Characterization of the Functions of Starch Synthase IIIb Expressed in the Vegetative Organs of Rice (Oryza sativa L.).

Rice is the model C3 crop for investigating the starch biosynthesis mechanism in endosperm because of its importance in grain production. However, little is known about starch biosynthesis in the vegetative organs of rice. In this study, we used novel rice mutants by inserting Tos17 into the starch synthase (SS) IIIb gene, which is mainly expressed in the leaf sheath (LS) and leaf blade (LB), and an ss1 mutant to clarify the differences in roles among SS isozymes during starch biosynthesis. Native polyacrylamide gel electrophoresis (PAGE)/activity staining for SS, using LS and LB of ss mutants, revealed that the lowest migrating SS activity bands on the gel were derived from SSIIIb activity and those of two ss3b mutants were not detected. The apparent amylose content of LS starch of ss3b mutants increased. Moreover, the chain-length distribution and size-exclusion chromatography analysis using ss mutants showed that SSIIIb and SSI synthesize the B2-B3 chain and A-B1 chain of amylopectin in the LS and LB respectively. Interestingly, we also found that starch contents were decreased in the LS and LB of ss3b mutants, although SSI deficiency did not affect the starch levels. All these results indicated that SSIIIb synthesizes the long chain of amylopectin in the LS and LB similar to SSIIIa in the endosperm, while SSI synthesizes the short chain in the vegetative organ as the same in the endosperm.

Phenotypic Plasticity Conditions the Response of Soybean Seed Yield to Elevated Atmospheric CO2 Concentration.

Selection for cultivars with superior responsiveness to elevated atmospheric CO2 concentrations (eCO2) is a powerful option for boosting crop productivity under future eCO2. However, neither criteria for eCO2 responsiveness nor prescreening methods have been established. The purpose of this study was to identify traits responsible for eCO2 responsiveness of soybean (Glycine max). We grew 12 Japanese and U.S. soybean cultivars that differed in their maturity group and determinacy under ambient CO2 and eCO2 for 2 years in temperature gradient chambers. CO2 elevation significantly increased seed yield per plant, and the magnitude varied widely among the cultivars (from 0% to 62%). The yield increase was best explained by increased aboveground biomass and pod number per plant. These results suggest that the plasticity of pod production under eCO2 results from biomass enhancement, and would therefore be a key factor in the yield response to eCO2, a resource-rich environment. To test this hypothesis, we grew the same cultivars at low planting density, a resource-rich environment that improved the light and nutrient supplies by minimizing competition. Low planting density significantly increased seed yield per plant, and the magnitude ranged from 5% to 105% among the cultivars owing to increased biomass and pod number per plant. The yield increase due to low-density planting was significantly positively correlated with the eCO2 response in both years. These results confirm our hypothesis and suggest that high plasticity of biomass and pod production at a low planting density reveals suitable parameters for breeding to maximize soybean yield under eCO2.

Combining mapping of physiological quantitative trait loci and transcriptome for cold tolerance for counteracting male sterility induced by low temperatures during reproductive stage in rice.

Male sterility induced by low temperatures (LTs) during the reproductive stage is a major constraint for temperate zone rice. To detect physiological quantitative trait loci (QTLs), we modeled genotypic variation in the physiological processes involved in low temperature spikelet sterility on the basis of anther length (AL), a proxy for microspore and pollen grain number per anther. The model accounted for 83% of the genotypic variation in potential AL at normal temperature and the ability to maintain AL at LT. We tested the model on 208 recombinant inbred lines of cold-tolerant 'Tohoku-PL3' (PL3) × cold-sensitive 'Akihikari' (AH) for 2 years. QTLs for spikelet fertility (FRT) at LT were detected on chromosomes 5 (QTL for Cold Tolerance at Reproductive stage, qCTR5) and 12 (qCTR12). qCTR12 was annotated with the ability to maintain AL under LTs. qCTR5 was in a region shared with QTLs for culm length and heading date. Genome-wide expression analysis showed 798 genes differentially expressed in the spikelets between the parents at LTs. Of these, 12 were near qCTR5 and 23 were near qCTR12. Gene expression analysis confirmed two candidate genes for qCTR5 (O-methyltransferase ZRP4, Os05g0515600; beta-1,3-glucanase-like protein, Os05g0535100) and one for qCTR12 (conserved hypothetical protein, Os12g0550600). Nucleotide polymorphisms (21 deletions, 2 insertions and 10 single nucleotide polymorphisms) in PL3 were found near the candidate conserved hypothetical protein (Os12g0550600) and upstream in PL3, but not in AH. Haplotype analysis revealed that this gene came from 'Kuchum'. The combination of mapping physiological QTLs with gene expression analysis can be extended to identify other genes for abiotic stress response in cereals.

Cooling water before panicle initiation increases chilling-induced male sterility and disables chilling-induced expression of genes encoding OsFKBP65 and heat shock proteins in rice spikelets.

In rice (Oryza sativa L.), chilling-induced male sterility increased when plants experienced low water temperature (Tw , 18 °C for 14 d) before panicle initiation. The number of mature pollen grains after chilling at the booting stage (12 °C for 5 d) was only 45% of total pollen grains in low-Tw plants, whereas it was 71% in normal-Tw plants (Tw not controlled; approximately 23 °C under air temperature of 26 °C/21 °C, day/night). Microarray and quantitative PCR analyses showed that many stress-responsive genes (including OsFKBP65 and genes encoding the large heat shock protein OsHSP90.1, heat-stress transcription factors and many small heat shock proteins) were strongly up-regulated by chilling in normal-Tw spikelets, but were unaffected or even down-regulated by chilling in low-Tw spikelets. OsAPX2 and genes encoding some other antioxidant enzymes were also significantly down-regulated by low Tw in chilled spikelets. The levels of lipid peroxidation products (malondialdehyde equivalents) were significantly increased in low-Tw spikelets by chilling. Ascorbate peroxidase activity in chilled spikelets was significantly lower in low-Tw plants than in normal-Tw plants. Our data suggest that an OsFKBP65-related chilling response, which protects proteins from oxidative damage, is indispensable for chilling tolerance but is lost in low-Tw spikelets.

A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance.

Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant's response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.

Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese.

Rice (Oryza sativa) is indispensable in the diet of most of the world's population. Thus, it is an important target in which to alter iron (Fe) uptake and homeostasis, so as to increase Fe accumulation in the grain. We previously isolated OsYSL2, a functional iron [Fe(II)]- and manganese [Mn(II)]-nicotianamine complex transporter that is expressed in phloem cells and developing seeds. We produced RNAi (OsYSL2i) and overexpression lines (OXOsYSL2) of OsYSL2. At the vegetative stage in an OsYSL2i line, the Fe and Mn concentrations were decreased in the shoots, and the Fe concentration was increased in the roots. At the reproductive stage, positron-emitting tracer imaging system analysis revealed that Fe translocation to the shoots and seeds was suppressed in OsYSL2i. The Fe and Mn concentrations were decreased in the seeds of OsYSL2i, especially in the endosperm. Moreover, the Fe concentration in OXOsYSL2 was lower in the seeds and shoots, but higher in the roots, compared with the wild type. Furthermore, when OsYSL2 expression was driven by the sucrose transporter promoter, the Fe concentration in the polished rice was up to 4.4-fold higher compared with the wild type. These results indicate that the altered expression of OsYSL2 changes the localization of Fe, and that OsYSL2 is a critical Fe-nicotianamine transporter important for Fe translocation, especially in the shoots and 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.

Introduction of the ZmDof1 gene into rice enhances carbon and nitrogen assimilation under low-nitrogen conditions.

The excessive application of nitrogen fertilizer to maximize crop yields causes negative environmental effects such as pollution and ecological imbalance. To overcome this problem, researchers have attempted to improve the nitrogen assimilation capacity of crops. Maize Dof1 (ZmDof1) is a plant-specific transcription factor shown to promote nitrogen assimilation in Arabidopsis thaliana (Arabidopsis) even under nitrogen-deficient conditions. The present study examines the effect of the introduction of the ZmDof1 gene on carbon and nitrogen assimilation in rice. ZmDof1 induced the expression of phosphoenolpyruvate carboxylase (PEPC) genes in transgenic rice plants and transactivated the PEPC promoters in protoplast transient assays, showing similar effects in rice as in Arabidopsis. Transgenic rice expressing ZmDof1 and grown in the presence of 360 µm (nitrogen-sufficient) or 90 µm (nitrogen-deficient) of nitrogen concentrations showed modulation of metabolite content and gene expression associated with the anaplerotic pathway for the TCA cycle, suggesting an increased carbon flow towards nitrogen assimilation. Furthermore, increases in carbon and nitrogen amounts per seedling were found in Dof1 rice grown under nitrogen-deficient conditions. Nitrogen deficiency also resulted in the predominant distribution of nitrogen to roots, accompanied by significant increases in root biomass and modification of the shoot-to-root ratio. Measurement of the CO2 gas exchange rate showed a significant increase in the net photosynthesis rate in Dof1 rice under nitrogen-deficient conditions. Taken these together, the present study displayed that ZmDof1 expression in rice could induce gene expressions such as PEPC genes, modulate carbon and nitrogen metabolites, increase nitrogen assimilation and enhance growth under low-nitrogen conditions.

Metabolic factors restricting sink strength in superior and inferior spikelets in high-yielding rice cultivars.

Many high-yielding rice cultivars with large sink size (total number of spikelet per unit area × mean grain weight) have been developed, but some japonica cultivars developed in Japan often fail to attain the expected high yield due to low sink strength of spikelets. Although there is natural variation in sink strength of spikelets among high-yielding cultivars, metabolic factors involved in the natural variation and relationships of sink strength in spikelets with final percentage of filled spikelets are not fully understood. In the present study, we examined cultivar differences in sink strength for superior and inferior spikelets (i.e. earlier fertilizing spikelets with faster growth and later fertilizing ones with slower growth, respectively) in a panicle, using each spikelet at 10 d after the onset of development (10 DAD) when starch accumulation in endosperm was actively proceeding. Nine high-yielding cultivars were used: five japonica-dominant and four indica-dominant cultivars. Cultivar differences were observed in starch contents at 10 DAD in each spikelet type, and indica cultivars had higher starch contents than japonica cultivars in both superior and inferior spikelets. In addition, starch contents at 10 DAD were closely related to percentage of filled grains at maturity in both spikelet types. The activities of sucrose synthase (SUS) and uridine diphosphoglucose pyrophosphorylase (UGP), and the protein levels of phosphorylase 1 (Pho1), were higher in indica than japonica cultivars, and were positively correlated with starch contents at 10 DAD for both superior and inferior spikelets; although metabolic states, revealed from relations between intermediate metabolites and starch contents, differed among spikelet types. Consequently, it was considered that SUS and UGP at the step from sucrose cleavage to adenosine diphosphoglucose synthesis, and Pho1 at the starch biosynthesis step, were key metabolic factors involved in cultivar differences of sink strength (ability to synthesize starch).

Selection of transcripts related to low-temperature tolerance using RNA sequencing from F2 plants between japonica and indica rice (Oryza sativa L.) cultivars.

At low temperatures (18°C), seedlings of an indica rice (Oryza sativa L.) cultivar Kasalath showed symptoms of chlorosis, although the leaves of a japonica cultivar Arroz da Terra remained green. In this study, transcripts related to the chlorophyll content of rice seedlings grown at 18°C were investigated using RNA-sequencing (RNA-Seq) data for F2 crosses between cultivars Arroz da Terra and Kasalath, as well as their parental cultivars. Differential expression analysis revealed that gene ontology terms related to 'photosynthesis' were significantly enriched in lowly expressed genes at 18°C than at 25°C in Kasalath. However, the gene ontology terms related to 'response to stress' were significantly enriched in highly expressed genes at 18°C than at 25°C in Kasalath. When the F2 plants were grown at 18°C, their chlorophyll contents varied. Transcripts with expression levels related to chlorophyll content were statistically selected using RNA-Seq data from 21 F2 plants. In regression models, frequently selected genes included four photosynthetic and two stress-responsive genes. The expression values of four photosynthetic and two stress-responsive genes in high-frequency selected genes were significantly correlated with chlorophyll content not only in plants analysed using RNA-Seq but also in 95 F2 plants.

GIS-Based Analysis for UAV-Supported Field Experiments Reveals Soybean Traits Associated With Rotational Benefit.

Recent advances in unmanned aerial vehicle (UAV) remote sensing and image analysis provide large amounts of plant canopy data, but there is no method to integrate the large imagery datasets with the much smaller manually collected datasets. A simple geographic information system (GIS)-based analysis for a UAV-supported field study (GAUSS) analytical framework was developed to integrate these datasets. It has three steps: developing a model for predicting sample values from UAV imagery, field gridding and trait value prediction, and statistical testing of predicted values. A field cultivation experiment was conducted to examine the effectiveness of the GAUSS framework, using a soybean-wheat crop rotation as the model system Fourteen soybean cultivars and subsequently a single wheat cultivar were grown in the same field. The crop rotation benefits of the soybeans for wheat yield were examined using GAUSS. Combining manually sampled data (n = 143) and pixel-based UAV imagery indices produced a large amount of high-spatial-resolution predicted wheat yields (n = 8,756). Significant differences were detected among soybean cultivars in their effects on wheat yield, and soybean plant traits were associated with the increases. This is the first reported study that links traits of legume plants with rotational benefits to the subsequent crop. Although some limitations and challenges remain, the GAUSS approach can be applied to many types of field-based plant experimentation, and has potential for extensive use in future studies.

Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary.

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA).

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4-10.2 times higher V(max) value and 15.9-43.1 times higher K(m) value for NH(4)(+), compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with (15)NH(4)(+) showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH(4)(+) absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

Short-Term Magnesium Deficiency Triggers Nutrient Retranslocation in Arabidopsis thaliana.

Magnesium (Mg) is essential for many biological processes in plant cells, and its deficiency causes yield reduction in crop systems. Low Mg status reportedly affects photosynthesis, sucrose partitioning and biomass allocation. However, earlier physiological responses to Mg deficiency are scarcely described. Here, we report that Mg deficiency in Arabidopsis thaliana first modified the mineral profile in mature leaves within 1 or 2 days, then affected sucrose partitioning after 4 days, and net photosynthesis and biomass production after 6 days. The short-term Mg deficiency reduced the contents of phosphorus (P), potassium, manganese, zinc and molybdenum in mature but not in expanding (young) leaves. While P content decreased in mature leaves, P transport from roots to mature leaves was not affected, indicating that Mg deficiency triggered retranslocation of the mineral nutrients from mature leaves. A global transcriptome analysis revealed that Mg deficiency triggered the expression of genes involved in defence response in young leaves.

Starch storage in the stems of wheat plants: localization and temporal changes.

BACKGROUND AND AIMS: Carbohydrate temporarily accumulates in wheat stems during the early reproductive growth phase, predominantly as water soluble carbohydrate (WSC), and is subsequently remobilized during grain filling. Starch has also been reported as a minor storage carbohydrate component in wheat stems, but the details are lacking. METHODS: The accumulation and localization of starch in wheat stem and leaf sheath tissue over a developmental period from 6 d before anthesis to 35 d after anthesis was investigated. KEY RESULTS: The region of the peduncle enclosed by the flag-leaf sheath, and the penultimate internode were the main tissues identified as containing starch, in which the starch grains localized to the storage parenchyma cells. In contrast, the exposed peduncle lacked starch grains. Starch grains were also found in the flag-leaf and second-leaf sheath. Plants grown in low-nitrogen conditions exhibited increased storage of both starch and WSC compared with plants grown in high-nitrogen supply. CONCLUSIONS: The major accumulation and decrease of starch occurred temporally independently to that for WSC, suggesting a different functional role for starch in wheat stems. Starch reutilization concomitant with peduncle growth, and the early development of the reproductive structures, suggested a role in provision of energy and/or carbon scaffolds for these growth processes.

Characterisation of a rice vacuolar invertase isoform, OsINV2, for growth and yield-related traits.

OsINV2, a rice vacuolar invertase isoform, was assessed for its functional roles in plant growth and development with key focus on its agronomic traits such as grain weight, grain filling percentage, grain number and dry weights at various stages until harvest. Lack of differences between the wild-type and the mutants with respect to any of the aforementioned traits tested revealed a possibility of functional compensation of OsINV2 in the mutants conceivably by its isoform. This was confirmed by OsINV2 promoter::GUS studies, where its spatial and temporal expression in the panicle elongation stages showed that although OsINV2 expression was observed from the stage with young panicles ~1 cm in length to the flag leaf stage, significant differences with respect to panicle and spikelet phenotypes between the wild-type and the mutant were not present. However, complement lines displaying an overexpression phenotype of OsINV2 possessed a higher stem non-structural carbohydrate content under both monoculm and normal tillering conditions. A trade-off between the spikelet number and grain weight in the complement lines grown under monoculm conditions was also observed, pointing towards the necessity of OsINV2 regulation for grain yield-related traits.

Genetic Evidence for the Role of a Rice Vacuolar Invertase as a Molecular Sink Strength Determinant.

BACKGROUND: Rice is a major crop feeding the majority of the global population, and increasing its sink strength is one of the modes to alleviate the declining availability of food for the rapidly growing world population. We demonstrate a role for an important rice vacuolar invertase isoform, OsINV3, in sink strength determination. RESULTS: OsINV3 mutants showed shorter panicles with lighter and smaller grains, owing to a smaller cell size on the outer and inner surfaces of the palea and lemma as observed by scanning electron microscopy. Further, strong promoter::GUS expression was observed in the palea, lemma and the rachis branches in the young elongating panicles, which supported the role of OsINV3 in cell expansion and thus, in spikelet size and panicle length determination. Size of the spikelet was found to directly influence the grain weight, which was confirmed by the lack of differences in weights of hulled grain for differently segregated alleles in the heterozygous lines. Assessment of field grown mutants not only revealed a drastic reduction in the percentage of ripened grain, 1000-grain weight and final yield, but also significantly reduced partitioning of assimilates to the panicles, whereby the total dry weight remained unaffected. Determination of the non-structural carbohydrate contents revealed a lower hexose-to-sucrose ratio in the panicles of the mutants from panicle initiation to 10 days after heading, a stage that identifies as the critical pre-storage phase of grain filling, whereas the starch contents were not affected. In addition, strong promoter::GUS expression was observed in the dorsal end of ovary during the pre-storage phase until 6 days after flowering, highlighting a function for OsINV3 in monitoring the initial grain filling stage. CONCLUSIONS: OsINV3 was found to regulate spikelet size by playing a key role in cell expansion, driving the movement of assimilates for grain filling by modulating the hexose-to-sucrose ratio, contributing in grain weight determination and thus, the grain yield.

Selection of Transcripts Affecting Initial Growth Rate of Rice Backcrossed Inbred Lines Using RNA Sequencing Data.

Seedling growth is an important factor for direct seeding of rice. However, the genetic and transcriptomic factors involved in this process are largely unknown. In this study, transcripts affecting shoot weight were identified in rice (Oryza sativa L.) using RNA sequencing (RNA-Seq) data from 20 backcrossed inbred lines (BILs) and their parental cultivars. The selection frequency of the genes for the regression model was determined using repeated analysis of random subsets of the transcriptome. The qLTG3-1gene, controlling low-temperature germinability, and short grain 1 gene (SG1), known to decrease organ elongation, showed high frequency. The quantitative trait loci (QTLs) analysis performed for BILs revealed that qLTG3-1 was included in the QTLs for shoot weight but SG1 was not. No nucleotide polymorphisms were found in the coding region of SG1 in either of the parental cultivars. Quantitative real-time PCR showed that SG1 expression was negatively correlated with shoot weight for all 104 BILs analyzed in this study. Expression QTL (eQTLs) analysis showed an eQTL for SG1 expression located in the same region as the QTL for shoot weight. However, no eQTLs were detected on the same chromosome as SG1, suggesting that nucleotide polymorphisms around the gene do not affect its expression in analyzed growth stage. Overall, these results indicate that RNA-Seq is a useful tool for identifying transcripts that can be related to seedling growth rate.

Differences in Viscosity of Superior and Inferior Spikelets of Japonica Rice with Various Percentages of Apparent Amylose Content.

Viscosity, a crucial characteristic for rice palatability, is affected by endosperm characters. We compared correlations between differences in viscosity of japonica rice with various palatability and endosperm characters. Changes in apparent amylose and protein contents (AAC% and PC%, respectively) and amylopectin side-chain distribution and the relationship of these traits with palatability were investigated in superior and inferior spikelets of good cultivars with low amylose content from Hokkaido and common cultivars from northeastern Japan, using rapid visco analyzer characteristics and rice-grain microstructures. Significant differences occurred in PC%, AAC%, breakdown, setback, peak time, and pasting temperature of different cultivars and grain positions. Amylopectin components showed remarkable differences in grain surfaces, surface layers, and section structure between the grain varieties. Hokkaido cultivars showed better viscosity than northeastern cultivars, particularly initial stage grains. Correlation analysis indicated viscosity was mainly AAC%-dependent, whereas differences in endosperm characteristics between spikelet positions were mainly due to grain-filling temperature.

A reduction of sucrose phosphate synthase (SPS) activity affects sucrose/starch ratio in leaves but does not inhibit normal plant growth in rice.

Sucrose phosphate synthase (SPS) has been shown to mediate sucrose/starch ratio in plant leaves through studies of 'starch leaf' species that mainly accumulate starch in their leaves. However, the contribution of SPS to sucrose/starch ratio in rice leaves, which mainly accumulate sucrose (i.e., 'sugar leaf'), has not been confirmed due to inconsistencies in the results of previous studies. In this study, we analyzed mutant lines with reduced SPS activity, which were generated using Tos17 insertion, RNAi, and the CRISPR/Cas9 system. The knockdown and knockout mutants of OsSPS1 showed a 29-46% reduction in SPS activity in the leaves, but the carbohydrate content in the leaves and plant growth were not significantly different from those of wild-type plants. In a double knockout mutant of OsSPS1 and OsSPS11 (sps1/sps11), an 84% reduction in leaf SPS activity resulted in higher starch accumulation in the leaves than in the wild-type leaves. However, the sps1/sps11 plants grew normally, which is in contrast to the inhibited growth of SPS mutants of Arabidopsis thaliana, a typical starch leaf plant. These results suggest that SPS has a smaller effect on the sucrose/starch ratio in leaves and growth of rice than on starch leaf species.