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First published online January 9, 2003; 10.1104/pp.015305 Plant Physiol, February 2003, Vol. 131, pp. 621-631
Fructan 1-Exohydrolases.
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
CONCLUSIONS
MATERIALS AND METHODS
LITERATURE CITED
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Graminan-type fructans are temporarily stored in wheat
(Triticum aestivum) stems. Two phases can be
distinguished: a phase of fructan biosynthesis (green stems) followed
by a breakdown phase (stems turning yellow). So far, no plant fructan
exohydrolase enzymes have been cloned from a monocotyledonous species.
Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 and
w2) from winter wheat stems. Similar to dicot plant
1-FEHs, they are derived from a special group within the cell wall-type
invertases characterized by their low isoelectric points. The
corresponding isoenzymes were purified to electrophoretic homogeneity,
and their mass spectra were determined by
quadrupole-time-of-flight mass spectrometry. Characterization of the
purified enzymes revealed that inulin-type fructans [
-(2,1)] are
much better substrates than levan-type fructans [-(2,6)]. Although
both enzymes are highly identical (98% identity), they showed
different substrate specificity toward branched wheat stem fructans.
Although 1-FEH activities were found to be considerably higher during
the fructan breakdown phase, it was possible to purify substantial
amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems,
suggesting that this isoenzyme might play a role as a -(2,1)-trimmer
throughout the period of active graminan biosynthesis. In this way, the
species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
CONCLUSIONS
MATERIALS AND METHODS
LITERATURE CITED
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Starch is the most prominent storage
carbohydrate in plants, but about 15% of flowering plant species use
fructan (a Fru polymer) as a storage compound (Hendry,
1993
). Inulin-type fructan consists of linear -(2,1)-linked
fructofuranosyl units and occur mainly in dicotyledonous species. Levan
consists of linear -(2,6)-linked fructofuranosyl units, but more
complex and branched fructan types (graminan, inulin neoseries, and
levan neoseries) are common in monocotyledonous species (Vijn
and Smeekens, 1999; Pavis et al., 2001b) mainly
belonging to the Poaceae (e.g. wheat [Triticum
aestivum], barley [Hordeum vulgare], oat
[Avena sativa], and temperate fodder grasses) and the
Liliaceae (e.g. onion, asparagus [Asparagus
officinalis]).
Next to their obvious role as reserve compounds, fructan might have
other functions in plants like stress protectants (drought and cold) or
osmoregulators (Vergauwen et al., 2000; Hincha et al., 2002, and refs. therein). Unlike starch, fructans are
water soluble and are believed to be stored in the vacuole
(Wiemken et al., 1986), although the exclusive vacuolar
localization has been questioned (Livingston and Henson,
1998).
Although the metabolism of inulin has become clear in dicotyledonous
species and the respective biosynthetic and breakdown enzymes have been
cloned (Edelman and Jefford, 1968; Van den Ende and Van Laere, 1996a; van der Meer et al., 1998;
Hellwege et al., 2000; Van den Ende et al.,
2000, 2001), fructan metabolism in monocots is
not yet completely unraveled. So far, four different fructosyltransferases, each with their own specificity, are believed to
be involved in monocot fructan biosynthesis. In addition to inulin
biosynthesis by Suc:Suc 1-fructosyl transferase (1-SST) and
fructan:fructan 1-fructosyl transferase (1-FFT), the 1-SST product
1-kestose is used as a substrate by the key enzyme fructan:fructan 6G-fructosyl transferase to produce
neokestose, which in turn can be further elongated by the
action of 1-FFT or Suc:fructan 6-fructosyl transferase (6-SFT) to
produce inulin or levan neoseries, respectively (Vijn and
Smeekens, 1999). Furthermore, it has become clear that 1-SST
and 6-SFT are the key enzymes for graminan biosynthesis in cereals like
barley and wheat (Sprenger et al., 1995; Kawakami and Yoshida, 2002). 6-SFT prefers 1-kestose as an acceptor
substrate, and thus mainly produces bifurcose (1&6 kestotetraose) from
Suc and 1-kestose. 1-FFT fulfills an elongation role during graminan biosynthesis (Jeong and Housley, 1992), and its cDNA has
recently been cloned from wheat (Kawakami et al., 2002).
For a long time, it has been a matter of debate whether the 6-kestose
necessary for levan-type fructan biosynthesis in cereals originates
from the direct action of a specific 6-SST (Penson and Cairns,
1994; Chatterton and Harrison, 1997) rather than
by the -(2,1)-hydrolysis of bifurcose (Bancal et al.,
1992), the main product of 6-SFT (Sprenger et al.,
1995). To explain the biosynthesis of the levan neoseries in
ryegrass (Lolium perenne; Pavis et al.,
2001a) and the authentic levan series in Poa secunda
(Wei et al., 2002), these authors suggest the existence
of 6-SST or 6-FT-like enzymes that might prefer fructans other than
1-kestose (e.g. neokestose or 6-kestose) as acceptor substrates.
However, so far these kinds of enzymes have never been fully characterized.
In wheat stems, a typical seasonal accumulation of fructan is observed.
Accumulation continues during stem growth and anthesis but fructan
content in stems strongly decreases during the later stages and
contributes to grain filling (Pollock and Cairns, 1991; Bancal and Triboï, 1993; Schnyder,
1996). Wheat stems accumulate low degree of polymerization (DP)
levan- and graminan-type fructans that are well characterized both on
C18 and anion-exchange chromatography with pulsed amperometric
detection (AEC-PAD; Bancal et al., 1993). In excised and
induced wheat leaves (Bancal et al., 1992) but not in
field-grown wheat stems (Bancal and Triboï,
1993), a trimming of graminan-type fructans by (a) specific
1-FEH(s) was suggested. Both fructan biosynthetic and breakdown enzymes
can be measured during graminan biosynthesis in wheat stems. This paper
reinforces the work on fructan metabolism in wheat stems with a special
attention to the putative role of 1-FEHs not only during the period of
fructan breakdown but also as a putative (2,1) trimmer during the
period of active fructan biosynthesis.
Plant 1-FEHs have been studied extensively in dicots like chicory
(Cichorium intybus) and Jerusalem artichoke
(Helianthus tuberosus; Van Laere and Van den Ende,
2002, and refs. therein). From monocots, only partially
purified preparations of 1-FEH were obtained from wheat (Jeong,
1991), barley (Henson, 1989), and Lolium
rigidum (Bonnett and Simpson, 1993). A fructan
6-exohydrolase (6-FEH) from ryegrass (Marx et al.,
1997b) and an FEH that preferentially hydrolyzes -(2
6)
(oat; Henson and Livingston, 1996) or multiple fructofuranosidic linkages (barley; Henson and Livingston,
1998) were purified more recently.
To our knowledge, no FEH cDNA has so far been cloned from a monocot
species. Three 1-FEHs have recently been cloned from chicory (Van den Ende et al., 2000, 2001). It is
surprising that in severe contradiction to fructan biosynthetic enzymes
that evolved from vacuolar-type invertases, dicot 1-FEHs apparently
evolved from cell wall-type invertases (Van den Ende et al.,
2002a).
To elucidate a putative role for 1-FEHs, not only during the period of fructan breakdown but also as a putative (2,1) trimmer during the period of active fructan biosynthesis, two isoforms of 1-FEH enzymes from wheat stems were purified and characterized. Their cDNAs were cloned and compared with other monocot glycosyl hydrolases and dicot 1-FEHs.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
CONCLUSIONS
MATERIALS AND METHODS
LITERATURE CITED
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1-FEH Activities during Wheat Stem Development
Fructans in wheat stems, as estimated by the increase of Fru after mild acid hydrolysis, accumulate to well after anthesis (green stems) but disappear during further ripening (stems turning yellow; Fig. 1A). 1-FEH activity can clearly be detected during the period of fructan biosynthesis but increases temporarily during the disappearance of fructans from the stems (Fig. 1B).
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Enzyme Characterization
Purification of 1-FEHs
Compared with young wheat seedlings, senescing wheat stems contain much lower invertase activities, which make them interesting tissues for 1-FEH purification. Two isoforms of 1-FEH (termed 1-FEH w1 and w2) were purified from senescing wheat stems (24 d after anthesis) by a combination of Concanavalin A (Con A) affinity chromatography and AEC at different pH. 1-FEH w1 and w2 were already separated after the first Mono Q column (Fig. 2), but for removal of contaminating bands after SDS-PAGE, one or two more runs at different pH were necessary (Table I). Comparable results were obtained with younger stems (at anthesis) except that, not surprisingly, enzyme activities were much lower (Fig. 2).
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1 protein, respectively. The concentration of
1-FEH w2 was apparently much higher than that of 1-FEH w1 (Fig. 2;
Table I). Invertase and 6-FEH activities were also detected (not
shown), but these could be separated from the two 1-FEH isoforms. The
molecular mass of both purified enzymes was estimated at about 70 kD by SDS-PAGE (Fig. 3).
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Enzymatic Properties
The pH optimum of both purified enzymes was between pH 4.5 and 5.5, and they were inactive above pH 7.5 (not shown). Both 1-FEHs had optimal activities between 30°C and 40°C, but activities remained surprisingly high at the lower temperature range (not shown). Similar properties were observed for a 1-FEH from barley (Henson, 1989) and a 6-FEH from ryegrass (Marx et al.,
1997b).
When using 1-kestose as a substrate, a Km
of 7 mM and a Vmax of
23.2 milliunits µg1 protein were obtained for
both 1-FEHs (Table II). The
Km is much lower and the
Vmax higher compared with the purified
1-FEH II from chicory (Km, 57.8 mM; Vmax, 11.1 milliunits µg1 protein; De Roover et
al., 1999). If inulin is used as a substrate, Km could not be determined accurately
because no sign of saturation was observed at 10% (w/v) inulin, which
is the maximum concentration that can be obtained in vitro. Both
enzymes were severely inhibited by Suc with an apparent inhibition
constant (Ki) of 0.90 mM at 12 mM inulin (not
shown). This Suc inhibition is much stronger than observed for chicory
1-FEH II (Ki, 5.9 mM;
De Roover et al., 1999).
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Substrate Specificities
Both isoforms were exohydrolases using a multichain mechanism of hydrolysis because no products other than Fru could be detected by using commercially available inulin as a substrate (not shown). Activity against Suc is minimal, indicating that these enzymes are fructan exohydrolases and not-fructofuranosidases or invertases (Table II). From all fructans tested, the purified enzymes most efficiently hydrolyzed -(2,1)-linkages: 1-Kestose and 1,1-nystose were the best substrates. 6-Kestose was hydrolyzed at least 100 times
slower than 1-kestose (Table II). Therefore, these enzymes can be
designated as 1-FEHs.
Incubation of graminans, isolated from wheat stems at anthesis,
with FEH w1 and w2 revealed a different degradation profile (Fig.
4A). Fructan structures are shown in
Figure 4B, available at www.plantphysiol.org. 1-FEH w1 hydrolyzed
6,1 kestotetraose (peak i), 6&1,1 kestopentaose (peak k), and 1&6,1
kestopentaose (peak l) faster than 1-FEH w2. Moreover, 6;6,1
kestopentaose and/or 6;1&6 kestopentaose (peak n+o; these compounds
cannot be separated on AEC-PAD) is readily hydrolyzed by 1-FEH w1 but
not by 1-FEH w2 (Fig. 4A).
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Cloning
Reverse transcriptase (RT)-PCR was performed on total RNA derived from senescing winter wheat stems (24 d after anthesis). Primers based on conserved cell wall invertase and dicot 1-FEHs were used to obtain two partial clones with high amino acid identity to cell wall-type invertases and 1-FEHs. A mixture of the latter was subsequently used as hybridization probe to screen a winter wheat cDNA library. Full-length 1-FEH w2 and partial 1-FEH w1 were obtained. A 1-FEH w2-based primer was subsequently used to derive the missing DNA sequence part of 1-FEH w1.
The 1-FEH w1 and 1-FEH w2 cDNAs encode polypeptides of 597 and 596 amino acids, respectively. The deduced amino acid sequences for wheat 1-FEH w1 and w2 are presented in Figure 5. The cDNA-derived pIs of 1-FEH w1 and w2 are calculated at 4.79 and 4.78, respectively. These values match the chromatographic behavior of the native proteins. Furthermore, both mature proteins contain four potential glycosylation sites (N-X-S/T; Fig. 5). The cDNA-derived molecular mass of both mature enzymes (61.2 kD) is lower than the 70 kD estimated from SDS-PAGE (Fig. 3), but this discrepancy can probably be explained by the glycosylation on at least two of four potential N-glycosylation sites (see below).
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Quadrupole-Time-of-Flight (TOF) Mass Spectrometric (MS) Analyses
Theoretical tryptic digests on the cDNA-derived 1-FEH w1 and w2
protein sequences yielded 49 and 48 peptides, respectively. These are
designated T1 to T49 from N to C terminus. Masses of tryptic
peptides were determined by Q-TOF and compared (Tables III and IV)
with the masses of theoretical cDNA-derived peptides (allowing for one
possible missed cleavage site and with the consideration of oxidized
Met). For 1-FEH w1, all except seven of the detected masses matched one
of the theoretical fragments within the acceptable mass measurement
error of ±0.1 D (see supplemental Table
III, available at
www.plantphysiol.org). For 1-FEH w2, all except two masses
matched the theoretical ones (see supplemental data Table IV, available at
www.plantphysiol.org). Collision-induced
dissociation MS/MS analysis yielded a number of sequence tags
(Mann and Wilm, 1994), which proved the identity of the
tryptic peptides (Tables III and IV). Five unexplained fragments of
1-FEH w1 proved to be glycosylated peptides after fragmentation. These
fragments fit perfectly with two of four potential
N-glycosylation sites (Fig. 5). The other unexplained masses
of 1-FEH w1 and w2 can be understood by the posttranslational cleavage
of the prepeptide region from the cDNA-derived sequence. For 1-FEH w2,
both peptides DPSPAVSTMYK or PSPAVSTMYK are the expected N-terminal
sequences of the mature enzyme because no K or R is directly in front
of this peptide region in the translated cDNA (Fig. 5). DPSPAVSTMYK is
accordingly the presumptive N-terminal sequence of the 1-FEH w1 enzyme.
Comparison with Other Glycosyl Hydrolases
The cDNA-derived amino acid sequences of 1-FEH w1 and w2 are 98% identical. Similarities to chicory 1-FEH I (50% identity) and 1-FEH IIa and b (48% identity) are lower. 1-FEH w1 and w2 are more similar to cell wall invertases (46%-54% identical amino acids) than to vacuolar invertases (39%-45% identity) and fructan biosynthetic enzymes (34%-41% identity). Similarities to microbial fructan hydrolases are much lower (13%-28% identity).
An unrooted radial tree of some members of cell wall-type glycosyl hydrolases is presented in Figure 6. Four distinct groups can be discerned: The first group (I) contains monocotyledonous and mainly basic cell wall invertases. The second group (II) contains dicotyledonous, basic cell wall-type invertases. A third group (III) also contains dicotyledonous and mainly basic cell wall-type invertases, with the exception of an acid cell wall invertase from Arabidopsis (INV5). A fourth group contains dicotyledonous (IVa) and monocotyledonous (IVb) enzymes. The IVa subgroup harbors the three chicory 1-FEH cDNAs and a fructosidase from Arabidopsis. 1-FEH w1 and w2 cluster together with cell wall invertases from rice and maize. All group IV members typically have an acidic pI, as is usually observed for vacuolar-type invertases.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
CONCLUSIONS
MATERIALS AND METHODS
LITERATURE CITED
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Properties of Plant FEHs
For the first time, to our knowledge, a completely purified
monocot 1-FEH was obtained as shown by the single protein band on
SDS-PAGE (Fig. 3). Purity was confirmed by Q-TOF MS analysis where all
observed peaks could be explained (Tables III and IV; Fig. 5). Also,
6-FEH and invertase activities were present in crude extracts, but they
eluted in different fractions. As observed for most other plant FEH
enzymes (Simpson and Bonnett, 1993; Marx et al.,
1997a, 1997b; De Roover et al.,
1999), the final 1-FEH w1 and w2 preparations showed negligible
invertase activity (Table II). In severe contrast to plant FEHs, which
are only capable of degrading fructans and not Suc,
-fructo(furano)sidases (EC 3.2.1.80) can degrade Suc as well as
fructans. Therefore, plant FEHs are clearly different from
-fructo(furano)sidases and cannot be classified under EC 3.2.1.80.
In our opinion, a new EC number should be appointed to be able to
properly classify this kind of enzyme.
The apparent molecular mass of 70 kD as estimated by SDS-PAGE is
in the same range of 1-FEH I from chicory (72 kD; Claessens et
al., 1990), 1-FEH II from chicory (64 kD; De
Roover et al., 1999), a 1-FEH from Jerusalem artichoke
(75 kD; Marx et al., 1997a), a 6-FEH from ryegrass (69 kD; Marx et al., 1997b) and a 1-FEH from barley (62.5 kD; Henson, 1989). However, considerably lower molecular
masses were reported for a purified FEH from barley (33 kD;
Henson and Livingston, 1998), a 6-FEH from oat (43 kD; Henson and Livingston, 1996), and a 6-FEH from
Dactylis glomerata (57 kD; Yamatoto and Mino,
1989). It is not clear whether the partially purified
preparation of wheat stem 1-FEH by Jeong (1991) is
identical to one of our purified enzymes because they estimated the
molecular mass at 63.7 kD. In contrast to the generally heterodimeric fructan biosynthetic enzymes, which are believed to have evolved from
vacuolar-type invertases (Van den Ende et al., 2002b),
all reported plant FEHs are monomeric enzymes.
It is widely accepted that cereal fructan metabolic enzymes, including
FEHs, are located within the vacuole (Wagner and Wiemken, 1986). Like FEHs in other grasses (Simpson and Bonnett,
1993; Marx et al., 1997b), wheat 1-FEH has an
acidic pH optimum supporting vacuolar localization. Glycosylation of
1-FEH w1 and w2, as proven by tryptic analysis (Table II) and binding
on Con A, is also consistent with a vacuolar localization. Also chicory
1-FEHs were suggested to be vacuolar because it was impossible to find
them in apoplastic fluid at levels higher than can be explained by
cellular leakage (Van den Ende et al., 2000,
2001).
Plant FEHs Are Derived from Cell Wall-Type Invertases
So far, no monocot FEH enzymes had been cloned. On the basis of sequences conserved in cell wall invertases and dicot 1-FEHs, two highly identical 1-FEH cDNAs were cloned from wheat stems. The tryptic peptides retrieved from both purified 1-FEHs covered 43% (1-FEH w1) and 21% (1-FEH w2) of the cDNA-derived sequence information with an identity of 100% (Tables III and IV; Fig. 5), convincingly demonstrating that the cDNAs code for the corresponding enzymes.
Like the genes encoding dicot 1-FEHs from chicory (Van den Ende
et al., 2002a), wheat 1-FEH w1 and w2 are most similar to cell
wall invertases. They cluster together in a group (IV in Fig. 6)
harboring fructosidases with low pIs. Therefore, most likely all plant
FEHs, in dicots as well as in monocots, have probably evolved from
genes of cell wall-type invertases that obtained a low pI and a
vacuolar targeting signal. It has now become clear that enzymes with a
totally different functionality (FEH versus invertase) and localization
(cell wall versus vacuole) can cluster together. Because only a
minority of the genes in Figure 6 have been identified by other means
than homology, it is precocious to call them invertases. Therefore to
prevent further confusion, it is important that future clones be
investigated more thoroughly before being classified simply as "cell
wall invertase." In this respect, it would be tempting to determine
the functionality of the Arabidopsis clone that groups together with
1-FEHs in group IV (Fig. 6).
Putative Functions of FEHs
Co-expression of 1-SST and 1-FEH has been found in primary leaves
of barley (Wagner and Wiemken, 1989) and in mature wheat stems (Bancal and Triboï, 1993). If both enzymes
are vacuolar, both fructan biosynthetic and breakdown enzymes would
operate simultaneously in cereal grasses. In dicots, co-expression of fructan biosynthetic and breakdown enzymes has never been reported (Van Laere and Van den Ende, 2002).
Suc and illumination drastically increases the 1-SST to
1-FEH ratio and fructan content (Wagner and Wiemken,
1989), and Suc stimulates 6-SFT expression in barley
(Nagaraj et al., 2001) and down-regulates the 1-FEH
genes. Suc export and fructan degradation by newly synthesized 6-FEH
enzymes was demonstrated in D. glomerata (Yamatoto
and Mino, 1985). Apart from the putative control of Suc on
gene expression, Suc directly inhibits wheat 1-FEH
w1 and w2, chicory 1-FEH II (De Roover et al.,
1999), and most
but not allmonocot FEHs in vitro
(Simpson and Bonnett, 1993; Marx et al.,
1997b). Therefore, FEH activity in vivo is probably controlled and/or modulated by the Suc concentration.
Defoliation induces expression of 1-FEH II in chicory (Van den
Ende et al., 2001) but very low 1-FEH to 1-SST ratios were found in control plants throughout the period of fructan biosynthesis (Van den Ende and Van Laere, 1996b). In temperate
grasses, a much higher 1-FEH to 1-SST ratio can be derived from data in
the literature (Wagner and Wiemken, 1989; Prud'homme et
al., 1992; Bancal and Triboï, 1993). It
can be speculated that temperate grass plants (at least in certain
parts like e.g. the stubble) might profit from maintaining relatively
high FEH activities that are ready to use but largely inhibited under
normal circumstances by the high Suc concentrations. Increased Suc
export, such as induced by grazing and defoliation (temperate fodder
grasses) or during grain filling, would almost immediately activate
these FEH enzymes. A later (slower) increase of FEH activities could
then be accomplished by up-regulating the genes.
Figure 1 clearly demonstrates the high basal level of 1-FEH activity
throughout the period of fructan biosynthesis in mature wheat stems.
Starting from about 1 week after anthesis, 1-FEH activities rise and
probably both 1-FEH and 6-FEH-type enzymes contribute to the complete
fructan breakdown in the stem during grain filling. The considerable
1-FEH activity in young, fructan biosynthesizing stems is not
attributable to minor activities of invertase because a specific 1-FEH
w2 could be purified from this stage (Figs. 2 and 3). Therefore, our
results support the idea that 1-FEHs might be involved as
-(2,1)-trimmers during graminan biosynthesis. In this way, they
might prevent formation of inulin-type fructans by 1-SST and 1-FFT or
further -(2,1)-elongation of branched graminans by 1-FFT. No inulin
oligomers above DP 5 can be detected in wheat. Moreover, it is striking
that FEH enzymes that preferentially degrade -(2,1)-linkages were
only reported from species that accumulate predominantly low-DP
-(2,6)-rich fructans (wheat, barley, and L. rigidum). Our
results, however, do not support the view (Bancal et al.,
1992) that small branched graminans can be selectively trimmed
by 1-FEH because branched molecules apparently are poor substrates for
1-FEH w1 and w2 (Fig. 5). Therefore, it seems unlikely that 6-kestose
formation in wheat stems might originate from breakdown of bifurcose.
The inability of the 1-FEHs to efficiently hydrolyze small branched
graminans might explain why these types of fructan accumulate in wheat.
6-FEH activity is also measurable in young wheat stems. Purification
and characterization of this enzyme is still a subject of ongoing
research. The presence of both 1-FEH and 6-FEH does not indicate a
general turnover of fructan. Winzeler et al. (1990) convincingly demonstrated that there is no fructan turnover in fructan-biosynthesizing wheat stems. The lack of fructan turnover is
consistent with a long-term reserve function, and this is in severe
contrast with findings in gramineous leaves where fructans turnover
very rapidly (Farrar and Farrar, 1985). Preliminary
experiments on partially purified 6-FEH showed even a stronger
inhibition by Suc compared with 1-FEH w1 and w2, and therefore the
activity of this enzyme might be nearly completely inhibited in vivo by the high Suc concentrations during the period of active fructan biosynthesis.
We propose that the species-specific fructan patterns in cereal
grasses, and in wheat in particular, are the result both of the
relative abundances and the specificities of fructan biosynthetic enzymes (1-SST, 1-FFT, 6-SFT, and/or other) and of fructan
exohydrolases acting as trimmers (1-FEH and 6-FEH). A third important
factor is most probably the Suc concentration at the site of fructan biosynthesis, acting as an activator/substrate (1-SST and 6-SFT) or
inhibitor (fructan exohydrolases) at the DNA and protein level. In
support of this idea, it was demonstrated (Bancal and
Triboï, 1993) that a much larger proportion of
-(2,1)-linked fructans accumulates in chilled leaf blade seedlings
of wheat (Suc content, 35 mg g1 fresh weight)
than in mature wheat culms (Suc content, 8 mg
g1 fresh weight). The effect of silencing 1-FEH
genes on DP and relative abundance of -(2,1)-linkages in wheat
graminans might further elucidate this point.
Different Substrate Specificities for 1-FEH w1 and w2
Despite the 98% identity at the amino acid level between 1-FEH w1
and w2, we were able to demonstrate a different specificity toward
endogenous, branched wheat stem fructans (Fig. 5). Bancal et al.
(1993) found that the partially purified 1-FEH from barley (Henson, 1989) also preferred inulin-type fructans over
branched fructans such as bifurcose. The steric hindrance by adjacent
-(2,6)-linked Fru, as suggested by Bancal et al.
(1993), is apparently more important for 1-FEH w1 than for
1-FEH w2 (Fig. 4). Moreover, 6-kestose-based branched carbohydrates
like 6;1&6 kestopentaose and/or 6;6,1 kestopentaose are substrates for
1-FEH w1 but not for 1-FEH w2 (Fig. 4). Further confirmation by
incubation with pure substrates is needed but is hampered by the fact
that these products are not commercially available, occur in only low
concentrations in plant tissues, and are very difficult to separate
(e.g. 6;1&6 kestopentaose and/or 6;6, 1 kestopentaose). Nevertheless,
it would be very interesting to compare both enzymes by site-directed mutagenesis.
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CONCLUSIONS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
CONCLUSIONS
MATERIALS AND METHODS
LITERATURE CITED
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Two highly identical 1-FEHs (1-FEH w1 and w2) from wheat stems
were characterized at the protein and DNA level. Besides their function
during fructan breakdown, results indicate that at least 1-FEH w2 might
be involved as a -(2,1)-trimmer during graminan biosynthesis.
Therefore, 1-FEHs might play a crucial role in determining the fructan
pattern and final DP in cereals. Despite being highly identical, both
isoenzymes have different activity toward endogenous branched-type
fructans. This first-time cloning of 1-FEHs from a monocot species
should aid in cloning of other FEHs (e.g. 6-FEHs) from cereals and
temperate fodder grasses and should contribute to our understanding of
FEHs regulation. As a future goal, control of 1-FEHs in wheat grains or
leaves of temperate fodder grasses by genetic engineering might enhance
the levels of graminan-type fructans in food and feed. These soluble
fibers might have even better beneficial effects than inulin
(Roberfroid et al., 1998) as a prebiotic throughout the colon.
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MATERIALS AND METHODS |
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INTRODUCTION
RESULTS
DISCUSSION
CONCLUSIONS
MATERIALS AND METHODS
LITERATURE CITED
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Plant Material
Wheat (Triticum aestivum cv Pajero) was sown and grown in local fields with sandy loam soil during the growing seasons in 1999, 2000, and 2001. Field-grown stems (first 10 cm below the ear) were collected two to three times per week covering the periods of heading (Feekes scales 10.1-10.5), flowering (Feekes scales 10.51-10.54), and grain ripening. The first sample was taken when the first ears became just visible (Feekes scale 10.1). The whole sampling period covered about 9 weeks. The samples were used for carbohydrate analyses, for enzyme activity determinations, and for enzyme purification purposes.
Purification of 1-FEH w1 and w2
Extraction
Wheat stems were sampled at anthesis (fructan biosynthesis in stems) and 24 d after anthesis (ripening grain and fructan breakdown in stems) and cut in very small pieces. One kilogram was immersed in liquid nitrogen and was subsequently homogenized dry with a Waring blender. Thereafter, a second dry homogenization was performed in a smaller Waring blender until a fine powder was obtained. Finally, this powder was dissolved in 1.5 L of 50 mM sodium-acetate buffer, pH 5, containing 1 mM EDTA, 10 mM NaHSO3, 1 mM mercaptoethanol, and 0.1% (w/v) Polyclar AT (Serva, Heidelberg), and a final homogenization step was performed. The homogenate was squeezed through cheesecloth.Purification and Electrophoresis
Ammonium sulfate was added to a saturation of 30% and gently stirred on ice for 30 min. After centrifugation for 20 min at 40,000g and 4°C, precipitated protein was discarded. Again ammonium sulfate was added to the supernatant to a final saturation of 80%. After a second centrifugation (20 min at 40,000g and 4°C), the precipitate was collected and redissolved in 150 mL of 50 mM sodium-acetate buffer, pH 5.0. Undissolved material was spun down for 15 min at 40,000g and 4°C. The supernatant was applied to a Con A Sepharose column (25 × 100 mm) and eluted as described (Van den Ende et al., 1996). Active fractions were
pooled, adjusted to pH 7.0 with concentrated Tris, and applied on Mono
Q1 (HR 5/5, Pharmacia AB, Uppsala) equilibrated with 20 mM
Tris-HCl buffer, pH 7.0. Proteins were eluted as described (Van
den Ende et al., 1996). 1-FEH w1 (fraction 15) and 1-FEH w2
(fractions 18-20) were diluted five times with 20 mM
His-HCl buffer, pH 6.0, and subsequently loaded again on Mono Q2 (1-FEH
w1) and Mono Q3 (1-FEH w2), equilibrated with 20 mM His-HCl
buffer, pH 6.0. Finally, the active fractions 17 and 18 from Mono Q3
were diluted five times in 50 mM sodium-acetate buffer, pH
5.2, and loaded on Mono Q4 equilibrated with 50 mM sodium-acetate buffer, pH 5.2. Whenever possible, enzymes were kept on
ice throughout the whole purification. Sodium-azide (0.02%, w/v) was
added to all buffers to prevent microbial growth. SDS-PAGE was
performed on 12.5% (w/v) polyacrylamide gels and stained with Coomassie Brilliant Blue-R250 as described (Van den Ende et al., 1996).
Carbohydrate Analyses and Enzyme Activity Determinations
Protein extracts of field-grown wheat stems were precipitated with ammonium sulfate, redissolved, and desalted to remove endogenous substrates as described (Van den Ende and Van Laere, 1996b). To measure the activities of 1-FEH, 6-FEH, and
invertase, aliquots were incubated with 3% (w/v) commercial chicory
root inulin (Sigma-Aldrich, St. Louis), 10 mM levan, and
100 mM Suc in 50 mM sodium-acetate buffer, pH
5.0, for different time intervals at 30°C. Sodium-azide (0.02%, w/v)
was added to all buffers to prevent microbial growth. Fru formation was
determined by AEC-PAD, and proteins were determined as described
(Van den Ende and Van Laere, 1996b). Throughout enzyme purification, aliquots were taken for activity determination. Enzymatic
activity is expressed in units, defined as the amount of enzyme that
formed 1 µmol Fru min1.
Fructan Substrates and Structure Determination
Wheat stem low-DP fructan (anthesis) was obtained as follows. Boiling water extracts were prepared. After centrifugation (20 min at 40,000g and 4°C), yeast
-glucosidase was added to
the supernatant (1.5 units mL1) and incubated overnight
at 37°C to specifically degrade Suc. The sample was subsequently
loaded and eluted from a Ca-Dowex column as described by
Timmermans et al. (2001). Hexose-free fractions were
pooled and used as a substrate for wheat 1-FEH enzymes. 1-Kestose and
1,1-nystose were prepared from Neosugar P (Beghin-Meiji Industries, Paris) by preparative reversed-phase HPLC (Nucleosil 7 C18,
250 × 12.7 mm) with water as solvent and a flow rate of 2 mL
min1. Manually collected fractions were pooled and
lyophilized. Low-Mr levan and 6-kestose were
generous gifts from Dr. M. Iizuka (Iizuka et al., 1993).
Neokestose was kindly provided by Dr. N.J. Chatterton. Levanbiose
(F2) was purified as described by Timmermans et al. (2001). Structures of branched wheat fructans were derived from the work of Bancal et al. (1993), but reconfirmation
occurred by manual collection of AEC-PAD peaks and enzymatic hydrolysis by the purified wheat 1-FEHs.
Total fructan content throughout wheat stem development was estimated
by total Fru liberated after mild acid hydrolysis in 0.06 N
HCl at 75°C for 1.5 h. Fru and Suc present before hydrolysis were subtracted.
Q-TOF Analyses on Tryptic Fragments
The SDS-PAGE protein bands of 1-FEH w1 and w2 (both 70 kD) exhibiting 1-FEH activity were subjected to MS identification. The Coomassie Brilliant Blue-stained protein bands were excised, trypsinized, extracted, desalted, and analyzed on Q-TOF as previously described (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. To assist MS/MS-based
glycosylation characterization, we have developed a software
application named Sweet Substitute (S. Clerens, W. Van den Ende, P.D.
Verhaert, L. Geenen, F. Vandesande, and L. Arckens, unpublished data).
RNA Isolation, RT-PCR, and Cloning
Total RNA was isolated from wheat stems (24 d after anthesis) by using the RNeasy Plant Mini kit (Qiagen USA, Valencia, CA). The conserved amino acid sequences HFQP (N-terminal), AFNN, and VFNN (C-terminal) were used to make the degenerated primers HFQP (5'-GSWTWYCAYTTYCARCC-3'), CTERMA (5'-GTCNCCR-TTRTTRAANGC-3'), and CTERMV (5'-GTCNCCRTTRTTRAANAC-3') occurring in dicot 1-FEHs and in plant cell wall invertases. One-step RT-PCR was performed (Access RT-PCR System, Promega, Madison, WI) with HFQP-CTERMA and HFQP-CTERMV. RT reaction was at 48°C. For PCR, the following conditions were used: 94°C, 3 min; followed by 35 cycles: 94°C, 40 s; 48°C, 40 s; and 72°C, 2 min. Final extension was at 72°C, 10 min (PCR Access kit, Promega). Only in the HFQP-CTERMA condition, a very weak band of about 1,500 bp was visible. Therefore, seminested PCR was attempted on this PCR product with primers ECPD (derived from the conserved amino acid sequence WECPD: 5'-GAATGTGGGARTGYCCNGA-3') and CTERMA. The 960-bp band was ligated in the TOPO-TA vector and transformed to Escherichia coli (TOPO-TA cloning kit, Invitrogen, Groningen, The Netherlands). Plasmid was extracted using Wizard Plus SV Minipreps (Promega). Partial sequencing yielded two types of clones with high similarity to cell wall-type invertases and dicot 1-FEHs (named 12.16 and 12.18). A mixture of PCR products derived from these clones was subsequently used as hybridization probes to screen a winter wheat cDNA library by plaque hybridization (described by Kawakami and Yoshida [2002]). Sequences were determined and analyzed as described (Kawakami and Yoshida, 2002).
One of the positive clones (named B62) resembled 12.16 but was still a
partial cDNA (5' part missing). Therefore, the cDNA library was
subsequently rescreened using this partial cDNA as probe, and then a
full-length sequence clone (named C42) corresponding to the 12.16 sequence of the original probe was obtained. Later, protein
purification and molecular mass determination of tryptic fragments of
the purified proteins revealed that the protein 1-FEH w2 corresponds to
clone C42 and 1-FEH w1 corresponds to clone B62. For unknown reasons,
efforts to obtain full-length B62 by screening the wheat cDNA library
remained unsuccessful. Therefore, the missing 5'-sequence part of 1-FEH
w1 was cloned by two-step RT-PCR and the use of Pfu polymerase as
previously described (Van den Ende et al., 2001). A
degenerate sense primer (5'-GCCGCCATGGCNCARGC-3') was chosen based on
the start codon region in 1-FEH w2. This was combined with a 1-FEH
w1-specific antisense primer (5'-AACAACAACTGCTTGCCATT-3'). Sequence
analysis revealed a perfect match with the B62 partial sequence.
Sequences were deposited in the EMBL sequence library (accession no.
AJ516025 for 1-FEH w1 and accession no. AJ508387 for 1-FEH w2).
Statistics
Values in the graphs represent means of three replicates on one enzyme preparation. The corresponding SE is indicated. Experiments were repeated on different enzyme preparations with consistent results.
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ACKNOWLEDGMENTS |
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We thank Edgard Nackaerts for his technical assistance. The help offered by Pierre Bancal for fructan structure determination on AEC-PAD is highly appreciated.
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FOOTNOTES |
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Received September 26, 2002; returned for revision October 23, 2002; accepted October 31, 2002.
1 This work was supported by the Fund for Scientific Research Flanders. W.V.d.E. is a Postdoc supported by the Fund for Scientific Research Flanders.
* Corresponding author; e-mail wim.vandenende{at}bio.kuleuven.ac.be; fax 32-16-321967.
[w] The online version of this article contains Web-only data. The supplemental material is available at www.plantphysiol.org.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.015305.
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ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
CONCLUSIONS
MATERIALS AND METHODS
LITERATURE CITED
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-2,6 fructans.
Plant Physiol
110: 639-644[Abstract] (2,1) fructan fructan fructosyl transferase activity.
Plant Physiol
100: 199-204-fructosidase (FEH) activity and characterization of a -(2,1)-linkage specific FEH.
New Phytol
135: 267-277[CrossRef]-(2,6)-linkage specific fructan--fructosidase from stubble of Lolium perenne.
New Phytol
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|---|---|---|---|
-(2,1)-Trimmers during Graminan
Biosynthesis in Stems of Wheat? Purification, Characterization, Mass
Mapping, and Cloning of Two Fructan 1-Exohydrolase
Isoforms
) and at
anthesis (
), respectively. Activity profile of the different
fractions. 1-FEH activity was measured by the Fru production from 3%
(w/v) commercial chicory inulin.
