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Plant Physiol. (1998) 117: 1507-1513
Cloning of Sucrose:Sucrose 1-Fructosyltransferase from Onion and
Synthesis of Structurally Defined Fructan Molecules from
Sucrose1
Irma Vijn2, *,
Anja van Dijken2,
Marcel Lüscher,
Antoine Bos,
Edward Smeets,
Peter Weisbeek,
Andres Wiemken, and
Sjef Smeekens
Departments of Molecular Cell Biology (I.V., A.v.D., A.B., E.S.,
P.W., S.S.), and Botanical Ecology and Evolutionary Biology (S.S.),
University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands; and University of Utrecht, Padualaan 8, 3584 CH Utrecht, The NetherlandsDepartment of Botany, University of Basel, Hebelstrasse 1, CH-4056
Basel, Switzerland (M.L., A.W.)
 |
ABSTRACT |
Sucrose
(Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant
fructan biosynthesis, since it catalyzes de novo fructan synthesis from
Suc. We have cloned 1-SST from onion (Allium cepa) by
screening a cDNA library using acid invertase from tulip (Tulipa
gesneriana) as a probe. Expression assays in tobacco
(Nicotiana plumbaginifolia) protoplasts showed the
formation of 1-kestose from Suc. In addition, an onion acid invertase
clone was isolated from the same cDNA library. Protein extracts of
tobacco protoplasts transformed with this clone showed extensive
Suc-hydrolyzing activity. Conditions that induced fructan accumulation
in onion leaves also induced 1-SST mRNA accumulation, whereas the acid
invertase mRNA level decreased. Structurally different fructan
molecules could be produced from Suc by a combined incubation of
protein extract of protoplasts transformed with 1-SST and protein
extract of protoplasts transformed with either the onion
fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan
6-fructosyltransferase.
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INTRODUCTION |
Fructans (polyfructosylsucrose) consist of polymers of Fru
attached to Suc and serve as an important storage carbohydrate in
approximately 15% of flowering plant species (Hendrey and Wallace, 1993). The Fru residues are either linked by a (2-1)
 -D-glycosidic bond, as in inulin derived from
Cichorium intybus L. (Bonnett et al., 1994 ), or by a (2-6)
-D-glycosidic bond, as in levans (e.g. Phleum
pratense L.; Suzuki and Pollock, 1986). In most grasses branched
fructans containing both types of linkages are produced (e.g.
Triticum aestivum L.; Carpita et al., 1989). In plants of the Liliales, to which onion (Allium cepa) and
tulip (Tulipa gesneriana) belong, a special type of fructan
is produced, the inulin neoseries. In this type of fructan the Glc
moiety of Suc contains fructosyl residues on both C1 and C6, resulting
in a polymer with (2-1)-linked fructosyl chains on either end of
the Suc molecule (Shiomi, 1989).
For synthesis of the linear inulin at least two enzymes are required
(Pollock and Cairns, 1991): 1-SST initiates fructan synthesis by
catalyzing the transfer of a fructosyl residue from Suc to another Suc
molecule, resulting in the formation of the trisaccharide 1-kestose
(G1-2F1-2F, also called isokestose), and 1-FFT elongates the
fructosyl chain. It has been shown that incubation of a combination of
purified 1-SST and 1-FFT from Jerusalem artichoke with Suc results in
the production of inulin with a polymer length of up to 20 fructosyl
residues (Koops and Jonker, 1996; Lüscher et al., 1996). For
production of the inulin neoseries, the type of fructan found in onion,
a third enzyme is needed, 6G-FFT (Shiomi, 1989; Wiemken et al., 1995).
Recently, we cloned the gene encoding the 6G-FFT from onion (Vijn et
al., 1997). This fructosyltransferase catalyzes the transfer of a Fru
residue of 1-kestose to C6 of the Glc moiety of Suc, forming neokestose
(F2-6G1-2F). Transgenic chicory plants harboring the onion 6G-FFT
under the control of the cauliflower mosaic virus 35S RNA promoter
produce inulin of the neoseries in addition to linear inulin (Vijn et
al., 1997).
In this paper we describe the cloning and functional characterization
of onion cDNA clones encoding a 1-SST and an acid invertase. Furthermore, we show that with specific combinations of
fructosyltransferases, structurally different fructan molecules can be
produced from Suc.
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MATERIALS AND METHODS |
Preparing and Screening of a cDNA Library of Onion
Onion (Allium cepa L., BMCCB) seeds were
germinated on soil and grown for 4 weeks at 21°C with a light/dark
cycle of 16 h/8 h. Subsequently, leaves were cut off (2 h after the
dark-to-light switch), placed in a 5% Suc solution, and illuminated
continuously for up to 24 h. Samples were taken after 0, 4, 8, 12, 16, and 24 h. mRNA isolation systems (PolyATtract I and II,
Promega) were used to isolate poly(A+) RNA from
leaves illuminated for 4 h. A cDNA library was made using a
cloning kit (ZAP-cDNA Gigapack III Gold Cloning Kit, Stratagene). The
amplified cDNA library was screened with a random
32P-labeled 1340-bp 5 fragment of the acid
invertase cDNA from tulip (Tulipa gesneriana) (accession no.
X95651; a kind gift of Dr. A.D. de Boer, ATO-DLO, Wageningen,
The Netherlands) according to the Stratagene protocol. Overnight
hybridization was performed in buffer containing 5× Denhardt's
solution, 6× SSC, and 0.5% SDS at 55°C, and the filters were washed
three times in 0.5× SSC and 0.1% SDS at 55°C. Sequencing of
the clones was performed with an automatic DNA-sequencing
apparatus (48 cm, model 373, Applied Biosystems) using a
dye-terminator cycle-sequencing kit (Prism, with Amplitaq DNA
polymerase, Applied Biosystems).
Expression of Isolated cDNAs in Tobacco Protoplasts
The complete inserts of the isolated cDNA clones were subcloned
into pMon999 (Monsanto, St. Louis, MO), which contains the cauliflower
mosaic virus 35S RNA promoter and a nopaline synthase terminator
sequence. Isolation and transformation of tobacco (Nicotiana plumbaginifolia) protoplasts was performed according to the
method of Goodall et al. (1990). Prior to PEG transformation of 6 × 105 protoplasts with 50 µg of plasmid DNA
the protoplasts were washed four times by floating in wash buffer (0.4 M Suc, 5 mM KCl, 125 mM
CaCl2 2H2O, and 5 mM Glc, pH 5.8). After overnight incubation at 22°C in
the dark, protoplasts were pelleted and resuspended in 50 mM Mes buffer, pH 5.6. Protoplasts were lysed by freezing in liquid nitrogen. Cell debris were removed by centrifugation at
13,000g for 5 min. Protein extracts were desalted by passing the supernatants over Biogel P-10 columns at 350g for 5 min
(Simmen et al., 1993). Finally, 135 µL of purified protein
extract was obtained per protoplast transfection. Total protein was
measured using the method of Bradford (1976), with BSA as the standard.
In Vitro Fructosyltransferase Activity Assay and Detection of
Products
Protein extracts from transformed protoplasts were incubated in
the presence of 0.02% NaN3 with an appropriate
sugar substrate (see ``'') in 50 mM Mes buffer, pH
5.6, at 27°C for up to 24 h. The reaction was stopped by heating
the samples at 95°C for 4 min. For fructosyltransferase activity
assays with a single transfection extract, 45 µL of purified protein
extract was used. The total amount of protein corresponding to this
volume was 15 µg from transfections with empty vector, onion 6G-FFT,
and barley 6-SFT, and 22 µg from transfections with AcN2 and AcT1.
For the combined enzyme incubations, 18 µL of each protein extract
was used. The end volume of all incubations was 50 µL. Products
formed were analyzed by HPLC using a CarboPac PA-100 column (Dionex,
Sunnyvale, CA) and a DX-500 gradient chromatography system with a
pulsed amperometric detector (Dionex). Five microliters from each
incubation was injected, including 0.5 µg of trehalose as an internal
standard (final injection volume, 10 µL).
Induction of Fructan Accumulation in Onion Leaves
Leaves of 4-week-old onion plants grown at 21°C with a
light/dark cycle of 16 h/8 h were cut off (2 h after the dark-to-light switch), placed in a 5% Suc solution, and then illuminated
continuously for up to 24 h. Sugars were isolated by extracting
the leaves three times with 80% ethanol at 60°C. The extracts were
vacuum desiccated and the sugar pellet was dissolved in water at 0.5 µL/mg fresh weight. Sugar composition was determined by TLC. One microliter of the sugar solution was spotted on TLC foil (Silicagel F1500, Schleicher & Schuell), which was developed three times in
acetone:water (87:13, v/v) (Wagner et al., 1983) and
then stained with a Fru-specific urea-phosphoric acid spray (Wise et
al., 1955).
RNA Extraction and Northern Analysis
Total RNA was isolated according to the method of Verwoerd et al.
(1989). The RNA gel-blot analysis was performed according to the
protocol for Hybond N membrane (version 2.0, Amersham). 5 Fragments of
the fructosyltransferase encoding cDNAs were used as probes: a 910-bp
EcoRI/XhoI fragment of pAcN2 (1-SST), a 577-bp EcoRI/KpnI fragment of pAcT1 (acid invertase), a
582-bp EcoRI/PstI fragment of pAc2 (6G-FFT) (Vijn
et al., 1997), and a 1500-bp fragment containing the coding region of
the 18S rRNA of Arabidopsis (18SrRNA) (Pruitt and
Meyerowitz, 1986). The northern blots were hybridized overnight at
65°C in the same hybridization buffer used for screening of the cDNA
library and were washed three times in 0.5× SSC and 0.1% SDS at
65°C.
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RESULTS |
Isolation of Onion cDNA Clones Encoding Fructosyltransferases
Based on the known homology between fructosyltransferases and
invertases (Sprenger et al., 1995; Vijn et al., 1997), an onion cDNA
library prepared from leaves induced for fructan synthesis was screened
with a 32P-labeled insert of an acid invertase
cDNA clone from tulip (accession no. X95651). Fourteen positive clones,
although containing cDNA inserts with different lengths, encoded the
same gene. The clone with the longest cDNA insert, called pAcN2, was
sequenced completely. pAcN2 has an insert of 2202 bp and contains a
poly(A+) stretch at the 3 end. It contains one
long open reading frame encoding a 623-amino acid polypeptide and a
63-bp 5-untranslated region. The deduced polypeptide contains five
potential glycosylation sites and has a pI of 5.2. The amino acid
sequence of AcN2 shows 49% identity to the chicory 1-SST polypeptide,
53% identity to the acid invertase of tulip, and 58% identity to the
onion 6G-FFT polypeptide (Fig. 1),
suggesting that AcN2 encodes a fructosyltransferase as well.
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| Figure 1.
Comparison of deduced amino acid sequence of AcN2
and AcT1 from onion with onion 6G-FFT, chicory 1-SST, and acid
invertase of tulip. AcSST, Deduced amino acid sequence
of AcN2 (accession no. AJ006066); AcInv, deduced amino
acid sequence of AcT1 (accession no. AJ007067);
Ac6G-FFT, from onion (Vijn et al., 1997; accession no.
Y07838); CiSST, 1-SST from chicory (de Halleux and Van
Cutsem, 1997; accession no. U81520); TgInv, vacuolar
invertase from tulip (accession no. X95651). The N-terminal end of the
mature 1-SST protein of chicory is indicated by an arrow. Identical
amino acids are shaded. The putative glycosylation sites in
AcSST and AcInv are underlined.
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Two other positive cDNA clones, also containing different-sized cDNA
inserts, encoded a gene that is highly homologous, but not identical to
the AcN2-encoded gene. The clone containing the longest cDNA insert,
pAcT1, was sequenced completely. pAcT1 has an insert of 2438 bp and
contains a poly(A+) stretch at the 3 end. It has
one long open reading frame encoding a 690-amino acid polypeptide and a
59-bp 5-untranslated region. The deduced polypeptide contained three
potential glycosylation sites and the pI was 4.7. The amino acid
sequence of AcT1 shows 47% identity to chicory 1-SST, 61% identity to
AcN2 and the acid invertase of tulip, and 63% identity to the onion
6G-FFT polypeptide (Fig. 1), suggesting that AcT1 also encodes a
fructosyltransferase.
The N-terminal end of mature chicory 1-SST has been determined (Van den
Ende et al., 1996) and starts at the 98th codon (de Halleux and Van
Cutsem, 1997) (Fig. 1). The high homology of AcN2 and AcT1 with the
fructosyltransferases starts downstream of the 98th codon of chicory
1-SST, suggesting that these proteins will also be processed from a
larger preproprotein.
Determination of Enzymatic Activity in Tobacco Protoplasts
The amino acid sequence comparisons suggest that both AcN2 and
AcT1 encode fructosyltransferases, but do not indicate specific enzymatic activities. Therefore, the cDNA inserts of pAcN2 and pAcT1
were placed under control of the cauliflower mosaic virus 35S RNA
promoter and transiently expressed in tobacco protoplasts. Protein
extracts of transformed protoplasts were incubated with either 20 or
100 mM Suc for 24 h at 27°C, and the sugar products were analyzed by HPLC. Several independent protoplast transformations and enzyme activity assays were performed, with comparable results.
The product formed by AcN2-transformed protoplasts after incubation
with 20 mM Suc was 1-kestose (Fig.
2A). Higher levels of 1-kestose were
produced when the protein extract was incubated with 100 mM
Suc (Fig. 2D). These results strongly suggest that AcN2 encodes a
1-SST. Incubation of AcT1-transformed protoplasts with 20 mM Suc resulted in a complete Suc hydrolysis showing
invertase activity (Fig. 2B). It is well known that at elevated Suc
levels invertase has some fructosyltransferase activity (Straathof et al., 1986; Obenland et al., 1993), and this was observed when AcT1
protein extract was incubated with 100 mM Suc (Fig. 2E). These results strongly suggest that AcT1 encodes an invertase. According to the high sequence homology to the acid invertase of tulip
(Fig. 1) and the low pI (4.7) of the deduced AcT1 polypeptide, AcT1
most likely codes for an acid invertase.
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| Figure 2.
Analysis of sugar products generated by protein
extracts of transformed protoplasts after incubation with different Suc
concentrations. Sugar products were analyzed by HPLC after a 24-h
incubation with 20 mM (A, B, and C) or 100 mM
(D and E) Suc at 27°C. A and D, Protein extract from protoplasts
transformed with AcN2 under control of the 35S promoter; B and E,
protein extract from protoplasts transformed AcT1 under control of the
35S promoter; C, protein extract from protoplasts transformed with
empty vector; and F, sugar extract from chicory root. Peaks in the HPLC
chromatograms: T, trehalose (internal standard); G, Glc; F, Fru; S, Suc
(G1-2F); 1-K, 1-kestose (G1-2F1-2F); DP4-6, inulin fructan with a
degree of polymerization of 4 to 6.
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mRNA Levels of 1-SST and Invertase during Induced Fructan
Accumulation in Onion Leaves
Normally, onion leaves do not accumulate fructan. However, by
supplying the leaves with excessive Suc, fructan synthesis can be
induced (Vijn et al., 1997). Excessive Suc can be supplied by removing
the sink tissue and feeding the leaves with Suc, by placing them under
continuous illumination, or both. mRNA accumulation of 1-SST (AcN2) and
invertase (AcT1) during fructan accumulation in onion leaves was
determined by northern blotting. Fructan accumulation was induced in
4-week-old onion leaves from which the sinks had been removed and that
were placed in 5% Suc solution and illuminated continuously for up to
24 h. After 4 h of continuous illumination, 1-SST mRNA was
clearly detected and increased up to 12 h, after which time it
stabilized (Fig. 3B). This expression
pattern corresponded with 1-kestose production, which was detected
after 4 h and increased up to 12 h, after which time it
stabilized (Fig. 3A). Invertase mRNA was present in the leaves at the
moment they were cut and placed in the Suc solution (Fig. 3B). During
fructan accumulation the mRNA level decreased (Fig. 3B). The expression
of onion 6G-FFT, which catalyzes the formation of neokestose from Suc
and 1-kestose, corresponded with neokestose formation. 6G-FFT mRNA was
detected at 12 h and increased after this time (Fig. 3B).
Neokestose production was detected at 16 h and increased up to
24 h (Fig. 3A).
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| Figure 3.
A, Fructan accumulation in onion leaves. TLC of
sugars extracted from onion leaves after several time intervals of
continuous illumination and feeding of 5% Suc. F, Fru; G, Glc; S, Suc;
N-K, neokestose; 1-K, 1-kestose; Nys, nystose; DP1-DP6, inulin fructan
with a degree of polymerization of 1 to 6; O, sugar products of onion
bulb; C, inulin from chicory. B, mRNA levels of 1-SST, invertase, and
6G-FFT during fructan accumulation in onion leaves. Northern blots
contain approximately 20 µg of total RNA from onion leaves treated
similarly as the ones used in A. cDNA clones encoding onion 1-SST,
invertase, and 6G-FFT were used as probes. An Arabidopsis
18S rRNA probe was used as a loading control.
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In Vitro Synthesis of Structurally Different Fructan Molecules
The cloning of 1-SST enables synthesis of fructan molecules from
Suc. Here we show that 1-SST in combination with other
fructosyltransferases for which the specific enzymatic activity is
known can synthesize structurally different fructan molecules from Suc.
Combined incubation of protein extracts of protoplasts transformed with
onion 1-SST and onion 6G-FFT with 100 mM Suc resulted in
the formation of 1-kestose and neokestose (Fig.
4A). Incubation of only protein extract
of onion 6G-FFT transformed protoplasts with 100 mM Suc did
not give any additional products (data not shown), but when this
protein extract was incubated with 20 mM Suc and 20 mM 1-kestose as the substrates, neokestose, nystose, and
some products with a higher degree of polymerization were formed (Fig.
4B). This shows that in vitro, 1-SST and 6G-FFT together are capable of producing fructan of the inulin neoseries from Suc. The combined incubation of protein extracts with only Suc has a higher
Suc-to-1-kestose ratio than the single incubation of 6G-FFT protein
extract with 20 mM Suc and 20 mM 1-kestose. A
low Suc concentration is preferable for 6G-FFT activity, which explains
the presence of products with a higher degree of polymerization in
Figure 4B.
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| Figure 4.
Analysis of sugar products generated by either
single or combined incubation of protein extracts from transformed
protoplasts with different substrates. Sugar products were analyzed by
HPLC after 24 h of incubation at 27°C. A, Combined incubation of
protein extracts from protoplasts transformed with onion 1-SST and
6G-FFT with 100 mM Suc; B, single incubation of protein
extract from protoplasts transformed with onion 6G-FFT with 20 mM Suc and 20 mM 1-kestose; C, sugar products
from onion bulb; D, combined incubation of protein extracts from
protoplasts transformed with onion 1-SST and barley 6-SFT with 100 mM Suc; E, single incubation of protein extract from
protoplasts transformed with barley 6-SFT with 100 mM Suc;
and F, single incubation of protein extract from protoplasts
transformed with onion 1-SST with 100 mM Suc. Peaks in the
HPLC chromatograms: T, trehalose (internal standard); G, Glc; F, Fru;
S, Suc (G1-2F); 1-K, 1-kestose (G1-2F1-2F); N-K, neokestose
(F2-6G1-2F); N, nystose (G1-2F1-2F1-2F); 6-K, 6-kestose
(G1-2F6-2F); BiF, bifurcose (G1-2F1[6-2F]1-2F).
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The combination of protein extracts of protoplasts transformed with
onion 1-SST and with barley 6-SFT resulted in the formation of
1-kestose, 6-kestose, and bifurcose from Suc (Fig. 4D). The single
incubation of protein extract of 6-SFT-transformed protoplasts with Suc
resulted in the production of 6-kestose, a low amount of 1-kestose
production, and a very low amount of bifurcose (Fig. 4E). These results
show that the cloning of 1-SST enables synthesis of structurally
different fructan molecules from Suc when combined with other
fructosyltransferases
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DISCUSSION |
In this paper we describe the isolation of the onion cDNA clone
pAcN2, which encodes the key enzyme in fructan synthesis: 1-SST. This
enzyme initiates de novo fructan synthesis in plants by catalyzing the
transfer of a fructosyl residue from Suc to another Suc molecule,
resulting in the production of 1-kestose and Glc. We also isolated the
cDNA clone pAcT1, which encodes an acid invertase.
The cDNA clones were isolated by screening an onion cDNA library with a
cDNA encoding a tulip acid invertase. The deduced amino acid sequence
of AcN2 shows 49% identity to chicory 1-SST, 53% identity to tulip
acid invertase, and 58% identity to 6G-FFT of onion. The deduced amino
acid sequence of AcT1 shows 61% identity to tulip acid invertase and
to onion 1-SST polypeptide and 63% identity to onion 6G-FFT. These
high sequence homologies only show that the cDNAs most likely encode
fructosyltransferases, but do not discriminate between specific
enzymatic activities, so the cDNAs were transiently expressed in
tobacco protoplasts. Incubation of protein extract of AcN2-transformed
protoplasts with Suc showed the formation of 1-kestose. When protein
extract of AcT1-transformed protoplasts was incubated with 20 mM Suc, a complete Suc hydrolysis was observed, whereas
with 100 mM Suc, in addition to hydrolysis of the Suc, some
1-kestose was also produced. This is in agreement with the enzymatic
activity of purified barley acid invertase, which also has 1-SST
activity at high Suc concentrations (Obenland et al., 1993). Since in
these enzymatic assays the same amount of total protein in the AcN2 and
AcT1 protoplast extracts was used, these results strongly suggest that
AcN2 encodes a 1-SST, whereas AcT1 encodes an invertase. Whether the
cloned 1-SST can also catalyze fructosyl transfer to water is not
known. The expression system used to characterize the enzymatic
activities of the cloned genes does contain endogenous invertases that
compete for the same substrate. To characterize the enzymatic
activities of the cloned 1-SST and invertase in more detail, it will be
necessary either to purify the proteins or to express them in a system
lacking -fructosidase activity.
During induced fructan accumulation in onion leaves, 1-SST (AcN2) mRNA
accumulation corresponded with Suc accumulation and 1-kestose
production, whereas the invertase (AcT1) mRNA level decreased upon Suc
accumulation. The accumulation of 6G-FFT mRNA corresponded to
neokestose production. Most likely, mRNA synthesis of 1-SST is induced
by a high Suc concentration, whereas for the induction of 6G-FFT mRNA
synthesis an additional signal is necessary. Furthermore, these results
strongly suggest that in vivo the enzymes encoded by AcN2 and AcT1 have
the same enzymatic activity as that determined in vitro.
Combined incubation of protein extracts of protoplasts
transformed with the onion 1-SST and 6G-FFT with 100 mM Suc
resulted in the formation of 1-kestose and neokestose. The combined
incubation of protein extracts of protoplasts transformed with onion
1-SST and barley 6-SFT resulted in the formation of 1-kestose,
6-kestose, and bifurcose. In chicory it has been shown that
introduction of the onion 6G-FFT or barley 6-SFT results in the
production of inulin of the neoseries and branched fructans,
respectively, in addition to linear inulin (Sprenger et al., 1997; Vijn
et al., 1997). These findings showed that the type of
fructan made by a plant can be changed by introduction of a
fructosyltransferase with a different enzymatic activity than the
endogenous fructosyltransferases. Here we show that it is possible to
synthesize in vitro structurally defined fructan molecules from Suc by
combining fructosyltransferases with specific enzymatic activities. By
introducing specific sets of fructosyltransferases, it will now be
possible to accumulate structurally defined fructan molecules in plants
that normally do not accumulate fructan.
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FOOTNOTES |
1
This work was supported in part by the
Netherlands Foundation for Chemical Research with financial aid from
the Netherlands Technology Foundation.
2
These authors contributed equally to this paper.
*
Corresponding author; e-mail i.m.c.vijn{at}bio.uu.nl; fax
31-30-25-13-65-5.
Received March 17, 1998;
accepted May 15, 1998.
The accession numbers for the sequences reported in this article are
AJ006066 (AcN2 and 1-SST) and AJ006067 (AcT1 and acid invertase).
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ABBREVIATIONS |
Abbreviations:
1-SST, Suc:Suc 1-fructosyltransferase.
6G-FFT, fructan:fructan 6G-fructosyltransferase.
6-SFT, Suc:fructan
6-fructosyltransferase.
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ACKNOWLEDGMENTS |
We thank Tony van Kampen (Agricultural University, Wageningen,
The Netherlands) for providing excellent sequence data of the isolated
cDNAs, Douwe de Boer (ATO-DLO, Wageningen, The
Netherlands) for his kind gift of the acid invertase cDNA clone of
tulip, and M. Hirayama (Meiji Seika Kaisha, Ltd., Saitama, Japan) for
the gift of 1-kestose.
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Abstract
Introduction