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The present study was conducted to characterize the functions of the major starch synthase (SS) isozymes SSI, SSIIa, and SSIIIa in rice endosperm and their functional interaction with starch branching enzyme (BE), by using their purified recombinant proteins. All the SS isozymes had similarly significant activities toward branched glucans such as amylopecin and glycogen whereas they scarcely showed activities toward maltohexaose. In vitro studies indicate that SSI mainly attacked A and B chains with degree of polymerization (DP) of 6 and 7 in their external segments and elongated them to DP8. It is likely that SSIIa and SSIIIa produced wider ranges of intermediate chains and long chains, respectively. This study also revealed that without addition of exogenous primer, the glucan synthesis of SSI in the presence of ≧0.3 M citrate was accelerated by the addition of any of the rice BE isozymes- BEI, BEIIa, or BEIIb, whereas no such interaction occurred between SSIIa or SSIIIa with any of the BEs. The SSI-BE unprimed glucan synthesis absolutely required citrate. The interaction between SSI and BE was established by stimulation of SSI activity with BE and by activation of the BE activity by SSI., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa (I), coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35-70% in the transgenic SSIIa (I)/isa1 lines. Thus, the α-glucans from the SSIIa (I)/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIa (I)/isa1 insoluble > SSIIa (I)/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the β-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIa (I)/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIa(I), whereas the expression of SSIIa(I) did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIa(I).

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F(2) generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ∼90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm.

To our knowledge the present paper shows for the first time the kinetic parameters of all the three starch branching enzyme (BE) isozymes, BEI, BEIIa and BEIIb, from rice with both amylopectin and synthetic amylose as glucan substrate. The activities of these BE isozymes with a linear glucan amylose decreased with a decrease in the molar size of amylose, and no activities of BEIIa and BEIIb were found when the degree of polymerization (DP) of amylose was lower than at least 80, whereas BEI had an activity with amylose of a DP higher than approximately 50. Detailed analyses of debranched products from BE reactions revealed the distinct chain length preferences of the individual BE isozymes. BEIIb almost exclusively transferred chains of DP7 and DP6 while BEIIa formed a wide range of short chains of DP6 to around DP15 from outer chains of amylopectin and amylose. On the other hand, BEI formed a variety of short chains and intermediate chains of a DP

In monocots, starch branching enzyme II (BEII) was functionally differentiated into BEIIa and BEIIb after separation from the dicots, and in cereals BEIIb plays a distinct role in amylopectin biosynthesis in the endosperm. The present study was conducted to examine to what extent a green algal BEII has an overlapping function with BEIIb in starch biosynthesis by introducing the Chlorella BEII gene into an amylose-extender (ae) mutant of rice. Chlorella BEII was found to complement the contribution of the rice endosperm BEIIb to the structures of amylopectin and starch granules because these mutated phenotypes were recovered almost completely to those of the wild type by the expression of Chlorella BEII. When the recombinant BE enzymes were incubated with the rice ae amylopectin, the branching pattern of Chlorella BEII was much more similar to that of rice BEIIb rather than rice BEIIa. Detailed analyses of BE reaction products suggests that BEIIb and Chlorella BEII only transfer chains with a degree of polymerization (DP) of 6 and 7, whereas BEIIa preferably transfers short chains with a DP of about 6-11. These results show that the Chlorella BEII is functionally similar to rice BEIIb rather than BEIIa.