Dataset Information

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

ABSTRACT: 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 ?-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional ?-amylase genes, identification of the ?-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify ?-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight ?-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 ?-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 ?-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 ?-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 ?-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.

SUBMITTER: Nakata M

PROVIDER: S-EPMC5723670 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Nakata Masaru M Fukamatsu Yosuke Y Miyashita Tomomi T Hakata Makoto M Kimura Rieko R Nakata Yuriko Y Kuroda Masaharu M Yamaguchi Takeshi T Yamakawa Hiromoto H

Frontiers in plant science 20171206

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 effici ...[more]

PMID: 29270189

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Project description:BackgroundGlobal 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).ResultsAppearance 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.ConclusionTogether 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.

Project description:High temperature impairs starch biosynthesis in developing rice grains and thereby increases chalkiness, affecting the grain quality. Genome encoded microRNAs (miRNAs) fine-tune target transcript abundances in a spatio-temporal specific manner, and this mode of gene regulation is critical for a myriad of developmental processes as well as stress responses. However, the role of miRNAs in maintaining rice grain quality/chalkiness during high daytime temperature (HDT) stress is relatively unknown. To uncover the role of miRNAs in this process, we used five contrasting rice genotypes (low chalky lines Cyp, Ben, and KB and high chalky lines LaGrue and NB) and compared the miRNA profiles in the R6 stage caryopsis samples from plants subjected to prolonged HDT (from the onset of fertilization through R6 stage of caryopsis development). Our small RNA analysis has identified approximately 744 miRNAs that can be grouped into 291 families. Of these, 186 miRNAs belonging to 103 families are differentially regulated under HDT. Only two miRNAs, Osa-miR444f and Osa-miR1866-5p, were upregulated in all genotypes, implying that the regulations greatly varied between the genotypes. Furthermore, not even a single miRNA was commonly up/down regulated specifically in the three tolerant genotypes. However, three miRNAs (Osa-miR1866-3p, Osa-miR5150-3p and canH-miR9774a,b-3p) were commonly upregulated and onemiRNA (Osa-miR393b-5p) was commonly downregulated specifically in the sensitive genotypes (LaGrue and NB). These observations suggest that few similarities exist within the low chalky or high chalky genotypes, possibly due to high genetic variation. Among the five genotypes used, Cypress and LaGrue are genetically closely related, but exhibit contrasting chalkiness under HDT, and thus, a comparison between them is most relevant. This comparison revealed a general tendency for Cypress to display miRNA regulations that could decrease chalkiness under HDT compared with LaGrue. This study suggests that miRNAs could play an important role in maintaining grain quality in HDT-stressed rice.

Project description:The gelatinization temperature of endosperm starch in most japonica rice cultivars is significantly lower than that in most indica rice cultivars. This is because three single nucleotide polymorphisms in the Starch synthase (SS) IIa gene in japonica rice cultivars (SSIIaJ ) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ? 12 compared to indica rice cultivars (SSIIaI ). SSIIa forms a trimeric complex with SSI and starch branching enzyme (BE) IIb in maize and japonica rice, which is likely important for the biosynthesis of short and intermediate amylopectin chains (DP ? 24) within the amylopectin cluster. It was unknown whether the complete absence of SSIIa further increases amylopectin short chains and reduces gelatinization temperature and/or forms altered protein complexes due to the lack of a suitable mutant. Here, we identify the SSIIa-deficient mutant rice line EM204 (ss2a) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to N-methyl-N-nitrosourea mutagenesis. The SSIIa gene in EM204 was mutated at the boundary between intron 5 and exon 6, which generated a guanine to adenine mutation and resulted in deletion of exon 6 in the mRNA transcript. SSIIa activity and SSIIa protein in developing endosperm of EM204 were not detected by native-PAGE/SS activity staining and native-PAGE/immunoblotting, respectively. SSIIa protein was completely absent in mature seeds. Gel filtration chromatography of soluble protein extracted from developing seeds showed that the SSI elution pattern in EM204 was altered and more SSI was eluted around 300 kDa, which corresponds with the molecular weight of trimeric complexes in wild type. The apparent amylose content of EM204 rice grains was higher than that in its parent Kinmaze. EM204 also had higher content of amylopectin short chains (DP ? 12) than Kinmaze, which reduced the gelatinization temperature of EM204 starch by 5.6°C compared to Kinmaze. These results indicate that EM204 starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.

Project description:Grain chalkiness is a key quality trait of the rice grain, whereas its underlying mechanism is still not thoroughly understood because of the complex genetic and environmental interactions. We identified a notched-belly (NB) mutant that has a notched-line on the belly of grains. The line dissects the endosperm into two distinct parts, the upper translucent part, and the bottom chalky part in the vicinity of the embryo. Using this mutant, our previous studies clued the negative influence of embryo on the biochemical makeup of the endosperm, suggesting the need for the in-depth study of the embryo effect on the metabolome of developing endosperm. This study continued to use the NB mutant to evolve a novel comparison method to clarify the role of embryo in the formation of a chalky endosperm. Grain samples of the wild-type (WT) and NB were harvested at 10, 20, and 30 days after fertilization (DAF), and then divided into subsamples of the embryo, the upper endosperm, and the bottom endosperm. Using non-targeted metabolomics and whole-genome RNA sequencing (RNA-seq), a nearly complete catalog of expressed metabolites and genes was generated. Results showed that the embryo impaired the storage of sucrose, amino acid, starch, and storage proteins in the bottom endosperm of NB by enhancing the expression of sugar, amino acids, and peptide transporters, and declining the expression of starch, prolamin, and glutelin synthesis-related genes. Importantly, the competitive advantage of the developing embryo in extracting the nutrients from the endosperm, transformed the bottom endosperm into an "exhaustive source" by diverting the carbon (C) and nitrogen (N) metabolism from synthetic storage to secondary pathways, resulting in impaired filling of the bottom endosperm and subsequently the formation of chalky tissue. In summary, this study reveals that embryo-induced metabolic shift in the endosperm is associated with the occurrence of grain chalkiness, which is of relevance to the development of high-quality rice by balancing the embryo-endosperm interaction.

Dataset Information

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

Publications

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

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