Replacement of the Endogenous Starch Debranching Enzymes ISA1 and ISA2 of Arabidopsis with the Rice Orthologs Reveals a Degree of Functional Conservation during Starch Synthesis

Sebastian Streb,Samuel C Zeeman,Joerg Fettke

doi:10.1371/journal.pone.0092174

Abstract

This study tested the interchangeability of enzymes in starch metabolism between dicotyledonous and monocotyledonous plant species. Amylopectin - a branched glucose polymer - is the major component of starch and is responsible for its semi-crystalline property. Plants synthesize starch with distinct amylopectin structures, varying between species and tissues. The structure determines starch properties, an important characteristic for cooking and nutrition, and for the industrial uses of starch. Amylopectin synthesis involves at least three enzyme classes: starch synthases, branching enzymes and debranching enzymes. For all three classes, several enzyme isoforms have been identified. However, it is not clear which enzyme(s) are responsible for the large diversity of amylopectin structures. Here, we tested whether the specificities of the debranching enzymes (ISA1 and ISA2) are major determinants of species-dependent differences in amylopectin structure by replacing the dicotyledonous Arabidopsis isoamylases (AtISA1 and AtISA2) with the monocotyledonous rice (Oryza sativa) isoforms. We demonstrate that the ISA1 and ISA2 are sufficiently well conserved between these species to form heteromultimeric chimeric Arabidopsis/rice isoamylase enzymes. Furthermore, we were able to reconstitute the endosperm-specific rice OsISA1 homomultimeric complex in Arabidopsis isa1isa2 mutants. This homomultimer was able to facilitate normal rates of starch synthesis. The resulting amylopectin structure had small but significant differences in comparison to wild-type Arabidopsis amylopectin. This suggests that ISA1 and ISA2 have a conserved function between plant species with a major role in facilitating the crystallization of pre-amylopectin synthesized by starch synthases and branching enzymes, but also influencing the final structure of amylopectin.

Highlights

Starch is the major storage carbohydrate in plants and an important renewable resource for both the food and non-food industry sectors
To identify species with significant differences in amylopectin structure and exclude variability resulting from the different techniques used between different laboratories, we analysed the structure of amylopectin from starch extracted from two monocot species and two dicot species (Arabidopsis and potato)
We used the well-established method of chain length distribution (CLD) analysis, where the a-1,6-bonds of amylopectin are enzymatically hydrolysed, resulting in linear chains that can be separated and quantified by HPAEC-PAD

Summary

Introduction

Starch is the major storage carbohydrate in plants and an important renewable resource for both the food and non-food industry sectors. Starch is comprised of two glucose polymers (amylopectin and amylose) and accumulates in plant tissues as semi-crystalline granules. Amylopectin accounts for the majority of the granule mass (around 60% to 90%, depending on the botanical source). It is a branched molecule in which a-1,4-linked glucan chains are connected via a-1,6-bonds [1,2] resulting in a tree-like structure. The arrangement of branch points is thought to be non-random, such that linear chain segments can align together to form double helices that pack into stable, semi-crystalline lamellae. The branch points are concentrated in the amorphous regions between these crystalline lamellae [1]

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Conclusion