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Plant Physiol. (1998) 116: 337-347
Identification and Partial Characterization of the Pectin
Methyltransferase "Homogalacturonan-Methyltransferase" from
Membranes of Tobacco Cell Suspensions1
Florence Goubet,
Leona N. Council, and
Debra Mohnen*
Complex Carbohydrate Research Center and Department of Biochemistry
and Molecular Biology, University of Georgia, 220 Riverbend Road,
Athens, Georgia 30602-4712
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ABSTRACT |
A membrane preparation from tobacco
(Nicotiana tabacum L.) cells contains at least one
enzyme that is capable of transferring the methyl group from
S-adenosyl-methionine (SAM) to the C6 carboxyl of
homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from
methyltransferases that catalyze methyletherification of the pectic
polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A
trichloroacetic acid precipitation assay was used to measure HGA-MT
activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead
to high and variable background radioactivity in the product pellet.
Attempts to reduce the incorporation of the 14C-methyl
group from SAM into pectin by the addition of the alternative methyl
donor 5-methyltetrahydrofolate were unsuccessful, supporting the role
of SAM as the authentic methyl donor for HGA-MT. The pH optimum for
HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM
was 38 µm, and the maximum initial velocity was 0.81 pkat
mg 1 protein. At least 59% of the radiolabeled product
was judged to be methylesterified homogalacturonan, based on the
release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The
released radioactivity eluted with a retention time identical to that
of methanol upon fractionation over an organic acid column. Cleavage of
the radiolabeled product by endopolygalacturonase into
fragments that migrated as small oligomers of HGA during thin-layer
chromatography, and the fact that HGA-MT activity in the membranes is
stimulated by uridine 5 -diphosphate galacturonic acid, a substrate for
HGA synthesis, confirms that the bulk of the product recovered from
tobacco membranes incubated with SAM is methylesterified HGA.
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INTRODUCTION |
The cell wall gives shape to cells and plays critical roles in
plant development (Carpita and Gibeaut, 1993 ). Primary cell walls,
those walls surrounding growing plant cells, are composed mainly of the
polysaccharides cellulose, hemicellulose, and pectin. Three classes of
pectin have been detected in plant cell walls: HGA, RG-I, and RG-II
(Jarvis, 1984; O'Neill et al., 1990). HGA, the most abundant pectic
polysaccharide, is a linear homopolymer of  -1,4-linked
d-galacturonic acid that is partially derivatized by
methylesterification at C-6, by acetylation at the C-2 or C-3 hydroxyl
(De Vries et al., 1986; Ishii, 1995), and in some plants by
xylosylation (De Vries et al., 1986; Schols et al., 1995).
The degree of methylesterification of HGA varies during cell culture,
and it is believed that the amount and pattern of HGA methylation is
important for wall function in growth and development (Jarvis et al.,
1988; Schaumann et al., 1993). This belief is supported by the
observation that HGA in the walls of young cells is highly
methylesterified, whereas HGA in the walls of older cells has a lower
degree of esterification (Schaumann et al., 1993). The differences in
the degree of methylesterification of pectins are believed to be
controlled by the activities of PMT in the Golgi apparatus (Vannier et
al., 1992) and PME in the cell wall (Gaffe et al., 1992).
PMT activity has previously been identified in mung bean
(Phaseolus aureus L.) seedlings (Kauss et al., 1969). The
fact that the PMT from mung bean catalyzed the transfer of
14CH3 from SAM to produce a
product that released [14C]methanol after
treatment with PME or base (Kauss et al., 1967) provided good evidence
that the described enzyme methylated HGA. Furthermore, the rate of PMT
activity in mung bean membranes increased in the presence of UDP-GalUA,
suggesting that HGA synthesized in the membranes was the methyl
acceptor (Kauss and Swanson, 1969). However, no
sensitivity of the methylated product to cleavage by EPGase was
reported and no subsequent work on the solubilization and purification
of the mung bean PMT has been published. Putative PMT activity has also
been detected in flax (Linum usitatissimum L.) hypocotyls
(Vannier et al., 1992) and flax suspension-cultured cells (Schaumann et
al., 1993). The PMT in flax was shown to synthesize a product from
which [14C]methanol was released after
treatment with a high concentration of base (1 m NaOH)
(Vannier et al., 1992). However, further studies to prove that the
enzyme transfers 14CH3 from
SAM specifically to HGA have not been reported. Recently, the PMT from
flax was partially purified and characterized, but the specificity of
this enzyme for the type of pectin substrate methylated was not
ascertained (Bruyant-Vannier et al., 1996). The fact that the
solubilized flax enzyme is activated by exogenous PGA supports its
identity as a transferase that methylates homogalacturonan (Bruyant-Vannier et al., 1996).
Bourlard et al. (1995) observed that different types of pectins (e.g.
rhamnogalacturonan versus HGA) stimulate the incorporation of methyl
groups into pectin at different pH optima. Therefore, it is possible
that unique PMTs exist that specifically methylate the different pectic
polysaccharides HGA, RG-I, and RG-II to form methylesters or
methylethers. Examples of methyl etherification include
2-O-methyl Xyl and 2-O-methyl Fuc in RG-II
(Darvill et al., 1978; O'Neill et al., 1996) and 4-O-methyl
GlcUA in a side branch of RG-I (An et al., 1995). Thus, unique
methyltransferases must exist that incorporate methyl groups into these
different pectic polysaccharides.
We report here the identification and characterization, in membranes
from tobacco (Nicotiana tabacum L.) cell suspensions, of a
PMT that methylates HGA, and we refer to this enzyme as HGA-MT. Tobacco
cell suspensions were used, because the enzyme that synthesizes HGA,
PGA-GalAT, has been identified and studied in these cells (Doong et
al., 1995). A comparison of the characteristics of the HGA-MT from
tobacco with the PMTs previously described from flax (Vannier et al.,
1992) and mung bean (Kauss et al., 1969) is also presented.
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MATERIALS AND METHODS |
Chemicals
The chloride salt of SAM, the ammonium salt of UDP-GalUA, pectins
of 31 to 93% degree of esterification, and 5-methyltetrahydrofolic acid were purchased from Sigma. Polygalacturonic acid was purchased from Sigma and ICN. Dextran standards were purchased from Pharmacia. Fluoral-P (4-amino-3-penten-2-one) was purchased from Acros
(Pittsburgh, PA). [14C]Methyl SAM (specific
activity, 55 mCi/mm) was purchased from American
Radiolabeled Chemicals (St. Louis, MO) and Amersham.
Plant Material
Tobacco (Nicotiana tabacum L. cv Samsun)
cell-suspension cultures were originally isolated from pith callus
tissue (Eberhard et al., 1989). The cells were grown on Murashige and
Skoog medium supplemented with 4.5 µm 2,4-D and 90 mm Suc, and subcultured every 12 d (Doong et al.,
1995).
Preparation of Membranes from Tobacco Cell-Suspension
Cultures
Membranes were prepared by a modification of the method of
Villemez et al. (1966). Three- to 4-d-old tobacco cells (75 g) were
collected by filtration and homogenized with a polytron in 100 mL of
grinding buffer (50 mm Tris-HCl, pH 7.3, 0.4 m
Suc, 1% [w/v] BSA, and 1 mm EDTA). The homogenate was
strained through a nylon cloth (50-µm pore size) and the filtrate
centrifuged at 3,500g for 15 min. The supernatant was
centrifuged at 100,000g for 1 h to yield a membrane
pellet and the pellet was resuspended in 5 mL of storage buffer (0.4 m Suc, 50 mm Tris-HCl, pH 6.8). HGA-MT activity
was measured in membranes (4-6 mg mL1 protein)
that were either assayed immediately after preparation or frozen and
stored at  80°C until use. Protein content was determined using a
Bradford assay (Bradford, 1976) with BSA as a standard.
HGA-MT Assay
The HGA-MT assay was a modification of that previously described
by Kauss and Hassid (1967). The membranes (25 µL, 100-150 µg of
protein) were incubated in 25 µL of reaction buffer (50 mm Tris-Mes, pH 8.5, 8 µm
[14C]methyl SAM [0.01 µCi], and 12 µm SAM) at 30°C for times ranging from 5 min to 4 h. The reaction was stopped by the addition of 50 µL of 20% TCA to
precipitate the methylated products. These mixtures were centrifuged
for 5 min at 4000g. Unincorporated SAM was removed by
washing the pellets twice with 200 µL of 2% TCA. The washed pellets
were resuspended in 300 µL of water, and the radioactivity
incorporated into the product was measured by liquid-scintillation counting using Scintiverse BD scintillation cocktail (Fisher
Scientific).
Chemical Extraction of Radiolabeled Product
The pellets obtained after TCA precipitation were partially
solubilized with boiling water, 0.5% boiling EDTA, 0.5% boiling ammonium oxalate, 0.5 m imidazole-HCl (pH approximately
6.0) at 25°C, or 0.1 n NaOH at 25°C. After one
treatment with 0.5% boiling ammonium oxalate, the pellets were further
treated with 0.5% boiling ammonium oxalate containing 1% Triton
X-100. All of these treatments were performed for 1 to 2 h, except
for the treatment with NaOH, which was performed for 4 to 12 h. After
treatment the suspensions were centrifuged and the amount of
radioactivity in the supernatant and pellet was measured. Uronic acids
were measured by a meta-hydroxybiphenyl assay, as adapted from
Blumenkrantz and Asboe-Hansen (1973).
PME Assay
PME activity was detected by the production of methanol
(Wojciechowski and Fall, 1996). The assay was modified by incubating 25 µL of membranes, 610 µg of 93% esterified pectin, 90 µg of Fluoral-P, 4 units of alcohol oxidase (ICN), and 153 mm
KH2PO4, pH 6.0, in the
absence or presence of 0.01% Triton X-100. The change in optical
density at 405 nm indicated the reaction of formaldehyde (produced by
the oxidation of methanol by alcohol oxidase) with
4-amino-3-penten-2-one (Fluoral-P) to yield
3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine and was a measure of PME
activity (Wojciechowski and Fall, 1996).
EPGase and PME Digestion of Product
Methylated product was demethylesterified and/or hydrolytically
cleaved by complete digestion with a cloned Aspergillus
aculeatus PME expressed in Aspergillus oryzae
(Christgau et al., 1996) (gift of Hans Peter Heldt-Hansen, Novo
Nordisk, Bagsvaerd, Denmark) and/or an EPGase from Aspergillus
niger that were purified from the culture filtrates (gifts of Carl
Bergmann, Complex Carbohydrate Research Center, Athens, GA). Methylated
product was resuspended in 200 µL of 50 mm sodium
acetate, pH 5.0, and treated with 1 to 4 units of EPGase and/or PME for
4 to 12 h at 30°C. The reaction products were analyzed by TLC or
HPLC.
TLC of 14C-Labeled Products
TLC was performed using precoated TLC plates (Silica Gel 60 WF254, EM Science, Gibbstown, NJ) run vertically
in water:isopropanol:hexyltriethylammonium phosphate (2:1:0.02, v/v)
(Q6 ion pair cocktail, Regis Chemical Co., Morton Grove, IL).
Radiolabeled product was visualized by exposing the TLC plates to
storage phosphor screens (Molecular Dynamics, Sunnyvale, CA). The
exposed screens were autoradiographed using a phosphor imager (model
425F, Molecular Dynamics). Sugars were detected by spraying the plates
with orcinol reagent (Sigma).
HPLC of 14C-Labeled Products
Radiolabeled methanol released from the
14C-labeled product by treatment with PME or NaOH
was collected by distillation and separated over a Rezex ROA-organic
acid column (Phenomenex, Torrance, CA) in water by HPLC using a
chromatography system (flow rate, 0.6 mL min1;
DX 500, Dionex, Sunnyvale, CA). Nonradiolabeled methanol was detected
by pulsed amperometric detection with postcolumn addition of 400 mm NaOH (approximately 0.2 mL
min1). Fractions containing radioactivity were
measured by liquid-scintillation counting using Scintiverse BD
scintillation cocktail. Samples were filtered through 5000 molecular
weight pore-size microfilterfuge tubes before chromatography. The
amperometric detector was operated with the following pulse sequences:
E1 = 0.05 V (duration, 400 ms); E2 = 0.75 V (duration, 210 ms); and E3 = 0.15 V (duration, 390 ms). The sampling period was
200 ms and the response time was 1 s.
Size-Exclusion Chromatography of 14C-Labeled Products
Radioactive product solubilized by treatment of the pellet for
1 h with 0.5% ammonium oxalate (oxalate fraction) was dialyzed against water at 4°C for 24 h, and the resulting dialysate was lyophilized, resuspended in water (100 µL, 1600 cpm), spin filtered, and separated over a Superose 12 HR 10/30 fast-protein liquid chromatography size-exclusion column (Pharmacia) in 50 mm
sodium acetate, pH 5.0, and 10 mm EDTA (flow rate, 0.48 mL
min1). Fractions (0.48 mL) containing
radioactivity were measured by liquid-scintillation counting using
Scintiverse BD scintillation cocktail. The elution times of dextran
molecular mass standards, pectin, and PGA were determined by pulsed
amperometric detection.
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RESULTS |
Establishment of an Assay for HGA-MT
Precipitation of Methylated Product
A major obstacle to the development of an assay for the
HGA-MT-catalyzed methylation of HGA by [14C]SAM
was the nonspecific binding of SAM to the product and the resulting
high background radioactivity. Vannier et al. (1992) previously
reported that the precipitation of methylated pectins in 95% ethanol
and subsequent washing of the product with 1 m NaCl in 60%
ethanol was successful in decreasing [14C]SAM
background. In our hands, however, these conditions were not sufficient
to reduce background cpm, and the resulting pellets had very high
backgrounds and variable cpm (Table I).
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Table I.
Comparison of different procedures for the
precipitation and washing of 14C products
Membranes were incubated with [14C]SAM, and the reaction
was stopped by the addition of 50 µL of 10 to 50% TCA, 20% TCA-65% ethanol, 4 n LiCl or 8 m guanidine or 450 µL
of 95% ethanol or methanol:chloroform (1:1, v/v). Each pellet was
washed twice with 200 µL of 2 to 20% TCA, methanol:chloroform (1:1,
v/v), 65% ethanol, 65% ethanol-1 m NaCl, 65% ethanol-2%
TCA, 2 n LiCl, or 4 m guanidine. N, No
activity; S, same value (1000-1500 cpm) as in the standard conditions;
L, low incorporation (<100 cpm) compared with standard conditions; H,
very high background; V, variable results from experiment to
experiment. The boxed S indicates the selected standard method to
analyze the HGA-MT activities.
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We tested the efficacy of a number of different treatments to
precipitate methylated products produced in tobacco membranes and to
remove unincorporated [14C]SAM. The treatments
included methanol:chloroform (1:1, v/v), as used for the precipitation
of HGA (Doong et al., 1995); 4 n LiCl and 8 m
guanidine, as used for precipitation of DNA or RNA (Ausubel et al.,
1996); and TCA, as used for the precipitation of proteins (Ausubel et
al., 1996) and of some polysaccharides (Kauss and Hassid, 1967). The
use of 20 to 50% TCA for precipitation was the only system that
resulted in the recovery of methylated compounds. The lowest background
and the most reproducible cpm were achieved by washing the pellet twice
with 2% TCA after precipitation. This assay method is most similar to
that of Kauss and Hassid (1967). Table II
shows the recovery of radioactivity in the pellet at each step of the
HGA-MT assay. No incorporation of radioactivity into the pellet above
background levels was obtained in control reactions containing the
buffer and boiled or heat-treated membranes (Table II). Thus, the
incorporation of radioactivity detected using the HGA-MT assay has the
characteristics expected for an enzyme-catalyzed reaction.
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Table II.
The recovery of radiolabeled product after each
step of the HGA-MT assay
Membranes were incubated with [14C]SAM (18,000 cpm) for
30 min at 30°C. The reaction was stopped by the addition of 50 µL
of 20% TCA and the pellet was washed twice with 2% TCA. The
background cpm are those recovered when TCA was added to reaction
buffer before the addition of enzymes (i.e. a time-0 control). The cpm recovered from control reactions containing membranes pretreated by
boiling for 5 min or heating at 60°C for 1 h are also shown. The
results are the average cpm ± sd from duplicate
samples from two or three independent experiments.
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Nature of the Methyl Donor
The methylation of HGA occurs by the transfer of a methyl group
from a methyl donor to HGA. The methyl donor SAM has been reported to
be the donor for HGA-MT (Kauss et al., 1967; Vannier et al., 1992).
However, the possibility exists that other methyl donors such as
5-methyltetrahydrofolate may be the direct donor. To test this
possibility the amount of the 14C-methyl group
incorporated into the product in reactions containing 10 µm [14C]SAM in the presence and
absence of 10 µm nonradiolabeled 5-methyltetrahydrofolate was determined. There was no decrease in the amount of radioactivity (98 ± 6% of controls, average ± se from
duplicate samples from two experiments) incorporated into the product
in the presence of 5-methyltetrahydrofolate, indicating that
5-methyltetrahydrofolate is not a methyl donor for HGA.
Kauss and Hassid (1967) obtained comparable results using radiolabeled
methyltetrahydrofolate. We conclude that SAM is the direct methyl donor
for the HGA-MT studied here.
Characterization of HGA-MT
Initial attempts to identify HGA-MT activity using membranes
prepared from tobacco by the method of Doong et al. (1995) did not
generate detectable HGA-MT activity. Membranes containing detectable
HGA-MT activity were obtained by the method of Vannier et al. (1992).
It was determined that the homogenization and storage buffers used by
Doong et al. (1995) contained inhibitors of membrane-bound HGA-MT
(0.1%  -mercaptoethanol, 25% glycerol, and 25 mm KCl)
that did not allow the detection of the HGA-MT activity.
Effect of Cations on HGA-MT Activity
The presence of 25 mm KCl in the homogenization buffer
inhibited HGA-MT activity by 40%. An inhibition of mung bean PMT by 2.5 mm KCl has been reported by Kauss and Hassid (1967).
The presence of 0.1 to 25 mm MgCl2
did not affect the incorporation of methyl groups into product by
microsomal membranes, as previously demonstrated for mung bean PMT by
Kauss and Hassid (1967). There was also no effect of 0.1 to 50 mm MnCl2 on HGA-MT activity.
Effect of Temperature on HGA-MT Activity
The temperature optimum for HGA-MT is between 25 and 40°C (Fig.
1A). This optimum is comparable to that
of other plant membrane-bound enzymes (McNab et al., 1968; Misawa et
al., 1996). The HGA-MT activity is reduced by more than 50% at
temperatures above 55°C. A thermal-inactivation curve for HGA-MT at
60°C is shown in Figure 1B. HGA-MT undergoes an exponential decay of
activity at 60°C, with a 50% reduction in activity after 5 min and a
complete inactivation after 40 min.
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| Figure 1.
Effect of temperature on HGA-MT activity in
membranes from tobacco cells. A, Temperature-optimum curve. Tobacco
membranes were incubated with [14C]SAM for 5 min at the
temperatures indicated and product was recovered by the HGA-MT assay.
The data represent the average cpm ± sd of product
recovered from duplicate samples from two independent experiments. B,
Thermal-inactivation curve. Tobacco membranes were incubated at 60°C
for the times indicated and then incubated with [14C]SAM
for 5 min at 25°C. Product was recovered by the HGA-MT assay. The
data represent the average cpm from duplicate samples from one
experiment.
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Effect of pH on HGA-MT Activity
The effect of reaction pH ranging from 5.0 to 9.3 on HGA-MT
activity was determined (Fig. 2). A major
peak of activity was obtained at pH 7.8 to 8.0. An apparent minor pH
optimum (7.0-7.3), most apparent in the 5-min reaction, is the same as
the reported pH optimum for flax (Bruyant-Vannier et al., 1996) and
mung bean PMT (Kauss and Hassid, 1967).
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| Figure 2.
Effect of pH on HGA-MT activity in membranes from
tobacco cells. Tobacco membranes were incubated with
[14C]SAM for 5 ( ) or 15 ( ) min in reaction buffer
adjusted to the indicated pH by the appropriate mixture of Tris and Mes
buffers. Product was recovered by the HGA-MT assay. The data represent the average cpm ± sd of product recovered from
duplicate samples from three to seven independent experiments.
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Effect of Triton X-100 on HGA-MT
Triton X-100 was used to permeabilize the membranes in an attempt
to increase HGA-MT activity by increasing the amount of SAM accessible
to the enzyme. The HGA-MT activity was not modified in the presence of
0.01% Triton X-100, a concentration commonly used to permeabilize
membranes (Goubet et al., 1994). When a high concentration of 0.1%
Triton X-100 was used, HGA-MT activity was inhibited by 30%.
Time Course of PME and HGA-MT Activities in Membranes
Kauss et al. (1969) and Bruyant-Vannier et al. (1995) have
reported that PME activity is present in microsomal membranes isolated from mung bean and flax. We therefore tested whether any PME was present in the tobacco membranes. Tobacco cell suspensions were harvested 0, 2, 3, 4, 6, 9, 12, and 16 d after transfer of cells to fresh media, and membranes were isolated and analyzed for PME and
HGA-MT activities (Table III). PME
activity was detected in membranes prepared from 0- and 6- to 12-d-old
cells. The PME may represent PME from the cell wall that bound to the
membranes during tissue homogenization. Alternatively, PME may be
located inside the Golgi apparatus or ER. To test whether a significant
proportion of PME activity was located inside the membrane vesicles,
membranes were permeabilized by treatment with 0.01% Triton X-100 and
PME activity was measured. Permeabilization of the membranes did not result in an increase in PME activity. Thus, no direct evidence for the
existence of PME within the membrane vesicles was obtained.
The highest HGA-MT activity was expressed at 2 to 4 and 9 to 12 d
after the transfer of the cells (Table III). The activity observed
during the lag phase has been described for flax cells (Schaumann et
al., 1993); however, a second optimum later during culture has not been
reported. Based on results from at least 20 experiments, the average
HGA-MT activity in membranes from 2- to 4-d-old cells is 0.14 ± 0.02 pmol s1 g1 cell.
The HGA-MT activity from 9- to 12-d-old cells is more variable (0.11 ± 0.07 pmol s1
g1 cell). This variability can be explained by
the presence of PME in the membranes of these cells (Table III). An
attempt was made to increase HGA-MT activity by permeabilizing
membranes in the presence of the putative exogenous acceptor PGA.
Incubation of membranes in the presence of 0.01% Triton X-100, with or
without PGA, caused no increase in HGA-MT activity (data not shown).
Higher concentrations of Triton X-100 (0.1%), with or without PGA,
inhibited HGA-MT by 30% (data not shown). These results suggest that
the endogenous acceptor may be present in excess and thus exogenous pectin does not increase HGA-MT activity. The standard HGA-MT assay is
performed using 3- to 4-d-old cells in the absence of permeabilizing
agents.
Reaction Kinetics
A time course of the incorporation of 14C
into the product is shown in Figure 3.
The rate of the reaction is linear during the first 10 to 15 min. The
initial velocity is 0.17 pkat mg1 protein,
which is significantly greater than the initial velocity of 0.005 pkat
mg1 protein reported for PMT from flax
(Bruyant-Vannier et al., 1996). The amount of HGA-MT activity at 60 min
at 30°C was proportional to the amount of membrane protein assayed
through 40 µg of protein per reaction. The apparent
Km of HGA-MT for SAM is 38 µm, and the Vmax ± sd is 0.81 ± 0.05 pkat mg1
protein at pH 7.8 (Fig. 4). The
Vmax of 0.045 pkat
mg1 protein reported for the PMT from mung bean
(our calculation from data reported by Kauss et al. [1969]) is lower
than the Vmax for HGA-MT from tobacco. The
reported apparent Km of 30 µm
SAM for PMT from flax cells (Bruyant-Vannier et al., 1996) and 60 µm SAM for PMT from mung bean (Kauss and Hassid, 1967)
are similar to the value reported here for HGA-MT in tobacco.
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| Figure 3.
Time course of the incorporation of the
14C-methyl into precipitable products. Membranes were
incubated with [14C]SAM (18,000 dpm) and product was
recovered by the HGA-MT assay. The data represent the average cpm ± sd of product recovered from duplicate samples from
three independent experiments.
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| Figure 4.
Hanes-Woolf plot of the kinetics of methyl
incorporation into precipitable product in tobacco cell membranes.
[SAM]/V0 (initial velocity) is the
concentration of SAM (µm) divided by
V0 (pmol methyl incorporated
s1 mg1 protein). Membranes were incubated
with [14C]SAM for 15 min and the product was recovered by
the HGA-MT assay. The data represent the average of duplicate samples
from two independent experiments.
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Effects of Different Potential Oligosaccharide and
Polysaccharide Substrates on HGA-MT Activity
To determine whether HGA-MT activity was dependent on a
PGA-GalAT-catalyzed synthesis of homogalacturonan (i.e. stimulated by
UDP-GalUA) or dependent on exogenous HGA or pectin substrates, we
tested whether the addition of UDP-GalUA, OGA, or pectin could stimulate HGA-MT activity. The addition of OGA, PGA, or pectin with
different degrees of esterification had no effect on the HGA-MT
activity (data not shown). A similar lack of stimulation by PGA or
pectin was also observed for PMT from mung bean (Kauss and Swanson,
1969) and flax (Bruyant-Vannier et al., 1996). The lack of stimulation
of HGA-MT by the pectic oligosaccharides and polysaccharides was
observed, regardless of whether the membranes were permeabilized by
0.01% Triton X-100 or not. UDP-GalUA, however, stimulated the
incorporation of methyl groups into HGA (Fig.
5). The greatest amount of product was
recovered in the presence of 20 to 50 µm UDP-GalUA. The
activation of tobacco HGA-MT by UDP-GalUA, however, was only 20%
compared with the reported 200% stimulation of the PMT from mung bean
by UDP-GalUA (Kauss et al., 1969). The presence of 0.01% Triton X-100
to permeabilize the membranes did not affect the stimulatory effect of
UDP-GalUA on HGA-MT activity (data not shown).
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| Figure 5.
Effect of UDP-GalUA on HGA-MT activity in tobacco
cell membranes. Membranes were incubated with [14C]SAM
for 15 min and the product was recovered by the HGA-MT assay. The data
represent the average cpm ± sd of product recovered
from duplicate samples from seven independent experiments.
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EDTA, a chelator of calcium ions, solubilizes some pectins (Selvendran
and O'Neill, 1995) and could potentially activate HGA-MT activity by
allowing better access of HGA substrates to HGA-MT. The HGA-MT of
tobacco was inhibited at EDTA concentrations greater than 2 mm, and no activation of HGA-MT was observed at any EDTA concentrations tested (0.1-10 mm). In contrast, Kauss and
Hassid (1967) have shown that 2 mm EDTA activates the PMT
activity from mung bean.
Characterization of the Product of HGA-MT
Radiolabeled Product Is Solubilized from the Pellet by Chemical
Extractions and Enzymatic Hydrolysis
SAM donates methyl groups to DNA, RNA, protein, lipid, and
carbohydrate acceptors (Chiang et al., 1996); therefore, it was necessary to determinate what fraction of the recovered product was
methylated HGA. The total product was treated with boiling water,
boiling EDTA, boiling ammonium oxalate, or imidazole (Table IV) in an attempt to solubilize pectin
from the pellet (Mort et al., 1991; Schaumann et al., 1993; Schols et
al., 1995; Selvendran and O'Neill, 1995). These treatments released at
most 24% of the radioactive product. Similarly, only 24% of the total
product was solubilized by treatment with EPGase, an enzyme specific
for HGA (data not shown).
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Table IV.
Chemical and enzymatic solubilization of total
14C-methylated products
After incubation of the membranes with [14C]SAM, the
methylated products were precipitated by 20% TCA and the pellets
washed twice with 2% TCA. The resulting pellets were treated with
boiling water, 0.5% boiling EDTA, 0.5% boiling ammonium oxalate, or
0.5 m imidazole-HCl (pH 6.0) for 1 to 2 h. The
radioactivity was measured in the supernatant and the pellet, and the
percentage of radioactivity solubilized from the pellet in each
fraction was calculated. The data represent the average
percentage of radioactivity solubilized ± sd from
duplicate samples from 3 to 12 independent experiments.
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Because TCA precipitates polysaccharides, proteins, and lipids, we
reasoned that a mixture of macromolecules was co-precipitated by the
TCA, thus making it difficult to solubilize pectin from the complex
pellet. The pellet was therefore first treated with ammonium oxalate to
solubilize the easily accessible pectin, yielding a supernatant
referred to as the oxalate fraction (Fig.
6). The remaining pellet was then treated
with ammonium oxalate containing 1% Triton X-100 to yield a second
supernatant called the oxalate-Triton fraction (Fig. 6). Triton X-100
was used to solubilize any protein-lipid-polysaccharide complex present
in the pellet. The combined oxalate and oxalate-Triton fractions
contained 82% of the radioactivity from the total pellet and 7.9% of
the total proteins (Fig. 6).
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| Figure 6.
Flow diagram of how the radiolabeled methylated
product was fractionated. The table inset gives the percentage of
radioactivity in each fraction and the percentage of total membrane
protein. ND, Not determined.
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The oxalate and oxalate-Triton fractions were dialyzed and analyzed for
uronic acid-positive material. Approximately 40 and 72 µg of uronic
acid-positive material was recovered in the oxalate and oxalate-Triton
fractions, respectively, from each initial product pellet.
Solubilization of Methylated Product by EPGase and PME
To confirm that the radiolabeled product was methylated HGA, we
tested the sensitivity of the product in the oxalate and oxalate-Triton fractions to fragmentation by purified EPGase and PME. PME removes methyl groups specifically from the C-6 methylester of HGA and EPGase
hydrolyzes the -(1 4)-GalUA linkages in HGA that has at least four
contiguous nonmethylesterified GalUAs (Chen and Mort, 1994; Benen et
al., 1996).
A representative thin-layer chromatogram showing the separation of
intact product and enzyme-treated product from the oxalate fraction is
shown in Figure 7. Comparable results
were obtained for product in the oxalate-Triton fraction (data not
shown). The intact product in the oxalate fraction remained at the
origin at a location comparable to PGA and pectin standards.
Orcinol-positive material was also detected at the origin (orcinol
reagent detects sugars). Digestion of the oxalate fraction with EPGase
resulted in a loss of 52% of the radioactive product at the origin and the migration of the radioactivity to positions similar to di-, tri-,
and heptagalacturonic acid. Digestion of the oxalate fraction with PME
resulted in a loss of 80% of the radioactive product at the origin.
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| Figure 7.
Representative thin-layer chromatogram of intact,
EPGase- and PME-treated product in the oxalate fraction. Membranes were incubated with [14C]SAM, and the pellet obtained after
TCA precipitation and washing was treated for 1 h with 0.5%
ammonium oxalate. After treatment, the solutions were centrifuged and
the supernatant was dialyzed and lyophilized. Equal amounts of
radioactive products were treated with 1 unit of EPGase or PME. The
samples were separated by TLC. Radiolabeled product was visualized by
exposing the TLC plate to storage phosphor screens for 3 to 4 weeks.
Similar results were obtained in three individual TLCs from three
separate experiments. The arrows represent the location of
digalacturonic acid (DiGalA), trigalacturonic acid (TriGalA), and
heptagalacturonic acid (HeptaGalA) standards. Commercially available
PGA and pectin standards remained at the origin.
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The average amount of radioactivity released by enzymatic treatment of
product in the oxalate and oxalate-Triton fractions from at least three
independent experiments is shown in Table V. EPGase treatment of the oxalate and
oxalate-Triton fractions resulted in the fragmentation of 60 and 35%
of the product, respectively, into oligosaccharides that migrated away
from the origin during TLC. Thus, at least 35% of the original product
pellet contained radioactivity in HGA. This value, however, is a
minimum estimate, because any EPGase products that remained at the
origin would not have been included. Considerably more radioactivity
was released from the oxalate fraction (82%) and the oxalate-Triton
fraction (67%) by PME. Because PME is specific for the C-6 methylester of HGA, these results show that at least 59% of the radioactivity incorporated into the intact product was HGA. Similar results were
obtained by treatment of the corresponding fractions with 0.1 n NaOH for 10 to 12 h at 25°C, confirming that the
methyl groups were present in a methylester linkage. The radioactivity released by PME or NaOH (data not shown) was not detected by TLC, because the methanol evaporated when the samples were lyophilized or at
the time of their application to TLC. In contrast, when the product
treated by PME or NaOH was directly separated or first distilled and
then separated by HPLC using a Rezex ROA-organic acid column, a single
peak of radioactivity with a retention time identical to that of
methanol was detected (data not shown). These results confirm that at
least 59% of the radioactive product produced in tobacco membranes was
methylated HGA.
Size-Exclusion Chromatography of Radiolabeled Product
Size-exclusion chromatography of product solubilized from the
intact pellet by ammonium oxalate resulted in several peaks of
radioactivity that eluted from the column from 16 to 40 min (Fig.
8). The size of the products, compared
with dextran standards, ranged from approximately 200 to 1.7 kD. The
broad, overlapping peaks eluting from 16 to 28 min (40-200 kD)
represent 39% of the radioactivity and elute with retention times
similar to those of pectin standards of either 90 or 30%
esterification. The two small peaks eluting from 29 to 33 min (6-40
kD) represent 10% of the radioactivity and elute with retention times
comparable to those of commercially available pectin and PGA. The large
peak from 34 to 40 min represents 51% of the radioactivity and elutes similarly to commercially available PGA. We do not know why some PGA
elutes relatively late during size-exclusion chromatography, although
an interaction between PGA and the column matrix is a possibility. The
anomalous behavior of GalUA-containing polymers during chromatography
has previously been noted (Mort et al., 1991). It is likely that the
methylated products are greater in size than a decagalacturonide,
because the radiolabeled product in the oxalate fraction remained at
the origin during TLC under conditions in which homogalacturonans of
degrees of polymerization less than 11 migrate away from the origin
(see Fig. 7). We interpret the complexity of the size-exclusion
chromatography profile to indicate that the methylated products are
heterogeneous in size and/or in their degree of methylesterification.
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| Figure 8.
Size-exclusion chromatography of products
solubilized from intact pellet by ammonium oxalate (oxalate fraction).
Membranes were incubated with [14C]SAM (70,000 dpm) for
60 min, and the product was recovered by the HGA-MT assay. Products
were separated by size-exclusion chromatography over a Superose 12 HR
10/30 column and fractions (0.48 mL) were collected for 50 min. The
elution times of dextran molecular mass standards (480, 70, 40, 10, and
6 kD) are indicated by arrows. The resolution limit for the dextran was
calculated as 1.7 kD. The range of elution times for 30 and 90%
esterified pectins (- - - -) and for two commercially available sources
of PGA ( ) are indicated.
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DISCUSSION |
A PMT in tobacco cell membranes that methylates HGA (HGA-MT) has
been identified. The product synthesized by membranes incubated with
[14C]SAM was shown to contain at least 59% HGA
based on the hydrolysis of the 14C-methyl group
by PME. The HGA-MT has a pH optimum of 7.8, a
Km for SAM of 38 µm, and a
Vmax of 0.81 pkat
mg1 protein. The characteristics of the tobacco
HGA-MT are compared with previously described PMTs from mung bean
(Kauss et al., 1969) and flax (Vannier et al., 1992) in Table
VI. The previously described PMTs have
a similar Km for SAM but a different pH
optimum than the HGA-MT from tobacco.
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|
Table VI.
Comparison of the kinetics and product of the PMTs
studied from mung bean (Kauss et al., 1969) and flax (Bruyant-Vannier
et al., 1996) with the HGA-MT from tobacco
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The identification of HGA-MT was complicated by the nonspecific binding
of SAM to compounds in the product pellet and by the presence of PME in
the microsomal membranes. The use of a modification of the
TCA-precipitation method of Kauss and Hassid (1967) eliminated most of
the nonspecific binding of SAM. Bruyant-Vannier et al. (1996) assayed
the PMT from flax membranes by ethanol precipitation and washing of the
methylated products. This technique did not work with tobacco
membranes. It is possible that differences between the flax and tobacco
PMTs or differences in the methylated pectins produced may account for
the requirement of different assays for flax versus tobacco PMTs. The
use of 2- to 4-d-old tobacco cells, which express no PME activity,
overcame the problems associated with the demethylesterification of the
methylated HGA product by PME in older cells. The PME may have been
introduced as a contaminant from the cell wall during membrane
preparation. Alternatively, it is possible that PME resides inside the
membrane vesicles, although the lack of latency of the PME activity
provides no evidence to support this. Previously, Bruyant-Vannier et
al. (1995) reported that PME was associated with flax membranes, and
these authors suggested that PME is naturally present inside membranes.
The presence of PME inside the membranes would not be totally
unexpected, because proteins to be transported to the wall are
processed through the ER and the Golgi apparatus.
Maximum HGA-MT activity was detected during the lag phase of growth,
when the fresh or dry cell weight does not change but cell division has
probably started. A similar result has been obtained in flax cell
suspensions (Schaumann et al., 1993). The methylated pectins are
synthesized and integrated in the cell wall during cell elongation and
division (Schaumann et al., 1993). The variability of HGA-MT activity
during the exponential phase of cell growth can be explained by the
presence of PME activity in the membranes.
The pH optimum for HGA-MT was 7.8, with a minor optimum at pH 7.3. Although we have only described the HGA-MT activity at pH 7.8 in this
paper, similar analyses were performed for HGA-MT activity at pH 7.3 (F. Goubet and D. Mohnen, unpublished results) in an effort to
determine whether the minor pH optimum at 7.3 represented an HGA-MT
isoenzyme. No evidence was obtained to support the hypothesis that the
HGA-MT activity at these two pH values originated from unique enzymes.
To study the relationship between HGA-MT and PGA-GalAT, the effect of
the nucleotide sugar UDP-GalUA (i.e. the substrate for PGA-GalAT) and
HGA (the putative substrate for HGA-MT) on HGA-MT activity was tested.
The addition of UDP-GalUA to membranes stimulates HGA-MT by 20%. The
activation of HGA-MT by UDP-GalUA has previously been described (Kauss
and Swanson, 1969). In contrast, OGA, PGA, and pectin of different
degrees of esterification have no effect on HGA-MT activity. Kauss and
Swanson (1969) and Bruyant-Vannier et al. (1996) have also shown no
stimulation of membrane-bound PMT by exogenous pectins. Four factors
can explain the absence of a stimulatory effect of HGA, PGA, or pectin
on HGA-MT activity. First, the endogenous pectin substrates may be
present in excess. Second, the exogenous pectin may be either larger or
smaller than the size required for recognition by HGA-MT. Third, the
PGA and the HGA may not penetrate the membranes. Attempts to overcome the latter potential limitation by permeabilization of the membranes using 0.01 or 0.1% Triton X-100 did not yield any stimulation of
HGA-MT activity by exogenous PGA or pectin. Fourth, the HGA-MT may
require that HGA is being actively synthesized. Evidence to support the
fourth possibility is the stimulation of HGA-MT by the addition of
UDP-GalUA, a substrate for HGA synthesis by PGA-GalAT.
It is not known whether one enzyme both synthesizes and methylates HGA
or whether PGA-GalAT and HGA-MT exist as separate enzymes. The fact
that UDP-GalUA increases the amount of [14C]SAM
incorporated into HGA suggests that GalUA from UDP-GalUA is first
incorporated into HGA and that the HGA is subsequently methylesterified. The inability of exogenous SAM to stimulate the in
vitro incorporation of UDP-GalUA into HGA (Kauss and Hassid, 1967;
Doong et al., 1995) suggests that methylesterification is not directly
linked to the synthesis of HGA. To determine if one or more enzymes is
required for HGA synthesis and methylation, it will be necessary to
purify the enzymes.
At least 59% of the radiolabeled product recovered by TCA
precipitation is methylated HGA. The remainder of the radioactivity is
insoluble material present in the pellet after TCA treatment (18%) and
radioactive compounds solubilized by oxalate or oxalate-Triton X-100
but not fragmented by EPGase, PME, or NaOH (23%). The radioactive product that was not hydrolyzed by either NaOH or PME may contain methyl groups incorporated into a nonmethylester linkage such as a
methylether linkage (Vannier et al., 1992).
In conclusion, HGA-MT has been detected in tobacco cells and a
procedure to study the activity of this enzyme has been described. The
identification of HGA-MT will facilitate its solubilization and
purification to better understand how pectin is synthesized and to
study the role of HGA-MT in plant growth and development.
| |
FOOTNOTES |
1
This work was supported by a grant from
Hercules, Inc. (Wilmington, DE).
*
Corresponding author; e-mail dmohnen{at}ccrc.uga.edu; fax
1-706-542-4412.
Received August 29, 1997;
accepted October 12, 1997.
| |
ABBREVIATIONS |
Abbreviations:
EPGase, endopolygalacturonase.
HGA, homogalacturonan.
HGA-MT, homogalacturonan-methyltransferase.
OGA, oligogalacturonides of
degree of polymerization of 7 to 23.
PGA, polygalacturonate.
PGA-GalAT, polygalacturonate-4--galacturonosyltransferase.
PME, pectin
methylesterase.
PMT, pectin methyltransferase.
RG-I and RG-II, rhamnogalacturonan I and II, respectively.
SAM, S-adenosyl-l-Met.
| |
ACKNOWLEDGMENTS |
We thank Carl Bergmann for the gifts of purified EPGase from
A. niger and the purified PME and for critical reading of
the manuscript, Hans Peter Heldt-Hansen for the gift of cloned PME, Stefan Eberhard for the gift of tobacco cell suspensions, Karen Liljebjelke for technical help with the characterization of the radiolabeled substrate, Carol L. Gubbins Hahn for drawing the figures,
and our colleagues at the Complex Carbohydrate Research Center for
their helpful discussions.
| |
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Top
Abstract
Introduction