The present investigation revealed that the alk and gel(t) genes, which cause the differences between a japonica rice variety Nipponbare and an indica rice variety Kasalath in terms of the disintegration of endosperm starch granules in alkali solution and their gelatinisation in a 4 M urea solution, respectively, cosegregated in backcross inbred lines derived from a cross between the two varieties. The segregation pattern of the profile for amylopectin chain-length, which was distinguished by enrichment in short chains of DP<==11 and depletion in intermediate-size chains of 12<==DP<==24 in japonica as compared with indica, was exactly the same as those of the above physico-chemical properties of starch granules, and the gene was designated as acl(t). Gene-mapping analysis showed that the starch synthase IIa ( SSIIa) gene is located at the alk locus on chromosome 6 in the rice genome. These results lead us to the possibility that different alleles of the SSIIa gene are responsible for differences in amylopectin structure between the two varieties, in that SSIIa plays a distinct role in the elongation of short chains within clusters (A+B(1) chains) of amylopectin. It is proposed that the activity of SSIIa in japonica rice is reduced in amount or functional capacity relative to the activity of this enzyme in indica rice. This, in turn, would explain why starch from japonica rice has a lower gelatinisation temperature than starch from indica rice and is more susceptible to disintegration in alkali or urea. The evidence for this hypothesis is that the alk(t), gel(t), acl(t) and SSIIa genes all map to the same locus.

With the ever-increasing global demand for high quality rice in both local production regions and with Western consumers, we have a strong desire to understand better the importance of the different traits that make up the quality of the rice grain and obtain a full picture of rice quality demographics. Rice is by no means a ‘one size fits all’ crop. Regional preferences are not only striking, they drive the market and hence are of major economic importance in any rice breeding / improvement strategy. In this analysis, we have engaged local experts across the world to perform a full assessment of all the major rice quality trait characteristics and importantly, to determine how these are combined in the most preferred varieties for each of their regions. Physical as well as biochemical characteristics have been monitored and this has resulted in the identification of no less than 18 quality trait combinations. This complexity immediately reveals the extent of the specificity of consumer preference. Nevertheless, further assessment of these combinations at the variety level reveals that several groups still comprise varieties which consumers can readily identify as being different. This emphasises the shortcomings in the current tools we have available to assess rice quality and raises the issue of how we might correct for this in the future. Only with additional tools and research will we be able to define directed strategies for rice breeding which are able to combine important agronomic features with the demands of local consumers for specific quality attributes and hence, design new, improved crop varieties which will be awarded success in the global market.

In this study, our goal was to evaluate the role of starch debranching enzymes in the determination of the structure of amylopectin. We screened mutant populations of Arabidopsis for plants with alterations in the structure of leaf starch by using iodine staining. The leaves of two mutant lines stained reddish brown, whereas wild-type leaves stained brownish black, indicating that a more highly branched polyglucan than amylopectin was present. The mutants were allelic, and the mutation mapped to position 18.8 on chromosome 1. One mutant line lacked the transcript for a gene with sequence similarity to higher plant debranching enzymes, and both mutants lacked a chloroplastic starch-hydrolyzing enzyme. This enzyme was identified as a debranching enzyme of the isoamylase type. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch in this mutant line when compared with the wild type and in the accumulation of the highly branched water-soluble polysaccharide phytoglycogen. Both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure.

The Arabidopsis thaliana genome encodes three ␣-amylase-like proteins (AtAMY1, AtAMY2, and AtAMY3). Only AtAMY3 has a predicted N-terminal transit peptide for plastidial localization. AtAMY3 is an unusually large ␣-amylase (93.5 kDa) with the C-terminal half showing similarity to other known ␣-amylases. When expressed in Escherichia coli, both the whole AtAMY3 protein and the C-terminal half alone show ␣-amylase activity. We show that AtAMY3 is localized in chloroplasts. The starch-excess mutant of Arabidopsis sex4, previously shown to have reduced plastidial ␣-amylase activity, is deficient in AtAMY3 protein. Unexpectedly, T-DNA knock-out mutants of AtAMY3 have the same diurnal pattern of transitory starch metabolism as the wild type. These results show that AtAMY3 is not required for transitory starch breakdown and that the starch-excess phenotype of the sex4 mutant is not caused simply by deficiency of AtAMY3 protein. Knockout mutants in the predicted non-plastidial ␣-amylases AtAMY1 and AtAMY2 were also isolated, and these displayed normal starch breakdown in the dark as expected for extraplastidial amylases. Furthermore, all three AtAMY double knock-out mutant combinations and the triple knock-out degraded their leaf starch normally. We conclude that ␣-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves.

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.

The starch synthase IIa (SSIIa) gene of rice (Oryza sativa L.) has been shown to be the alk gene that controls alkali disintegration of rice grains, although the effects of naturally occurring alk mutant alleles on enzyme function have yet to be determined. We genotyped 60 rice cultivars for two single-nucleotide polymorphisms (SNPs) in rice SSIIa, including one that results in an amino acid substitution. Incorporating data for three other SNPs previously genotyped in rice SSIIa, five haplotypes were found. We analysed the association of these SSIIa haplotypes with the chain-length distribution of amylopectin, the gelatinisation temperature of rice flour, the alkali spreading score, and the starch association of the enzyme. It was determined that two SNPs resulting in amino acid changes close to the C-terminus most likely alter SSIIa both in terms of activity and starch granule association. This in turn alters the branch-length distribution of amylopectin and the gelatinisation properties of starch.

The structure of endosperm amylopectin was compared between two rice varieties, Kinmaze (subspecies japonica) and IR36 (subspecies indica), as well as their waxy mutants, all grown under controlled temperature. The distinct varietal difference in chain length distribution of amylopectin was confirmed by high performance anion‐exchange chromatography equipped with pulsed amperometric detection. Amylopectin from Kinmaze contains more very short chains with degree of polymerization (DP) between 6 and 10 and less chains with DP from 13 to 22 than amylopectin from IR36, while there is little difference in the distribution of longer chains with DP > 24 between the two varieties. Waxy mutation had little effect on chain length distribution of endosperm amylopectin. The temperature during grain‐filling affected the chain length distribution of amylopectin in both varieties in a similar way; grain‐filling at lower temperatures lead to an increased proportion of chains of DP 6—13 and decreased the percentage of chains with DP 20—27 and DP 44—54. However, the temperature‐dependent changes in chain length distribution of amylopectin were within the range of varietal difference between Kinmaze and IR36. These results strongly suggest that factors regulating the varietal difference in patterns of chain length of amylopectin are dissimilar to those causing the temperature effects on amylopectin fine structure in rice endosperm.

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (Mr) of 102,069 Da, similar in size to its Mr of about 100,000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3'-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.