Journal of Experimental Botany, Vol. 57, No. 11, pp.

2719–2734, 2006
doi:10.1093/jxb/erl034 Advance Access publication 25 July, 2006

RESEARCH PAPER

Molecular and functional characterization of a cDNA

encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/
fructan:fructan 1-fructosyltransferase (1-FFT) from
perennial ryegrass (Lolium perenne L.)

Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011
Bertrand Lasseur1, Jérémy Lothier1, Abdelmadjid Djoumad2, Barbara De Coninck4, Sjef Smeekens3,
André Van Laere4, Annette Morvan-Bertrand1, Wim Van den Ende4 and Marie-Pascale Prud’homme1,*
1
UMR INRA-UCN 950 EVA Ecophysiologie Végétale, Agronomie et Nutritions NCS, Université de Caen,
Esplanade de la Paix, F-14032 Caen cedex, France
2
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbooke, QC, Canada
3
Department of Molecular Plant Physiology, University Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
4
Department of Biology, Laboratory for Molecular Plant Physiology, Botany Institute, KULeuven, Kasteelpark
Arenberg 31, B-3001 Leuven, Belgium

Received 24 January 2006; Accepted 24 April 2006

Abstract regulation of expression. Lp6G-FFT gene expression

Fructans are the main storage compound in Lolium
perenne. To account for the prevailing neokestose-
based fructan synthesis in this species, a cDNA library
of L. perenne was screened by using the onion (Allium
cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT)
as a probe. A full length Lp6G-FFT clone was isolated
with significant homologies to vacuolar type fructosyl-
transferases and invertases. The functionality of the
cDNA was tested by heterologous expression in Pichia
pastoris. The recombinant protein demonstrated both
6G-FFT and fructan:fructan 1-fructosyltransferase ac-
tivities (1-FFT) with a maximum 6G-FFT/1-FFT ratio
of two. The activity of 6G-FFT was investigated with res-
pect to developmental stage, tissue distribution, and
alterations in carbohydrate status expression and com-
pared to sucrose:sucrose 1-fructosyltransferase (1-SST).
Lp6G-FFT and Lp1-SST were predominantly expres-
sed in the basal part of elongating leaves and leaf
sheaths. Expression of both genes declined along the
leaf axis, in parallel with the spatial occurrence of
fructan and fructosyltransferase activities. Surpris-
ingly, Lp6G-FFT was highly expressed in photosyn-
thetically active tissues where very low extractable
fructosyltransferase activity and fructan amounts
were detected, suggesting a post-transcriptional
increased only in elongating leaves following similar
increases of sucrose content in blades, sheaths, and
elongating leaf bases. Regulation of Lp6G-FFT gene
expression depends on the tissue according to its
sink–source status.
Key words: Fructan, fructan:fructan 6G-fructosyltransferase,
gene expression, heterologous expression, Lolium perenne,
Pichia pastoris, sucrose:sucrose 1-fructosyltransferase.
Introduction
Fructans are sucrose-derived fructose polymers. Fructan
types differ from one another by their length (degree of
polymerization, DP), branching, the linkage type between
adjacent fructose and the position of the glucose residue.
Fructans are known for their role as a carbohydrate reserve
found in approximately 15% of the flowering plant species
(Hendry and Wallace, 1993). Many of these plant species
belong to economically important orders such as Asterales
[chicory (Cichorium intybus), Jerusalem artichoke (Heli-
anthus tuberosus)], Liliales [onion (A. cepa), asparagus
(Asparagus officinalis)], and Poales [barley (Hordeum
vulgare), wheat (Triticum aestivum), ryegrass (L. perenne)].

* To whom correspondence should be addressed. E-mail: marie-pascale.prudhomme@unicaen.fr

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
2720 Lasseur et al.
From a human point of view, fructans are one of the most
promising ingredients for functional foods since they act
as prebiotics (Rao, 1999) and have favourable effects in
the prevention of cardiovascular diseases, colon cancer,
and osteoporosis (Kaur and Gupta, 2002).
In Asterales such as chicory and Jerusalem artichoke,
inulin series fructans (inulins) are found, which consists of
linear (b2-1) linked fructose residues with a terminal
glucose residue. Inulins are synthesized by the concerted
action of two enzymes. Sucrose:sucrose 1-fructosyltransferase
(1-SST) catalyses fructosyl transfer from one sucrose mole-
cule to the fructosyl residue of another sucrose molecule via
a (b2-1) linkage, resulting in the trisaccharide 1-kestose.
Fructan:fructan 1-fructosyltransferase (1-FFT) elongates 1-
kestotriose with additional (b2-1)-linked fructose units
(Edelman and Jefford, 1968; Koops and Jonker, 1996;
Lüscher et al., 1996; Van den Ende and Van Laere, 1996).
In Liliales such as onion and asparagus, inulins and
inulin neoseries fructans are observed. Inulin neoseries
have neokestose or 6G-kestotriose as a backbone. 6G-
kestotriose is based on the linkage of fructosyl residue on
both C1 and C6 of glucose. Adding further fructosyl
moieties on both terminal fructose residues of 6G-kestotriose
results in the inulin neoseries type of polymers containing
(b2-1)-linkages between adjacent fructosyl residues (Ernst
et al., 1998). The enzyme fructan:fructan 6G-fructosyl-
transferase (6G-FFT) initiates the formation of the 6G-
linked chain. It uses 1-kestotriose as a fructosyl donor and
transfers the fructose unit to the glucose moiety of sucrose
or to low DP-inulin via a (b2-6) linkage (Shiomi, 1989;
Vijn et al., 1997). The 6G-kestotriose produced is the
shortest fructan of the inulin neoseries. In asparagus, three
enzymes, 1-SST, 1-FFT, and 6G-FFT participate in syn-
thesis of fructans (Shiomi, 1989), whereas in onion only
two enzymes operate, 1-SST and 6G-FFT. Indeed, 6G-FFT
purified from onion bulbs had 1-FFT activity (Fujishima
et al., 2005). The recombinant enzyme expressed in
tobacco plants, protoplasts, or BY2 cells, was also able to
synthesize DP4 inulin from 1-kestotriose, indicating 1-FFT
like activity (Vijn et al., 1997; Ritsema et al., 2003).
Moreover, the introduction of onion 1-SST and 6G-FFT
into sugar beet resulted in a fructan profile closely re-
sembling that from onion (Weyens et al., 2004).
Members of the Poales contain fructans with (b2-6)
linked fructose units. The glucose residue can be terminal
or internal to the molecule, leading to fructans of the levan
series or the levan neoseries, respectively. Many of the
fructan molecules from Avena (Livingston et al., 1993) and
Lolium species (Sims et al., 1992; Pavis et al., 2001a)
belong to the levan neoseries while some fructans from
T. aestivum (Bancal et al., 1992), Dactylis glomerata
(Chatterton et al., 1993a), and Bromus tectorium (Chatterton
et al., 1993b) belong to the levan series. In some species
such as wheat and barley, fructans called graminans are
structurally more complex and consist of (b2-6) linked
fructose units with (b2-1) branches (Bancal et al., 1992).
Based on studies with barley, Wiemken et al. (1995)
proposed the 1-SST/6-SFT model for graminan biosynth-
esis. Sucrose:fructan 6-fructosyltransferase (6-SFT) catal-
yses the formation of bifurcose (1&6 kestotetraose), using
sucrose as fructosyl donor and 1-kestotriose, produced by
1-SST, as fructosyl acceptor. The 6-SFT also catalyses the
elongation of fructan molecules by transferring fructosyl
units from sucrose to fructan via (b2-6) linkages (Duchateau
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et al., 1995). The first 1-FFT gene in members of Poales
has been cloned recently from wheat (Kawakami and
Yoshida, 2005). It produces (b2-1) branches in fructans
synthesized by 6-SFT. 6-SFT is probably a key enzyme in
fructan biosynthesis in many cereal crops (wheat, barley)
and cool-season grasses (crested wheatgrass, big blue-
grass) (Sprenger et al., 1995; Wei and Chatterton, 2001;
Wei et al., 2002). In Lolium species, however, 6-SFT might
not be the enzyme responsible for the (b2-6) linkages
since the bifurcose is absent (Pavis et al., 2001a). In-
stead, fructans of the two neoseries prevail, suggesting an
important role for the 6G-FFT enzyme. 6G-FFT activities
and fructan levels peaked in the basal segment of elongat-
ing leaves and mature leaf sheaths (Pavis et al., 2001b).
Regardless of the agronomic importance of L. perenne as
a major grassland species, only one fructosyltransferase
cDNA of Lolium is functionally characterized: 1-SST has
been identified and expressed in Pichia pastoris (Chalmers
et al., 2003). Traditional plant-breeding approaches have
been successful in producing high-fructan ryegrasses
that have been shown to improve the nutritional value
for ruminants (Miller et al., 2001). However, fructan
biosynthesis regulation in L. perenne is still poorly under-
stood. Greater understanding of the underlying mechanisms
of fructan accumulation will benefit future breeding
programmes.
The aim of this study was to investigate whether 6G-FFT
represents a key enzyme for the biosynthesis of fructans of
the inulin and levan neoseries in L. perenne. To do this, a
cDNA clone encoding 6G-FFT from Lolium stubble,
composed of elongating leaf bases and mature leaf sheaths,
was isolated and characterized by heterologous expression
in P. pastoris. The effects of developmental stage and
carbohydrate status on the expression and tissue localiza-
tion of this gene are described and compared with the gene
expression pattern of 1-SST, along with the corresponding
enzyme activities and fructan levels.
Materials and methods
Plant material
Seeds of Lolium perenne cv. Bravo were germinated in 9 dm3 pots
and grown hydroponically for 8 weeks on a nutrient solution as
previously described by Prud’homme et al. (1992). The nutrient
solution was aerated continuously and replaced every week. Plants
were grown in a greenhouse with day/night temperatures of 22/18 8C

and a photoperiod of 16 h of natural light supplemented by a
photosynthetic photon flux density of 110 lmol photons mÿ2 sÿ1
(Phyto tubes, Claude, GTE, Puteaux, France).
After 8 weeks of growing, plants were harvested. Based on the
presence of the ligule, mature leaves were separated from elongating
leaves. Sheaths and elongating leaf bases previously enclosed by the
sheaths were dissected longitudinally into five segments [four310 mm
segments (0–40 mm) from the leaf base and a fifth variable length
segment of about 40 mm]. Blades and emerged part of the elongating
leaves were divided into three equal parts.
Synthesis of fructan was induced in the plants 8 weeks after
sowing, according to the method used by Smouter and Simpson
(1991). Plants were maintained under continuous light with a photo-
synthetic photon flux density of 150 lmol photons mÿ2 sÿ1 for up to
72 h, with roots and shoot meristems in the nutrient solution cooled at
5 8C. Control swards were grown under the original plant growth
conditions, with a daylength of 16 h. Plants were divided into three
parts; the sheaths of mature leaves, the blades of mature leaves
together with the emerged parts of elongating leaves, and elongating
leaf bases.
Each sampling was done in triplicate. One part of the harvested
tissues was used immediately for enzyme extraction whereas the
remainder was frozen, stored at ÿ80 8C for RNA extraction, or
freeze-dried for soluble carbohydrate extraction.
Extraction and analysis of water-soluble carbohydrates
Soluble carbohydrates were extracted from 25 mg (elongating
leaves), 50 mg (leaf sheaths) or 100 mg (emerged parts of elongating
leaves and blades) of freeze-dried tissues as described previously
(Morvan-Bertrand et al., 2001). The purification protocol used to
remove charged compounds was performed according to Amiard
et al. (2003). Glucose, fructose, sucrose, and fructans were quantified
by high-performance liquid chromatography (HPLC) on a cation
exchange column (Sugar-Pak 300 mm36.5 mm, Millipore Waters,
Milford, MA, USA) eluted with 0.1 mM CaEDTA in water using
mannitol as the control standard (Guerrand et al., 1996).
Protein extraction and in vitro fructosyltransferase
activity assays
Leaf segments (1 g FW for elongating leaf bases, 2 g FW for blades
and sheaths) were ground at 4 8C at a ratio of 2.5 cm3 gÿ1 fresh wt
in 80 mM citrate phosphate buffer (pH 5.5) containing 5 mM
dithiothreitol (DTT). The homogenate was centrifuged at 20 000 g for
10 min and the supernatant was desalted on Sephadex G50, which
also eliminated small soluble carbohydrates from the extract.
Extraction was done in triplicate.
For 1-SST assay, the mixture consisted of 100 mm3 enzyme extract
and 100 mm3 sucrose 100 mM in 80 mM citrate phosphate buffer pH
5.5. After incubation at 30 8C for 2 h, 100 mm3 mannitol (1 g dmÿ3)
was added as an internal standard and the reaction was stopped by
incubation in a boiling water bath for 3 min. For each extraction,
triplicate samples were run together with duplicate enzyme blanks
(no substrate). The assay mixtures were passed through a column
containing ion exchange resins (Amberlite IR 120, Amberlite IRA
416) and PVPP as described by Amiard et al. (2003). The column
was eluted with water. Carbohydrates of the assay mixture were
separated and quantified by HPLC on the Sugar-PAK column, under
the same conditions as for water-soluble carbohydrate analysis. 1-
SST activity was defined as the amount of 1-kestotriose formed per
unit of time and gÿ1 fresh tissue upon incubation of the enzyme
preparation in 50 mM sucrose.
For 6G-FFT assay, the mixture consisted of 50 mm3 enzyme
extract, 25 mm3 sucrose at 200 mM, and 25 mm3 1-kestotriose at
200 mM in 80 mM citrate phosphate buffer pH 5.5. After incubation
at 30 8C for 4 h, 20 mm3 trehalose (60 mg dmÿ3) was added as an
6G-FFT/1-FFT from perennial ryegrass 2721
internal standard and the reaction was stopped by incubation in
a boiling water bath for 3 min. For each extraction, triplicate samples
were run together with duplicate enzyme blanks (no substrate). The
assay mixtures were passed through a column containing ion
exchange resins (Amberlite IR 120, Amberlite IRA 416) and PVPP
as described by Amiard et al. (2003). The column was eluted with
water. Carbohydrates of the assay mixture were separated and
quantified by high-performance anion exchange chromatography
and pulsed amperometric detection (HPAEC-PAD DX-300, Dionex,
Sunnyvale, CA, USA) on an analytical CarboPac PA100 column
(4 mm3250 mm) using a sodium acetate gradient (1 cm3 minÿ1) in
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90 mM NaOH. Using solutions A (90 mM NaOH) and B (90 mM
NaOH and 500 mM NaOAc), the following running profile was
applied: T=0, 100% A; T=4 min, 100% A; T=10 min, 98% A, 2% B;
T=20.1 min, 100% B; T=25 min 100% B; T=25.1 min, 100% A;
T=30 min, 100% A. Fructan structures were deduced from their
retention times on anion exchange chromatography by comparison
with those of known standards. 6G-FFT activity was defined as the
sum of both 6G-kestotriose and 1&6G-kestotetraose amounts formed
per unit of time gÿ1 fresh tissue upon incubation of the enzyme
preparation in 50 mM sucrose and 50 mM 1-kestotriose. In order to
draw the calibration curves, 6G-kestotriose and 1&6G-kestotetraose
were kindly provided by NJ Chatterton and N Shiomi, respectively.
Preparation and screening of a cDNA library of
Lolium perenne
Leaf sheaths and elongating leaf bases (1.5 g FW) of 8-week-old
plants treated to accumulate fructans (Smouter and Simpson, 1991)
were ground in liquid nitrogen. The powder obtained was used to
purify poly (A+) RNA with Dynabeads oligo (dT)25 kit (Dynal,
France) by following the manufacturer’s recommendation. Double-
stranded cDNA was synthesized from poly (A+) RNA and a cDNA
library was constructed using a Lambda-Zap cDNA library kit and
the Gigapack III Gold Cloning Kit (Stratagene, France). The cDNA
library was screened with a fragment of 1330 bp of onion 6G-FFT
(Vijn et al., 1997). This fragment was labelled with [a-32P] dCTP by
using the random priming method with NEBlot kit (Biolabs, France).
Membranes were hybridized overnight at 42 8C and washed twice
in 23 SSC, 0.5% SDS for 15 min at room temperature, then rinsed
twice in the same buffer at 56 8C. After three rounds of purification,
positive clones were excised and recircularized in a pBluescript
vector (Stratagene, USA). Sequencing of positive clones was made
by Genome Express (Meylan, France). Nucleotide sequences were
compared with sequences available in the NCBI Databank.
Expression of isolated cDNA in Pichia pastoris
The putative coding region of mature Lp6GFFT (determined by
sequence homology with the N-terminal sequence of the 6-SFT of
barley (Sprenger et al., 1995) and the 1-SST of F. arundinacea
(Lüscher et al., 2000) was amplified by PCR with the primers
PIC6GFFT-F (59-GTCCGGAATTCGCCGGAGGGTTCCCGTG-
GAGCAAC-39) and PIC 6GFFT-R (59-GAC GCTCTAGACTAT-
GAGTCCTTAACCATGACGGT-39). EcoRI and XbaI sites are
indicated in bold in the primers. PCR was performed with Pfu
Proofreading Polymerase (Promega, France). PCR conditions were:
1 min at 95 8C; 5 cycles of 30 s at 95 8C, 30 s at 56 8C, and 4 min at
72 8C followed by 30 cycles of 30 s at 95 8C, 30 s at 68 8C, and 4 min
at 72 8C; with a final extension for 5 min at 72 8C. PCR products and
pPICZalpha A (expression vector) were digested with restriction
enzymes corresponding to restriction sites introduced by PCR and
purified with Nucleospin Extract Kit (Macherey-Nagel, Germany).
The digested vector was dephosphorylated with CIAP (Stratagene,
USA), and then PCR products were cloned in frame behind the alpha-
factor signal of the pPICZalpha A vector. The plasmids were
transformed into Escherichia coli competent cells as described by
2722 Lasseur et al.

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Fig. 1. Comparison of the deduced amino acid sequence of 6G-FFT from Lolium perenne, cultivar Bravo (Lp6G-FFT, EMBL accession no. AF492836)
with the putative 6G-FFT isolated from Lolium perenne, cultivar Aberystwyth S23 (Lp6G-FFT?, NCBI accession no. AB125218), the 6G-FFT from
Allium cepa (Ac6G-FFT, EMBL accession no. AY07838), the 6G-FFT from Asparagus officinalis (Ao6G-FFT, EMBL accession no. AB084283) and
the 1-SST of Lolium perenne (Lp1-SST, EMBL accession no. AY245431). Asterisks, colons, and periods indicate identical residues, conserved

Van den Ende et al. (2001). Cells were plated on 23 YT medium
supplemented with zeocine as a selection marker. Positive clones
were used for vector amplification. The P. pastoris wild-type strain
X33 was transformed by electroporation with 20 lg PmeI-linearized
pPICZaA-Lp6G-FFT. Transformants were selected on YPDS/
zeocine plates.
Production and characterization of the recombinant 6G-FFT
In order to produce the recombinant 6G-FFT enzyme for character-
ization, a 90 cm3 preculture medium (BMGY) was inoculated with
single colonies and incubated overnight at 30 8C, 200 rpm. Cells were
harvested by centrifugation and resuspended in 20 cm3 of induction
medium (BMMY) and incubated for 4 d at 29 8C. Methanol was
replenished every day to a final concentration of 2%. Protein
purification was done by following the protocol described by De
Coninck et al. (2005).
Aliquots were incubated at 30 8C with sucrose (final concentration
100 mM), sucrose and 1-kestotriose (100 mM and 50 mM, re-
spectively), 1-kestotriose (final concentration 50 mM), and with 6G-
kestotriose (final concentration 100 mM). For linkage analysis of the
products, they were further incubated with heterologous 1-FEH IIa
purified from chicory roots and previously expressed in P. pastoris
(Verhaest et al., 2004). The carbohydrates produced were analysed
by HPAEC (DX-300, Dionex, Sunnyvale, CA, USA) equipped with
a CarboPac PA100 anion-exchange column (4 mm3250 mm) and
pulsed amperometric detection. Carbohydrates of the assay mixture
were separated and quantified by high-performance anion exchange
chromatography and pulsed amperometric detection (HPAEC-PAD
DX-300, Dionex, Sunnyvale, CA, USA) on an analytical CarboPac
PA100 column (4 mm3250 mm) using a sodium acetate gradient
(1 cm3 minÿ1) in 90 mM NaOH. Using solutions A (90 mM NaOH)
and B (90 mM NaOH and 500 mM NaOAc), the following
running profile was applied: T=0, 100% A; T=4 min, 100% A;
T=10 min, 98% A, 2% B; T=20.1 min, 100% B; T=25 min 100% B;
T=25.1 min, 100% A; T=30 min, 100% A. Identities were assigned
to peaks by comparison with commercial standards and identified
onion carbohydrates.
In the pH experiment, 100 mM sodium citrate buffer (pH 3.4 to
5.4) and 100 mM sodium phosphate buffer (pH 5.75 to 8.25) were
used. Aliquots of the enzyme were incubated with 100 mM sucrose
and 50 mM 1-kestotriose (final concentrations) for 45 min at 30 8C.
In the temperature-optimum experiment, reactions were performed
at pH 5.4 at different temperatures (0–60 8C). 6G-FFT activity was
based on 6G-kestotriose production. Experiments were done in
triplicate.
RNA isolation and RT-PCR analysis
Plant tissues were ground in liquid nitrogen and suspended in
a prewarmed (80 8C) solution consisting of 750 mm3 phenol and
750 mm3 extraction buffer (0.1 M LiCl, 100 mM TRIS–HCl, 10 mM
EDTA, 1% (w/v) SDS, pH 8.0). After shaking, 750 mm3 of
chloroform-isoamylalcohol (24:1 v:v) were added and the solution
was centrifuged for 5 min (4 8C) at 20 000 g. Total RNA was
precipitated with LiCl (final concentration 2 M) overnight at 4 8C.
Following centrifugation for 30 min (4 8C) at 20 000 g, the pellet was
suspended in 250 mm3 of water treated with diethylpyrocarbonate
(0.1%, v/v) and 250 mm3 of phenol-chloroform-isoamylalcohol
(25:24:1 by vol.), mixed, and centrifuged for 5 min. RNA in the
substitutions, and semi-conserved substitutions, respectively. Putative start of the large subunit and of the small subunit is indicated by an arrow.
Potential glycosylation sites are underlined. The b-fructosidase motif and the cysteine catalytic site are boxed. Inverted triangles above the sequence
alignments indicate the three carboxylic acids that are crucial for enzyme catalysis (Verhaest et al., 2005). Differences between the two 6G-FFT from
Lolium perenne are indicated by circled amino acids.
6G-FFT/1-FFT from perennial ryegrass 2723
3
supernatant was precipitated again with 1 cm of absolute ethanol
and 50 mm3 of Na-acetate buffer (3 M; pH 5.6) and stored for 1 h at
ÿ80 8C. After centrifugation for 20 min (4 8C) at 20 000 g, the pellet
was resuspended in 100 mm3 RNase free-water. Samples were then
treated by following the Clean-up protocol of the RNeasy Minikit
(Quiagen) coupled to a DNase treatment (RNase free-DNase;
Quiagen, France).
One lg was used for retrotranscription by using the i-script cDNA
synthesis kit (Bio-Rad, France). cDNA was then amplified by
polymerase chain reaction (PCR) using 59-GCCAGGTCATCCT-
GCTCTAC-39 and 59-CCGGCATGAGCTCGTAGTT-39 as specific
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primers for 1-SST (Chalmers et al., 2003), 59-TCTCAACTCTTCG-
GACATCGA-39 and 59-TACATGTCGTCAGCCAAGAAG-39 as
specific primers for 6G-FFT (Lasseur et al., 2002), 59-CGGA-
TAACCGTAGTAATTCTAG-39 and 59-GTACTCATTCCAAT-
TACCAG AC-39 as primers for 18S rRNA. Amplification was
achieved in the following conditions so that for each cDNA, products
are analysed during the exponential phase of the PCR curve: 5 min at
95 8C; 30 cycles (1-SST, 6G-FFT) or 13 cycles (18S rRNA) of
denaturation at 95 8C for 30 s, annealing at 60 8C for 30 s and
elongation at 72 8C for 1 min. PCR products were analysed by
agarose gel electrophoresis and sequenced.
Results
Molecular characterization of Lolium perenne
fructosyltransferase
A perennial ryegrass cDNA library, prepared from stubble
of plants induced to accumulate fructan-related enzymes,
was screened with a 32P-labelled insert of the onion 6G-
FFT (Vijn et al., 1997). After repeated screenings, several
positive clones were picked up. The longest cDNA was
fully sequenced and consisted of 2508 bp, containing an
open reading frame (ORF) of 1938 bp and a poly(A)
sequence at its 39 end. The ORF encoded a polypeptide of
645 amino acids (Fig. 1) with six potential N-glycosylation
sites, a calculated pI of 4.84 and an estimated molecular
weight of 60.6 kDa for the mature protein. The cDNA was
first termed ‘putative 1-SST’ (Lasseur et al., 2002; acces-
sion no. AF492836) because of its high identity (81%) to
the 1-SST polypeptide of Festuca arundinacea. It shares
a similar identity (80%) with the deduced amino acid
sequence of L. perenne 1-SST (Chalmers et al., 2003).
Interestingly, the cDNA shows 98.3% identity at the
nucleotide level and 99.4% identity at the amino acid level
with a sequence isolated from another cultivar of L. perenne
(Aberystwyth S23, accession no. AB125218) identified as
a putative 6G-FFT. As far as is known, however, functional
analysis of the encoded enzyme has not been published.
When compared with other vacuolar-type fructosyltrans-
ferases and invertases, the cDNA had greater identity with
sequences from gramineous plants such as barley 6-SFT
2724 Lasseur et al.
(61%) and wheat invertase (61%) than with sequences for
1-SSTs, 6G-FFTs and invertases from liliaceous plants
(53–59%) or 1-FFTs and 1-SSTs from asteraceous plants
(47–51%) (Fig. 2). Furthermore, the sequence of the
‘putative 1-SST’ showed greater homology with vacuolar-
type than with cell-wall type invertases belonging to the
glycoside hydrolase family 32. The cDNA contains the
so-called sucrose-binding box NDPNG.

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Fig. 2. Unrooted phylogenetic tree of protein sequences of some invertase and fructan metabolism genes including the newly cloned Lolium perenne
6G-FFT (in bold). Their respective accession numbers are: A. cepa invertase AJ006067, A. cepa 1-SST AJ006066, A. cepa 6G-FFT AY07838, A.
sativum 1-SST AY098442, A. thaliana invertase AY039610, A. thaliana invertase AY046009, A. officinalis invertase AF002656, A. officinalis 6G-FFT
AB084283, B. vulgaris invertase AJ277455, B. oleracea invertase AF274298, B. oleracea invertase AF274299, C. annuum invertase P93761, C.
intybus invertase AJ419971, C. intybus 1-SST U81520, C. intybus 1-FFT U84398, C. scolymus 1-SST Y09662, C. scolymus 1-FFT AJ000481, D.
carota invertase Q42722, D. carota invertase X75352, F. arundinacea 1-SST AJ297369, H. tuberosus 1-SST AJ009757, H. tuberosus 1-FFT AJ009756,
H. vulgare 6-SFT X83233, I. batatas invertase AF017082, I. batatas invertase AY037937, L. perenne invertase AY082350, L. perenne 1-SST
AY245431, L. perenne 6G-FFT AF492836, L. perenne putative 6-FT AF494041, L. perenne LpFT1 AF481763, L. esculentum invertase P29000, O.
sativa invertase AF276703, O. sativa invertase AF276704, P. vulgaris invertase O24509, P. ampla 6-SFT AF192394, P. cerasus invertase AY048579,
S. officinarum invertase AY302083, T. officinale 1-SST AJ250634, T. aestivum invertase AJ635225, T. aestivum 6-SFT AB029887, T. aestivum
1-SST AB029888, T. gesneriana invertase X97642, V. faba invertase Q43857, V. vinifera invertase Q9S943, V. vinifera invertase QS944, Z. mays
invertase P49175.

Expression of recombinant protein in Pichia pastoris
The ‘putative 1-SST’ cDNA was expressed in P. pastoris
to investigate the enzymatic properties of the protein. The
recombinant protein showed fructosyltransferase activity
(see below) and displayed optimum activity at pH 5.4,
consistent with a vacuolar localization (Fig. 3). The protein
showed a broad temperature optimum range, between
30 8C and 40 8C. Interestingly, the enzyme was still active
at low temperatures.
Incubation with 100 mM sucrose alone showed the
formation of small amounts of 1-kestotriose and 6G-
kestotriose, products of 1-SST and 6G-FFT activities,
respectively (Fig. 4A). So far 6G-FFTs with 1-SST activity
have not been described. The small amount of 1-kestotriose
produced makes it unlikely that the recombinant protein
is a genuine 1-SST. Probably the enzyme first produces
some 1-kestotriose from sucrose and then preferentially
uses 1-kestotriose as a donor substrate to biosynthesize
6G-kestotriose. Also fructose was found, indicating that
100
Relative 6G-FFT activity (%)
80
60
40
20
0
3 4 5 6
pH
100
Relative 6G-FFT activity (%)
80
60
40
20
0 disappeared progressively while 6G-kestotriose, fructose,
0 10 20 30
Temperature (°C)
Fig. 3. Effect of pH and temperature (pH 5.4) on 6G-FFT activity
estimated by production of 6G-kestotriose generated by the recombinant
protein produced in Pichia pastoris after incubation during 45 min
with 100 mM sucrose and 50 mM 1-kestotriose in 100 mM sodium
citrate buffer (closed circles) or in 100 mM sodium phosphate buffer
(open circles).
6G-FFT/1-FFT from perennial ryegrass 2725
some fructosyl residues were transferred to water rather
than to sucrose.
The production of 6G-kestotriose in these reactions was
probably limited by the amount of 1-kestotriose formed in
the incubation medium. Indeed, when the recombinant
protein was incubated with both sucrose (100 mM) and 1-
kestotriose (50 mM), 6G-kestotriose was produced within
10 min, together with nystose (1,1-kestotetraose) and
1&6G-kestotetraose (4c; FGFF) (Fig. 4B). Sucrose did
Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011
not act as a fructosyl donor since no glucose was released
during the first two hours. Nystose is the product of 1-FFT
activity which transfers a fructose residue of 1-kestotriose
to the terminal fructose unit of another 1-kestotriose.
1&6G-Kestotetraose (FGFF) could either be produced by
a 6G-FFT activity or a 1-FFT activity, depending on the
acceptor, 1-kestotriose or 6G-kestotriose, respectively. The
fructan profile formed after 24 h resembles that of onion
fructans. DP5 inulin and fructans from the DP5 inulin
neoseries were formed. Consequently, the recombinant
protein of L. perenne is able to synthesize fructans from
the inulin series and fructans from the inulin neoseries
simultaneously. It possesses both 6G-FFT and 1-FFT
activities. The relative amounts of the three initial products
suggest that in this enzyme, 6G-FFT is at most twice as
active as 1-FFT.
Incubation with 1-kestotriose (50 mM) as a sole sub-
strate led to the production of 6G-kestotriose, nystose, and
1&6G-kestotetraose (FGFF) within 10 min. Fructose and
sucrose were also released indicating a 1-kestosidase like
activity (Fig. 4C). The amount of 6G-kestotriose was
similar to that produced by the recombinant protein
incubated with both sucrose and 1-kestotriose (Fig. 4B).
The amount of nystose and 1&6G-kestotetraose (FGFF),
7 8 however, were higher, probably because of the lack of
competition between the acceptors sucrose and 1-kestotriose.
Consequently, fructans of DP >4 were synthesized more
rapidly, i.e. within 30 min rather than 24 h.
Sucrose slows down the reaction. Fructans were pro-
duced within 45 min on condition that the sucrose
concentration was smaller than the 1-kestotriose concen-
tration (Fig. 4D, E).
In order to assess whether only (b2-1) linkages were
produced by the recombinant protein, products of an
overnight incubation with 1-kestotriose were boiled and
further incubated for up to 3 h with the recombinant fructan
1-exohydrolase IIa (1-FEH IIa) from chicory expressed in
P. pastoris (Verhaest et al., 2004) (Fig. 4F). Fructans
40 50 60 70 and sucrose increased. 6G-kestotriose is a poor substrate
for 1-FEH IIa and thus was expected to accumulate. Fruc-
tose and sucrose are products of 1-FEH IIa. Sucrose is not
degraded further since 1-FEH IIa has almost no invertase
activity. Removal of fructans other than 6G-kestotriose and
accumulation of 6G-kestotriose indicate that the recombi-
nant protein catalyses the formation of (b2-1) linkages.
2726 Lasseur et al.

G S G S
A F
B F 1-K 6G-K 4b
5a
Nys
6G-K 5c, 5d
1-K 4c
24h onion

24h
8h
2h
4h
1h
2h
30min

Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011
1h 10min

0h 0h
0 5 10 15 20 0 5 10 15 20

GF S S 1-K

C 6G-K D Nys
4b 5a 4c
1-K Nys
5c, 5d
4c 6G-K
onion 500mM

24h 250mM

2h 100mM
Detector response (arbitrary units)

1h 50mM

30min 25mM

10min 10mM

0h 5mM
0 5 10 15 20 0 5 10 15 20
S F S
1-K
E F
500mM G 6G-K

250mM
3h
100mM

50mM 90min

25mM 1-K
30min
10mM

5mM 0h

0 5 10 15 20 0 5 10 15 20
G F S 6G-K

4c 4b
24h

2h

1h

30min

10min

0h
0 5 10 15 20

Retention time (min)

Fig. 4. High performance anion-exchange chromatograms of the reaction products generated by the Pichia pastoris expressed protein. Reaction
mixtures containing the recombinant protein were incubated for up to 24 h at 30 8C with 100 mM sucrose (A), 100 mM sucrose and 50 mM 1-kestotriose
(B), 50 mM 1-kestotriose (C), 100 mM 6G-kestotriose, for 45 min at 30 8C with 100 mM sucrose and 5–500 mM 1-kestotriose (D), 20 mM 1-kestotriose
and 5–500 mM sucrose (E) or 100 mM 6G-kestotriose (G). Extract previously incubated for 1 h with sucrose and 1-kestotriose (100 mM each)

When 6G-kestotriose was given as the main substrate
(Fig. 4G), 1,6G-kestotetraose (4b; FFGF) and sucrose were
produced while the amount of initial 1&6G-kestotetraose
(4c; FGFF) decreased. 1,6G-kestotetraose (FFGF) could be
produced by fructosyl transfer from 1&6G-kestotetraose
(FGFF) to 6G-kestotriose as described for asparagus
(Ueno et al., 2005). However, glucose-linked fructosyl
retrieval from 1&6G-kestotetraose (FGFF) would liberate
1-kestotriose, but 1-kestotriose was not released in the
medium. The transfer of fructose-linked fructosyl from
1&6G-kestotetraose to 6G-kestotriose liberating 6G-
kestotriose is another possibility. 6G-Kestotriose can also
act as a fructosyl donor towards another 6G-kestotriose
molecule (Fig. 4B). This is probably the major pathway for
the synthesis of 1,6G-kestotetraose (FFGF) since an im-
portant release of sucrose occurs. The observed decrease
of the 1&6G-kestotetraose (FGFF) amount (Fig. 4G) makes
it unlikely that it is produced from 6G-kestotriose. No
fructan of DP5 (5c, 5d) were produced, indicating that 1-
kestotriose and/or nystose are necessary for their synthesis.
From the results of these experiments, a possible path-
way for oligosaccharides of inulins and inulin neoseries
formed by the recombinant 6G-FFT from L. perenne is
summarized in Fig. 5A.
Effect of leaf developmental stage on soluble
carbohydrates, enzyme activities and gene expression
pattern of Lp1-SST and Lp6G-FFT
Leaf growth in grasses is confined to the basal region,
which is enclosed by the sheaths of mature leaves
(Schnyder et al., 1990). Cells are displaced away from their
origin as a result of continued production and elongation
of new cells. The tissue that emerged from the enclosing
leaf sheaths is almost fully differentiated and photosyn-
thetically active (Wilhelm and Nelson, 1978). Four31 cm
segments and a fifth longer segment were cut, starting from
the base of growing leaves and leaf sheaths. Leaf blades
and the emerged parts of growing leaves were divided
into three parts of equal length (Fig. 6A). From each
segment, carbohydrates, proteins, and total RNA were ex-
tracted. A 300 bp fragment of Lp1-SST cDNA, a 501 bp
fragment of Lp6G-FFT cDNA and a 383 bp fragment of
18S rRNA were amplified by RT-PCR from each sample
adjusted to contain equal amounts of RNA. In elongating
leaves and in mature leaf sheaths, activity of 1-SST and
6G-FFT was greatest in the basal segment where the
accumulation of fructan and sucrose was maximal and
where transcripts levels of Lp1-SST and Lp6G-FFT were
amplified in largest amounts (Fig. 6B, C). Together with
(F, 0 h) was boiled for 5 min, incubated for up to 3 h with 1-FEH II purified from chicory roots and further expressed in Pichia pastoris (F). G, glucose; F,
fructose; S, sucrose; 1-K, 1-kestotriose; 6G-K, 6G-kestotriose; Nys, nystose (1,1-kestotetraose); 4c, 1&6G-kestotetraose; 4b, 1,6G-kestotetraose; 5a,
1,1,1-kestopentaose; 5c, 1,1&6G-kestopentaose; 5d, 1&1,6G-kestopentaose. Elution pattern of onion saccharides are presented (B, C, Ernst et al., 1998).
6G-FFT/1-FFT from perennial ryegrass 2727
fructan and corresponding enzyme activities, transcripts of
Lp1-SST and Lp6G-FFT dropped subsequently along the
axis of leaf sheaths and of enclosed parts of elongating
leaves, so that they became barely detectable in the fifth
segment of each tissue (Fig. 6C). In photosynthetically
active tissues (Fig. 6B, segments 6, 7, 8), fructans were
present at lower amounts than in sink tissues. Fructan
synthesizing activities were also very low and Lp1-SST
mRNA fragments were not detected (Fig. 6C, segments 6,
Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011
7, 8). Surprisingly, Lp6G-FFT mRNA fragments were
amplified, especially in central part segments (Fig. 6C,
segment 7) where they reached similar amounts as in basal
segments (Fig. 6C, segment 1). Glucose and fructose levels
peaked in the third segment of elongating leaves (Fig. 6B),
probably as a result of high invertase (data not shown) and
FEH activity found in the corresponding maturation zone
(Morvan-Bertrand et al., 2001).
Transcript levels of Lp6G-FFT and Lp1-SST, enzyme
activities and carbohydrate levels upon induction of
fructan accumulation in leaves
Accumulation of large quantities of fructans can be induced
in leaves of grasses by cooling the roots and continuous
illumination of the shoots (Smouter and Simpson, 1991;
Guerrand et al., 1996; Pavis et al., 2001b; Wei et al., 2001).
After several time intervals, shoots were harvested and
dissected into leaf blades, leaf sheaths, and elongating leaf
bases. Fructans accumulated first in elongating leaf bases
and in leaf sheaths (Fig. 7A, B). After 72 h, fructans
increased slightly in leaf blades (Fig. 7C). The first 6 h of
treatment had no effect on the fructan pattern, but after 6 h,
fructans increased about 5-fold within 66 h in both
elongating leaf bases and in leaf sheaths to reach high
levels, 250 mg gÿ1 DW and 230 mg gÿ1 DW, respectively.
By contrast, fructans only slightly increased in leaf blades
after 48 h of treatment. Surprisingly, the fructan precursor
sucrose showed a similar accumulation pattern in all leaf
tissues, including leaf blades. It increased slightly dur-
ing the first 6 h of treatment, rapidly accumulated within
the following 18 h to reach about 70 mg gÿ1 DW, and
decreased thereafter. Glucose and fructose followed similar
but tissue-specific patterns, glucose levels being always
slightly higher than fructose levels in tissues where fructan
accumulated. Proteins and total RNA were extracted from
the Lolium leaves from which carbohydrates were isolated.
Both 1-SST and 6G-FFT activities strongly increased in
elongating leaf bases between 6 h and 24 h of treatment
whereas 1-SST activity only slightly increased in leaf
sheaths. Initial in vitro activities were lower in leaf sheaths
2728 Lasseur et al.

A S
Nys Nys Lp6G-FFT were higher than that of Lp1-SST but remained
(GFFF) 1-K
constant throughout the experiment.
6G 1-K
1-K -F
T FT S
FF
1-

1-K 1-K S 5c (FGFFF) Discussion

(GFF)
S
6G
-F
1-K
FF
T Lp6G-FFT has 1-FFT activity
S FT 1-
4c In this paper, the isolation and the functional character-
6G-FFT (FGFF) ization of the first grass 6G-FFT/1-FFT obtained from

S 1-K
6G-K 1-
S The primary structure of known enzymes catalysing
6G-K FF
6G-K T the different transfructosylations in one plant family seems
1-FFT
(FGF) 5d (FFGFF)
1-
S
6G-K
FF
T enzymes catalysing the same reaction in other plant
4b
(FFGF)
B The recombinant protein obtained from P. pastoris has
6-(S/F)FT 6G-FFT/1-FFT
Levan neoseries 6G-KESTOTRIOSE
T
-FF
+S 6G-FFT/ T/1
1-FFT G-FF
6
SUCROSE
1-SST
1-KESTOTRIOSE
6G-FFT/1-FFT
6-(S/F)FT
6-KESTOTRIOSE
Fig. 5. (A) Possible pathway for production of fructo-oligosaccharides
detected in the reaction of the recombinant protein produced in Pichia
pastoris. Reactions are due to the 6G-FFT or the 1-FFT activity of the
protein. Bold lines indicate major pathways. S, sucrose; 1-K, 1-
kestotriose; 6G-K, 6G-kestotriose; Nys, nystose (1,1-kestotetraose); 4c,
1&6G-kestotetraose; 4b, 1,6G-kestotetraose; 5c, 1,1&6G-kestopentaose;
5d, 1&1,6G-kestopentaose. (Adapted from Ueno et al., 2005) (B) Model
of the synthesis of the different Lolium-type fructans by sucrose:sucrose
1-fructosyltransferase (1-SST), fructan:fructan 6G-fructosyltransferase/
fructan:fructan 1-fructosyltransferase (6G-FFT/1-FFT), and sucrose or
fructan:fructan 6-fructosyltransferase (6-(S/F)FT).
than in elongating leaf bases on a fresh wt basis, but they
were similar in both tissues when expressed on a protein
wt basis (data not shown). In leaf blades, 6G-FFT activity
remained constant. 1-SST activity increased slightly and
transiently during the first 6 h of treatment, but after 24 h,
the activity was low and similar to that in leaf blades of
control plants. The gene expression patterns of Lp1-SST
and Lp6G-FFT followed the curve of corresponding
enzyme activities in leaf sheaths and elongating leaf bases
upon the induction of fructan accumulation. In elongating
leaf bases, the amount of Lp1-SST and Lp6G-FFT trans-
cripts slightly decreased during the first 6 h of treatment,
but increased again thereafter. In leaf sheaths, the trans-
cripts levels of Lp1-SST increased within the first 24 h
while the amount of Lp6G-FFT transcripts was not affected
by the treatment. In leaf blades, the transient small increase
in 1-SST activity during the first 6 h of treatment was not
correlated with an increase in Lp1-SST transcripts. Instead,
Lp1-SST transcripts increased in leaf blades of illuminated
plants after 24 h of treatment while transcript levels of
Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011
L. perenne are described.
to be more closely related to one another than those of
families. The Lp6G-FFT also shares more identity with
fructosyltransferases and invertases of gramineous plants
than with the two known 6G-FFTs of liliaceous plants.
Inulin neoseries
a predominant 6G-FFT activity. This enzyme is therefore
a key enzyme in L. perenne since it catalyses the formation
Inulin series of fructan of the inulin neoseries. In addition, the enzyme
catalyses the biosynthesis of inulins as well as the formation
of (b2-1) linkages onto fructans of the inulin neoseries.
Previously, these fructans were reported to be synthesized
by separate 6G-FFT and 1-FFT activities in asparagus
(Shiomi, 1981, 1982, 1989) or in onion (Shiomi et al.,
1997). Recently, such a 1-FFT activity has been reported
for the native 6G-FFT of onion (Fujishima et al., 2005) or
asparagus (Ueno et al., 2005). The recombinant protein of
perennial ryegrass has a relative 1-FFT to 6G-FFT activity
(at most 0.5) similar to that of native onion 6G-FFT/1-FFT
enzyme (0.48, Fujishima et al., 2005), whereas the re-
combinant protein of asparagus has only a 1-FFT activity
13 times lower than the 6G-FFT activity (Ueno et al.,
2005). In this species, the native 6G-FFT has a negligible
1-FFT activity (Shiomi, 1981). The general properties of
L. perenne 6G-FFT resemble those of 6G-FFT from onion
bulbs. Both enzymes are bifunctional, almost free of
1-kestotriose hydrolysing activity and free from invertase
activity. When incubated with 1-kestotriose as sole sub-
strate, both enzymes produced 1&6G-kestotetraose (4c;
FGFF), nystose and small amounts of 6G-kestotriose with
the liberation of sucrose at the initial reaction time.
Recombinant Lp6G-FFT and native onion 6G-FFT also
show differences. Indeed, native onion 6G-FFT, but not
Lp6G-FFT, produced 1-kestotriose and 1&6G-kestotetraose
(4c; FGFF) in the presence of 6G-kestotriose. Moreover,
native onion 6G-FFT has no 1-SST activity with sucrose as
the sole substrate, whereas Lp6G-FFT formed small
amounts of 1-kestotriose with the liberation of glucose.
However, the existence of a triple-function 6G-FFT/1-FFT/
1-SST protein in L. perenne is unlikely and a separate 1-
SST protein has been recently cloned and characterized
from this species (Chalmers et al., 2003). To determine
whether these properties are artefactual, for example, due to
 6G-FFT/1-FFT from perennial ryegrass 2729

Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011

Fig. 6. Tissue distribution of fructans, sucrose, glucose, and fructose (mg gÿ1 dry wt) (B), 1-SST and 6G-FFT activities (nkat gÿ1 fresh wt) (C) as well
as 1-SST and 6G-FFT transcripts as compared with 18S rRNA transcripts (C). After 8 weeks of growing, plants were harvested and the mature leaves
(open circles) separated from the elongating leaves (closed circles). Sheaths and elongating leaf bases previously enclosed by the sheaths were dissected
longitudinally into five segments (four310 mm segments (0–40 mm) from the leaf base and a fifth variable length segment of about 40 mm). Blades and
emerged part of the elongating leaves were divided into three equal parts (A). Gene expression was determined by PCR using gene-specific primers and
visualized by ethidium bromide staining. Vertical bars represent 6SE (n=3).
 Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011
2730 Lasseur et al.

slight differences in folding or glycosylation pattern
(Hochstrasser et al., 1998), it will be necessary to purify
the native enzyme from L. perenne or to express the gene in
another expression system. A partially purified FFT from
Lolium rigidum, free of invertase activity, was able to use
1-kestotriose and 6G-kestotriose as sole substrates, and
produced 6G-kestotriose as well as nystose and 1&6G-
kestotetraose (4c; FGFF) when assayed with both 1-
kestotriose and sucrose (St John et al., 1997a, b). This
native Lolium FFT had similar properties as the recombinant
Lp6G-FFT. Interestingly, it produced 1&6G-kestotetraose
and small amounts of 1-kestotriose with 6G-kestotriose
as the sole substrate and it was free of 1-SST activity.
These data indicate that the small differences between
onion and ryegrass 6G-FFT properties are possibly due
to the yeast expression system.
The Lp6G-FFT enzyme synthesized fructans with only
(b2-1) linked fructosyl residues. All possible reaction
products of the 6G-FFT and 1-FFT activity of Lp6G-FFT
up to DP5 and DP4, respectively, were found in extracts
from leaves and roots of L. perenne (Pavis et al., 2001a):
6G-kestotriose (FGF), nystose (GFFF), 1,6G-kestotetraose
(4b; FFGF), 1&6G-kestotetraose (4c; FGFF), 1,1&6G-
kestopentaose (5c; FGFFF), 1&1,6G-kestopentaose (5d;
FFGFF). The only oligofructans not synthesized by Lp6G-
FFT were the DP4 and the DP5 with (b2-6) linkages. Since
Lp6G-FFT has a clear 1-FFT activity, there might be no
need for a separate 1-FFT protein, as is the case in onion
(Ritsema et al., 2003). By contrast, a separate 1-FFT does
exist in asparagus (Ueno et al., 2005). Conclusively,
fructans present in L. perenne might be synthesized by a
three-enzyme system instead of a four-enzyme system as
previously suggested (Pavis et al., 2001a): 1-SST, 6G-FFT
together with 1-FFT and 6-(S/F)FT (Fig. 5B). However, the
sequence of a putative 1-FFT homologue has been sub-
mitted to the NCBI database. Functional analysis of the
encoded enzyme might reveal whether an independent
1-FFT function exists in L. perenne. As far as 6G-FFT is
concerned, several sequences might exist (Hisano et al.,
2004). The sequences could represent the same gene in
different L. perenne varieties. They could also be alleles
of the same gene or members of a multigene family of
6G-FFT in the L. perenne genome.
In this species, the enzyme that produces the predomi-
nant (b2-6) is not known yet.
The sucrose binding-box of the Lp6G-FFT protein
contains the b-fructosidase motif NDPNG highly con-
served in invertases, but also invariably found in 1-SST of
F. arundinacea (Lüscher et al., 2000), T. aestivum, and
Fig. 7. Concentrations of fructans, sucrose, glucose, and fructose (mg gÿ1 dry wt), 1-SST and 6G-FFT activities (nkat gÿ1 fresh wt), expression of
1-SST and 6G-FFT genes in elongating leaf bases (A), leaf sheaths (B), and leaf blades (C) of Lolium perenne plants cultivated for 8 weeks with
a photoperiod of 16 h at day/night temperatures of 22/18 8C were further subjected to the same conditions (control, closed circles) or to continuous light
with roots at 4 8C (illuminated, open circles) for 72 h. Vertical bars represent 6SE (n=3). Expression of 1-SST and 6G-FFT transcripts are compared with
18S rRNA transcript expression. Gene expression was determined by PCR using gene specific primers and visualized by ethidium bromide staining.
6G-FFT/1-FFT from perennial ryegrass 2731
L. perenne (Chalmers et al., 2003). The motif is present
with some variations in other fructosyltransferases (Rit-
sema et al., 2004). NDPSG instead of NDPNG was found
in the two other known 6G-FFTs from onion and asparagus.
Consequently, Ser in the b-fructosidase motif is not an
important amino acid for 6G-FFT activity. This corrobo-
rates the recent finding of Ritsema et al. (2004) who
demonstrated that the shift from NDPSG to NDPNG by
site-directed mutagenesis in onion 6G-FFT did not affect
Downloaded from http://jxb.oxfordjournals.org/ at Sistema de Bibliotecas y de Información Universidad de Buenos Aires on November 16, 2011
the enzyme specificity.
Regulation of 6G-FFT expression is tissue-specific
In several grasses, large fructan stores are deposited
temporarily in the growth zone at the base of elongating
leaves and in mature leaf sheaths (Lüscher and Nelson,
1995; Roth et al., 1997; Schnyder and Nelson, 1989; Pavis
et al., 2001b). The expression pattern of Lp6G-FFT was
studied in leaf tissues using semi-quantitative RT-PCR and
compared with that of Lp1-SST.
In heterotrophic leaf tissues: The largest amount of Lp6G-
FFT and Lp1-SST fragments were amplified from the first
segment of the base of elongating leaves and leaf sheaths.
It declined thereafter. This is consistent with findings for
other gramineae such as F. arundinacea (Lüscher et al.,
2000), L. perenne cv. Citadel (Chalmers et al., 2003), and
L. temulentum (Gallagher et al., 2004). In the latter spe-
cies, the putative fructosyltransferase FT 2:2 was mostly
expressed in tiller bases where fructans were generally
localized. In growing leaf bases of F. arundinacea, 1-SST
transcript levels matched the 1-SST activity (Lüscher and
Nelson, 1995) and the fructan content (Schnyder and
Nelson, 1989), suggesting that 1-SST activity was mainly
regulated at the transcriptional level (Lüscher et al., 2000).
Similar results were obtained in the present study (Fig. 6).
Indeed, the amount of messages in elongating leaf bases
and leaf sheaths followed the curve of 1-SST and 6G-
FFT activities and fructan content. This result indicates
that in sink tissues Lolium fructosyltransferases are also
predominantly controlled at the transcriptional level. Post-
transcriptional regulation might also occur since Lp6G-
FFT and Lp1-SST transcripts were generally more abundant
in leaf sheaths than in elongating leaf bases while the
corresponding activities measured in vitro were much
higher in elongating leaf bases than in leaf sheaths (Fig.
6C). It was demonstrated before that addition of cyclohex-
imide and actinomycin D could decrease the amount of
extractable 1-SST activity from leaf sheaths of illuminated
Lolium plants, suggesting that activity increases occurred
2732 Lasseur et al.
by de novo synthesis of proteins (Guerrand et al., 1996).
The present results further support this point of view.
Similarly, increases in 6G-FFT activity and transcripts
also occurred in elongating leaf bases by the exposure of
plants to low root temperatures and continuous illumina-
tion (Fig. 7A). It is therefore hypothesized that the cor-
responding protein is de novo synthesized. However, for
a definite answer, the protein level of the two fructosyl-
transferases should be followed over time.
1-SST initiates fructan biosynthesis by producing 1-
kestotriose. 6G-FFT is one of the elongating enzymes
that use 1-kestotriose to produce longer fructans of spe-
cific types. In barley, where no fructan of the neo-
series accumulate, 6-SFT and 1-FFT are the elongating
enzymes. Roth et al. (1997) suggested that 1-FFT might
also play a role in fructan degradation by modifying
the size distribution of the fructan chain since it occurred
in the elongating leaf zone where fructans are degraded.
In L. perenne, 6G-FFT does not seem to fulfil this func-
tion as the activity decreased continuously along the axis
of the growing leaf including the maturation zone where
fructans are degraded.
In autotrophic leaf tissues: Surprisingly, Lp6G-FFT was
expressed in mature leaf blades and the emerged parts
of elongating leaves where extractable 6G-FFT activity
was very low and fructans barely accumulate (Fig. 6).
Here, Lp1-SST transcripts were not detected. Lp6G-FFT
transcripts were more abundant in the central region
than in other parts of the leaves. Here, Lp6G-FFT levels
are not indicative of enzymatic activity, suggesting post-
transcriptional regulation of expression. Interestingly, similar
results have been reported for the putative fructosyltrans-
ferase FT 2:2 of L. temulentum. Transcripts were detected
in photosynthetically active tissue but did not correlate
with fructan synthesis (Gallagher et al., 2004). Moreover,
Lp1-SST transcripts accumulate in leaf blades that were
continuously illuminated but no increase in 1-SST activity
was observed (Fig. 7C). Therefore, in photosynthetically
active tissue, fructan metabolism genes including Lp6G-
FFT and Lp1-SST, appear to be mainly regulated post-
transcriptionally. It is currently unclear why Lp6G-FFT
and Lp1-SST mRNA accumulate in this tissue.
In conclusion, this is the first time a grass 6G-FFT cDNA
has been cloned and functionally characterized. This
enzyme has an important 1-FFT activity and produces
both inulins and inulin neoseries. Regulation of Lp6G-
FFT gene expression depends on the tissue according to
its sink–source status.
Acknowledgements
The authors wish to thank N Shiomi and NJ Chatterton for their
kind gifts of 1&6G-kestotetraose and 6G-kestotriose, respectively,
and K Le Roy and R Vergauwen for their helpful advice. They ack-
nowledge the valuable technical help by A Bré.
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