IRE1-mediated cytoplasmic splicing and regulated IRE1-dependent decay of mRNA in the liverwort Marchantia polymorpha.

The unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response is a homeostatic cellular response conserved in eukaryotes to alleviate the accumulation of unfolded proteins in the ER. In the present study, we characterized the UPR in the liverwort Marchantia polymorpha to obtain insights into the conservation and divergence of the UPR in the land plants. We demonstrate that the most conserved UPR transducer in eukaryotes, IRE1, is conserved in M. polymorpha, which harbors a single gene encoding IRE1. We showed that MpIRE1 mediates cytoplasmic splicing of mRNA encoding MpbZIP7, a M. polymorpha homolog of bZIP60 in flowering plants, and upregulation of ER chaperone genes in response to the ER stress inducer tunicamycin. We further showed that MpIRE1 also mediates downregulation of genes encoding secretory and membrane proteins in response to ER stress, indicating the conservation of regulated IRE1-dependent decay of mRNA. Consistent with their roles in the UPR, Mpire1 ge and Mpbzip7 ge mutants exhibited higher sensitivity to ER stress. Furthermore, an Mpire1 ge mutant also exhibited retarded growth even without ER stress inducers, indicating the importance of MpIRE1 for vegetative growth in addition to alleviation of ER stress. The present study provides insights into the evolution of the UPR in land plants.

Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer-like 1 in Arabidopsis.

Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.

Genetic engineering of eggplant accumulating ß-carotene in fruit.

KEY MESSAGE: Genetic engineering of eggplant using fruit-specific EEF48 promoter-driven bacterial PSY gene, crtB, confers ß-carotene accumulation in fruit. Eggplant (Solanum melongena L.) is globally cultivated especially in Asia and is an important source of nutrients in the diets of low-income consumers in developing countries. Since fruits of eggplant have low provitamin A carotenoid content, it is expected to develop eggplant with high carotenoid content for combatting vitamin A deficiency. To achieve this, the present study implemented a metabolic engineering strategy to modify the carotenoid biosynthetic pathway in eggplant. Expression analysis of carotenogenic genes in eggplant tissues showed that the expression of the endogenous phytoene synthase (PSY) was low in fruit and callus. Orange-colored calluses were generated from ectopic expression of crtB gene, which encodes bacterial PSY, in eggplant cells. The orange calluses accumulated > 20 µg g-1 FW of ß-carotene, which was approximately 150-fold higher than that of the untransformed calluses. These observations suggest that the PSY expression is the rate-limiting step for ß-carotene production in callus and fruit. Since the orange calluses did not regenerate plants, we chose eggplant EEF48 gene, which is presumably expressed in fruit. We amplified its promoter region by TAIL-PCR and showed that the EEF48 promoter is indeed active in eggplant fruit. Subsequently, transgenic eggplant lines having EEF48 promoter-driven crtB were produced. Among the transgenic lines produced, one line set fruit containing 1.50 µg g-1 FW of ß-carotene, which was 30-fold higher than that of the untransformed fruits (0.05 µg g-1 FW). The self-pollinated progenies showed a 3:1 segregation ratio for the presence and absence of the transgene, which was linked to the ß-carotene accumulation in fruit. These results provide a strategy for improvement of carotenoid content in eggplant fruit.

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

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.

Deficiency in the double-stranded RNA binding protein HYPONASTIC LEAVES1 increases sensitivity to the endoplasmic reticulum stress inducer tunicamycin in Arabidopsis.

OBJECTIVE: microRNA (miRNA) is a small non-coding RNA that regulates gene expression by sequence-dependent binding to protein-coding mRNA in eukaryotic cells. In plants, miRNA plays important roles in a plethora of physiological processes, including abiotic and biotic stress responses. The present study was conducted to investigate whether miRNA-mediated regulation is important for the endoplasmic reticulum (ER) stress response in Arabidopsis. RESULTS: We found that hyl1 mutant plants are more sensitive to tunicamycin, an inhibitor of N-linked glycosylation that causes ER stress than wild-type plants. Other miRNA-related mutants, se and ago1, exhibited similar sensitivity to the wild-type, indicating that the hypersensitive phenotype is attributable to the loss-of-function of HYL1, rather than deficiency in general miRNA biogenesis and function. However, the transcriptional response of select ER stress-responsive genes in hyl1 mutant plants was indistinguishable from that of wild-type plants, suggesting that the loss-of-function of HYL1 does not affect the ER stress signaling pathways.

CRISPR/Cas9-mediated homologous recombination in tobacco.

KEY MESSAGE: Co-transformation of multiple T-DNA in a binary vector enabled CRISPR/Cas9-mediated HR in tobacco. HR occurred in a limited region around the gRNA target site. In this study, CRISPR/Cas9-mediated homologous recombination (HR) in tobacco (Nicotiana tabacum L. 'SR-1') was achieved using binary vectors comprising two (T1-T2) or three (T1-T2-T3) independent T-DNA regions. For HR donor with the tobacco acetolactate synthase gene, SuRB, T-DNA1 contained ΔSuRBW568L, which lacked the N-terminus region of SuRB and was created by three nucleotide substitutions (ATG to GCT; W568L), leading to herbicide chlorsulfuron (Cs) resistance, flanked by the hygromycin (Hm)-resistant gene. T-DNA2 consisted of the hSpCas9 gene and two gRNA inserts targeting SuRB and An2. For the 2nd HR donor with the tobacco An2 gene encoding a MYB transcription factor involved in anthocyanin biosynthesis, T-DNA3 had a 35S promoter-driven An2 gene lacking the 3rd exon resulting in anthocyanin accumulation after successful HR. After selecting for Hm and Cs resistance from among the 7462 Agrobacterium-inoculated explants, 77 independent lines were obtained. Among them, the ATG to GCT substitution of endogenous SuRB was detected in eight T1-T2-derived lines and two T1-T2-T3-derived lines. Of these mutations, four T1-T2-derived lines were bi-allelic. All the HR events occurred across the endogenous SuRB and 5' homology arm of the randomly integrated T-DNA1. HR of the SuRB paralog, SuRA, was also found in one of the T1-T2-derived lines. Sequence analysis of its SuRA-targeted region indicated that the HR occurred in a limited (< 153 bp) region around the gRNA target site. Even though some T1-T2-T3-derived lines introduced three different T-DNAs and modified the An2 gRNA target site, no signs of HR in the endogenous An2 could be observed.

Constitutive BiP protein accumulation in Arabidopsis mutants defective in a gene encoding chloroplast-resident stearoyl-acyl carrier protein desaturase.

The unfolded protein response (UPR) occurs when protein folding and maturation are disturbed in the endoplasmic reticulum (ER). During the UPR, a number of genes including those encoding ER-resident molecular chaperones are induced. In Arabidopsis, BiP3 has been used as a UPR marker gene whose expression is strongly induced in response to ER stress. In this study, we mutagenized Arabidopsis plants expressing ß-glucuronidase (GUS) gene under the control of BiP3 promoter and isolated a mutant that exhibits strong GUS activity without treatment with ER stress inducers. By whole genome sequencing, we identified a causal gene in the mutant as SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY2 (SSI2), which encodes stearoyl-acyl carrier protein desaturase that converts stearic acids to oleic acids in the chloroplasts. In addition to GUS proteins, the ssi2 mutant accumulates endogenous BiP3 proteins without treatment by any stress reagents. Interestingly, although the degree of endogenous BiP3 protein accumulation in the ssi2 mutant was comparable to that in wild-type plants treated with the ER stress inducer tunicamycin, much less BiP3 transcripts were detected in the ssi2 mutant compared to tunicamycin-treated wild-type plants. Our finding suggests a genetic link between fatty acid metabolism in the chloroplasts and ER functions.

DNA double-strand breaks promote endoreduplication in radish cotyledon.

KEY MESSAGE: DSBs differently affect endoreduplication and organ size in radish cotyledons and hypocotyls in different light conditions, suggesting that DSBs-mediated endoreduplication varies based on different developmental and environmental cues. Endoreduplication induced by DNA double strand breaks (DSBs) in Arabidopsis thaliana roots and cultured cells has been reported in recent years. In this study, we investigated whether DSBs-mediated endoreduplication also occurs in other tissues, such as cotyledons and hypocotyls of radish (Raphanus sativus var. longipinnatus) plants. To induce DSBs, UV irradiation and Zeocin treatment were applied to in vitro-cultured radish seedlings, and ploidy distribution of the treated tissues was analyzed by flow cytometry. Consequently, frequencies of the higher ploidy (8C) cells and cycle values in the cotyledon tissues increased with increasing doses of UV irradiation and concentrations of Zeocin, irrespective of light conditions. UV-stimulated endoreduplication was also observed in four Brassica species. In hypocotyls, UV treatments decreased the frequencies of higher ploidy (32C) cells and cycle values in dark-grown seedlings, whereas Zeocin treatments increased the frequencies of higher ploidy (16C and 32C) cells and cycle values in light- and dark-grown seedlings. Among the treatments, organ sizes did not simply correlate with cycle values. The effects of treatments on endoreduplication and organ size differed based on organ and light conditions, indicating that DSBs-mediated endoreduplication may involve a multifaceted response to different developmental and environmental cues.

A 64-bp sequence containing the GAAGA motif is essential for CaMV-35S promoter methylation in gentian.

This study investigated sequence specificity and perenniality of DNA methylation in the cauliflower mosaic virus (CaMV) 35S promoter of transgenic gentian (Gentiana triflora×G. scabra) plants. Unlike conventional transgene silencing models, 35S promoter hypermethylation in gentian is species-specific and occurs irrespective of the T-DNA copy number and genomic location. Modified 35S promoters were introduced into gentian, and single-copy transgenic lines were selected for methylation analysis. Modified 35S promoter lacking a core (-90) region [35S(Δcore)] in gentian conferred hypermethylation and high levels of de novo methylation of the CpHpH/CpCpG sites in the 35S enhancer regions (-298 to -241 and -148 to -85). Therefore, promoter transcription may not be an absolute requirement for the methylation machinery. In vitro, de novo methylation persisted for more than eight years. In another modified 35S promoter, two "GAAGA" motifs (-268 to -264 and -135 to -131) were replaced by "GTTCA" in the two highly de novo methylated regions. It did not support hypermethylation and showed transgene expression. A 64-bp fragment of the 35S enhancer region (-148 to -85) was introduced into gentian and the resultant transgenic lines analyzed. The 64-bp region exhibited hypermethylation at the CpG/CpWpG sites, but the CpHpH/CpCpG methylation frequency was lower than those of the unmodified 35S- and 35S(Δcore) promoters. Nevertheless, a distinct CpHpH/CpCpG methylation peak was found in the 64-bp region of all single-copy transgenic lines. These results suggest that the 64-bp region may contain an element required for 35S methylation but insufficient for high de novo methylation compared with those in the unmodified 35S and 35S(Δcore) promoters.

Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P.

Inositol-requiring enzyme 1 affects meristematic division in roots under moderate salt stress in Arabidopsis.

The unfolded protein response (UPR) mitigates stress caused by accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) is the most conserved sensor of the UPR with ribonuclease activity that mediates cytoplasmic splicing and decay of mRNA encoding secretory and membrane proteins. In the present study, we demonstrate that the Arabidopsis mutant defective in two IRE1 genes exhibit retarded growth of primary roots under moderate salt stress, although such grow retardation is not observed in wild type plants. Microscopic observation showed decrease in the number of meristematic cells in the mutant under salt stress. This finding suggests that IRE1 plays a role in the maintenance of root meristems under salt stress. Possible connections between the function of IRE1 and the salt sensitivity are discussed.

Effect of light and auxin transport inhibitors on endoreduplication in hypocotyl and cotyledon.

KEY MESSAGE: Enhancement of endoreduplication in dark-grown hypocotyl is a common feature in dicotyledonous polysomatic plants, and TIBA-mediated inhibition of the endoreduplication is partially due to abnormal actin organization. Many higher plant species use endoreduplication during cell differentiation. However, the mechanisms underlying this process have remained elusive. In this study, we examined endoreduplication in hypocotyls and cotyledons in response to light in some dicotyledonous plant species. Enhancement of endoreduplication was found in the dark-grown hypocotyls of all the polysomatic species analyzed across five different families, indicating that this process is a common feature in dicotyledonous plants having polysomatic tissues. Conversely, endoreduplication was enhanced in the light-grown cotyledons in four of the five species analyzed. We also analyzed the effect of a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA) on endoreduplication in hypocotyl and cotyledon tissues of radish (Raphanus sativus L. var. longipinnatus Bailey). TIBA was found to inhibit and promote endoreduplication in hypocotyls and cotyledons, respectively, suggesting that the endoreduplication mechanism differs in these organs. To gain insight into the effect of TIBA, radish and spinach (Spinacia oleracea L.) seedlings were treated with a vesicle-trafficking inhibitor, brefeldin A, and an actin polymerization inhibitor, cytochalasin D. Both of the inhibitors partially inhibited endoreduplication of the dark-grown hypocotyl tissues, suggesting that the prominent inhibition of endoreduplication by TIBA might be attributed to its multifaceted role.

Tunicamycin-induced inhibition of protein secretion into culture medium of Arabidopsis T87 suspension cells through mRNA degradation on the endoplasmic reticulum.

The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants.

Arabidopsis tRNA ligase completes the cytoplasmic splicing of bZIP60 mRNA in the unfolded protein response.

Arabidopsis bZIP60 is a major transcription factor that activates the unfolded protein response and is regulated by cytoplasmic splicing. Two Arabidopsis inositol-requiring 1s (IRE1A and IRE1B) cleave bZIP60 mRNA; however, the ligase that connects the two half-molecules of the split bZIP60 mRNA has not yet been identified. We aimed to determine whether the Arabidopsis tRNA ligase RLG1 catalyzes the ligation of cleaved bZIP60 mRNA. Recombinant IRE1B containing the ribonuclease domain correctly cleaved synthetic RNA covering the cleaved site of bZIP60 in vitro. Recombinant RLG1 then ligated the two cleaved fragments. The cytoplasmic form of RLG1 was expressed in a T-DNA insertion mutant whose homozygote exhibited a lethal phenotype and when the transgene was substituted with endogenous RLG1, the plants grew normally. RLG1 proteins derived from transgene were mainly found in the cytoplasm; however, some were in the microsomal fraction, possibly on the ER membrane. This intracellular distribution of RLG1 is discussed.

CaMV-35S promoter sequence-specific DNA methylation in lettuce.

KEY MESSAGE: We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

Exogenous salicylic acid activates two signaling arms of the unfolded protein response in Arabidopsis.

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.

The polar auxin transport inhibitor TIBA inhibits endoreduplication in dark grown spinach hypocotyls.

We addressed the question of whether an additional round of endoreduplication in dark-grown hypocotyls is a common feature in dicotyledonous plants having endopolyploid tissues. Ploidy distributions of hypocotyl tissues derived from in vitro-grown spinach (Spinacia oleracea L. cv. Atlas) seedlings grown under different light conditions were analyzed by flow cytometry. An additional round of endoreduplication (represented by 32C cells) was found in the dark-grown hypocotyl tissues. This response was inhibited by light, the intensity of which is a crucial factor for the inhibition of endoreduplication. The higher ploidy cells in cortical tissues of the dark-grown hypocotyls had larger cell sizes, suggesting that the additional round of endoreduplication contributes to hypocotyl elongation. More importantly, a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), strongly inhibits endoreduplication, not only in spinach but also in Arabidopsis. Because other polar auxin transport inhibitors or an auxin antagonist show no or mild effects, TIBA may have a specific feature that inhibits endoreduplication.

An attempt to detect siRNA-mediated genomic DNA modification by artificially induced mismatch siRNA in Arabidopsis.

Although tremendous progress has been made in recent years in identifying molecular mechanisms of small interfering RNA (siRNA) functions in higher plants, the possibility of direct interaction between genomic DNA and siRNA remains an enigma. Such an interaction was proposed in the 'RNA cache' hypothesis, in which a mutant allele is restored based on template-directed gene conversion. To test this hypothesis, we generated transgenic Arabidopsis thaliana plants conditionally expressing a hairpin dsRNA construct of a mutated acetolactate synthase (mALS) gene coding sequence, which confers chlorsulfuron resistance, in the presence of dexamethasone (DEX). In the transgenic plants, suppression of the endogenous ALS mRNA expression as well as 21-nt mALS siRNA expression was detected after DEX treatment. After screening >100,000 progeny of the mALS siRNA-induced plants, no chlorsulfuron-resistant progeny were obtained. Further experiments using transgenic calli also showed that DEX-induced expression of mALS siRNA did not affect the number of chlorsulfuron-resistant calli. No trace of cytosine methylation of the genomic ALS region corresponding to the dsRNA region was observed in the DEX-treated calli. These results do not necessarily disprove the 'RNA cache' hypothesis, but indicate that an RNAi machinery for ALS mRNA suppression does not alter the ALS locus, either genetically or epigenetically.

Defects in IRE1 enhance cell death and fail to degrade mRNAs encoding secretory pathway proteins in the Arabidopsis unfolded protein response.

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 up-regulated by tunicamycin, indicating the connection between the UPR and the cytosolic protein response.

Epigenetic modifications of the 35S promoter in cultured gentian cells.

Our previous studies found strict gene silencing associated with CaMV-35S promoter-specific de novo methylation in transgenic gentian plants. To dissect the de novo methylation machinery, especially in association with histone modification, 35S-driven sGFP-expressing and -silenced gentian cultured cell lines that originated from a single transformation event were produced and used for epigenetic analyses. A sGFP-expressing primarily induced cell suspension culture (PS) was hypomethylated in the 35S promoter region, although a low level of de novo methylation at the 35S enhancer region (-148 to -85) was detected. In contrast, a sGFP-silenced re-induced cell suspension culture (RS), which originated from leaf tissues of a transgenic plant, was hypermethylated in the 35S promoter region. Chromatin immunoprecipitation analysis showed that in RS, histone H3 of the silenced 35S promoter region was deacetylated and also dimethylated on lysine 9. Interestingly, in the silenced 35S promoter 3' region, dimethylation of histone H3 lysine 4 was also observed. When hypomethylation and histone H3 acetylation of the 35S region occurred in PS, de novo methylation at the 35S enhancer region had already taken place. The de novo methylation status was also resistant to 5-aza-2'-deoxycytidine treatment. These results suggest that de novo methylation of the enhancer region is a primitive process of 35S silencing that triggers histone H3 deacetylation.