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...

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...

SummaryThe biochemical and molecular basis of the wrinkledseeded phenotype of rug4 mutants of pea (Pisum sativum L.) has been investigated. Mutant embryos have reduced starch contents and only 5% of the sucrose synthase activity of wild-type embryos during development. Activities of other enzymes involved in the conversion of sucrose to starch are unaffected. A gene encoding an isoform of sucrose synthase expressed in the embryo co-segregates with the rug4 locus, and one of the three mutant alleles has been show to carry a point mutation in this gene that converts a highly conserved arginine residue to a lysine residue. It is highly likely that the reduced starch content of the mutant embryo is a direct consequence of the loss of sucrose synthase activity. The mutations reduce the activity of sucrose synthase in the testa and the leaf by 50% or less, but activity in Rhizobium-infected root nodules is reduced by 85%. Although the nodules of mutant plants contain metabolically active bacteroids, the N content and δ 15 N values of these plants in the field indicate that, unlike wild-type plants, they derive little of their N from N 2 fixation via Rhizobium. Sucrose synthase thus appears to be essential for the supply of carbon for bacteroid metabolism and/or ammonia assimilation during nitrogen assimilation.

The subcellular location of activity and protein of ADP-glucose pyrophosphorylase (AGPase) in developing tomato (Lycopersicon esculentum) fruit was determined following a report that the enzyme might be present inside and outside the plastids in this organ. Plastids prepared from crude homogenates of columella and pericarp, the starch-accumulating tissues of developing fruit, contained 8% to 18% of the total activity of enzymes known to be confined to plastids, and 0.2% to 0.5% of the total activity of enzymes known to be confined to the cytosol. The proportion of the total activity of AGPase in the plastids was the same as that of the enzymes known to be confined to the plastid. When samples of plastid and total homogenate fractions were subjected to immunoblotting with an antiserum raised to AGPase, most or all of the protein detected was plastidial. Taken as a whole, these data provide strong evidence that AGPase is confined to the plastids in developing tomato fruit.

Isoforms of starch synthase (EC 2.4.1.21) in pea (Pisum sativum L.) leaves have been identi®ed and compared with those in developing pea embryos. Puri®cation and immunoprecipitation experiments show that most of the soluble starch synthase activity of the leaf is contributed by a novel isoform (SSIII) that is antigenically related to the major soluble isoform of the potato tuber. The major soluble isoform of the embryo (SSII) is also present in the leaf, but contributes only 15% of the soluble activity. Study of the leaf starch of lam mutant peas, which lack the abundant granule-bound isoform responsible for amylose synthesis in the embryo (GBSSI), indicates that GBSSI is not responsible for the synthesis of amylose-like material in the leaf. Leaves appear to contain a novel granule-bound isoform, antigenically related to GBSSI. The implications of the results for understanding of the role of isoforms of starch synthase are discussed.

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