Analysis of the functional interaction of Arabidopsis starch synthase and branching enzyme isoforms reveals that the cooperative action of SSI and BEs results in glucans with polymodal chain length distribution similar to amylopectin.

Henrike Brust,Christophe D'Hulst,Tanja Lehmann,Joerg Fettke,Frederik Börnke

doi:10.1371/journal.pone.0102364

Henrike Brust, Christophe D'Hulst + Show 3 more

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https://doi.org/10.1371/journal.pone.0102364

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Abstract

Starch synthase (SS) and branching enzyme (BE) establish the two glycosidic linkages existing in starch. Both enzymes exist as several isoforms. Enzymes derived from several species were studied extensively both in vivo and in vitro over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from in vitro studies including both interaction of leaf derived and heterologously expressed SS and BE isoforms. We found that SSI activity in native PAGE without addition of glucans was dependent on at least one of the two BE isoforms active in Arabidopsis leaves. This interaction is most likely not based on a physical association of the enzymes, as demonstrated by immunodetection and native PAGE mobility analysis of SSI, BE2, and BE3. The glucans formed by the action of SSI/BEs were analysed using leaf protein extracts from wild type and be single mutants (Atbe2 and Atbe3 mutant lines) and by different combinations of recombinant proteins. Chain length distribution (CLD) patterns of the formed glucans were irrespective of SSI and BE isoforms origin and still independent of assay conditions. Furthermore, we show that all SS isoforms (SSI-SSIV) were able to interact with BEs and form branched glucans. However, only SSI/BEs generated a polymodal distribution of glucans which was similar to CLD pattern detected in amylopectin of Arabidopsis leaf starch. We discuss the impact of the SSI/BEs interplay for the CLD pattern of amylopectin.

Highlights

Starch is the major storage carbohydrate in plants and forms insoluble granules consisting of two glucose polymers, amylose and amylopectin
We demonstrated that SSI activity in native PAGE is dependent on at least one of the branching enzyme (BE)-isoforms and independent of external applied glucans
Characterization of starch synthase activities Starch synthase activity was analysed in Arabidopsis leaf extracts (Fig. 1)

Summary

Introduction

Starch is the major storage carbohydrate in plants and forms insoluble granules consisting of two glucose polymers, amylose and amylopectin. In both a-1,4-glycosidic bonds are present but only the latter contains significant amounts of a-1,6-glycosidic bonds responsible for branching of the glucan chains [1,2]. Amylopectin, as the major glucose polymer, accounts for 70–80% of the starch and forms a semi-crystalline structure with alternating crystalline and amorphous lamellae having a conserved dimension of 9– 10 nm [3,4] The basis of this semi-crystalline structure is related to the high order organization of branch points within the starch [5]. Long chains (B2, B3), which interconnect different clusters, are thought to alternate tangentially or radially [8,9,10]

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