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.).
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.
Lasseur
Bertrand
B
UMR INRA-UCN 950 EVA Ecophysiologie Végétale, Agronomie et Nutritions NCS, Université de Caen, Esplanade de la Paix, F-14032 Caen cedex, France.
Lothier
Jérémy
J
Djoumad
Abdelmadjid
A
De Coninck
Barbara
B
Smeekens
Sjef
S
Van Laere
André
A
Morvan-Bertrand
Annette
A
Van den Ende
Wim
W
Prud'homme
Marie-Pascale
MP
D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D018076:DNA, Complementary / Q000737:chemistry; D005630:Fructans / Q000096:biosynthesis; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007444:Inulin / Q000096:biosynthesis; D008129:Lolium / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D016415:Sequence Alignment; D013395:Sucrose / Q000378:metabolism
D018076:DNA, Complementary; D005630:Fructans; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D007444:Inulin; D006602:Hexosyltransferases; C105700:fructan - fructan 6G-fructosyltransferase