Fructan biosynthesis in excised leaves of Lolium temulentum L.

Abstract

Analyses of the kinetic properties and products of furctosyl transferases from Lolium temulentum L. suggest that these enzymes are artifacts of conventional extraction and/or assay methods. An alternative approach to the investigation of fructan biosynthesis in cell-free extracts has been developed. This approach employs the sensitivity of radiotracers to overcome the limitations of conventional quantitative methods for the analysis of fructan. Homogenates of leaves were prepared, which contained enzyme protein, endogenous sucrose and fructan. These extracts catalysed the rapid and efficient incorporation of radioactivity from [U-14C]sucrose into material with a degree of polymerization greater than 2. The chromatographic pattern of these labelled products was identical to the pattern of native leaf fructan. Kestose, which does not accumulate to high concentrations in vivo but was a major product of our previous enzyme assay procedures, was not a product of these homogenates. The flux of radioactivity into oligofructan was sufficient to account for observed rates of fructan accumulation in the leaf and fructosyl transfer was observed at sucrose concentrations between 15 and 30 mol m-3, which is within the probable physiological range of substrate concentration for L. temulentum leaf cells. Extracts derived from leaves which were not actively accumulating fructan were unable to label oligofructan. Labelling of fructan was not due to non-enzymic isotope exchange. Leaf homogenates contained invertase and fructan hydrolase activities. Invertase activity was inhibited by pyridoxal HCl and pyridoxine HCl when these compounds were included in reactions at 20 mol m-3. Inhibition of invertase activity was correlated with a marked stimulation of fructosyl transfer, suggesting competition for substrate. Because this system functions at physiologically relevant sucrose concentrations and produces a pattern of labelled products identical to native leaf fructan, we suggest that it represents a more convincing starting point for studies of the enzymology of fructan biosynthesis than previous cell-free extracts.