Fructan exohydrolase from grasses

Abstract

SUMMARYIn grasses, fructan reserves are mobilized from vegetative plant parts during seasonal growth, after defoliation during grazing and from stems during seed filling. Well-illuminated leaves show a diurnal pattern of fruetan accumulation during the light and mobilization during the dark. In expanding leaves, fructans are accumulated in cells of the elongation zone and when mobilized are considered to contribute assimilate for synthetic processes. Even in leaves which do not contain high fructan concentrations, high rates of fructan turnover occur. The process of fructan mobilization appears to be regulated in relation to ontogenic events, demand for assimilate during growth and in response to environmental stress.Hydrolysis of fructans in bacteria is catalyzed by both endo-and exohydrolases. However, in higher plants only fructan exohydrolases (FEH) (EC 3.2.1.80) have been reported. FEH has been extracted from only a limited number of grass species. The pH optimum of FEH activities varies between pH 4-5-5-5, the temperature optimum ranges from 25-40 °C and FEH is considered to be entirely localized in vacuoles. Estimates of the K^ for FEH assayed using high molecular weight fructan substrates vary widely and should be considered carefully because most substrates are ill-defined. Many studies indicate that crude and partially-purified FEH activity is highest when assayed using a fructan substrate extracted from the species that was the source of the enzyme activity. Inulin extracted from members of the Asteraceae is generally less readily hydrolyzed and levans from bacteria are relatively poor substrates for FEH from grasses. Glycosidic-linkage-specific hydrolysis has been demonstrated for an FEH activity extracted from barley. This FEH activity hydrolyzed /?-2,l-glycosidie linkages more rapidly than /?-2,6-linkages. Most other studies are less conclusive because ill-defined fructan substrates were used. Two isoforms of FEH are reported in leaves of Lolium spp., but the roles of isoforms and their kinetic characteristics are not known.FEH activity in different tissues may be regulated by metabolic concentrations, sucrose (5-10 mM) being a strong inhibitor in vitro of FEH from some species. Results of experiments with Dactylis glomerata indicate control of expression of FEH activity at the gene level. In stem bases, FEH activity increased after defoliation. The increase was abolished by applications of inhibitors of protein synthesis and was apparently repressed by application of various sugars.Although the rates of fructan hydrolysis measured in vitro are sufficient to explain the in vivo rates of fructan hydrolysis, it is yet to be shown whether fructan hydrolysis in vivo is due to the activity of FEH exclusively, or FEH and invertase-like activities.The overriding conclusion is that the various studies of FEH from grasses present a confusing and incomplete picture of the function, activity and kinetics of this enzyme. This is due in part to the lack of defined, commercially-available substrates. The chromat...

Mobilization of fructan reserves and changes in enzyme activities in wheat stems correlate with water stress during kernel filling

“…The current findings in relation to water stress and the earlier observations concerned with the response to a change in the source-sink balance (Willenbrink et al, 1998) tend to support the suggestion by Schnyder (1993) that the onset of carbon (C) loss from the stem fructan pools requires the induction of FEH as observed in other grass species. However there were similar changes in the activity of INV and this enzyme could also be involved in fructan metabolism (Simpson & Bonnett, 1993).…”

“…The overall sucrose content did not appear to fall and on this basis a sucrose control system (Simpson & Bonnett, 1993) appears not to be operating. However sucrose concentration might vary widely between different tissues in the stem, often occurring in greatest amounts in the vascular tissue (Wardlaw, 1990 ;Hendrix, 1995 ;Verscht et al, 1998).…”

“…The procedures used for the determination of enzyme activity were based on those used in earlier studies Bancal & Triboı$ , 1993 ;Simpson & Bonnett, 1993) and the level of activity of both FEH and INV in the stems of control plants was similar to those reported previously for wheat (Bancal & Triboı$ , 1993).…”

“…Aliquots (0.3 ml) of the filtrate, with 0.15 ml of fructan substrate (2% (w\v) in citrate\phosphate buffer, pH 5.5) were incubated for 30 min or 60 min, at 30mC. This amount of substrate did not saturate the reaction as shown when a comparison was made using 4% (w\v) fructan (Simpson & Bonnett, 1993), although paired measurements using 4 and 2 % fructan on a range of samples showed that the relative enzyme activities from different organs were not altered by the change in concentration. Incubation was stopped by transferring the sample tubes to boiling water for 3 min then holding these on ice.…”

Carbohydrate composition and invertase activity of the leafy liverwort Porella platyphylla

“…Our results suggest the invertase activity is similarly sensitive to inhibition by pyridoxal (64 % inhibition by 10 mol m −$ ) as true invertases. Some invertases also have fructan exohydrolase activity (FEH) Simpson and Bonnett, 1993) although sucrose inhibitable FEH without invertase activity has been characterized . The activity of the P. platyphylla enzyme against fructan has not yet been determined so its possible role in fructan breakdown needs further investigation.…”