• Fructan : fructan 6G-fructosyltransferase (6G-FFT) has been proposed to be the enzyme essential for the production of neo-series inulin. Transfer of a fructose unit from short chain inulins to the C6 of the glucose residue of sucrose or inulin was proposed to be its most important characteristic. Here, we investigate the activity of 6G-FFT from onion (Allium cepa) more thoroughly.
• Tobacco BY2 suspension cultures were employed as an expression system for the fructosyltransferase 6G-FFT. Activity was measured using 1-kestose as a substrate and products were detected using high-performance anion exchange chromatography (HPAEC).
• 6G-FFT showed multiple activities. An array of fructans of the inulin series and inulin neo-series were produced. First 1,1-kestotetraose and 1 and 6G-kestotetraose were synthesized, as well as 6G-kestotriose. Prolonged incubation produces a complex fructan series with a higher degree of polymerization.
• The fructan pattern observed after incubation of onion 6G-FFT with 1-kestose closely resembles the complex fructan pattern found in onion. These results questions the need for a separate fructan : fructan 1-fructosyltransferase (1-FFT) activity in onion.
Fructans are stored in onion bulbs as a reserve carbohydrate and are used for regrowth of the plant in spring. Fructans start to accumulate at the onset of bulbing, which in its turn coincides with increased fructosyltransferase activity (Shiomi et al., 1997; Kahane et al., 2001). During growth the fructan contents of bulbs increases, but by the end of the growth season a decline in fructan content is observed (Shiomi et al., 1997). Storage of onion bulbs leads to a further decrease in fructan levels and sprouting is induced (Pak et al., 1995). This phenomenon severely reduces storage times for onion. A simple plant fructan is inulin, which is present in members of the Asteraceae such as chicory and artichoke. Inulin consists of linear β(1–2) linked fructose residues with one terminal glucose residue. Two enzymes involved in inulin synthesis were isolated from plants, sucrose : sucrose 1-fructosyltransferase (1-SST, EC 126.96.36.199) and fructan : fructan 1-fructosyltransferase (1-FFT, EC 188.8.131.52). The former is essential for synthesis of the trisaccharide 1-kestose (1-kestotriose) from sucrose, while the latter needs 1-kestose to produce inulin with a higher degree of polymerization (DP) (Edelman & Jefford, 1968; Koops & Jonker, 1996; Lüscher et al., 1996; van den Ende et al., 1996).
In Liliaceae, such as onion and asparagus, a different type of inulin is present, namely the inulin neo-series. In the inulin neo-series, two β(1–2) linked fructose chains are attached to the sucrose starter unit. One chain is linked to the C1 of the fructose residue, as is the case in inulin. The other chain is linked to the C6 of the glucose residue. Therefore, several types of fructan chains are distinguished in onion (Ernst et al., 1998): an inulin series which is elongated at the fructose of the starter sucrose, designated Ix (x represents the degree of polymerization); an neokestose-based series with elongation only at the glucose residue of the starter sucrose, designated Nx; another neokestose-based series with elongation on both the fructose and glucose residue of the starter sucrose, designated Nx; and a series without a glucose residue, present after storage of onions, designated Fx.
Fructan : fructan 6G-fructosyltransferase (6G-FFT) initiates the formation of the 6G-linked chain. It uses 1-kestose as a fructose donor and transfers the fructose unit to the glucose residue of sucrose or oligofructan via a β(2–6) linkage (Shiomi, 1989; Vijn et al., 1997). The trisaccharide neo-kestose (6G-kestotriose) is the shortest fructan of the inulin neo-series and can be elongated on both terminal fructose residues with β(1–2)-linked fructose units.
Fructan : fructan 6G-fructosyltransferase was first described by Henry and Darbyshire (Henry & Darbyshire, 1980) as an enzyme from onion that produces the trisaccharide neokestose. Subsequently, Shiomi purified the 6G-FFT enzyme from asparagus roots (Shiomi, 1981). Enzymatic characterization revealed that not only 1-kestose, but also that low-DP inulin is used as fructosyl donor by 6G-FFT. Low-DP inulin and sucrose can be used as fructosyl acceptors (Shiomi, 1981; Shiomi, 1982). 6G-FFT from onion was cloned by Vijn et al. (1997), who showed that introduction of 6G-FFT from onion into chicory causes the production of inulin neo-series fructans, in addition to the inulin naturally present in chicory root. Vijn and coworkers also expressed 6G-FFT in tobacco plants and protoplasts. Tobacco has no native fructan production, and extracts from transformed plants or protoplasts were used for enzymatic assays with 1-kestose as a substrate. Assays of 6G-FFT-transformed tobacco plants showed the formation of neo-kestose and DP4 and DP5 fructans of unknown structure from 1-kestose. The same assays on tobacco protoplasts resulted in the formation of DP4 inulin and an unknown fructan of the inulin neo-series. The conclusions from these articles are that 6G-FFT is able to initiate the formation of the inulin neo-series fructans due to the transfer of a fructosyl residue to the C6 of the glucose moiety of sucrose or low DP inulin. 6G-FFT needs low DP inulin as a substrate to donate a fructosyl residue; sucrose can be used as a fructosyl acceptor only. Furthermore, it seems that 6G-FFT is also be able to synthesize DP4 inulin from 1-kestose, indicating 1-FFT-like activity.
The expression systems described above resulted in low activity of the enzyme in protoplasts and tobacco plants, or ambiguous results because of the interfering activities of other fructosyltransferases in chicory. Tobacco BY2 cells form a fast-growing suspension culture derived from Nicotiana tabacum cv. Bright Yellow 2. These cells have been used successfully to express foreign proteins (for review see Fisher et al., 1999). Therefore, we used BY2 cells to study the activity of the 6G-FFT enzyme in more detail. We show that 6G-FFT is able to synthesize a range of fructans when 1-kestose is provided as a substrate. The fructan-pattern obtained shows that the enzymatic activity of 6G-FFT on 1-kestose can produce the complex array of fructan molecules observed from onion.
Materials and Methods
Cloning of 6G-FFT
The cloning of 6G-FFT in pMON999 is described by Vijn et al. (Vijn et al., 1997). To enable Agrobacterium-mediated transformation the cassette containing the gene and 35S-promotor and NOS-terminator sequence was excised from pMON999 using NotI, partly filled with G and ligated to pBin19 (Bevan, 1984) which was cut with XmaI and partly filled with C. The resulting plasmid is called pBinT24P3.
Growth and transformation of BY2 cells
BY2 cells are grown as a suspension culture in modified Linsmaier and Skoog medium supplemented with 0.2 mg ml−1 2,4-dichlorophenoxyacetic acid (2,4-D) as described by Nagata et al. (1992); the medium contains 3% sucrose. The cells were grown at 27°C at 150 r.p.m. in the dark and subcultured once a week by a 70-fold dilution in fresh medium.
Agrobacterium-mediated transformation of BY2 cells was performed according to An (1985) and Genschik et al. (1998). Briefly, BY2 cells were incubated for 3 d at 27°C in the dark (without shaking) with Agrobacterium tumefaciens strain LBA4404 containing pBinT24P3. Subsequently, cells were put on plates with medium containing two antibiotics, vancomycin (750 µg ml−1) to kill off Agrobacterium and kanamycin (100 µg ml−1) to select for transformed cells. Transformed cells appear after 4–6 wk as a callus on plates and were maintained as a callus by transferring to a fresh plate once a month. From transformed callus a suspension culture was initiated by addition of a small callus clump to 5 ml of culture medium. Once a suspension was observed, this was diluted in 20 ml and, after growth, to 100 ml. Eventually, transgenic cells in suspension grew as fast as wild-type BY2 cells and were subcultured once a week.
Fructosyltransferase activity assay
A 2 ml suspension of cells was spun for 5 min at 2300 Rcf and the supernatant was removed. Callus was put in an Eppendorf tube of 2 ml. 6G-FFT was released from the cells by shaking them twice in a dismembrator (B. Braun Biotech International Grublt, Melsungen, Germany) for 1 min at 2800 r.p.m. in the presence of glass beads (two at 2 mm diameter and two at 4 mm diameter). Debris was spun down at 15 700 Rcf for 15 min. Forty microlitres of the supernatant was combined with 5 µl of an 0.5 M (mol/1) 2-(N-morpholino) ethanesulfonic acid (MES) buffer pH 5, 7 and 5 µl of substrate to a total volume of 50 µl, and incubated at 28°C. After incubation, products were analysed using silica thin layer chromatography (TLC) run three in 90% acetone, or a Dionex HPAEC-PAD (high-performance anion exchange chromatography with pulsed amperometric detection) system with a PA100 column, both described by Vijn et al., 1997). To obtain a better separation of onion fructans on the HPAEC-PAD system the sodium acetate (NaAc) gradient was changed. Using solutions A (water), B (0.5 m NaOH) and C (1 m NaAc), the following running profile was applied: T = 0, 80% A 20%, B; T = 5 min, 50% A, 50% B; T = 15 min, 40% A, 50% B, 10% C; T = 20 min, 33% A, 50% B, 17% C; T = 35 min, 50% B, 50% C. We observed the same peaks in onion as reported previously by Shiomi et al. (1997) and Ernst et al. (1998). We use peak identification as proposed by Ernst et al. (1998), and nomenclature as proposed by Lewis (1993) and Waterhouse & Chatterton (1993).
Expression of 6G-FFT
We used an onion 6G-FFT cDNA clone (NCBI GenBank accession number: Y07838) under the control of the 35S promotor. The construct contains the native vacuolar signal sequence of 6G-FFT. Transformation was performed using A. tumefaciens and kanamycin was used as a selection marker. Transformed cells were selected on plates containing both kanamycin and vancomycin. The latter antibiotic is used to select against the bacteria. Transformed cells formed calli, which could be transferred to new plates and maintained as a callus, or used to initiate a suspension cell line.
Activity of 6G-FFT could be detected in suspension-cultured transformants after disruption of the cells and incubation with 1-kestose. Several individually transformed lines were tested and all showed 6G-FFT activity. The fast-growing line P3 was used in subsequent experiments. The activity is stably maintained under kanamycin selection. For cells grown in suspension, early logarithmic cultures showed a lower enzyme activity than late logarithmic or early stationary-phase cultures (Fig. 1). Most of the enzyme activity was recovered in the supernatant after cell lysis, which was typical for a soluble protein.
The activity of extracts from the 6G-FFT-transformed line P3 incubated with 200 mm 1-kestose as a substrate showed synthesis of an extended range of products (Fig. 2a). The peak annotation, as proposed by Ernst et al. (1998) for onion fructans is indicated in Fig. 2. No activity was observed in extracts of untransformed BY2 cells (Fig. 2b).
Activity of 6G-FFT was also observed in cell-free extracts obtained from transformed calli. Feeding of 1-kestose to extracts from callus cells leads to production of the same compounds as feeding 1-kestose to extracts from suspension cells (Fig. 3). Fast-growing callus gave the highest 6G-FFT activity (data not shown). Thus, transformed callus can be used as a source of 6G-FFT.
6G-FFT synthesizes a range of fructans as present in onion
Comparison of the profile of products from 6G-FFT activity (Fig. 2a) with onion extract (Fig. 2c) shows high similarity. In particular, all lower DP fructans from onion are present in the graph representing 6G-FFT activity. Peaks representing the highest DP fructans in onion were absent from the 6G-FFT graph. This is presumably due to the relatively high amount of 1-kestose available in the 6G-FFT assay. The production of a range of products by 6G-FFT with the same retention time on HPAEC as those in the onion extract, indicates that 6G-FFT is a main determinant of the onion fructan pattern.
Lowering the 1-kestose concentration from 200 mm to 50 mm still resulted in considerable 6G-FFT activity (Fig. 4). The same range of products could be seen, only amounts were reduced. At 20 mm 1-kestose the activity of 6G-FFT was severely diminished (Fig. 4).
Onion-type fructans build up in time during incubation of 6G-FFT with 1-kestose
Incubation of 6G-FFT with 1-kestose shows an accumulation of products over time (Fig. 3). After 1 h of incubation, 1 and 6G-kestotetraose (peak N4) is the major product formed. This is a fructan of the inulin neo-series, which is the result of the transfer of the terminal fructose residue from a 1-kestose-donor molecule to the C6 of the terminal glucose residue of another 1-kestose acceptor molecule (Fig. 5). 1,1-Kestotetraose (I4) is also formed; this is an inulin that similarly results from the transfer of a fructose residue between two 1-kestose molecules. Furthermore, the smallest inulin neo-series molecule 6G-kestotriose (N3) is formed, for which sucrose is used as an acceptor (Fig. 5).
Longer fructans can be seen after 4 h of incubation. The first DP5 product formed, 1,1 and 6G-kestopentaose (N5a) and 1 and 1,6G-kestopentaose (N5b) can both directly be synthesized from the relative abundant 1 and 6G-kestotetraose. Other DP5 products in addition to the DP4 product 1,6G-kestotetraose (N4) accumulate subsequently, followed by higher-DP fructans (Figs 3 and 5).
The fast-growing tobacco suspension culture cell-line BY2 can be used to functionally express fructosyltransferases. BY2-derived 6G-FFT produces a fructan pattern similar to onion, containing a range of fructans with higher DPs than previously reported by Vijn et al. (1997). This indicates the production of more protein or a more active protein in this system compared with transiently transformed protoplasts and transgenic tobacco plants used before. Furthermore, the range of products produced by 6G-FFT resembled the onion pattern more closely than was observed previously (Vijn et al., 1997). Therefore, expression in BY2 cells is the preferred system to study activity of this enzyme.
When sucrose was added as a substrate formation of only trace amounts of 1-kestose could be found from extracts of cells transformed with 6G-FFT as well as untransformed BY2 cells (data not shown). Such activity was previously reported for vacuolar invertases at high sucrose concentrations (Straathof et al., 1986; Vijn et al., 1998).
The HPAEC-PAD peak areas can be compared between samples for the same compound only, and not for different compounds in one sample. No attempt was made to quantify the individual amounts. Reduction of the 1-kestose peak area over time shows that considerable amounts of the substrate were used, indicating high activity of the enzyme (data not shown).
The observed activities of 6G-FFT would imply that the enzyme is not only capable of 6G-fructosyltransferase activity, but is also able to elongate fructans by repetitive coupling of fructose residues via β(1–2) linkages. This was previously proposed by Vijn et al. (1997) who reported the formation of DP4 inulin by 6G-FFT expressed in tobacco protoplasts. Here, we show that 6G-FFT is able to produce higher DP fructans of the inulin and the inulin neo-series from 1-kestose. The fructan series produced both in onion and by 6G-FFT from 1-kestose are: an inulin series which is elongated at the fructose of the starter sucrose, Ix; a neokestose-based series with elongation only at the glucose residue of the starter sucrose, Nx; and another neokestose-based series with elongation on both the fructose and glucose residue of the starter sucrose, Nx (Ernst et al., 1998). A fourth series in onion is a series without a glucose residue, Fx. This series was only present after storage of onion and is probably a breakdown product. We did not observe this series in the profile obtained from the 6G-FFT assays, indicating the absence of breakdown.
Dual activities can now be assigned to 6G-FFT: the coupling of a fructosyl residue to the terminal glucose C6; and a 1-FFT-like activity, namely the coupling of a fructosyl residue to the C1 of a terminal fructose residue, regardless whether the fructose residue ultimately connects to the glucose or the fructose residue from the sucrose starter unit.
BY2-expressed 6G-FFT seems to determine the onion fructan pattern. However, it does not produce the greatest amount DP fructans from the onion profile. This observation has been made previously for FFTs. The activity of 1-FFT from Cynara scolymnus expressed in potato has been reported to polymerize up to DP 40, whereas in extracts from C. scolymnus, fructans up to DP 200 are reported (Praznik & Beck, 1985; Hellwege et al., 2000). Moreover, in vitro activity of 1-FFT resulted in synthesis up to DP 23 inulin only (Hellwege et al., 1998). Also, 1-FFT of Helianthus tuberoses and Cichorium intybus were unable to produce inulin in vitro with as high a DP as was found in vivo (Praznik & Beck, 1985; van den Ende & van Laere, 1996; Hellwege et al., 1998). This is most likely due to suboptimal reaction conditions.
By analogy with the situation in asparagus, where Shiomi isolated SST, 6G-FFT and FFT enzymes (Shiomi & Izawa, 1980; Shiomi, 1981; Shiomi et al., 1985) it was proposed that 1-FFT would also be present in onion. However, the 1-FFT enzyme or clone could not be isolated from onion. Since 6G-FFT is clearly able to produce the three fructan types present in onion from 1-kestose, we suggest that a separate 1-FFT activity is absent from onion.
If the only fructosyltransferases present in onion are 1-SST and 6G-FFT, the situation in onion is analogous to that of the inulin-producing species chicory and Jerusalem artichoke, where 1-SST and 1-FFT are the two fructosyltransferases present (Lüscher et al., 1996; van den Ende & van Laere, 1996; van der Meer et al., 1998). In these situations 1-SST initiates fructan biosynthesis by production of 1-kestose from sucrose. 1-Kestose is subsequently used by an elongating enzyme to produce longer fructans of specific types. In the case of the inulin-producing species 1-FFT is the elongating enzyme; in the case of onion, which produces inulin neo-series, 6G-FFT is the elongating enzyme.
We thank Dr Masao Hirayama, Meiji Seika Kaisha Ltd, Japan for his kind gift of 1-kestose. T. Ritsema and J. Joling are indebted to the Netherlands Organization for Scientific Research (NWO) for funding of the CW/STW project 790.35.075.