Evaluation of protein production in rice seedlings under dark conditions.

Although plants have several advantages for foreign protein production, cultivation of transgenic plants in artificial plant growth facilities involves the use of a great amount of electricity for lightning and air conditioning, reducing cost-effectiveness. Protein production in plants grown in darkness can overcome this problem, but the amount of protein produced in the dark is unknown. In this study, the total amount of soluble protein produced in rice seedlings germinated and grown in light or darkness were examined at several time points after germination and under different temperature, nutritional, and seedling density conditions. Our results indicate that rice seedlings grown in darkness produce a comparable amount of total soluble protein to those grown in light. Furthermore, we found that the best conditions for protein production in dark-grown rice seedlings are large seeds germinated and grown for 10-12 days at 28 °C supplemented with Murashige and Skoog medium and 30 g/l sucrose in dense planting. Therefore, our results suggest that foreign proteins can be produced in rice seedlings in the dark, with a reduced electricity use and an increase in cost-effectiveness.

Interaction of ONION2 ketoacyl CoA synthase with ketoacyl CoA reductase of rice.

BACKGROUND: Fatty acid elongases (FAEs), which catalyse elongation reactions of a carbon chain of very-long-chain fatty acids, play an important role in shoot development in rice. The elongation reactions consist of four sequential reactions catalysed by distinct enzymes, which are assumed to form an elongation complex. However, no interacting proteins of ONION1 (ONI1) and ONI2, which are ketoacyl CoA synthase catalyzing the first step and are required for shoot development in rice, are reported. METHODS AND RESULTS: In this study ketoacyl CoA reductase (KCR) that interacts with ONI1 and ONI2 was searched. A database search identified 10 KCR genes in the rice genome. Among the genes, the expression pattern of KCR1 was similar to that of ONI2. Yeast two-hybrid analysis showed interaction of ONI2 with KCR1, which was confirmed by GST pull-down assay. No interacting partner of ONI1 was identified. CONCLUSIONS: Our results suggest that ONI2 and KCR1 form an FAE complex that may play a role in biosynthesizing VLCFAs during shoot development.

Cytokinin-induced expression of OSH1 in a shoot-regenerating rice callus.

The expression of a KNOX class 1 gene OSH1 is induced by cytokinin during regeneration of shoots from callus in Oryza sativa L. (rice). This cytokinin-induced expression was enhanced by overexpression of homologues of cytokinin-signalling phosphorelay genes such as a histidine kinase gene OHK3, a phosphotransmitter gene OHP2 and a response regulator gene ORR1 in cultured cells. Regionally overlapped expression of these genes and OSH1 was observed in shoot apex. These results suggest that these cytokinin-signalling genes are positive regulators of the expression of OSH1, and mediate the OSH expression upon shoot regeneration from callus in rice.

Expression of a fungal laccase fused with a bacterial cellulose-binding module improves the enzymatic saccharification efficiency of lignocellulose biomass in transgenic Arabidopsis thaliana.

Delignification is effective for improving the saccharification efficiency of lignocellulosic biomass materials. We previously identified that the expression of a fungal laccase (Lac) fused with a bacterial cellulose-binding module domain (CBD) improved the enzymatic saccharification efficiency of rice plants. In this work, to evaluate the ability of the Lac-CBD fused chimeric enzyme to improve saccharification efficiency in a dicot plant, we introduced the chimeric gene into a dicot model plant, Arabidopsis thaliana. Transgenic plants expressing the Lac-CBD chimeric gene showed normal morphology and growth, and showed a significant increase of enzymatic saccharification efficiency compared to control plants. The transgenic plants with the largest improvement of enzymatic saccharification efficiency also showed an increase of crystalline cellulose in their cell wall fractions. These results indicated that expression of the Lac-CBD chimeric protein in dicotyledonous plants improved the enzymatic saccharification of plant biomass by increasing the crystallinity of cellulose in the cell wall.

Molecular characterization and targeted quantitative profiling of the sphingolipidome in rice.

Recent advances in comprehensive metabolite profiling techniques, the foundation of metabolomics, is facilitating our understanding of the functions, regulation and complex networks of various metabolites in organisms. Here, we report a quantitative metabolomics technique for complex plant sphingolipids, composed of various polar head groups as well as structural isomers of hydrophobic ceramide moieties. Rice (Oryza sativa L.) was used as an experimental model of monocotyledonous plants and has been demonstrated to possess a highly complex sphingolipidome including hundreds of molecular species with a wide range of abundance. We established a high-throughput scheme for lipid preparation and mass spectrometry-based characterization of complex sphingolipid structures, which provided basic information to create a comprehensive theoretical library for targeted quantitative profiling of complex sphingolipids in rice. The established sphingolipidomic approach combined with multivariate analyses of the large dataset obtained clearly showed that different classes of rice sphingolipids, particularly including subclasses of glycosylinositol phosphoceramide with various sugar-chain head groups, are distributed with distinct quantitative profiles in various rice tissues, indicating tissue-dependent metabolism and biological functions of the lipid classes and subclasses. The sphingolipidomic analysis also highlighted that disruption of a lipid-associated gene causes a typical sphingolipidomic change in a gene-dependent manner. These results clearly support the utility of the sphingolipidomic approach in application to wide screening of sphingolipid-metabolic phenotypes as well as deeper investigation of metabolism and biological functions of complex sphingolipid species in plants.

Enhanced production of reducing sugars from transgenic rice expressing exo-glucanase under the control of a senescence-inducible promoter.

We generated transgenic rice plants that express EXG1 exo-glucanase under the control of a senescence-inducible promoter. When a GUS coding sequence was connected to a promoter region of STAY GREEN (SGR) gene of rice and introduced into rice, GUS activity was specifically observed along with senescence. When an EXG1 cDNA was connected to the SGR promoter and introduced into rice, higher cellulase activities were detected after senescence. The EXG1 transgenic plants showed enhanced enzymatic saccharification efficiencies after senescence, but no significant difference of saccharification efficiencies was observed before senescence. The saccharification efficiencies were correlated with the cellulase activities in the transgenic plants. The EXG1 transgenic plants showed neither morphological abnormality nor sterility, both of which were observed when EXG1 was constitutively overexpressed. These results indicate that expression of cell wall degrading enzymes such as cellulase by a senescence-inducible promoter is one of the ways to enhance the saccharification ability of cellulosic biomass without affecting plant growth for efficient production of biofuels.

A candidate factor that interacts with RF2, a restorer of fertility of Lead rice-type cytoplasmic male sterility in rice.

BACKGROUND: The pollen function of cytoplasmic male sterile (CMS) plants is often recovered by the Restorer of fertility (Rf) gene encoded by the nuclear genome. An Rf gene of Lead rice type CMS, Rf2, encodes a small mitochondrial glycine-rich protein. RF2 is expected to function by interacting with other proteins, because RF2 has no motifs except for glycine-rich domain. FINDINGS: To elucidate the protein that interacts with RF2, we performed yeast two-hybrid screening. We identified four genes and named RF2-interacting candidate factors (RIF1 to RIF4). A study of subcellular localization demonstrated that only RIF2 was targeted to mitochondria. A pull-down assay using E. coli-produced recombinant GST-tagged RF2 and His-tagged RIF2 confirmed that RF2 interacted with RIF2. RIF2 encodes ubiquitin domain-containing protein. CONCLUSIONS: These results suggest that RIF2 is a candidate factor of a fertility restoration complex of RF2.

Organ fusion and defective shoot development in oni3 mutants of rice.

Maintenance of organ separation is one of the essential phenomena for normal plant development. We have identified and analyzed ONION3 (ONI3), which is required for avoiding organ fusions in rice. Loss-of-function mutations of ONI3, which were identified as mutants with ectopic expression of KNOX genes in leaves and morphologically resembling KNOX overexpressors, showed abnormal organ fusions in developing shoots. The mutant seedlings showed fusions between neighboring organs and also within an organ; they stopped growing soon after germination and subsequently died. ONI3 was shown to encode an enzyme that is most similar to Arabidopsis HOTHEAD and is involved in biosynthesis of long-chain fatty acids. Expression analyses showed that ONI3 was specifically expressed in the outermost cell layer in the shoot apex throughout life cycle, and the oni3 mutants had an aberrant outermost cell layer. Our results together with previous studies suggest that long-chain fatty acids are required for avoiding organ fusions and promoting normal shoot development in rice.

Application of fungal laccase fused with cellulose-binding domain to develop low-lignin rice plants.

We observed a reduction of lignin content linked to the expression of fungal laccase in rice plants. The lignin content of L-4, which showed the highest LAC activity among transgenic lines produced, was lower than that of the control line. However, this change was not reflected to the saccharification efficiency.

ONION2 fatty acid elongase is required for shoot development in rice.

A plant's surface is covered with epicuticular wax, which protects plants from inappropriate environmental conditions such as drought and pathogen attack. Very-long-chain fatty acids (VLCFAs) are the main component of epicuticular wax on the surface of above-ground organs. Here we show that a fatty acid elongase catalyzing an elongation reaction of VLCFAs is required for shoot development in rice. onion2 (oni2) mutants produced very small shoots in which leaves were fused to each other, and ceased growing after germination. The midrib of oni2 leaf blades was not developed correctly. Molecular cloning showed that ONI2 encodes a fatty acid elongase, which catalyzes the first step of elongation reactions of a carbon chain of VLCFAs, and oni2 had a reduced amount of VLCFAs. Expression analysis showed that ONI2 is specifically expressed in the outermost cell layer of young lateral organs. These results suggest that ONI2 is a layer 1-specific gene required for development of the entire shoot and that VLCFAs play an essential role in normal shoot development in rice.

Enhanced saccharification of rice straw by overexpression of rice exo-glucanase.

BACKGROUND: Efficient production of carbon-neutral biofuels is key to resolving global warming and exhaustion of fossil fuels. Cellulose, which is the most abundant biomass, is physically strong and biochemically stable, and these characteristics lead to difficulty of efficient saccharification of cellulosic compounds for production of fermentable glucose and other sugars. RESULTS: We transformed rice with overexpressing constructs of rice genes encoding each of three classes of cellulases. The exo-glucanase overexpressing plants showed various abnormalities in leaf such as division of leaf blade, crack on leaf surface, excess lacunae in midrib structure and necrotic colour change. The overexpressing plants also showed sterility. Noticeably, these plants showed enhanced saccharification of stems after maturation. These results indicate that overexpression of the exo-glucanase gene brought about various developmental defects associated with modification of cell wall and enhanced saccharification in rice. On the other hand, endo-glucanase-overexpressing plants could not be obtained, and overexpression of ß-glucosidase brought about no effect on plant growth and development. CONCLUSIONS: Our results indicate that genetic engineering of cellulosic biomass plants by overexpressing cellulase genes will be one of the approaches to confer enhanced saccharification ability for efficient production of cellulosic biofuels such as ethanol.

Positive autoregulation of a KNOX gene is essential for shoot apical meristem maintenance in rice.

Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM.

Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice.

We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice.

Altered expression of auxin-related genes in the fatty acid elongase mutant oni1 of rice.

VLCFAs are the main components of cuticular wax, which covers and protects plants from physical and biological stresses. However, the effect of fatty acid composition or the physiological role of VLCFAs on plant development under normal growth conditions is not well understood. We analyzed loss-of-function mutants of ONION1 (ONI1) which encodes fatty acid elongase (ß-ketoacyl CoA synthase) catalyzing an elongation reaction of a carbon chain of VLCFAs. We showed that oni1 shoot contained a reduced amount of VLCFAs, and differentiation and functionality of an outermost cell layer (L1) were highly perturbed in oni1 shoot. In spite of the L1-specific expression of ONI1, the effects of the oni1 mutation were not restricted to L1, but expanded to inner cells, so that the entire shoot development was impaired including failure of the maintenance of the SAM and ectopic expression of SAM-specific KNOX genes in leaf. Thus, ONI1 function is cell non-autonomous, and signaling from L1 to inner cells may support proper development of inner cells. Here we report that expression of auxin-related genes was affected in oni1 shoot, and we speculate the existence of improper auxin distribution due to a lack of normal L1 in oni1 shoot.

Fatty acid elongase is required for shoot development in rice.

Organisms are covered extracellularly with cuticular waxes that consist of various fatty acids. In higher plants, extracellular waxes act as indispensable barriers to protect the plants from physical and biological stresses such as drought and pathogen attacks. However, the effect of fatty acid composition on plant development under normal growth conditions is not well understood. Here we show that the ONION1 (ONI1) gene, which encodes a fatty acid elongase (ß-ketoacyl CoA synthase) involved in the synthesis of very-long-chain fatty acids, is required for correct fatty acid composition and normal shoot development in rice. oni1 mutants containing a reduced amount of very-long-chain fatty acids produced very small shoots, with an aberrant outermost epidermal cell layer, and ceased to grow soon after germination. These mutants also showed abnormal expression of a KNOX family homeobox gene. ONI1 was specifically expressed in the outermost cell layer of the shoot apical meristem and developing lateral organs. These results show that fatty acid elongase is required for formation of the outermost cell layer, and this layer is indispensable for entire shoot development in rice.

COE1, an LRR-RLK responsible for commissural vein pattern formation in rice.

Leaf veins have a complex network pattern. Formation of this vein pattern has been widely studied as a model of tissue pattern formation in plants. To understand the molecular mechanism governing the vascular patterning process, we isolated the rice mutant, commissural vein excessive1 (coe1). The coe1 mutants had short commissural vein (CV) intervals and produced clustered CVs. Application of 1-N-naphthylphthalamic acid and brefeldin A decreased CV intervals, and application of 1-naphthaleneacetic acid increased CV intervals in wild-type rice; however, coe1 mutants were insensitive to these chemicals. COE1 encodes a leucine-rich repeat receptor-like kinase, whose amino acid sequence is similar to that of brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and which is localized at the plasma membrane. Because of the sequence similarity of COE1 to BAK1, we also examined the involvement of brassinosteroids in CV formation. Brassinolide, an active brassinosteroid, decreased the CV intervals of wild-type rice, and brassinazole, an inhibitor of brassinosteroid biosynthesis, increased the CV intervals of wild-type rice, but coe1 mutants showed insensitivity to these chemicals. These results suggest that auxin and brassinosteroids regulate CV intervals in opposite directions, and COE1 may regulate CV intervals downstream of auxin and brassinosteroid signals.

Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter.

An OsWRKY11 gene, which encodes a transcription factor with the WRKY domain, was identified as one of the genes that was induced by both heat shock and drought stresses in seedlings of rice (Oryza sativa L.). To determine if overexpression of OsWRKY11 confers heat and drought tolerance, OsWRKY11 cDNA was fused to the promoter of HSP101 of rice and introduced into a rice cultivar Sasanishiki. Overexpression of OsWRKY11 was induced by heat treatment. After heat pretreatment, the transgenic lines showed significant heat and drought tolerance, as indicated by the slower leaf-wilting and less-impaired survival rate of green parts of plants. They also showed significant desiccation tolerance, as indicated by the slower water loss in detached leaves. Our results indicate that the OsWRKY11 gene plays a role in heat and drought stress response and tolerance, and might be useful for improvement of stress tolerance.

Identification, characterization and interaction of HAP family genes in rice.

A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members.

Identification and characterization of cytokinin-signalling gene families in rice.

We identified four histidine kinase (HK) genes of a cytokinin receptor family, two histidine-containing phosphotransmitter (HPt) genes, thirteen A-type response regulator (RR) genes and six B-type RR genes in the rice genome. The HK genes (OHK2, OHK3, OHK4 and OHK5 for Oryza sativa HK), the HPt genes (OHP1 and OHP2 for O. sativa HPt) and the B-type RR genes (ORR1, ORR2, ORR3, ORR4 and ORR6 for O. sativa RR) except one (ORR5) showed expression in various organs. ORR5 was expressed in callus and flower. Three A-type RR genes (OsRR4, OsRR9 and OsRR10 for O. sativa RR) showed cytokinin-induced expression, and three (OsRR8, OsRR12 and OsRR13) showed expression in flower. We also identified two other genes named OHK1 and CHARK (CHASE domain Receptor-like serine/threonine Kinase). OHK1 encodes an HK similar to Arabidopsis CKI1, which is involved in female gametophyte development. CHARK encodes a protein with an extracellular cytokinin-perceiving CHASE domain and a cytoplasmic serine/threonine kinase domain which are connected with a single transmembrane domain. The presence of all four gene families and CHARK in the rice genome suggests that a cytokinin signal is transduced by the phosphotransfer mechanism as is the case in Arabidopsis, and that rice may have an additional novel signalling pathway involving serine/threonine phosphorylation.

Expression of SERK family receptor-like protein kinase genes in rice.

Some SERK-family receptor-like protein kinase genes have been shown to confer embryonic competence to cells. In this study, we isolated two novel rice genes, OsSERK1 and OsSERK2, belonging to the SERK-family. OsSERK2 showed constitutive expression. The OsSERK1 promoter showed reporter gene activities in some specific tissues in a germinating seed, leaf and root, but not in a developing embryo. This promoter activity suggests that OsSERK1 may have roles in non-embryonic tissues rather than in the embryo.