Yasunori Nakamura

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.

Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm.

We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) ≥ 37 and short chains with DP 12 to 21, marked increase in short chains with DP ≤ 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2–3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.

The fine structure of amylopectin in endosperm from Asian cultivated rice can be largely classified into two classes

1 The present investigations have been performed to characterize the structure of amylopectin in endosperm of rice plants cultivated in Asian countries, and to determine the relationship between the amylopectin structure and physicochemical properties of the starch. The results indicated that almost all of rice amylopectin was distinctly classified into L-type or S-type. The L-type amylopectin was diffrent from the S-type amylopectin in that the numbers of short α-1, 4-glucan chains of DP ≤ 10 were less than 20% of the total α-1, 4-glucan chains of DP ≤ 24 in a single cluster in the former and those were higher than 24% in the latter, while no significant difference between both types was found in the proportion of B2 and longer chains of DP ≥ 25. Strikingly, among the 129 rice varieties examined, only a single variety cv. Khauk Yoe belonging to the Tropical Japonica rice group had an intermediate amylopectin structure between L-type and S-type, called M-type amylopectin. Apparently, the proportion of chains with DP ≤10 to those with DP ≤ 24 in amylopectin molecules was negatively correlated with the onset temperature of gelatinization of the starch, whereas no such correlation was observed between the amylose content and the thermal properties of starch. All these results show that the varietal difference in amylopectin structure is predominantly due to difference in lengths of A chains of the cluster and plays a critical role in determining the physicochemical properties of starch in the rice endosperm.

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.

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Abstract. Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type.

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.

Changes in enzyme activities associated with carbohydrate metabolism during the development of rice endosperm

Abstract Changes in activities of 18 enzymes involved in carbohydrate metabolism from sucrose to starch were analyzed in relation to amounts of starch, soluble sugars, and soluble protein, as well as grain weight and water content during endosperm development of Japonica rice ( Oryza sativa L., cv. Fujihikari). The developmental patterns of ADPglucose pyrophosphorylase and starch branching enzyme (Q-enzyme) differed from the other enzymes examined in that they exhibited the greatest enhancement during endosperm development. In addition, both ADPglucose pyrophosphorylase and Q-enzyme activities increased 7–8 days after anthesis, when activities of the other enzymes including soluble starch synthase had already reached their highest levels. The accumulation patterns for the former two enzymes correlated well with the curve for starch production. The increases in ADPglucose pyrophosphorylase, soluble starch synthase and Q-enzyme were distinct, suggesting that synthesis of these enzymes are independently modulated genetically. The results strongly suggest that both ADPglucose pyrophosphorylase and Q-enzyme play key roles in starch biosynthesis of rice endosperm.

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.

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.

Functional Interaction Between Plastidial Starch Phosphorylase and Starch Branching Enzymes from Rice During the Synthesis of Branched Maltodextrins

The present study established the way in which plastidial α-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules.