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Starch Synthase IIa-Deficient Mutant Rice Line Produces Endosperm Starch With Lower Gelatinization Temperature Than Japonica Rice Cultivars

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Starch Synthase IIa-Deficient Mutant Rice Line Produces Endosperm Starch With Lower Gelatinization Temperature Than Japonica Rice Cultivars

Satoko Miura et al. Front Plant Sci.

Abstract

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.

Keywords: amylopectin; amylose; endosperm starch; low gelatinization temperature; protein complex; rice; starch synthase IIa.

Figures

FIGURE 1
FIGURE 1
Immunoblotting of mature and developing endosperm using anti-SSIIa serum. Total, total proteins; SP+LBP, soluble and loosely bound protein; TBP, tightly bound protein; IR36, indica rice cultivar, SSIIaI/GBSSII; #1110-290, SSIIaI/GBSSIJ; Kinmaze, japonica rice cultivar, SSIIaJ/GBSSIJ; EM204, ss2a/GBSSIJ. Arrows show the possible truncated SSIIa bands in EM204.
FIGURE 2
FIGURE 2
Structure of the OsSSIIa gene [mRNA (accession No. AB115916.1 and genomic DNA (accession No. AP003509.3)] and identification of the mutation site in EM204.
FIGURE 3
FIGURE 3
Pleiotropic effects on starch biosynthetic enzymes revealed by native-PAGE/activity staining. (A) Starch synthase (SS) I and SSIIIa activity was detected using glycogen as primer. It is noted that SSI derived from Indica rice migrate faster than that of japonica rice (Chen and Bao, 2016; Itoh et al., 2017). (B) SSIIa activity was detected using maize amylopectin as primer; reaction was performed at pH 10 to minimize hydrolase activity (see Section “Materials and Methods”). (C) Branching enzyme (BE) activity. BEIIb and Phosphorylase 1 (Pho1) activity bands were overlapped. (D) Debranching enzyme (DBE) activity. Each isozyme labeled with arrowheads has been identified with corresponding mutant rice lines. IR36, indica rice cultivar, SSIIaI/GBSSII; #1110-290, SSIIaI/GBSSIJ; Kinmaze, japonica rice cultivar, SSIIaJ/GBSSIJ; EM204, ss2a/GBSSIJ.
FIGURE 4
FIGURE 4
Isozyme distribution in protein fractions [Total protein (Total), soluble protein (SP), loosely bound protein (LBP), and tightly bound protein (TBP)] extracted from developing endosperm (10–15 DAF). Immunoblotting of each fraction was performed using anti-SSI, SSIIa, SSIIIa, SSIVb, GBSSI, BEI, BEIIa, and BEIIb antibodies. IR36, indica rice cultivar, SSIIaI/GBSSII; #1110-290, SSIIaI/GBSSIJ; Kinmaze, japonica rice cultivar, SSIIaJ/GBSSIJ; EM204, ss2a/GBSSIJ.
FIGURE 5
FIGURE 5
Molecular weight distribution of starch synthase (SS) (A–C) and branching enzyme (BE) (D,E) analyzed by native-PAGE/activity staining (A,D) and immunoblotting of the corresponding gel (B,C,E) loaded with the fractions obtained from gel filtration chromatography of soluble extract from rice developing endosperm. #1110-290, SSIIaI/GBSSIJ; Kinmaze and Nipponbare, japonica rice cultivar, SSIIaJ/GBSSIJ; EM204, ss2a/GBSSIJ. The left lane of each native-PAGE/activity staining gel and immunoblot is the loading control (IR36 for SS and Nipponbare for BE), except for D and E in Kinmaze, which are blanks. Total is the crude extract before gel filtration chromatography loaded with different amounts of extract (1 and ¼) for semi-quantitative comparisons. More mobile purple bands in EM204 of BE activity staining (D) are also BEIIa.
FIGURE 6
FIGURE 6
Morphology of rice grains observed using a stereo-microscope with overhead light (upper panels) and on a light box (lower panels). The parts that are chalky or opaque in the seeds appeared dark on a light box. Note that central region of some of EM204 seeds were chalky. IR36, indica rice cultivar, SSIIaI/GBSSII; #1110-290, SSIIaI/GBSSIJ; Kinmaze, japonica rice cultivar, SSIIaJ/GBSSIJ; EM204, ss2a/GBSSIJ.
FIGURE 7
FIGURE 7
Capillary electrophoresis analysis of amylopectin molecular structure. (A) Amylopectin chain-length distribution patterns in mature endosperm. (B) Differential plots of Kinmaze (SSIIaJ), EM204 (ss2a), and #1110-290 (SSIIaI) expressing japonica-type GBSSI. Each panel shows one typical representative data set of at least three replications. DP, degree of polymerization. Data from Crofts et al. (2017b).

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References

    1. Abe N., Asai H., Yago H., Oitome N. F., Itoh R., Crofts N., et al. (2014). Relationships between starch synthase I and branching enzyme isozymes determined using double mutant rice lines. 14:80. 10.1186/1471-2229-14-80 - DOI - PMC - PubMed
    1. Ahmed Z., Tetlow I. J., Ahmed R., Morell M. K., Emes M. J. (2015). Protein-protein interactions among enzymes of starch biosynthesis in high-amylose barley genotypes reveal differential roles of heteromeric enzyme complexes in the synthesis of A and B granules. 233 95–106. 10.1016/j.plantsci.2014.12.016 - DOI - PubMed
    1. Asai H., Abe N., Matsushima R., Crofts N., Oitome N. F., Nakamura Y., et al. (2014). Deficiencies in both starch synthase IIIa and branching enzyme IIb lead to a significant increase in amylose in SSIIa-inactive japonica rice seeds. 65 5497–5507. 10.1093/jxb/eru310 - DOI - PMC - PubMed
    1. Chen Y., Bao J. (2016). Underlying mechanisms of zymographic diversity in starch synthase I and pullulanase in rice-developing endosperm. 64 2030–2037. 10.1021/acs.jafc.5b06030 - DOI - PubMed
    1. Crofts N., Abe K., Aihara S., Itoh R., Nakamura Y., Itoh K., et al. (2012). Lack of starch synthase IIIa and high expression of granule-bound starch synthase I synergistically increase the apparent amylose content in rice endosperm. 19 62–69. 10.1016/j.plantsci.2012.05.006 - DOI - PubMed

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