Natural variation in rice starch synthase IIa affects enzyme and starch properties

Abstract

The natural variation in starch synthase IIa (SSIIa) of rice (Oryza sativa L.) was characterised using near-isogenic lines (NILs). SSIIa is a candidate for the alk gene regulating the alkali disintegration of rice grains, since both genes are genetically mapped at the same position on chromosome 6 and related to starch properties. In this study, we report that the alkali-susceptible cultivar Nipponbare lacked SSIIa activity in endosperm. However, the activity was detected with NILs having the alk allele of alkali-tolerant Kasalath. SSIIa protein was present even in Nipponbare endosperm, but it was not associated with starch granules at the milky stage of endosperm. Three single-nucleotide polymorphisms (SNPs) predicting amino acid substitutions existed between the cDNA sequences of SSIIa of Nipponbare and Kasalath were genotyped with 65 rice cultivars and four wild relatives of cultivated rice. The results obtained explain the potential importance of two of the amino acid residues for starch association of rice SSIIa. An analysis of the chain-length distribution of ␤-limit dextrin of amylopectin showed that without SSIIa activity, the relative number of A-chains (the short chains without branches) increased and that of B1-chains (the short chains with branches) decreased. This suggests that, given the SSIIa defect, short A-chains could not reach a sufficient length for branching enzymes to act on them to produce B1-chains.

Effects of Variations in Starch Synthase on Starch Properties and Eating Quality of Rice

“…NIL(Wx a ) had more short chains with a degree of polymerization (dp) of 6-15 and fewer with dp 19-30 than Nipponbare. NIL(Alk) showed very similar profiles to those of Kasalath in having fewer short chains of dp 6-11 and more mid-length chains of dp 13-23, as reported by Umemoto et al (2004). The chain-length distribution profile of NIL(SSI k ) was the most similar to that of Nipponbare.…”

“…Each lane contained protein from 1 mg of starch. Immunoblotting for SSI and SSIIa was conducted as reported by Umemoto et al (2004) and Umemoto and Aoki (2005) with antisera raised against synthetic polypeptides specific to each. Starch synthases in the soluble fraction of developing endosperm at the milk-ripe stage were separated by electrophoresis in glycogen-containing native gels, the gels were incubated with ADP-glucose, and synthase activities were detected, as described by Okamoto et al (2002).…”

“…NIL(Wx a ) and NIL(SSI k ) were originally developed for research on heading date as NIL(Hd3b) and NIL(Hd3a), respectively (Monna et al, 2002). NIL(Alk) was one of four NILs developed to evaluate SSIIa variation (Umemoto et al, 2004).…”

“…The gelatinization properties of rice flour were measured with a Rapid Visco Analyzer (RVA; RVA-4, Newport Scientifi c, Sydney, NSW, Australia) according to Umemoto et al (2004). We analyzed the thermal properties of polished rice by differential scanning calorimetry (DSC; Micro DSCIII, Setaram, Caluire, France): 10 polished rice grains and 1.3 times their weight in water were added to a DSC pan, and the DSC pan was allowed to equilibrate for 1 h at 25ºC before measurement.…”

Characterization Methods for Starch-Based Materials: State of the Art and Perspectives

“…Starch with a CLD containing a lower portion of DP of 6 to 12 and higher portion of DP of 12 to 24 exhibits high GT. [66] Mutations in SSIIa have been associated with these differences: [67] e.g. a plant with inactive SSIIa produces starch with a lower GT.…”