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Plant Physiol. (1998) 116: 137-144
Effects of Sulfanilamide and Methotrexate on 13C
Fluxes through the Glycine Decarboxylase/Serine
Hydroxymethyltransferase Enzyme System in Arabidopsis1
Vikram Prabhu*,
K. Brock Chatson,
Helen Lui,
Garth D. Abrams, and
John King
Department of Biology, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada S7N 5E2 (V.P., H.L., G.D.A., J.K.); and Plant
Biotechnology Institute, National Research Council of Canada,
Saskatoon, Saskatchewan, Canada S7N 0W9 (K.B.C.)
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ABSTRACT |
In C3 plants large
amounts of photorespiratory glycine (Gly) are converted to serine by
the tetrahydrofolate (THF)-dependent activities of the Gly
decarboxylase complex (GDC) and serine
hydroxymethyltransferase (SHMT). Using 13C
nuclear magnetic resonance, we monitored the flux of carbon through the
GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate
synthase inhibitor, showed little reduction in flux through GDC/SHMT.
Two other sulfonamide analogs were tested with similar results,
although all three analogs competitively inhibited the partially
purified enzyme. However, methotrexate or aminopterin, which are
confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased
the flux through the GDC/SHMT system by 60% after 48 h and by
100% in 96 h. The uptake of [ -13C]Gly was not
inhibited by either drug class. The specificity of methotrexate action
was shown by the ability of 5-formyl-THF to restore flux through the
GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with
sulfonamides strongly suggest that the mitochondrial THF pool has a
long half-life. The studies with methotrexate support the additional,
critical role of dihydrofolate reductase in recycling THF oxidized in
thymidylate synthesis.
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INTRODUCTION |
THF coenzymes are required in the synthesis of thymidylate, purine
nucleotides, amino acids, and in organellar protein synthesis (Cossins,
1987 ; Appling, 1991). In many cellular systems the single carbons
involved in THF-dependent processes are derived from the  -carbon of
Ser (Schirch, 1984; Narkewicz et al., 1996). SHMT catalyzes the
transfer of a methylene group from Ser to THF for direct use in
thymidylate synthesis; alternatively, CH2-THF is reduced to CH3-THF by methylene-THF reductase or
oxidized to HCO-THF by C1-THF-synthase (Appling, 1991; Nour and
Rabinowitz, 1991) and then used in other THF-dependent cellular
processes. In C3 plants the GDC/SHMT enzyme
system is considered to be the major pathway for the generation of
single-carbon units via Ser. This is a consequence of photorespiration,
which requires large amounts of Gly to be metabolized by this
enzyme system (Oliver, 1994). In Arabidopsis thaliana the
flux of single carbons into Ser via the GDC/SHMT pathway is 4-fold
greater than that through the alternative C1-THF synthase/SHMT pathway
(Prabhu et al., 1996a). Below we describe some biochemical
relationships between enzymes that regenerate the cofactor THF (Fig.
1) and carbon flux through the
THF-dependent GDC/SHMT enzyme system in A. thaliana (L.)
Heynh. Columbia wild type.
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| Figure 1.
The enzymatic reactions and intermediates in the
biosynthesis of THF. HPPK, 6-Hydroxymethyl-7,8-dihydropterin
pyrophosphokinase; DHPS, dihydropteroate synthase (EC 2.5.1.15); DHFS,
dihydrofolate synthase (EC 6.3.2.12); DHFR (EC 1.5.1.3); and FPGS,
folylpolyglutamate synthetase (EC 6.3.2.17).
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Antagonists of THF biosynthesis have been developed for clinical use as
antiproliferative and antimicrobial agents, which exploit the critical
role that THF coenzymes play in cellular metabolism (Pratt and Taylor,
1990). Sulfanilamide and its analogs (Fig.
2) inhibit DHPS (Fig. 1), whereas
methotrexate (Fig. 2) and its analogs inhibit DHFR (Fig. 2) isolated
from a number of organisms (Cossins, 1987; Pratt and Taylor, 1990;
Schweitzer et al., 1990). DHPS is involved in the de novo pathway of
THF biosynthesis, whereas DHFR has a dual role (Fig. 1); it is involved
in the reduction of dihydrofolate to THF from both the de novo pathway
and that arising from the oxidation of THF during TS activity. These
drugs may be useful in manipulating the availability of THF in higher plants, hence permitting a better understanding of its
metabolism.
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| Figure 2.
Structures of folate analogs. Note that the
commercial leucovorin used in our experiments contained both the
physiological stereoisomer depicted here and the nonphysiological
stereoisomer.
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We previously used a combined dosage of sulfanilamide and methotrexate
with qualitative NMR observations to demonstrate the requirement of THF
for Ser synthesis via the GDC/SHMT and C1-THF synthase/SHMT pathways in
Arabidopsis (Prabhu et al., 1996a). Combined dosages have also been
used to demonstrate the THF-dependent metabolism of Ser through the
C1-THF synthase/SHMT pathway in yeast (Pasternack et al., 1994). The
Arabidopsis study suggested that THF metabolism in this organism is
organized differently from that in other eukaryotes. Recent studies of
pea leaves suggested that THF metabolism is compartmented largely in
the mitochondria (Neuburger et al., 1996), unlike that in other
organisms (Appling, 1991). Here, using NMR, we quantify the fluxes
of carbon through the GDC/SHMT enzyme system in Arabidopsis, as
influenced by exposure to drugs that inhibit DHPS and DHFR, which are
key enzymes that produce and maintain cellular THF levels. The results
shed new light on interactions between the THF pathway and
THF-dependent Gly metabolism in plant mitochondria.
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MATERIALS AND METHODS |
[-13C]Gly and sodium salt
3-(trimethylsilyl)propionic-2,2,3,3-d4 acid
(TSP), were purchased from Cambridge Isotopes Laboratories Inc.
(Andover, MA). All other chemicals were from Sigma.
Arabidopsis thaliana (L.) Heynh. Columbia Culture
Conditions and 13C-Labeling Procedures
The procedures for the growth and 13C
labeling of plants and the preparation of leaf extracts were
essentially as described previously (Prabhu et al., 1996a). All
experiments were repeated three or more times and the data are
presented as the means with respective se values.
NMR Parameters
NMR spectra were collected on an AMX 500-MHz instrument (Bruker,
Billerica, MA). 13C chemical shifts from
natural abundance spectra of authentic samples were first obtained in
100 mm phosphate buffered to pH 7.3 and 25°C, internally
referenced to 2,2-dimethyl-2-silapentane-5-sulfonic acid, sodium salt
(Prabhu et al., 1996b). Assignments of resonances in the extracts were
confirmed by spiking the extracts with authentic samples (Prabhu et
al., 1996a).
Quantitative analyses of 13C-enriched compounds
in the plants were accomplished using extracts. Two sets of acquisition
parameters were used: (a) inverse-gated decoupled experiments with a
20-s delay time and (b) standard broad-band experiments, details of which have been described previously (Prabhu et al., 1996a). All peaks
were calibrated relative to a solution of 2 mm sodium
[13C]formate in 100 mm potassium
phosphate (pH 7.3), sealed in a capillary and inserted concentrically
into the 5-mm tube containing the sample. The
13C-13C multiplets versus
13C-12C center-line
singlets of the protonated carbons of interest were quantitated using
integration and isotopomer analyses; their intensities are independent
of potential differences in T1 and nuclear Overhauser effects (London
et al., 1975; Suzuki et al., 1975), especially under conditions of
gated decoupling with long delay times (Gadian, 1982), as used here.
However, the differential intensities of the
-13C versus the -13C
in Ser under broad-band decoupling were corrected for using data
generated from standard curves of authentic compounds obtained under
similar experimental conditions.
1H spectra were acquired using a 9.7-µs (90°)
pulse, a spectral width of 7812 Hz, an acquisition time of 2 s,
and a delay time of 1 s. The sample temperature was maintained at
25°C and 32,000 data points were acquired for each sample.
Attenuation of the water resonance was achieved by presaturation (2 s
at 40 dB). One hundred twenty scans were acquired for each sample and a
line broadening of 0.3 Hz was used in the processing of the free
induction decay. Chemical shifts from natural abundance spectra of
authentic samples were first obtained in 100 mm phosphate
buffered to pH 7.3 and 25°C, referenced internally to
2,2-dimethyl-2-silapentane-5-sulfonic acid and externally to a 10 mm solution of TSP in 100 mm potassium phosphate (pH 7.3), sealed in a capillary and inserted concentrically into the 5-mm tube containing the sample. The spectra of the plant extracts were acquired with the external standard, TSP only; the singlet of the trimethyl resonance of TSP was set to zero. Assignments of peaks in the extracts were confirmed by spiking the sample with pure
compounds.
Extraction and Assay of DHFR
Root material from plants grown in liquid culture was harvested,
blotted dry with paper towels, and frozen at  80°C. The tissue was
then ground in a mortar on ice using a small amount of acid-washed sand. A buffer containing 100 mm potassium phosphate (pH
7.5), 1 mm EDTA, 1 mm PMSF, 10% (v/v)
glycerol, and 20 mm 2-mercaptoethanol was added while
grinding until a fine slurry was achieved. The slurry was filtered
through three layers of cheesecloth and centrifuged at
10,000g for 10 min in a JA-25.5 rotor using an Avanti J-25 instrument (Beckman). The supernatant was treated with solid ammonium sulfate, and protein precipitating between 30 and 60% ammonium sulfate
saturation was removed by centrifugation as described above and stored
overnight at 20°C. Storage overnight resulted in better activity
than if the enzyme was assayed directly after salt precipitation. The
protein was desalted over a Sephadex G-25 column (Pharmacia) and its
concentration was estimated by the method of Bradford (1976). DHFR
activity was assayed spectrophotometrically (Misra et al., 1961). The
assay was done in a final volume of 500 µL containing 100 mm potassium phosphate (pH 7.5), 100 µm NADPH, 100 µm dihydrofolic acid, 500 µg of protein, and
various concentrations of methotrexate or aminopterin. The enzyme
activity was monitored by recording the change in
A340 once every 30 s for 10 min, using
a photodiode array spectrophotometer (DU 7400, Beckman). Controls
lacking dihydrofolate were used to subtract nonspecific oxidation of
NADPH. Specific activity was expressed as nanomoles of dihydrofolate
reduced to THF per milligram of protein per hour.
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RESULTS |
Metabolism of [-13C]Gly to Ser as Detected by
13C NMR
The rationale for the 13C-enrichment
patterns of Ser in Arabidopsis plants supplied with
[-13C]Gly has been described previously
(Prabhu et al., 1996a). For this study only
[,-13C]Ser and
[-13C]Ser, the two species unequivocally
ascribable to synthesis via GDC/SHMT activities, were taken into
account; [-13C]Ser could be synthesized
using the alternative C1-THF synthase pathway (Prabhu et al., 1996a).
For a measure of flux through the GDC/SHMT enzyme system the
13C-enriched isotopomers of Ser described above
were quantitated after 6 h of supply of
[-13C]Gly. This was based on the relative
patterns and concentrations of isotopomers described from time-course
experiments in which we did not detect signals from potential products
of further metabolism of Ser such as hydroxypyruvate or other sugars
(Prabhu et al., 1996a). Other NMR studies of Gly metabolism in plant
cells also noted little metabolism of the Ser produced via the GDC/SHMT
pathway in comparable periods (Ashworth and Mettler, 1984; Neeman et
al., 1985). In some animal cellular systems the de novo incorporation of amino acids into proteins has been shown to be sufficient to warrant
correction in measurements of flux for specific pathways (Flogel et
al., 1997). However, even in hydrolyzed extracts of our samples we did
not see NMR signals that would indicate incorporation of
13C-enriched compounds into macromolecules (V. Prabhu, B. Chatson, G. Abrams, and J. King, unpublished data). Thus,
our quantitative measurements of Ser accumulation are a good
representation of flux through the GDC/SHMT enzyme system, which we
express as micromoles of 13C-enriched Ser/g fresh
weight/6 h.
Individual Effects of Methotrexate and Sulfanilamide
Experiments were first performed with sulfanilamide (Fig.
3a) or methotrexate (Fig. 3b) at a
range of concentrations to evaluate their use in subsequent
experiments. Nonlimiting concentrations were then used to examine
potential differences in the effects of the two classes of drugs.
Methotrexate-treated plants displayed a strong reduction in the flux of
13C through the GDC/SHMT system, whereas
with sulfanilamide only a small effect was observed (Table
I). After 96 h of exposure to the
drugs, this flux could no longer be detected in methotrexate-treated plants, whereas with sulfanilamide only a slight reduction with respect
to the control was detected.
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| Figure 3.
Effects of varying concentrations of inhibitors
on the flux of [-13C]Gly through the GDC/SHMT enzyme
system in Arabidopsis leaves. a, Sulfanilamide; b, methotrexate. Plants
were exposed to inhibitors for 48 h and then supplied with
[-13C]Gly for 6 h.
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Table I.
Relative effectiveness of methotrexate (100 µm) versus sulfanilamide (2 mm) in reducing
flux through the GDC/SHMT enzyme system
The data are expressed as the means and respective ses of
flux.
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Time-Dependent Effects of Simultaneous Exposure to Methotrexate and
Sulfanilamide
Plants exposed simultaneously to methotrexate (100 µm) and sulfanilamide (2 mm) showed a
time-dependent reduction in the flux of 13C
through the GDC/SHMT system, although the cellular concentrations of
[-13C]Gly did not decrease (Fig.
4). The reduction was approximately 50%
in the first 24 h, but a 96-h period was required to completely reduce the Ser signal to that of the background. No additive effect of
sulfanilamide to methotrexate was obvious, because the results were
similar to those obtained with methotrexate alone (Table I). At 0 h of drug exposure the concentration of
[-13C]Gly was slightly lower than at other
time points, coinciding with the maximum flux. Quantitative isotopomer
analyses showed that the exposure to antifolates also resulted in an
alteration in the pools of single carbon (Table
II). The proportion of the dually
enriched [,-13C]Ser was reduced, whereas
those of the singly enriched species, [-13C]Ser and
[-13C]Ser, increased. Based on the
experiments described in Table I, it appears that the changes in
isotopomer proportions were caused by methotrexate alone (not shown).
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| Figure 4.
Time-dependent effects of combined exposure to
methotrexate plus sulfanilamide on flux through the GDC/SHMT enzyme
system in Arabidopsis leaves.
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Table II.
Relative proportions of the three species of
13C-enriched Ser after treatment with methotrexate (100 µm) plus sulfanilamide (2 mm)
The data are expressed as the percentage of total
13C-enriched Ser and the experimental details are as
described for Figure 5. There are no data for 96 h because the
[13C]Ser signal was not distinguishable from the baseline
noise.
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Comparison of the Effects of Alternative Analogs
We examined the effect of aminopterin (Fig. 2), which like
methotrexate is a competitive inhibitor for DHFR, on reduction of THF
availability for the operation of the GDC/SHMT enzyme system in
Arabidopsis. A 48-h exposure to aminopterin, as with methotrexate, reduced the flux by about 60% (Table
III), and after exposures of 96 h,
the resonance of 13C-enriched Ser could not be
distinguished above the background in the NMR spectra from either
treatment (not shown). The uptake of Gly by plants was unaffected by
either aminopterin or methotrexate treatment (not shown).
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Table III.
Comparison of the effectiveness of alternative
analogs of sulfanilamide and methotrexate in reducing flux through the
GDC/SHMT enzyme system after 48 h of drug exposure
The concentrations of sulfonamides were 4 mm, whereas those
of methotrexate and aminopterin were 200 µm.
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A partially purified preparation of DHFR enzyme activity from
Arabidopsis leaves was completely inhibited by 1 µm
methotrexate or aminopterin (data not shown). However, high levels of a
nonspecific NADPH-oxidizing activity were present in this leaf extract,
which did not allow nonlimiting concentrations of NADPH to be
maintained for the determination of I50 values.
These experiments were then attempted using root preparations that had
reduced levels of the nonspecific NADPH-oxidizing activity. Controls
were included to determine the nonspecific oxidation of NADPH.
Methotrexate and aminopterin were strong inhibitors of the root DHFR
preparation, with I50 values of less than 10 nm for both analogs (Fig.
5). Germination of seeds was
completely inhibited, and growth (change in fresh mass) of 3-week-old
plants was severely reduced in flask culture in the presence of both
analogs (data not shown).
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| Figure 5.
Inhibition of DHFR enzyme activity in in vitro
assays by methotrexate and aminopterin. The 100% activity was 75 ± 5 nmol (mean ± se, n = 3) of
THF mg 1 protein h1.
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Experiments in which the effect of sulfadiazine (Fig. 2) was compared
with that of sulfanilamide revealed that this analog also did not
reduce flux through the GDC/SHMT enzyme system in vivo (Table III). A
third analog, sulfacetamide, was also tested and yielded similar
results (not shown). We used 1H NMR spectroscopy
to determine whether individual sulfonamides were present in the
shoots. When plants were supplied with sulfanilamide for 48 h, the
accumulation of this compound in the shoots could be detected from the
resonances of the aromatic hydrogens (Fig. 6). The endogenous metabolite fumarate
was present in all of the samples that were examined. The spectra are
plotted using the intensity of the fumarate peak as a convenient
internal standard; the intensity of the fumarate peak and that of the
external reference in replicates of control and sulfonamide-treated
plants were not significantly different from each other. When
sulfacetamide was supplied to Arabidopsis, this compound could be
detected in the shoots from the aromatic resonances, as well as the
methyl resonance of the acetyl group (not shown). These experiments
confirmed that the supplied sulfonamides were transported to and
accumulated in quantity in the shoot tissues.
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| Figure 6.
1H NMR detection of the accumulation
of sulfanilamide in Arabidopsis shoots. a, Spectrum of control plants
showing the area of interest that contains the endogenous metabolite
fumarate. b, Plants supplied with 5 mm sulfanilamide for
48 h; the aromatic proton resonances of this compound are
indicated in the spectrum. c, Spectrum of the sample shown in b spiked
with sulfanilamide.
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In vitro experiments using a partially purified preparation of the DHPS
from Arabidopsis showed that all three analogs were highly potent
inhibitors, with I50 values of less than 20 µm (Prabhu et al., 1997). Germination of seeds was
completely inhibited and growth of 3-week-old plants was reduced in
flask culture in the presence of all three analogs added singly (data
not shown).
Specificity of the Inhibition by Methotrexate
To determine if the decrease in flux after methotrexate exposure
was attributable to depletion of THF levels by inhibition of DHFR, or
rather to direct inhibition of the GDC/SHMT enzymes by polyglutamylated
forms of methotrexate, we performed rescue experiments using leucovorin
(Fig. 2). Figure 7 shows that the supply
of leucovorin after complete methotrexate inhibition restored flux
through the GDC/SHMT enzyme system. During a 24-h period the plants
supplied with leucovorin showed a measurable flux, indicating that
there was little direct inhibition of these enzymes by methotrexate
(Table IV). The proportions of the
individual species of 13C-enriched Ser in the
leucovorin-rescued plants were considerably different from those in the
control (Table V). (Note that the quantities of the isotopomers of [-13C]Ser
versus [-13C]Ser were corrected for
differential intensities and do not correspond directly with the
intensities of the individual peaks in Fig. 7.) In
methotrexate-plus-leucovorin-treated plants, the proportions of both
the singly enriched species, [-13C]Ser and
[-13C]Ser, increased relative to that of
[,-13C]Ser. This was similar to the
observation recorded in Table II.
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| Figure 7.
Leucovorin rescue of methotrexate inhibition of
flux through the GDC/SHMT enzyme system in Arabidopsis leaves. a,
Broad-band decoupled spectra of control plants supplied with
[-13C]Gly for 24 h. b, Plants exposed to 200 µm methotrexate for 96 h, transferred to
methotrexate-free media for 12 h, and then supplied with
[-13C]Gly for 24 h. c, Plants exposed to 200 µm methotrexate for 96 h, transferred to
methotrexate-free media containing 1 mm leucovorin for
12 h, and then supplied with [-13C]Gly for
24 h.
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Table IV.
Leucovorin rescue after methotrexate inhibition of
Ser synthesis
The methotrexate-treated plants were transferred to fresh media lacking
methotrexate, with or without leucovorin for 12 h. Subsequently,
[-13C]Gly (1 mm) was supplied for 24 h and the flux through the GDC/SHMT enzymes was determined.
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Table V.
Relative proportions of the three species of
13C-enriched Ser in leucovorin rescue experiments
The treatments are the same as those described in Table IV. The data
are expressed as the percentage of total 13C-enriched Ser.
There are no data for the methotrexate-only treatment (Table IV).
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DISCUSSION |
The sulfonamide class of inhibitors caused only a slight reduction
in THF-dependent flux through the GDC/SHMT enzyme system in
Arabidopsis. Studies of the target enzyme showed that the sulfonamide analogs were strong inhibitors of the Arabidopsis DHPS (Prabhu et al.,
1997). 1H NMR studies confirmed that under the
conditions of our experiments, the supplied sulfonamides were present
in the shoot tissue. The continued metabolism of Gly in plants exposed
to sulfonamides is unlikely to have occurred using de novo-synthesized
THF. Therefore, our results suggest that the mitochondrial THF pool is
either large and/or has a long half-life.
High levels of folate were recorded in mitochondria isolated from pea
leaves (Neuburger et al., 1996); the large mitochondrial folate pools
likely reflect the requirement of C3 plants to
metabolize large amounts of Gly to Ser (Oliver, 1994). Studies of the
partially purified DHPS from Arabidopsis (Prabhu et al., 1997) lend
some indirect support to the idea that THF levels in Arabidopsis may reach high or nonlimiting levels. The product of the biosynthetic reaction, dihydropteroic acid, competitively inhibits the enzyme activity. The DHPS from pea leaves was also found to be inhibited in
this manner (Rebeille et al., 1997). The existence of such a mechanism
indicates that product feedback inhibition could regulate the flow of
metabolites when THF levels are sufficient to meet cellular
requirements.
Methotrexate may inhibit THF-dependent enzymes in vivo after undergoing
polyglutamylation as observed in animal cells (McGuire and Coward,
1984; Kim et al., 1993). However, our leucovorin rescue experiments
strongly suggested that the effects of methotrexate were restricted to
the inhibition of DHFR and that there was little, if any, direct
inhibition of GDC/SHMT enzymes. Leucovorin is widely used in animal
systems as a rescue agent from methotrexate toxicity (Stover and
Schirch, 1993). Because it is a fully reduced form of folic acid, its
supply can alleviate the inability of methotrexate-inhibited DHFR to
supply THF. Also, in previous studies of the uptake and metabolism of
methotrexate by plant cells it was found that methotrexate was a poor
substrate for folylpolyglutamate synthetase (see Fig. 1) from
Datura innoxia in in vitro assays, and that methotrexate polyglutamylation in the cells themselves was only slight (Wu et al.,
1993, 1994). Thus, unlike in animal cells, polyglutamylation in plant
cells may not be a significant factor in the action of methotrexate.
DHFR plays an important role in the cellular functioning of all
organisms. Our studies strengthen the idea of a crucial role for DHFR
in the reduction of dihydrofolate produced by oxidation of THF by TS
activity. In plants, including Arabidopsis, DHFR and TS are encoded by
bifunctional genes coding for bifunctional polypeptides (Lazar et al.,
1993; Luo et al., 1993, 1997; Wang et al., 1996). The direct channeling
of dihydrofolate between the TS and DHFR domains in bifunctional
polypeptides has been proposed (Knighton et al., 1994). In plants
thymidylate synthesis may occur exclusively in the mitochondria
(Neuburger et al., 1996). This could explain the rapid depletion of the
THF required for mitochondrial Gly metabolism in Arabidopsis when DHFR
activity is inhibited by methotrexate. Thus, methotrexate treatment had a more dramatic effect than the sulfonamides on reducing the
availability of THF for mitochondrial Gly metabolism.
Methotrexate is a useful drug to manipulate THF availability in plants.
Exposure of Arabidopsis plants to methotrexate resulted in changes in
the isotopomers of [13C]Ser produced by the
GDC/SHMT system. The change in isotopomers could reflect an alteration
in the pools of active THF (HCO-, CH-, CH2-, and
CH3-) in the cellular system arising from
methotrexate action. In mammalian cells rapid alterations in the
proportions of one-carbon THF occur after exposure to methotrexate,
along with an increase in the amount of dihydrofolates (Allegra et al., 1986).
In Arabidopsis methotrexate treatment reduced THF availability, which
in turn reduced the flux through the GDC/SHMT system. The reduced flux
caused endogenous (photorespiratory) Gly concentrations to rise and
increased the
12CH2-THF:13CH2-THF
ratio; this was recorded by NMR as a decrease in the proportion of
[,-13C]Ser relative to the singly
enriched species. The ratio of [-13C]Ser
to [-13C]Ser was approximately 2:1 in
control and methotrexate-treated plants. The 2:1 ratio in control
plants is reflective of the added flux through the C1-THF-synthase
pathway, resulting in higher levels of
[-13C]Ser (Prabhu et al., 1996a).
The maintenance of the 2:1 ratio in methotrexate-treated plants
suggests that the GDC activity was not directly inhibited by
methotrexate. Furthermore, the pools of THF used by the two pathways
are well equilibrated, because reduction in the
[-13C]Ser signal paralleled that of
[-13C]Ser. These suggestions are also
supported by the leucovorin rescue experiments, which showed the
ability to restore flux through the GDC/SHMT and C1-THF-synthase
pathways (again 2:1 ratio).
Our antimetabolite experiments suggest several additional ideas about
THF metabolism in Arabidopsis. The lack of reduction in Gly metabolism
by sulfonamides, and the long time period required for complete
inhibition by methotrexate, indicate that cellular folate pools have a
relatively long half-life. The leucovorin rescue experiments indicate
that reduced folates can be transported across mitochondrial membranes
because the supplied leucovorin was able to restore mitochondrial
metabolism of Gly. In other eukaryotes the parallel paths of THF
metabolism in the cytosol and mitochondria are largely interconnected
by transport of single carbons in the form of formate, Gly, or Ser
between these two cellular compartments (Appling, 1991). However,
information on the compartmentation of THF metabolism and thymidylate
synthesis in plants is limited, and the few individual studies have
suggested quite different pictures of these processes in plants (Cella
et al., 1991; Huangpu et al., 1996; Neuburger et al., 1996; Luo et al.,
1997).
To our knowledge, this study demonstrated for the first time in a
higher plant, and as far as we know in any cellular system, the
distinctly different effects of methotrexate and sulfanilamide on
THF-dependent metabolism. The NMR approach to examine these problems
was particularly useful. The results shed new light on the regulation
of THF metabolism in plants and accentuate the dual role played by DHFR
in contrast to that of DHPS. The direct monitoring of the effects of
these drugs on the major pathway producing carbons for use in other
THF-dependent processes is our first step toward understanding the
relationships between THF biosynthesis and THF-dependent cellular
metabolism in plants. Studies of THF-dependent processes that occur
outside the mitochondria should lead to a better understanding of the
compartmentation of THF metabolism in higher plants.
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FOOTNOTES |
1
This study was supported in part by
grants-in-aid of research from the Natural Sciences and Engineering
Research Council of Canada to J.K.
*
Corresponding author; e-mail prabhuv{at}duke.usask.ca; fax
1-306-966-4461.
Received June 20, 1997;
accepted September 22, 1997.
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ABBREVIATIONS |
Abbreviations:
CH2-THF, 5,10-methylene-THF.
CH3-THF, 5-methyl-THF.
DHFR, dihydrofolate reductase.
DHPS, dihydropteroate synthase.
GDC, Gly decarboxylase complex.
HCO-THF, 10-formyl-THF.
I50, concentration of inhibitor resulting in
50% reduction in enzyme activity.
leucovorin, 5-formyl-THF.
SHMT, Ser
hydroxymethyltransferase.
THF, tetrahydrofolate.
TS, thymidylate
synthase.
| |
ACKNOWLEDGMENTS |
We acknowledge the useful suggestions of the anonymous
reviewers.
| |
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Abstract
Introduction