JXB Advance Access originally published online on January 31, 2006
Journal of Experimental Botany 2006 57(4):775-789; doi:10.1093/jxb/erj065
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RESEARCH PAPER |
Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes
1KU Leuven, Laboratory of Molecular Plant Physiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium
2KU Leuven, Laboratory of Neuroplasticity and Neuroproteomics, Naamsestraat 59, B-3000 Leuven, Belgium
* To whom correspondence should be addressed. E-mail: wim.vandenende{at}bio.kuleuven.ac.be
Received 9 May 2005; Accepted 15 November 2005
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Abstract |
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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.
Key words: 1-FFT, biotechnology, Echinops ritro, fructan, heterologous expression, inulin, Pichia pastoris
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fructans are Fru-based oligomers and polymers occurring as reserve carbohydrates in several plant families (Hendry, 1993
). Fructans are believed to accumulate mainly in vacuoles, but their presence in the apoplast has been reported (Livingston and Henson, 1998; Van den Ende et al., 2005). Fructans have quickly gained importance as healthy food ingredients (Ritsema and Smeekens, 2003) and are commercially extracted from chicory roots (Van den Ende et al., 2004). Like the raffinose family of oligosaccharides (RFOs), fructans are excellent prebiotics since they selectively promote beneficial colon bacteria like lactobacilli and bifidobacteria (Cummings et al., 2001). Both fructans and RFOs might be implicated in frost and drought tolerance (Pilon-Smits et al., 1995; Amiard et al., 2003; Parvanova et al., 2004; Hisano et al., 2004; Tapernoux-Luthi et al., 2004), most probably by stabilizing membranes (Vereyken et al., 2001; Hincha et al., 2002, 2003).
Several types of fructans occur in plants (Van den Ende et al., 2004). The family of the Asteraceae typically accumulates inulin-type fructan in which ß (2,1) linkages occur between the Fru units (Lewis, 1993). Inulin is biosynthesized in vitro from Suc by the combined action of two fructosyl transferases: Suc:Suc 1-fructosyl transferase (1-SST; EC 2.4.1.99
[EC]
; G-F+G-F
G+G-F-F) and fructan:fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100
[EC]
G-Fm+G-Fn
G-F(m–1)+G-F(n+1) with m >1 and n 
1) (Edelman and Jefford, 1968; Van Laere and Van den Ende, 2002). 1-FFTs elongate a pre-synthesized chain at the expense of another pre-synthesized chain. These kinds of disproportionation reactions should be clearly distinguished from ‘de novo synthesis’. The validity of the 1-SST/1-FFT model in vivo was supported by simultaneous expression of heterologous 1-SST and 1-FFT transforming potato (Hellwege et al., 2000) or sugar beet (Sévenier et al., 1998) into an inulin-producing crop. However, the 1-SST/1-FFT model is still a subject of criticism (Cairns, 2003).
Remarkably, within the Asteraceae, globe thistle (Echinops ritro, Vergauwen et al., 2003), artichoke (Cynara scolymus; Hellwege et al., 2000), and Viguiera discolor (Itaya et al., 1997) biosynthesize fructans with a considerably higher mean and maximal DP than other well-known species such as Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. In this study, the term ‘high DP fructan’ (hDP fructan) will be reserved for plant-derived fructan fractions with a mean DP >20. However, it should be clear that hDP plant fructans are much smaller than microbial levans or inulins.
Several plant 1-FFTs have been (partially) purified, characterized, and cloned as recently reviewed by Vergauwen et al. (2003). In contrast to 1-SSTs, 6-SFTs, invertases, and bacterial levansucrases, 1-FFT enzymes cannot use Suc as a donor substrate. The high DP inulin profile observed in globe thistle (Echinops ritro) can be attributed to the presence of a high DP 1-FFT (hDP 1-FFT). Kinetic comparisons with the chicory low DP 1-FFT (lDP 1-FFT) demonstrated that the globe thistle hDP 1-FFT preferred longer inulin chains as acceptors while the chicory lDP 1-FFT had a higher affinity for short carbohydrates (Suc, Fru, and 1-K) as acceptor substrates (Vergauwen et al., 2003).
In this paper, the cloning and functional analysis of an hDP 1-FFT from Echinops ritro, a native meditteranean species particularly adapted to drought (Chatto, 1982) is reported. The recombinant enzyme has similar properties as the native hDP 1-FFT and produces hDP inulin-type fructans in vitro resembling those found in vivo.
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Materials and methods |
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Plant material
Echinops ritro was sown (10 May 2003) and grown in a local garden. On 23 September 2003, plants were harvested for purification and RNA extraction.
Purification of the native 1-FFT and Q-TOF MS
E. ritro roots (0.6 kg) were harvested in September, at the stage of maximal DP, when 1-SST activity was low and 1-FEH activity was not yet induced. The hDP 1-FFT was purified as described by Vergauwen et al. (2003). The SDS-PAGE protein bands of 52 and 19 kDa (Fig. 1) were subjected to mass spectrometric (MS) identification. The Coomassie Brilliant Blue stained protein bands were excised, trypsinized, extracted, desalted, and analysed on Q-TOF as described earlier by Van den Ende et al. (2001). Sequence information was derived from the MS/MS spectra with the aid of the MaxEnt 3 (deconvoluting and deisotoping of data) and PepSeq software from the Micromass BioLynx software package.
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RNA preparation, cloning and sequencing
Roots from three different plants were washed, peeled, cut in small pieces, and mixed. Total RNA was isolated from this material by using the Rneasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Two degenerate primers XFOR and XREV (Table 1) were constructed based on peptide sequence information of the native 1-FFT (Table 2). One-step RT-PCR with these primers (Access RT-PCR System, Promega, Madison, WI, USA) was performed. RT reaction was at 48 °C. PCR conditions: 94 °C, 3 min; followed by 35 cycles: 94 °C, 30 s; 48 °C, 30 s, and 72 °C, 2 min. Final extension was at 72 °C, 10 min. A first PCR product of 850 bp was obtained after agarose electrophoresis. The PCR mixture was ligated in the TOPO-TA vector and transformed to E. coli (TOPO-TA cloning kit, Invitrogen, Groningen, The Netherlands). Plasmids were extracted using the Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA) and the insert was sequenced. Some specific primers were subsequently chosen and combined with an oligo dT-based primer in an attempt to clone the 3' part of the cDNA. However, no specific PCR products could be obtained. Therefore, one of the specific primers (XHDP1, Table 1) was combined with the CTERMINV1 primer which is conserved among vacuolar invertases and fructosyl transferases. PCR conditions were identical to those during the first PCR (see above). A second PCR product of 1100 bp was obtained, subcloned, and sequenced. Alignment of both PCR products showed an identical overlapping region of 440 bp indicating that they originated from the same gene. Subsequently, two new specific primers EFFTF1 and EFFTF2 were chosen close to the C-terminal part of the second PCR product. EFFT1 was combined with an oligo dT primer, but a complex band pattern appeared. On this PCR mixture, a semi-nested PCR was attempted with EFFT2 and an oligo dT primer resulting in a single band representing the 3' part of the cDNA. The 5' part of the cDNA was partially cloned by RT-PCR by combining FFTDT (a conserved 1-FFT primer) and EFFTR (35 cycles, annealing temperature 50 °C). The resulting 600 bp fragment was subcloned and sequenced.
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Heterologous expression in Pichia pastoris
The mature protein part of E. ritro was estimated based on FEHs from chicory and sugar beet which were successfully expressed in Pichia pastoris (Van den Ende et al., 2001
, 2003). An RT-PCR was performed by using the primers PicEF and PicER (Table 1). Pfu Proofreading polymerase (Promega, Madison, WI, USA) was used (annealing temperature 57 °C for 35 cycles). Three independently amplified PCR fragments were first subcloned in the TOPO-XL vector and fully sequenced (TOPO-XL cloning kit, Invitrogen, Groningen, The Netherlands). After vector amplification in E. coli, the DNA fragment was cut out with PstI and NotI and ligated in the pPICZ
B vector (Invitrogen, Groningen, The Netherlands). The resulting expression plasmid pEr1-FFT contains the putative mature protein part of E. ritro 1-FFT in frame behind the -factor signal sequence. This plasmid was transformed to E. coli competent cells as described (Van den Ende et al., 2001). Cells were plated on 2xYT medium supplemented with zeocine as a selection marker. Positive colonies were used for vector amplification. P. pastoris strain X33 was subsequently transformed by using electroporation with both 20 µg of PmeI linearized pEr1-FFT and with empty vector (as a control). Further selection and handling was as described (Kawakami and Yoshida, 2002), except that a concentration of 2% (v/v) methanol was maintained in the Pichia expression media.
Purification of the heterologous hDP 1-FFT
At 96 h post-induction, the culture was centrifuged and proteins were precipitated for 1.5 h in 80% ammonium-sulphate saturated citrate-phosphate buffer (10 mM final, pH 5.0) on ice. Finally, a centrifugation occurred (40 000 g) for 15 min at 4 °C. The precipitate was redissolved in 100 ml TRIS-HCl buffer pH 7.5. Undissolved material was spun down for 10 min at 40 000 g and 4 °C. The supernatant was applied to a Q Sepharose column (25x100 mm). Bound proteins were eluted with Na-Ac buffer pH 5.0 also containing 100 mM NaCl and 0.02% Na-azide. Active fractions were loaded onto the HiLoadTM 16/60 Superdex 75 column which was previously equilibrated with Na-Ac buffer pH 5.0 also containing 100 mM NaCl and 0.02% Na-azide. The fractions containing enzymatic activity were applied on a Mono Q anion exchange column (Pharmacia HR 5/5, Uppsala, Sweden), which was equilibrated with 20 mM TRIS-HCl buffer pH 7.5. Proteins were eluted using a linear gradient from 0 M to 0.3 M NaCl in 30 min (flow rate 1 ml min–1). Active fractions were subjected to SDS-PAGE on 12.5% polyacrylamide gels and stained with Coomassie Brilliant Blue-R250 as described by Vergauwen et al. (2003).
Carbohydrate analyses, enzyme assays and acid hydrolysis
Neosugar P was a gift from Beghin-Meiji Industries, Paris, France. 1-K was kindly provided by Dr Iizuka (Iizuka et al., 1993). Carbohydrates, as well as products of enzymatic reactions, were analysed by AEC-PAD (CarbopacTM PA-100, Dionex, Sunnyvale, CA, USA). A Na-acetate gradient was applied as follows: 0–6 min, 10 mM; 6–16 min, 10–100 mM; 16–26 min, 100–175 mM; 26–36 min, 175–230 mM, 36–61 min, 230–315 mM. Regeneration was 5 min with 500 mM Na-acetate. The column was re-equilibrated for 9 min with 90 mM NaOH. Shorter or longer methods (but with identical gradient slope) were also applied.
Enzymatic activity is expressed in nanokatal (nkat), defined as the amount of enzyme that transferred 1 nmol of Fru s–1. A suitable amount of heterologous 1-FFT was incubated with 50 mM 1-K and 2% (w/v) Neosugar P or 4% (w/v) Neosugar P in 60 mM Na-MES buffer pH 6.25 at 0 °C and 30 °C, respectively. Please refer to the figure legends for further details on the incubation conditions. The reaction was stopped by keeping an aliquot for 5 min in a boiling water bath.
Total acid hydrolysis for mean DP estimation was performed as described by Vergauwen et al. (2003). Partial acid hydrolysis was performed in a similar way, but a shorter incubation time (10 min) and a lower HCl concentration (10 mM) was used.
All experiments were repeated at least three times with consistent results.
Induction of fructan biosynthesis in excised leaves of E. ritro
Fresh leaves (18 in total, three different plants, six leaves per plant) were taken at the end of the dark period (12 October, 06.30 h). The 18 leaves were recombined into three groups of six leaves (each consisting of two leaves of each plant). One group of leaves was immediately immersed in liquid nitrogen and served as a control. The two other groups of leaves were incubated under continuous light (for the conditions, see Nagaraj et al., 2004) in 200 mM Suc and water, respectively. The incubation time was 54 h. Thereafter, samples were immersed in liquid nitrogen. The leaves were homogenized with mortar and pestle (under liquid nitrogen). Aliquots of the fine powder were used for RNA extraction. Total RNA was isolated by using the Rneasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Ninety ng of total RNA was subsequently used in an RT-PCR (28 cycles) with the primers FFDT and EFFTR and with a rRNA primer couple (Van den Ende et al., 2002). Carbohydrate extracts were prepared as described above. After keeping these extracts for 1 week at 4 °C, visible precipitating material was only detected in the extract of the Suc-induced leaves but not in the other two extracts. After centrifugation, a huge white pellet was only formed in the extract of the Suc-induced leaves. The three pellets were resolubilized in hot water and aliquots of these were analysed with AEC-PAD as described above.
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Results |
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Purification and mass fingerprint of the native hDP 1-FFT from E. ritro
The native high DP 1-FFT enzyme was purified as described by Vergauwen et al. (2003)
. As observed for many other vacuolar invertases and fructosyl transferases, it appeared as a heterodimer after SDS–PAGE showing bands of approximately 52 kDa (large subunit) and 19 kDa (small subunit) (Fig. 1, right lane). The two bands were excised, trypsinized in-gel, and the masses of ZipTip-eluted tryptic peptides were determined by Q-TOF. A theoretical tryptic digest of the cDNA-derived hDP 1-FFT protein sequence (obtained later, see further Fig. 3) yielded 47 peptides (designated T1–T47 from N- to C-terminus). Most masses matched, within the acceptable mass measurement error of ±0.2 Da, with one of the theoretical fragments (Table 2). Special attention was given to the unexplained fragments. After fragmentation, some fragments appeared to contain a missed cleavage, an oxidized methionine, or a cysteine-bound mercaptoethanol (Table 2). However, most of the unexplained fragments were found to be glycosylated peptides, of which the MS/MS spectra matched the predictions of the Sweet Substitute program (Clerens et al., 2004). The carbohydrate composition and chain length was highly diverse (Table 2). Including the peptide backbones of the glycopeptides, 41.68% coverage of the protein sequence was found.
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Cloning a cDNA encoding Echinops ritro hDP 1-FFT
A functional hDP 1-FFT cDNA from E. ritro was cloned by RT-PCR and PCR. The peptide sequences TAYHYQPAK (T4) and VTWGYVAESDSGDQDR (T24) obtained from the native hDP 1-FFT enzyme (Table 2) were used to develop degenerated sense and antisense primers. The partial cDNA fragment obtained was sequenced and encoded the peptides T8, T9, T16, and T20 previously detected from the native enzyme (Table 2). Using this sequencing information, new specific primers were derived for 3' RACE RT-PCR (Table 1). A near complete cDNA was obtained by 5' RACE RT-PCR by using a 1-FFT conserved primer (Table 1).
An unrooted phylogenetic tree was constructed containing the E. ritro 1-FFT cDNA-derived amino-acid sequence (see arrow, Nr. 22 in Fig. 2) and many other vacuolar-type plant invertases and fructosyl transferases. Three distinct groups can clearly be distinguished (Fig. 2). Groups I and II contain dicotyledonous enzymes and group III contains monocotyledonous enzymes. Vitis vinifera and Daucus carota contain two vacuolar invertases belonging to group I and group II, respectively. By contrast, the two vacuolar invertases of Arabidopsis thaliana and Brassica oleracea cluster in group I, suggesting that the group II gene was lost during its evolution and the group I gene was duplicated in the Brassicaceae. Group I only contains invertases, but group II splits further into two subgroups. The subgroup IIa only harbours invertases, but the second subgroup, IIb, harbours dicotyledonous fructan biosynthetic enzymes. 1-SST and 1-FFT enzymes can be clearly distinguished. The E. ritro enzyme groups together with other Asteracean 1-FFTs. Four subgroups (IIIa–d) can be differentiated within the monocots. The subgroups IIIa and c only contain invertases. Like Arabidopsis, rice contains two vacuolar invertases: one locates in subgroup IIIa and the other in IIIc. Subgroup IIIb contains both invertases and fructan biosynthetic enzymes from Asparagales. These enzymes are more homologous to subgroup IIIa invertases than to subgroup IIIc invertases. Subgroup IIId mainly contains fructan biosynthetic enzymes from Poaceae, but the presence of invertases in this subgroup can not be excluded. This subgroup further splits up in enzymes that biosynthesize ß (2,1) Fru-Fru linkages (1-SSTs) and those that make ß (2,6) linkages (6-SFTs and a putative 6-FT).
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The E. ritro hDP 1-FFT cDNA-derived amino-acid sequence (EMBL accession no. AJ811624) is aligned with other Asteracean 1-FFTs in Fig. 3. Five potential glycosylation sites [N-X-(S/T)] were detected (Fig. 3). The hDP 1-FFT from E. ritro (Cardueae tribe) was 76% identical to the hDP 1-FFT from globe artichoke (Cardueae tribe), 74% identical to the lDP 1-FFT from chicory (Cichorieae tribe), and 71% identical to the lDP 1-FFT from Helianthus tuberosus (Heliantheae tribe). The estimated molecular weight, without accounting for probable glycosylations, was predicted at 60.8 kDa. Its predicted pI was 4.92 which is typical for vacuolar fructosyl transferases and invertases.
Heterologous expression of Echinops ritro hDP 1-FFT cDNA
Heterologous expression in Pichia pastoris proved a valuable system with which to judge the main functionality of plant fructosyl transferases (Kawakami and Yoshida, 2002, and references therein), FEHs, and invertases (De Coninck et al., 2005). A partial cDNA containing the estimated mature protein part of the hDP 1-FFT from E. ritro was ligated in-frame behind the -factor signal sequence and heterologously expressed in Pichia pastoris. The recombinant hDP 1-FFT enzyme was purified from the yeasts supernatant and incubated together with 2% Neosugar P and 50 mM 1-K, respectively. Products were analysed with AEC-PAD and followed as a function of time at two incubation temperatures (0 °C and 30 °C). After 20 min, Suc and 1,1 nystose are formed from 1-K (Fig. 4A). After 80 min, the DP5 inulin-type fructan peak increased. Magnification of the chromatogram (see inset) revealed the presence of DP6 as well as higher DP inulin type fructans, some of them only eluting from the column with 500 mM Na-Acetate (see arrow). The high DP inulin formation further increased between 80 min and 6 h. However, the high DP inulin peak disappeared again after 24 h of incubation at 30 °C while the DP6, DP7, and Fru concentrations further increased. The high DP inulin peak eluting with 500 mM Na-acetate was higher at 0 °C compared with 30 °C after 6 h of incubation. A much shorter lag-phase was observed for high DP inulin formation when starting from Neosugar P as a substrate since the higher DP inulins (arrow) are clearly visible after 20 min at 30 °C (Fig. 4B). As for 1-K, the high DP inulin formation further increased at 0 °C, but not at 30 °C after 6 h. No extra Glc formation was detected during a 24 h incubation demonstrating that the enzyme was free from invertase or 1-SST activities (Fig. 4A, B). However, after long-term incubation, Fru consistently increased in the reaction mixtures, indicating that this recombinant 1-FFT has an intrinsic 1-K hydrolysing and/or 1-FEH activity, similar to the properties observed for the native 1-FFT enzymes from chicory (Van den Ende et al., 1996) and Echinops (Vergauwen et al., 2003).
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The recombinant enzyme produces a typical high DP inulin profile in vitro, closely resembling the in vivo carbohydrate pattern of E. ritro (Fig. 4C). Figures 4C and D demonstrate that the heterologous enzyme produces inulin-type fructans and nothing else. Indeed, a partial acid hydrolysate of a 66% (v/v) acetone fraction of the in vitro-synthesized material (Fig. 4C) shows the presence of Fru and inulo-n-ose type fructans (Fig. 4D). A nearly identical pattern was obtained for a partial acid hydrolysate of commercial chicory root inulin (Fig. 4D). The inulin nature of the in vitro-synthesized material was further confirmed by incubation with heterologous 1-FEH IIa from chicory and sugar beet 6-FEH (Fig. 4D).
No 1-FFT enzymatic activity and inulin formation was detected in the supernatants of Pichia pastoris strains carrying an empty vector as a control (data not shown).
Purification and mass fingerprint of the recombinant hDP 1-FFT from E. ritro
The heterologously expressed enzyme was isolated from the Pichia pastoris growth medium by ammonium sulphate precipitation. Further purification included anion exchange chromatography and gel exclusion chromatography. SDS-PAGE showed a single band of 75 kDa (Fig. 1, left lane) demonstrating that the heterologous enzyme is not proteolitically cleaved in large and small subunits as observed for the native enzyme (Fig. 1, right lane). Q-TOF MS analysis on the recombinant enzyme yielded many tryptic fragments, most of which were identical to the ones observed for the native enzyme (Table 2). However, a number of unique fragments were detected (Table 3) such as the N-terminus of the heterologous enzyme (EAEAAEFDR) and many glycosylated peptides. Including the peptide backbones of the glycopeptides, 36.23% coverage of the protein sequence was found.
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Comparison of native and heterologous glycosylations
The glycopeptides discovered in the digests of the native subunits proved glycosylations on three sites: T7 (AVPVNTSDPLLIDWVR) and T12 (NNTGLVLVYHTK) in the large subunit, and T45 (LFLFNNATDITVK) in the small subunit. Considerable heterogeneity was observed: on T12, typical plant glycosylation types (complex and paucimannosidic, Maia and Leite, 2001
; Lerouge et al., 1998) were found [(GlcNAc)0–2(Xyl)0–1(Fuc)0–1(Man)3(GlcNAc)2], as well as glycans of which the pentasaccharide core was trimmed [(Xyl)0–1(Fuc)0–1(Man)1–3(GlcNAc)2]. T45 only contained high-mannose type glycosylation [(Man)0–3(Man)3(GlcNAc)2].
In the digest of the heterologously expressed enzyme, glycosylations on T7, T12, and T45 were found. As expected from Pichia-derived glycoproteins (Bretthauer and Castellino, 1999), these were all of the high-mannose type, with up to 11 mannoses attached [(Man)2–11(Man)3(GlcNAc)2]. Tables 2 and 3 summarize the different glycans that were found.
Kinetics of native and heterologous 1-FFTs: a comparison
The best donor substrate of Asteracean 1-FFTs is 1-K (Edelman and Jefford, 1968; Van den Ende et al., 1996; Vergauwen et al., 2003). It was shown before that higher DP inulin-type fructans are the best acceptor substrates for the native 1-FFT from Echinops ritro (Vergauwen et al., 2003). A comparison of 1-K substrate saturation kinetics for both the heterologous and native 1-FFT enzymes is presented in Fig. 5. Comparable Lineweaver–Burk plots were obtained showing that both enzymes have similar properties. The apparent 1-K donor Kms of the native and heterologous enzymes were estimated at 15 mM and 22 mM, respectively. These values are in accordance with the overall concentrations of 1-K in root or induced leaf tissues (10–15 mM).
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Induction of fructan biosynthesis in leaves and 1-FFT expression analysis
It was demonstrated before that leaves of chicory do not accumulate fructans, but fructan biosynthesis and fructan accumulation can be induced after incubating excised leaves in Suc solutions (Vijn et al., 1997
). The same principle was used here to induce high DP fructan accumulation in leaves of Echinops ritro. Leaves were incubated in 200 mM Suc or in water (as a control) and compared with untreated leaves. High DP inulin auto-precipitated from a carbohydrate extract of Suc-induced leaves (Fig. 6A, arrow in panel 2) but not from untreated (panel 1) or water-induced leaves (panel 5). A magnification of panel 2 is presented in panel 3 showing that 1-K, 1,1 nystose, and higher inulin-type fructans are also present in low concentrations in this fraction. Panel 5 demonstrates that a huge amount (different scale) of Fru is liberated after mild acid treatment of this fraction.
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The RT-PCR in Fig. 6B clearly shows that the cloned 1-FFT gene is detected after only 28 PCR cycles in the Suc-induced leaves (panel 2) but not in the untreated (panel 1) or water-incubated (panel 3) leaves, suggesting that 1-FFT gene transcription is greatly induced by the Suc treatment. A 400 bp rRNA band was considered as a control (panels 4–6).
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Discussion |
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Low DP versus high DP plant fructosyl transferases
Cynara scolymus (artichoke), Echinops ritro (globe thistle), and Viguiera discolor are examples of Asteracean species accumulating inulins with a considerably higher DP (Itaya et al., 1997
; Hellwege et al., 2000, Vergauwen et al., 2003). The difference in DP is attributed to the presence of special fructosyl transferases termed hDP 1-FFTs. These enzymes belong to glycosyl hydrolase family 32 (http://afmb.cnrs-mrs.fr/CAZY; Henrissat and Davies, 1997), of which all members share a common 5-bladed ß-propeller fold (Verhaest et al., 2005, and references therein) and are believed to work via a ping-pong mechanism as also observed in the related glycosyl hydrolase family 68 (Dedonder, 1972; Chambert et al., 1974; Vergauwen et al., 2003). The hDP 1-FFTs from E. ritro and C. scolymus clearly have different kinetic properties compared with lDP 1-FFTs (Hellwege et al., 2000; Vergauwen et al., 2003). Compared with the lDP chicory 1-FFT, the globe thistle hDP 1-FFT strongly prefers longer inulins as acceptor substrates (Vergauwen et al., 2003). Substrate saturation kinetics (Fig. 5) were performed. The apparent 1-K donor Km of the native enzyme was estimated at 15 mM. It was found that the concentrations of 1-K in root tissues or induced leaf tissues ranges between 10–15 mM. Thus, the measurable 1-K concentrations in the tissue are sufficient to make the enzyme active. Moreover, expression of the cloned 1-FFT gene in fructan-accumulating and non-fructan-accumulating tissues of Echinops ritro (Fig. 6) have been compared. The results show that 1-FFT gene expression (Fig. 6B) correlates well with high DP fructan accumulation (Fig. 6A), suggesting that the fructosyl transferase, which is responsible for hDP inulin production in planta, has indeed been cloned.
So far, the advantage of storing high DP versus low DP inulins in perennial plants like Echinops ritro, Viguiera discolor, and Cynara scolymus remains unclear although it is worth mentioning that these species are all tolerant to drought. It can be postulated that high DP fructans directly or indirectly play a role in osmoregulation processes or are able to stabilize proteins and/or membranes causing increased resistance to drought-induced stress. It was shown that fructans are able to insert between the headgroups of phospholipids and stabilize them under drought conditions (Vereycken et al., 2001). Fructans have been found in the apoplast (Livingston and Henson, 1998; Van den Ende et al., 2005). Since they are apparently not biosynthesized in the apoplast (Van den Ende et al., 2005), they need to be transported from the vacuole to the apoplast by an up-to-now elusive mechanism. It is tempting to speculate that a vesicle-mediated fructan transport (exocytosis, endocytosis) occurs between the vacuole and the plasma membrane (Echeverria, 2000; Etxeberria et al., 2005). Interestingly, inulin spherocrystals have been detected in the xylem of V. discolor (Itaya et al., 1997), probably indicating the presence of high DP inulin in the extracellular space that could putatively interact with plasma membranes protecting them under drought.
Some perennial monocots like Lolium perenne (Pavis et al., 2001), Dactylis glomerata (Volaire and Lelièvre, 1997) and Phleum pratense (Thorsteinsson et al., 2002) also can accumulate high DP type fructans of the levan or neo-levan type. Unfortunately, the respective 6-FT enzymes biosynthesizing these high DP levans have not been characterized so far.
The cloning of the E. ritro 1-FFT cDNA (see below) and other plant hDP FT enzymes will allow studies on transgenic plants in which the gene is overexpressed or suppressed. Most probably, these studies will clarify the putative advantage of high DP fructans over low DP fructans and their roles under drought stress.
Cloning and molecular analysis of a hDP 1-FFT cDNA from E. ritro
The cloning of a hDP 1-FFT from Echinops ritro by using peptide sequence information of the native enzyme and performing RT-PCR is described here for the first-time (Tables 1, 2). Figure 2 indicates that fructan biosynthetic enzymes of the Asparagales, Poaceae, and Asteraceae arose from vacuolar invertases via strictly independent evolutionary events. Fructan biosynthesis may have arisen twice in Angiosperm evolution. Most likely, dicot fructan biosynthetic enzymes are more ancient than monocot fructan biosynthetic enzymes. Indeed, the similarity between the latter enzymes and their corresponding invertases is much greater. It can be further speculated that high DP 1-FFT enzymes evolved from a low DP 1-FFT ancestor but this remains to be further demonstrated.
The derived translated hDP 1-FFT cDNA (Fig. 3) has properties similar to other vacuolar type invertases and fructosyl transferases: a long prepropeptide region for vacuolar targeting, a low pI (4.77) and a calculated molecular mass of about 61 kDa (Van den Ende et al., 2002). The apparent molecular mass of 75 kDa (recombinant enzyme, Fig. 1) and 52+19 kDa (native enzyme, Fig. 1) is clearly higher than the calculated molecular mass of 61 kDa, but the difference can be explained by the extensive glycosylation being present on at least three out of five putative glycosylation sites (Table 2). Generally, fructan biosynthetic enzymes and a number of vacuolar invertases occur as heterodimers (Van den Ende et al., 1996) containing a large subunit (N-terminal end) of about 50–55 kDa and a small subunit (C-terminal end) of about 17–20 kDa. Surprisingly, the mature protein regions show no clear-cut different amino-acids between the high DP 1-FFTs from Echinops and Cynara on the one hand and the low DP 1-FFTs from Helianthus and Cichorium on the other hand (Fig. 3).
Heterologous expression in Pichia pastoris
As previously mentioned, the Pichia expression system is suitable to discriminate between invertases, FEHs, and fructosyl transferases. The successful heterologous expression of a highly evolved transferase like the hDP 1-FFT from E. ritro further confirms that the Pichia pastoris expression system is probably useful to judge the main functionality of all GH 32 enzymes. Therefore, the Pichia expression system is an ideal tool to judge the functionality of novel GH 32 members.
The present study shows that the recombinant hDP 1-FFT is fully functional (Fig. 4) and has similar properties to its native, heterodimeric counterpart (Fig. 5). This fact indicates that the proteolytic cleavage into large and small subunits in the native enzyme (Fig. 1) is not a prerequisite for obtaining an active enzyme. Overall, the physiological significance of proteolytic cleavage of fructosyl transferases into different subunits in planta is unclear (Van den Ende et al., 1996). The difference in glycosylation between native and recombinant enzyme does not seem to affect the properties of the enzyme in a significant way (Fig. 5). This is also consistent with the fact that glycosylations are situated far from the active site of the enzyme (1-FFT 3-D structure modelling with FEH as a scaffold). Glycosylations might affect enzyme stability rather than enzyme catalysis.
All native 1-FFTs are unable to use Suc as a donor substrate (Van den Ende et al., 1996; Vergauwen et al., 2003) and the recombinant E. ritro enzyme has the same property (Fig. 4A, B). The recombinant 1-FFT produces hDP inulin-type fructan profiles in vitro resembling those found in vivo from physiologically relevant 1-K concentrations (Fig. 4C). A high DP fraction of the in vitro-synthesized material was subjected to partial acid hydrolysis together with native chicory root inulin as a control. Exactly the same ß (2,1)-type fructo-oligosaccharides could be observed in both cases and no unknown peaks could be detected, demonstrating the inulin nature of the in vitro-synthesized material (Fig. 4C, D). Furthermore, incubations of the in vitro-synthesized material with 1-FEH showed Fru liberation while no hydrolysis occurred with 6-FEH (Fig. 4D).
In a batch system where no substrate is added as a function of incubation time, a maximal hDP inulin production is observed after a certain incubation time (Fig. 4A, B). So far, a maximal DP of 55 could be obtained in this batch system (data not shown). Similarly, as observed before for the chicory 1-FFT, 1-K substrate depletion and Suc accumulation after longer incubation times leads to an unfavourable 1-K/Suc ratio (Van den Ende and Van Laere, 1996) resulting in fructan depolymerization by using high DP inulin as donor and Suc and low DP inulins as acceptor substrates. The results suggest that a favourable 1-K/Suc ratio is necessary for efficient inulin production in vivo, even in Echinops ritro containing a 1-FFT enzyme which prefers longer inulin chains as acceptors. 1-SST most likely fulfils a crucial role in vivo by maintaining a 1-K/Suc ratio favourable for fructan polymerization (Vergauwen et al., 2003).
In contrast to carbohydrate profiles of chicory (Fig. 4A, B), where a slowly decreasing peak height is observed with increasing DP, the profiles of E. ritro show a strong decrease from DP4 to DP6 (Fig. 4C). However, from DP6 on, peak height remains roughly constant or decreases only slowly with higher DP. The latter pattern strongly suggests that the E. ritro hDP 1-FFT enzyme binds up to six Fru moieties of the acceptor substrate. Five fructosyl binding sites were proposed for Aspergillus awamori 1-FEH (Kulminskaya et al., 2003) while five to seven Glc binding sites are typical for amylases (Yamamoto, 1995).
Applications and perspectives
Fructans are prebiotics since they selectively promote beneficial colon bacteria like lactobacilli and bifidobacteria. Moreover, fructans stimulate Ca2+ resorption from the colon and possibly play an important role in preventing colon cancer (Roberfroid and Delzenne, 1998). As such, fructans quickly gain importance as healthy food ingredients. So far mainly lower DP inulin-type fructosyl oligo-saccharides (FOS), extracted from tap roots of industrial chicory varieties are used as food and feed additives. However, low DP inulins are quickly fermented in the proximal colon, only stimulating beneficial bacteria in this part of the colon. Longer DP inulins or branched fructans which are degraded more slowly throughout the colon might overcome this problem (Roberfroid et al., 1998). Higher DP fructans (Van Waes et al., 1998) are more suitable as fat replacers or needed for specific non-food applications requiring derivatization (Verraest et al., 1996). Different applications require fructans with a different DP. The recombinant 1-FFT described in this manuscript not only has the capacity to synthesize higher DP inulins, by adjusting reaction time, temperature (Fig. 4), and the ratio of 1-K/Suc or Neosugar P/Suc, the same enzyme will mainly produce FOS. Therefore, this enzyme might be of biotechnological value for a whole range of applications. Enzyme immobilization and continuous production of high DP inulins from Neosugar P as the sole substrate could be attempted. Alternatively, the introduction of the high DP 1-FFT gene in chicory might be tried. However, an additional boost in 1-SST activity might be necessary to lower the endogenous Suc concentration and optimize the 1-K/Suc ratio.
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Acknowledgements |
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W Van den Ende is a Postdoc supported by the FSR, Flanders. We thank the Laboratory of Functional Biology, KU Leuven (Professor J Winderickx) for the use of their equipment.
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Footnotes |
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Abbreviations: 1-FEH, fructan exohydrolase; 1-FFT, fructan:fructan 1-fructosyl transferase; 1-K, 1-kestose; 1-SST, sucrose:sucrose 1-fructosyl transferase; AEC-PAD, anion exchange chromatography with pulsed amperometric detection; DP, degree of polymerization; Glc, glucose; Fru, fructose; Suc, sucrose; Q-TOF MS: quadrupole time-of-flight mass spectrometry; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase–polymerase chain reaction.
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|---|
Amiard V, Morvan-Bertrand A, Billard J-P, Huault C, Keller F, Prud'homme M-P. 2003. Fructans, but not the sucrosyl-galactosides, raffinose and loliose, are affected by drought stress in perennial ryegrass. Plant Physiology 132, 2218–2229.
Bretthauer RK, Castellino FJ. 1999. Glycosylation of Pichia pastoris-derived proteins. Biotechnology and Applied Biochemistry 30, 193–200.
Cairns AJ. 2003. Fructan biosynthesis in transgenic plants. Journal of Experimental Botany 54, 549–567.
Chambert R, Treboul G, Dedonder R. 1974. Kinetic studies of levansucrase of Bacillus subtilis. European Journal of Biochemistry 41, 285–300.[Web of Science][Medline]
Chatto B. 1982. The Dry Garden. Orion Publishing.
Clerens S, Van den Ende W, Verhaert PD, Geenen L, Arckens L. 2004. Sweet Substitute: a software tool for in silico fragmentation of peptide-linked N-glycans. Proteomics 4, 629–632.[CrossRef][Web of Science][Medline]
Cummings JH, Macfarlane GT, Englyst HN. 2001. Prebiotic digestion and fermentation. American Journal of Clinical Nutrition 73, 415–420.
De Coninck B, Le Roy K, Francis I, Clerens S, Vergauwen R, Halliday A, Smith SM, Van Laere A, Van den Ende W. 2005. Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificities. Plant, Cell and Environment 28, 432–443.[CrossRef]
Dedonder R. 1972. Role and mechanisms of transglycosylation reactions. In: Piras R, Pontis HG, eds. Biochemistry of the glycosidic linkage. New York: Academic Press, 21–78.
Echeverria E. 2000. Vesicle-mediated solute transport between the vacuole and the plasma membrane. Plant Physiology 123, 1217–1226.
Edelman J, Jefford T. 1968. The mechanism of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus. New Phytologist 67, 517–531.[CrossRef][Web of Science]
Etxeberria E, Baroja-Fernandez E, Munoz FJ, Pozueta-Romero J. 2005. Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells. Plant and Cell Physiology 46, 474–481.
Hellwege EM, Czapla S, Jahnke A, Willmitzer L, Heyer AG. 2000. Transgenic potato (Solanum tuberosum) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke (Cynara scolymus) roots. Proceedings of the National Academy of Sciences, USA 97, 8699–8704.
Hendry G. 1993. Evolutionary origins and natural functions of fructans. A climatological, biogeographic and mechanistic appraisal. New Phytologist 123, 3–14.[Web of Science]
Henrissat B, Davies G. 1997. Structural and sequence-based classification of glycoside hydrolases. Current Opinion in Structural Biology 7, 637–644.[CrossRef][Web of Science][Medline]
Hincha DK, Zuther E, Hellwege EM, Heyer AG. 2002. Specific effects of fructo-and gluco-oligosaccharides in the preservation of liposomes during drying. Glycobiology 12, 103–110.
Hincha DK, Zuther E, Heyer AG. 2003. The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions. Biochimica et Biophysica Acta 1612, 172–177.[Medline]
Hisano H, Kanazawa A, Kawakami A, Yoshida M, Shimamoto Y, Yamada T. 2004. Transgenic perennial ryegrass plants expressing wheat fructosyltransferase genes accumulate increased amounts of fructan and acquire increased tolerance on a cellular level to freezing. Plant Science 167, 861–868.[CrossRef]
Iizuka M, Yamaguchi H, Ono S, Minamiura N. 1993. Production and isolation of levan by use of levansucrase immobilized on the ceramic support SM-10. Bioscience, Biotechnology and Biochemistrry 57, 322–324.
Itaya NM, Buckeridge MS, Figueiredo-Ribeiro RCL. 1997. Biosynthesis in vitro of high-molecular-mass fructan by cell-free extracts from tuberous roots of Viguiera discolor (Asteraceae). New Phytologist 136, 53–60.[CrossRef]
Kawakami A, Yoshida M. 2002. Molecular characterization of sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated with fructan accumulation in winter wheat during cold hardening. Bioscience Biotechnology and Biochemistrry 66, 2297–2305.
Kulminskaya AA, Arand M, Eneyskaya EV, Ivanen DR, Shabalin KA, Shishlyannikov SM, Saveliev AN, Korneeva OS, Neustroev KN. 2003. Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis. Biochimica et Biophysica Acta 1650, 22–29.[Medline]
Lerouge P, Cabanes-Macheateau M, Rayon C, Fischette-Lainé AC, Gomord V, Faye L. 1998. N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Molecular Biology 38, 31–48.[CrossRef][Web of Science][Medline]
Lewis DH. 1993. Nomenclature and diagrammatic representation of oligomeric fructans. A paper for discussion. New Phytologist 124, 583–594.[CrossRef]
Livingston DP, Henson CA. 1998. Apoplastic sugars, fructans, fructan exohydrolase, and invertase in winter oat: responses to second-phase cold hardening. Plant Physiology 116, 403–408.
Maia IG, Leite A. 2001. N-glycosylation in sugarcane. Genetics and Molecular Biology 24, 1–4.
Nagaraj VJ, Altenbach D, Galati V, Luscher M, Meyer AD, Boller T, Wiemken A. 2004. Distinct regulation of sucrose: sucrose-1-fructosyltransferase (1-SST) and sucrose: fructan-6-fructosyl transferase (6-SFT), the key enzymes of fructan synthesis in barley leaves: 1-SST as the pacemaker. New Phytologist 161, 735–748.[CrossRef]
Parvanova D, Ivanov S, Konstantinova T, Karanov E, Atanassov A, Tsvetkov T, Alexieva V, Djilianov D. 2004. Transgenic tobacco plants accumulating osmolytes show reduced oxidative damage under freezing stress. Plant Physiology and Biochemistry 42, 57–63.[Medline]
Pavis N, Boucaud J, Prud'homme MP. 2001. Fructans and fructan-metabolizing enzymes in leaves of Lolium perenne. New Phytologist 150, 97–109.[CrossRef][Web of Science]
Pilon-Smits EAH, Ebskamp MJM, Jeuken MJW, Weisbeek PJ, Smeekens SCM. 1995. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiology 107, 125–130.[Abstract]
Ritsema T, Smeekens S. 2003. Fructans: beneficial for plants and humans. Current Opinion in Plant Biology 6, 223–230.[CrossRef][Web of Science][Medline]
Roberfroid MB, Delzenne NM. 1998. Dietary fructans. Annual Review of Nutrition 18, 117–143.[CrossRef][Web of Science][Medline]
Roberfroid MB, Van Loo JAE, Gibson GR. 1998. The bifidogenic nature of chicory inulin and its hydrolysis products. Journal of Nutrition 128, 11–19.
Sevenier R, Hall RD, van der Meer IM, Hakkert HJ, van Tunen AJ, Koops AJ. 1998. High level fructan accumulation in a transgenic sugar beet. Nature Biotechnology 16, 843–846.[CrossRef][Web of Science][Medline]
Tapernoux-Luthi EM, Bohm A, Keller F. 2004. Cloning, functional expression, and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan: galactan galactosyltransferase, from common bugle leaves. Plant Physiology 134, 1377–1387.
Thorsteinsson B, Harrison PA, Chatterton NJ. 2002. Fructan and total carbohydrate accumulation in leaves of two cultivars of timothy (Phleum pratense Vega and Climax) as affected by temperature. Journal of Plant Physiology 159, 999–1003.[CrossRef]
Van den Ende W, De Coninck B, Clerens S, Vergauwen R, Van Laere A. 2003. Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping, and functional analysis of a novel ‘cell wall invertase-like’ specific 6-FEH from sugar beet (Beta vulgaris L.). The Plant Journal 36, 697–710.[CrossRef][Web of Science][Medline]
Van den Ende W, De Coninck B, Van Laere A. 2004. Plant fructan exohydrolases: a role in signaling and defense? Trends in Plant Science 9, 523–528.[CrossRef][Web of Science][Medline]
Van den Ende W, Michiels A, Le Roy K, Van Laere A. 2002. Cloning of a vacuolar invertase from Belgian endive leaves (Cichorium intybus L.). Physiologia Plantarum 115, 504–512.[Medline]
Van den Ende W, Michiels A, Van Wonterghem D, Clerens S, De Roover J, Van Laere A. 2001. Defoliation induces 1-FEH II (fructan 1-exohydrolase II) in Witloof chicory roots. Cloning and purification of two isoforms (1-FEH IIa and 1-FEH IIb). Mass fingerprint of the 1-FEH II enzymes. Plant Physiology 126, 1186–1195.
Van den Ende W, Van Laere A. 1996. De novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose:sucrose fructosyl transferase and fructan:fructan fructosyl transferase) from chicory roots (Cichorium intybus L.). Planta 200, 335–342.
Van den Ende W, Van Wonterghem D, Verhaert P, Dewil E, Van Laere A. 1996. Purification and characterization of fructan:fructan fructosyl transferase from chicory roots (Cichorium intybus L.). Planta 199, 493–502.
Van den Ende W, Yoshida M, Clerens S, Vergauwen R, Kawakami A. 2005. Cloning, characterization and functional analysis of novel 6-kestose exohydrolases (6-KEHs) from wheat (Triticum aestivum L.). New Phytologist 166, 917–932.[CrossRef][Web of Science][Medline]
Van Laere A, Van den Ende W. 2002. Inulin metabolism in dicots: chicory as a model system. Plant, Cell and Environment 25, 803–815.[CrossRef]
Van Waes C, Baert J, Carlier L, Van Bockstaele E. 1998. A rapid determination of the total sugar content and the average inulin chain length of in roots of chicory (Cichorium intybus L.). Journal of the Science of Food and Agriculture 76, 107–110.[CrossRef]
Vereyken IJ, Chupin V, Demel RA, Smeekens SCM, De Kruijff B. 2001. Fructans insert between the headgroups of phospholipids. Biochimica et Biophysica Acta, Biomembranes 1510, 307–320.[CrossRef]
Vergauwen R, Van Laere A, Van den Ende W. 2003. Properties of fructan:fructan 1-fructosyltransferase (1-FFT) from Cichorium intybus L. and Echinops ritro L., two Asteracean plants storing greatly different types of inulin. Plant Physiology 133, 391–401.
Verhaest M, Van den Ende W, Le Roy K, De Ranter CA, Van Laere A, Rabijns A. 2005. X-Ray diffraction structure of a plant glycosyl hydrolase family 32 protein: fructan 1-exohydrolase IIa of Cichorium intybus. The Plant Journal 41, 400–411.[Web of Science][Medline]
Verraest DL, Peters JA, van Bekkum H, van Rosmalen GM. 1996. Carboxymethyl inulin: a new inhibitor for calcium carbonate precipitation. Journal of the American Oil Chemists Society 73, 55–62.
Vijn I, Van Dijken A, Sprenger N, Van Dun K, Weisbeek P, Wiemken A, Smeekens S. 1997. Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase. The Plant Journal 11, 387–398.[CrossRef][Web of Science][Medline]
Volaire F, Lelièvre F. 1997. Production, persistence, and water-soluble carbohydrate accumulation in 21 contrasting populations of Dactylis glomerata L. subjected to severe drought in the south of France. Australian Journal of Agricultural Research 48, 933–944.[CrossRef]
Yamamoto T. 1995. Enzyme chemistry and molecular biology of amylases and related enzymes. Boca Raton, Florida: The Amylase Research Society of Japan, CRC Press.
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