23 research outputs found

    Underlying Mechanisms of Zymographic Diversity in Starch Synthase I and Pullulanase in Rice-Developing Endosperm

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    Amylopectin is synthesized by the coordinated actions of many (iso)­enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SSs), branching enzymes (BEs), and debranching enzymes (DBEs). Here, two polymorphic forms of starch synthase I (SSI) and pullulanase (PUL) in rice-developing seeds, designated as SSI-1/SSI-2 and PUL-1/PUL-2, were discovered for the first time by zymographic analysis. The SSI and PUL polymorphisms were strongly associated with the <i>SSI</i> microsatellite marker (<i>p</i> = 3.6 × 10<sup>–37</sup>) and <i>PUL</i> insertion/deletion (InDel) markers (<i>p</i> < 3.6 × 10<sup>–51</sup>). Western blotting and mass spectrometric analysis confirmed that the polymorphic bands were truly the SSI and PUL enzymes. Only one non-synonymous variation in <i>SSI</i> DNA sequence (the SNP A/G) causing the change of the amino acid K438 to E438 was observed, which coincided well with the polymorphic forms of SSI. Nine non-synonymous variations were found between PUL-1 and PUL-2. Two non-synonymous variations of PUL (F316L and D770E) were identified by mass spectrometric analysis, but all of the variations did not change the structure of PUL. The co-immunoprecipitation results revealed the differences in protein–protein interaction patterns, i.e., strong or weaker signals of SSI–BEI and SSI–BEIIb, between the two forms of SSI. The results will enhance our understanding of SSI and PUL properties and provide helpful information to understand their functions in starch biosynthesis in rice endosperm

    Comparative Phosphoproteomic Analysis of the Developing Seeds in Two Indica Rice (<i>Oryza sativa</i> L.) Cultivars with Different Starch Quality

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    Protein phosphorylation plays important roles in regulation of various molecular events such as plant growth and seed development. However, its involvement in starch biosynthesis is less understood. Here, a comparative phosphoproteomic analysis of two indica rice cultivars during grain development was performed. A total of 2079 and 2434 phosphopeptides from 1273 and 1442 phosphoproteins were identified, covering 2441 and 2808 phosphosites in indica rice 9311 and Guangluai4 (GLA4), respectively. Comparative analysis identified 303 differentially phosphorylated peptides, and 120 and 258 specifically phosphorylated peptides in 9311 and GLA4, respectively. Phosphopeptides in starch biosynthesis related enzymes such as AGPase, SSIIa, SSIIIa, BEI, BEIIb, PUL, and Pho1were identified. GLA4 and 9311 had different amylose content, pasting viscosities, and gelatinization temperature, suggesting subtle difference in starch biosynthesis and regulation between GLA4 and 9311. Our study will give added impetus to further understanding the regulatory mechanism of starch biosynthesis at the phosphorylation level

    Genome-wide Association Mapping of Polyphenol Contents and Antioxidant Capacity in Whole-Grain Rice

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    Whole grains contain various bioactive phytochemicals including phenolic acids, and consumption of whole grains may provide desirable health benefits and reduce the risks of chronic diseases due to their antioxidant activities. In this study, we qualified and quantified 9 bound phenolic compounds in 32 red and 88 white pericarp accessions of rice. A genome-wide association study (GWAS) was conducted for free (FP) and bound (BP) phenolic compounds and their antioxidant capacities with high-quality single-nucleotide polymorphisms (SNPs) in two colored grain panels and the whole panel. <i>Rc</i> was detected for all FP and antioxidant capacities in the whole panel. Three loci (chr.1:30970095, chr.6:24392269, and chr.9:6670223) for more than five phenolic-related traits, two loci (chr.4:34120529 and chr.11:28947480) for more than two FP-related traits, and one locus (chr.11:23220681) for ferulic acid detected in two panels were potentially new genes that are valuable for further gene cloning. Overall, this study increases our understanding on the genetics of phenolic acid biosynthesis in the phenylpropanoid pathway

    Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization

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    <div><p>To uncover the genetics of rice grain weight, we constructed an RIL population derived from a cross between a large grain accession M201 and a small size variety JY293. Specific Locus Amplified Fragment Sequencing (SLAF-Seq) technology was used to genotype two bulked DNA pools made from individual DNA of the heaviest 30 lines and the lightest 30 lines according to the 1000 grain weight (TGW). Bulked segregant analysis (BSA) was used to identify SLAFs strongly associated with TGW. Two marker-intensive regions at 24,600,000–24,850,000 bp and 25,000,000–25,350,000 bp on chromosome 3 were identified tightly related to the TGW. Then a linkage map of chromosome 3 was constructed with SSR markers and some SLAF derived single nucleotide polymorphisms (SNPs). Quantitative trait locus (QTL) mapping for TGW, grain length, grain width, and grain thickness revealed one major QTL in the second hot-region and two other minor QTLs for grain weight. These three QTLs displayed hierarchical effects on grain length and grain weight in order of <i>qTGW3</i>.<i>2</i> (<i>qGL3</i>) <i>qTGW3</i>.<i>1</i> (<i>GS3</i>) <i>qTGW3</i>.<i>3</i>. Multiple comparisons of means among the eight combinations of 3 QTLs revealed that the lines with two of three QTLs deriving from M201 displayed a large grain weight phenotype (TGW 40.2g, average data of three years) and lines with both <i>qTGW3</i>.<i>1</i> and <i>qTGW3</i>.<i>3</i> alleles from M201 (42.5g) had similar grain weight to the <i>qTGW3</i>.<i>2</i> (40.8g) alone. Two strategies with similar effectiveness were proposed to improve grain weight by marker-assisted selection (MAS). One is to introduce the novel <i>qTGW3</i>.<i>2</i> allele alone, and the other is to pyramid <i>qTGW3</i>.<i>1</i> and <i>qTGW3</i>.<i>3</i> alleles together. One new allele of <i>GS3</i> (39 bp deletion in intron 1) and two SNPs in coding sequence of <i>qGL3</i> identified in this study from M201 are useful in pyramiding elite alleles for molecular breeding for improvement of rice yield.</p></div

    A simplified scheme for SLAF-Seq.

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    <p>(a) Thirty lines with largest TGW and 30 lines with lightest TGW were pooled and used for SLAF-Sequencing (the histogram was drawn based on the data collected at the F<sub>7</sub> generation) (b) All SLAFs (black lines) distributed on 12 chromosomes (c) Polymorphic SLAFs (black lines) distributed on 12 chromosomes (d) Polymorphic markers (black lines) between two DNA bulks distributed on 12 chromosomes with intensive markers distributed in the block of chromosome 3. (e) Identification of the hot region for TGW. The reference genome is in the middle of the map, the green box means intergenic regions, orange box means gene regions, yellow box means repeat regions. Red and blue lines represent the relative abundance of the maternal and paternal alleles between two bulked DNA pools (Pool 1 and Pool 2).</p

    Genetic linkage map of chromosome 3 and QTL mapping for TGW.

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    <p>The linkage map was constructed with 32 markers, covering 179.4 cM, with an average distance of the adjacent markers of 5.61 cM (left); interval mapping for TGW was conducted and a plot of LOD scores was shown with three distinct peaks corresponding to <i>qTGW3</i>.<i>1</i>, <i>qTGW3</i>.<i>2</i> and <i>qTGW3</i>.<i>3</i> (right).</p

    Relationships among Genetic, Structural, and Functional Properties of Rice Starch

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    We determined the relationships among the structural properties, in vitro digestibility, and genetic factors in starches of 14 rice cultivars. Weight-based chain-length distributions in amylopectin ranged from 18.07% to 24.71% (fa, DP 6–12), 45.01% to 55.67% (fb<sub>1</sub>, DP 13–24), 12.72% to 14.05% (fb<sub>2</sub>, DP 25–36), and 10.80 to 20.72% (fb<sub>3</sub>, DP > 36), respectively. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) ranged from 78.5% to 87.5%, 1.2% to 6.0%, and 10.1% to 18.0%, respectively. AAC was negatively correlated with RDS content but positively correlated with RS content in rice starch. The proportion of short chains in amylopectin, i.e. the amount of fraction IIa (FrIIa) fractionated by gel permeation chromatography (GPC), was positively correlated with RDS. Starch synthase IIa (SSIIa) gene controlled the degree of crystallinity, the amount of fa chains of amylopectin. SSIIIa gene controlled the amount of fb<sub>1</sub> chains. <i>Wx</i> gene controlled the FrI, FrIIa, RDS, and RS. Starch debranching enzyme isoamylase II (ISA2) gene also controlled the RDS, which may suggest that RDS was also affected by amylopectin structure, although no correlation between them was found. This study indicated that genetics (i.e., starch biosynthesis related genes) controlled the structural properties of starch, and both amylose content and amylopectin fine structure determined functional properties of rice starch (i.e., the digestion), each in a different way. Understanding the “genetics–structure–function” relationships in rice starches will assist plant breeders and food processors in developing new rice varieties and functional foods

    Association Mapping of Quantitative Trait Loci for Mineral Element Contents in Whole Grain Rice (<i>Oryza sativa</i> L.)

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    Mineral elements in brown rice grain play an important role in human health. In this study, variations in the content of iron (Fe), zinc (Zn), selenium (Se), cadmium (Cd), and lead (Pb) in 378 accessions of brown rice were investigated, and association mapping was used to detect the quantitative trait loci (QTLs) responsible for the variation. Among seven subpopulations, the mean values of Zn and Cd in the <i>japonica</i> group were significantly higher than in the <i>indica</i> groups. The population structure accounted for from 5.7% (Se) to 22.1% (Pb) of the total variation. Correlation analyses showed that Pb was positively correlated with the other minerals (<i>P</i> < 0.001) except for Se. For the five mineral elements investigated, 20 QTLs, including some previously reported and new candidate loci, were identified. Particularly, three cases of QTL colocalization, i.e. Cd and Pb on chromosome 5, Zn and Pb on chromosome 7, and Se and Pb on chromosome 11, were observed. This study suggested that the identified markers could feasibly be used to enhance desired micronutrients while reducing the heavy metal content in whole rice grain by marker-assisted selection (MAS)

    SLAFs data summary for parents and bulked DNA pools.

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    <p>SNP: single nucleotide polymorphism. EnzymePos SNP: SNP at restriction enzyme cleavage position. InDel: insertion/deletion</p><p>SLAFs data summary for parents and bulked DNA pools.</p
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