Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.)

Abstract

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.

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

“…Production of glucans by the different isoforms was visualized by staining with Lugols solution. Native gels staining for SS activity showed three bands, as is normal for potato tubers [10] (Figure 1). The band migrating fastest was the SSI isoform, that migrating slowest was the SSIII isoform, whereas the SSII isoform migrated at intermediate speed.…”

“…Both the activity of the soluble SS, and the production of nondenaturing gels used to visualize the SS activities of the different isoforms, were measured using methods published previously [10] except that 500 mM sodium citrate was added to the assay buffer for the soluble SS activity. GBSS activity was measured using the same method as for the soluble activity.…”

“…The starch content of the potato tubers was measured using a previously published method [10]. The amylose content of the purified starch was measured by two different methods ; first by quantitatively precipitating the amylopectin using the lectin Concanavalin A according to the instructions from a kit (Megazyme International), and secondly by an iodine-binding assay [18].…”

“…Both immunoprecipitation and antisense experiments have shown that one isoform (SSIII) [10,11] contributes the major activity (80 % of the total) in potato tubers, with a second one (SSII) [5] being responsible for the majority (15 % of the total) of the remainder. When the activity of either of these isoforms is reduced using antisense techniques no differences are found in the constituent chains within starch [5,10,12]. In this paper we report on the reduction in activity of both of these isoforms simultaneously, which leads to a gross alteration in the amylopectin.…”

Interaction with amylopectin influences the ability of granule-bound starch synthase I to elongate malto-oligosaccharides

“…First, the amylopectin-synthesizing isoforms have Nterminal domains which GBSSI lacks (SSI [3,4] ; SSII [5,6] ; SSIII [7][8][9]). The amino acid sequences of the N-terminal domains are not highly conserved between orthologues from different plant species, and their function is unknown.…”