Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

Abstract

Several studies have suggested that debranching enzymes (DBEs) are involved in the biosynthesis of amylopectin, the major constituent of starch granules. Our systematic analysis of all DBE mutants of Arabidopsis thaliana demonstrates that when any DBE activity remains, starch granules are still synthesized, albeit with altered amylopectin structure. Quadruple mutants lacking all four DBE proteins (Isoamylase1 [ISA1], ISA2, and ISA3, and Limit-Dextrinase) are devoid of starch granules and instead accumulate highly branched glucans, distinct from amylopectin and from previously described phytoglycogen. A fraction of these glucans are present as discrete, insoluble, nanometer-scale particles, but the structure and properties of this material are radically altered compared with wild-type amylopectin. Superficially, these data support the hypothesis that debranching is required for amylopectin synthesis. However, our analyses show that soluble glucans in the quadruple DBE mutant are degraded by a-and b-amylases during periods of net accumulation, giving rise to maltose and branched malto-oligosaccharides. The additional loss of the chloroplastic a-amylase AMY3 partially reverts the phenotype of the quadruple DBE mutant, restoring starch granule biosynthesis. We propose that DBEs function in normal amylopectin synthesis by promoting amylopectin crystallization but conclude that they are not mandatory for starch granule synthesis.

Significance of Light, Sugar, and Amino Acid Supply for Diurnal Gene Regulation in Developing Barley Caryopses

“…7,8,and 10). A similar function was recently demonstrated for isoamylase paralogs in Arabidopsis (Streb et al, 2008). The specific roles of HvISA1 and HvISA2 remain unclear (Burton et al, 2002), and neither seems to be under diurnal control (Table I).…”

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.…”

“…Thus, ISA1 and ISA2 appear to have been adapted primarily for activity in starch biosynthesis rather than degradation. The role of these DBEs appears to be remodeling of a soluble glucan precursor by the removal of certain branch linkages, thus facilitating the transition to the semicrystalline state Myers et al, 2000;Streb et al, 2008).…”

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

“…First, Streb et al (2008) reported that in a particular mutant background lacking starch DBE, the presence of an additional mutation in a particular plastidial a-amylase restored starch synthesis in Arabidopsis leaves. This may imply that starch granule aggregation could dispense with the postulated debranching mechanism, although the crystallinity of this residual material still needs to be ascertained.…”