A nascent polypeptide synthesized on the rough endoplasmic reticulum (ER) is translocated and folded with the assistance of molecular chaperones and other folding factors such as glycosylation ⁄ modification enzymes and disulfide oxidoreductases within the ER. However, the folding of nascent polypeptides occasionally does not occur, resulting in the accumulation of unfolded or misfolded proteins in the ER (ER stress). To solve this problem, eukaryotic cells sense ER stress and induce a set of genes called unfolded protein response (UPR) genes. In the budding yeast Saccharomyces cerevisiae, ER transmembrane protein kinase ⁄ riboendonuclease Ire 1p is activated by ER stress [1,2], and nonconventionally splices mRNA of basic leucine zipper transcription factor Hac 1p [3][4][5]. Hac 1p is translated from the spliced mRNA and induces the UPR genes, having a UPR cis-acting regulatory element [6][7][8]. On DNA microarray analysis, 381 genes have been identified as UPR ones induced by both tunicamycin (TM) and dithiothreitol [9]. These comprise 6% of the total yeast genes encoding 173 unknown proteins and 208 proteins related to folding, glycosylation ⁄ modification, translocation, protein degradation, Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). In this case, so-called unfolded protein response (UPR) genes are induced. We determined the transcriptional expression of Arabidopsis thaliana UPR genes by fluid microarray analysis of tunicamycin-treated plantlets. Two hundred and fifteen up-regulated genes and 17 down-regulated ones were identified. These genes were reanalyzed with functional DNA microarrays, using DNA fragments cloned through fluid microarray analysis. Finally, 36 up-regulated and two downregulated genes were recognized as UPR genes. Among them, the up-regulation of genes related to protein degradation (HRD1, SEL-1L ⁄ HRD3 and DER1), regulation of translation (P58 IPK ), and apoptosis (BAX inhibitor-1) was reconfirmed by real-time reverse transcriptase-PCR. The induction of SEL-1L protein in an Arabidopsis membrane fraction on tunicamycin-treatment was demonstrated. Phosphorylation of initiation factor-2a, which was inhibited by P58 IPK , was decreased in tunicamycin-treated plantlets. However, regulatory changes in translation caused by ER stress were not detected in Arabidopsis. Plant cells appeared to have a strategy for overcoming ER stress through enhancement of protein folding activity, degradation of unfolded proteins, and regulation of apoptosis, but not regulation of translation.Abbreviations AARE, amino acid response element; ATF6, activating transcription factor 6; AZC, L-azetidine-2-carboxylic acid; BI-1, Bax inhibitor-1; eIF2a, initiation factor-2a; Endo H, endoglycosidase H; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; ERSE, ER stress response element; MS, Murashige and Skoog medium; PDI, protein disulfide isomerase; PKR, double stranded RNA-activated protein kinase; P-UPRE, plant-specific UPR element; RAMP4, riboso...

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Secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER) in eukaryotic cells. Nascent polypeptide chains, which are translated on the rough ER, are translocated to the ER lumen and folded into their native conformation. When protein folding is inhibited because of mutations or unbalanced ratios of subunits of hetero‐oligomeric proteins, unfolded or misfolded proteins accumulate in the ER in an event called ER stress. As ER stress often disturbs normal cellular functions, signal‐transduction pathways are activated in an attempt to maintain the homeostasis of the ER. These pathways are collectively referred to as the unfolded protein response (UPR). There have been great advances in our understanding of the molecular mechanisms underlying the UPR in yeast and mammals over the past two decades. In plants, a UPR analogous to those in yeast and mammals has been recognized and has recently attracted considerable attention. This review will summarize recent advances in the plant UPR and highlight the remaining questions that have yet to be addressed.

Many proteins that are synthesized in the endoplasmic reticulum (ER) are folded with an accompanying formation of intramolecular disulfide bonds, aided by protein disulfide isomerase (PDI) and related proteins, which are characterized by thioredoxin motifs within their primary structure [1,2]. Both yeast and mammalian PDIs are known to be multifunctional folding catalysts and molecular chaperones, which catalyze the formation and rearrangement of disulfide bonds between correct pairs of cysteine residues in nascent polypeptide chains within the ER [3]. Mammalian PDI functions not only as a catalytic enzyme, but also as a subunit of both microsomal triacylglycerol transfer protein [4] and prolylhydroxylase [5]. The mammalian PDI family, ER-60 ⁄ ERp57, which also has a protein oxidoreductase activity, interacts and cooperates with calnexin and cal-reticulin for oxidative folding of N-glycosylated proteins [6-8]. The genes of these PDI families are unfolded protein response (UPR) genes, which are induced by the accumulation of unfolded proteins in the ER [9]. Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a¢, and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by con-focal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions. Abbreviations Ab, amyloid b-peptide; AZC, L-azetidine-2-carboxylic acid; DSP, dithiobis(succinimidylpropionate); ER, endoplasmic reticulum; PDI, protein disulfide isomerase; PSV, protein storage vacuole; UPR, unfolded protein response.

Wild-type human lysozyme (hLZM) is secreted when expressed in mouse L cells, whereas misfolded mutant hLZMs are retained and eventually degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem. 210, 591-599). These misfolded mutant hLZMs are associated with protein disulfide isomerase (Otsu, M., Omura, F., Yoshimori, T., and Kikuchi, M. (1994) J. Biol. Chem. 269, 6874-6877). From the observation that this degradation is sensitive to cysteine protease inhibitors, such as N-acetyl-leucyl-leucyl-norleucinal and N-acetyl-leucyl-leucyl-methioninal, but not to the serine protease inhibitors, 1-chloro-3-tosylamido-7-amino-2-heptanone and (p-amidinophenyl)methanesulfonyl fluoride, it was suggested that some cysteine proteases are likely responsible for the degradation of abnormal proteins in the endoplasmic reticulum (ER). ER-60 protease (ER-60), an ER resident protein with cysteine protease activity (Urade, R., Nasu, M., Moriyama, T., Wada, K., and Kito, M. (1992) J. Biol. Chem. 267, 15152-15159), was found to associate with misfolded hLZMs, but not with the wild-type protein, in mouse L cells. Furthermore, denatured hLZM is degraded by ER-60 in vitro, whereas native hLZM is not. These results suggest that ER-60 could be a component of the proteolytic machinery for the degradation of misfolded mutant hLZMs in the ER.