title: Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne).
abstract: The main storage compounds in Lolium perenne are fructans with prevailing β(2-6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.
authors.affiliation: UMR INRA-UCBN 950 EVA Ecophysiologie Végétale, Agronomie & nutritions NCS, Université de Caen, Esplanade de la Paix, 14032 Caen Cedex, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Lasseur
authors.forename: Jérémy
authors.initials: J
authors.lastname: Lothier
authors.forename: Andres
authors.initials: A
authors.lastname: Wiemken
authors.forename: André
authors.initials: A
authors.lastname: Van Laere
authors.forename: Annette
authors.initials: A
authors.lastname: Morvan-Bertrand
authors.forename: Wim
authors.initials: W
authors.lastname: Van den Ende
authors.forename: Marie-Pascale
authors.initials: MP
authors.lastname: Prud'homme
mesh_terms: D000595:Amino Acid Sequence; D005630:Fructans / Q000096:biosynthesis*; D018506:Gene Expression Regulation, Plant; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D008129:Lolium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology / Q000254:growth & development; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D001426:Bacterial Proteins
chemical_list: D005630:Fructans; D006602:Hexosyltransferases; D010940:Plant Proteins; D011994:Recombinant Proteins; C021451:inulosucrase; D001426:Bacterial Proteins