Dataset Information

Contributions of Three Starch Branching Enzyme Isozymes to the Fine Structure of Amylopectin in Rice Endosperm.

ABSTRACT: Three starch branching enzyme (BE) isozymes, BEI, BEIIa, and BEIIb, are involved in starch biosynthesis in rice endosperm. Past in vivo and in vitro studies have suggested that each BE isozyme plays a distinct role in forming the fine structure of amylopectin. To elucidate more details of their roles, we prepared DNA constructs in which all the possible combinations of the expressions of these three isozymes were suppressed in developing rice endosperm. Analysis of the chain-length distributions of amylopectin produced under these various conditions confirmed the contributions of the individual BE isozymes to the fine structure of amylopectin in rice endosperm. Among these isozymes, the impact of loss of BEIIb activity on amylopectin fine structure was most remarkable and indicated that it plays a specific role in the synthesis of short chains with a 6-13 degree of polymerization (DP). The contribution of BEI to the amylopectin synthesis was unclear when only BEI activity was reduced. It was clear, however, when both BEI and BEIIb activities were substantially inhibited. The DP11-22 intermediate chains were markedly reduced in the ?BEI/BEIIb line compared with the ?BEIIb line, indicating that BEI plays a distinct role in the synthesis of these intermediate chains. Although no substantial change in amylopectin chain profile was detected in the ?BEIIa line, the role of BEIIa could be deciphered by analyzing amylopectin fine structure from the ?BEI/BEIIa/BEIIb line in comparison to that from ?BEI/BEIIb line. This strongly suggests that BEIIa compensates for the role of BEI, rather than that of BEIIb, by forming intermediate chains of DP11-22. In addition, the new possibility that BEIIa is involved in the formation of starch granules in rice endosperm was suggested because the onset temperature for gelatinization of starch granules in the ?BEIIa/BEIIb line was significantly higher than that in the ?BEIIb line. In summary, the present study highlights the distinct roles of BEI, BEIIa, and BEIIb in the synthesis of amylopectin in developing rice endosperm.

SUBMITTER: Sawada T

PROVIDER: S-EPMC6206275 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Contributions of Three Starch Branching Enzyme Isozymes to the Fine Structure of Amylopectin in Rice Endosperm.

Sawada Takayuki T Itoh Mizuho M Nakamura Yasunori Y

Frontiers in plant science 20181023

Three starch branching enzyme (BE) isozymes, BEI, BEIIa, and BEIIb, are involved in starch biosynthesis in rice endosperm. Past <i>in vivo</i> and <i>in vitro</i> studies have suggested that each BE isozyme plays a distinct role in forming the fine structure of amylopectin. To elucidate more details of their roles, we prepared DNA constructs in which all the possible combinations of the expressions of these three isozymes were suppressed in developing rice endosperm. Analysis of the chain-length ...[more]

PMID: 30405671

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Project description:Amylopectin, the major component of starch, is synthesized by synergistic activity of multiple isozymes of starch synthases (SSs) and branching enzymes (BEs). The frequency and length of amylopectin branches determine the functionality of starch. In the rice endosperm, BEIIb generates short side chains of amylopectin and SSI elongates those branches, which can be further elongated by SSIIa. Absence of these enzymes greatly affects amylopectin structure. SSI, SSIIa, and BEIIb associate with each other and with other starch biosynthetic enzymes although SSIIa is low activity in japonica rice. The aim of the current study was to understand how the activity of starch biosynthetic enzyme complexes is compensated in the absence of SSI or BEIIb, and whether the compensatory effects are different in the absence of BEIIb or in the presence of inactive BEIIb. Interactions between starch biosynthetic enzymes were analyzed using one ss1 null mutant and two be2b japonica rice mutants (a mutant producing inactive BEIIb and a mutant that did not produce BEIIb). Soluble proteins extracted from the developing rice seeds were separated by gel filtration chromatography. In the absence of BEIIb activity, BEIIa was eluted in a broad molecular weight range (60-700 kDa). BEIIa in the wild-type was eluted with a mass below 300 kDa. Further, majority of inactive BEIIb co-eluted with SSI, SSIIa, and BEI, in a mass fraction over 700 kDa, whereas only small amounts of these isozymes were found in the wild-type. Compared with the be2b lines, the ss1 mutant showed subtle differences in protein profiles, but the amounts of SSIIa, SSIVb, and BEI in the over-700-kDa fraction were elevated. Immunoprecipitation revealed reduced association of SSIIa and BEIIb in the ss1 mutant, while the association of BEIIb with SSI, SSIIa, SSIVb, BEI, and BEIIa were more pronounced in the be2b mutant that produced inactive BEIIb enzyme. Mass spectrometry and western blotting revealed that SSI, SSIIa, SSIIIa, BEI, BEIIa, starch phosphorylase 1, and pullulanase were bound to the starch granules in the be2b mutants, but not in the wild-type and ss1 mutant. These results will aid the understanding of the mechanism of amylopectin biosynthesis.

Dataset Information

Contributions of Three Starch Branching Enzyme Isozymes to the Fine Structure of Amylopectin in Rice Endosperm.

Publications

Contributions of Three Starch Branching Enzyme Isozymes to the Fine Structure of Amylopectin in Rice Endosperm.

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