Mutations in the Gene Encoding Starch Synthase II Profoundly Alter Amylopectin Structure in Pea Embryos

Abstract

Mutations at the rug5 ( rug osus 5 ) locus have been used to elucidate the role of the major soluble isoform of starch synthase II (SSII) in amylopectin synthesis in the developing pea embryo. The SSII gene maps to the rug5 locus, and the gene in one of three rug5 mutant lines has been shown to carry a base pair substitution that introduces a stop codon into the open reading frame. All three mutant alleles cause a dramatic reduction or loss of the SSII protein. The mutations have pleiotropic effects on the activities of other isoforms of starch synthase but apparently not on those of other enzymes of starch synthesis. These mutations result in abnormal starch granule morphology and amylopectin structure. Amylopectin contains fewer chains of intermediate length (B 2 and B 3 chains) and more very short and very long chains than does amylopectin from wild-type embryos. The results suggest that SSII may play a specific role in the synthesis of B 2 and B 3 chains of amylopectin. The extent to which these findings can be extrapolated to other species is discussed. INTRODUCTIONThe starch synthases that catalyze the synthesis of the branched amylopectin component of the starch granule are poorly understood. It is well established that a specific class of granule-bound starch synthases (known as granulebound starch synthase I or GBSSI) is responsible for the synthesis of the unbranched amylose component of the granule. Two or more distinct isoforms other than GBSSI are present in storage organs of the species examined to date, and these together with starch branching enzymes (SBEs) are responsible for the synthesis of amylopectin. However, it is not clear whether different isoforms play qualitatively distinct roles in amylopectin synthesis . Study of the roles of such isoforms is hampered by a lack of mutations that affect them specifically and exclusively. Analysis of transgenic potato plants in which activities of specific isoforms have been reduced (Edwards et al., 1995;Abel et al., 1996;Marshall et al., 1996) has thus far not revealed whether these isoforms have qualitatively distinct roles.In this study, we elucidate the role of the major isoform of starch synthase present in the soluble fraction of the developing pea embryo. Starch synthase II (SSII) is a protein of 77 kD that accounts for 60 to 70% of the soluble starch synthase activity of the pea embryo. It is also present within the matrix of the starch granule (Smith, 1990;Denyer and Smith, 1992; Dry et al., 1992; Denyer et al., 1993;Edwards et al., 1996). Analysis of mutant lines of peas from which GBSSI is absent has shown conclusively that SSII is not involved in amylose synthesis (Denyer et al., 1995a). It is reasonable to assume, therefore, that SSII is important in the synthesis of amylopectin.Amylopectin is a highly branched polymer consisting of linear chains of ␣ (1,4)-linked glucose residues joined together by ␣ (1,6)-linkages. Within the granule, the chains are thought to be arranged in clusters at intervals of 9 nm, within which chains associate t...

Natural variation in rice starch synthase IIa affects enzyme and starch properties

“…The chain-length distribution of amylopectin from the grain of Nipponbare, which lacked functional SSIIa, is typical in that short chains of DP 7-11 are enriched and those of DP 13-23 are suppressed compared to NILs(Alk) and Kasalath having the functional SSIIa allele. This difference in chain length has been observed in starch from the SSII mutant of the pea embryo (Craig et al 1998), SSII-antisensed potato tuber (Edwards et al 1999), SGP-1 (SSIIa protein) lacking wheat endosperm (Yamamori et al 2000;Li et al 1999), and the SSIIa mutant of barley endosperm (Morell et al 2003). These observations support the view that the rice SSIIa gene is the causative gene for differences in the chain-length distribution of amylopectin between japonica and indica varieties.…”

The action of starch synthase II on 6′′′‐α‐maltotriosyl‐maltohexaose comprising the branch point of amylopectin

“…The pea low-amylose (lam ) mutant line (SIM 503) [16] was derived from round-seeded wild-type pea Pisum sativum L. (line BCl/RR) as described by Denyer et al [17]. Plants were grown as described previously [17].…”

A minor form of starch branching enzyme in potato ( Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A

“…Studies involving leaf starch synthesis in peas also suggest that SBE may not be the most important factor controlling the polymodal distribution of glucan chain lengths in amylopectin (Tomlinson et al, 1997). Recently, mutations in starch synthases were identified in pea (rug5) and maize (dull1) and were shown to dramatically affect the amylopectin structure and chain length distribution suggesting that starch synthases may be more important than SBE in this respect (Craig et al, 1998;Gao et al, 1998).…”