Transgenic potato tubers accumulate high levels of 1-kestose and nystose: functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke (Cynara scolymus) blossom discs.
By screening a cDNA library of artichoke (Cynara scolymus) blossom discs for fructosyltransferases, we isolated a clone designated Cy21. The deduced amino acid sequence shows homology to acid beta-fructosyl hydrolases and to the sucrose-fructan 6-fructosyltransferase (6-SFT) of barley. Transiently expressed in Nicotiana tabacum protoplasts, the Cy21 gene-product synthesized 1-kestose, indicating that Cy21 codes for a sucrose sucrose 1-fructosyltransferase (1-SST). The enzyme worked at physiologically relevant sucrose concentrations (25 mM sucrose). In the protoplast system, 1-kestose seemed to be the only fructan product of the 1-SST. The enzyme activity was not affected by pyridoxal-HCl, an inhibitor of both the beta-fructosyl hydrolase and the fructosyltransferase activity of invertases. The fructosyltransferase activity of the Cy21 gene-product, however, could be inhibited by Zn2+, Ag+ and Cu2+ ions. In artichoke plants the Cy21 transcript was highly abundant in primary roots and blossom discs. Transgenic potato tubers expressing Cy21 contain high levels of 1-kestose along with nystose and traces of fructosyl-nystose, supporting the conclusion that the Cy21 clone encodes a sucrose sucrose 1-fructosyltransferase.
Hellwege
E M
EM
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm, Germany. hellwege@mpimp-golm.mpg.de
Gritscher
D
D
Willmitzer
L
L
Heyer
A G
AG
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015723:Gene Library; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D018517:Plant Roots; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011523:Protoplasts / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011198:Solanum tuberosum / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism; D014026:Nicotiana; D014162:Transfection; D014312:Trisaccharides / Q000096:biosynthesis*; D014675:Vegetables / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D006602:Hexosyltransferases; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013395:Sucrose; D014312:Trisaccharides; C072433:1-kestose; C043600:fungitetraose; C097181:sucrose - fructan 6-fructosyltransferase; C021451:inulosucrase; D001426:Bacterial Proteins