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.

Homologs of PROTEIN TARGETING TO STARCH Control Starch Granule Initiation in Arabidopsis Leaves

“…However, ISA1-containing isoamylase is known to differ in the endosperms of rice and other cereals compared with Arabidopsis leaves and other dicots like potato. In rice endosperms, ISA1 is present primarily as a homodimer, whereas in Arabidopsis, it is exclusively present as an ISA1/ISA2 complex (Delatte et al, 2005;Utsumi and Nakamura, 2006;Streb and Zeeman, 2014).…”

Biosynthesis of Reserve Starch

“…5.4a and 5.5) was reported by James et al (1995) using the gene-tagging method. Since then the essential roles of DBEs in amylopectin synthesis have been verified by extensive investigations of a variety of mutants and transformants from maize (James et al 1995;Beatty et al 1999;Dinges et al 2001;Dinges et al 2003;Kubo et al 2010;Lin et al 2012Lin et al , 2013, rice (Nakamura et al , 1997Kubo et al 1999Kubo et al , 2005Fujita et al 2003Fujita et al , 2009Utsumi et al 2011), potato (Hussain et al 2003;Bustos et al 2004), barley (Burton et al 2002), Arabidopsis (Zeeman et al 1998;Dellate et al 2005;Wattebled et al 2005Wattebled et al , 2008Streb et al 2008;Pfister et al 2014;Streb and Zeeman 2014), Chlamydomonas (Mouille et al 1996;Colleoni et al 1999aColleoni et al , 1999bDauvillée et al 2000Dauvillée et al , 2001Sim et al 2014), and Cyanobacterium sp. CLg1 (Cenci et al 2013) (also see review by Hennen-Bierwagen et al 2012), although in this chapter experimental results with maize and rice endosperm are mainly described.…”

Starch Biosynthesis in Leaves and Its Regulation

“…The isa1 isa2 double mutant accumulates large amounts of phytoglycogen while the starch content is strongly reduced. Expression of ISA1 from maize or rice in the isa1 isa2 mutant restores wild-type starch synthesis (Facon et al 2013;Streb and Zeeman 2014). This result indicates that ISA1 from cereals can function without ISA2 even in a heterologous physiological context.…”