Makoto Hakata

Atlas of Rice Grain Filling-Related Metabolism under High Temperature: Joint Analysis of Metabolome and Transcriptome Demonstrated Inhibition of Starch Accumulation and Induction of Amino Acid Accumulation

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°C/28°C) and a control temperature (25°C/20°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.

A Novel Factor FLOURY ENDOSPERM2 Is Involved in Regulation of Rice Grain Size and Starch Quality

The authors clone the rice FLOURY ENDOSPERM2 (FLO2) gene; flo2 mutants have aberrant endosperm, and FLO2 overexpressors have enlarged grains. Gene expression and protein interaction studies indicate that FLO2, a novel tetratricopeptide repeat containing protein, regulates storage starch and protein gene expression in rice endosperm development and may also play a role in heat tolerance. 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.

Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice

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.

Suppression of α-amylase genes improves quality of rice grain ripened under high temperature.

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.

Ectopic Overexpression of The Transcription Factor OsGLK1 Induces Chloroplast Development in Non-Green Rice Cells

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.

Increase in Cellulose Accumulation and Improvement of Saccharification by Overexpression of Arabinofuranosidase in Rice

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.

Overexpression of a rice TIFY gene increases grain size through enhanced accumulation of carbohydrates in the stem.

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.

Stress Tolerance Profiling of a Collection of Extant Salt-Tolerant Rice Varieties and Transgenic Plants Overexpressing Abiotic Stress Tolerance Genes

Environmental stress tolerance is an important trait for crop improvement. In recent decades, numerous genes that confer tolerance to abiotic stress such as salinity were reported. However, the levels of salt tolerance differ greatly depending on growth conditions, and mechanisms underlying the complicated nature of stress tolerance are far from being fully understood. In this study, we investigated the profiles of stress tolerance of nine salt-tolerant rice varieties and transgenic rice lines carrying constitutively expressed genes that are potentially involved in salt tolerance, by evaluating their growth and viability under salt, heat, ionic and hyperosmotic stress conditions. Profiling of the extant varieties and selected chromosome segment substitution lines showed that salt tolerance in a greenhouse condition was more tightly correlated with ionic stress tolerance than osmotic stresses. In Nona Bokra, one of the most salt-tolerant varieties, the contribution of the previously identified sodium transporter HKT1;5 to salt tolerance was fairly limited. In addition, Nona Bokra exhibited high tolerance to all the stresses imposed. More surprisingly, comparative evaluation of 74 stress tolerance genes revealed that the most striking effect to enhance salt tolerance was conferred by overexpressing CYP94C2b, which promotes deactivation of jasmonate. In contrast, genes encoding ABA signaling factors conferred multiple stress tolerance. Genes conferring tolerance to both heat and hyperosmotic stresses were preferentially linked to functional categories related to heat shock proteins, scavenging of reactive oxygen species and Ca(2+) signaling. These comparative profiling data provide a new basis for understanding the ability of plants to grow under harsh environmental conditions.

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.

Turgor-responsive starch phosphorylation in Oryza sativa stems: A primary event of starch degradation associated with grain-filling ability

Grain filling ability is mainly affected by the translocation of carbohydrates generated from temporarily stored stem starch in most field crops including rice (Oryza sativa L.). The partitioning of non-structural stem carbohydrates has been recognized as an important trait for raising the yield ceiling, yet we still do not fully understand how carbohydrate partitioning occurs in the stems. In this study, two rice subspecies that exhibit different patterns of non-structural stem carbohydrates partitioning, a japonica-dominant cultivar, Momiroman, and an indica-dominant cultivar, Hokuriku 193, were used as the model system to study the relationship between turgor pressure and metabolic regulation of non-structural stem carbohydrates, by combining the water status measurement with gene expression analysis and a dynamic prefixed 13C tracer analysis using a mass spectrometer. Here, we report a clear varietal difference in turgor-associated starch phosphorylation occurred at the initiation of non-structural carbohydrate partitioning. The data indicated that starch degradation in Hokuriku 193 stems occurred at full-heading, 5 days earlier than in Momiroman, contributing to greater sink filling. Gene expression analysis revealed that expression pattern of the gene encoding α-glucan, water dikinase (GWD1) was similar between two varieties, and the maximum expression level in Hokuriku 193, reached at full heading (4 DAH), was greater than in Momiroman, leading to an earlier increase in a series of amylase-related gene expression in Hokuriku 193. In both varieties, peaks in turgor pressure preceded the increases in GWD1 expression, and changes in GWD1 expression was correlated with turgor pressure. Additionally, a threshold is likely to exist for GWD1 expression to facilitate starch degradation. Taken together, these results raise the possibility that turgor-associated starch phosphorylation in cells is responsible for the metabolism that leads to starch degradation. Because the two cultivars exhibited remarkable varietal differences in the pattern of non-structural carbohydrate partitioning, our findings propose that the observed difference in grain-filling ability originated from turgor-associated regulation of starch phosphorylation in stem parenchyma cells. Further understanding of the molecular mechanism of turgor-regulation may provide a new selection criterion for breaking the yield barriers in crop production.