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.

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).

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.

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.

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.

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.

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.

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.

A decrease in phytic acid content substantially affects the distribution of mineral elements within rice seeds.

Phytic acid (myo-inositol hexakisphosphate; InsP6) is the storage compound of phosphorus and many mineral elements in seeds. To determine the role of InsP6 in the accumulation and distribution of mineral elements in seeds, we performed fine mappings of mineral elements through synchrotron-based X-ray microfluorescence analysis using developing seeds from two independent low phytic acid (lpa) mutants of rice (Oryza sativa L.). The reduced InsP6 in lpa seeds did not affect the translocation of mineral elements from vegetative organs into seeds, because the total amounts of phosphorus and the other mineral elements in lpa seeds were identical to those in the wild type (WT). However, the reduced InsP6 caused large changes in mineral localization within lpa seeds. Phosphorus and potassium in the aleurone layer of lpa greatly decreased and diffused into the endosperm. Zinc and copper, which were broadly distributed from the aleurone layer to the inner endosperm in the WT, were localized in the narrower space around the aleurone layer in lpa mutants. We also confirmed that similar distribution changes occurred in transgenic rice with the lpa phenotype. Using these results, we discussed the role of InsP6 in the dynamic accumulation and distribution patterns of mineral elements during seed development.

Elevated atmospheric CO2 levels affect community structure of rice root-associated bacteria.

A number of studies have shown that elevated atmospheric CO2 ([CO2]) affects rice yields and grain quality. However, the responses of root-associated bacteria to [CO2] elevation have not been characterized in a large-scale field study. We conducted a free-air CO2 enrichment (FACE) experiment (ambient + 200 µmol.mol(-1)) using three rice cultivars (Akita 63, Takanari, and Koshihikari) and two experimental lines of Koshihikari [chromosome segment substitution and near-isogenic lines (NILs)] to determine the effects of [CO2] elevation on the community structure of rice root-associated bacteria. Microbial DNA was extracted from rice roots at the panicle formation stage and analyzed by pyrosequencing the bacterial 16S rRNA gene to characterize the members of the bacterial community. Principal coordinate analysis of a weighted UniFrac distance matrix revealed that the community structure was clearly affected by elevated [CO2]. The predominant community members at class level were Alpha-, Beta-, and Gamma-proteobacteria in the control (ambient) and FACE plots. The relative abundance of Methylocystaceae, the major methane-oxidizing bacteria in rice roots, tended to decrease with increasing [CO2] levels. Quantitative PCR revealed a decreased copy number of the methane monooxygenase (pmoA) gene and increased methyl coenzyme M reductase (mcrA) in elevated [CO2]. These results suggest elevated [CO2] suppresses methane oxidation and promotes methanogenesis in rice roots; this process affects the carbon cycle in rice paddy fields.

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.

Analysis of gene-disruption mutants of a sucrose phosphate synthase gene in rice, OsSPS1, shows the importance of sucrose synthesis in pollen germination.

The molecular function of an isoform of sucrose phosphate synthase (SPS) in rice, OsSPS1, was investigated using gene-disruption mutant lines generated by retrotransposon insertion. The progeny of the heterozygote of disrupted OsSPS1 (SPS1(+/-)) segregated into SPS1(+/+), SPS1(+/-), and SPS1(-/-) at a ratio of 1:1:0. This distorted segregation ratio, together with the expression of OsSPS1 in the developing pollen revealed by quantitative RT-PCR analysis and promoter-beta-glucuronidase (GUS) fusion assay, suggested that the disruption of OsSPS1 results in sterile pollen. This hypothesis was reinforced by reciprocal crosses of SPS1(+/-) plants with wild-type plants in which the disrupted OsSPS1 was not paternally transmitted to the progeny. While the pollen grains of SPS(+/-) plants normally accumulated starch during their development, pollen germination on the artificial media was reduced to half of that observed in the wild-type control. Overall, our data suggests that sucrose synthesis via OsSPS1 is essential in pollen germination in rice.

Phenotypic analyses of rice lse2 and lse3 mutants that exhibit hyperaccumulation of starch in the leaf blades.

BACKGROUND: To identify genes that potentially regulate the accumulation, mobilization, and transport of photoassimilates in rice (Oryza sativa L.) leaves, we recently screened a mutant collection of rice by iodine staining to visualize leaf starch contents. From this screening, we isolated a rice mutant that exhibits hyperaccumulation of starch in leaves and designated it as the Leaf Starch Excess 1 (LSE1) mutant. Here, we report two other rice LSE mutants, LSE2 and LSE3. RESULTS: Unlike lse1 plants, lse2 and lse3 plants displayed retarded growth; lse2 showed an extremely dwarf phenotype and rarely survived in paddy fields; lse3 showed inhibited growth with pale green leaf blades, low tiller numbers, reduced height, and low grain yield. In lse2 and lse3 plants, the mature source leaves contained larger amounts of starch and sucrose than those in wild-type and lse1 plants. Furthermore, microscopic observations of leaf transverse sections indicated that hyperaccumulation of starch in chloroplasts of mesophyll and bundle sheath cells occurred in lse2 and lse3 plants, while that in vascular cells was noticeable only in lse3 leaves. CONCLUSIONS: The distinct phenotypes of these three LSE mutants suggest that the LSE2 and LSE3 mutations occur because of disruption of novel genes that might be involved in the path of sucrose transport from mesophyll cells to phloem sieve elements in rice leaves, the mechanism for which has not yet been elucidated.

Suppression of starch synthesis in rice stems splays tiller angle due to gravitropic insensitivity but does not affect yield.

In rice (Oryza sativa L.), tiller angle - defined as the angle between the main culm and its side tillers - is one of the important factors involved in light use efficiency. To clarify the relationship between tiller angle, gravitropism and stem-starch accumulation, we investigated the shoot gravitropic response of a low stem-starch rice mutant which lacks a large subunit of ADP-glucose pyrophosphorylase (AGP), called OsAGPL1 and exhibits relatively spread tiller angle. The insensitive gravitropic response exhibited by the mutant led us to the conclusion that insensitivity of gravitropism caused by stem-starch reduction splayed the tiller angle. Furthermore, since another AGP gene called OsAGPL3 was expressed at considerable levels in graviresponding sites, we generated a double mutant lacking both OsAGPL1 and OsAGPL3. The double mutant exhibited still lower stem-starch content, less sensitive gravitropic response and greater tiller angle spread than the single mutants. This indicated that the expansion of the tiller angle caused by the reduction in starch level was intense according to the extent of the reduction. We found there were no significant differences between the double mutant and wild-type plants in terms of dry matter production. These results provided new insight into the importance of stem-starch accumulation and ideal plant architecture.

Disruption of a rice gene for α-glucan water dikinase, OsGWD1, leads to hyperaccumulation of starch in leaves but exhibits limited effects on growth.

To identify potential regulators of photoassimilate partitioning, we screened for rice mutant plants that accumulate high levels of starch in the leaf blades, and a mutant line leaf starch excess 1 (LSE1) was obtained and characterized. The starch content in the leaf blades of LSE1 was more than 10-fold higher than that in wild-type plants throughout the day, while the sucrose content was unaffected. The gene responsible for the LSE1 phenotype was identified by gene mapping to be a gene encoding α-glucan water dikinase, OsGWD1 (Os06g0498400), and a 3.4-kb deletion of the gene was found in the mutant plant. Despite the hyperaccumulation of starch in their leaf blades, LSE1 plants exhibited no significant change in vegetative growth, presenting a clear contrast to the reported mutants of Arabidopsis thaliana and Lotus japonicus in which disruption of the genes for α-glucan water dikinase leads to marked inhibition of vegetative growth. In reproductive growth, however, LSE1 exhibited fewer panicles per plant, lower percentage of ripened grains and smaller grains; consequently, the grain yield was lower in LSE1 plants than in wild-type plants by 20~40%. Collectively, although α-glucan water dikinase was suggested to have universal importance in leaf starch degradation in higher plants, the physiological priority of leaf starch in photoassimilate allocation may vary among plant species.

The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose.

Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs) suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter-luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real-time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light-dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both P OsSPS1 ::LUC and P OsSPS11 ::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the two OsSPS genes.