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.

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M(r) of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers.

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia co/i), and a fungus (Neurospora crassa).These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.The three aromatic amino acids are synthesized via the shikimate pathway in microorganisms and plants (Fig. 1). Although the reaction sequence in the prechorismate pathway is identical in all organisms investigated so far, there are considerable differences in enzyme organization, as well as regulation, between organisms of different taxonomic groups. In Escherichia coli, the seven enzymatic activities of the prechorismate pathway are associated with monofunctional polypeptides. In contrast, in (reviewed in ref. 4). In Euglena gracilis, the organization of the prechorismate pathway appears to resemble that found in fungi. The first and the last steps are catalyzed by single enzymes (DAHP-synthase and chorismate synthase, respectively), whereas activities 2 to 6 form a large complex resembling the fungal arom complex (27). Euglena and fungi are strikingly similar in other biochemical pathways as well. Thus, both groups of organisms use the L-a-aminoadipate pathway for lysine biosynthesis, whereas bacteria, algae, and plants follow the diaminopimelate route (30). On the basis of such findings, a close evolutionary relationship was proposed for euglenoids and fungi. But aromatic biosynthesis shows several features that, among all organisms studied so far, are unique for E. gracilis. Anthranilate synthase, the first enzyme in the tryptophan branch ( Fig. 1), is monomeric in Euglena, whereas in all other anthranilate synthases characterized so far the chorismate and glutamine binding sites reside on distinct polypeptide chains (16). The synthesis of tryptophan from anthranilate is catalyzed by a single protein in Euglena (17), but in all other known examples tryptophan synthase is separable from the other activities of the tryptophan branch (17). Unlike ot...

The primary transcript of one of the two chorismate synthase genes (LeCS2) of tomato is differentially processed due to an alternative splicing of the third intron. A novel observation was made when the abundances of the two LeCS2-specific transcripts in different organs were analysed. The ratio of these two transcripts differs in RNA populations from different organs. Possible explanations for this finding and its potential physiological impact for plant metabolism are discussed.

To gain an increased understanding of the role of isoamylase (EC 3.2.1.68) in amylopectin synthesis, we studied the tissue-specific distribution and subcellular localisation of this enzyme in wheat (Triticum aestivum L.). A cDNA for wheat isoamylase was isolated from an endosperm-specific library and the missing 5' end was amplified by anchored polymerase chain reaction. Isoamylase transcripts were detected in reproductive and vegetative tissues, with the highest levels occurring in developing kernels. Wheat kernels were then dissected into embryo, endosperm, pericarp and chlorophyll layer, and subjected to protein blot analysis. Isoamylase was most abundant in the endosperm. Within the endosperm, the vast majority of isoamylase was soluble. A much smaller amount of the enzyme was associated with starch granules. Isoamylase was not trapped within starch granules and was absent from dry seeds. Isoamylase was also present in green tissue, which suggests a role in the synthesis of both reserve and leaf starches.