[Show abstract][Hide abstract] ABSTRACT: Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against α-(1,5)-linked l-Ara (LM6) and β-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol.
PLoS ONE 11/2013; 8(11):e78269. DOI:10.1371/journal.pone.0078269 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Screening of rice full-length cDNA overexpressing (FOX) lines allowed the identification of a TIFY gene, TIFY11b, as a growth-promoting gene whose overexpression increased plant height and seed size. The grains of TIFY11b-overexpressing plants exceeded those of non-transformants in length, width and thickness, resulting in 9-21% increases in grain weight. The increase was achieved by overexpressing the gene in the whole plant body, but not by seed-restricted expression, indicating that seed enlargement is attributable to overexpression in vegetative organs such as the leaf. The whole-body overexpressing plants developed longer leaves along with higher levels of starch and sucrose in the leaf sheath and culm at the heading stage than the non-transformants. Although overexpression of TIFY11b did not alter the photosynthetic rate per leaf area before and after heading, it caused an accumulation of higher levels of the carbohydrate assimilate, probably due to increased photosynthesis per plant, suggesting that the increase in grain size and weight is attained by enhanced accumulation and translocation of the carbohydrate in the culms and leaf sheaths of the transgenic plants. Thus, TIFY11b is a novel grain-size increasing gene.
[Show abstract][Hide abstract] ABSTRACT: High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism-related genes. Here we report the involvement of a starch-hydrolyzing enzyme, α-amylase, in high temperature-triggered grain chalkiness. In developing seeds, high temperature induced the expression of α-amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α-amylase activity, while it decreased an α-amylase-repressing plant hormone, ABA, suggesting starch to be degraded by α-amylase in developing grains under elevated temperature. Furthermore, RNAi-mediated suppression of α-amylase genes in ripening seeds resulted in fewer chalky grains under high-temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α-amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.
[Show abstract][Hide abstract] ABSTRACT: Calcium acts as a messenger in various signal transduction pathways in plants. Calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components in calcium signaling pathways. In rice, the CDPKs constitute a large multigene family consisting of 29 genes, but the biological functions and functional divergence or redundancy of most of these genes remain unclear. Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, we generated 250 independent transgenic rice plants overexpressing individual rice CDPKs (CDPK FOX-rice lines). These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type (WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants. These results suggest that OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress.
[Show abstract][Hide abstract] ABSTRACT: After the completion of its genomic sequencing, rice (Oryza saliva L.) has become firmly established as the premiere model plant among monocot crops. Various genetic resources have been developed for rice to accelerate the identification and characterization of rice genes with as-yet unknown functions. These resources include collections that carry natural variations and progeny derived from chemical and irradiation mutagenesis. Collections of rice strains with natural or artificial DNA insertions that create loss-of-function mutations have been of great importance for gene discovery and tagging. However, it is often difficult to observe mutant phenotypes for functionally duplicated genes and genes essential for plant growth and development. To overcome this disadvantage, gain-of-function strategies have been developed, including activation tagging and full-length cDNA overexpressing gene (FOX)-hunting systems. In this review, we summarize the current status and discuss the advantages and disadvantages of the gain-of-function approaches, and then, perspectives on accelerating discovery of rice genes.
[Show abstract][Hide abstract] ABSTRACT: We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic and genome-wide functional analysis of rice genes. In this study, we constructed a novel binary vector carrying the Gateway site-specific recombination cassette and then constructed rice FOX libraries containing a maximum of 13,823 independent, full-length cDNAs (fl-cDNAs) that correspond to approximately half the total number of rice fl-cDNA clones. By introducing the FOX libraries via Agrobacterium, we generated 2,586 FOX-rice lines exhibiting various visible alterations (e.g., plant height, tillers, leaves, and heading dates). The introduced fl-cDNAs, integrated into individual transgenic rice genomes, were amplified by genomic PCR and identified using sequencing analysis. The fl-cDNAs were PCR-amplified in 2,251 (94.2%) of the 2,389 FOX-rice lines that were examined, identifying 1,920 independent fl-cDNAs in the FOX lines. In addition to the previously generated FOX-rice plants, our new collection of FOX-rice lines produced through the Gateway system should be a useful tool for the efficient identification of gene functions in rice. Moreover, this Gateway-based technology should be applicable to other species in which a collection of fl-cDNA clones is available.
[Show abstract][Hide abstract] ABSTRACT: Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.
The Plant Cell 10/2010; 22(10):3280-94. DOI:10.1105/tpc.109.070821 · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High temperature impairs grain filling by inhibiting the deposition of storage materials such as starch and protein. To comprehend its impact on grain filling metabolism in rice (Oryza sativa), levels of metabolites and transcripts related to central pathways of metabolism were simultaneously determined in developing caryopses exposed to high temperature (33 degrees C/28 degrees C) and a control temperature (25 degrees C/20 degrees C) during the milky stage. A capillary electrophoresis-based metabolomic analysis revealed that high temperature increased the accumulation of sucrose and pyruvate/ oxaloacetate-derived amino acids and decreased levels of sugar phosphates and organic acids involved in glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle, respectively. A transcriptomic analysis using a whole genome-covering microarray unraveled the possible metabolic steps causing the shortage of storage materials under the elevated temperature. Starch deposition might be impaired by down-regulation of sucrose import/degradation and starch biosynthesis, and/or up-regulation of starch degradation as well as inefficient ATP production by an inhibited cytochrome respiration chain, as indicated by the response of gene expression to high temperature. Amino acid accumulation might be attributed to the heat-stable import of amino acids into the caryopsis and/or repression of protein synthesis especially the tRNA charging step under high temperature. An atlas showing the effect of high temperature on levels of metabolites and gene expression in the central metabolic pathways is presented.
[Show abstract][Hide abstract] ABSTRACT: For systematic and genome-wide analyses of rice gene functions, we took advantage of the full-length cDNA overexpresser (FOX) gene-hunting system and generated >12 000 independent FOX-rice lines from >25 000 rice calli treated with the rice-FOX Agrobacterium library. We found two FOX-rice lines generating green calli on a callus-inducing medium containing 2,4-D, on which wild-type rice calli became ivory yellow. In both lines, OsGLK1 cDNA encoding a GARP transcription factor was ectopically overexpressed. Using rice expression-microarray and northern blot analyses, we found that a large number of nucleus-encoded genes involved in chloroplast functions were highly expressed and transcripts of plastid-encoded genes, psaA, psbA and rbcL, increased in the OsGLK1-FOX calli. Transmission electron microscopy showed the existence of differentiated chloroplasts with grana stacks in OsGLK1-FOX calli cells. However, in darkness, OsGLK1-FOX calli did not show a green color or develop grana stacks. Furthermore, we found developed chloroplasts in vascular bundle and bundle sheath cells of coleoptiles and leaves from OsGLK1-FOX seedlings. The OsGLK1-FOX calli exhibited high photosynthetic activity and were able to grow on sucrose-depleted media, indicating that developed chloroplasts in OsGLK1-FOX rice calli are functional and active. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to plantlet regeneration medium. These results strongly suggest that OsGLK1 regulates chloroplast development under the control of light and phytohormones, and that it is a key regulator of chloroplast development.
[Show abstract][Hide abstract] ABSTRACT: The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.
[Show abstract][Hide abstract] ABSTRACT: Synthesis of storage starch and protein accumulation is the main action of endosperm organogenesis in term of the economic importance of rice. This event is strongly disturbed by abiotic stresses such as high temperature; thus, the upcoming global warming will cause a crisis with a great impact on food production^1,2^. The enzymes for the protein storage and starch synthesis pathway should work in concert to carry out the organogenesis of rice endosperm^3-5^, but the regulatory mechanism is largely unknown. Here we show that a novel regulatory factor, named OsCEO1, acts as the conductor of endosperm organogenesis during the rice grain filling stage. The physiological properties of _floury-endosperm-2_ (_flo2_) mutants showed many similarities to symptoms of grains developed under high-temperature conditions, suggesting important roles of the responsible gene in sensitivity to high-temperature stress. Our map-based cloning identified the responsible gene for the _flo2_ mutant, _OsCEO1_, which has no homology to any genes of known function. The _OsCEO1_ belongs to a novel conserved gene family and encodes a protein composed of 1,720 amino acid residues containing a TPR (tetratricopeptide repeat) motif, which is considered to mediate a protein-protein interaction. The yeast two-hybrid analysis raised an unknown protein showing homology to a late embryogenesis abundant protein and a putative basic helix-loop-helix protein as candidates for the direct interactor for _OsCEO1_, whereas no enzyme genes for the synthesis of storage substances were detected. The _flo2_ mutant exhibited reduced expression of several genes for putative regulatory proteins as well as many enzymes involved in storage starch and proteins. These results suggest that _OsCEO1_ is a superior conductor of the novel regulatory cascade of endosperm organogenesis and may have important roles in the response to high-temperature stress.