Kei-ichiro Mishiba

Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor

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.

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.

Two different transposable elements inserted in flavonoid 3′,5′-hydroxylase gene contribute to pink flower coloration in Gentiana scabra

Pink-flowered gentian plants (Gentiana scabra) have been bred from spontaneous mutations of blue-flowered gentian plants, but the formation mechanism(s) is unknown so far. To investigate the process, two independent pink-flowered gentian plant lines were analyzed by a molecular biological approach. HPLC analysis showed that petals of the blue-flowered cultivar contained a small amount of cyanidin derivatives and major delphinidin derivatives, whereas pink petals had only a small amount of cyanidin derivatives. To find the causal factor(s) of this change, we focused on flavonoid 3′,5′-hydroxylase (F3′,5′H), which is a key enzyme for delphinidin biosynthesis in the flavonoid biosynthetic pathway. Molecular analyses confirmed that the loss of delphinidin synthesis could be attributed to the insertions of different transposable elements in the F3′,5′H gene in each independent pink-flowered gentian plant. Sequence analysis showed that these transposable elements were classified into an hAT superfamily and terminal-repeat retrotransposon in miniature (TRIM), by which normal F3′,5′H transcripts were interrupted. Southern blot analysis indicated that they belong to high copy number elements and are also found in a related gentian species (G. triflora). These results suggest that the transposable elements inserted in F3′,5′H are the source of the mutations and may also play a substantial role in the genomic evolution of the genus Gentiana.

Heterologous Expression of Two Gentian Cytochrome P450 Genes can Modulate the Intensity of Flower Pigmentation in Transgenic Tobacco Plants

Two different heterologous expression systems, microsomal fractions of Saccharomyces cerevisiae and transgenic tobacco plants, were used to investigate the enzymatic activities of flavonoid 3′-hydroxylase (GtF3′H) and flavone synthase II (GtFSII) homologues isolated from gentian petals. Recombinant GtF3′H expressed in yeast showed hydroxylation activities in the 3′ position with several flavonoid substrates, while recombinant GtFSII was able to produce flavone from flavanone. GtF3′ H-expressing transgenic tobacco plants showed a slight increase in anthocyanin content and flower color intensity, and conversion of the flavonol quercetin from kaempferol. On the other hand, GtFSII-expressing plants showed a remarkable reduction in anthocyanin content and flower color intensity, and additional accumulation of flavone, especially luteolin derivatives. We demonstrated that two cytochrome P450s from gentian petals have F3′H and FSII enzymatic activities both in vitro and in vivo, and might therefore be useful in modification of flower color using genetic engineering.

Strict de novo methylation of the 35S enhancer sequence in gentian.

A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora × G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (−90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (−148 to −85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the −149 to −124 and −107 to −83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants.

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.

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.

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.

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.