IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.

The unfolded protein response (UPR) is a cellular response highly conserved in eukaryotes to obviate accumulation of misfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) catalyzes the cytoplasmic splicing of mRNA encoding bZIP transcription factors to activate the UPR signaling pathway. Arabidopsis IRE1 was recently shown to be involved in the cytoplasmic splicing of bZIP60 mRNA. In the present study, we demonstrated that an Arabidopsis mutant with defects in two IRE1 paralogs showed enhanced cell death upon ER stress compared with a mutant with defects in bZIP60 and wild type, suggesting an alternative function of IRE1 in the UPR. Analysis of our previous microarray data and subsequent quantitative PCR indicated degradation of mRNAs encoding secretory pathway proteins by tunicamycin, DTT, and heat in an IRE1-dependent manner. The degradation of mRNAs localized to the ER during the UPR was considered analogous to a molecular mechanism referred to as the regulated IRE1-dependent decay of mRNAs reported in metazoans. Another microarray analysis conducted in the condition repressing transcription with actinomycin D and a subsequent Gene Set Enrichment Analysis revealed the regulated IRE1-dependent decay of mRNAs-mediated degradation of a significant portion of mRNAs encoding the secretory pathway proteins. In the mutant with defects in IRE1, genes involved in the cytosolic protein response such as heat shock factor A2 were upregulated by tunicamycin, indicating the connection between the UPR and the cytosolic protein response.heat stress | protein folding | bioinformatics T he unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response is a cellular response that is highly conserved in eukaryotes to obviate accumulation of misfolded proteins and to alleviate protein overload in the ER (1-3). Inositol-requiring enzyme 1 (IRE1), which is the primary transducer of the UPR in various organisms, catalyzes the unconventional or cytoplasmic splicing of mRNAs encoding bZIP transcription factors to up-regulate the UPR-related genes, such as genes for the ER-resident molecular chaperones, through its ribonuclease domain. The cytoplasmic splicing by IRE1 activates the bZIP transcription factors HAC1, XBP1, and bZIP60 in yeast, animals, and plants, respectively, by producing active proteins. Although the fundamental mechanism of the cytoplasmic splicing by IRE1 appears to be highly conserved, the mechanism of transcription factor activation is diverse among organisms (4).In addition to the cytoplasmic splicing of mRNAs for transcription factors, other functions of metazoan IRE1 have been reported. One such function is the degradation of mRNAencoding proteins in the secretory pathway referred to as regulated IRE1-dependent decay (RIDD) (5-7). RIDD is considered to contribute to reducing the amount of proteins entering the ER in the UPR. The metazoan UPR has an alternative mechanism to reduce the amount of protein entering the ER, and this mechanism is regulated by PKR-li...

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SummaryIn this study, no transgenic gentian (Gentiana triflora · Gentiana scabra) plants produced via Agrobacteriummediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5¢ coding regions of the transgenes 35S-bar and 35S-GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2¢-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing.

The unfolded protein response (UPR) is a highly conserved cellular response that prevents abnormal maturation of proteins in the endoplasmic reticulum (ER). The expression of genes encoding ER chaperones is induced during the UPR. In the Arabidopsis UPR, two membrane-bound transcription factors, bZIP60 and bZIP28, activate the expression of those genes. bZIP60 is regulated by unconventional cytoplasmic splicing catalyzed by inositol requiring enzyme 1 (IRE1), which is located in the ER membrane. bZIP28 is regulated by intramembrane proteolysis. Pathogen infection and salicylic acid (SA) have been reported to induce the expression of some UPR genes. Here, we show that UPR genes including BiP3, a marker gene of the Arabidopsis UPR, are induced by exogenous SA treatment and activation of bZIP60 in an IRE1-dependent manner. The induction of gene expression and activation of bZIP60 were independent of NPR1 and HsfB1 under these experimental conditions. We generated antibodies to detect the proteolytic products of bZIP28 after SA treatment. An assay using these antibodies showed that SA activated bZIP28, as well as activating bZIP60 through IRE1. Together, these results show that exogenous SA treatment activates two signaling arms of the Arabidopsis UPR. We propose a possible mechanism of activation of the UPR machinery by SA.