Fructan biosynthesis in Lolium perenne: appraisal of soluble and insolubleenzymatic pathways

Previous work on Lolium perenne showed that sucrose:sucrose fructosyltransf erase (SST) activity did not increase concomitantly with fructan synthesis in regrowing leaves or in mature leaf blades of plants that have been subje cted to carbohydrate-accumulating conditions. This was contrary to the patt ern of SST activity in roots and stubble. To obtain further insight into th e fructan synthesizing activities and to explain this discrepancy, total fr uctosyltransferase activity (FT) was assayed by increasing the sucrose and the soluble enzyme concentrations and was compared to sedimentable phlein s ucrase activity (PS) throughout the regrowth period following defoliation i n leaves, stubble and roots. Before analysis on 2-month-old plants, PS acti vity was characterized in dark-grown coleoptiles, using [U-C-14]sucrose. PS activity had a pH optimum of 6.0 and produced 1-kestose in addition to hig h molecular weight fructans with a mean DP of 9. In 2-month-old plants, sed imentable PS and FT soluble reactions contained an initial sucrose concentr ation of 160 mM and 400 mM and proteins equivalent to 1.4 and 2.1 g f. wt o f tissue, respectively. In stubble and roots, the FT preparation catalysed the synthesis of large fructans, and the overall pattern resembled the nati ve fructans when separated by anion exchange HPLC. In regrowing leaves, the FT preparation produced low-DB fructans relatively more than in vivo but s ynthesized the high-DP fructan characteristic of the tissue. Moreover, FT a ctivity did not remain at a low level like SST activity but increased from day 5 after defoliation when fructans began to accumulate. PS activity form ed very few low-DP fructans and 1-kestose was the main product. Trisacchari des generated by PS activity represented 2-5% of the total trisaccharide sy nthesis. High-DB fructans were detectable only when the products of the rea ction were concentrated 100 times. Results are discussed with respect to th e relative contribution of FT and PS activities for the synthesis of 1-kest ose and fructans in roots, stubble and leaves of Lolium perenne.