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Structure and Properties of Starch in Rice Double Mutants Lacking Starch Synthase (SS) IIa and Starch Branching Enzyme (BE) IIb.

ABSTRACT: Starch biosynthetic enzymes form multi-protein complexes consisting of starch synthase (SS) I, SSIIa, and starch branching enzyme (BE) IIb, which synthesize amylopectin clusters. This study analyzed the starch properties in two double mutant rice lines lacking SSIIa and BEIIb, one of which expressed an inactive BEIIb protein. The ss2a be2b lines showed similar or greater seed weight than the be2b lines, and plant growth was not affected. The ss2a line showed increased short amylopectin chains resulting in a lower gelatinization temperature. Starch granule morphology and A-type crystallinity were similar between the ss2a line and the wild type, except for a mild chalky seed phenotype in the ss2a line. However, the starch phenotype of the ss2a be2b lines, which was similar to that of be2b but not ss2a, was characterized by increased long amylopectin chains, abnormal starch granules, and B-type crystallinity. The similarity in phenotype between the ss2a be2b and be2b lines may be attributed to the inability of the be2b mutants to generate short amylopectin branches, which serve as primers for SSIIa. Therefore, the presence or absence of SSIIa hardly affected the amylopectin structure under the be2b background. The amylose content was significantly higher in the ss2a be2b lines than in the be2b lines. Starch crystallinity was greater in ss2a be2b lines than in be2b lines, despite the fact that starch crystallinity is generally negatively correlated with amylose content. This suggests that the formation of a double helix between long amylopectin chains and amylose affects starch crystallinity in the ss2a be2b mutants.

SUBMITTER: Ida T

PROVIDER: S-EPMC8367641 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Amylopectin, the major component of starch, is synthesized by synergistic activity of multiple isozymes of starch synthases (SSs) and branching enzymes (BEs). The frequency and length of amylopectin branches determine the functionality of starch. In the rice endosperm, BEIIb generates short side chains of amylopectin and SSI elongates those branches, which can be further elongated by SSIIa. Absence of these enzymes greatly affects amylopectin structure. SSI, SSIIa, and BEIIb associate with each other and with other starch biosynthetic enzymes although SSIIa is low activity in japonica rice. The aim of the current study was to understand how the activity of starch biosynthetic enzyme complexes is compensated in the absence of SSI or BEIIb, and whether the compensatory effects are different in the absence of BEIIb or in the presence of inactive BEIIb. Interactions between starch biosynthetic enzymes were analyzed using one ss1 null mutant and two be2b japonica rice mutants (a mutant producing inactive BEIIb and a mutant that did not produce BEIIb). Soluble proteins extracted from the developing rice seeds were separated by gel filtration chromatography. In the absence of BEIIb activity, BEIIa was eluted in a broad molecular weight range (60-700 kDa). BEIIa in the wild-type was eluted with a mass below 300 kDa. Further, majority of inactive BEIIb co-eluted with SSI, SSIIa, and BEI, in a mass fraction over 700 kDa, whereas only small amounts of these isozymes were found in the wild-type. Compared with the be2b lines, the ss1 mutant showed subtle differences in protein profiles, but the amounts of SSIIa, SSIVb, and BEI in the over-700-kDa fraction were elevated. Immunoprecipitation revealed reduced association of SSIIa and BEIIb in the ss1 mutant, while the association of BEIIb with SSI, SSIIa, SSIVb, BEI, and BEIIa were more pronounced in the be2b mutant that produced inactive BEIIb enzyme. Mass spectrometry and western blotting revealed that SSI, SSIIa, SSIIIa, BEI, BEIIa, starch phosphorylase 1, and pullulanase were bound to the starch granules in the be2b mutants, but not in the wild-type and ss1 mutant. These results will aid the understanding of the mechanism of amylopectin biosynthesis.

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.

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Structure and Properties of Starch in Rice Double Mutants Lacking Starch Synthase (SS) IIa and Starch Branching Enzyme (BE) IIb.

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