Takayuki Umemoto

Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties

Abstract 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+B1 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.

Single-nucleotide polymorphisms in rice starch synthase IIa that alter starch gelatinisation and starch association of the enzyme

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.

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

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 β-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.

Differences in Amylopectin Structure Between Two Rice Varieties in Relation to the Effects of Temperature During Grain-Filling

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) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene

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 102069 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.

Activity of starch synthase and the amylose content in rice endosperm

Abstract The content of amylose in endosperm of non-waxy japonica rice ( Oryza sativa cv Akitakomachi) was increased by lowering the growth temperature from 25° to 15° during the ripening period. The activities of sucrose synthase, ADPglucose pyrophosphorylase, starch branching enzyme (Q-enzyme) and soluble starch synthase in endosperm developed at 15° were lower than or similar to those at 25°, when compared on a endosperm basis at the similar ripening stage. In contrast, the activity of starch granule-bound starch synthase, which is considered to be indispensable for amylose synthesis, was higher by 3–3.5-fold in the endosperm developed at the low temperature than that at the high ambient temperature. The results suggest that the low temperature specifically accelerates the expression of the bound starch synthase gene (waxy gene) in rice endosperm, which resulted in elevated amylose biosynthesis in the endosperm when developed at lower temperatures.

Activity of granule-bound starch synthase is an important determinant of amylose content in rice endosperm

Effects of temperature on amylose synthesis in rice endosperm were investigated using 13 cultivars known to differ in amylose contents in endosperm. Both amylose content and granule-bound starch synthase (GBSS) activity was increased when endosperms were developed under lower temperature in low- and medium-amylose cultivars, while this was not the case for high-amylose cultivars. Amylose content in high-amylose cultivars was stable under different temperature, with varying GBSS activity. A nearly linear correlation between GBSS activity and amylose content was observed as far as activity levels about 250 nmol min-1 g-1 DW. These results suggest that GBSS activity is an important determinant of amylose content in endosperm of low- and medium-amylose cultivars, while factors other than the enzyme activity limit amylose synthesis in the high-amylose cultivars.

Effect of grain location on the panicle on activities involved in starch synthesis in rice endosperm

Abstract Starch metabolism in rice endosperm of both the superior and inferior caryopses in the panicle was characterized by comparing changes in activities of five major enzymes associated with starch synthesis during endosperm development; sucrose synthase, ADPglucose pyrophosphorylase, Q-enzyme (starch branching enzyme), and soluble and starch granule-bound forms of starch synthase. Activities of ADPglucose pyrophosphorylase of both the superior and inferior caryopses increased markedly between 11 and 14 days after pollination. The developmental patterns of Q-enzyme activities were quite different between the superior and inferior caryopses. In addition, it was found that eight-14 days after pollination the activity of the starch granule-bound starch synthase in the inferior caryopsis was markedly lower than that in the superior caryopsis. These results suggest that the low activity of the granule-bound starch synthase is related to the lower content of amylose in the inferior caryopsis.

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

Abstract We evaluated the effects of functional variation in three starch synthases in rice (Oryza sativa L.)−granule-bound starch synthase I (GBSSI, wx), starch synthase I (SSI, SSI), and starch synthase IIa (SSIIa, alk)−between indica cultivar Kasalath and japonica cultivar Nipponbare on starch properties and eating quality. We used three near-isogenic lines−NIL(Wxa), NIL(SSI k), and NIL(Alk)−containing chromosomal segments of Kasalath on a Nipponbare genetic background. The Wxa allele explained most of the difference in amylose content between the two cultivars, and decreased the peak viscosity and breakdown to less than half of those of Nipponbare. These changes reduced the quality of cooked rice both just after cooking and after storage at 5ºC. The variation in SSIIa also affected the eating quality after storage of cooked rice at 5ºC : NIL(Alk) became harder and less sticky than Nipponbare, although the rices were comparable just after cooking. Differential scanning calorimetry revealed faster retrogradation of the once-gelatinized starch in NIL(Alk). The variation in SSI alleles hardly affected these properties.

Genetic Analysis of Long Chain Synthesis in Rice Amylopectin

The amount of long chains (LC) of amylopectin in high-amylose rice is thought to be one of the important determinants of its quality when cooked. A wide range of differences in LC content have been reported in rice varieties, which can be clearly divided into four classes based on LC and apparent amylose content: namely, amylose and LC-free, low or medium-amylose and low-LC, high-amylose and medium-LC, high-amylose and high-LC. However, genetic factors controlling LC content have not been fully understood. Here, we performed quantitative trait loci (QTL) analysis of LC content using 157 recombinant inbred lines (RILs) derived from a cross of a low-LC cultivar, Hyogokitanishiki, and a high-LC line, Hokuriku 142. By analyzing randomly selected 15 RILs, it was shown that high LC content (≥11%) was associated with high setback viscosity (≥200 RVU), and that low LC (≤ 3%) was associated with low setback viscosity (≤ 130 RVU), as measured by a Rapid Visco Analyzer. With setback viscosity as an indicator for LC content, QTL analysis was conducted using 60 DNA markers including a CAPS marker that distinguished Wxa and Wxb alleles coding for granule-bound starch synthase I (GBSSI or Wx protein), the enzyme working for amylose biosynthesis. Only one QTL with a peak log of likelihood score at the wx locus was detected, and no line showing setback viscosity corresponding to the medium-LC class appeared. The fact that wx mutants of Hokuriku 142 lacked LC in their rice starch supports the view that the functional Wx allele is indispensable for LC synthesis in addition to amylose synthesis in rice endosperm. We suggest three possible reasons why no line with medium-LC content was observed. First, the locus (loci) responsible for generation of medium-LC may be located very close to the wx locus and not able to be dissected by the population and DNA markers we used. Second, there may be special QTLs for medium-LC cultivars that do not exist in low- or high-LC cultivars. Third, medium-LC cultivars may have an as-yet unidentified Wx allele with lower capability in LC synthesis compared to the Wx allele in high-LC cultivars.