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.
Fructans, which are beta-(2,1) and/or beta-(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) 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 beta-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.
Lothier
Jérémy
J
UMR INRA-UCBN 950 EVA Ecophysiologie Végétale, Agronomie et Nutritions NCS, Université de Caen, Esplanade de la Paix, 14032 Caen cedex, France.
Lasseur
Bertrand
B
Le Roy
Katrien
K
Van Laere
André
A
Prud'homme
Marie-Pascale
MP
Barre
Philippe
P
Van den Ende
Wim
W
Morvan-Bertrand
Annette
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005630:Fructans / Q000096:biosynthesis* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008129:Lolium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016415:Sequence Alignment
D005630:Fructans; D010940:Plant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C044118:fructan beta-fructosidase