Plant Physiol. (1998) 118: 885–894
Fucosyltransferase and the Biosynthesis of Storage and
Structural Xyloglucan in Developing Nasturtium Fruits1
Darrell Desveaux, Ahmed Faik, and Gordon Maclachlan*
Biology Department, McGill University, 1205 Docteur Penfield Avenue, Montreal, Quebec, Canada H3A 1B1
chromatography and PAD (Fanutti et al., 1996; Faik et al.,
1997a).
The typical structural XG in primary walls contains Fuc
and less Gal than storage XG. Nevertheless, Gal is an
essential part of wall XG because terminal Fuc residues are
attached to it by an a-1,2 linkage in a three-sugar side
chain. Such side chains facilitate XG binding to cellulose
(Levy et al., 1991, 1997). Small amounts of Fuc have been
detected in hydrolysates of nasturtium seed extracts, but it
was not shown to derive from structural components of the
wall (Ruel et al., 1990). If expanding nasturtium fruit cells
also contain fucosylated XG in primary walls, cotyledons
must be capable of synthesizing two forms of XG with
quite different compositions and extracellular locations.
Recently, we detected (Faik et al., 1997b) XG-dependent
fucosyltransferase activity in extracts of particulate membranes from developing nasturtium fruits. This raises the
question of how or whether the great bulk of NXG avoids
being fucosylated in vivo. There are several possible explanations. Assuming that XG:fucosyltransferase is localized
and active in the Golgi toward the end of the secretory
process, either in trans cisternae or secretory vesicles
(Brummell et al., 1990) or in the trans Golgi network (Zhang
and Staehelin, 1992; Driouich et al., 1993), it could be that
two forms of XG are synthesized at the same time but in
different Golgi compartments, with XG:fucosyltransferase
confined to the site that leads to wall XG. It is also possible
that structural and storage XG are formed at different times
during cell expansion, or in separate cells or tissues, and
that fucosyltransferase is active only when or if the wall is
incorporating XG.
An alternative and more speculative explanation is that
newly synthesized NXG is fucosylated, but as a transitory
decoration with Fuc cleaved from the polymer before or
during the time it is deposited in periplasmic spaces.
This would require the action of an a-fucosidase with
the capacity to defucosylate XG. However, those plant
a-fucosidases that have been studied to date, those in
extracts of germinated nasturtium seeds and pea epicotyls
(Farkas et al., 1991; Augur et al., 1993), are only able to
hydrolyze Fuc from XG oligosaccharide when it is free in
solution, not when it is combined as a subunit in intact XG.
Young, developing fruits of nasturtium (Tropaeolum majus L.)
accumulate large deposits of nonfucosylated xyloglucan (XG) in
periplasmic spaces of cotyledon cells. This “storage” XG can be
fucosylated by a nasturtium transferase in vitro, but this does not
happen in vivo, even as a transitory signal for secretion. The only
XG that is clearly fucosylated in these fruits is the structural fraction
(approximately 1% total) that is bound to cellulose in growing
primary walls. The two fucosylated subunits that are formed in vitro
are identical to those found in structural XG in vivo. The yield of
XG-fucosyltransferase activity from membrane fractions is highest
per unit fresh weight in the youngest fruits, especially in dissected
cotyledons, but declines when storage XG is forming. A block
appears to develop in the secretory machinery of young cotyledon
cells between sites that galactosylate and those that fucosylate
nascent XG. After extensive galactosylation, XG traffic is diverted to
the periplasm without fucosylation. The primary walls buried beneath accretions of storage XG eventually swell and lose cohesion,
probably because they continue to extend without incorporating
components such as fucosylated XG that are needed to maintain
wall integrity.
XG that accumulates in cotyledons of developing nasturtium (Tropaeolum majus L.) seeds as a temporary “storage”
polysaccharide (NXG) differs from primary wall “structural” XG of most dicots in three major respects: (a) NXG is
deposited in massive amounts (up to 20% of seed dry
weight) in periplasmic spaces between plasma membranes
and primary walls, i.e. in apposition to the wall (Hoth et al.,
1986; Hoth and Franz, 1986; Ruel et al., 1990). It is not
mobilized until about 8 d after germination (Edwards et al.,
1985) in an auxin-dependent event (Hensel et al., 1991). (b)
NXG is readily extracted with hot water (Hsu and Reeves,
1967; Hoth et al., 1986) or dilute alkali (Edwards et al., 1985;
Hensel et al., 1991), whereas wall XG is so well integrated
between and even into the cellulose framework (Hayashi,
1989; Carpita and Gibeaut, 1993; Edelmann and Fry, 1992)
that the microfibril:XG complex must be swollen and hydrogen bonds broken (e.g. by 24% KOH) before this bound
XG will dissolve. (c) NXG contains Glc, Xyl, and Gal in a
molar ratio of 4:3:1.7, but no trace of Fuc, as determined by
sensitive analyses using high-performance anion-exchange
Abbreviations: Chaps, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; NXG and TXG, storage xyloglucans containing no Fuc derived from nasturtium and tamarind seed, respectively; PAD, pulsed amperometric detection; XG, xyloglucan.
Oligosaccharide subunits of XG are abbreviated according to the
nomenclature proposed by Fry et al. (1993).
1
This study was funded by the Natural Sciences and Engineering Research Council of Canada via a scholarship (to D.D.) and a
research grant (to G.M.).
* Corresponding author; e-mail [email protected];
fax 1–514 –398 –5069.
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Desveaux et al.
Moreover, there is no evidence that terminal fucosylation
of XG is required as a signal for XG secretion; in fact,
mutants of Arabidopsis that are unable to synthesize Fuc
continue to incorporate normal levels of XG into cell walls
(Reiter et al., 1993). Therefore, one aim of this study was to
clarify how developing nasturtium fruits can harbor an
active XG:fucosyltransferase and also generate large
amounts of nonfucosylated storage XG.
With respect to the timing of the deposition of storage
XG in relation to cotyledon growth, Hoth and Franz (1986)
reported the first visible periplasmic deposits in electron
micrographs of cells from developing nasturtium cotyledons at 23 d after anthesis. The cotyledons continue to
grow rapidly while generating protein bodies, depositing
NXG and greatly increasing dry weight (Hoth et al., 1986).
The periplasmic deposits stain with the Thiery silver proteinate reagent for polysaccharide and with a polyclonal
antibody to XG (Ruel et al., 1990). They also stain strongly
with Coomassie brilliant blue (Hoth et al., 1986), indicating
the presence of protein in these accretions. The light micrographs in the latter study show enough cotyledon cells
to calculate statistically significant values for average cell
size (cross-sectional area). Sizes increased between cells of
sections observed before NXG deposition, those measured
during deposition with some naked primary wall still visible, and those measured after heavy deposition with the
entire periplasm filled with accretions, leaving no intercellular connections. Thus, the ratio of the relative sizes of
tissue cells cut at 18, 26, and 35 d after anthesis was
1:1.6:2.5, respectively. Clearly, the deposition of periplasmic XG does not restrict substantial cell expansion, although it may erect a barrier to the incorporation of new
wall materials.
In the present study HPLC and PAD were used to identify the XG subunits that are present in cellulase digests of
NXG and nasturtium wall XG, and to compare the XG
subunits that are fucosylated in vivo and in vitro. The point
during fruit development at which NXG begins to be deposited was estimated from dry:fresh weight ratios and
direct examination of electron micrographs. Levels of fucosyltransferase activity with or without added TXG were
measured in detergent extracts of membranes from homogenates of whole fruits prepared before and after endogenous NXG began to be generated. The activity was also
compared in extracts of excised cotyledons versus pericarp
tissue. The results demonstrate that structural XG in primary walls of nasturtium fruits is fucosylated, and that the
level of membrane-bound, XG-dependent fucosyltransferase activity declines when cotyledons form storage XG.
MATERIALS AND METHODS
Plant Materials and Chemicals
Dry nasturtium (Tropaeolum majus L. var Climbing Giants) seeds were purchased from W.H. Perron Ltd. (Montreal, Canada), washed for 10 min in 10% commercial
bleach, soaked in water overnight, and germinated in moist
vermiculite at room temperature. After about 1 month,
seedlings were transferred to pots containing sand, Pro-
Plant Physiol. Vol. 118, 1998
Mix (Pharmacia), and black earth in a ratio of 2:1:1, plus
lime (1 g L21). Plants were grown in a growth chamber
under 16 h of light (21°C) and 8 h of dark (18°C) with 70%
RH throughout the day. Flowers were self-pollinated and
the first fruits appeared about 10 d after anthesis. Three
fruits often develop from one flower. The term “fruit” as
used here refers to the seed with cotyledons enveloped by
integument plus the outer pericarp, composed mainly of
green, spongy parenchyma.
Individual fruits were harvested from their first appearance (,1 mm diameter, 50 mg fresh weight) to the time
about 30 d after anthesis when they were fully grown
(about 1.4 cm in diameter, 1 g fresh weight). They were
weighed at daily intervals, surface sterilized for 5 min in
10% (v/v) commercial bleach, washed, and frozen for later
use. Dry weight was measured after freeze drying. To
obtain growth curves for developing fruits, the diameters
of individual fruits were estimated daily with calipers at
the widest point, and fresh and dry weights were calculated from standard curves obtained by relating diameters
to weights. These weights were plotted versus age in days
after anthesis, using 15 d as the reference point, which is
when the fruits, including the seeds with integuments,
began their most rapid period of expansion (Hoth et al.,
1986).
The substrate for fucosyltransferase, GDP-l-[U-14C]Fuc,
was purchased from New England Nuclear/DuPont (8.3
GBq mmol21). The NXG provided as the potential Fuc
acceptor was prepared from mature seeds by the method
described by Edwards et al. (1985), except that extraction
was in 2 n NaOH, 0.01% NaBH4 at room temperature
without heat to avoid possible peeling reactions. TXG and
partially purified cellulase from Trichoderma sp. were purchased from Megazyme International (Bray, Ireland). BSA,
Chaps, DTT, leupeptin, Pipes buffer, PMSF, Pronase, tosyl
Lys chloromethyl ketone, and tosyl Phe chloromethyl ketone were purchased from Sigma. CarboPac PA-100 columns for HPLC and the apparatus for PAD were from
Dionex (Sunnyvale, CA). Sepharose CL-6B was from Pharmacia and Bio-Gel P2 was from Bio-Rad.
Solubilization of XG-Dependent
Fucosyltransferase Activity
The method used to extract and assay fucosyltransferase
activity from nasturtium fruit particulate membrane was a
modification of the procedures described previously for
enzyme from pea epicotyls (Hanna et al., 1991; Faik et al.,
1997a). Frozen whole fruits or excised cotyledons and pericarp tissues were homogenized in an Osterizer blender
(Sunbeam, Canada) (4 3 30 s at 1-min intervals) in 2 volumes of cold extraction buffer composed of 0.1 m PipesKOH, pH 6.8, 0.4 m Suc, 5 mm MgCl2, 5 mm MnCl2, 1 mm
DTT, plus 10 mm leupeptin, 1 mm PMSF, 0.1 mm tosyl Lys
chloromethyl ketone, and 0.1 mm tosyl Phe chloromethyl
ketone. The resulting mixture was filtered through nylon
cloth to remove cell wall debris, and the filtrate was centrifuged (model L8–80 centrifuge, Beckman) at high speed
(100,000g) with an angle rotor (50 titanium) for 60 min at
4°C to obtain pellets containing total particulate mem-
Xyloglucan:Fucosyltransferase in Developing Nasturtium Fruits
branes. Enzyme was solubilized from particulate pellets
derived from 10 g fresh weight of whole fruits, or a minimum of 3 g fresh weight of cotyledons, by stirring at 4°C
for 30 min in one-third volume of extraction buffer 6 0.3 to
0.4% (w/v) Chaps detergent, and removing insoluble material by recentrifugation (100,000g). In some experiments
this was followed by a second extraction with Chaps. The
detergent extracts were analyzed for fucosyltransferase activity, as described below. Protein concentrations were
measured using an assay kit (Bio-Rad) with BSA as a
standard.
Enzymic Assays
The basic assay medium for fucosyltransferase was composed of 0.1 m Hepes-KOH, pH 6.8, 25 mm MnCl2, 5 mm
DTT, and 0.5 m Suc. Standard reaction mixtures (50 mL total
volume) contained 20 mL of assay medium, 10 mL of GDP[14C]Fuc (92.5 pmol, 85,000 dpm), 10 mL of 1% XG (100 mg
of NXG or TXG), and 10 mL of enzyme (up to 10 mg of
protein). The final concentration of substrate GDP-[14C]Fuc
was 1.85 mm. Water replaced XG in controls. Reactions
were initiated by the addition of enzyme and terminated
after 30 min of incubation at room temperature. To avoid
precipitation of insoluble salts of the labeled substrate, and
to precipitate protein but not XG, reaction mixtures were
terminated first by the addition of cold 10% TCA (for at
least 1 h at 4°C) and centrifugation, followed by the addition of cold ethanol to the supernatant (final concentration
67%). The mixtures were chilled to 220°C and centrifuged
to precipitate XG and other insoluble materials. The pellets
were washed three times with 67% ethanol, redissolved/
suspended in 200 mL of water, and radioactivity was determined by liquid-scintillation spectroscopy.
Oligosaccharide subunits of XG were prepared by digesting the reaction-mixture components that were insoluble in 67% ethanol with Trichoderma sp. cellulase (0.5 mg
mL21) in 50 mm sodium acetate buffer, pH 5.0, at 35°C for
16 h. The reaction was terminated by boiling. The products
were fractionated on columns (1.1 3 126 cm) of Bio-Gel P2
with 0.01% NaN3 as an eluent. In this system, [14C]Fuclabeled XG oligosaccharides eluted at the high-Mr end of
the carbohydrate peak (Maclachlan et al., 1992).
a-Fucosidase activity was assayed versus TXG that had
been fucosylated by incubation with GDP-[14C]Fuc in solubilized pea fucosyltransferase (Hanna et al., 1991), or
versus a mixture of oligosaccharides generated from this
labeled XG by partial hydrolysis with Trichoderma sp. cellulase. The assay procedure (Farkas et al., 1991) used paper
chromatography to measure the initial rate of release of
free [14C]Fuc from these substrates by various enzyme
preparations.
Primary Wall XG
The buffer-insoluble debris that were retained by nylon
filters from enzyme homogenates of developing nasturtium fruits were collected frozen from several experiments
using a wide size range of fruits. The combined debris from
100 g fresh weight of fruits were stirred in approximately 5
887
volumes of 2 n NaOH, 0.01% NaBH4 at room temperature
overnight. This was repeated four times to ensure that all
traces of starch, NXG, and other unbound wall matrix
materials had been dissolved (see also Edwards et al., 1985;
Hanna et al., 1991).
The insoluble residue remaining from the debris after
this scouring treatment was stirred overnight in 24% KOH
and 0.1% NaBH4 to dissociate any residual cellulose:XG
macromolecular complex. The mixture was centrifuged to
remove cellulose and the primary cell wall XG was precipitated from the supernatant with 2 volumes of cold ethanol
(Hayashi and Maclachlan, 1984). The precipitate was redissolved in hot water and reprecipitated as an insoluble
XG:copper complex by adding Fehling’s solutions (Rao,
1959). The blue pellet was resuspended in 0.5 m EDTA, pH
6.5, and the XG was precipitated with 2 volumes of ethanol.
Washes were repeated until copper was completely eluted
from the flocculent wall XG. Total carbohydrate was assayed with phenol sulfuric acid (Dubois et al., 1956) and
XG specifically by the I-KI method of Kooiman (1960),
using commercial TXG as a standard.
Analysis of Monosaccharides and Oligosaccharides
Oligosaccharides obtained from NXG or nasturtium cell
wall XG, as described above, were concentrated by heating,
and 5 to 10 mg was injected into a CarboPac PA-100 column
attached to an apparatus for HPLC (Beckman). Carbohydrate was eluted with 30 mm sodium acetate in 0.1 m
NaOH (degassed with helium). Elution profiles were recorded automatically using PAD. To determine the distribution of 14C in oligosaccharides purified from 10 enzymic
reaction mixtures, fractions were collected manually as
they eluted from the PAD apparatus at times that related to
identifiable peaks. 14C was determined in neutralized fractions pooled from several injections until peaks containing
several hundred disintegrations per minute were recovered for accurate scintillation spectrometry. Monosaccharides were prepared from XG or oligosaccharides by complete hydrolysis with 2 n trifluoroacetic acid in sealed
tubes at 120°C for 1 h. The acid was removed by evaporation and aliquots (5 mg of carbohydrate) were analyzed by
elution from a CarboPac PA-100 column and PAD with
degassed 12 mm NaOH.
Electron Microscopy
Nasturtium seeds were collected at different stages of
development, and sections cut from cotyledons with a
razor were fixed for 2 h in 3.5% glutaraldehyde, 0.1 m
phosphate buffer, pH 7.2. The tissue was postfixed in 1%
OsO4 in the same buffer at 4°C for 1 h, dehydrated in cold
25% ethanol, followed by 50%, 75%, and 95% ethanol at
room temperature, and embedded in Spurr’s epoxy resin.
Thin sections (700 Å) were cut with a microtome (Ultra Cut
E, Reichert, Vienna, Austria), transferred to copper grids,
and stained with 2% uranyl acetate (10 min) followed by
lead citrate (20 min). Sections were viewed and photographed with an electron microscope (model EM 410, Philips, Eindhoven, The Netherlands).
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Desveaux et al.
RESULTS
Plant Physiol. Vol. 118, 1998
mol %, which can be calculated from the relative concentrations and Mr values of oligosaccharide peaks in Figure 1.
Composition of Primary Cell Wall XG from Developing
Nasturtium Fruits
Primary cell wall XG, as extracted from the filtered debris obtained from enzymic homogenates (“Materials and
Methods”), yielded 150 mg of XG per 100 g of nasturtium
fruit, which was similar to the value of 0.2% fresh weight
recorded for primary wall XG extracted from pea epicotyls
by a similar process (Hayashi and Maclachlan, 1984). The
purified wall XG was hydrolyzed to subunits with Trichoderma sp. cellulase, fractionated through a Bio-Gel P2 column, and the oligosaccharide peak was analyzed by HPLC.
Figure 1 presents the PAD profiles obtained for oligosaccharide subunits, with elution positions from the CarboPac
column of known XG oligosaccharides indicated by the
abbreviated nomenclature proposed by Fry et al. (1993).
The wall XG contained the following subunits in order of
their elution, along with the ratio of recoveries (mol %) in
parentheses: XXXG (24), XXFG (18), XLXG (17), XLFG (30),
and XXLG (11). Fucosylated subunits made up about onehalf of the XG components on a molar basis, which is
typical of primary wall XG oligosaccharide PAD profiles
reported for other dicots, e.g. Arabidopsis stem and apple
fruit (Zablackis et al., 1995; Vincken et al., 1996).
When nasturtium wall XG was totally hydrolyzed to
monosaccharides and analyzed by HPLC to obtain PAD
profiles, the only sugars detected were Glc, Xyl, Gal, and
Fuc, with a ratio of values for relative mol % close to
4:3:1:0.5. This corresponds exactly to the monosaccharide
XG-Dependent Fucosyltransferase Activity
Enzyme extracted with or without Chaps detergent from
particulate membranes of young, developing nasturtium
fruits was able to catalyze incorporation of label from
GDP-[14C]Fuc into product(s) soluble in 10% TCA but insoluble in 67% ethanol if reaction mixtures contained
added storage seed XG. As also observed with pea microsomal extracts (Hanna et al., 1991; Faik et al., 1997a), fucosyl transfer to insoluble products was distinctly greater
when detergent was used in the extraction medium. This
implies that at least some of the enzyme responsible was
intrinsically bound to membrane and that availability of
acceptor XG in these extracts was a limiting factor. We
could not detect any a-fucosidase activity in supernatants
or membrane extracts of fruit homogenates that could act
on intact fucosylated XG. The assay method that was used
readily measured XG nonasaccharide-dependent fucosidase activity in extracts of germinated nasturtium seeds
(Farkas et al., 1991).
Incorporation of [14C]Fuc into an ethanol-insoluble product was greater in the presence of XG from tamarind than
from nasturtium seed, presumably because of the differences in affinity of nasturtium fucosyltransferase for these
two acceptors, i.e. differences in their subunit composition.
This difference is most marked in the relative concentrations of octasaccharides, which could act as fucosyl acceptors: TXG contains mainly XXLG (galactosylated nearest
the unsubstituted Glc), whereas XLXG predominates in
NXG (Fanutti et al., 1996; Faik et al., 1997a; see also Fig. 1).
XXFG is the major fucosylated nonasaccharide in pea cell
wall XG (Hayashi and Maclachlan, 1984; Guillén et al.,
1995), which must have derived from XXLG. Thus, nasturtium fucosyltransferase may prefer TXG over NXG as an
acceptor because the former contains more of the octasaccharide needed to form XXFG.
Fucosylated Products
Figure 1. Profiles of oligosaccharide subunits produced by cellulase
hydrolysis of NXG and primary cell wall XG from developing nasturtium fruits. NXG and wall XG were extracted sequentially using 2
N NaOH and 24% KOH, respectively, as described in “Materials and
Methods.” XG was digested with Trichoderma sp. cellulase and the
resulting oligosaccharides were recovered from peak fractions after
gel filtration on Bio-Gel P2 columns. The two sets of oligosaccharides
were then fractionated one after the other by HPLC through a CarboPac PA-100 column and assayed using PAD, with calibration by
purified authentic XG subunits (Vincken et al., 1995; Faik et al.,
1997a). Subunits are designated according to the nomenclature devised by Fry et al. (1993).
In preliminary experiments, reaction mixtures incubated
in the absence of added storage XG and terminated without
adding TCA generated relatively substantial amounts of
14
C-labeled product that was insoluble in 67% ethanol. This
was not labeled XG or protein but eluted near the total
volume of effluent from a column of Sepharose CL-6B. It
cofractionated without further treatment on columns of
Bio-Gel P2 and CarboPac PA-100 with GDP-Fuc and sugar
phosphate. It dissolved readily in 10% TCA, like XG, but in
the presence of acid it was not precipitated by 67% ethanol.
This product was apparently a charged derivative of
[14C]Fuc that was dissociated by acid. It was probably a
divalent salt of the substrate or a phosphorylated degradation product because these are not soluble in neutral 67%
ethanol. In subsequent tests reaction mixtures were first
acidified and precipitated with 10% TCA and then precip-
Xyloglucan:Fucosyltransferase in Developing Nasturtium Fruits
itated with 67% ethanol. This treatment reduced values for
controls (without XG) to near zero by leaving salts of the
substrate and derivatives in the acidic ethanol supernatant.
The 67% ethanol-insoluble 14C product formed by nasturtium fruit extract in the presence of TXG was dissolved
in boiling water and fractionated with 0.1 n NaOH as a
solvent on a column of Sepharose CL-6B. The 14C profile
paralleled exactly the profile of TXG (phenol sulfuric acid
assay), with a peak eluting at a size equivalent to dextran
(.106 D). There was no shoulder corresponding to the peak
of protein in these preparations, and the profile was not
altered by incubation with protease (Pronase). However,
the Sepharose peak was completely degraded by treatment
with Trichoderma sp. cellulase to products that fractionated
on columns of Bio-Gel P2 at the upper end of the peak of
XG oligosaccharide subunits. Aliquots of these Bio-Gel P2
peaks were passed through a CarboPac PA-100 column for
oligosaccharide and 14C analysis, as described in “Materials and Methods.” Figure 2 represents the disintegrations
per minute recovered from TXG and NXG digests as a
function of retention time in this chromatographic system.
There were two peaks of 14C that corresponded to the
known elution positions of fucosylated nonasaccharide
(XXFG) and decasaccharide (XLFG). It is unlikely that these
889
two products were fucosylated on a different Gal unit (e.g.
XFXG and XFLG), because if such subunits existed, they
would be expected to elute from Dionex columns at different times than authentic XXFG and XLFG, just as the two
oligosaccharides XXLG and XLXG elute separately (Buckeridge et al., 1992; Vincken et al., 1995, 1996; Faik et al.,
1997a).
When NXG was the acceptor in these tests, the yield of
labeled XXFG was much lower than the yield of XLFG (1:8,
respectively; Fig. 2), as predicted above. This reflects the
relative concentrations of precursor subunits in reserve
NXG (XXLG ,, XLLG; see Fig. 1). There was no sign of a
third fucosylated product that might have formed from
XLXG, the main octasaccharide in NXG. In TXG, however,
the precursor octasaccharide for XXFG was predominant,
and with this as an acceptor, the XXFG:XLFG ratio (2:3; Fig.
2) reflected a much higher yield of nonasaccharide. It is
concluded that nasturtium XG fucosyltransferase preferentially transferred Fuc to the Gal residue closest to the
unsubstituted Glc of the oligosaccharides XXLG or XLLG,
and that the yields of nonasaccharide:decasaccharide depended on the relative availability of the appropriate precursor subunits in the XG acceptor.
XG Deposition during Nasturtium Fruit Development
Figure 2. Incorporation of label from GDP-[14C]Fuc into XG subunits
by fucosyltransferase from developing nasturtium fruits. 14C products
insoluble in 67% ethanol that were formed by Chaps extract of
particulate membrane in the presence of storage NXG or TXG were
hydrolyzed by Trichoderma sp. cellulase to generate labeled oligosaccharides subsequently isolated by fractionation through a Bio-Gel
P2 column. Individual oligosaccharides were resolved by HPLC
through a CarboPac PA-100 column. Arrows indicate the relative
retention times of subunit oligosaccharides in storage NXG and TXG
compared with those in fucosylated structural wall XG. 14C was
determined in fractions as they eluted from the PAD apparatus.
Figure 3 shows the fresh and dry weights of whole fruits
measured at daily intervals from when they first emerged
to when they abruptly stopped expanding and began senescing. A plot of dry versus fresh weight (inset) indicates
a point about midway into the period of most rapid expansion when the slope increased, i.e. at approximately 500 mg
fresh weight per fruit. This corresponds to 22 to 23 d after
anthesis, and is attributable to relatively rapid increments
of dry weight (storage reserves) after this time.
Electron micrographs of fruit sections that were approaching half-maximum size (400 mg fresh weight per
fruit, 21 d after anthesis; Fig. 4A) show cells from the fleshy
part of cotyledons that are fully vacuolated with typical
primary walls but no periplasmic XG. Two days later (600
mg fresh weight per fruit; Fig. 4B), cells contained many
fragmented vacuoles in the process of developing into
protein bodies (see also Hoth et al., 1986), plus prodigious
thickenings of electron-dense material between the plasma
membrane and wall. At a higher magnification (Fig. 4, C
and D), the Golgi and ER configurations were often seen
near these periplasmic thickenings. Adjacent cells generally deposited periplasmic accretions on opposite sides of
the primary wall (Fig. 4, B–D). Regions in which visible
plasmodesmata traverse adjacent walls seemed to be the
last to be invaded by the deposits. Periplasmic XG and
protein-body formation took place exactly when the major
increase in dry weight was observed (Fig. 3). A few days
later, when cells were fully grown, the extracellular deposits were even more extensive, completely covering most
intercellular connections, and preventing contact between
any part of the plasma membrane and wall. The primary
wall buried underneath the periplasmic thickenings tended
to swell and lose definition (Fig. 4, B and D) compared with
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Desveaux et al.
Plant Physiol. Vol. 118, 1998
Figure 3. Increases in fresh and dry weights of
nasturtium fruits during development. In a plot
of dry/fresh weight (inset), the slope increases at
a time when fruits are about one-half of their full
size, which is exactly when periplasmic XG and
protein bodies begin to be deposited (see Fig. 4).
the typical compact and cohesive walls observed when
they and the plasma membrane were in contact.
Fragmented vacuoles often contain electron-transparent
crystals (see Fig. 4B) that are also visible in light micrographs as irregular clear deposits within Coomassie bluestained protein bodies (Hoth et al., 1986). These inclusions
are probably insoluble salts (e.g. Ca21 phytate), which are
commonly found in seeds as a storage component confined
to protein bodies (Loewus, 1982; Bewley and Black, 1994).
This is a potential source of divalent ions that precipitated
the substrate GDP-[14C]Fuc, which was observed during
incubations with particulate nasturtium extracts.
XG:Fucosyltransferase Activity during Nasturtium
Fruit Development
XG-dependent fucosyltransferase activity recovered in
two sequential detergent washes of particulate fractions
from whole fruits was measured from the early growing
period (16–22 d after anthesis), before periplasmic XG appeared in cotyledons, to the period of continued growth
and the beginning of maturation, when the rate of periplasmic XG deposition was maximal (Figs. 3 and 4). The results
(Table I) show that dry weight increased most markedly
after the fruits reached an average fresh weight of about
400 g (22–24 d after anthesis). The recovery in both extracts
of fucosyltransferase activity per unit fresh weight of fruit,
assayed in the presence of added TXG, peaked before
this time and then declined during the surge of NXG
deposition.
Direct tests were carried out to determine whether any
part of the relatively low levels of XG-dependent fucosyltransferase activity that were recovered from fruits during
the last half of their development (Table I) was derived
from cotyledons. Fruits were selected at sizes at which they
had either not quite begun to deposit or were actively in
the process of depositing NXG. Enveloping pericarps were
separated manually from seeds, which were then dissected
into integuments (discarded) and the remaining embryonic
tissue (mainly cotyledons). Pericarps and cotyledons were
weighed, homogenized, and particulate membrane frac-
tions were extracted with detergent to produce separate
enzyme preparations for comparison of XG-dependent fucosyltransferase activity. The results (Table II) show that
extracts of cotyledons from 400-mg fruit were about 10
times richer in XG:fucosyltransferase activity on a freshweight basis than extracts of the much larger pericarp.
Chromatography (Bio-Gel P2) confirmed that the product formed by cotyledons was entirely digested by cellulase to labeled XG oligosaccharide. When whole fruits increased from 400 to 600 and 800 mg fresh weight, the
cotyledons expanded 7- and 12-fold, respectively. This
must have depended in part on cell division in the meristem followed by cell expansion, because the increase observed in average cell size of the fleshy part of the cotyledons (see Hoth et al., 1986; Fig. 4, A and B) was not
sufficient by itself to explain such a great increase in fresh
weight. During these developments the cotyledons lost
most of their XG:fucosyltransferase activity per unit fresh
weight. As observed with whole fruits (Table I), there was
no sign of any burst of this activity when NXG began or
continued to be deposited, whether calculated per unit
fresh weight or per fruit (Table II). By comparison, the
pericarp tissue was nearing the end of its growing period
during these tests, and fresh weight only increased by
about 40%. XG:fucosyltransferase activity from the pericarp, calculated per unit fresh weight or per fruit, increased
slightly and then declined.
DISCUSSION
Our results (Figs. 1–4) demonstrate that nasturtium fruits
deposit two kinds of XG during their development to maturity: storage XG specifically into periplasmic spaces of
cotyledon cells, and structural XG into growing primary
walls. Structural XG is fucosylated in the subunits XLFG
and XXFG, which make up 30 and 18 mol %, respectively,
of all subunits; the others are heptasaccharide (XXXG) and
two octasaccharides (XXLG and XLXG). The absence of
digalactosylated nonasaccharide (XLLG) in this wall preparation is notable, for it indicates that there was no contamination of the extracted wall XG by storage XG, in
Xyloglucan:Fucosyltransferase in Developing Nasturtium Fruits
891
Figure 4. Transmission electron micrographs of nasturtium cotyledon parenchyma examined when fruits were about
half-maximum size (400–600 mg per fruit). Seeds were sectioned, fixed, postfixed in OsO4, embedded in Spurr’s epoxy
resin, and thin sections (700 Å) of cotyledon tissues were stained with uranium and lead as described in “Materials and
Methods.” At 22 d after anthesis (A), no periplasmic deposits were detected, but by 24 d (B), deposits filled the spaces
between the plasma membrane and the primary wall (periplasm) to a thickness many times that of the walls. Most deposits
were first visible as accretions localized in adjacent cells on opposite sides of the wall (C). The walls under these deposits
were swollen and contained microfibrils that were visibly dispersed (D). c, Cytoplasm; fv, fragmented vacuole; G, Golgi;
nxg, periplasmic XG; pd, plasmodesmata; pw, primary wall; s, starch; and v, vacuole. Bars in C and D 5 1 mm.
which this is the most abundant subunit (Fig. 1). When the
fruits were about half-maximum size, cotyledons suddenly
began to deposit storage NXG, which contains relatively
more Gal than wall XG but no Fuc. Detergent extracts of
particulate membranes from whole nasturtium fruits had
the enzymic capacity to fucosylate storage XG to form the
same subunits that are found in structural XG. To our
knowledge, this is the first published report that identifies
892
Desveaux et al.
Plant Physiol. Vol. 118, 1998
Table I. Yields of XG-dependent fucosyltransferase activity in detergent extracts of particulate membranes of developing nasturtium fruits
Whole fruits were harvested over a period corresponding to 16 to 27 d after anthesis, which encompassed the time when growth was most
rapid and the age (21–23 d after anthesis, 400 – 600 g fresh weight per fruit; Figs. 3 and 4) when periplasmic XG began to be deposited in
cotyledons. Particulate pellets prepared by centrifugation (100,000g) of filtered homogenates were extracted twice in sequence with 0.3% and
0.4% Chaps detergent as indicated, and the two solubilized extracts were assayed for fucosyltransferase activity in reaction medium with or
without added TXG, as described in “Materials and Methods.” 14C was measured in products soluble in 10% TCA but insoluble in 67% ethanol.
The effect of TCA was to dissociate insoluble salts of the labeled substrate and render them soluble in ethanol, thereby reducing artifactual 14C
in controls to less than about 600 dpm h21 g21 fresh weight in all reactions.
Days after
Anthesis
Weight
Fresh
XG-Dependent Fucosyltransferase Activity
Dry
0.3% Chaps
mg fruit21
16
18
22
24
27
48
153
416
665
927
0.4% Chaps
dpm h21 g21 fresh wt
5
16
43
135
185
XXFG and XLFG as the products formed in vitro by fucosyltransferase activity from any plant source. The level of
this XG:fucosyltransferase activity per unit fresh weight is
highest in the youngest nasturtium fruits (Table I), especially cotyledons (Table II) before they develop the capacity
to form NXG. It then diminishes but does not disappear.
Thus, the question is what function(s) the XG:fucosyltransferase activity may have in nasturtium cotyledons, which
are especially known for their capacity to generate relatively large amounts of nonfucosylated storage XG.
Several observations in this study mitigate against any
requirement for the transitory fucosylation of storage XG
as a signal to facilitate its secretion. First, no Fuc was
detected in subunits of NXG (Fig. 1). Second, there was no
increase in XG:fucosyltransferase activity that coincided
with the burst of NXG biosynthesis (Tables I and II; compare with Fig. 4). Third, NXG was a relatively ineffectual
fucosyl acceptor for the nasturtium transferase, probably
because this enzyme preferentially fucosylated the octasaccharide subunit XXLG (Fig. 2), which is only a minor
constituent of NXG (Fig. 1). Finally, no a-fucosidase activity was detectable in nasturtium fruits, which could have
hydrolyzed Fuc from an XG precursor to form NXG. It is
concluded that nasturtium fruit XG:fucosyltransferase does
not act to fucosylate, even temporarily, any XG that is
destined for storage in periplasmic spaces.
12,100
18,800
13,700
7,100
6,800
8500
9400
8100
5300
3500
If we assume, therefore, that XG:fucosyltransferase activity only functions to catalyze the last step of biosynthesis of XG that is destined for integration into the primary wall, there are two possible uses for the reduced
levels of enzyme activity observed (Table II) after NXG
starts to fill periplasmic spaces. Cotyledons increase in
size at least 10-fold while this is happening (Table II), cell
division must continue for a time, and primary walls must
extend to keep pace. XG:fucosyltransferase would be expected to be active in new growing cells that coexist with
maturing cells that deposit NXG. In maturing cells it is
unlikely that any structural XG that might be formed
could diffuse through the periplasmic accretions to reach
the wall, although it could be directed to and secreted
near the shrinking regions of the plasma membrane,
where intercellular connections still remain. It is not surprising that these are the only parts of the primary wall
that continue to show a compact form and borders that
are as well defined as they were in earlier stages of
growth. Walls buried under NXG swell, and it is difficult
to discern their edges. Microfibrils visibly separate from
one another (Fig. 4D) as if they were being pulled apart
and are no longer tethered by sufficient structural XG or
other matrix-binding agents that are required to maintain
the integrity of the wall framework.
Table II. Recovery of XG-dependent fucosyltransferase activity in detergent extracts of particulate membranes from excised nasturtium cotyledons and pericarp
Whole fruits, weighing approximately 400, 600, or 800 mg each, were dissected on an iced tray to separate seeds minus integuments (mainly
cotyledons) from the fleshy protective pericarp. These were weighed, homogenized, filtered, centrifuged, and extracted once with 0.4% Chaps,
as described in “Materials and Methods.” Fucosyltransferase activity was assayed in the detergent extracts with or without added TXG and
expressed per unit fresh weight.
Fruits
Fresh Wt
Cotyledons
XG-Dependent Fucosyltransferase Activity
Pericarp
Cotyledons
mg fruit21a
400
600
800
a
13
92
153
Pericarp
dpm h21 g21 fresh wt
364
432
518
33,100
12,400
4,300
3400
5100
1700
The sum of values for cotyledons and pericarp do not add up to the initial weights of whole fruits because integuments (seed coats) were
discarded.
Xyloglucan:Fucosyltransferase in Developing Nasturtium Fruits
The basic problem for future research that is raised by
this study is to understand the mechanism of how maturing cotyledon cells suddenly increase the rate of XG biosynthesis and channel almost all of it into secretion of NXG,
effectively avoiding the final fucosylation step reserved for
biosynthesis of structural XG. It appears that nascent NXG
is processed in the secretory machinery only to the point at
which it is well galactosylated, and then it exits in traffic
directed toward quiescent regions of the plasma membrane, where periplasmic XG is secreted. NXG deposits
particularly on all sides of intercellular spaces and on
opposite sides of the primary walls of adjacent cells, where
there are no plasmodesmata (Fig. 4). The factors that trigger and regulate this diversion are unknown. Perhaps maturing cotyledon cells develop a block between the steps
that catalyze galactosylation of XG and terminal fucosylation, as is observed when the fungal antibiotic brefeldin A
is added to actively secreting plant cells. Driouich et al.
(1993) reported that suspension-cultured cells of sycamore
react to brefeldin A by blocking transfer of XG via vesicles
from the trans Golgi cisternae to the trans Golgi network,
with the result that fucosylation is inhibited but XG biosynthesis is not, and truncated XG accumulates in secretory
vesicles.
In any event, because nasturtium cotyledons do not extinguish the capacity to form wall XG when the biosynthesis of periplasmic XG becomes the predominant pathway,
XG fucosylation may continue in a transition period in a
few cells that are slow to differentiate or in a few strategically located Golgi configurations. It remains to be established whether storage and structural XG are ever assembled in the same Golgi at the same time.
ACKNOWLEDGMENT
We thank Kathy Hewitt (Electron Microscopy Centre, Biology
Department, McGill University) for preparing the electron
micrographs.
Received June 1, 1998; accepted August 3, 1998.
Copyright Clearance Center: 0032–0889/98/118/0885/10.
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