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

Takayuki Sawada,Mizuho Itoh,Yasunori Nakamura

doi:10.3389/fpls.2018.01536

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 ΔBEI/BEIIb line compared with the ΔBEIIb 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 ΔBEIIa line, the role of BEIIa could be deciphered by analyzing amylopectin fine structure from the ΔBEI/BEIIa/BEIIb line in comparison to that from ΔBEI/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 ΔBEIIa/BEIIb line was significantly higher than that in the ΔBEIIb line. In summary, the present study highlights the distinct roles of BEI, BEIIa, and BEIIb in the synthesis of amylopectin in developing rice endosperm.

Highlights

Starch branching enzyme (BE) is the only enzyme capable of forming the branch linkages in amylopectin, a major starch component, that usually comprises 65–85% of starch
Out of about 30 lines regenerated from each RNAi construct, 6–9 in which enzyme activities were suppressed as assessed by zymogram (Figure 2) were selected: #1, 2, 8, 12, 14, 19, and 25 for BEI lines; #2, 4, 5, 6, 8, 10, and 14 for BEIIa lines; #2, 5, 6, 8, 12, 13, 15, and 27 for BEIIb lines; #3, 11, 14, 17, 27, and 29 for BEI/BEIIa lines; #2, 5, 6, 9, 11, 13, 16, 22, and 27 for BEI/BEIIb lines; #4, 6, 7, 11, 12, and for BEIIa/BEIIb lines; and #4, 7, 9, 12, 24, and for BEI/BEIIa/BEIIb lines
When BEIIb activity is lost, the band color turns blue because only linear glucans are synthesized by phosphorylase 1 (Pho1) in the presence of glucose 1phosphate (Yamanouchi and Nakamura, 1992)

Summary

Introduction

Starch branching enzyme (BE) is the only enzyme capable of forming the branch linkages in amylopectin, a major starch component, that usually comprises 65–85% of starch. By using high-performance anion-exchange chromatographypulsed amperometric detection (HPAEC-PAD) or fluorophoreassisted carbohydrate electrophoresis (FACE), the contribution of each BE isozyme to the fine structure of amylopectin was analyzed by precisely measuring the chain-length distribution of amylopectin formed in mutants and transformants in which the activities of BE isozymes were singly or multiply modified (see the review by Nakamura, 2015, 2018; Tetlow and Emes, 2017) In these analyses, the lengths of α-1,4 chains [i.e., their degree of polymerization (DP) values] liberated after debranching the α1,6 glucosidic linkages (branches) of amylopectin with isoamylase (ISA) were measured by the FACE method. Chain-distribution analysis of glucans formed by in vitro BE enzymatic reactions confirmed the chain-length specificity of each isozyme and characterized its enzymatic properties, such as substrate specificity toward branched and linear glucans and maltooligosaccharides (Nakamura et al, 2010; Sawada et al, 2014; see the review by Nakamura, 2015)

Methods

Results

Conclusion