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Starch is glucose polymer linked by α-1,4 and α-1,6 glucosidic bonds. The balance of activity between starch synthases (SSs) and branching enzymes (BEs) is important for formation of amylopectin structure. SSI and SSIIIa isozymes account for 60 and 30%, respectively, of soluble SS activity in develop- ing rice endosperm. Complete loss of both SSI and SSIIIa activities induces sterility. By contrast, a double mutant (ss1 L /ss3a) generated by crossing the leaky ss1 mutant, whose SS activity is approximately ca. 16% of the wild type, and the ss3a null mutant is fertile. Although there is only a significant residual SS activity in the developing endosperm of ss1 L /ss3a, the yield and growth of the double-mutant line is sufficient. We analyzed the amylopectin chain-length distribution, thermal and pasting properties, morphologies of starch granules and amyloplasts, and crystallinity of endosperm starch in ss1 L /ss3a and the parental mu- tant lines. The chain-length distribution pattern of ss1 L /ss3a was similar to that of ss3a and there was an additive effect of the reduction of SSI activity on the chain-length of amylopectin in the ss3a background. These changes in the amylopectin chain-length distribution were considered to increase the gelatinization temperature of the starch. The gelatinized starch viscosity of the double-mutant and ss3a lines was very low. Most ss1 L /ss3a starch granules were round-shaped, and the starch granular size was smaller than those of the parental mutant lines and the wild type. The starch crystallinity of ss1 L /ss3a was slightly re- duced compared with that of the wild type.

The functions of the major isozymes related to the starch biosynthesis in cereal endosperm have been resolved based on the researches using the mutant lines. For further understanding of the relationships between isozymes, the analyses of double mutant lines must be very efficient. In this study, double mutant rice line (ss1L/isa1) between leaky starch synthase I mutant line (ss1L) and isoamylase 1 mutant line (isa1) was generated and structures of the stored α-glucans were characterized. Cross-section of the parent isa1 and ss1L/isa1 seeds was negative for iodine staining and more than 90% of the α-glucans was soluble in water. Chain-length distributions of the glucans were similar to each other between the two lines, and the differential plot obtained by subtraction of the chain-length distribution of isa1 from that of ss1L/isa1 was nearly identical to the corresponding differential plot of ss1L vs. wild type, indicating effect of SSI is simply additive on chain-length distribution of the product regardless of isoamylase1 activity. Analyses of β-amylase limit dextrins revealed slight, statistically significant difference in internal chain-length distributions of the two isa1 mutant lines in contrast to the previous result on isa1 mutant lines with or without exogenous indica-type SSIIa. This study demonstrated that the primary role of SSI in the isa1 background is outer-chain elongation with a little effect on branch-position distribution, which is the same role as the case in normal amylopectin biosynthesis in the presence of isoamylase 1. Such chain-elongation properties differ among the three major SS isozymes.

The present paper established new methods for determining the activities of starch branching enzyme (BE) and starch synthase (SS). The methods are based on the calculation of the data on chain- length distribution obtained from the fl uorophore-assisted carbohydrate capillary electrophoresis (FACE) method, in which the molar percentage of each linear chain comprising the branched glucan can be pre- cisely determined. The FACE method can give us two important results at the same time, namely the chain-length distribution and the enzyme activity, for characterization of BE and SS. Although the activ- ity of BE has been quantitatively determined by measuring the reducing power of the isoamylorysates of the reaction product and the substrate, this method has diffi culties in having a high background value when branched glucan such as amylopectin and glycogen is used as substrate in the in vitro reaction, whereas the FACE method has no such disadvantages.

The present paper shows the effects of reaction temperature on the activity and chain-transfer properties of all three starch branching enzyme (BE) isozymes, BEI, BEIIa and BEIIb, from rice towards amylopectin as a glucan substrate. The activity of BEI increased with an increase in the reaction temperature from 10 to 30oC, then the activity decreased at higher temperatures, and finally BEI lost its activity at 45oC. Both BEIIa and BEIIb activities were optimum at 25oC, and no significant activity was found at 45oC. The decrease in the enzymatic activity at high temperatures between 35-40oC was more significant in BEIIb than in BEIIa. Despite the temperature-dependent activity, no practical changes in distinct chain preferences of any of the three BE isozymes were found. These findings indicate that these responses could influence the starch production, structure and physicochemical properties affected by environmental temperatures during the growth of rice plants.

A reliable quantitative assay method for starch branching enzyme (BE) remains to be established whereas it has been required to characterize BE towards understanding the regulatory mechanism for the synthesis of starch in plant tissues. We describe a new quantitative assay method for BE activities with malto-oligosaccharides, amylose and amylopectin as substrates by using bicinchoninic acid (BCA) for measuring directly the reducing terminals of linear glucans formed after debranching the reaction products. The BCA method not only can be performed by simple procedures and easy handling with the use of cheap toxic-free reagents, but also shows highly color-stable properties after the treatment of the reaction mixture with the color-yielding reagents compared with the modified Park-Johnson method (Carbohydr. Res., 94, 205-213(1981)). The intensity and the spectrum of the purple color generated in the treated assay mixture are maintained in sugars and glucans in the range of glucose and amylose with degree of polymerization up to at least 1658. The BCA method can be also applied for characterization of BE when amylopectin is used as glucan substrate. In this paper we report this efficient, reproducible and quantitative BCA method by showing some experimental results obtained in an attempt to determine the kinetic parameters of BEIIb from rice endosperm.

SSI accounts for 60% of the total SS activity in the soluble fraction in the developing rice en- dosperm. Rice SSI-deficient mutants were identified by using reverse genetics, and the chain-length analysis of the endosperm starch showed that SSI distinctly synthesizes DP 8―12 chains from short DP 6―7 chains emerg- ing from the A chains and the branch points in the B1 chains of amylopectin. 1) In this study, to evaluate by in vitro study the function of recombinant SSI of rice (rSSI), the change in the chain-length distribution of glyco- gen or amylopectin in the activity band after rSSI enzymatic reactions in native-PAGE gel was examined. When glycogen was used as the substrate, the -glucan produced in the rSSI activity band on the native- PAGE gel had specifically fewer DP 6 chains and more DP 8 chains than unmodified glycogen did. When rice amylopectin was used as the substrate, the -glucan produced in the rSSI activity band showed the oscillation of a short turn in the range of DP 20 compared with the unmodified amylopectin; the chains with DP 6, 7, 10, 11, 13 and 17 decreased, while the extent of decrease was reduced in the chains with DP 8, 12 and 15. These results suggest that rSSI preferentially elongates the chains up to DP 8 from the short and long B1 and B2 chains with DP 6 or 7 from branch points to the non-reduced end as well as the A chains with DP 6 or 7 by adding one or two glucose moieties.

The tandem-cluster structure of amylopectin must be a refined product that higher plants have evolved from the cluster-free random branched structure of glycogen. During the evolutionary process, plants have increased the number of isozymes of three classes of enzymes, namely soluble starch synthase (SS), starch branching enzyme (BE) and starch debranching enzymes (DBE), by giving each individual enzyme a distinct function in constructing the amylopectin cluster structure. On the basis of biochemical and molecular analyses of starch mutants and transgenic plants of rice, the present paper presents a model for amylopectin cluster biosynthesis in rice endosperm to understand how each enzyme is involved in this process. The author also proposes a notion called “cluster-world” where by the structure of amylopectin varies through changes in several distinct patterns of the cluster structure, and alteration of activities of key enzymes such as SSIIa, BEIIb and isoamylase1 results in the structural variation within the fixed pattern depending on which enzyme activity is altered.

We isolated various kinds of mutants participating in the biosynthesis of endosperm starch by the MNU treatment of fertilized egg cells in japonica rice cultivars. There were at least 6 mutant loci lowering amylose content including the wx locus. These low amylose mutations altered the amylose content without affecting the amylopectin structure. The amylose content did not affect the initiation of gelatinization, but influenced greatly the swelling of gel, indicating that amylose content affects the swelling power of gel and the termination of gelatinization. BEIIb mutation decreased specifically the short chains with DP less than 17 in amylopectin whereas BEI mutation was characterized by a significant decrease in long chains with DP longer than 38 and short chains with DP 12 to 23, suggesting that BEIIb and BEI contributes to the synthesis of A chains, and B, and B3 chains of amylopectin, respectively. Notable alteration on the chain length profile was not observed in BEIIa mutation. Each BE mutation showed distinct effect on the thermal properties of endosperm starch. The sug2 mutation lowering the expression level of PUL increased shorter chains of amylopectin and accumulated a significant amount of the water-soluble polysaccharides as well as ISA (sugl ) mutation. The fl o2 mutation decreased simultaneously the expression levels of some of the enzymes including BEI, PUL and GBSS, suggesting that the wild type Flo2 gene probably encodes a regulatory protein that modulates the expression of the genes involved in starch biosynthesis. Some mutants involved in the biosyn thesis of ADP-glucose were also isolated.

Metabolic engineering of the structure of amylopectin in rice endosperm has been performed by manipulating the activities of the major isoforms of soluble starch synthase, starch branching enzyme, and starch debranching enzymes. Our results show that each isoform distinctly affects amylopectin cluster structure and that the change leads to the synthesis of novel starches with varied physicochemical properties . This paper describes these experimental results and discusses the advantage of the molecular approach for elucidation of the starch biosynthesis system and for production of novel starches.