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JXB Advance Access originally published online on April 24, 2007
Journal of Experimental Botany 2007 58(8):1969-1983; doi:10.1093/jxb/erm053
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© The Author [2007]. 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

RESEARCH PAPER

Cloning, gene mapping, and functional analysis of a fructan 1-exohydrolase (1-FEH) from Lolium perenne implicated in fructan synthesis rather than in fructan mobilization

Jérémy Lothier1, Bertrand Lasseur1, Katrien Le Roy2, André Van Laere2, Marie-Pascale Prud'homme1, Philippe Barre3, Wim Van den Ende2 and Annette Morvan-Bertrand1,*

1UMR INRA-UCBN 950 EVA Ecophysiologie Végétale, Agronomie et Nutritions NCS, Université de Caen, Esplanade de la Paix, 14032 Caen cedex, France
2Department of Biology, Laboratory for Molecular Plant Physiology, Institute of Botany and Microbiology, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium
3INRA-UGAPF 889, F-86600 Lusignan, France

* To whom correspondence should be addressed. E-mail: annette.bertrand{at}unicaen.fr

Fructans, which are ß-(2,1) and/or ß-(2,6) linked polymers of fructose, are important storage carbohydrates in many plants. They are mobilized via fructan exohydrolases (FEHs). The cloning, mapping, and functional analysis of the first 1-FEH (EC 3.2.1.153 [EC] ) from Lolium perenne L. var. Bravo is described here. By screening a perennial ryegrass cDNA library, a 1-FEH cDNA named Lp1-FEHa was cloned. The Lp1-FEHa deduced protein has a low iso-electric point (5.22) and it groups together with plant FEHs and cell-wall type invertases. The deduced amino acid sequence shows 75% identity to wheat 1-FEH w2. The Lp1-FEHa gene was mapped at a distal position on the linkage group 3 (LG3). Functional characterization of the recombinant protein in Pichia pastoris demonstrated that it had high FEH activity towards 1-kestotriose, 1,1-kestotetraose, and inulin, but low activity against 6-kestotriose and levan. Like other fructan-plant FEHs, no hydrolase activity could be detected towards sucrose, convincingly demonstrating that the enzyme is not a classic invertase. The expression pattern analysis of Lp1-FEHa revealed transcript accumulation in leaf tissues accumulating fructans while transcript level was low in the photosynthetic tissues. The high expression level of this 1-FEH in conditions of active fructan synthesis, together with its low expression level when fructan contents are low, suggest that it might play a role as a ß-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.

Key words: Fructan biosynthesis, fructan exohydrolase, gene mapping, Lolium perenne, Pichia pastoris

Received 9 January 2007; Revised 23 February 2007 Accepted 26 February 2007


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