Previous work on Lolium perenne showed that sucrose[ratio ]sucrose fructosyltransferase (SST) activity did not increase concomitantly with fructan synthesis in regrowing leaves or in mature leaf blades of plants that have been subjected to carbohydrate-accumulating conditions. This was contrary to the pattern of SST activity in roots and stubble. To obtain further insight into the fructan synthesizing activities and to explain this discrepancy, total fructosyltransferase activity (FT) was assayed by increasing the sucrose and the soluble enzyme concentrations and was compared to sedimentable phlein sucrase activity (PS) throughout the regrowth period following defoliation in leaves, stubble and roots. Before analysis on 2-month-old plants, PS activity was characterized in dark-grown coleoptiles, using [U-14C]sucrose. PS activity had a pH optimum of 6.0 and produced 1-kestose in addition to high molecular weight fructans with a mean DP of 9. In 2-month-old plants, sedimentable PS and FT soluble reactions contained an initial sucrose concentration of 160 mM and 400 mM and proteins equivalent to 1.4 and 2.1 g f. wt of tissue, respectively. In stubble and roots, the FT preparation catalysed the synthesis of large fructans, and the overall pattern resembled the native fructans when separated by anion exchange HPLC. In regrowing leaves, the FT preparation produced low-DP fructans relatively more than in vivo but synthesized the high-DP fructan characteristic of the tissue. Moreover, FT activity did not remain at a low level like SST activity but increased from day 5 after defoliation when fructans began to accumulate. PS activity formed very few low- DP fructans and 1-kestose was the main product. Trisaccharides generated by PS activity represented 2–5% of the total trisaccharide synthesis. High-DP fructans were detectable only when the products of the reaction were concentrated 100 times. Results are discussed with respect to the relative contribution of FT and PS activities for the synthesis of 1-kestose and fructans in roots, stubble and leaves of Lolium perenne.

Fructans are linear or branched polymers containing a single sucrose and repeating fructose residues. An early model for fructan biosynthesis in higher plants suggested that partial synthesis of the polymer occurred in the cell cytosol. The current model suggests that synthesis requires the interaction of two separate fructosyltransferases located in the vacuole. Tobacco lines containing a chemically induced promoter, directing expression of the Bacillus amyloliquefaciens SacB gene in the present study, provided an opportunity to regulate and target fructan synthesis to the cytosol of transgenic plants. Induced expression of the gene led to rapid destruction of leaf tissue. Amino acid substitution at a highly conserved site (Arg331) in the SacB gene reduced the fructosyltransferase efficiency without reducing the invertase activity of the enzyme. Expression of the mutant gene in transgenic tobacco also resulted in leaf damage. However, the appearance of necrotic tissue was greatly delayed. The results suggest that the phenotype is due to accumulation of fructan in the cytosol. Fructan metabolism in the cytosol of potato tubers was also detrimental to development. Tuber size and starch synthesis was significantly reduced in lines containing the untargeted gene. Transgenic tobacco and potato containing the SacB gene offer an opportunity to study the metabolism of fructan and the effect of accumulation on plant cell development.

The accumulation of fructo-oligosaccharides and the activities of fructosyltransferase (sucrose[ratio ]sucrose 1F-fructosyltransferase (SST), 1F-fructosyltransferase (1F-FT) and 6G-fructosyltransferase (6G-FT)) in the bulbs of three onion cultivars were investigated from June to September 1993. The total fructo-oligosaccharide content increased from June until August, then decreased in September, except in one cultivar. The levels of neokestose and its related tetrasaccharides (1F, 6G-di-β-D-fructofuranosyl sucrose and 6G (1-β-D-fructofuranosyl)2sucrose) were higher than those of 1-kestose and nystose throughout growth. The activities of 6G-FT, 1F -FT and SST were high in June and July, then decreased; SST activity was very low in September. The activity ratios of 6G-FT to 1F-FT varied between 1·86 and 2·65 over the growth period. Two trisaccharides, three tetrasaccharides and four pentasaccharides were identified, together with a mixture of hexa- and heptasaccharides, all of which were synthesized in vitro from 0·1 M sucrose by an enzyme preparation of onion bulbs harvested in August. Octa- and nonasaccharides other than the saccharides formed from sucrose were also synthesized from 0·1 M 1-kestose or 0·1 M neokestose. All the saccharides produced from sucrose, 1-kestose or neokestose by the crude enzyme prepared from onion bulbs were identical to the saccharides occurring naturally in onion bulbs.