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JXB Advance Access first published online on July 13, 2006
This version published online on July 25, 2006
Journal of Experimental Botany, doi:10.1093/jxb/erl034
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received January 24, 2006
Accepted April 24, 2006

RESEARCH PAPER

Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.)

Bertrand Lasseur 1, Jérémy Lothier 1, Abdelmadjid Djoumad 2, Barbara De Coninck 3, Sjef Smeekens 4, André Van Laere 3, Annette Morvan-Bertrand 1, Wim Van den Ende 3, and Marie-Pascale Prud'homme 1 *

1 UMR INRA-UCN 950 EVA Ecophysiologie Végétale, Agronomie et Nutritions NCS, Université de Caen, Esplanade de la Paix, F-14032 Caen cedex, France
2 Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbooke, QC, Canada
3 Department of Biology, Laboratory for Molecular Plant Physiology, Botany Institute, KULeuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium
4 Department of Molecular Plant Physiology, University Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

* To whom correspondence should be addressed.
Marie-Pascale Prud'homme, E-mail: marie-pascale.prudhomme{at}unicaen.fr


  Abstract

Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.

Keywords: Fructan; fructan:fructan 6G-fructosyltransferase; gene expression; heterologous expression; Lolium perenne; Pichia pastoris; sucrose:sucrose 1-fructosyltransferase.
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