Fructan biosynthesis in excised leaves of wheat (Triticum aestivum L.): a comparison of de novo synthesis in vivo and in vitro.

Carbohydrate accumulation by excised, continuously illuminated leaves of wheat (Triticum aestivum L.) was followed over a 24 h period. At 0 h, the tissue contained no detectable fructan. In the initial 6 h, only sucrose was accumulated. After 6 h the de novo synthesis of fructans was induced. Fructans accumulated in the sequence 1-kestose, bifurcose, nystose, oligofructans of apparent degree of polymerization (DP) up to 9 and finally, 6-kestose, which was first detected after 22 h. A cell-free protein extract from leaves illuminated for 24 h catalyzed the de novo synthesis of fructan from sucrose. The properties of this fructan synthetic activity (FSA) were characterized. The FSA was stable, exhibiting < 20% loss of activity when stored at 5 °C or 25 °C for 6 h. The FSA exhibited an apparent K of 114 mM, and an apparent pH optimum at 5.5. The in vitro synthesis of fructan of DP > 3 was not inhibited by sucrose even at 1000 mM. Pyridoxal-hydrochloride at 20 mM did not enhance rates of enzymatic fructan synthesis or significantly inhibit the release of free fructose in the optimized enzymatic reaction. The rate of oligofructan synthesis in the optimized reaction approximated to rates of accumulation in the leaf (1.35 mg g h and 1.18 mg g h respectively). The sequence of oligofructan synthesis in vitro was the same as that observed in the leaf, with the exception that 6-kestose was synthesized early in the time course, in parallel with 1-kestose and bifurcose. Fructans of apparent DP ≤ 8 were detected after 10 h of incubation. When incubated with bifurcose as sole substrate, the cell-free preparation liberated free monosaccharides, without the accumulation of trisaccharide or sucrose as intermediates. The results are discussed with reference to current, conflicting models for the biosynthesis of fructan in cereals.