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.
Highlights
Starch synthases (SSs) have a central role in starch biosynthesis
The SSIIa activity of recombinant SSIIaJ derived from the japonica rice cultivars Nipponbare and Kinmaze is estimated as ca. 10% of that of recombinant SSIIaI derived from the indica rice cultivar IR36 (Nakamura et al, 2005)
The SSIIa band was not detected in the mature endosperm of EM204 using immunoblotting with a polyclonal antiserum of SSIIa, it was detected in Kinmaze (SSIIaJ/GBSSI allele derived from japonica cultivars (GBSSIJ)) (Figure 1, Mature endosperm, Total)
Summary
Starch synthases (SSs) have a central role in starch biosynthesis. SSs elongate α-1,4-linked linear glucan chains of starch by transferring an ADP glucose (ADPG) residue to the nonreducing end of glucans. The rice SSIIa gene controls the chain-length distribution of endosperm amylopectin in indica and japonica rice cultivars and corresponds to the alk (alkali disintegration of starch granules) gene (Umemoto et al, 1999, 2002). The SSIIa activity of recombinant SSIIaJ derived from the japonica rice cultivars Nipponbare and Kinmaze is estimated as ca. Differences in the amylopectin chain-length distribution significantly affect the starch gelatinization temperature. 8◦C lower than that of indica rice starch (Nakamura et al, 2002) This is because long parallel chains of amylopectin in indica rice expressing SSIIaI can form a longer double helix; by contrast, short chains with DP ≤ 12 can only form a short double helix in japonica rice expressing SSIIaJ
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