MutMapPlus identified novel mutant alleles of a rice starch branching enzyme IIb gene for fine-tuning of cooked rice texture.

Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine-tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met-723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica-type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α-amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.

Suppression of α-amylase genes improves quality of rice grain ripened under high temperature.

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism-related genes. Here we report the involvement of a starch-hydrolyzing enzyme, α-amylase, in high temperature-triggered grain chalkiness. In developing seeds, high temperature induced the expression of α-amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α-amylase activity, while it decreased an α-amylase-repressing plant hormone, ABA, suggesting starch to be degraded by α-amylase in developing grains under elevated temperature. Furthermore, RNAi-mediated suppression of α-amylase genes in ripening seeds resulted in fewer chalky grains under high-temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α-amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.

Discovery of natural Miscanthus (Poaceae) triploid plants in sympatric populations of Miscanthus sacchariflorus and Miscanthus sinensis in southern Japan.

PREMISE OF THE STUDY: Looming petroleum shortages and projected negative impacts of human-induced climate change may be partly alleviated by the development and use of bioenergy feedstock crops. Miscanthus ×giganteus, a highly productive sterile triploid hybrid grass that was discovered in Japan several decades ago, has considerable potential as an alternative source of energy. Given the risks, however, involved in the reliance upon production of one clone of this hybrid, which is a natural cross between Miscanthus sacchariflorus and Miscanthus sinensis, for lignocellulosic bioenergy production, natural occurrences of triploidy were investigated in sympatric populations of tetraploid M. sacchariflorus and diploid M. sinensis in Japan. METHODS: Seeds were counted and DNA content was estimated by flow cytometry for plants of M. sacchariflorus and M. sinensis in several sympatric populations throughout Japan. Chromosomes were also counted for select plants. KEY RESULTS: Based on seed-set data, M. sacchariflorus has significantly lower seed set than M. sinensis in Japan. Putative triploid seeds were found on M. sacchariflorus plants in southern Japan. CONCLUSIONS: This is the first report of the natural occurrence of Miscanthus triploid plants in several decades. If found to be sterile and similar in productivity to the commonly cultivated clone of M. ×giganteus, these triploid plants might serve as additional sources of genetic variation for bioenergy production. Seed set data also indicates that other triploid plants might be found in more northern regions of Japan.

Ploidy distribution and DNA content variations of Lonicera caerulea (caprifoliaceae) in Japan.

Ploidy level and geographical distribution were investigated in Japanese Lonicera caerulea L. Flow cytometric analysis revealed the presence of DNA diploid and DNA tetraploid plants in Japan. Chromosome observation confirmed that diploid and tetraploid plants showed 2n = 2x = 8 and 2n =4x = 36, respectively. The DNA diploid populations were found only in lowland mires, Betsukai, Bekanbeushi, Kushiro and Kiritappu located in eastern Hokkaido. On the other hand, DNA tetraploid populations were distributed in a wide area of Hokkaido, and mainland of Japan. The habitats of DNA tetraploid plants were lowland to alpine region. The DNA content measurement with flow cytometry revealed significant differences in the relative DNA contents among DNA tetraploid populations. The relative DNA content within DNA tetraploid populations varied 1.157-fold at maximum, and might correlate with altitude indicating that DNA contents were smaller as altitude increases. The wide area of distribution in various environments of DNA tetraploid plants suggested the adaptability of the tetraploid plants. Although diploid and tetraploid populations were found, no triploid was detected, indicating crossing difficulty between diploid and tetraploid as confirmed by crossing experiment.

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains.

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.