Aspartokinase in Lemna Minor L

Plant Physiol. (1973) 51, 327-331

Aspartokinase in Lemna minor L.

STUDIES ON THE IN VIVO AND IN VITRO REGULATION OF THE ENZYME'

Received for publication August 17, 1972

KWAN F. WONG AND DAVID T. DENNIS Department of Biology, Queen's University, Kingston, Ontario, Canada

ABSTRACT shows feedback regulation by lysine alone and a concerted feedback inhibition by lysine and threonine. The growth of Lemna minor was followed by means of frond Dunham and Bryan (7, 8) have reported that the growth number, fresh weight, and dry weight measurements in the of Marchantia gemmalings is inhibited by several amino acids presence of various amino acids at a concentration 0.25 mM. of the above There was a pronounced inhibition Lysine inhibited growth but not to the same extent as threonine pathway. by lysine and threonine which they postulated as being a con- and homoserine. Isoleucine was also an inhibitor of growth. certed inhibition of aspartokinase. However, they did not In the presence of methionine there was some growth for 2 to present any direct evidence for 3 days, but most of the to this, since they were unable to by 5 days plants appeared be dead. isolate the enzyme. The data in the previous paper (16) When lysine and threonine were added there was no would together, indicate that such a mechanism be A growth at all, and the were may operating. compari- plants dead after 5 days. This effect son of lysine + threonine could be reversed methionine of the in vivo and in vitro properties of aspartokinase from by adding one species would demonstrate the or homoserine to the growth medium. importance of this enzyme The isolated aspartokinase from Lemna showed inhibition in regulation of the aspartate family of amino acids. In this by lysine and higher concentrations of threonine. When report it is shown that the growth of Lemna minor L. is greatly these inhibited by lysine and threonine when added together. amino acids were added together at low there This concentrations, inhibition can be relieved was a concerted inhibition of the It is by the addition of methionine or aspartokinase. suggested homoserine, a precursor of methionine. This that some effects of amino acids on the growth of L. minor can indicates that the in vivo behaves in a be explained on the basis of a concerted feedback control of enzyme similar manner to the isolated aspartokinase. enzyme. MATERIALS AND METHODS Growth of L. minor. Sterile fronds of L. minor were obtained by the method of Hillman (10). The plants were grown under sterile conditions in 250-ml Erlenmeyer flasks, each containing 100 ml of the following growth medium: MgSO, The amino acids isoleucine, threonine, homoserine, methio- 2 mM; Ca(NOJ),, 7 mm; KNO,, 5 mM; KHaPO, 2 mM; sucrose, nine, and lysine are all derived from aspartate in bacteria and 10 mM; FeNaEDTA, 38 1M; H.BO., 46 ,iM; MnCl,, 9.2 FiM; in plants (4-6, 14, 15). The branched pathway for the bio- CUSO4, 3.2 ,uM; ZnSO4, 1.8 p,m; Na,MoO4, 4.1 lAm; CoClk, 3 ,m; synthesis of these amino acids is shown in this scheme and each of the L-amino acids (0.25 mM) tested. The final pH was 6.0. In all cases, sterile conditions were achieved by auto- ASPARTATE claving the growth medium at 15 p.s.i. for 15 min and by transferring the Lemna in a sterile cabinet. -ASPARTYL- PHOSPHATE Usually, one plant consisting of three to four fronds was transferred to the growth medium, and the cultures were grown in a growth cabinet of 26 C and in continuous light -ASPARTIC-SEMIALDEHYDE with an intensity of 1000 ft-c. The growth of the plants was determined by the number of fronds, the fresh weight, and METHIONINE.*-- HOMOSERINE LYSINE dry weight. All tests were run in duplicates and the experiments were repeated three times. Preparation of THREONINE Aspartokinase. Fronds of L. minor were harvested, washed with distilled water, and dried of excess water. They were ground in a Waring Blendor for 1 min with ISOLEUCINE acetone precooled to -15 C. The homogenate was filtered through a Biichner funnel and washed thoroughly with cold The first enzyme of this pathway is aspartokinase (ATP-L- acetone until the filtrate was colorless. The slightly green aspartate 4-phosphotransferase, EC 2.7.2.4), and it was powder was dried and then stored at -15 C. shown in the previous paper that this enzyme from wheat germ The acetone powder was suspended in 0.05 M TES (pH 8.0) in a ratio of 1:20 w/v. After stirring for 30 min the suspen- sion was filtered through eight layers of cheesecloth and then 'This work was supported by Grant A5051 from the National centrifuged at 20,000g for 20 min. The crude extract was Research Council of Canada. fractionated with saturated ammonium sulfate, and the frac- 327 328 WONG AND DENNIS Plant Physiol. Vol. 51) 1973 tion precipitated between 0.78 M and 1.56 M was collected by 2.5 centrifugation as described above. The protein collected was dissolved in 0.05 M TES-30% glycerol (pH 8.0) and dialyzed o- Con t ro I thoroughly overnight in the same buffer. All procedures were *- Lysine carried out at 4 C. The enzyme preparation was used without 2.0 A- Homoserine further purification. o-Threonine Assay for Aspartokinase Activity. Aspartokinase activity from Lemna extract was assayed as previously described (16), *1.5 except that the reactions were carried out for 1 hr. Protein z was estimated by first precipitating the protein with 10% tri- chloroacetic acid and then dissolving the precipitate in 1 N 0 .1.0 NaOH. The solution was then estimated by the biuret method LL0 (9). 0,

RESULTS 0.5 Effect of Amino Acids on Growth of Lemna. The effects of six individual amino acids and combinations of amino acids on the growth of L. minor for a 10-day period are shown in 0 Table I. In the control medium where amino acids were not added, fronds were healthy, dark green, and roots reached the 0 2 4 6 8 10 bottom of the flask. Plants cultured in a medium containing D a y s aspartic acid were similar to the control plants except for a slightly rate of Medium FIG. 1. Effect of amino acids on the growth of L. minor. The higher growth. containing lysine plants were cultured as described in "Materials and Methods." The caused shorter root growth and a slower rate of over-all growth number of fronds in each flask were counted daily for 9 by about 25%. The effects of threonine and homoserine in- days. dividually appear to be similar. Both caused a decrease in 2.5 frond size and multiplication, a clumping of fronds for the first 6 to 7 days, and a shorter root growth. In time these *_ Lys-Thr- Hom plants turn to a yellowish green color. Plants grown in isoleucine showed a slight modification in the shape of the fronds 2 .0 o- Lys-Thr- Met and a slower growth rate. Methionine completely inhibited growth after 5 days. Occasionally, however, some plants sur- z vived, but their fronds were very small and their growth was 1o.5 X- LYs-Thrv very slow. 10 The most dramatic effect on the growth of Lemna was seen U. when plants were grown in a medium containing both lysine 0 and threonine. After 1 day the three fronds which make up the intact plant separated from each other and lost their ability to 0 multiply and grow. After 2 days the fronds showed signs of 0.5 5X-X-X~xX Table I. Effect of L-Arnino Acids oli the Growth of L. minlor in 10 Days L. minor were grown in the growth media containing the indi- cated amino acids. Each amino acid was at 0.25 mm. 0 2 4 6 8 10 Addition Frond Fresh Dry D ays FIG. 2. Effect of combination of amino acids on the growth of mtg L. minor. The plants were cultured as described in "Materials and None 373 433 51.5 Methods." The number of fronds in each flask were counted daily Aspartic acid 400 554 56.1 for 9 days. Lysine 230 275 37.9 Threonine 84 20 9.6 death. The color of the fronds changed from green to dark Homoserine 98 33 11.5 brown, yellow, and finally to cream color in about 8 days. Isoleucine 73 84 16.9 Exactly the same changes were observed for Lemna grown in Methionine D1 DI D' a medium containing lysine-threonine-aspartate and lysine- Lysine + threonine D2 D2 D2 threonine-isoleucine. Lysine + threonine + aspartic acid D2 D' D2 The separation of fronds from the intact plant, changes Lysine + threonine + isoleucine D2 D2 D2 in pigmentation, and death caused by lysine-threonine were Lysine + threonine + homoserine 203 120 28.2 prevented by the further addition of homoserine or methionine Lysine + threonine + methionine 48 41 12.2 to the medium. The plants remained intact, green, and have the ability to multiply. However, growth was slower than in 1 Plants showed signs of death after 5 days. Occasionally, some the control plants. Of the two amino acids, homoserine was plants will multiply, but fronds were very small and growth was more effective in relieving the concerted inhibitions exerted by very slow. lysine and threonine. The effect of amino acids on the growth 2 Plants died within 5 days. of L. minor is shown in Figures 1, 2, and 3. 329 Plant Physiol. Vol. 51, 1973 ASPARTOKINASE IN LEMNA MINOR L.

11.

FIG. 3. Effect of amino acids on the growth of L. minor after a 10-day period. 330 WONG AND DENNIS Plant Physiol. Vol. 51, 1973

Table II. Effect of L-Amino Acid on Aspartokinase Activity the wheat germ enzyme and the enzyme from L. minor are Enzyme activity was assayed as described in "Materials and due to changes in the enzyme during extraction. There is also Methods." Each assay contained 3.3 mg of protein. the possibility that isozymes are present with different regulatory properties and that the proportions of the various isozymes Addition Eazymic Activity change in different preparations and different tissues. So far there is no evidence for isozymes of aspartokinase in plants. A/lkr X 103 The effect of amino acids on the growth of L. minor is simi- None 16 lar to that reported for Marchantia gemmalings. Many of the Threonine (0.5 mM) 16 growth effects can be accounted for by an effect of the amino Lysine (0.3 mM) 10 acids on aspartokinase. When an external source of amino Lysine (0.3 mM) + threonine 6 acids is provided the rate at which each one is taken up may (0.5 mM) be different so that the actual internal concentrations of the Methionine (5.0 mM) 14 various amino acids may be different. Also there may be sec- Homoserine (5.0 mM) 14 ondary effects of the amino acids not related to feedback con- Isoleucine (5.0 mM) 14 trol of their synthesis. The amino acids used in this study are metabolically related to aspartic acid. Isoleucine and methionine show a pro- Growth of Lemna was also investigated in media containing nounced inhibition of growth which cannot be explained as a 0.05 mm and 0.5 mm of the amino acids. With 0.05 mm only simple feedback inhibition of aspartokinase. It is possible that methionine and lysine-threonine were slightly inhibitory. With they exert an effect by a repression of the enzyme such as that 0.5 mm lysine, threonine, homoserine, and isoleucine each in- reported for E. coli (2, 13). The action of homoserine in inhibited growth, whereas methionine and lysine-threonine killed hibiting growth is difficult to explain, since it has no known the plants. effect on any enzyme of the pathway. In some plants, such as Effect of Amino Acids on the Isolated Aspartokinase Ac- germinating peas (14), homoserine can accumulate to high tivity. Table II shows the effect of some L-amino acids on the concentrations. Growth inhibition by lysine or threonine is aspartokinase activity which was extracted from Lemna plants probably due to feedback inhibition of aspartokinase as was grown in the control medium. Methionine, homoserine, and shown in the isolated enzyme (Fig. 4). In addition, threonine isoleucine gave only a slight inhibition at 5 mm. Threonine at may inhibit homoserine dehydrogenase. Such an effect has 0.5 mm was without effect. Similar to the wheat germ en- been reported for the higher plant enzyme (1) and the bacterial zyme (16), lysine was a very potent inhibitor, a concentration enzyme (3). of 0.3 mm causing an inhibition of 37%. When 0.3 mm lysine The dramatic effect of lysine and threonine when added to- was combined with 0.5 mm threonine, the inhibition was in- gether can be attributed to their concerted inhibition of as- creased to 62%. Lysine and threonine appeared to inhibit the partokinase activity. Such an inhibition would result in a lack Lemna enzyme in a concerted manner. Greater details of of methionine which is required for protein synthesis and feedback inhibition by lysine and threonine is shown in Figure would result in death of the plant. The inhibition of growth by 4. Lysine showed a pronounced inhibition at low concentra- lysine-threonine could be partially overcome by the inclusion tions, and at 0.6 mm gave 80% inhibition. Threonine inhibited of methionine (or a precursor of methionine, homoserine) in the enzyme in a sigmoidal manner, very little inhibition being the growth medium. This again indicates that it is the inhibi- observed up to 1 mm. When the concentration of lysine was tion of aspartokinase by lysine-threonine and the subsequent varied in the presence of 0.5 mm threonine, a noninhibitory deficiency of methionine which is responsible for the death of concentration, a much greater inhibition was observed than in the plants. the presence of lysine alone. Lysine and threonine together In tobacco cells cultured with nitrate as the nitrogen source, can effectively regulate aspartokinase activity. Filner (9) found that threonine, isoleucine, methionine, and aspartate were inhibitory to growth and that this inhibition DISCUSSION was due to the repression of the formation of nitrate reductase. This study shows that aspartokinase isolated from L. minor L-Threonine (mM) has properties similar to the enzyme from wheat germ. It is 0 1 2 3 4 inhibited by lysine but the maximum amount of inhibition 16 which can be obtained is greater than in the wheat germ en- X 12 x0 - T h r zyme. Threonine shows little inhibition up to a concentration of 1 mm. However, at higher concentrations threonine be- 0- Ly + T comes much more a maximum inhibition of hAr inhibitory, giving = approximately 65%. The inhibition by threonine shows a sigmoidal response to threonine concentration. This inhibition by threonine was not found in the wheat germ enzyme to such an extent if at all. The concentrations of threonine required to produce a significant inhibition are high and it may have no physiological significance. None of the other amino acids tested shows any significant inhibition. The concerted inhibition by lysine and threonine at low o 0.2 0.4 0.6 0.8 concentrations was more pronounced than that in the wheat L-Lysi ne (mM) and indicates an effective mechanism for control germ enzyme FIG. 4. Effect of lysine and threonine on aspartokinase activity. of the aspartokinase from L. minor. In different preparations Enzynme activity was assayed as described in "Materials and the amount of inhibition by lysine and throonine was quanti- Methods." Each assay contained 3.7 mg of protein. x: Threonine tatively variable although the same pattern was always ob- varied; O: lysine varied; *: threonine kept constant at 0.5 mm and served. It is possible that the differences in properties between lysine varied. Plant Physiol. Vol. 51, 1973 ASPARTOKINASE IN LEMNA MINOR L. 331 This repression would effectively deprive the cells of nitrogen. 4. DOUGALL, D. K. 1965. The biosynthesis of protein amino acids in plant tissue cultures. I. Isotope competition experiments using glucose-U-14C and the He also found that lysine and arginine were derepressors. It is protein amino acids. Plant Physiol. 40: 891-897. clear that the response of L. minor to amino acids is different 5. DOUGALL, D. K. 1966. The biosynthesis of protein amino acids in plant tissue from that observed in tobacco cells since in Lemna lysine was cultures. II. Further isotope competition experiments using protein amino inhibitory whereas aspartic acid was not. Also, although acids. Plant Physiol. 41: 1411-1415. 6. DOUGALL, D. K. AND M. M. FULTON. 1967. Biosynthesis of protein amino acids threonine inhibited the growth of Lemna, lysine did not reverse in plant tissue cutures. IV. Isotope competition experiments using glucose- the inhibition but increased it. Joy (11) also suggested that the U-14C and potential intermediates. Plant Physiol. 42: 941-945. effect of amino acids on the growth of Lemna is not directly 7. DuNHAm, V. L. AND J. K. BRYAN. 1969. Synergistic effects of metabolically re- related to the nitrate reductase system. lated amino acids on the growth of a multicellular plant. Plant Physiol. 14: 1601-1608. This report suggests that L. minor contains an aspartokinase 8. DuNHAM, V. L. AND J. K. BRYAN. 1971. Synergistic effects of a metabolically similar to the aspartokinase from wheat germ. Since only related amino acids on the growth of a multicellular plant. II. Studies of 14C0 limited amounts of L. minor are available and the specific amino acid incorporation. Plant Physiol. 47: 91-97. activity of the extracts are low, a detailed kinetic analysis 9. FILNER, P. 1966. Regulation of nitrate reductase in cultured tobacco cells. could not be performed. Biochim. Biophys. Acta 118: 299-310. 10. HILLMAN, W. J. 1961. The Lemnaceae, or duckweeds. Bot. Rev. 27: 221-287. The data in this report suggest that some of the physiologi- 11. Joy, K. W. 1969. Nitrogen metabolism of Lemna minor. I. Growth, nitrogen cal responses to the exogenously supplied amino acids by sources and amino acid inhibition. Plant Physiol. 44: 845-848. Lemna is due to the regulation of aspartokinase. 12. LAYSNE, E. 1957. Spectrophotometric and turbidimetric methods for measur- ing proteins. In: S. P. Colowick and N. 0. Kaplan, eds., Methods in Enzy- mology, Vol. III. Academic Press, Inc., New York. LITERATURE CITED pp. 447-454. 13. PATTE, J. C., G. LE BRAs AND G. N. COHEN. 1967. Regulation by methionine 1. BRYAN, J. K. 1969. Studies on the catalytic and regulatory properties of homo- of the synthesis of a third aspartokinase and of a second homoserine dehy- serine dehydrogenase of Zea mays roots. Biochim. Biophys. Acta 171: 205- drogenase in Escherichia coli Ki. Biochim. Biophys. Acta 136: 245-257. 216. 14. NAYLOR, A. W., R. RABsoN, AND N. E. TOLBERT. 1958. Aspartic-C14 acid me- 2. COHEN, G. N. AND J. C. PATTE. 1963. Some aspects of the regulation of amino tabolism in leaves, roots, and stems. Physiol. Plant. 11: 537-547. acid biosynthesis in a branched pathway. Cold Spring Harbor Symp. Quant. 15. UNIBARGER, E. AND B. D. DAVIS. 1962. Pathway of amino acid biosynthesis. Biol. 28: 513-516. In: I. C. Gunsalus and R. Y. Stanier, eds., The Bacteria, Vol. III. Academic 3. DATTA, P. 1967. Regulation of homoserine biosynthesis by L-cysteine, a termi- Press, Inc., New York. pp. 167-251. nal metabolite of a link-ed pathway. Proc. Nat. Acad. Sci. U.S.A. 58: 635- 16. WONG, K. F. AND D. T. DENNcIS. 1973. Aspartokinase from wheat germ. Isola- 641. tion, characterization, and regulation. Plant Physiol. 51: 322-326.