Fructan:fructan 1-fructosyltransferase,a key enzyme for biosynthesis of graminan oligomers in hardened wheat

Fructans play important roles not only as a carbon source for survival under persistent snow cover but also as agents that protect against various stresses in overwintering plants. Complex fructans having both ß-(2,1)- and ß-(2,6)-linked fructosyl units accumulate in wheat (Triticum aestivum L.) during cold hardening. We detected fructan: fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) activity for catalyzing the formation and extension of ß-(2,1)-linked fructans in hardened wheat tissues, cloned cDNAs (wft3 and wft4) of 1-FFT, and analyzed the enzymatic properties of a wft3 recombinant protein (Wft3m) produced by yeast. Wft3m transferred ß-(2,1)-linked fructosyl units to phlein, an extension of sucrose through ß-(2,6)-linked fructosyl units, as well as to inulin, an extension of sucrose through ß-(2,1)-linked fructosyl units, but could not efficiently synthesize long inulin oligomers. Incubation of a mixture of Wft3m and another recombinant protein of wheat, sucrose:fructan 6-fructosyltransferase (6-SFT), with sucrose and 1-kestotriose produced fructans similar to those that accumulated in hardened wheat tissues. The results demonstrate that 1-FFT produces branches of ß-(2,1)-linked fructosyl units to phlein and graminan oligomers synthesized by 6-SFT and contributes to accumulation of fructans containing ß-(2,1)- and ß-(2,6)-linked fructosyl units. In combination with sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and 6-SFT, 1-FFT is necessary for fructan synthesis in hardened wheat.     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Factors affecting fructosyltransferases and fructan exohydrolase activities in <Emphasis Type="Italic">Agave tequilana</Emphasis> Weber var. azul

There is great interest in the fructosyltransferases (FTFs) involved in fructan metabolism and agents affecting their activity. Agaves accumulate fructans, fructose polymers linked by glycosidic β(2–1) and β(2–6) bonds in linear or branched configurations. In plants, fructans provide protection under stress conditions. The sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), fructan:fructan 6G-fructosyltransferase (6G-FFT), and fructan exohydrolase (FEH) activities were analyzed in micropropagated Agave tequilana plants in the absence and presence of HgCl₂, AgNO₃, MgCl_2, sodium deoxycholate (DNa), and sodium dodecyl sulfate (SDS). Kestose, nystose and neokestose were synthesized by the respective FTFs. HgCl₂ and AgNO₃ inhibited all FTFs, mainly up to 90 % in 1-SST and 1-FFT. DNa increased 1-SST (32 %) and 1-FFT (45 %) activities, and SDS increased 6G-FFT activity by 96 %. Finally, AgNO₃ inhibited FEH activity by 78 %. Our results might be relevant on the regulation of FTFs in agave and other crops, for instance by the increment the fructans synthesis in stressed plants.     

Variation in the in vitro generated fructan pattern from sucrose as a function of the purified chicory root 1-SST and 1-FFT concentrations

At low concentrations of purified chicory root 1-SST, only 1-kestosewas produced from a physiologically relevant sucrose concentration.As the 1-SST concentration increased, some higher oligofructanswere also detected, showing that 1-SST has some 1-FFT activityafter sucrose exhaustion in the reaction mixtures. The consequencesfor the interpretation of fructan synthesizing activities invitro are discussed. With a mixture of both purified 1-SST and1-FFT it was found that the higher the enzyme concentration,the higher the maximal DP of the fructans that could be synthesized.The higher the enzyme concentration, the higher the relativeabundance of the larger DP fructans and fructose in the reactionmixtures. Taken together with previous results, there is confidencethat the final fructan pattern obtained in vitro is a functionof the (1-SST+1-FFT)/sucrose ratio and suggest that the latterratio in situ could affect the highly variable tissue- or species-specificpattern of fructans produced in vivo. Key words: 1-FFT, 1-SST, chicory, enzyme concentration, sucrose     

Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes

Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.     

Molecular cloning and characterization of a high DP fructan: fructan 1-fructosyl transferase from Viguiera discolor (Asteraceae) and its heterologous expression in Pichia pastoris

Inulin-type fructans are stored in the tuberous roots of the Brazilian cerrado plant Viguiera discolor Baker (Asteraceae). In Cynara scolymus (artichoke) and Echinops ritro (globe thistle), the fructans have a considerably higher degree of polymerization (DP) than in Cichorium intybus (chicory) and Helianthus tuberosus (Jerusalem artichoke). It was shown before that the higher DP in some species can be attributed to the properties of their fructan: fructan 1-fructosyl transferases (1-FFTs; EC 2.4.1.100), enzymes responsible for chain elongation. Here, we describe the cloning of a high DP (hDP) 1-FFT cDNA from V. discolor and its heterologous expression in Pichia pastoris . Starting from 1-kestose and Neosugar P (a mixture of oligo-inulins from microbial origin) as substrates, the recombinant enzyme produces a typical hDP inulin profile in vitro, closely resembling the one observed in vivo. The enzyme shows no invertase activity and sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) activity in vitro. Pattern evolution during incubation suggests that inulins with DP ≥ 6 are much better substrates than sucrose or lower DP oligo-fructans. Because hDP inulin-type fructans show superior properties for specific food and non-food applications, the hDP 1-FFT gene from V. discolor has potential for the production of hDP inulin in vitro or in transgenic crops.     

Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus 'Colombia': I. Fructan: fructan fructosyl transferase

Fructan: fructan fructosyl transferase (FFT), one of the enzymesinvolved in the synthesis of ß-2,1 linked fructosepolymers has been purified 205-fold from tubers of Helianthustuberosus harvested in the accumulation phase. The molecularweight of the native as well as the SDS-denatured protein isapproximately 70 kDa. On IEF, the protein was separated intofive molecular species with pl values between pH 4.5–5.0.The optimum pH for fructosyl transfer activity was between 5.5–7.0.Temperature optimum was in the range of 25-35° C; the Q10value between 25 and 5° C was 1.14. FTT catalysed the self-transferof fructosyl groups with GF₂, GF₃, GF₄ or GF₅ as substrate andacceptor. The rate of elf-transfer with both GF₂ and GF₃ increasedlinearly with substrate concentration up to 100 mol m^–3and was still not saturated at 600 and 300 mol m^–3, respectively.FFT was unable to hydrolyse GF or to catalyse the self-transferwith GF but could mediate the transfer of fructosyl units frominulin on to GF. Key words: Fructan: fructan fructosyl transferase, Helianthus tuberosus, Jerusalem artichoke, purification, kinetics     

Effect of ethylene glycol and glycerol fructosides on the activity and product specificity of bacterial and plant fructosyltransferases

Fructosyltransferases (FTs) are key enzymes in plants and bacteria to synthesize fructans. To gain insight on the specificity of the hexose subsites in the active site of FTs, ethylene glycol fructoside (EGF) and glycerol fructoside (GF), containing fructose in the furanose configuration, were synthesized in vitro and used as substrates to study the effect on the activity of bacterial levansucrase (BsLS), chicory root sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT). The results demonstrated that EGF and GF, at physiologically relevant concentrations, were efficient acceptor substrates for BsLS and 1-FFT, but not for 1-SST. EGF and GF cannot be used as donor substrates for BsLS, 1-SST and 1-FFT. A model is proposed to explain the subsite specificity differences between the three FTs involved in this study.     

Localization of the Enzymes of Fructan Metabolism in Vacuoles Isolated by a Mechanical Method from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

Vacuoles isolated by a mechanical slicing method from developing tubers of Jerusalem artichoke (Helianthus tuberosus L.) contain activities of the two principal enzymes responsible for fructan synthesis: sucrose-sucrose fructosyl transferase and fructan-fructan fructosyl transferase. Both enzymes are associated with the vacuolar sap and not with the tonoplast. In vacuoles isolated from dormant tubers, the fructan-fructan fructosyl transferase activity remains in the vacuolar sap but the fructan exohydrolase activity is associated with the tonoplast. Fructan is hydrolysed by these vacuoles to fructose, which can be exported to the suspending medium. The localization of the enzymes of fructan metabolism in the vacuole has implications for the maintenance of fructan polymerisation.     

Properties of fructan:fructan 1-fructosyltransferases from chicory and globe thistle,two Asteracean plants storing greatly different types of inulin

Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism.     

Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase

Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase [1-SST], sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase [1-FFT], fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.     

Purification and Characterization of the Enzymes of Fructan Biosynthesis in Tubers of Helianthus tuberosus Colombia (II. Purification of Sucrose:Sucrose 1-Fructosyltransferase and Reconstitution of Fructan Synthesis in Vitro with Purified Sucrose:Sucrose 1-Fructosyltransferase and Fructan:Fructan 1-Fructosyltransferase)

Sucrose:sucrose 1-fructosyltransferase (1-SST), an enzyme involved in fructan biosynthesis, was purified to homogeneity from tubers of Helianthus tuberosus that were harvested in the accumulation phase. Gel filtration under native conditions predicted a molecular mass of about 67 kD. Electrophoresis or gel filtration under denaturing conditions yielded a 27- and a 55-kD fragment. 1-SST preferentially catalyzed the conversion of sucrose into the trisaccharide 1-kestose (GF2). Other reactions catalyzed by 1-SST at a lower rate were self-transfructosylations with GF2 and 1,1-nystose (GF3) as substrates yielding GF3 and 1,1,1-fructosylnystose, respectively, as products. 1-SST also catalyzed the removal of the terminal fructosyl unit from both GF2 and GF3, which resulted in the release of sucrose and GF2, respectively, and free Fru. The purified enzyme did not display [beta]-fructosidase activity. An enzyme mixture of purified 1-SST and fructan:fructan 1-fructosyltransferase, both isolated from tubers, was able to synthesize fructans up to a degree of polymerization of at least 13 with sucrose as a sole substrate.     

Low temperature and defoliation affect fructan-metabolizing enzymes in different regions of the rhizophores of Vernonia herbacea

In addition to the storage function, fructans in Asteraceae from floras with seasonal growth have been associated with drought and freezing tolerance. Vernonia herbacea, native of the Brazilian Cerrado, bears underground reserve organs, rhizophores, accumulating inulin-type fructans. The rhizophore is a cauline branched system with positive geotropic growth, with the apex (distal region) presenting younger tissues; sprouting of new shoots occurs by development of buds located on the opposite end (proximal region). Plants induced to sprouting by excision of the aerial organs present increased 1-fructan exohydrolase (1-FEH) activity in the proximal region, while plants at the vegetative stage present high 1-sucrose:sucrose fructosyltransferase (1-SST) in the distal region. The aim of the present study was to analyze how low temperature (5 °C) could affect fructan-metabolizing enzymes and fructan composition in the different regions of the rhizophores of intact and excised plants. 1-SST and 1-fructan:fructan fructosyltransferase (1-FFT) were higher in the distal region decreasing towards the proximal region in intact plants at the vegetative phase, and were drastically diminished when cold and/or excision were imposed. In contrast, 1-FEH increased in the proximal region of treated plants, mainly in excised plants subjected to cold. The ratio fructo-oligo to fructo-polysaccharides was significantly higher in plants exposed to low temperature (1.17 in intact plants and 1.64 in excised plants) than in plants exposed to natural temperature conditions (0.84 in intact vegetative plants and 0.58 in excised plants), suggesting that oligosaccharides are involved in the tolerance of plants to low temperature via 1-FEH, in addition to 1-FFT. Principal component analysis indicated different response mechanisms in fructan metabolism under defoliation and low temperature, which could be interpreted as part of the strategies to undergo unfavorable environmental conditions prevailing in the Cerrado during winter.     

Purification and Characterization of Wheat beta(2-->1) Fructan:Fructan Fructosyl Transferase Activity

Fructans are the major storage carbohydrate in vegetative tissues of wheat (Triticum aestivum L.). Fructan:fructan fructosyl transferase (FFT) catalyzes fructosyl transfer between fructan molecules to elongate the fructan chain. The objective of this research was to isolate this activity in wheat. Wheat (cv Caldwell) plants grown at 25°C for 3 weeks were transferred to 10°C to induce fructan synthesis. From the leaf blades kept at 10°C for 4 days, fructosyl transferase activity was purified using salt precipitation and a series of chromatographic procedures including size exclusion, anion-exchange, and affinity chromatography. The transferase activity was free from invertase and other fructan-metabolizing activities. Fructosyl transferase had a broad pH spectrum with a peak activity at 6.5. The temperature optimum was 30°C. The activity was specific for fructosyl transfer from β(2→1)-linked 1-kestose or fructan to sucrose and β(2→1) fructosyl transfer to other fructans (1-FFT). Fructosyl transfer from oligofructans to sucrose was most efficient when 1-kestose was used as donor molecule and declined as the degree of polymerization of the donor increased from 3 to 5. 1-FFT catalyzed the in vitro synthesis of inulin tetra- and penta-saccharides from 1-kestose; however, formation of the tetrasaccharide was greatly reduced at high sucrose concentration. 6-Kestose could not act as donor molecule, but could accept a fructosyl moiety from 1-kestose to produce bifurcose and a tetrasaccharide having a β(2→1) fructose attached to the terminal fructose of 6-kestose. The role of this FFT activity in the synthesis of fructan in wheat is discussed.     

Exogenous nitric oxide effect on fructan accumulation and FBEs expression in chilling-sensitive and chilling-resistant wheat

This study was to investigate the effect of exogenous nitric oxide (NO) on fructan accumulation and fructan biosynthesic enzymes (FBEs) expression in seedlings leaves of two wheat (Triticum aestivum L.) cultivars, winter wheat (Zhoumai18, ZM) and spring wheat (Yanzhan4110, YZ), under 4 °C. The seedlings of two wheat cultivars were subjected to different concentrations of sodium nitroprussiate (SNP) for 0, 24, 48, and 96 h. Relative water content (RWC) was increased by exogenous NO in YZ, but decreased in ZM. Except for glucose, fructose and fructans of degree of polymerization (DP) 3 in YZ, other soluble carbohydrates contents in the two wheat cultivars all increased to different degrees. The activities of FS (including sucrose: sucrose 1-fructosyltransferase (1-SST, EC: 2.4.1.99) and sucrose: fructan 6-fructosyltransferase (6-SFT, EC: 2.4.1.10)) were significantly higher than fructan: fructan 1-fructosyltransferase (1-FFT, EC: 2.4.1.100) in the seedlings of two wheat cultivars. The same phenomenon occurred to FBEs expression. In addition, sucrose content decreased while fructans content increased under low temperature, which was in accordance with the improved 1-FFT activity in ZM. Moreover, fructans content increased to a high level under high concentration of NO in ZM while kept at a constant low level in YZ. The expression levels of FBEs were universally higher in ZM than in YZ, which identified with the high frost resistance of the winter cultivar. It is concluded that exogenous NO treatment on wheat may be a good option to reduce chilling injury by regulating fructan accumulation in leaves. This is the first report owing that exogenous NO alleviated the negative effects of chilling stress by accumulating fructans in wheat.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.