Vikram Prabhu*, K. Brock Chatson, Garth D. Abrams, and John King. Department ...... Dickinson JR, Dawes IW, Boyd ASF, Baxter RL (1983) 13C NMR studies of ...
Plant Physiol. (1996) 112: 207-21 6
Nuclear Magnetic Resonance Detection of lnteractions of Serine Hydroxymethyltransferase with C1-TetrahydrofoIate Synthase and Glycine Decarboxylase Complex Activities in Arabidopsis'
13C
Vikram Prabhu*, K. Brock Chatson, Garth D. Abrams, and John King Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E2 (V.P., J.K.); and Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N OW9 (K.B.C., G.D.A.) ~
~
through GDC activity (reaction 1) for Ser biosynthesis by SHMT (reaction 2).
~~
I n C, plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (CDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance t o examine serine biosynthesis by these two pathways i n Arabidopsis fhaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the CDC/ SHMT system and observed directly i n a higher plant the flux of formate through the C1-THF synthase/SHMT system. l h e accumulation of 13C-enrichedserine over 24 h from the CDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed t o methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. l h e results suggest that continuous supply of THF i s essential to maintain high rates of serine metabolism. Nuclear magnetic resonance i s a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment. ~
~
~
Gly +NAD++THF -5,lO-methylene-THF
+NADH+NH,+ CO,
5,lO-Methylene-THF +Gly-Ser +THF
(1) (2)
In leaves of plants with C, photosynthesis large quantities of Gly formed as a consequence of photorespiration are converted to Ser in the mitochondria by this pathway (Oliver, 1994). The GDC activity is greater than that of SHMT in plant leaf mitochondria, which results in the maintenance of high levels of 5,lO-methylene-THF (Rebeille et al., 1994). Thus, the equilibrium of the SHMT reaction is shifted in favor of Ser synthesis, which enables recycling of THF for continuous operation of the GDC reaction during photorespiration. Formate is a potential alternative single-carbon source for the production of the 5,lO-methylene-THF required for Ser synthesis. The activation of formate to 5,10-methyleneTHF is accomplished in three enzyme-mediated reactions, catalyzed by 10-formyl-THF synthetase (reaction 3), 5,lOmethenyl-THF cyclohydrolase (reaction 4), and 5,lOmethylene-THF dehydrogenase (reaction 5).
~~
Folate coenzymes mediate single-carbon transfers in a variety of cellular processes such as purine biosynthesis, amino acid metabolism, thymidylate synthesis, and chloroplast and mitochondrial protein synthesis. In spite of the pivotal role THF plays in cellular metabolism, studies of THF biosynthesis and THF-dependent metabolism in plants remain sparse. We have recently begun wideranging studies of folate metabolism in Datuva cells and Arabidopsis plants (Wu et al., 1993, 1994; Prabhu et al., 1994). In this study we addressed the THF-dependent biosynthesis of Ser in intact plants of Arabidopsis tkaliana (L.) Heynh. Columbia wild type using I3C NMR. THF-dependent Gly and Ser metabolism are closely linked in a variety of organisms (Schirch, 1984). The use of a common pool of THF allows transfer of the a-C of Gly
Formate +THF +ATP-10-formyl-THF
+ADP +Pi
(3)
10-Formyl-THF+H+~5,10-methenyl-THF+H20(4) 5,lO-Methenyl THF+NADPH ++5,10-methylene-THF+NADP+
(5)
In most eukaryotes these three activities occur on a single polypeptide called the C1-THF synthase (Nour and Rabinowitz, 1992). The known exception to the eukaryotic trifunctional C1-THF synthase occurs in plants. Recent studies with spinach (Nour and Rabinowitz, 1991, 1992) and pea (Kirk et al., 1995) have shown that the synthetase
This study was supported by grants-in-aid of research from the Natural Sciences and Engineering Research Council of Canada and from the University of Saskatchewan to J.K. * Corresponding author; e-mail prabhuv8duke.usask.ca; fax
Abbreviations: DHFR, dihydrofolate reductase; GDC, Gly decarboxylase complex; ppm, parts per million; SHMT, Ser hydroxymethyl transferase; THF, tetrahydrofolate.
1-306 -966 -4461.
207
Prabhu et al.
208
activity occurs on a separate protein moiety from that of the cyclohydrolase and reductase, which occur on a bifunctional protein. In yeast, the activities of the C1-THF synthase are coupled in vivo (Pasternack et al., 1992, 1994). Previous studies with higher plants suggested that formate may be an important source of single-carbon units for Ser synthesis (Gifford and Cossins, 1982a, 1982b; Shingles et al., 1984). However, direct evidence for the interaction of C1-THF synthase with SHMT in plants is lacking. In light of these observations, we examined four aspects of folate-mediated Gly and Ser metabolism in planta using NMR spectroscopy with the model plant Arabidopsis tkaliuna. The first objective was to confirm that the GDC activity in Arabidopsis was coupled with SHMT activity through a common pool of THF in the synthesis of Ser. Second, we wished to determine whether formate could supply the third carbon for Ser synthesis through the enzyme activities of 10-formyl-THF synthetase, 5,lO-methenyl-THF cyclohydrolase, and 5,lO-methylene-THF dehydrogenase. A third objective was to investigate whether the synthesis of Ser by one pathway repressed the other. Our fourth goal was to examine the importance of THF supply to Ser synthesis; for this we examined Ser synthesis in plants exposed to sulfanilamide and methotrexate, powerful inhibitors of THF biosynthesis. 13C NMR spectroscopy is especially suited to examine problems such as those described above. It allows us to determine the exact position of the label in a given metabolite in planta, unlike 14C-labeling,with which such determination requires selective and sometimes uncertain degradation of isolated metabolites. This is especially important in studies of formate, Gly, and Ser metabolism, in which the potential is high for label to be incorporated into multiple positions of one metabolite, as well as into severa1 end products (Gifford and Cossins, 1982a, 1982b; Pasternack et al., 1994). Knowledge of the positions of the label in metabolites allows the biosynthetic activity of specific enzymes to be inferred. Finally, direct evidence for the in vivo enzyme interactions of activities such as C1-THF synthase and GDC with SHMT can be obtained in NMR studies (Brindle et al., 1995). Such interactions cannot be inferred with certainty from in vitro reactions with isolated enzymes. A preliminary account of this study was presented as an abstract (Prabhu et al., 1995). MATERIALS A N D METHODS
13C-enriched compounds were purchased from Cambridge Isotopes Laboratories (Andover, MA). Plant Culture Conditions and '3C-Labeling Procedures
Seeds of Aruhidopsis tkaliana (L.) Heynh. Columbia wildtype plants were surface-sterilized by soaking them for 10 min in a 30% (v/v) solution of commercial bleach (Javex; Colgate-Palmolive, Toronto, Ontario, Canada) followed by thorough rinsing in sterilized, deionized water. Approximately 15 seeds were then put into a 125-mL flask containing 50 mL of a pH 5.5 medium of 14.6 M SUC,10 mM KNO,, 2.5 mM KH,PO,, 2 mM Mg,SO,.7 H,O, 2 mM CaC1,*2H,O, 70 p~ H,BO,, 14 p~ MnCl,, 1 p~ ZnS04.7H,0, 10 p~
Plant Physiol. Vol. 11 2, 1996
NaC1, 0.5 p~ CuSO,, 0.2 p~ Na2Mo0,.2H20, 1 p~ CoCl,, and iron as sequestrene at 0.022 g/L. The flasks were shaken continuously at 150 rpm in a chamber at 25°C under continuous light with a PPFD of 103 2 20 pE m-' s-l recorded within the flask by inserting a quantum sensor through a hole in the bottom. By the 3rd week plants had grown upright, with their roots immersed in the swirling medium and their foliage in the atmosphere within the flask. At this time ',C-enriched compound(s) and/ or inhibitors were supplied aseptically to individual flasks at concentrations determined from preliminary experiments not to be limiting. Both [a-13C]Gly and unenriched Gly were supplied at 1 mM, and sodium [13C]formate was supplied at 2 mM. These concentrations were chosen from preliminary experiments in which the concentrations ranged from 250 p~ to 4 mM. Concentrations of the inhibitors methotrexate and sulfanilamide were 50 FM and 2 mM, respectively. Plants were incubated with these compounds for specific time intervals, and then removed from the flasks and washed thoroughly with deionized water to remove these compounds from their surfaces. For spectral acquisition of whole plants, approximately 1 g fresh weight of the washed plant material was introduced with a small amount of 'H,O into a 5-mm NMR tube, such that the window of sampling contained shoot material. Extracts of plant material were prepared using 1 g fresh weight of shoot material frozen at various times in liquid nitrogen. Perchloric acid extracts were prepared by grinding the tissue to a fine powder in a frozen mortar and pestle with a small amount of acid-washed sand. One milliliter of 5 or 25% (v/v) perchloric acid was added, and the mixture was allowed to thaw and then was ground further in the mortar on ice. The cellular debris was removed by centrifugation (10,OOOg) at 4"C, and the supernatant was neutralized to pH 7.0 to 7.5 with KOH. The precipitated KC10, was then removed by centrifugation (10,OOOg). The supernatant was frozen, lyophilized, and resuspended in 1 mL of 100 mM potassium phosphate buffer (pH 7.3) containing 5% (v/v) 'H,O. This perchloric acid extract was then placed in a 5-mm NMR tube for spectral acquisition. For phosphate-buffered extracts 1 mL of 100 mM potassium phosphate buffer (pH 7.3) was added to 1 g fresh weight of frozen tissue and ground in the mortar on ice. The ground extract of tissue was centrifuged directly after grinding or boiled first for 3 min and then centrifuged to remove cellular debris. The supernatant was frozen, lyophilized, resuspended in 1 mL of the phosphate buffer (pH 7.3) containing 5% (v/v) 2H,0, and placed in a 5-mm NMR tube. We did not detect any qualitative or quantitative differences between boiled and unboiled phosphate extracts; boiled extracts were used in a11 experiments. Control experiments were performed with pot-grown plants to determine whether metabolism of the supplied 13C-enriched compounds was different under photosynthetic conditions. For this plants were grown in commercial potting soil in a growth chamber with an 8-h photoperiod (PPFD of 900 pE mp2 s-l supplied by a 1:l mixture of fluorescent Sylvania Gro-Lite:incandescent Sylvania 60-W
13C NMR Detection of Ser Metabolism in Arabidopsis
lamps) and light/ dark temperature of 25°C. Three-weekold plants were carefully removed, and their roots were placed in 2-mL glass vials containing solutions of 13Cenriched formate or Gly. The plants were kept in these solutions for 24 h in the growth chamber under continuous light. The longer incubation periods were required because of the plants' slower uptake than in flask culture. Plants were then introduced into a 5-mm NMR tube as described above. NMR spectra of plants grown under photosynthetic conditions also showed the synthesis of the same 13Cenriched species of Ser as detected in plants in flask culture. The results presented are from experiments done in flask culture. A11 experiments were repeated three or more times. Representative spectra are included in "Results."
NMR Parameters
13C NMR spectra were collected on an AMX 500-MHz instrument (Bruker, Billerica, MA). Assignments of resonances of in planta spectra and those of extracts were made based on chemical shifts of added authentic amino acid samples. Chemical shifts from natural abundance spectra of authentic samples were first obtained in 100 mM phosphate at pH 7.3 and 25"C, internally referenced to 2,2-dimethyl-2-silapentane-5-sulfonicacid, sodium salt (Prabhu et al., 1996). Subsequently, in planta spectra of the authentic samples were obtained after supplying plants with 20 mM unenriched samples for 12 h. Assignments of resonances in the extracts were confirmed by spiking the extracts with authentic samples. For qualitative spectra of whole shoots, the acquisition parameters included a 5-ps (90") pulse with broad-band proton decoupling, a spectral width of 31,250 Hz, an acquisition time of 0.5 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. Twelve hundred scans were acquired for each sample, and a line broadening of 4 Hz was used in the processing of the free induction decay. Quantitative analyses of I3C-enriched Ser in the plants were accomplished using extracts. Relative to the amounts of Ser in phosphate extracts, 31 2 8% loss of enriched Ser in 5% (v/v) perchloric acid extracts and 37 ? 6% loss of enriched Ser in 25% (v/v) perchloric acid extracts (means ? SD, n = 4) were documented. The quantitation was done therefore using phosphate-buffered extracts. Two sets of acquisition parameters were used: inverse-gated decoupled experiments with a long delay time and standard broad-band experiments. The parameters for the broadband experiments were as described above for whole-shoot spectra. The parameters for the inverse-gated decoupled experiments included a 2.5-ps (45") pulse, a spectral width of 31,250 Hz, an acquisition time of 0.5 s, and a delay time of 20 s. The decoupling was achieved using the composite pulse WALTZ-16 only during the acquisition period. The sample temperature was maintained at 25"C, and 32,000 data points were acquired for each sample. Twelve hundred scans were acquired for each sample, and a line
209
broadening of 4 Hz was used in the processing of the free induction decay. The broad-band decoupled experiments were used to cross-check the quantities estimated from inverse-gated experiments, since the latter required long periods (approximately 7 h) during which sample degradation or alteration could occur. A11 peaks were calibrated relative to sodium formate, sealed in a capillary, and inserted into the 5-mm tube containing the sample. We could use externa1 formate as a reference, since in plants supplied with [13C]formate this substrate could not be unequivocally detected over the background noise in whole plants or in extracts even with a 20-s time delay. Furthermore, quantitation of these extracts was highly comparable using both the broad-band and inverse-gated experiments. Standard curves of concentrations ranging from 250 ~ L to M 2 mM of [13C]Ser (calculated from 1.1% natural abundance) were prepared for both broad-band and inverse-gated decoupled conditions. Since the intensities of the nonequivalent carbons were not comparable under broad-band decoupling, individual standard curves for the individual resonances were prepared. A11 standard curves were linear over the range. The estimates of the amount of 13Cenriched Ser in samples agreed well from both broad-band and inverse-gated experiments. RESULTS Metabolism of [ ( ~ - ' ~ C l Cto l y Ser
Through the use of 13C NMR it is possible to detect the combined activities of the GDC and SHMT enzyme systems in whole plants, because of the characteristic resonances of the selectively enriched Ser produced. The three potential labeling patterns in Ser when [a-13C]Gly is metabolized to Ser are depicted in Figure l. Thus, the NMR spectrum of plants that accumulated [c~-'~C]Sershould show a single resonance at approximately 59 ppm (Prabhu et al., 1996). Similarly, [p-13C]Ser should resonate at approximately 63 ppm, and [a-p-13C]Ser should resonate at both 59 and 63 ppm. However, the resonances of [a-p13C]Serwould appear as split signals, each centered on 59 and 63 ppm, since the adjacent 13C-labelednuclei are spinspin coupled (Gadian, 1982). When Arabidopsis plants were supplied with [ a-'3C]Gly, the NMR spectrum showed its accumulation in the plant and the Ser to which it was metabolized (Fig. 2b). The spectrum of the whole shoots was comparable to that of the phosphate-buffered extract (Fig. 2d) and the perchloric acid extracts of shoots (not shown). No other significant 13 C signals were detected above the background. Similar spectra were observed for pot-grown plants supplied with [a-13C]Gly (data not shown). Plants not supplied with [a-13C]Gly did not give significant signals above the background (Fig. 2, a and c). The expanded regions of the spectra (Fig. 2, top) show the presence of a11 three potential 13C-enriched species of Ser diagrammed in Figure 1. [/3-13C]Ser was synthesized from endogenous Gly and 5,1O-l3C-methylene-THF from GDC activity on [a-13C]Gly (Fig. 1, a). [c~-P-'~C1Serwas
Prabhu et al.
210 Figure 1. Scheme of possible single-carbon transfers in formate, Gly, and Ser metabolism. Silhouetted C, I 3 C label.
Plant Physiol. Vol. 112, 1996
l39H,-COOH ~
\
1
3
~
0
0
~
e: : : /
Decahoxylase
C1-THF Synthase
13CH2-THF D
F H2-COO/; NH2
/
,
\
\i c
Sedne Hydroxymethyltranslerase
HO-'3CH2-CH-COOH
136H,-COOH
NH2
HO-'36H,-'3qH-COOH CH,-THF P , - C NH,O O H
[ap-"Cl serine
[p-'3c]serine
'
dually enriched because the a-13C was derived directly from the supplied [a-13C]Gly, whereas the P-I3C was derived indirectly from [ ~ ~ ~ C l via G l y5,10-13C-methyleneTHF by GDC action on [~x-~~C]Gly (Fig. 1, c). The NMR signal of [a-p-13C]Ser is evident as two doublets due to one bond coupling (J = 38 Hz) between the adjacent 13Cs; the
doublet representing the CX-'~C is centered on the resonance of [a-13C]Ser,whereas the doublet representing the P-13C is centered on the resonance of [P-13C]Ser. The presence of both [P-'3C]Ser and [a-P-l3C]Ser indicate that GDC and SHMT activities interacted in vivo through a common pool of THF in the transfer of the 13C from Gly to Ser (Fig. 1). Phosphate E x t r a c t s
I n t a c t Shoots
p - Ser
a-Ser
a-Gly
L
-1
L
...
... mmm
N v v
omp.
m w w N w N
[c~-'~C]serine
mrl0
v1LoyI
\I I
p -Ser
1
a-Ser
a-Gly
1
1
I
d
b
a c
Figure 2. Metabolism of [ C Y - ' ~ C ] Ga, I ~ Complete . I3C spectrum of control plants showing background signal. b, Complete l3C spectrum with expanded regions of interest of whole shoots of plants supplied [ ( ~ - ' ~ c ] Cfor l y 1 2 h, showing the accumulation of [ ( ~ - ' ~ C l Gand l y its metabolism to [c~-'~C]Ser, [P-I3C]Ser, and [ ( ~ - p - ' ~ C ] S(pairs er of doublets). c, Complete 13Cspectrum of a phosphate extract of control plants showing background signal. d, Complete I 3 Cspectrum with expanded regions of interest of a phosphate extract of whole shoots of plants supplied [a-13C]GIy for 12 h. All spectra are from broad-band decoupled experiments as described in "Materiais and Methods."
13C NMR Detection of Ser Metabolism in Arabidopsis
21 1
However, the presence of the third species, [a-13C]Ser, is not necessarily indicative of GDC activity since the p-C supplied from 5,lO-methylene-THF could have had an endogenous origin other than Gly (Fig. 1). The existence of such an alternative pathway is demonstrated in the next experimental section involving Cl-THF- synthase.
T
Metabolism of ['3ClF~rmateto [P-l3C1Ser
The supply of [13C]formate should result in the activation of endogenous THF to 5,10-13C-methylene-THF if reactions 3 to 5 are coupled to each other. The 5,1O-I3Cmethylene-THF, if then available to SHMT along with unenriched Gly, would result in the synthesis of [p-13C]Ser (Fig. 1, a). When Arabidopsis plants were supplied with [13C]formate and unenriched Gly, [p-13C]Ser was the only significant metabolite detected in the NMR spectrum (Fig. 3 ) . Thus, the C1-THF-synthase and SHMT activities interacted through a common pool of THF in the activation and transfer of the formate carbon for Ser synthesis. Figure 3, a and b, shows the accumulation of Ser in intact shoots and phosphate-buffered extracts of shoots in experiments with a 1-s delay under broad-band decoupled conditions. The spectrum of the 5% perchloric acid extract was also similar (not shown). No major peak attributable to formate was detected in any of the spectra, although pure solutions of
--.*
o
O
C
I a
200
l
180
'
l
160
'
140
l
~
120
l
100
'
80
l
'
60
l
40
'
l
'
l
2 0 ppm
Figure 3. Metabolism of formate to Ser in intact plants. a, Complete broad-band decoupled 13C spectrum pulsed at 1-s intervals of whole shoots of plants supplied with [l3C1formate and unenriched Cly for 1 2 h. The only strong resonance is that of [P-l3C1Ser. b, Complete broad-band decoupled I 3 C spectrum, pulsed at 1-s intervals, of a phosphate extract of shoots of plants supplied with I'3Clformate and unenriched Cly for 12 h. c, Complete inverse-gated decoupled 13C spectrum, pulsed at 20-s intervals, of a phosphate extract of shoots of plants supplied with [l3C1formate and unenriched Cly for 1 2 h. The expansion of the singlet is shown above. The resonance of [p-I3C]Ser in the phosphate extracts occurred at 63.0 ppm.
I
I
I
I
12
15
18
21
I 24
Quantitation of the Synthesis of Ser via the GDC/SHMT and C1 -THF Synthase/SHMT Pathways
I n t a c t Shoots 1 s delay
~
I
9
[13C]formate and [I3C]formate added to the flask medium could be unequivocally detected under the same instrument parameters (not shown). When spectra were acquired with a 20-s time delay with inverse-gated decoupling (Fig. 2c), the nuclear Overhauser effect was minimized and the intensity of the [p-13C]Ser resonance appears smaller than in the broad-band, decoupled spectra. A few small peaks, evident just over the background, were observed between 170 and 180 ppm (Fig. 2c), although again no strong signal attributable to I3C-enriched formate was evident. The fact that the enriched (99% I3C) [13C]formate signal was not distinguishable over that of endogenous metabolites (1.1% 13C) indicated that free formate was maintained at extremely low concentrations in the plant.
Phosphate E x t r a c t 2 0 s d e l a y
~
I
6
Figure 4. Relationships between hours of supply of substrates and 13C-enriched Ser accumulation in phosphate extracts of shoots of plants by the CDC/SHMT and C1-THF synthase/SHMT enzymes systems. SDS for the Ser quantities from C1-THF synthase/SHMT activities were to0 small to be displayed on this scale. f.w., Fresh weight.
1
Phosphate E x t r a c t 1 s d e l a y
3
Time (h)
p - Ser
I
C1 -Synthase/SHMT
,*A
~
lTo 'quantitate l ~ ~ the relative accumulations of 13C-enriched Ser from the fluxes through the two pathways, plants were supplied with either [a-13C]Gly or [13C]formate and unenriched Gly. Throughout a 24-h period the accumulation of I3C-enriched Ser ascribed to the GDC/ SHMT pathway was about 4-fold greater than that from the C1-THF synthase/ SHMT pathway (Fig. 4). In the presence of [a-13C]Gly, three species of I3C-enriched Ser were synthesized via the GDC /SHMT pathway. Quantitative analyses of the individual species showed that after about 6 h of [a-13C]Gly supply, the majority of Ser synthesized was the dually enriched species (Fig. 5 ) . Furthermore, the proportions of
212
Prabhu et al.
Plant Physiol. Vol. 112, 1996
fanilamide (2 mM) and methotrexate (50 p ~ )a, sharp decrease in Ser synthesis occurred within 24 h of exposure to these antifolates (Fig. 7). The incorporation of label into Ser from both [I3C]formate and [a-13C]Gly was similarly reduced. However, up to 84 h of pretreatment with antifolates was necessary to reduce the Ser signal to near that of the background. Antifolate exposure did not reduce the signal of the Gly supplied to the plant, indicating that uptake of substrate was unaffected (spectra not shown). DlSCUSSlON
O
I
I
I
3
6
9
I
I
I
I
I
12
15
18
21
24
I
Time (h) Figure 5. Accumulation of proportions of the different species of
'3C-enrichedSer over time when [a-'3ClGly was supplied. the three species of Ser were maintained between 6 and 24 h (Fig. 5), whereas the total amount of Ser continued to increase during this period (Fig. 4).
NMR is powerful because it allows the simultaneous monitoring of severa1 enzymatic reactions by the appearance of supplied label in different positions of the same metabolite. Here we have begun to provide a framework of information concerning THF-dependent Ser synthesis in intact plants using NMR. The activities of THF-dependent enzymes appear to be strongly influenced by the number of polyglutamate residues attached to the THF molecule (McGuire and Coward, 1984). Which species of polyglutamylated THF is optimal for which THF-dependent enzyme is often not known, however, and is difficult and costly to determine. For example, the affinity constant of pea leaf SHMT decreases by an order of magnitude when the THF substrate used in vitro to measure the activity of this enzyme contains three or more glutamate residues (Besson et al., 1993). The study of THF-dependent metab-
lndependent Operation of the GDC/SHMT and C1-THF Synthase Pathways
Combinations of the I3C-enriched and unenriched substrates, formate and Gly, were used to examine the synthesis of Ser by one pathway in the presence or absence of the substrate of the other. When only [13C]formate was supplied, it was metabolized to [p-13C]Ser(Fig. 6a), and when [l3C]formate and unenriched Gly were supplied together the height of this resonance appeared to increase (Fig. 6b). Thus, the supply of Gly did not repress but instead appeared to enhance the synthesis of Ser via this pathway. Subsequently, the activity of the GDC / SHMT system was monitored in the presence or absence of formate. When only [cy-l3C]Gly was supplied, the two l3C-enriched species of Ser indicative of the GDC/SHMT reactions, [a+13C]Serand [P-13C]Ser,were evident along with [a-13C]Ser in the NMR spectrum (Fig. 6c). When [a-'"CIGly and unenriched formate were supplied together, [a-/3-13C]Serand [p-13C]Ser were still produced (Fig. 6d), showing that the operation of the GDC/SHMT pathway was not repressed by the presence of formate. This combination of experiments strongly suggests that the two pathways operate independently . 7 63 PPm 59
PPm
Effects of Antifolate Preexposure on Ser Synthesis from Formate and Gly
Figure 6. Detection of Ser synthesis when combinations of 13C-
Since Gly and Ser metabolism are THF-dependent, inhibition of the synthesis of THF should result in the reduction and ultimate cessation of their metabolism. When Arabidopsis plants were treated simultaneously with sul-
enriched and unenriched Gly and/or formate were supplied to plants for 12 h. Plants were supplied with [l3C1formate (a), ['3Clformate and unenriched Gly (b), [ ~ x - ' ~ C ] G l(c), y and [ ~ x - ' ~ C ] G land y unenriched formate (d). Chemical shift assignments are as described in previous figures.
13C
NMR Detection of Ser Metabolism in Arabidopsis
AA
24
60
59
72
84
PPm
Figure 7. The effects of pretreatment time with methotrexate and
sulfanilamide on Ser synthesis from formate and Gly. Plants were supplied with [13C]formate,unenriched Gly, and [ ( ~ - ' ~ C l Gconcurly rently after the pretreatment. The Ser signal represents the Ser produced during 1 2 h following the pretreatment with antifolates. The periods of pretreatment with antifolates are indicated beside the spectra. Chemical shift assignments are as described in previous figures.
olism thus poses a unique problem, and NMR is suited to examining THF-dependent metabolism in the cellular environment where the natural range and relative quantities of THF-polyglutamylation exist. Our results confirm that GDC and SHMT activities are closely coupled through a common pool of THF in Arabidopsis. Second, we have detected unequivocally in intact plants the flux of the single carbon of formate into Ser via the sequential activities of C1-THF synthase and SHMT enzymes. Third, our experiments strongly suggest that the operation of the GDC/SHMT pathway is independent of that of the C1-THF-synthase/SHMT pathway. Fourth, we have shown that the continued supply of THF is critica1 to allow for Ser metabolism in Arabidopsis. The significance of these results is discussed in more detail below. Metabolism of [ ( ~ - ' ~ C l G to l y Ser
Our experiments do not reflect actual photorespiratory conditions; although the shoots of our plants grew in the atmosphere above the liquid medium, they likely obtained the majority of their carbon from the SUCin the medium. However, our control experiments with plants grown un-
21 3
der photosynthetic conditions gave similar results. Since experiments with inhibitors required treatments that lasted up to 5 d, it was not possible to keep the plants growing in vials under photosynthetic conditions. Furthermore, nonaseptic supply of inhibitors for such long periods resulted in microbial contamination, which could alter their effectiveness. Flask culture allowed the uniform supply of inhibitors aseptically for the long durations needed for some of the experiments. No metabolites other than Ser were detected in our NMR spectra; it is possible that NMR was not sensitive to metabolites of low concentrations such as purines. However, it does not appear that purine biosynthesis in Arabidopsis derives single carbons from GDC activity. Arabidopsis plants deficient in GDC activity grow normally at high atmospheric carbon dioxide concentrations and are not auxotrophic for purines or their precursors; thus, GDC activity appears to be restricted to photorespiratory (Ser) metabolism (Artus et al., 1994), and other THF-dependent reactions must therefore not obtain single-carbon units directly from GDC activity. It has been postulated that 5,lO-methylene-THF is channeled directly between the T protein of the GDC complex and SHMT without being released from enzyme protein surfaces (Rebeille et al., 1994). Certainly, in NMR studies with tobacco cell cultures the coupling of the two sets of reactions through a common pool of THF was strongly suggested (Ashworth and Mettler, 1984). The strong intensity of the Ser signals in our study suggested that if 5,lOI3C-methylene-THF was first released from GDC before being used in Ser synthesis, it should have been present in the cell in sufficiently high concentration for its NMR signa1 to be detected, at least in extracts. This did not occur. In their studies of GDC / SHMT-coupled reactions in vitro, Rebeille et al. (1994) found that 5,lO-methylene-THF was released into their assay medium. However, Rebeille et al. (1994) also pointed out that the concentrations of proteins used in the in vitro assay were about 400 times lower than those recorded in the matrix of plant mitochondria. Our in planta NMR experiments strengthen the suggestion that in intact plants a high protein to THF ratio results in THF not being released from enzyme protein surfaces but instead being channeled from the T protein of the GDC complex directly to SHMT. Metabolism of [13C]Formate to [P-l3C1Ser
Our experiments showed that formate can contribute directly to the active THF single-carbon pool in Arabidopsis and clearly demonstrated that formate is incorporated into the third carbon of Ser. Previous studies of formate metabolism in plants showed the incorporation of radiolabel from formate into Ser, Asp, Ala, and Gly, as well as in severa1 other compounds (Gifford and Cossins, 1982a, 1982b). However, the possibility of nonenzymatic label incorporation by 3H exchange could not be discounted in one study (Gifford and Cossins, 1982a).Furthermore, since the positions of label in the metabolites were not evident, the biosynthetic route could not be clearly inferred. We did not observe the labeling of Asp, Ala, Gly, or organic acids.
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Formate has the potential to be incorporated as the a-C of Gly via 5,lO-methylene-THF through the reverse, synthase activity of the GDC complex. When Arabidopsis plants were supplied with [13C]formate, only [p-13C]Ser was detected in the NMR spectra. If [13C]formate activated to 5,1O-l3C-methylene-THF was utilized in the synthesis of Gly, then [ ~x-’~C]Gly and / or [a-p-13C]Ser should have been detected. In yeast cells [13C]formate was metabolized to [ ~x-’~C]Gly and [a-p-13C]Serinvolving the synthase activity of the GDC complex (Pasternack et al., 1992). It appears that in this respect metabolism in Arabidopsis differs from that in yeast. This difference is most likely due to the unique phenomenon of photorespiration in plants, which favors the equilibrium of the GDC / SHMT reactions to be in the direction of Ser synthesis (Oliver, 1994; Rebeille et al., 1994). The flux of formate through the CI-THF synthase/ SHMT system into Ser in our experiments with Arabidopsis is similar to that observed in yeast (Pasternack et al., 1992, 1994). The C1-THF synthase in spinach (Nour and Rabinowitz, 1991, 1992) and pea (Kirk et al., 1995) consists of monofunctional synthetase and bifunctional cyclohydrolase and dehydrogenase proteins. It is therefore likely that the Arabidopsis enzymes are also similarly organized, although this remains to be investigated. It appears that, although their structural organization may be different, the functional activity of the three enzymes in plants is similar to that in other eukaryotes. Formate dehydrogenase activity may regulate the availability of formate for activation of THF in plant tissue (Oliver, 1981; Colas des Francs-Small et al., 1993). The expression of formate dehydrogenase is greatest in nongreen tissues (Colas des Francs-Small et al., 1993) and therefore could regulate the transport and accumulation of formate in leaf tissue. Ser biosynthesis using formate in roots requires further investigation. Quantitation of the Synthesis of Ser via the CDC/SHMT and C1-THF Synthase Pathways
Extracts of tissues prepared with concentrations of perchloric acid ranging from 4 to 70% have been used in NMR studies to quantitate many metabolites, including amino acids (Chang and Roberts, 1989; Sumegi et al., 1992; Gout et al., 1993). Qualitatively, our extracts made with phosphate buffer and perchloric acid were similar. However, in quantitative analyses of our samples, extracts utilizing perchloric acid at a concentration of 5% resulted in large losses in 13C-enriched Ser relative to phosphate-buffered extracts. We did not see any enriched metabolites unique to the perchloric acid extracts. Phosphate-buffered extracts were also used in NMR studies of Ser synthesis in yeast (Pasternack et al., 1992, 1994). The proportions of each enriched species in a mixture of isotopomers when quantitated can give valuable information about the relative contributions of specific enzymatic processes. The intensities of 13C-13Cmultiplets versus 13C12C center Iine singlets in mixtures of isotopomers are independent of any potential differences in TI and nuclear Overhauser effects for protonated carbons (London et al., 1975; Suzuki et al., 1975; Dickinson et al., 1983), especially
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when spectra are acquired under gated, decoupling conditions with an adequate time delay (Gadian, 1982). We found that estimates of the Ser concentrations in our samples agreed well under both the broad-band and gated decoupling conditions and that the amounts of Ser accumulated in our plants were in close agreement with other studies of plant cells by NMR (Ashworth and Mettler, 1984). The accumulation of Ser in plants via the GDC/SHMT pathway was about 4-fold greater than that through the C1-THF synthase/SHMT pathway. It is possible that the accumulation of Ser does not reflect the actual flux through the pathways because of differential metabolism of the two pools of Ser in different compartments. However, based on the very high activity of GDC (Besson et al., 1993; Oliver, 1994) and mitochondrial SHMT (Besson et al., 1995), the accumulation of Ser most likely reflects the potential of flux through the two pathways. lndependent Operation of GDC/SHMT and C1 -1HF Synthase/SHMl Pathways
We found the synthesis of Ser via the GDC/SHMT and C1-THF-synthase / SHMT pathways to occur independently of each other in Arabidopsis. Although the GDC/ SHMT system is located in mitochondria (Oliver, 1994), the intracellular location of Ser synthesis from formate remains unknown in Arabidopsis. In yeast, mitochondrial and cytoplasmic isozymes of both CI-THF synthase and SHMT exist, but about 90% of the total utilization of formate for Ser synthesis occurs via the cytosolic form of C1-THF synthase (Pasternack et al., 1992, 1994). Higher plants contain mitochondrial, cytosolic, and plastidic isoforms of SHMT (Somerville and Ogren, 1981; Walton and Woolhouse, 1986; Turner et al., 1992; Besson et al., 1995). However, the mitochondrial SHMT in plants and in Arabidopsis in particular (Somerville and Ogren, 1981) does not appear to serve any function other than that in photorespiratory metabolism, since mutant plants deficient in mitochondrial SHMT grow normally under nonphotorespiratory conditions. Thus, in plants, Cl-THF synthase may play a broader role in supplying the single carbons for cellular THFdependent metabolism than in other types of organisms. Through the use of mutant lines deficient in mitochondrial GDC or SHMT (Somerville and Ogren, 1981; Artus et al., 1994), we hope to examine the ability of different sources to satisfy single-carbon requirements in Arabidopsis. NMR studies of the metabolism of these 13C-enriched compounds in the mutant lines should help clarify some aspects of compartmentation. Effects of Antifolate Preexposure on Ser Synthesis from Formate and Gly
The antifolates sulfanilamide and methotrexate effectively inhibited the THF-dependent synthesis of Ser when supplied to whole Arabidopsis plants. Indeed, in vitro studies have shown that the two target enzymes DHFR and dihydropteroate synthase from Arabidopsis are strongly
13C NMR Detection of Ser Metabolism in Arabidopsis inhibited by methotrexate and sulfanilamide, respectively (V. Prabhu, H . Lui, and J. King, unpublished results). The combined action of these inhibitors was also effective i n experiments w i t h yeast in vivo (Pasternack e t al., 1994). Whereas t h e GDC a n d C1-THF synthase reactions for Ser synthesis could use different pools of THF because of compartmentation of metabolism (Appling, 1991), in Arabidopsis the synthesis of Ser f r o m formate a n d Gly was inhibited similarly, suggesting that t h e pools of THF i n b o t h pathways were equilibrated rapidly. The s h a r p decrease in Ser synthesis in Arabidopsis after a 24-h exposure to sulfanilamide and methotrexate added together reflects inhibition of the de n o v o p a t h w a y of THF biosynthesis. Methotrexate inhibition of DHFR w o u l d also inhibit recycling of dihydrofolate resulting f r o m thymidylate synthase activity. Our results suggest t h a t t h e continuous supply of THF is critica1 t o Ser metabolism. The continued, albeit very much reduced, synthesis of Ser bet w e e n 24 and 84 h of antifolate exposure suggested that either THF continued t o be mobilized from existing pools d u r i n g this extended period or reduced biosynthesis of THF continued despite the presence of antifolate inhibitors. Low levels of THF could also be recycled from dihydrofolate released d u r i n g thymidylate synthesis, if DHFR activity w a s not completely inhibited. In yeast, just 3 h of antifolate e x p o s u r e was sufficient t o inhibit completely Ser synthesis from f o r m a t e (Pasternack e t al., 1994), w h i c h i s reflective of the high g r o w t h rates and metabolic flux in t h a t organism. T h e complex cellular organization of higher p l a n t s perhaps allows m e t a b o l i s m to c o n t i n u e for long periods before lethal d r u g s have a complete effect.
ACKNOWLEDCMENTS We thank Dr. Francis Martin, Centre de Institut National de la Recherche Agronomique de Nancy (France) for critica1 reading of a previous version of this manuscript, and we acknowledge the helpful suggestions of two anonymous reviewers. We also thank Helen Lui for technical assistance with the growth of Arabidopsis plants in flask culture and Dennis Dyck for assistance with the figures. J
Received March 21, 1996; accepted June 6, 1996. Copyright Clearance Center: 0032-0889/96/112/0207/ 10.
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