Ion exchange chromatography of water extracts was used to classify the fructans in leaves of 40 temperate dicots and 110 temperate grass species. Of the 150 species studied, only Poa ampla (Merrill) contained a single β-2,6 linked fructan series. All other species grown at 10/5°C day/night temperatures (16 h day) contained some β-1,2 linked oligofructans, even when the major fructan series was β-2,6 linked. Leaves of P. ampla plants grown at 15/10°C day/night temperatures contained some β-1,2 linked fructans and raffinose in addition to the dominant β-2,6 series, but did not contain significant amounts of 1-kestose or bifurcose (1&6-kestotetraose). Leaves of Poa arctica R.Br. and Poa bulbosa L. contained significant amounts of raffinose and various β-1,2 linked oligomers but no 1-kestose. In vivo synthesis of 6-kestose in leaves of P. ampla grown at 10/5°C proceeds in the absence of significant amounts of either 1-kestose or bifurcose. Thus P. ampla probably has a specific 6-SST (6-sucrose-sucrosefructosyl transferase) and might provide a ready source of β-2,6 linked grass fructan.

To test the hypothesis that fructan structure can be used as a taxonomic character for the Poaceae we examined the accumulation of the linear, β-2,6-linked fructan series with a terminal glucose residue within the tribes Aveneae and Poeae. Only Dactylis glomerata L. (Poeae) has been shown to contain this series, making it unique among fructan structures and a potential taxonomic marker. To this end, 13 members of the tribes Aveneae and Poeae were surveyed for the presence of this fructan series by comparing extracts of water-soluble carbohydrate by TLC with an extract of D. glomerata. The fructans of four species were similar to those of D. glomerata, as determined by TLC. Further comparison by anion-exchange chromatography and linkage analysis demonstrated that Phalaris aquatica L., Puccinellia stricta (Hook. f.) C. H. Blom and Holcus lanatus L. contained a linear, β-2,6-linked series of fructan with a terminal glucose residue but that Lagurus ovatus L., although containing predominantly β-2,6-linked fructan was composed of more than one series of fructan, one with a terminal glucose residue and one with an internal glucose residue. A linear β-2,6-linked series of fructans with a terminal glucose residue was the dominant fructan accumulated by some species of genera belonging to both the Aveneae (Phalaris and Holcus) and the Poeae (Dactylis and Puccinellia). As both of these tribes also contain genera that accumulate fructans with an internal glucose residue, e.g. Avena and Lagurus (Aveneae) and Lolium (Poeae), structure of fructan cannot be used to distinguish these tribes. Review of the literature, however, showed that it is possible to separate the supertribes Triticodae and Poodae on the basis of the structure of the fructan that they accumulate.

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.

Fructans (polyfructosylsucrose) are synthesized by a number of plants and micro-organisms. Plant fructans are localized in the vacuole and have a low degree of polymerization (DP), whereas the fructans synthesized by micro-organisms are usually much bigger. There is an increasing interest in fructans for both food and non-food applications. In order to accumulate fructans of high DP in the plant vacuole, the levansucrase protein of Bacillus subtilis was fused to the vacuolar targeting sequence of sporamin and expressed in plants. Transgenic tobacco plants in which this fusion gene is expressed accumulate fructans to levels up to 21% of the d. wt. They showed a reduced translocation of carbohydrates, bleaching of the leaves, stunted growth and increased levels of hexoses and starch. The levansucrase protein was not translocated to the plant vacuole, but retained in the endomembrane system, even though the same targeting signal was able to translocate the E. coli GUS protein to the plant vacuole.

Sucrose[ratio ]sucrose fructosyltransferase (SST) activity was partially purified from whole shoots of Lolium rigidum by a combination of affinity chromatography, gel filtration and anion-exchange chromatography. The SST activity co-eluted with some fructan[ratio ]fructan fructosyltransferase (FFT) and invertase activities and consequently the partially purified preparation was termed the fructosyltransferase (FT) preparation.

The SST-like activity in the FT preparation was purified 214-fold and had an apparent molecular mass of 84000. The FT preparation contained several peptides with an apparent pI of 4·6–4·7. When assayed with sucrose concentrations up to 600 mM, the FT preparation synthesized 1-kestose at all concentrations, and synthesized 6-kestose at concentrations of 150 mM and greater. The Km of 1-kestose production was 0·2 M. When the FT preparation was assayed at a concentration of activity approximately half that measured in fresh tissue with 100 mM sucrose, 1-kestose, or 6G-kestose as substrates, fructans of degree of polymerization (DP) [les ]5 were synthesized.

A partially purified FFT activity, free of SST and invertase activities, which synthesized β-2,1-glycosidic linked oligofructans of DP [les ]6, was combined in vitro with the FT preparation (FFT-FT preparation) to give a ratio of SST[ratio ]FFT activities similar to that measured in crude enzyme extracts from L. rigidum. The FFT-FT preparation synthesized oligofructans when assayed with 100 mM concentrations of sucrose, 1-kestose or 6G-kestose, but was not able to synthesize fructans of DP [ges ]6 even after extended assays of up to 10 h. The FFT-FT preparation was also assayed with 100 mM sucrose with small amounts of concentrated sucrose added periodically during the assay to maintain the substrate concentration. In this assay, the FFT-FT preparation synthesized fructans up to an apparent DP of 17 or greater. The fructans of DP [ges ]6 synthesized in the assay appeared to form two molecular series containing both β-2,1- and β-2,6-glycosidic linked fructosyl residues with terminal or internal glucosyl residues. The apparent rate of SST activity in the assay of the FFT-FT preparation was greater than that measured in a similar assay of the FT preparation alone which did not result in fructans with DP [ges ]6. It was concluded that the FFT-FT preparation, when assayed with a continual supply of sucrose, contained a factor which promoted synthesis of fructans of DP [ges ]6 and synthesis of β-2,6-glycosidic linkages between fructosyl residues.

Tuberous roots of Viguiera discolor accumulate up to 80% of their dry mass as fructans. The distribution pattern of oligomers suggests the predominance of anabolic reactions at the beginning of dormancy, when a continuous series of fructans between sucrose and higher polymers is present. This paper describes the synthesis in vitro of fructans of high molecular mass by enzyme extracts prepared from growing tuberous roots of V. discolor at the beginning of dormancy. Sucrose[ratio ]sucrose fructosyl transferase activity was characterized by the synthesis of 1-kestose from sucrose and fructan[ratio ]fructan fructosyl transferase activity by the production of nystose from 1-kestose. The optimal temperature for both activities was 30°C and the optimal pH range was between 5·5 and 6·0. The apparent Km for sucrose with respect to 1-kestose synthesis was 173 mol m−3, whereas the Km for nystose synthesis from 1-kestose was much higher (592 mol m−3). Long incubation periods (up to 264 h) with sucrose, 1-kestose or nystose produced fructans of high molecular mass in vitro that resembled those found in vivo. Our data show that the higher the molecular mass of the substrate, the higher the average molecular mass of the product that was formed.

A partially purified enzyme preparation from leaves of Lolium temulentum L. was previously shown to catalyse the net synthesis of oligofructans and polyfructans from sucrose. Here the same preparation is shown to catalyse the hydrolysis of both sucrose and oligofructans. The magnitude and properties of these hydrolytic activities have been determined. The significance of these catabolic activities for studies of fructan polymerization both in vitro and in tissues in a physiologically anabolic state are discussed.

The preparation hydrolysed 1-kestose, 6-kestose, neokestose, inulin oligosaccharides of low degree of polymerization (DP 4 and 5) and endogenous oligofructans from L. temulentum, with the concomitant release of monosaccharide. The preparation also released reducing sugar at low rates from high molecular weight inulin but had no detectable activity against bacterial levan. Simultaneous incubation of sucrose and Neosugar (a commercially available mixture of predominantly β-2, 1 linked tri-, tetra- and penta-saccharides) showed that sucrose was preferentially hydrolysed by the preparation, with Neosugar fructans being protected from hydrolysis at sucrose concentrations >30 mol m−3. The kinetic properties for hydrolysis of both sucrose and Neosugar were determined. For sucrose and Neosugar respectively, Michaelis constants at 30°C and pH 6·0 were 7·7±0·5 and 14·1±1·1 mol m−3 (as terminal fructose) and maximum velocities were 6·5±0·1 and 2·7±0·1 mg g−1 fr. wt h−1 (equivalent to 10·0 and 4·2 nkat g−1 as reducing sugar release). Maximal temperatures for activity were 45 and 44°C, and Arrhenius activation energies were 39·9 and 46·9 kJ mol−1. Preincubations for 1 h at 49 and 48°C caused 50% loss of activity in subsequent assays at 30°C. The pHs for maximal activity for the two substrates were 5·2±0·1 and 5·5±0·1.

Using size exclusion chromatography (SEC), an activity catalysing the formation of fructan oligosaccharides and another catalysing sucrose hydrolysis, were not fully resolved, but exhibited distinct profiles of elution indicating Mr of 57 and 133 kD respectively. When assayed for the hydrolysis of Neosugar, the SEC eluate exhibited two peaks of activity indicative of Mr values of 57 and 133 kD.

To study the role of fructan in leaf development of barley seedlings, fructan contents and the activities of enzymes involved in fructan metabolism were measured in segments of the third leaf at the stage of maximal growth. Fructans amounted to more than 10% of the d. wt in the growth zone at the base of the developing leaf and decreased sharply in the zone above. The specific activities of sucrose[ratio ]sucrose 1-fructosyltransferase (1-SST) and sucrose[ratio ]fructan 6-fructosyltransferase (6-SFT), two key enzymes in fructan synthesis, also peaked in the growth zone but decreased more slowly in the zone above. By contrast, the specific activities of invertase and fructan exohydrolase (FEH) were low in the growth zone at the leaf base and had their peak just above it. The specific activity of fructan[ratio ]fructan 1-fructosyltransferase (1-FFT) was approx. constant over the whole leaf. These results indicate that transitory accumulation of fructans in the growth zone plays a key role in the development of barley leaves.

The objectives of this study were (i) to evaluate the relative involvement of fructans from leaf sheaths and from elongating leaf bases to regrowth after defoliation of Lolium perenne L. by following fructan exohydrolase (FEH), sucrose-sucrose fructosyl transferase (SST) and invertase (INV) activities and (ii) to examine whether gibberellins could be implicated in regulation of FEH activity. In stubble, 36% of fructans are located in leaf bases and 64% in leaf sheaths. During the first phase of regrowth, the depletion of carbohydrates was mainly due to the decline of fructans, which represented 76% and 50% of the carbohydrates mobilized from leaf sheaths and elongating leaf bases respectively. Despite a decrease in protein content, FEH activity increased 2·5-fold in leaf sheaths and six-fold in elongating leaf bases, so that the fructan-hydrolysing activity was four times higher in growing leaves than in leaf sheaths, 2 d after defoliation. INV activity also increased, whereas the pattern of SST activity was inversely related to the variations of both hydrolysing activities. SST activity, which is highest in growing leaves, declined approximately threefold in leaf sheaths and elongating leaf bases at the beginning of regrowth.

The increase in activity of FEH in stubble was strongly inhibited by an inhibitor of the biosynthesis of gibberellin, uniconazole. FEH activity was decreased to c. 67%, 45% and 33% of the level in nontreated plants 24, 48 and 72 h following defoliation, respectively. The inhibition could be overcome by a subsequent treatment with gibberellic acids (GAs). For the first time, data are provided to support the view that GAs might play a role in the regulation of FEH activity, and the implication of this result is discussed.

A novel extracellular fructanhydrolase was isolated from the culture filtrate of Lactobacillus paracasei ssp. paracasei P 4134 grown on a mineral medium supplemented with fructan extracted from Timothy (Phleum pratense L.) as the only carbon source. The enzyme was purified by a combination of ammonium sulphate precipitation, affinity chromatography, preparative isoelectric focusing and anion-exchange chromatography. As a result of these procedures, the specific enzyme activity increased 93-fold, with a final yield of 28·4%. The substrate-specific activities against different fructan types were determined by incubating the enzyme fractions with fructan extracted from Timothy (predominantly β-2,6 fructosyl-fructose linkages), inulin from Dahlia tubers (mostly β-2,1 fructosyl-fructose linkages) and sucrose. The purified enzyme catalysed the hydrolysis of β-2,6-linked fructan more rapidly than the β-2,1 linkages of inulin. Additionally, the enzyme showed low ability to hydrolyse sucrose. Fructose was the main product of the degradation of Timothy fructan and inulin, indicating a high exohydrolytic activity of the enzyme. It is proposed that the fructan-degrading enzyme from L. paracasei ssp. paracasei P 4134 is a β-D-fructan-fructohydrolase (EC 3.2.1.80). The enzyme preparation showed a single protein band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a mobility corresponding to molecular weight of c. 42 kDa. It was concluded that only one molecular weight of fructan-degrading enzyme exists in L. paracasei ssp. paracasei P 4134.

After cooling the roots and shoot apex of barley, there is a reduction in sucrose export from, and an increased total accumulation of carbohydrate and induction of fructan synthesis in, source leaves. We investigated carbohydrate accumulation in individual leaf cells. Using the microsampling technique and microfluorometric enzymatic assay, the amounts of various carbohydrates were measured in mesophyll, bundle sheath and epidermal cells. Epidermal cells take no part in carbohydrate partitioning and have very low concentrations of sugars even when leaf export is reduced. Even without cooling, however, there are remarkable changes in sucrose concentration (up to 200 mM) in mesophyll and bundle sheath cells during the photoperiod. The activity of acid invertase in samples from individual cells was very low, c. 3 μmol sucrose (g f. wt)−1 h−1. Interrelationships between these cell types and the control of fructan metabolism are discussed.

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.

Possible limitations of sink strength for fructan storage in tubers of Jerusalem artichoke (Helianthus tuberosus L. cv. Waldspindel) were studied in 2-yr field experiments. Treatments included the removing of side branches and the breaking of apical dominance in unbranched plants. Data from both treatments and the comparison of results obtained for 2 yr, which differed in temperature and total irradiance, indicated that fructan storage in tubers during the storage phase was not limited by supply of assimilate (sucrose). Moreover, fructan concentrations were very constant despite large variations in tuber yields and yield components (e.g. number of tubers per plant, tuber length). However, stimulation of cell expansion and diameter of tubers increased fructan concentrations, probably owing to a larger vacuolar volume in the storage cells. We concluded that fructan storage in tubers of Jerusalem artichoke is not source-limited as far as carbon import into tubers is concerned. However, a strong shoot control, most probably regulated via phytohormones, limits sink strength. Fructan concentrations in tubers might not only be increased by sink activity (fructan-synthesizing enzymes) but also by sink capacity (vacuolar volume). In this respect the shoot might play a decisive role not only as a temporary storage pool for carbohydrates but also as a source of native growth-regulators which control sink capacity.

Two species of Bromus adapted to different climatic conditions were studied to determine their responses to chilling and water stress. Enzymes of sucrose and fructan metabolism as well as fructoside content were measured in roots and shoots. The results indicate that Bromus pictus (adapted to a cold desert area) has a constitutive fructan synthesis, whilst B. auleticus (adapted to a warmer northern climate) only accumulated fructan under cold stress. Microscopic observations of roots exposed to fluorescent probes ascertained cell viability and showed that the apex of roots in the northern species were more affected by the water-deficit treatment than the southern species. The presence of fructans in B. pictus and their absence in B. auleticus support the hypothesis that fructans play a role in plant tolerance to environmental stress.

Barley (Hordeum vulgare L. cv. Agneta) was cultivated hydroponically under continuous light, constant temperature and relative humidity. The relative growth rate (RGR) was kept at 25% by an exponential supply of complete medium for 2 wk. The cultures were then transferred to phosphorus-free media and the amounts of total P, fructans, starch, sucrose, glucose and fructose in sink and source leaves were measured at 0, 12, 24, 48, 72, 120 and 156 h. The capacity of net fructan synthesis (i.e. total activity of sucrose[ratio ]sucrose 1-fructosyltransferase (1-SST) and sucrose[ratio ]fructan 6-fructosyltransferase (6-SFT)), the activity of fructan exohydrolase (FEH), as well as the activity of acid invertase were also measured in the two types of leaves. Omission of P from the growth medium caused a decrease in the P content in both sink and source leaves, whereas the RGR was not affected during the 156 h of the experiment. The fructan concentration increased in low-P plants whilst there was little or no effect on the concentrations of starch, sucrose, glucose and fructose. There was a slight increase in the capacity of net fructan synthesis in both sink and source leaves at 156 h after the onset of P-starvation. There was no change in the activity of FEH or acid invertase. The results indicate that in barley leaves the mechanism for carbon partitioning into fructans is more sensitive towards low P conditions than the mechanism for carbon partitioning into starch.

In the present work we compare responses to hypoxia of the carbohydrate content of related plant species, which grow naturally on sites prone to flooding (Senecio aquaticus Hill., Myosotis palustris (L.) Lehm. Rchb.) with plants from habitats with only a low risk of oxygen shortage (Senecio jacobaea L., Myosotis arvensis (L.) Hill.). Whilst the amounts of glucose, fructose and sucrose changed only slightly following hypoxia and peaked at a maximum approximately double that of the aerated control specimens, the fructan content increased fivefold to tenfold. In nearly all the plants studied, fructan became the main polysaccharide reserve. For example, the flooding-tolerant Senecio aquaticus accumulated the highest amount, in particular fructans with a degree of polymerization up to 35 compared with 10 under control conditions. Nearly 70% of the soluble carbohydrates were fructans, compared with 30% under aerated conditions.

In all species tested the starch fraction marginally increased or remained constant.

Fructans were found to accumulate as a response to oxygen deficiency in both flooding-tolerant and intolerant species but with higher absolute values and ratios between fructan and starch in the flooding-tolerant species. At 24 h after the onset of hypoxic treatment, the sugar content rose, in spite of the diminished photosynthetic rates. The ability to accumulate fructans seems to vary in plants coping with oxygen shortage but could be interpreted as advantageous in comparison to starch synthesis in terms of (i) the location of the fructan metabolism in the vacuoles, with no negative feedback in the photosynthetic apparatus and (ii) the possibility of storage of sucrose in the form of fructose polymers without the intermediate stages and energy-using processes which are indispensable for starch synthesis. These results might indicate that fructan metabolism could play a role in the tolerance of oxygen deficiency.

Wheat stems serve as a store for fructans to buffer the plant against nutritional and environmental influences. It has been suggested that fructan storage influences yield stability and tolerance of environmental factors. Near infra-red spectroscopy (NIR) analysis provides a rapid and accurate assessment of the fructan content of the wheat stem, as well as allowing detection of growth-limiting nutrient stresses, and so is proving to be a useful technique for making crop management decisions. Commercial laboratories using NIR analysis have been tissue-testing crops in the eastern Australian wheat belt since 1993. In healthy, normally developing crops not under stress there is a predictable relationship between nitrogen and fructan. Investigation of the nitrogen and fructan concentrations in commercial crops has confirmed an inverse relationship between these two constituents. The function: Fructan (%)=a+bN%+cN%2 accounted for up to 81% of variation in tissue fructan concentration. In commercial tissue-testing this relationship is used to detect crops under stresses other than nitrogen deficiency. If the fructan concentration deviates by more than 4%, cereal growers are advised that their crop might be subject to other stresses which might reduce its response to applied nitrogen.

The growth and fructan contents of plants of Vernonia herbacea (Vell.) Rusby regenerated from fragments of the underground organ (rhizophore) were analysed during two developmental cycles. When cultivated away from their natural ecosystem, these plants went through the same phenological phases as those naturally grown in the cerrado. The first aerial shoots started developing from initial fragments before the new underground system, the former acting as the main metabolic sink for the fructan present in the initial fragments. The new rhizophores started to develop by the sixth month, as photosynthates were transformed into fructans. Growth parameters measured reached higher values in the second growth cycle, exhibiting growth curves synchronized with those obtained for fructan contents in the new rhizophores. Fructo-oligosaccharides were available mostly during the period of fast growth of aerial organs whilst the fructo-polysaccharides were reduced. Fructan variation indicates that these compounds are metabolized during development. Additionally, the rapid growth, intense production of rhizophores and high fructan content make Vernonia herbacea a promising source of fructan for commercial use.