Tatsuro Hirose

Comprehensive Expression Profiling of Rice Grain Filling-Related Genes under High Temperature Using DNA Microarray

To elucidate the effect of high temperature on grain-filling metabolism, developing rice (Oryza sativa) ‘Nipponbare’ caryopses were exposed to high temperature (33°C/28°C) or control temperature (25°C/20°C) during the milky stage. Comprehensive gene screening by a 22-K DNA microarray and differential hybridization, followed by expression analysis by semiquantitative reverse transcription-PCR, revealed that several starch synthesis-related genes, such as granule-bound starch synthase I (GBSSI) and branching enzymes, especially BEIIb, and a cytosolic pyruvate orthophosphate dikinase gene were down-regulated by high temperature, whereas those for starch-consuming α-amylases and heat shock proteins were up-regulated. Biochemical analyses of starch showed that the high temperature-ripened grains contained decreased levels of amylose and long chain-enriched amylopectin, which might be attributed to the repressed expression of GBSSI and BEIIb, respectively. SDS-PAGE and immunoblot analysis of storage proteins revealed decreased accumulation of 13-kD prolamin, which is consistent with the diminished expression of prolamin genes under elevated temperature. Ripening under high temperature resulted in the occurrence of grains with various degrees of chalky appearance and decreased weight. Among them, severely chalky grains contained amylopectin enriched particularly with long chains compared to slightly chalky grains, suggesting that such alterations of amylopectin structure might be involved in grain chalkiness. However, among high temperature-tolerant and sensitive cultivars, alterations of neither amylopectin chain-length distribution nor amylose content were correlated to the degree of grain chalkiness, but rather seemed to be correlated to grain weight decrease, implying different underlying mechanisms for the varietal difference in grain chalkiness. The possible metabolic pathways affected by high temperature and their relevance to grain chalkiness are discussed.

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 worlds 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.

Cellular localisation and function of a sucrose transporter OsSUT1 in developing rice grains

We previously reported the cloning and tissue-specific expression of a gene encoding a sucrose/proton symporter in rice (Oryza sativa L.), OsSUT1 (Hirose et al. 1997, Plant Cell Physiology38, 1389–1396). This gene is expressed at high levels in the filling grain, leaf sheath and stem. Expression in these tissues occurred only after heading i.e. during the development of the reproductive structure as a major sink. In this paper, we report localisation of the transcript and protein to specific cells in the filling rice grain by in situ hybridisation with a probe from theOsSUT1 cDNA, and immunolocalisation of OsSUT1 and proton-pumping ATPase (H+-ATPase). An OsSUT1 cDNA probe recognises a transcript of approximately 2.4 kb, and the SUT1 antibody recognises a protein of approximately 55 kDa in total membrane protein extracts from the filling grain, leaf sheath and stem. In the developing grain, OsSUT1 is expressed at low levels before heading, with expression reaching a peak approximately ten days after emergence of the panicle from the sheath. Transcript is then present throughout seed development, with expression falling substantially after about 25 days post-heading. Both transcript and protein are localised to the aleurone cells of the developing grain, and are also detected in the maternal tissue, particularly the nucellus, vascular parenchyma tissue and the nucellar projection. Tissue slices from filling rice grain showed high rates of sucrose uptake that were inhibited by pCMBS. The role of OsSUT1 in sucrose transport to the filling grain endosperm is discussed.

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.

Tissue specificity and diurnal change in gene expression of the sucrose phosphate synthase gene family in rice

The rice genome contains 5 isogenes for sucrose phosphate synthase (SPS), the key enzyme in sucrose synthesis; however, little is known about their transcriptional regulation. In order to determine the expression patterns of the SPS gene family in rice plants, we conducted an expression analysis in various tissues and developmental stages by real-time quantitative RT-PCR. At the transcript level, the rice SPS genes, particularly SPS1, were preferentially expressed in source tissues, whereas SPS2, SPS6, and SPS8 were expressed equally in source and sink tissues. We also investigated diurnal changes in SPS gene expression, SPS activity, and soluble sugar content in leaf blades. Interestingly, the expression of all the SPS genes, particularly that of SPS1 and SPS11, tended to be higher at night when the activation state of the SPS proteins was low, and the mRNA levels of SPS1 and SPS6 were negatively correlated with sucrose content. Furthermore, the temporal patterns of SPS gene expression and sugar content under continuous light conditions suggested the involvement of endogenous rhythm and/or sucrose sensing in the transcriptional regulation of SPS genes. Our data revealed differential expression patterns in the rice SPS gene family and part of the complex mechanisms of their transcriptional control.

Starch reduction in rice stems due to a lack of OsAGPL1 or OsAPL3 decreases grain yield under low irradiance during ripening and modifies plant architecture

Starch accumulated in rice (Oryza sativa L.) stems before heading as nonstructural carbohydrates (NSCs) is reported to be important for improving and stabilising grain yield. To evaluate the importance of stem starch, we investigated a retrotransposon (Tos17) insertion rice mutant lacking a gene encoding a large subunit of ADP-glucose pyrophosphorylase (AGP) called OsAGPL1 or OsAPL3. The AGP activity and starch contents of the mutant were drastically reduced in the stem (i.e. leaf sheath and culm) but not in the leaf blade or endosperm. This starch reduction in the leaf sheaths of the mutant was complemented by the introduction of wild-type OsAGPL1. These results strongly suggest that OsAGPL1 plays a principal role in stem starch accumulation. Field experimentations spanning 2 years revealed that the mutant plants were shorter than the wild-type plants. Moreover, the tiller number and angle were larger in the mutant plants than the wild-type plants, but the dry weight at heading stage was not different. The grain yield was slightly lower in control plots without shading treatment. However, this difference increased substantially with shading. Therefore, stem starch is indispensable for normal ripening under low irradiance conditions and probably contributes to the maintenance of appropriate plant architecture.

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.

Sucrose Transport in Higher Plants: From Source to Sink

Sucrose transport in plants has been as area of intense interest for many years, particularly following the cloning of the first sucrose/proton symporter, or sucrose transporter, from spinach more than 15 years ago. Much debate and research has focused on phloem loading, particularly the issue of apoplasmic versus symplasmic pathways of loading and in the apoplasmic loaders, on the location of the sucrose transporter protein and mRNA. This chapter focuses on pointing out the remaining unanswered questions in phloem loading and sucrose transport in general rather than extensively reviewing the literature. We discuss in more detail the long-distance transport pathway from source to sink and post-phloem unloading in sink tissue such as dicot seed, cereal grain, sink leaves, roots and tubers.

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.

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 POsSPS1::LUC and POsSPS11::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.