Nozomu Koizumi

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, Mpire1ge and Mpbzip7ge mutants exhibited higher sensitivity to ER stress. Furthermore, an Mpire1ge 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.

Dicer-Like1 (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 other hand, 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.

Key message Genetic engineering of eggplant using fruit-specific EEF48 promoter-driven bacterial PSY gene, crtB, confers β-carotene accumulation in fruit. Abstract 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 β-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 β-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 β-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 β-carotene accumulation in fruit. These results provide a strategy for improvement of carotenoid content in eggplant fruit.

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.

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 β‐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.

Under the unfolded protein response (UPR), transcripts encoding the endoplasmic reticulum (ER) chaperones are increased and those encoding proteins synthesized in the ER are decreased. To reproducibly detect such changes of an expression profile, homogeneous growth of plants is desired. In addition, uniform treatment with drugs inducing the UPR is also necessary. Here we describe our methods of plant culture and drug treatment, and procedure to detect gene expression by quantitative RT-PCR.

Summary: 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.

The unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response occurs when folding and maturation of secretory and membrane proteins are impaired in the ER. The UPR induces a number of genes that encode ER-localized molecular chaperones and folding enzymes to increase folding capacity in the ER. We have identified Tunicamycin Induced 1 (TIN1), an Arabidopsis gene that is highly induced during the UPR. We have shown that TIN1 protein is localized in the ER but its physiological function remains to be elucidated. In the present study we generated and analyzed transgenic Arabidopsis plants expressing TIN1 under CaMV35S promoter to obtain insights into the physiological role of TIN1. We found that although TIN1-overexpressing plants grew as did wild-type plants under ambient laboratory conditions, their pollen grains exhibited abnormal surface morphology. The result suggests a specific role of TIN1 in secretion of proteins and/or lipids during pollen development.

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.

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.