding UPR-specific transcription factors, such as HAC1 in yeasts (Sidrauski & Walter, 1997), XBP1 in metazoans (Yoshida et al, 2001), and bZIP60 in Arabidopsis (Deng et al, 2011; Nagashima et al, 2011). Activated IRE1 degrades mRNAs encoding secretory pathway proteins, designated as the regulated IRE1-dependent decay (RIDD) of mRNAs in fission yeast (Kimmig et al, 2012), metazoans (Hollien & Weissman, 2006; Iqbal et al, 2008; Han et al, 2009; Hollien et al, 2009), and plants (Mishiba et al, 2013; Hayashi et al, 2016). Distinct catalytic mechanisms between cytoplasmic splicing and RIDD has been reported (Tam et al, 2014), how IRE1 outputs these two modules during physiological and developmental processes is still unclear (Maurel et al, 2014)
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