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

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.

Starch Biosynthesis in the Developing Endosperms of Grasses and Cereals

“…SBEs catalyse a non-reversible reaction generating α-(1→6) branch linkages in α-(1→4)-linked glucans by hydrolytic cleavage of internal α-(1→4) bonds and transfer of the released reducing ends to C6 hydroxyls to form the branched structure of amylopectin [259]. The action of SBE creates a new non-reducing end oligosaccharide chain, which can be elongated by SSs, and this action can also stimulate SS activity [260,261]. The actions of different SBE isoforms in conjunction with specific SSs (above) has a profound impact on amylopectin cluster characteristics and granule architecture [262][263][264][265], and genetic studies in Arabidopsis have shown that specific SBE isoforms are required to complement the loss of endogenous SBEs in order to form crystalline competent starch [266].…”

Biosynthesis of Reserve Starch

“…It was also established that SS and BE were much more preferentially reactive toward branched glucans than linear MOS and dextrins/glucans (Nakamura et al 2010 and many references therein), suggesting that the actual glucan substrates for SS and BE during in vivo starch synthesis are the dual parallel chains or the double helical chains, respectively, of the branched glucans/dextrins, as proposed by Borovsky et al (1979) for BE. Recently, it was found that SSI from rice and SS from Arabidopsis (Brust et al 2014) could efficiently synthesize glucans by interacting closely with BE isozymes, possibly forming the protein complex, in the absence of added primer as found in the interaction between Pho1 and BE . It is noted that these interactions were mediated by branched glucans, but not by linear glucans Brust et al 2014).…”

“…Recently, it was found that SSI from rice and SS from Arabidopsis (Brust et al 2014) could efficiently synthesize glucans by interacting closely with BE isozymes, possibly forming the protein complex, in the absence of added primer as found in the interaction between Pho1 and BE . It is noted that these interactions were mediated by branched glucans, but not by linear glucans Brust et al 2014). These results strongly suggest that at least SSI, BEs, and Pho1 can closely interact with their counterparts including many types of glucans varied under different physiological conditions.…”

Protein-Protein Interactions During Starch Biosynthesis

“…Previous work also showed that purified SS was stimulated by SBE (Hawker et al 1974;Boyer and Preiss 1979;Pollock and Preiss 1980), probably through an increase in nonreducing ends provided by branching enzyme activity. Studies using recombinant forms of Arabidopsis starch synthase isoforms 1-4, combined with either recombinant BE2 or BE3 (both equivalent to SBE class II branching enzymes of cereals), produced polyglucans of varying structure (Brust et al 2014). Interestingly, when combining recombinant SSI with either form of SBE, the chain length distribution (CLD) pattern, following isoamylase debranching of the glucan product, closely resembled that of wild-type starch extracted from leaves.…”