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

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.

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

“…The 8% reduction observed in the total starch content of the shrunken lines and the slight reduction in peak gelatinization temperature (at least 2 • C) of all shrunken and opaque lines tested also support the hypothesis that the residual SSIIa in Nipponbare has functional roles because its downregulation leads to significantly altered starch biosynthesis in the rice grain. The reduction of the peak gelatinization temperature was consistent with the earlier report by Miura et al (2018), which observed a reduction of about 6 • C in the gelatinization temperature of the null SSIIa line.…”

“…In this study, the already low expression of weakly active japonica-type SSIIa in Nipponbare endosperm was further downregulated to determine any impact on its possible roles in amylopectin biosynthesis and starch structure. Our results, using a different japonica rice line (Nipponbare) and a different technique (RNA silencing), are in broad agreement with the results obtained by Miura et al (2018).…”

“…starch from these SSIIa-null mutants all exhibited increased proportions of short amylopectin chains (approximately DP 6-12) and decreased levels of medium chains (approximately DP 13-36) compared to their respective parents. Similar changes in the chain length distribution profile were observed in Nipponbare compared with NILs(Alk), a near-isogenic line of Nipponbare with introgressed indica SSIIa (Haplotype 1) from Kasalath (Umemoto et al, 2004) and a recent study of an SSIIa null mutation in japonica rice (Miura et al, 2018). The 8% reduction observed in the total starch content of the shrunken lines and the slight reduction in peak gelatinization temperature (at least 2 • C) of all shrunken and opaque lines tested also support the hypothesis that the residual SSIIa in Nipponbare has functional roles because its downregulation leads to significantly altered starch biosynthesis in the rice grain.…”

“…In rice, an SSIIa mutant from the japonica line Kinmaze was identified completely devoid of SSIIa expression. This SSIIa null mutant, generated using N-methyl-N-nitrosourea, had a 4% increased amylose content, and a reduction of about 6 • C in the gelatinization temperature (Miura et al, 2018). The 4% increase of amylose in the SSIIa null japonica rice resulted in the same levels of amylose comparable to that of indica rice lines.…”

“…et al, 2012). Clearly, therefore, SSIIa has diverse roles in starch biosynthesis by virtue of its enzymatic, scaffolding and stromal distribution functions during starch biosynthesis in cereals (Miura et al, 2018). All these findings highlight the importance of SSIIa in determining fine amylopectin structure and the resulting functional properties of rice grain.…”