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...

Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin

“…This is somewhat similar to results found with two mutants affected in SS activity in pea [7] and Chlamydomonas [9] and, therefore, this may be a general feature of a reduction in SS activity. Unlike the two mutants, however, reductions in the relative proportion of chains of dp between 20 and 40 within the amylopectin of the transgenic lines were not so apparent.…”

“…In the present study, as in the pea rug5 mutant and the C. rheinhardtii sta3 mutant [7,23], the alteration in SS activity led to a change in the shape of the starch granule ( Figure 2). As was suggested for the pea mutant we think that this is likely to be due to the alteration in branching structure of the amylopectin, which may lead to a change in the crystallinity of the granule.…”

“…In these lines, as the SS activity was reduced, the number of chains eluting in fraction 2 was also reduced but, also, specific chains of dp between approximately 20 and 40 in length within fraction 1 were decreased in number ( Figures 3D-3F and 4D-4F). As such, these lines produce amylopectin that is more similar to that found in the rug5 and sta3 mutants [7,9] than when only the SSIII or SSII isoform is reduced.…”

“…In pea the mutation has been shown to be in a gene coding for a SS isoform that is similar to SSII from potato [7]. Studies have shown that this enzyme contributes up to 60 % of the SS activity in the pea embryo [24,25].…”

“…Recently, two mutant genes have been cloned from different plant species, one in pea (rug5) [7] and one in maize (dull1) [8], both of which were shown to be mutations in SS enzymes. In addition, a mutation in the green alga Chlamydomonas rheinhardtii (sta3) [9], has been isolated which causes the organism to have a reduced SS activity.…”