Dataset Information

Functional Genomic Validation of the Roles of Soluble Starch Synthase IIa in Japonica Rice Endosperm.

ABSTRACT: The enzyme starch synthase IIa (SSIIa) in cereals has catalytic and regulatory roles during the synthesis of amylopectin that influences the functional properties of the grain. Rice endosperm SSIIa is more active in indica accessions compared to japonica lines due to functional SNP variations in the coding region of the structural gene. In this study, downregulating the expression of japonica-type SSIIa in Nipponbare endosperm resulted in either shrunken or opaque grains with an elevated proportion of A-type starch granules. Shrunken seeds had severely reduced starch content and could not be maintained in succeeding generations. In comparison, the opaque grain morphology was the result of weaker down-regulation of SSIIa which led to an elevated proportion of short-chain amylopectin (DP 6-12) and a concomitant reduction in the proportion of medium-chain amylopectin (DP 13-36). The peak gelatinization temperature of starch and the estimated glycemic score of cooked grain as measured by the starch hydrolysis index were significantly reduced. These results highlight the important role of medium-chain amylopectin in influencing the functional properties of rice grains, including its digestibility. The structural, regulatory and nutritional implications of down-regulated japonica-type SSIIa in rice endosperm are discussed.

SUBMITTER: Jr VMB

PROVIDER: S-EPMC7142255 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Functional Genomic Validation of the Roles of Soluble Starch Synthase IIa in Japonica Rice Endosperm.

Jr Vito M Butardo VMB Luo Jixun J Li Zhongyi Z Gidley Michael J MJ Bird Anthony R AR Tetlow Ian J IJ Fitzgerald Melissa M Jobling Stephen A SA Rahman Sadequr S

Frontiers in genetics 20200402

The enzyme starch synthase IIa (SSIIa) in cereals has catalytic and regulatory roles during the synthesis of amylopectin that influences the functional properties of the grain. Rice endosperm SSIIa is more active in indica accessions compared to japonica lines due to functional SNP variations in the coding region of the structural gene. In this study, downregulating the expression of japonica-type SSIIa in Nipponbare endosperm resulted in either shrunken or opaque grains wit ...[more]

PMID: 32300357

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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:Starch, which is composed of amylose and amylopectin, is the key determinant of rice quality. Amylose is regulated by the Waxy (Wx) gene, whereas amylopectin is coordinated by various enzymes including eight soluble starch synthases (SSSs), of which SSSI accounts for ∼70% of the total SSS activity in cereal endosperm. Although great progress has been made in understanding SSSI gene expression and function, allelic variation and its effects on gene expression, rice physicochemical properties and qualities, and interactions with the Wx gene remain unclear. Herein, SSSI nucleotide polymorphisms were analyzed in 165 rice varieties using five distinct molecular markers, three of which reside in an SSSI promoter and might account for a higher expression of the SSSIi allele in indica ssp. than of the SSSIj allele in japonica ssp. The results of SSSI promoter-Beta-Glucuronidase (β-GUS) analysis were consistent with the expression results. Moreover, analysis of near isogenic lines (NILs) in the Nipponbare (Nip) background showed that Nip (SSSIi ) and Nip (SSSIj ) differed in their thermal properties, gel consistency (GC), and granule crystal structure. Knockdown of SSSI expression using the SSSI-RNA interference (RNAi) construct in both japonica and indica backgrounds caused consistent changes in most tested physicochemical characteristics except GC. Moreover, taste value analysis (TVA) showed that introduction of the SSSI allele in indica or knockdown of SSSI expression in japonica cultivars significantly reduced the comprehensive taste value, which was consistent with the superior taste of japonica against indica. Furthermore, to test the potential interaction between SSSI and different Wx alleles, three NILs within the Wx locus were generated in the indica cv. Longtefu (LTF) background, which were designated as LTF (Wxa ), LTF (Wxb ), and LTF (wx). The SSSI-RNAi construct was also introduced into these three NILs, and physiochemical analysis confirmed that the knockdown of SSSI significantly increased the rice apparent amylose content (AAC) only in the Wxa and Wxb background and caused different changes in GC in the NILs. Therefore, the effect of SSSI variation on rice quality also depends on its crosstalk with other factors, especially the Wx gene. These findings provide fundamental knowledge for future breeding of rice with premium eating and cooking qualities.

Dataset Information

Functional Genomic Validation of the Roles of Soluble Starch Synthase IIa in Japonica Rice Endosperm.

Publications

Functional Genomic Validation of the Roles of <i>Soluble Starch Synthase IIa</i> in <i>Japonica</i> Rice Endosperm.

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