Yoichi Hashida

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.

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.

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.

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.

A simple method for squeezing juice from rice stems and its use in the high-throughput analysis of sugar content in rice stems

Abstract Sugar content in rice (Oryza sativa L.) stem is an agronomically important trait for rice used in straw silage or whole-crop silage. However, the mechanisms underlying sugar accumulation in rice stems remain unclear, mainly due to the time-consuming method for measuring sugar content. Here, we established a simple method for squeezing stem juice from rice plants, similar to that used during breeding selection in sugarcane or sorghum. The Brix value of the stem juice, which can easily be measured using a portable refractometer, significantly correlated with the soluble sugar contents in the stem juice and tended to correlate with those in stem tissues. This indicates that the Brix value of the stem juice can be used for estimating the sugar content in rice stems. This simple estimation method will be a useful tool for high-throughput analysis of sugar content in rice stems during mutant screening, QTL analysis, and breeding selection.

Mutation of the SP1 gene is responsible for the small-panicle trait in the rice cultivar Tachisuzuka, but not necessarily for high sugar content in the stem

Abstract Tachisuzuka, a rice cultivar grown for whole-crop silage, is characterized by the small-panicle trait and high-stem sugar content. To investigate the interrelationship between the two features, we attempted to identify the gene responsible for the small-panicle trait in Tachisuzuka, and also to examine the function of the gene using a knockout mutant line. A functionally disruptive deletion of the nucleotide sequence was found in the gene SP1 (Short-Panicle 1; Os11g0235200) in Tachisuzuka, which has been reported as a candidate gene for the small-panicle trait. A gene knockout mutant of SP1 obtained from the cultivar Nipponbare showed a small-panicle phenotype similar to that observed in Tachisuzuka. However, soluble sugar content in the stem did not increase in the knockout line, whereas starch content increased significantly. Overall, disruption of SP1 is responsible for the small-panicle phenotype of Tachisuzuka, but it is only partially associated with the high-stem sugar content.

Suppression of starch accumulation in ‘sugar leaves’ of rice affects plant productivity under field conditions

Abstract While many plants accumulate the majority of their photoassmilates as starch during the daytime, some plants accumulate sucrose. Although the existence of these high-sucrose leaves, called ‘sugar leaves’, has long been known, the physiological characteristics of sugar leaves compared to ‘starch leaves’ remain unclear. In this study, the physiological roles of starch accumulation in rice, which has typical sugar leaves, were investigated using a mutant with suppressed leaf-starch biosynthesis. When grown under controlled conditions with light intensity of 400 μmol m−2 s−1, the initial growth of the mutant was similar to that of the wild-type plant, even with a 6-h-light/18-h-dark photoperiod in which carbon resources for growth are required during the night. This finding indicates that rice does not rely on leaf starch as a carbon resource during the night. By contrast, under field conditions, the grain yields of the mutant were significantly lower than those of the wild type only when the plants were exposed to full sunlight during the ripening period. These results may indicate that starch accumulation in sugar leaves plays an important role in maintaining a high source capacity under sufficient light conditions rather than as a carbon resource for the plant’s growth at night.

Characterization of sugar metabolism in the stem of Tachisuzuka, a whole-crop silage rice cultivar with high sugar content in the stem

Abstract Tachisuzuka, a rice cultivar for whole-crop silage, is characterized by a small panicle and high sugar content in the stem. Our previous study suggests that the high sugar content in the stem of Tachisuzuka is due to a factor other than the small panicle. To characterize sugar metabolism in the stem of Tachisuzuka, here we compared carbohydrate content, enzyme activity, and the expression of genes involved in sugar metabolism in the stem between Tachisuzuka and its parental variety, Kusanohoshi. Thinning the panicles of Kusanohoshi increased the starch content in the leaf sheath and internode but did not increase the sucrose content in the leaf sheath to the same level as that of Tachisuzuka. This suggests that Tachisuzuka has high potential to accumulate sucrose in its leaf sheath. Comparison of enzyme activity showed that the hexokinase activities in the leaf sheath tended to be higher in Tachisuzuka than Kusanohoshi or panicle-thinned Kusanohoshi, suggesting that glucokinase or fructokinase affects sugar accumulation in the stem of Tachisuzuka. Comparative transcriptome analysis revealed the differences in expression levels of carbohydrate-related genes between Tachisuzuka and Kusanohoshi. In particular, the expression levels of ISA2, which encodes starch-debranching enzyme, and TMT2, which encodes tonoplast monosaccharide transporter – both of which maybe involved in sugar accumulation in grass stems – were higher in Tachisuzuka than Kusanohoshi. Thus, these enzymes and transporters may contribute to the high sugar content in the stem of Tachisuzuka.