A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6–22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5–8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9–16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx^a) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx^a.

Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the hst1 gene, transferring salinity tolerance from “Kaijin” into high-yielding “Yukinko-mai” (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC₃F₃ population, named “YNU31-2-4”, in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC₃F₂ genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC₃F₂ variants along with field assessment data indicated that “YNU31-2-4” plants carrying the hst1 gene had similar agronomic traits to the WT under normal growth condition. “YNU31-2-4” seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that “YNU31-2-4” seedlings avoided Na⁺ accumulation in shoots under salt stress. The “YNU31-2-4” plants showed an improved phenotype with significantly higher net CO₂ assimilation and lower yield decline than WT under salt stress at the reproductive stage. “YNU31-2-4” is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.

The coat protein (CP) gene of rice stripe virus was introduced into two japonica varieties of rice by electroporation of protoplasts. The resultant transgenic plants expressed the CP at high levels (up to 0.5% of total soluble protein) and exhibited a significant level of resistance to virus infection. Plants derived from selfed progeny of the primary transformants also expressed the CP and showed viral resistance, indicating stable transmission of the CP gene and the trait of resistance to the next generation. Moreover, the virally encoded strip disease-specific protein was not detected in transgenic plants expressing CP 8 weeks after inoculation, indicating protection before viral multiplication. These studies demonstrated that CP-mediated resistance to virus infection can be extended to cereals and to the viruses transmitted by an insect vector (planthopper).

To determine the role of α-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed expression or overexpression of α-amylase I-1. In the lines with suppressed expression of α-amylase I-1 at both the mRNA and protein levels, seed germination and seedling growth were markedly delayed in comparison with those in the wild-type plants. However, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in the plants overexpressing α-amylase I-1. In immunocytochemical analysis with specific anti-α-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts and extracellular space in young leaf cells. We further examined the expression and targeting of α-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of the expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of α-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, we concluded that α-amylase I-1 targets the chloroplasts through the endoplasmic reticulum–Golgi system and plays a significant role in the starch degradation in rice leaves.

The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. Two functional Wx alleles of rice exist: Wx^a, which produces a large amount of amylose, and Wx^b, which produces a smaller amount of amylose because of the mutation at the 5′ splice site of intron 1. Wx^b is largely distributed in Japonica cultivars, and high amylose cultivars do not exist in Japonica cultivars. We introduced the cloned Wx^a cDNA into null-mutant Japonica rice (wx). The amylose contents of these transgenic plants were 6–11% higher than that of the original cultivar, Labelle, which carries the Wx^a allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle. We also observed a gene-dosage effect of the Wx^a transgene on Wx protein expression, but a smaller dosage effect was observed in amylose production with over 40% of amylose content in transgenic rice. Moreover, one transgenic line carrying eleven copies of the transgene showed low levels of Wx expression and amylose in the endosperm. This suggested that the integration of excessive copies of the transgene might lead to gene silencing.

Global climate models predict an increase in global mean temperature and a higher frequency of intense heat spikes during this century. Cereals such as rice (Oryza sativa L.) are more susceptible to heat stress, mainly during the gametogenesis and flowering stages. During periods of high temperatures, grain filling often causes serious damage to the grain quality of rice and, therefore, yield losses. While the genes encoding enzymes involved in carbohydrate metabolism of chalky grains have been established, a significant knowledge gap exists in the proteomic and glycomic responses to warm temperatures in situ. Here, we studied the translucent and opaque characters of high temperature stressed chalky grains of 2009 and 2010 (ripening temperatures: 24.4 and 28.0 °C, respectively). Appearance of chalky grains of both years showed some resemblance, and the high-temperature stress of 2010 remarkably extended the chalking of grain. Scanning electron microscopic observation showed that round-shaped starch granules with numerous small pits were loosely packed in the opaque part of the chalky grains. Proteomic analyzes of rice chalky grains revealed deregulations in the expression of multiple proteins implicated in diverse metabolic and physiological functions, such as protein synthesis, redox homeostasis, lipid metabolism, and starch biosynthesis and degradation. The glycomic profiling has shown slight differences in chain-length distributions of starches in the grains of 2009-to-2010. However, no significant changes were observed in the chain-length distributions between the translucent and opaque parts of perfect and chalky grains in both years. The glucose and soluble starch contents in opaque parts were increased by the high-temperature stress of 2010, though those in perfect grains were not different regardless of the environmental changes of 2009-to-2010. Together with previous findings on the increased expression of α-amylases in the endosperm, these results suggested that unusual starch degradation rather than starch synthesis is involved in occurring of chalky grains of rice under the high-temperature stress during grain filling period.