Relationships between starch synthase I and branching enzyme isozymes determined using double mutant rice lines

Abstract

BackgroundStarch is the most important carbohydrate in plant storage tissues. Multiple isozymes in at least four enzyme classes are involved in starch biosynthesis. Some of these isozymes are thought to interact and form complexes for efficient starch biosynthesis. Of these enzyme classes, starch synthases (SSs) and branching enzymes (BEs) play particularly central roles.ResultsWe generated double mutant lines (ss1/be1 and ss1 L /be2b) between SSI (the largest component of total soluble SS activity) and BEI or BEIIb (major BEs in developing rice endosperm) to explore the relationships among these isozymes. The seed weight of ss1/be1 was comparable to that of wild type, although most ss1/be2b seeds were sterile and no double recessive plants were obtained. The seed weight of the double recessive mutant line ss1 L /be2b, derived from the leaky ss1 mutant (ss1 L ) and be2b, was higher than that of the single be2b mutant. Analyses of the chain-length distribution of amylopectin in ss1/be1 endosperm revealed additive effects of SSI and BEI on amylopectin structure. Chain-length analysis indicated that the BEIIb deficiency significantly reduced the ratio of short chains in amylopectin of ss1 L /be2b. The amylose content of endosperm starch of ss1/be1 and ss1 L /be2b was almost the same as that of wild type, whereas the endosperm starch of be2b contained more amylose than did that of wild type. SSI, BEI, and BEIIb deficiency also affected the extent of binding of other isozymes to starch granules.ConclusionsAnalysis of the chain-length distribution in amylopectin of the double mutant lines showed that SSI and BEI or BEIIb primarily function independently, and branching by BEIIb is followed by SSI chain elongation. The increased amylose content in be2b was because of reduced amylopectin biosynthesis; however, the lower SSI activity in this background may have enhanced amylopectin biosynthesis as a result of a correction of imbalance between the branching and elongation found in the single mutant. The fact that a deficiency of SSI, BEI, or BEIIb affected the affinity of other starch biosynthetic isozymes for the starch granule implies that there is a close interaction among SSI, BEI and BEIIb during amylopectin biosynthesis in rice endosperm.

Protein-Protein Interactions During Starch Biosynthesis

“…This adds considerable weight to the argument that it is the heteromeric complexes which are the functionally active components during amylopectin biosynthesis. Similar changes in the starch granule proteome have been observed in rice mutants (Abe et al 2014) where mutation in SBEIIb led to the appearance of SBEI in granules. In wheat, barley and rice mutants which lack a functional SSIIa (Yamamori et al 2000;Morell et al 2003;Umemoto and Aoki 2005), the granules also become devoid of SSI and SBEII class proteins.…”

Biosynthesis of Reserve Starch

“…These include reactivity toward various glucans including branched glucans (amylopectin and glycogen), amylose, and malto-oligosaccharides (MOS) (Tetlow 2012); chemicals such as cyclodextrins (Vikso-Nielsen and Blennow 1998), phosphorylated metabolites (Morell et al 1997), and citrate (Hamada et al , 2002; temperature dependence of activities Hamada et al 2002;Ohdan et al 2011;Sawada et al 2013); and association with starch granules (Rahman et al 1995;Mu-Forster et al 1996;Hamada et al 2001Hamada et al , 2002Grimaud et al 2008;Liu et al 2012b;Abe et al 2014). For other properties of BE, see review by Tetlow (2012).…”

Rice Endosperm Starch Phosphorylase (Pho1) Assembles with Disproportionating Enzyme (Dpe1) to Form a Protein Complex That Enhances Synthesis of Malto-oligosaccharides

“…In rice endosperm, protein-protein interactions occur between or among starch synthases (SSs), branching enzymes (BEs), pullulanase, and OsPho1 (21). In wheat, it has been reported that Pho1 interacts with BEI and BEIIb in an ATP-dependent manner in soluble extracts of developing endosperms harvested at mid-to-late developmental stage (14).…”