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Background: We previously developed a transgenic rice that contains seven linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens Cry j 1 and Cry j 2. Oral administration of 80 g of transgenic rice for 20 weeks suppressed allergen-specific T-cell proliferation in participants with JC pollinosis, but their clinical symptoms did not improve. Objective: We examined the clinical efficacy of low-dose (5 g and 20 g) intake of the transgenic rice administered for two successive seasons. Methods: In this randomized, double-blind, placebo controlled study, transgenic rice seeds (5 g or 20 g) were orally administered to the participants for 24 weeks in each of two successive JC pollen seasons. We analyzed T-cell proliferation and cytokine expression, and monitored symptom and medication scores during the pollen season. Quality of life (QOL) was evaluated by using the Japanese Allergic Rhinitis Quality of Life Standard Questionnaire (JRQLQ). Results: Specific T-cell proliferation after stimulation with 7Crp, Cry j 1, and Cry j 2 was significantly suppressed in the second JC pollen season. No significant differences were found among the three groups (5 g, 20 g, and placebo) with regard to clinical symptoms or medication scores in the first season. However, the medication scores and face scale for overall condition of JRQLQ improved in the 5-g transgenic rice group in the second season, although careful re-examination with a large sample size is necessary to confirm the results. Conclusion: Low-dose oral administration of transgenic rice that contains 7Crp significantly reduced allergen-specific T-cell responses and improved medication scores during the second season of administration. Thus, oral intake of the transgenic rice has the potential to induce immune tolerance to JC pollen allergens when administered for at least two successive seasons.

Human transforming growth factor-β1 (hTGF-β1) was produced in transgenic rice seeds. To boost its production yield and to extract it simply, it was expressed under the control of seed-specific promoters along with the simultaneous suppression of endogenous seed storage proteins (SSPs) through RNA interference (RNAi). When driven by the 26 kDa α-globulin endosperm-specific promoter, it accumulated up to the markedly high level of 452 μg/grain. However, exchange with other seed-specific promoters such as 18 kDa oleosin and AGPase promoters resulted in remarkable reduction to the levels of 62 and 48 μg/grain, respectively, even though endogenous SSPs were reduced to the similar level. These production levels were almost similar to those (42 and 108 μg/grain) produced by the glutelin GluB-1 endosperm-specific promoter and the maize ubiquitin constitutive promoter without reduction of SSPs, respectively. When extracted from these transgenic rice seeds with reduced SSPs with various buffers, it could be solubilized with denaturant solution, which was in remarkable contrast with those without depressed SSPs which required further supplementation of reducing agent for extraction. This difference was associated with the fact that it was mainly deposited to ER-derived structures though self-aggregation or interaction with remaining prolamin via intermolecular disulfide bonds.

Japanese cedar (Cryptomeria japonica) pollinosis (JCP) is one of the major seasonal IgE-mediated type I allergies from February to April each year. Not only human patients but also Japanese monkeys (Macaca fuscata) are afflicted with this pollinosis in Japan, which exhibit similar clinical allergic symptoms such as allergenic rhinitis and conjunctivitis. Therefore, monkeys naturally sensitized to JC pollen allergens are expected to serve as a suitable animal model for exploiting the allergy vaccine for JCP, since allergen-specific immunotherapy is the only curative treatment for allergy diseases. We generated transgenic rice containing the hypoallergenic JC pollen Cry j 1 and Cry j 2 allergen derivatives as tolerogen. In this study, safety and efficacy of transgenic rice seed were evaluated by oral administration to Japanese monkeys. Healthy monkeys were not sensitized to Cry j 1 and Cry j 2 allergens, even when administered for one to ten months. By contrast, peripheral blood mononuclear cell (PBMC) proliferation and IgE antibody specific to these allergens were reduced in Japanese monkeys with JCP. Especially, suppression of allergen-specific PBMC proliferation was observed within only two months after administration. These findings indicate that this transgenic rice acts as effective tolerogen to induce oral immune tolerance against JC allergens.

Summary Reduction of the levels of arsenic (As) in rice grains is an important challenge for agriculture. A recent study reported that the OsABCC1 transporter prevents As accumulation in grains by sequestering As–phytochelatin complexes into vacuoles in the upper nodes. However, how phytochelatins are provided in response to As remains unclear. Here, we show that the phytochelatin synthase OsPCS1 plays a crucial role in reducing As levels in rice grains. Using a forward genetic approach, we isolated two rice mutants (has1 and has2) in which As levels were much higher in grains but significantly lower in node I in comparison with the wild type. Map-based cloning identified the responsible genes as OsABCC1 in has1 and OsPCS1 in has2. The levels of As in grains and node I were similar between the two mutants, suggesting that OsABCC1 preferentially cooperates with OsPCS1 to sequester As, although rice has another phytochelatin synthase, OsPCS2. In vitro phytochelatin synthesis assay indicated that OsPCS1 was more sensitive to activation by As than by cadmium, whereas OsPCS2 was more weakly activated by As than by cadmium. Transgenic plants highly expressing OsPCS1 showed significantly lower As levels in grains than wild-type plants did. Our results provide new knowledge of the relative contribution of rice PCS paralogs to As sequestration and suggests a good candidate tool to reduce As levels in rice grains. This article is protected by copyright. All rights reserved.

Galactinol synthase (GolS) is considered to be a key regulator of the biosynthesis of Raffinose family oligosaccharides (RFOs). Accumulation of RFOs has been reported to play a role in protection against abiotic stresses. We identified two cDNAs encoding galactinol synthase from wheat (Triticum aestivum L.), which we designated as TaGolS1 and TaGolS2. Expression of the two TaGolS genes was induced by cold stress but not by drought, heat stress or ABA treatment in wheat. We generated transgenic lines of rice (Oryza sativa L.) constitutively overexpressing TaGolS1 or TaGolS2. These transgenic plants accumulated significantly higher levels of galactinol and raffinose than did wild-type plants and exhibited enhanced cold-stress tolerance. The results demonstrate the involvement of galactinol and raffinose in the development of chilling stress in rice and indicate that the genetic modification of the biosynthesis of RFOs by transformation with GolS genes could be an effective method for enhancing chilling-stress tolerance in rice.

Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

Development of a large-scale transformation procedure is essential for generating T-DNA insertion lines and FOX libraries and for gene targeting studies. Suspension culture may be one of the best sources for producing a large number of transgenic cells; however, suspension-cultured cell clusters have been reported to have low Agrobacterium-mediated transformation frequencies in rice. In this investigation, we found that rice calli produce some unidentified substance critical for Agrobacterium-mediated transformation of rice that is released in the liquid culture media. By co-cultivating 3-day old suspension-cultured cells with Agrobacterium on filter paper moistened with enriched N6 media containing suspension-cultured cell media, more than 104 stable transformants were routinely obtained from 1 g of suspension-cultured cell clusters. Transformation efficiency was about 60-times higher than that obtained from calli co-cultured in N6 media alone. Also, judging from the average weight of the cell clusters, using suspension-cultured cell clusters resulted in about 10-fold enhancement of the transformation efficiency compared with that of calli subcultured on solid media.

Technologies for transformation of rice have been developed to meet the requirements of functional genomics in order to enable the production of transgenic rice plants with useful agricultural characters. However, many rice varieties are not efficiently transformed by Agrobacterium. We have succeeded in establishing a highly efficient transformation system in rice by co-cultivating rice calli with Agrobacterium on three filter papers moistened with enriched N6 or DKN media instead of using solid media. Rice calli immersed in Agrobacterium suspension (EHA101, Agrobacterium concentration of OD600=0.04) were co-cultured on three pieces of filter paper (9cm in diameter) moistened with 5.5mL of N6 or DKN liquid co-cultivation medium supplemented with 2,4-d (2mg/L), proline (10mM), casein hydrolysate (300mg/L), sucrose (30g/L), glucose (5g/L), l-cysteine (100mg/L) and acetosyringone (15mg/L) at 25°C for 3 days in the dark. Compared with the transformation efficiency of calli co-cultivated on solid media, transformation efficiency was increased by about fivefold by using the filter paper method for many varieties of rice, including those that previously yielded much poor transformation rates.

Transgenic rice plants that accumulate the hypocholesterolemic pentapeptide lactostatin (IIAEK) as a fusion protein with the endosperm seed storage protein glutelin were developed. Plants were selected from Agrobacterium-mediated transformants using a mutated-rice acetolactate synthase (mALS) marker protein expressed under the control of the rice callus-specific promoter (CSP) (CSP:mALS). Lactostatin accumulation levels were compared in mature seeds of 78 independent transgenic rice lines selected for either the CSP:mALS gene cassette or hygromycin phosphotransferase (HPT) under the control of the CaMV 35S promoter (35S:HPT). Transgenic rice seeds harboring a CSP:mALS gene cassette accumulated more (approximately 120-200% on average) lactostatin than those with a 35S:HPT gene cassette, and accumulation was largely independent of transgene copy number. Furthermore, transgenic rice seeds that were selected by HPT under the control of CSP (CSP:HPT) also accumulated more lactostatin (approximately 160% on average) than transgenic rice seeds with the 35S:HPT gene cassette. These results indicate that high-value bioactive peptides such as lactostatin can be produced at higher levels using callus-specific selection than by conventional constitutive selection. Specific expression of a marker gene at the selection stage may thus result in increased target peptide accumulation levels in seeds.

We have developed an efficient rice transformation system that uses only rice genome-derived components. The transgenic 'Koshihikari' rice, low-glutelin mutant a123, is capable of accumulating large amounts of bioactive peptides in the endosperm. Agrobacterium-mediated transformation using the mutated-acetolactate synthase (mALS) gene expressed under the control of the callus-specific promoter (CSP) as a selectable marker was used to introduce GFP and an anti-hypertensive hexapeptide into 'Koshihikari' a123. The CSP:mALS gene cassette confers pyrimidinyl carboxy herbicide resistance to transgenic rice callus, but is not expressed in regenerated plants. Transformation efficiency of transgenic rice line a123 was improved from about 10% to about 30% by modifying callus induction, callus selection and regeneration media conventionally used for rice tissue culture.

In some eukaryotes, endoplasmic reticulum (ER) stress induces regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) of mRNAs. Recently, the expression levels of the mRNAs encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD have been insufficiently investigated due to difficulty of in planta analyses. Here, the RIDD susceptibilities of various mRNAs that are difficult to analyze in planta were examined using transient expression analyses of rice protoplasts. In this system, the mRNAs encoding three rice seed storage proteins (SSPs) - namely α-globulin, 16-kDa prolamin and 10-kDa prolamin - were downregulated in response to ER stress. The rapid ER stress-induced degradation of these mRNAs was repressed in cells in which the ribonuclease activity of IRE1 was specifically abolished by genome editing, suggesting that the mRNAs encoding certain SSPs are strong targets of RIDD. Furthermore, we investigated whether these RIDD targets are substrates of the IRE1 ribonuclease using a recombinant IRE1 protein, and identified candidate IRE1-mediated cleavage sites. Overall, the results demonstrate the existence of a post-transcriptional mechanism of regulation of SSPs, and illustrate the basic and multifaceted characteristics of RIDD in higher plants.

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.

To obtain a better understanding of the function of mitochondrial uncoupling protein (UCP) in higher plants, the wheat gene for mitochondrial uncoupling protein (WhUCP) in rice was overexpressed by Agrobacterium-mediated transformation with a construct containing the WhUCP ORF under control of the 35S promoter. The transgenic rice plants showed a significant increase in tolerance against oxidative stress promoted by exogenous hydrogen peroxide at the seedling stage. The transgenic rice plants overexpressing WhUCP also exhibited greater tolerance against cold stress than did the wild-type plants. These results demonstrated that the mitochondrial UCP in higher plants is positively involved in the pathway for abiotic stress tolerance, probably through a decrease in cellular oxidative damage, and that controlled uncoupling by UCP could be used for improvement of stress tolerance in higher plants.

Rice varieties vary in their capacity for callus induction, growth, and regeneration. We identified the locus and candidate gene which conferred good callus growth and regenerative ability in the rice variety Koshihikari, a notorious poor rice line for genetic transformation. In addition, we succeeded in establishment of a new selectable marker system using the NiR gene for Agrobacterium-mediated transformation of rice, c.v. “Koshihikari.” The locus was mapped onto chromosome 1, and the nearest RFLP marker was C0178. A total of 500 segregating individuals (BC6F2 seeds) were screened for recombination by PCR-based screening and its location narrowed to a 540-kb region that had been sequenced by the International Rice Genome Sequencing Project. One ORF encoded a putative ferredoxin-nitrite reductase (NiR), which has been suggested to be required for callus induction and growth. The growth and regeneration ability of the calli initiated from Koshihikari was improved through integration of the NiR gene from Konansou. Analysis of ORFs and the promoter region of NiR indicated that the promoter region of NiR gene is responsible for growth and regeneration ability of calli in rice, c.v. Koshihikari. We established a NiR selection system for Agrobacterium-mediated transformation in rice, c.v. Koshihikari, by integration of the NiR gene isolated from rice c.v. Konansou. Transgenic rice plants regenerated from selected calli exhibited β-glucuronidase (GUS) activity. A transformation frequency of 9% was obtained. The results indicated that the NiR selection system is devoid of the disadvantages and concerns of using foreign genes (antibiotics and herbicide resistant) for selection.

The human gene for CYP2B6, a cytochrome P450 monooxygenase that inactivates xenobiotic chemicals, was introduced into Oryza sativa cv. Nipponbare by Agrobacterium-mediated transformation. At germination, R(1) seeds of transgenic rice plants expressing CYP2B6 (CYP2B6 rice) showed a high tolerance to 5 microM metolachlor, a preemergence herbicide that is degraded by CYP2B6. Thin-layer chromatography after culture with (14)C-labeled metolachlor revealed that the amounts of residual metolachlor decreased in plant tissues and the medium of CYP2B6 rice faster than those of untransformed Nipponbare. CYP2B6 rice plants were able to grow in the presence of 13 out of 17 herbicides: five chloroacetamides and mefenacet, pyributicarb, amiprofos-methyl, trifluralin, pendimethalin, norflurazon, and chlorotoluron. These herbicides differ in their modes of action and chemical structures. Transgenic rice expressing a xenobiotic-degrading human CYP2B6, which has broad substrate specificity, should be good not only for developing herbicide tolerant rice but also for reducing the environmental impact of agrochemicals.

We compared the transcription patterns of calli initiated from the cultivars Koshihikari, Sasanishiki and Konansou and from isogenic lines with the aim of identifying genes correlated with good regeneration ability of the callus in rice by mRNA differential display. Koshihikari and Sasanishiki isogenic lines exhibited the same good regeneration ability as that of Konansou, and Koshihikari and Sasanishiki exhibited poor regeneration ability compared with that of Konansou. One full-length cDNA ( Os22A ) whose expression was elevated in the rice calli that exhibited vigorous plant regeneration was isolated. Sequence analysis revealed that Os22A is identical to the EST clones expressed in Oryza sativa in the endosperm 10 days after pollination (accession number BI797481 ) and in the panicle at the ripening stage (accession number AU172698 ). Os22A is highly homologous to a barley gene coding for glucose dehydrogenase (accession number S72926 ) that is specifically expressed in developing embryos. Os22A encodes a transcript of 1391 nucleotides and is specifically expressed in calli with good regeneration ability in rice. Expression of Os22A mRNA decreased or disappeared with decrease in regeneration ability. In order to characterize the function of Os22A , we integrated Os22A into rice calli ( O. sativa cv . Koshihikari). Calli initiated from Koshihikari exhibited poor regeneration ability but the transformed calli showed good regeneration ability in the regeneration medium. The regeneration ability of transformed calli clearly improved by integration of Os22A . Our results suggest that Os22A is involved in plant regeneration ability of the callus.

OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. Green fluorescent protein-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.

CEBiP, a plasma membrane-localized glycoprotein of rice, directly binds with chitin elicitors (CE), and has been identified as a receptor for CE by using CEBiP-RNAi rice cells. To further clarify the function of CEBiP, we produced CEBiP-disrupted rice plants by applying an efficient Agrobacterium-mediated gene-targeting system based on homologous recombination, which has recently been developed for rice. Homologous recombination occurred at the CEBiP locus in ~0.5 % of the positive/negative selected calli. In the self-pollinated next generation, it was confirmed that the first exon of CEBiP was replaced with the hygromycin selection cassette as designed, and that the expression of CEBiP was completely deficient in homozygous cebip lines. Affinity-labeling analysis using biotinylated N-acetylchitooctaose demonstrated that CEBiP is the major CE-binding protein in rice cultured cells and leaves, which was consistent with the result that the response to CE in cebip cells was greatly diminished. Nevertheless, we observed a significant decrease in disease resistance against Magnaporthe oryzae, the causal agent of rice blast disease, only when the cebip leaf sheaths were inoculated with a weakly virulent strain, suggesting that CE perception during the infection process of M. oryzae is limited. The response to peptidoglycan and lipopolysaccharides in cebip cells was not affected, strongly suggesting that CEBiP is a CE-specific receptor.

The endoplasmic reticulum (ER) stress sensor, IRE1, contains a kinase domain and a ribonuclease domain. Ribonuclease mediates the unconventional splicing of mRNA encoding the transcription factor AtbZIP60 in Arabidopsis, or OsbZIP50 in rice, and thereby transduces signals from stressed ER. Here, we demonstrate the additional roles of plant IRE1 using genetically modified rice plants. Using a gene targeting system based on homologous recombination, genomic IRE1 was replaced with two types of missense alleles, leading to a defect in the kinase or ribonuclease activity of IRE1. Genetic analysis of these alleles demonstrated that the kinase activity of IRE1 plays a vital role independent of ribonuclease activity. Furthermore, the existence of ribonuclease substrates other than OsbZIP50 mRNA is demonstrated for the first time. This study provides new insights into higher plant signalling using a gene targeting approach.

Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271-282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large number of transformants are required to generate T-DNA insertion and FOX libraries as well as gene-targeting studies. To overcome these challenges, we established a high-efficiency transformation system in rice by cocultivating rice calli with Agrobacterium on filter papers moistened with enriched liquid media instead of using solid media (Ozawa, Plant Sci. 176:522-527, 2009; Ozawa and Takaiwa, Plant Sci. 179:333-337, 2010). In this system, the transformation efficiency of the calli is almost 100% in many varieties.

Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271-282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large number of transformants are required to generate T-DNA insertion and FOX libraries as well as gene-targeting studies. To overcome these challenges, we established a high-efficiency transformation system in rice by cocultivating rice calli with Agrobacterium on filter papers moistened with enriched liquid media instead of using solid media (Ozawa, Plant Sci. 176:522-527, 2009; Ozawa and Takaiwa, Plant Sci. 179:333-337, 2010). In this system, the transformation efficiency of the calli is almost 100% in many varieties.

Rice seeds, a rich reserve of starch and protein, are a major food source in many countries. Unlike the seeds of other plants, which typically accumulate one major type of storage protein, rice seeds use two major classes, prolamines and globulin-like glutelins. Both storage proteins are synthesized on the endoplasmic reticulum (ER) and translocated to the ER lumen, but are then sorted into separate intracellular compartments. Prolamines are retained in the ER lumen as protein bodies whereas glutelins are transported and stored in protein storage vacuoles. Mechanisms responsible for the retention of prolamines within the ER lumen and their assembly into intracisternal inclusion granules are unknown, but the involvement of RNA localization has been suggested. Here we show that the storage protein RNAs are localized to distinct ER membranes and that prolamine RNAs are targeted to the prolamine protein bodies by a mechanism based on RNA signal(s), a process that also requires a translation initiation codon. Our results indicate that the ER may be composed of subdomains that specialize in the synthesis of proteins directed to different compartments of the plant endomembrane system.

Suspension cultures in which cell clusters were small and had a high capacity for somatic embryogenesis (about 60%) were established from immature panicles of F1 plants from a cross between Oryza sativa and Oryza latifolia The cells were subcultured at seven-day intervals in a modified N6 medium. The cell clusters were quite small (approximately 30–200μm in diameter) after culture for two months in this medium. When small clusters of cells were transferred to N6 medium, that had been diluted with an equal volume of water and supplemented with α-naphthalenacetic acid (53 nM), 4-pyridylurea (2.2 μM) and sucrose (30 gl-1), somatic embryogenesis occurred at high frequency (about 60%). The system established in the present work is useful for biochemical and molecular biological research of the somatic embryogenesis in the Gramineae.

Background The endoplasmic reticulum (ER) stress response is widely known to function in eukaryotes to maintain the homeostasis of the ER when unfolded or misfolded proteins are overloaded in the ER. To understand the molecular mechanisms of the ER stress response in rice (Oryza sativa L.), we previously analyzed the expression profile of stably transformed rice in which an ER stress sensor/transducer OsIRE1 was knocked-down, using the combination of preliminary microarray and quantitative RT-PCR. In this study, to obtain more detailed expression profiles of genes involved in the initial stages of the ER stress response in rice, we performed RNA sequencing of wild-type and transgenic rice plants produced by homologous recombination in which endogenous genomic OsIRE1 was replaced by missense alleles defective in ribonuclease activity. Results At least 38,076 transcripts were investigated by RNA sequencing, 380 of which responded to ER stress at a statistically significant level (195 were upregulated and 185 were downregulated). Furthermore, we successfully identified 17 genes from the set of 380 ER stress-responsive genes that were not included in the probe set of the currently available microarray chip in rice. Notably, three of these 17 genes were non-annotated genes, even in the latest version of the Rice Annotation Project Data Base (RAP-DB, version IRGSP-1.0). Conclusions Therefore, RNA sequencing-mediated expression profiling provided valuable information about the ER stress response in rice plants and led to the discovery of new genes related to ER stress.