title: 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.
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.
authors.affiliation: Institute for Agricultural Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Streb
authors.affiliation: Institute for Agricultural Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Zeeman
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002236:Carbohydrate Conformation; D017124:Conserved Sequence; D015870:Gene Expression; D005816:Genetic Complementation Test; D007517:Isoamylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D013213:Starch; C000589120:ISA1 protein, Arabidopsis; C000589121:ISA2 protein, Arabidopsis; D007517:Isoamylase