Jan 7, 1981 - rable amounts. At longer labeling times, soluble 3S-methionine. EE. E. ~~~~~~~~~~~~~ui. 1 MINUTE. 4 MINUTE. 200400. 400. 100 53300. 300.
Plant Physiol. (1981) 68, 619-625 0032-0889/81/68/0619/07/$00.50/0
Homocysteine Biosynthesis in Green Plants: Physiological Importance of the Transsulfuration Pathway in Lemna paucicostata Received for publication January 7, 1981 and in revised form March 23, 1981
PETER K. MACNICOL', ANNE H. DATKO, JOHN GIOVANELLI, AND S. HARVEY MUDD2 National Institute of Mental Health, Laboratory of General and Comparative Biochemistry, Bethesda, Maryland 20205 least 97% of the total net homocysteine synthesis (12). To extend these studies to higher plants, we have now carried out similar To permit an assessment of the relative contributions of the transsulexperiments with Lemna paucicostata Hegelm. 6746. This plant furation and the direct sulfhydration pathways for homocysteine biosynhas significant advantages for rapid radioactive labeling studies thesis, the time course of incorporation of 35S from 36SO4- into Various its small (e.g. size, simple structure, and aquatic habit as well as sulfur-containing compounds in Lemna pauicostaa has been determined. Plants were grown with either low (4.5 micromolar) or ample (1,000 the ease of obtaining reproducible populations of vegetatively micromolar) sulfate in the medium. At the shortest labeling times, 655. reproducing plants growing rapidly under axenic conditions [7, cystathionine was the predominant "S-conti organic sulfur com- 15]). Considerable background knowledge about the sulfur metabpound. The flux of sulfur into cystathionine was sufficient to sustain the olism of Lemna is now available (1-3, 6, 8, 10, 16). Further, we known rate of methionine biosynthesis. It was calculated that transsulfur- have developed a phytostat which permits the growth of this ation accounted for at least 90 and 85% of the total homocysteine synthesis microphyte in semicontinuous culture with sulfate maintained at in low and ample sulfate-grown plants, respectively (and may have ac- very low, but constant, concentrations in the growth medium (9). counted for 100%). No marked rise in the 3S-soluble cysteine:3S-homo- Under these conditions the inorganic sulfate pool of the plants is cysteine ratio was observed even at the shortest labeling times, but it is lowered (8) and, as will be shown presently, radioactivity from argued that this may be due to (a) the observed compartmentation of 35so42- enters more rapidly into the intermediates in the methiosoluble cysteine, and (b) the impracticality of using labeling times shorter nine biosynthetic pathway. The results ofthese studies are reported than 17 seconds. Additional evidence supporting the importance of trans- here. sulfuration in Lemna is briefly described. ABSTRACT
MATERIALS AND METHODS
Green plants possess the enzyme activities to synthesize homocysteine, the immediate precursor of methionine, by either of two pathways (13). Transsulfuration involves the transfer of sulfur from cysteine to homocysteine via the intermediate, cystathionine
(4, 11): Cysteine + O-phosphohomoserine -- cystathionine + Pi (1) Cystathionine + H20-* homocysteine + pyruvate + NH3 (2) Cysteine + O-phosphohomoserine (1)+(2) + H20 -. homocysteine + pyruvate + NH3 + Pi The alternative is the direct sulfhydration of O-phosphohomoserine (6): H2S + O-phosphohomoserine -. homocysteine + Pi (3) Study of the rates at which sulfur from 35so42- accumulated in various sulfur compounds in Chlorella sorokiniana permitted us recently to estimate that for this alga growing in a steady state with limiting sulfate the transsulfuration pathway contributes at ' Present address: Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, Canberra, A. C. T., Australia. 2To whom requests for reprints should be sent.
619
Chemicals. Chemicals used in the growth medium for Lemna were obtained as described (9). Commercially available reagent grade chemicals, chosen because according to the supplier's specifications they contained the lowest amounts of sulfate contamination, were used for preparation of the medium. During the course of the experiments it became clear that the medium contained more sulfate than had been anticipated. The source of the contaminating sulfate was traced to the preparation of EDTA (9). Values for sulfate concentrations given in this paper have been corrected for this contamination. Sources and preparation of nonradioactive and radioactive chemicals were as in previous work (12). Plant material. Lemna paucicostata Hegelm. 6746 was grown in stock culture as described (9). Experimental colonies were grown photoautotrophically either at 4.5 IM sulfate (in a phytostat with the input medium containing 5.3 lsM sulfate and the flow rate maintained at 10 ml/colony .24 h) (9) or at 1 mm sulfate (batchwise in 1200 ml medium contained in 2-liter flasks) (9). Precautions Against Contamination. Freedom of the plant samples from microorganisms was ensured by culturing samples of medium and homogenized plants at frequent intervals (9). Any contaminated cultures were discarded. General Methods. To avoid cross-contamination by trace amounts of radioactive amino acids, all glassware used for extraction and assay was either new or had been soaked overnight in 10%1o HNO3 in H2SO4. 35S in individual amino acids was assayed by addition of an exactly known amount of authentic tritiated
620
MACNICOL ET AL.
amino acid to the sample and purification to constant 3S:3H ratio by successive paper electrophoresis and/or chromatography. When a constant 3S:3H ratio was reached, the 3S content of the compound was calculated as the product of this ratio and the amount of tritium added (12). Further details concerning these methods are described in the cited papers from this laboratory. The following electrophoresis buffers were employed: buffer 1, 1.16 M acetic acid (pH 2.5); buffer 2, 2% (0.48 N) formic acid, 10 mM 2-mercaptoethanol; buffer 3, 25 mm Na-acetate, 10 mM acetic acid, 1 mM Na2EDTA. The following solvents were used: solvent 1, methanol:pyridine:HCl (1.25 M) (37:4:8); solvent 2, 2-propanol: formic acid (88%) (6:4); solvent 3, 1-butanol:acetic acid:water (12: 3:5); solvent 4, 2-propanol:formic acid (88%):water (7:1:2); solvent 5, acetone: 0.5% (w/v) urea (3:2, v/v) containing 10 mm 2-mercaptoethanol. For the assay of cystathionine, the papers for electrophoresis and chromatography were prewashed with glacial acetic acid and water. Short-Term Labeling Experiments. Incubations of Lemna with 35S-sulfate for periods of 17 s to 60 min were performed in baskets made of fine-mesh stainless steel gauze with sides 2.5 cm high and handles. The floor of the baskets varied in area from 9.5 to 91.5 cm2, depending upon the number of colonies to be incubated. The baskets were contained in a 19-cm crystallizing dish equipped with a sparger and filled to just below the tops of the baskets with sterile presparged "holding medium." In the case of the plants grown in the phytostat this holding medium was 4.5 pM in sulfate. Plants were rapidly counted from the phytostat into these baskets. This and subsequent operations were performed in a laminar flow hood at 25 C at a vertical light intensity of 50 to 60 ,uE m 2 s-1 of photosynthetically active light. The medium was continuously sparged with sterile 1% (v/v) C02 in air, and the same gas mixture was gently blown over the plants through a sterile nozzle with many perforations. When all the baskets were filled with plants to confluence, each basket was briefly lifted up, drained, placed on several layers of filter paper while the few colonies hanging on the sides were gently pushed to the bottom with a spatula, and the basket was transferred to a sterile empty 19-cm dish. As soon as this dish was filled with the baskets, 200 ml of presparged, temperature-equilibrated 35S-sulfate-containing medium was added through a funnel between the baskets and a stopwatch was started. The dish was gently swirled and C02/air blown over the plants as before. Just prior to termination ofthe desired incubation time, the appropriate basket was removed, shaken to remove most of the excess medium, and the reaction was terminated by dropping the basket into a beaker of boiling 80%o ethanol containing 10 mm acetic acid. The beaker was returned to the hotplate and the ethanol allowed to simmer for approximately 3 min. After cooling, the plants with supernatant solution were transferred to a stoppered jar and stored at -40 C pending extraction. In the case of the plants grown in medium containing 1 mm sulfate, the holding medium was 1 mm in sulfate. Immediately prior to incubation with 3SO42, this medium was replaced with presparged medium, 4.5 pM in sulfate, by aspirating the original medium away through a sterile pipet, adding 4.5 Am sulfate medium to the same level, swirling thoroughly, aspirating away, and apain adding fresh 4.5 pm sulfate medium. Incubation with 35SO4 - medium then followed, using the same procedure as for plants grown at 4.5 uM sulfate. When necessary in order to obtain sufficient material, replicate incubations were carried out and the samples combined. Intermediate-Term Labeling Experiment. Plants were grown in the phytostat at 4.5 pM sulfate to about 80%o of confluence and then harvested back to approximately 800 colonies. At zero time the medium was aspirated out and replaced with 2 liters of sterile, presparged, temperature-equilibrated, 3S-sulfate-containing medium, 4.5 uM in total sulfate. Pumping of medium of the same specific activity, but 5.3 ,UM in sulfate into the phytostat was then
Plant Physiol. Vol. 68, 1981
immediately commenced. About 5 min before the end of each desired labeling time (2-15 h) approximately 60 colonies were transferred into presparged, 4.5 jLM MS-medium at 25 C in a beaker, and from these the required number of colonies was counted out into the same medium in another beaker. At the required time the radioactive medium was aspirated off and boiling 80%o ethanol, 10 mm acetic acid was poured over the plants, which were then treated as described above. Extraction of Lemna and Fractionation of Extracts. Each sample of killed plants in 80%o ethanol, 10 mM acetic acid was treated essentially as described by Datko et al. (8) ("initial procedure for extraction of tissue and assay of 35S compounds") with omission of the methanol-chloroform-water extraction of the pellet and, in later experiments, addition of a 5% TCA extraction of the pellet to remove coprecipitated inorganic sulfate (12). The water soluble fraction from each preparation was used for analysis of 3S amino acids after addition of suitable tritiated markers, and the pellet was used for analysis of 35S-protein cysteine3 and 35S-protein methionine, essentially as previously described (8, 12). Cysteic, homocysteic, y-glutamylcysteic and GSH sulfonic acids were isolated from the "acidic oxidation products" fraction by paper electrophoresis in buffer 1. Each compound was then purified by successive paper chromatography. For cysteic and homocysteic acids the solvents used were 1, 2, and 3 (see under "Materials and Methods"), in that order. rGlutamylcysteic acid and GSH sulfonic acid migrated together during chromatography with solvents 1 and 2. These compounds were then separated by prolonged chromatography in solvent 3 (8). Methionine sulfone was isolated from the "neutral oxidation products" fraction by successive paper chromatography in solvents 4 and 2, in that order. Cystathionine was isolated from an independent aliquot of the water soluble fraction by Dowex 50-H+ chromatography (12) followed by successive electrophoresis in buffer 2, chromatography in solvent 4 containing 10 mm 2-mercaptoethanol (3 MM paper), repeat electrophoresis in buffer 2, and finally chromatography in solvent 5. Because of interference by yellow phenolic material, the loading on the paper was reduced to not more than 50 Lemna colonyequivalents per cm lane width. Even so, movement on the first electrophoretograms was retarded and the tritiated marker cystathionine split into two zones, the larger of which was nearly immobile. These fractions were chromatographed separately in solvent 4. Inasmuch as they had the same RF, they were recombined for the remaining steps. Assay of 3"S in Protein Cysteine and Protein Methionine. Each protein-containing pellet was oxidized with performic acid and hydrolyzed, and the hydrolysate was separated into acidic and neutral oxidation products as described (12). Cysteic acid (in the acidic oxidation fraction) was purified, after addition of tritiated marker, by successive electrophoresis in buffer 3 and chromatography in solvents 4 (3 MM paper), 2 and 3, in that order. For the electrophoresis step the loading was reduced to approximately 15 colony-equivalents per cm width of lane; it was then increased by a factor of two for each succeeding step. Methionine sulfone was purified after addition of tritiated marker by electrophoresis in buffer 2 and chromatography in solvents 4 (3 MM paper) and 3, in that order. The loading was reduced to approximately 25 colony-equivalents per cm for the first step and 50 per cm for the second. RESULTS During the present studies, Lemna colonies growing photoautotrophically on media containing fixed concentrations of sulfate
3For convenience, "cysteine" and "homocysteine" as used in this paper in connection with compounds present in intact plants do not necessarily imply a distinction between the reduced (sulfhydryl) forms and the oxidized (disulfide) forms.
Plant Physiol. Vol. 68, 1981
TRANSSULFURATION IN LEMNA
621
were exposed continuously to 35So42- for varying periods. Because of the wide range in the durations of the labeling periods, several separate experiments were carried out, similar in principle, but differing in the specific radioactivity of the 35So42- in the medium and in the details of the experimental design (as described under "Materials and Methods" under "short-term" and "intermediate- E E term," and in reference [8]). The 35S which had accumulated in a a variety of compounds in the plants during these labeling periods was then measured as described under "Materials and Methods." The results of most importance for an assessment of the relative , . contributions of the transsulfuration and direct sulfiydration 15 20 25 30 DISTANCE FROM ORIGIN, cm pathways were obtained by measurements of 35S-cystathionine. Any cystathionine present in a plant sample would have been FIG. 2. Distribution of MS after chromatography of neutral oxidation found (as its oxidation product) in the neutral oxidation products products a sample of plants labeled to an isotopic steady state by fractions obtained during the purification procedure (8, 12). Figure growth infrom had been grown for 5.8 doublings in 4.5 5so42-. 1 illustrates the distribution of 3S after various samples of the JUM 3SO42- (see TableTheI inplants reference [8]). Solvent 4 was used to develop the neutral oxidation products fractions had been subjected to paper The 3H peak at cm 27 to 28 was due to authentic 3Hchromatography. For comparison, in Figure 2 is shown a repre- chromatogram. methionine sulfone; the 3H peak at cm 4, due to the oxidation product of sentation of a similar chromatogram of the neutral oxidation authentic 3H-cystathionine. products derived from plants labeled to an isotopic steady state by growth in the presence of 35SO42-. These Figures show that when the plants had been incubated for short periods with 35s42- (as exemplified here by the "1 minute" sample) most of the 35S in the I me cystathionine neutral oxidation products fraction travelled in a single, slowly 60 -600 moving peak (centered about cm 6 in these chromatograms). It will be shown below that this peak was due to the presence of 35S50 -500 cystathionine (assayed here as its oxidation product). At 1-min labeling time, the amount of 35S in soluble methionine (assayed z 40 -400 here as its sulfone, and indicated by the 3H peak at cm 21 to 23) was much less than the amount of 35S present in cystathionine 30- -300 oxidation product. After 4 min of labeling with $o42, 35S C,, cystathionine and soluble 3S-methionine were present at compaz 20- -200 rable amounts. At longer labeling times, soluble 3S-methionine 0 C. ,-;,;.
-
..,, .
.
10 EE
E
E
~~~~~~~~~~~~~ui 1
200400 100
MINUTE
4
800
MINUTE 400
53300
300 -600
150-200
200- -400
50 -100
100
60 MINUTE
15 HOUR
2000-4000
400-
1500 --3000
300
1000-2000
200
500-1000
100
1
-200
5
10
15
20
25
30
1
DISTANCE FROM
5
ORIGIN,
10
15
20
25
30
cm
FIG. 1. Distribution of 3S after chromatography of neutral oxidation products. Neutral oxidation products fractions were prepared from plant samples which had been incubated with 5so42- for the indicated periods of time. Aliquots of these fractions were subjected to chromatography with solvent 4, and the distribution of radioactivity on the chromatograms was determined. The peak of 'H radioactivity (approximately cm 21 to 23) indicates the position of an internal standard of 'H-methionine sulfone. Due to differences in the aliquots used for chromatography and in the specific radioactivity of the 5SO42- in the medium during labeling, in these representations the relative rates of accumulation of 3S may be judged directly for different compounds on the same chromatogram, but not for the compounds on different chromatograms. The prominent peak between 15 and 20 cm at later incubation times was predominantly Smethylmethionine sulfonium.
41 46 18 23 DISTANCE FROM ORIGIN, cm
28
100
33
FIG. 3. Distribution of radioactivity in final chromatograms of isolates of soluble methionine (sulfone) (left side), and cystathionine (right side) from plants labeled to an isotopic steady state with 35so42- Conditions of labeling are described in the legend to Figure 2. Purification procedures were as described under "Materials and Methods," with omission of the repeat electrophoresis in buffer 2.
greatly predominated over 35S-cystathionine until, in the sample from plants labeled to isotopic steady state, no discrete peak of S corresponding to cystathionine oxidation product (indicated by the 3H peak at cm 4) could be discerned (Fig. 2). As described above, cystathionine, soluble methionine (as the sulfone), and a number of other 3S-containing compounds were each purified from each plant sample, along with authentic tritiated markers, the presence of which made it possible to establish the radiopurity of each final isolate and to correct for losses during purification. Figure 3 shows chromatograms of the final preparations of methionine (sulfone) and cystathionine isolated from plants labeled to isotopic steady state. These results show that even when the starting material was as relatively impure as the 35S-cystathionine in the isotopic steady state labeled plants (Fig. 2), the purification methods used led to the isolation of 35Scontaining compounds which co-chromatographed with the corresponding authentic 3H-labeled compound and were reasonably free of contaminating 35S, and therefore suitable for calculation of the amount of 35S-compound in the original plant sample. The results obtained from several experiments in which plants were labeled for various periods are summarized in Table I. To permit comparison of the values in this Table, the 3S content of
622
MACNICOL ET AL
Plant Physiol. Vol. 68, 1981
Table I. Accumulation of 35S in Various Compounds The techniques for performing the "short term" labeling and "intermediate term" labeling are described under "Materials and Methods." The concentration of 3SO42- in the labeling medium was 4.9 ylM in Experiments A and D, 5.1 tM in Experiment B, and 4.5 pi in Experiment C. Labeling Conditions
Sulfate Concn. in Growth Medium
Num-
3SO4in medium
berof colo-
Time
Soluble Cys- Cystathioteine nine
Homocysteine
rltml
Soluble Me- ysteine aYnd thionine Glutathione
Protein
Protein Me-
Cysteine
thionine
nies
dpm/10'l8
dpm/lO08 mol 3SO4t-
s dpm/colony mol Experiment A-"short term" 4.5 0.354 17 1,502 3.48 21.8 1.35 1.02 2.49 9.32 ND(
