Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants

Abstract

It has been generally assumed that the [alpha]-(1->4)-linked and [alpha]-(1->6)-branched glucans of starch are generated by the coordinated action of elongation (starch synthases) and branching enzymes. We have identified a novel Chlamydomonas locus (STA7) that when defective leads to a wipeout of starch and its replacement by a small amount of glycogen-like material. Our efforts to understand the enzymological basis of this phenotype have led us to determine the selective disappearance of an 88-kD starch hydrolytic activity. We further demonstrate that this enzyme is a debranching enzyme. Cleavage of the [alpha]-(1->6) linkage in a branched precursor of amylopectin (preamylopectin) has provided us with the ground rules for understanding starch biosynthesis in plants. Therefore, we propose that amylopectin clusters are synthesized by a discontinuous mechanism involving a highly specific glucan trimming mechanism.

Functional Interactions between Starch Synthase III and Isoamylase-Type Starch-Debranching Enzyme in Maize Endosperm

“…Residual starch granules in ISA1-deficient mutants are abnormal in size, shape, and number and contain structurally altered amylopectin. These observations are remarkably consistent across species and tissues, including maize (Zea mays), rice (Oryza sativa), and barley (Hordeum vulgare) endosperm, potato (Solanum tuberosum) tuber, Arabidopsis (Arabidopsis thaliana) leaf, and Chlamydomonas reinhardtii cells (James et al, 1995;Mouille et al, 1996;Zeeman et al, 1998;Kubo et al, 1999;Dauvillée et al, 2001a;Burton et al, 2002;Bustos et al, 2004;Delatte et al, 2005;Wattebled et al, 2005Wattebled et al, , 2008Streb et al, 2008). Thus, ISA1 and ISA2 appear to have been adapted primarily for activity in starch biosynthesis rather than degradation.…”

Biochemical Characterization of the Chlamydomonas reinhardtii α-1,4 Glucanotransferase Supports a Direct Function in Amylopectin Biosynthesis1

“…5, A and B). We found no evidence of oligosaccharide production by spotting the treated samples on TLC plates as described in Mouille et al (1996). The amount of amyloglucosidaseresistant material (cyclic glucans) remained under 5% of the total polysaccharide amounts used in these experiments, suggesting that incubation times exceeding 12 h and higher enzyme activities are required to produce these structures.…”

“…Our zymogram procedures were established to detect starch hydrolases in starch-containing gels in Mouille et al (1996). The oligosaccharide-incorporation zymogram technique was modified from these procedures as follows.…”

“…Following the technique established in Mouille et al (1996), polysaccharide was eluted from the bands present in the amylopectin-containing gels and subjected to 1 H-NMR analysis. A similar analysis was performed after 135-fold purification of the enzyme activity (see above) and treatment of amylopectin in purification buffer.…”

Convergent Evolution of Polysaccharide Debranching Defines a Common Mechanism for Starch Accumulation in Cyanobacteria and Plants

“…Clues as to the nature of the biochemical mechanism distinguishing starch from glycogen synthesis came from the study of glycogen-accumulating mutants of Chlamydomonas and cereals that proved defective for the same GH13-type (for Glycosyl Hydrolase family 13, according to the carbohydrate active enzyme (CAZy) classification) of debranching enzyme (DBE; James et al, 1995;Mouille et al, 1996;Kubo et al, 1999). In the green algae, the substitution of starch by glycogen was complete, thereby hinting that the absence of DBE prevented amylopectin synthesis altogether.…”