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ATPases from the Roots of Agrostis tenuis Sibth.,: Effect of pH, Mg2+, Zn2+, and Cu2+
ATPases from the Roots of Agrostis tenuis Sibth.: Effect of pH, Mg2+, Zn2+, and Cu 2+ WERNER VELTRUP
Institut fiir Angewandte Botanik, Hindenburgplatz 55, D-4400 Miinster, F.R.G. Received July 20,1982' Accepted October 21,1982
Summary ATPases from the roots of two ecotypes of Agrostis tenuis Sibth. showed an activity optimum at neutral pH. Magnesium is needed as a cofactor, but the relation between Mg2+ and ATP is not always one. With respect to the effect of heavy metals it could be found that ATPases from an ecotype of a soil with a normal supply with heavy metals as well as some from a zinc-ecotype showed enhanced sensitivity to zinc and copper with decreasing activity. Only in the zinc-ecotype could ATPases be found which were stimulated by zinc in vitro.
Key words: Agrostis tenuis, A TPases, effect ofpH, Mj+, Zn 2 +, and Cu2 +.
Introduction Among higher plants there are some which adapt at to soils contaminated by heavy metals by the evolution of particular ecotypes, e.g. Agrostis tenuis (Bradshaw, 1952), Silene inflata (Baumeister, 1954), Armeria maritima var. halleri (Ernst, 1974), as well as Agrostis gigantea (Hogan et a!., 1977). The adaptations are not to be found in specific key enzymes of the cell metabolism, which are tolerant to the metals (Mathys, 1975; Ernst, 1975), but in processes of complex formation and transport (Ernst et a!., 1975; Mathys, 1977; Rauser and Curvetto, 1980). With respect to processes of transport, Leonard and Hodges (1973), Leigh et a!. (1974), Pohlman-Nepveu et al. (1979), as well as Stout and Cleland (1982) tried to establish a connection between the transport of ions and the activity of A TPases. Starting with these interpretations and the possible importance of transport processes for the mechanisms of tolerance to heavy metals, it was attempted to isolate and to characterize A TPases from the roots of Agrostis tenuis from soils with a normal supply of heavy metals as well as from a zinc-ecotype.
Materials and Methods Plants from Agrostis tenuis Sibth. came from the coarse heavy metal habitat Greven (52 0 19' N07°39' E) and the heavy metal contaminated site Elpetal (51°19'N08°26'E). The plants were cleaned of soil particles with tap water, and afterwards washed carefully with deionized water. Dabbing the plant material with blotting paper removed excess water. Only the excised roots were used for the following experiments. Roots which were to be used for enzymatic research were shock frozen with liquid nitrogen and stored at about -18°C.
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For the determination of the content of cations, the roots were dried at 105°C until constant weight. Then they were digested in perchloric acidlnitric acid (1/7 v/v), after which the samples were made up to constant volumes with deionized water. The cation content of the soils was measured in two ways. For the determination of the total content, the samples of air-dried soils were digested in aqua regia. Exchangeable parts were determined by extraction of the air-dried soils with 1 mol· m- 3 ammonium acetate solution (lOll w/v). The cations were identified by atomic absorption spectrophotometry. The preparation of the crude extracts and the measurement of the activities of the ATPases were performed according to Veltrup (1978). A further purification and isolation of the enzymes was done with ammonium sulfate precipitations followed by separation with Sephacryl S 300 using a tris/acetate buffer (pH 7.2). The fractionation was done with 3 ml samples, using a flow of 18 ml . h -I. The length of the column was 90 cm, the diameter 1.6 cm. The preparation of the enzymes was performed at 4°C. The Pi liberated was determined according to Marsh (1959), and that of the proteins according to Lowry et al. (1951). The data were calculated from three independent measurements, using independent samples, and mean values as well as the standard deviations were calculated. To find significant differences between the data, the F- and t-test were used (Fisz, 1970; Sachs, 1972). The line regression was performed according to Sachs (1972) and Retzlaff et al. (1975).
Results Soil analysis showed that the habitat Elpetal is characterized by high amounts of zinc, manganese, and lead and is therefore distinct from that of Greven (Tab. 1). However, for the flora the exchangeable fractions are more decisive, because these fractions that are available to the plants (Ernst, 1965, 1966). In this connection the heavy metal zinc was found to be highly important for the habitat Elpetal. Table 1: Content of cations in the soils from Greven and Elpetal. t ~ total content, a ~ content of cations extractable with ammonium acetate solution. Data mmol· kg-I air-dried soil. n.d. = not detectable. Cu Greven t 0.57 a 0.05 Elpetal t 1.18 a 0.12
Zn 2.17 0.15 257 16.8
Fe
Mn
Pb
474 0.34
4.73 0.33
n.d. n.d.
244 0.20
110 0.22
4.92 0.12
Ca 54.9 13.0 105 185
Mg
Na
K
123 0.62
3.79 0.65
48.6 1.12
57.6 0.70
8.88 0.91
46.0 1.69
The cation content of the soils is reflected in that of the roots (Tab. 2). It is especially striking that in the case of Elpetal high amounts of zinc could be found in the Table 2: Content of cations in the roots of Agrostis tenuis Sibth. from different, contaminated soils. Data in mmol· kg-I dry weight. n.d. = not detectable.
Greven Elpetal
Cu
Zn
Fe
Mn
Pb
Ca
Mg
Na
K
0.10 0.19
2.32 50.5
9.53 7.84
1.87 1.31
n.d. 0.12
86.3 79.3
32.5 31.3
24.8 10.2
92.1 162
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roots, in contrast to Greven. Plants from the habitat Elpetal must be zinc-ecotypes (Mathys, 1973), while those from Greven must be characterized as sensitive ecotypes. In the following experiments A TPases were isolated from the roots of Agrostis tenuis, to determine the activity of enzymes and the in vitro effect of zinc and copper. The crude extracts were therefore treated with ammonium sulfate. The 40-80 % as well as the 80-100 % precipitations were used for the studies instead of high activities of the ATPases. The fractions collected after separation with Sephacryl S 300 could be characterized in the following way: Greven
1) Elution of the 40-80 % precipitate showed a protein peak after 57 ml buffer (measured at 280 nm), while the maximum of the ATPases activity was found after 66-69 ml. According to the protein adjustment of the column, the molecular weight could be assigned to 45,000-86,000 dalton. The in vitro effect of zinc and copper must be described as being inhibitory. The volume activity in the presence of 1 mmol· m- 3 magnesium was 6.7 {tmol Pi .1- 1 • min-I. 2) Elution of the 80-100 % precipitate showed a protein peak at 57, 72 and 93 ml. The maximum activity of the ATPases could be found after 78 ml buffer. Therefore the molecular weight must be greater than 86,000 dalton. Zinc and copper inhibited the activity of the enzymes. The volume activity was 1.2 {tmol Pi .1- 1 • min-I, when 1 mmol· m -3 Mg2+ was present in the assay. Elpetal
1) With respect to the 40-80% precipitation the protein peak was found after 57 ml, and the maximum ATPases activity could be measured after 105 ml with a volume activity of 62.6{tmol Pi ,1- 1 • min-I, when measured in the presence of 1 mmol· m- 3 Mg2+. The molecular weight could therefore be stated at 45,000 dalton. The fractions 102-108 ml were used for the further experiments. These samples contained ATPases which were inhibited by zinc and copper, when testing the in vitro effect. 2) The 80-100 % ammonium sulfate fraction also showed a protein peak after 57 ml, and two further ones after 72 and 95 ml. The maximum ATPase activity could be found after 93 ml with 10.6 {tmol Pi ,1- 1 . min -1 in the presence of 1 mmol· m- 3 Mg2+. The samples 81-84 showed an in vitro stimulation of the ATPase activity by zinc. The molecular weight must be put near 86,000 dalton. The volume activity was measured with 3.2 {tmol Pi ,1- 1 . min- 1 in the presence of 1 mmol· m- 3 magnesium. According to the in vitro effects of zinc upon the fractions, they can be distinguished, on the one hand, as those fractions which showed decreased activities. They should be named inhibited ATPases, and on the other hand, as those which showed increased activities. The latter should be named stimulated ATPases. Z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
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WERNER VELTRUP
Inhibited A TPases For the determination of an optimal pH of the test medium, 1 mmol· m -3 ATP and 1 mmol· m- 3 Mg2+ were added to the assay according to Lindberg et al. (1974). The resultant activity of the ATPases is drawn in Fig. 1.
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Fig. 1: Effect of the pH of the test medium upon the activity of inhibitable ATPases from the roots of different Agrostis ecotypes at 25°C. 0 - - 0 sensitive, x--x Zn-resistant plants.
In the case of the sensitive ecotype, only small activity of the A TPases resulted, and the optimum was found at 6.9. Alkalinization of the test medium led to significantly reduced activities. On the other hand inhibited A TPases from zinc-ecotypes showed a pH optimum at 7.0, not significantly distinguished from the values obtained at 6.9 and 7.1. Here, too, an increase of the pH led to significant reductions of the activities. Accordingly the tests were adjusted to pH 6.9 in the case of the sensitive ecotypes, and to pH 7.0 in the case of zinc-ecotypes. The effect of magnesium is shown in Fig. 2. In the case of the sensitive ecotypes the addition of 0.25 mmol· m- 3 Mg2+ to the test medium enhanced significantly the activity of the A TPases. A further increase of the magnesium concentration caused a decreased activity. The resultant pattern can be characterized by the line y = -0.22 x + 2.1 (r = 0.99) in a significant manner. ATPases from zinc-ecotypes could also be stimulated by Mg2+ up to a concentration of 0.75 mmol· m- 3. A further increase led to a significant loss of activity. In
z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
ATPases from the roots of Agrostis
461
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Fig, 2: Effect of Mg2+ in the test medium upon the activity of inhibitable ATPases from the roots of different Agrostis ecotypes at 25°C. 0 - - 0 sensitive, x - - x Zn-resistant plants.
accordance with these optima 0.25, on the one hand, and 0.75 mmol· m- 3 Mg2+ on the other hand were used in the following experiments. If zinc or copper was added to the assay, an inhibition of the ATPases could be observed (Fig. 3). In higher concentration ranges (> 200 /-tmol· m -3) copper was more inhibitory than zinc. In the case of the sensitive ecotypes 325 /-tmol· m -3 Zn2+ led to 37 %, and 325 /-tmol· m -3 Cu 2 + to a 41 % reduction of the activity, while in the case of the zinc-ecotypes zinc reduced only about 27 % and copper only about 39 %.
Stimulated A TPases Finally the fraction which was stimulated by the in vitro presence of zinc has to be characterized. With respect to the pH of the test medium an optimum condition was found at 7.2 (Fig. 4). In the following experiments the pH was adjusted accordingly. As suggested by to the effect of Mg2+ it was found that the addition of
Z. Pjlanzenphysiol. Ed. 108. S. 457-469. 1982.
462
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1 mmol· m -3 A TP and 1 mmol· in-3 Mg2+ resulted in an optimal activity (Fig. 4). A further increase of the magnesium concentration led to a significant loss of activity, compared with the optimum, although this activity is still greater than the basic activity (Ommo! ' m- 3 Mg2+). The in vitro effect of copper was tested using an optimal pH and Mg2+ concentration (Fig. 5). Even 100 /Lmol · m -3 Cu 2+ in the test medium led to a 17 % reduction of the ATPase activity, while 325 /Lmol· m- 3 Cu2+ resulted in a 55 % reduction which proved to be significant. Finally the effect of zinc upon these A TPases was tested, firstly without magnesium and secondly with 1 mmol· m- 3 Mg2+. In both cases showed a stimulating effect was obtained (Fig. 6). The figure shows that part of the activity which is attributed solely to the in vitro effect of zinc. The basic activity, i.e. the activity with the effect of Mg2+ alone, was substracted from the total activity. The data were transformed according to Lineweaver and Burk (1934) and the kinetic constants were calculated using the method of least squares. A kinetic evaluation of the data (Fig. 6) showed that the influence of zinc alone led to positive results only in the range of 25-125 /Lmol· m -3. The resulting pattern Z. Pf/anzenphysiol. Ed. 108. S. 457-469. 1982.
A TPases from the roots of Agrostis
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can be described by y = 52.9x + 6.9 (r = 0.99) and the kinetic constants can be given with Vmax = 12.9±2.14/tmol Pdmg proteint 1. h- 1 and Km = 683 ± 113 /tmol· m- 3 . The range above 175 /tmol· m- 3 led to negative kinetic constants. If one examines the pattern under a simultaneous effect of magnesium, one can find that this has to be divided into three sections, which is also obvious from the correlation ceofficients. The range 25-75 /tmol· m-3 is represented by the line y = 8.8x + 0.25 (r = 0.99) with Vmax = 3.9±1.7 /tmol Pi'(mg proteint1·h- 1 and Km = 34.7 ± 22.3 /tmol· m-3, that of 75-225 /tmol· m -3 Zn2+ by y = 25.1 x + 0.062 (r = 0.995) with Vrnax = 16.1±1.6/tmol Pi'(mg proteint1·h- 1 and Km = 407± 160/tmol· m- 3 • An evaluation of the range above 225/tmol'm- 3 Zn2+ led to negative kinetic constants. This range will not be considered further. With respect to the stimulation by zinc and the simultaneous influence of magneZ. Pjlanzenphysiol. Rd. 108. S. 457-469. 1982.
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Fig. 5: Effect of increasing concentrations of copper upon Zn-stimulated ATPases from the roots of Agrostis tenuis (zinc-ecotype) at 25°C.
sium it can be pointed out that with increasing concentrations of the heavy metal a decrease of the affinity and an increase of the maximum velocity resulted.
Discussion A high dependence of the activity of plant A TPases upon the pH of the test medium was already demonstrated by Hall (1973), Leonard and Hotchkiss (1976), as well as by Stok et al. (1981). This is typical for crop and wild plants (Hill and Hill, 1973; Kuiper and Kuiper, 1979 a, b). Inhibited as well as stimulated fractions of A TPases from the two ecotypes studied showed an optimum in neutral ranges (Fig. 1 and 4). This is in agreement with the results on other ATPases (Hall and Butt, 1969; Kuiper et al., 1974; Kawasaki et al., 1979; Kuiper and Kuiper, 1979 a). A slight shifting of the optimum between the different fractions might depend upon the different soils and the resultant different ion contents of the roots (Tab. 1 and 2). This is also stressed by Kuiper and Kuiper (1979 a, b) in the case of Plantago species. Besides the pH dependence, plant A TPases also show stimulation in the presence of magnesium (Hansson and Kylin, 1969; Leonard and Hodges, 1973; Hendrix and Kennedy, 1977). The investigations of Lindberg et al. (1974), Lindberg (1976), as well as Z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
A TPases from the roots of Agrostis
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Fig. 6: Effect of increasing concentrations of zinc upon the activity of Zn-stimulated A TPases from the roots of Agrostis tenuis (zinc-ecotype) at 25°C. The upper part of the figure represents the double reciprocal ~lot of the data in the lower part. +--+ without Mg2+ and 0 - - 0 with 1 mmol· m -3 Mg + in the test medium.
Erdei and Kuiper (1980) showed an optimal activity of the ATPases if the relation between magnesium and A TP was one. In the case of the inhibited A TPases from Agrostis tenuis roots this relation is less than one (Fig. 2)_ With respect to the stimulated fractions (Fig. 4)
z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
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WERNER VELTRUP
must be noted that Mg2+ must be considered as a cofactor for the A TPases studied, as was already stressed by Kylin and Lundborg (1980). With respect to the effect of zinc and copper upon the inhibited ATPases a significantly decreased activity could be found. Similar phenomena were reported for phosphatases. This was shown by Cox and Thurman (1978) who extracted enzymes from the roots of Anthoxantum plants, as well as by Cox and Hutchinson (1980) who studied phosphatases from the roots of Deschampsia plants. The inhibition of the activity might, on the one hand, be explained by the formation of copper-ATP-complexes, which could only be transformed to a small extent according to the comments of Chaberek and Martell (1959), and possibly, on the other hand, by direct interactions between heavy metals and the enzymes (Vallee and Ulmer, 1972; Foy et aI., 1978). The stimulated fractions showed that copper also inhibited, while only zinc led to positive reactions (Figs. 5 and 6). In the investigations of Leonard and Hodges (1973) as well as Perlin and Spanswick (1981) that part of the activity which depends on the presence of zinc was characterized further. Mathys (1975) could demonstrate that the nitrate reductase from the leaves of Silene cucubalus (zinc-ecotype) showed 20 % of the control activity under the influence of 15 p.mol· m -3 Zn 2+, the malate dehydrogenase 60 % under the influence of 1 mmol· m- 3 , and the isocitrate dehydrogenase only 40% of the control activity in the presence of 150 p.mol· m- 3 Zn 2+. Therefore it might be possible that in the case of Agrostis tenuis no tolerance of the enzymes may occur either, and, in consequence, higher concentration ranges should not be relevant from an eco-physiological view. In ecologically important concentration ranges the presence of magnesium is needed, especially in the lower concentration ranges of zinc. Here, a high affinity to zinc could be found because of low Km-values. Furthermore, it could be demonstrated, on account of the lower Pi liberation, that zinc cannot substitute for magnesium. This means that A TPases stimulated by zinc also require magnesium, as do other A TPases (Kylin and Gee, 1970; Lindberg et aI., 1974; Kuiper and Kuiper, 1979 b; Erdei and Kuiper, 1980). Mathys (1973) found in experiments with zinc-ecotypes of Agrostis tenuis that increasing concentrations of zinc led to an increased uptake. The investigations of Turner (1970), Auquier and de Laval (1974), Wainwright and Woolhouse (1975), and Brookes et aI. (1981), demonstrated that grasses do not possess exclusion mechanisms with respect to heavy metals. Considering the discussion concerning the participation of A TPases in ion transport processes (d. Hodges, 1976), the zinc stimulated ATPases might possibly be connected with the zinc transport. This might suggest further aspects of zinc tolerance mechanisms, but more investigations and results are needed.
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467
References AUQUIER, P. andJ. DE LEVAL: Teneur en zinc, plomb et manganese, in situ et en culture experimentale, des Festuca des terrains calaminaires Belges. Bull. Soc. roy. Bot. Belg. 107, 375-386 (1974). BAUMEISTER, W.: Dber den EinfluB des Zinks bei Silene inflata Smith. Ber. Deutsch. Bot. Ges. 67, 205-213 (1954). BRADSHAW, A. D.: Populations of Agrostis tenuis resistant to lead and zinc poisoning. Nature 169, 1098 (1952). BROOKES, A., J. C. COLLINS, and D. A. THURMAN: The mechanism of zinc tolerance in grasses. J. Plant Nutr. 3,695-705 (1981). CHABEREK, S. and A. E. MARTELL: Organic sequestering agents. J. Wiley and Sons, Inc., New York,1959. CHRISTIENSEN, H. H. and A. KYLIN: Substrates and modifiers of Mg- and Ca-activated ATPases from wheat roots. I. Congress ofFESPP, pp. 130-131. Edinburgh, Scotland, 1978. Cox, R. M. and T. C. HUTCHINSON: Multiple metal tolerances in the grass Deschampsia cespitosa (L.) Beauv. from the Sudbury smelting area. New Phytol. 84, 631-647 (1980). Cox, R. M. and D. A. THURMAN: Inhibition by zinc of soluble and cell wall acid phosphatases of zinc-tolerant and non-tolerant clones ofAnthoxanthum odoratum. Ibid. 80,17-22 (1978). ERDEI, L. and P. J. C. KUIPER: Substrate-dependent modulation of ATPase activity by Na+ and K+ in roots of Plantago species. Physiol. Plant. 49, 71-77 (1980). ERNST, W.: Okophysiologisch-soziologische Untersuchungen der Schwermetall-Pflanzengesellschaften Mitteleuropas unter EinschluB der Alpen. Abh. Landesmuseum Naturk. Miinster 27, 1-54 (1965). - Okologisch-soziologische Untersuchungen der Schwermetall-Pflanzengesellschaften Siidfrankreichs und des ostlichen Harzvorlandes. Flora B 156, 301-318 (1966). - Schwermetallvegetation der Erde. Fischer Verlag, Stuttgart, 1974. - Physiology of heavy metal resistance in plants. Int. Conf. Heavy Metals in the Environment, II, 121-136. Toronto, Canada, 1975. ERNST, W., W. MATHYS, and P. JANIESCH: Physiologische Grundlagen der Schwermetallresistenz-Enzymaktivitaten und organische Sauren. Forsch.-Ber. Land Nordrhein-Westfalen 2496, 1-38 (1975). FISZ, M.: Wahrscheinlichkeitsrechnung und mathematische Statistik. VEB Deutscher Verlag der Wissenschaften, Berlin, 1970. Foy, C. D., R. L. CHANEY, and M. C. WHITE: The physiology of metal toxicity in plants. Ann. Rev. Plant Physiol. 29, 511-566 (1978). HALL, J. L.: Enzyme localization and ion transport. In: ANDERSON, W. P. (ed.): Ion transport in plants, pp. 11-24. Academic Press, London, New York, 1973. HALL, J. L. and V. S. BUTT: Adenosine triphosphatase activity in cell wall preparations and excised roots of barley. J. Exp. Bot. 20, 751-762 (1969). HANSSON, G. and A. KYLIN: ATPase activities in homogenates from sugar beet roots, relation to Mg2+ and (Na+ + K+)-stimulation. Z. Pflanzenphysiol. 60, 270-275 (1969). HENDRIX, D. L. and R. M. KENNEDY: Adenosine triphosphatase from soybean callus and root cells. Plant Physiol. 59, 264-267 (1977). HILL, B. S. and A. E. HILL: Enzymatic approaches to chloride transport in the Limonium salt gland. In: ANDERSON, W. P. (ed.): Ion transport in plants, pp. 379-384. Academic Press, London, New York, 1973. HODGES, T. K.: ATPases associated with membranes of plant cells. In: LUTTGE, U. and M. G. PITMAN (eds.): Transport in plants. II. Part A Cells, pp. 260-283. Springer-Verlag, Berlin, Heidelberg, New York, 1976. Z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
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HOGAN, G. D., G. M. COURTIN, and W. E. RAUSER: Copper tolerance in clones of Agrostis gigantea from a mine waste site. Can. J. Bot. 55, 1043-1050 (1977). HOGAN, G. D. and W. E. RAUSER: Role of copper binding, absorption and translocation in coppertolerance in Agrostis gigantea Roth. J. Exp. Bot. 32, 27-36 (1981). KAwASAKI, I., M. KXHR, and A. KYLIN: Interactions of divalent cations in their action on ATPases from Cucumber roots. Physiol. Plant. 45, 437-439 (1979). KUIPER, D. and P. J. C. KUIPER: Ca2+_ and Mi+-stimulated ATPases from roots of Plantago major and Plantago maritima: response to alterations of the level of mineral nutrition and ecological significance. Ibid. 45, 1-6 (1979 a). - - Ca2+- and Mg2+ -stimulated ATPases from roots of Plantago lanceolata, Plantago media and Plantago coronopus: response to alterations of the level of mineral nutrition and ecological significance. Ibid. 45, 240-244 (1979 b). KUIPER, P. J. c., M. KXHR, C. E. E. STUIVER, and A. KYLIN: Lipid composition of whole roots and of Ca2+, Mg2+ -activated adenosine triphosphatases from wheat and oat as related to mineral nutrition. Ibid. 32,33-36 (1974). KYLIN, A. and R. GEE: Adenosine triphosphatase activities in leaves of the mangrove Avicennia nitida JACQ. Influence to sodium and potassium ratios and salt concentrations. Plant Physiol. 45,169-172 (1970).
KYLIN, A. and T. LUNDBORG: Transport ATPases - usefullness, limitations and perspectives of concept. II. Congress of FESPP, pp. 72-73. Santiago de Compostela, Spain, 1980. LEIGH, R. A., R. G. WYN JONES, and F. A. WILLIAMSON: The possible role of vesicles and ATPases in ion uptake. In: ZIMMERMANN, U. and J. DAINTY (eds.): Membrane transport in plants, pp. 307-316. Springer-Verlag, Berlin, Heidelberg, New York, 1974. LEONARD, R. T. and T. K. HODGES: Characterization of plasma membrane associated adenosine triphosphatase activity in oat roots. Plant Physiol. 52, 6-12 (1973). LEONARD, R. T. and C. W. HOTCHKISS: Cation-stimulated adenosine triphosphatase activity and cation transport in corn roots. Ibid. 58, 331-335 (1976). liNDBERG, S.: Kinetic studies of a (Na+ + K+ + Mg2+) ATPase in sugar beet roots. II. Activation by Na+ and K+. Physiol. Plant. 36,139-144 (1976). LINDBERG, S., G. HANSSON, and A. KYLIN: Kinetic studies of a (Na+ + K+ + Mg2+) ATPase in sugar beet roots. I. Mg-dependence. Ibid. 32, 103-107 (1974). LINEWEAVER, H. and D. BURK: The determination of enzyme dissoziation constants. J. Amer. Chern. Soc. 56, 658-666 (1934). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MARSH, B. B.: The estimation of inorganic phosphate in the presence of adenosine triphosphate. Biochim. Biophys. Acta 32, 357-361 (1969). MATHYS, W.: Vergleichende Untersuchungen der Zinkaufnahme von resistenten und sensitiven Populationen von Agrostis tenuis Sibth. Flora 162, 492-499 (1973). - Enzymes of heavy metal-resistant and non-resistant populations of Silene cucubalus and their interaction with some heavy metals in vitro and in vivo. Physiol. Plant. 33,161-165 (1975). - The role of malate, oxalate, and mustard oil glucosides in the evolution of zinc-resistance in herbage plants. Ibid. 40, 130-136 (1977). PERLIN, D. S. and R. M. SPANSWICK: Characterization of ATPase activity associated with corn leaf plasma membranes. Plant Physiol. 68, 521-526 (1981). POHLMAN-NEPVEU, J., M. KXHR, A. KYLIN, C. E. E. STUIVER, and P. J. C. KUIPER: Uptake and translocation of Ca2+ and Mg2+ ions in seedlings of oat and wheat, and its correlation with Ca2+_ and Mg2+ -activated ATPase from the roots. Physiol. Plant 45, 347-350 (1979). RAUSER, W. E. and N. R. CURVETTO: Metallothionein occurs in roots of Agrostis tolerant to excess copper. Nature 287, 563-564 (1980).
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RETZLAFF, G., G. RUST, and J. WAIBEL: Statistische Versuchsplanung. Verlag Chemie, Weinheim, 1975. SACHS, L.: Statistische Methoden. Springer-Verlag, Berlin, Heidelberg, New York, 1972. STOK, B. P., O. G. TANCZOS, M. KxHR, C. E. E. STUIVER, A. KYLIN, and P. J. C. KUIPER: Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition. Physiol. Plant. 52,115-123 (1981). STOUT, R. G. and R. E. CLELAND: Evidence for a Cl--stimulated MgATPase proton pump in oat root membranes. Plant Physiol. 69, 798-803 (1982). TURNER, R. G.: The subcellular distribution of zinc and copper within the roots of metal-tolerant clones of Agrostis tenuis Sibth. New Phytol. 69, 725-731 (1970). VALLEE, B. L. and D. D. ULMER: Biochemical effects of mercury, cadmium and lead. Ann. Rev. Biochem. 41, 91-128 (1972). VELTRUP, W.: Der EinfluB von Cu2+ und Mg2+ auf die Aktivitat von ATPasen aus Gerstenwurzeln. Biochem. Physiol. Pflanzen 173,17-22 (1978). WAINWRIGHT, S. J. and H. W. WOOLHOUSE: Physiological mechanisms of heavy metal tolerance in plants. In: CHADWICK, M. J. and G. T. GOODMAN (eds.): The ecology of resource degradation and renewal, pp. 231-257. Blackwell Sci. Publ., Oxford, 1975.
z. Pjlanzenphysiol. Ed. 108. S. 457-469. 1982.
Institut fiir Angewandte Botanik, Hindenburgplatz 55, D-4400 Miinster, F.R.G. Received July 20,1982' Accepted October 21,1982
Summary ATPases from the roots of two ecotypes of Agrostis tenuis Sibth. showed an activity optimum at neutral pH. Magnesium is needed as a cofactor, but the relation between Mg2+ and ATP is not always one. With respect to the effect of heavy metals it could be found that ATPases from an ecotype of a soil with a normal supply with heavy metals as well as some from a zinc-ecotype showed enhanced sensitivity to zinc and copper with decreasing activity. Only in the zinc-ecotype could ATPases be found which were stimulated by zinc in vitro.
Key words: Agrostis tenuis, A TPases, effect ofpH, Mj+, Zn 2 +, and Cu2 +.
Introduction Among higher plants there are some which adapt at to soils contaminated by heavy metals by the evolution of particular ecotypes, e.g. Agrostis tenuis (Bradshaw, 1952), Silene inflata (Baumeister, 1954), Armeria maritima var. halleri (Ernst, 1974), as well as Agrostis gigantea (Hogan et a!., 1977). The adaptations are not to be found in specific key enzymes of the cell metabolism, which are tolerant to the metals (Mathys, 1975; Ernst, 1975), but in processes of complex formation and transport (Ernst et a!., 1975; Mathys, 1977; Rauser and Curvetto, 1980). With respect to processes of transport, Leonard and Hodges (1973), Leigh et a!. (1974), Pohlman-Nepveu et al. (1979), as well as Stout and Cleland (1982) tried to establish a connection between the transport of ions and the activity of A TPases. Starting with these interpretations and the possible importance of transport processes for the mechanisms of tolerance to heavy metals, it was attempted to isolate and to characterize A TPases from the roots of Agrostis tenuis from soils with a normal supply of heavy metals as well as from a zinc-ecotype.
Materials and Methods Plants from Agrostis tenuis Sibth. came from the coarse heavy metal habitat Greven (52 0 19' N07°39' E) and the heavy metal contaminated site Elpetal (51°19'N08°26'E). The plants were cleaned of soil particles with tap water, and afterwards washed carefully with deionized water. Dabbing the plant material with blotting paper removed excess water. Only the excised roots were used for the following experiments. Roots which were to be used for enzymatic research were shock frozen with liquid nitrogen and stored at about -18°C.
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For the determination of the content of cations, the roots were dried at 105°C until constant weight. Then they were digested in perchloric acidlnitric acid (1/7 v/v), after which the samples were made up to constant volumes with deionized water. The cation content of the soils was measured in two ways. For the determination of the total content, the samples of air-dried soils were digested in aqua regia. Exchangeable parts were determined by extraction of the air-dried soils with 1 mol· m- 3 ammonium acetate solution (lOll w/v). The cations were identified by atomic absorption spectrophotometry. The preparation of the crude extracts and the measurement of the activities of the ATPases were performed according to Veltrup (1978). A further purification and isolation of the enzymes was done with ammonium sulfate precipitations followed by separation with Sephacryl S 300 using a tris/acetate buffer (pH 7.2). The fractionation was done with 3 ml samples, using a flow of 18 ml . h -I. The length of the column was 90 cm, the diameter 1.6 cm. The preparation of the enzymes was performed at 4°C. The Pi liberated was determined according to Marsh (1959), and that of the proteins according to Lowry et al. (1951). The data were calculated from three independent measurements, using independent samples, and mean values as well as the standard deviations were calculated. To find significant differences between the data, the F- and t-test were used (Fisz, 1970; Sachs, 1972). The line regression was performed according to Sachs (1972) and Retzlaff et al. (1975).
Results Soil analysis showed that the habitat Elpetal is characterized by high amounts of zinc, manganese, and lead and is therefore distinct from that of Greven (Tab. 1). However, for the flora the exchangeable fractions are more decisive, because these fractions that are available to the plants (Ernst, 1965, 1966). In this connection the heavy metal zinc was found to be highly important for the habitat Elpetal. Table 1: Content of cations in the soils from Greven and Elpetal. t ~ total content, a ~ content of cations extractable with ammonium acetate solution. Data mmol· kg-I air-dried soil. n.d. = not detectable. Cu Greven t 0.57 a 0.05 Elpetal t 1.18 a 0.12
Zn 2.17 0.15 257 16.8
Fe
Mn
Pb
474 0.34
4.73 0.33
n.d. n.d.
244 0.20
110 0.22
4.92 0.12
Ca 54.9 13.0 105 185
Mg
Na
K
123 0.62
3.79 0.65
48.6 1.12
57.6 0.70
8.88 0.91
46.0 1.69
The cation content of the soils is reflected in that of the roots (Tab. 2). It is especially striking that in the case of Elpetal high amounts of zinc could be found in the Table 2: Content of cations in the roots of Agrostis tenuis Sibth. from different, contaminated soils. Data in mmol· kg-I dry weight. n.d. = not detectable.
Greven Elpetal
Cu
Zn
Fe
Mn
Pb
Ca
Mg
Na
K
0.10 0.19
2.32 50.5
9.53 7.84
1.87 1.31
n.d. 0.12
86.3 79.3
32.5 31.3
24.8 10.2
92.1 162
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459
roots, in contrast to Greven. Plants from the habitat Elpetal must be zinc-ecotypes (Mathys, 1973), while those from Greven must be characterized as sensitive ecotypes. In the following experiments A TPases were isolated from the roots of Agrostis tenuis, to determine the activity of enzymes and the in vitro effect of zinc and copper. The crude extracts were therefore treated with ammonium sulfate. The 40-80 % as well as the 80-100 % precipitations were used for the studies instead of high activities of the ATPases. The fractions collected after separation with Sephacryl S 300 could be characterized in the following way: Greven
1) Elution of the 40-80 % precipitate showed a protein peak after 57 ml buffer (measured at 280 nm), while the maximum of the ATPases activity was found after 66-69 ml. According to the protein adjustment of the column, the molecular weight could be assigned to 45,000-86,000 dalton. The in vitro effect of zinc and copper must be described as being inhibitory. The volume activity in the presence of 1 mmol· m- 3 magnesium was 6.7 {tmol Pi .1- 1 • min-I. 2) Elution of the 80-100 % precipitate showed a protein peak at 57, 72 and 93 ml. The maximum activity of the ATPases could be found after 78 ml buffer. Therefore the molecular weight must be greater than 86,000 dalton. Zinc and copper inhibited the activity of the enzymes. The volume activity was 1.2 {tmol Pi .1- 1 • min-I, when 1 mmol· m -3 Mg2+ was present in the assay. Elpetal
1) With respect to the 40-80% precipitation the protein peak was found after 57 ml, and the maximum ATPases activity could be measured after 105 ml with a volume activity of 62.6{tmol Pi ,1- 1 • min-I, when measured in the presence of 1 mmol· m- 3 Mg2+. The molecular weight could therefore be stated at 45,000 dalton. The fractions 102-108 ml were used for the further experiments. These samples contained ATPases which were inhibited by zinc and copper, when testing the in vitro effect. 2) The 80-100 % ammonium sulfate fraction also showed a protein peak after 57 ml, and two further ones after 72 and 95 ml. The maximum ATPase activity could be found after 93 ml with 10.6 {tmol Pi ,1- 1 . min -1 in the presence of 1 mmol· m- 3 Mg2+. The samples 81-84 showed an in vitro stimulation of the ATPase activity by zinc. The molecular weight must be put near 86,000 dalton. The volume activity was measured with 3.2 {tmol Pi ,1- 1 . min- 1 in the presence of 1 mmol· m- 3 magnesium. According to the in vitro effects of zinc upon the fractions, they can be distinguished, on the one hand, as those fractions which showed decreased activities. They should be named inhibited ATPases, and on the other hand, as those which showed increased activities. The latter should be named stimulated ATPases. Z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
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Inhibited A TPases For the determination of an optimal pH of the test medium, 1 mmol· m -3 ATP and 1 mmol· m- 3 Mg2+ were added to the assay according to Lindberg et al. (1974). The resultant activity of the ATPases is drawn in Fig. 1.
18
..
.c
15
E 12 0:.-
0
~ 2,0 ::t
1,0
o
L-~~_~~~_~-'-~---' 7,4 7,2 6,8 7,0
pH of Ihll IllsI mlldium
Fig. 1: Effect of the pH of the test medium upon the activity of inhibitable ATPases from the roots of different Agrostis ecotypes at 25°C. 0 - - 0 sensitive, x--x Zn-resistant plants.
In the case of the sensitive ecotype, only small activity of the A TPases resulted, and the optimum was found at 6.9. Alkalinization of the test medium led to significantly reduced activities. On the other hand inhibited A TPases from zinc-ecotypes showed a pH optimum at 7.0, not significantly distinguished from the values obtained at 6.9 and 7.1. Here, too, an increase of the pH led to significant reductions of the activities. Accordingly the tests were adjusted to pH 6.9 in the case of the sensitive ecotypes, and to pH 7.0 in the case of zinc-ecotypes. The effect of magnesium is shown in Fig. 2. In the case of the sensitive ecotypes the addition of 0.25 mmol· m- 3 Mg2+ to the test medium enhanced significantly the activity of the A TPases. A further increase of the magnesium concentration caused a decreased activity. The resultant pattern can be characterized by the line y = -0.22 x + 2.1 (r = 0.99) in a significant manner. ATPases from zinc-ecotypes could also be stimulated by Mg2+ up to a concentration of 0.75 mmol· m- 3. A further increase led to a significant loss of activity. In
z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
ATPases from the roots of Agrostis
461
20
.
.c
,
c
]! o
Ci.
15
'"
E -.,.0:-
0
~ 2,0 :l..
1.0
o
o
2 mM
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Mg2+ in Ihq Irsl mqdium
Fig, 2: Effect of Mg2+ in the test medium upon the activity of inhibitable ATPases from the roots of different Agrostis ecotypes at 25°C. 0 - - 0 sensitive, x - - x Zn-resistant plants.
accordance with these optima 0.25, on the one hand, and 0.75 mmol· m- 3 Mg2+ on the other hand were used in the following experiments. If zinc or copper was added to the assay, an inhibition of the ATPases could be observed (Fig. 3). In higher concentration ranges (> 200 /-tmol· m -3) copper was more inhibitory than zinc. In the case of the sensitive ecotypes 325 /-tmol· m -3 Zn2+ led to 37 %, and 325 /-tmol· m -3 Cu 2 + to a 41 % reduction of the activity, while in the case of the zinc-ecotypes zinc reduced only about 27 % and copper only about 39 %.
Stimulated A TPases Finally the fraction which was stimulated by the in vitro presence of zinc has to be characterized. With respect to the pH of the test medium an optimum condition was found at 7.2 (Fig. 4). In the following experiments the pH was adjusted accordingly. As suggested by to the effect of Mg2+ it was found that the addition of
Z. Pjlanzenphysiol. Ed. 108. S. 457-469. 1982.
462
WERNER VELTRUP
25
20 ~ -!-
c:
' jjj 0
I-
a.
0
....
2
01
E
a:-
COpplZr
15
'0
E
:l.
cop p~r
o
o
300 200 100 fmol of hCQvy meta.ls In th~ test INIdlum
Fig. 3: Effect of increasing concentrations of zinc and copper upon inhibitable ATPases from the roots of sensitive (0 , .) and Zn-resistant (0 , x) Agrostis tenuis plants at 25°C.
1 mmol· m -3 A TP and 1 mmol· in-3 Mg2+ resulted in an optimal activity (Fig. 4). A further increase of the magnesium concentration led to a significant loss of activity, compared with the optimum, although this activity is still greater than the basic activity (Ommo! ' m- 3 Mg2+). The in vitro effect of copper was tested using an optimal pH and Mg2+ concentration (Fig. 5). Even 100 /Lmol · m -3 Cu 2+ in the test medium led to a 17 % reduction of the ATPase activity, while 325 /Lmol· m- 3 Cu2+ resulted in a 55 % reduction which proved to be significant. Finally the effect of zinc upon these A TPases was tested, firstly without magnesium and secondly with 1 mmol· m- 3 Mg2+. In both cases showed a stimulating effect was obtained (Fig. 6). The figure shows that part of the activity which is attributed solely to the in vitro effect of zinc. The basic activity, i.e. the activity with the effect of Mg2+ alone, was substracted from the total activity. The data were transformed according to Lineweaver and Burk (1934) and the kinetic constants were calculated using the method of least squares. A kinetic evaluation of the data (Fig. 6) showed that the influence of zinc alone led to positive results only in the range of 25-125 /Lmol· m -3. The resulting pattern Z. Pf/anzenphysiol. Ed. 108. S. 457-469. 1982.
A TPases from the roots of Agrostis
463
20
15
c
'o" o L-
a.
o
Ol
1
2
3
mM Mg2+ in Ihcz Iczsl
~
medium
0:-
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E
:l.
20
15
Fig. 4: Effect of pH and Mg2+ upon the activity of Zn-stimulated ATPases from the roots of Ag· rostis tenuis (zinc-ecotype) at 25°C.
o
6,8
7,0
7,2
7,4
pH of th« tnt mltdium
can be described by y = 52.9x + 6.9 (r = 0.99) and the kinetic constants can be given with Vmax = 12.9±2.14/tmol Pdmg proteint 1. h- 1 and Km = 683 ± 113 /tmol· m- 3 . The range above 175 /tmol· m- 3 led to negative kinetic constants. If one examines the pattern under a simultaneous effect of magnesium, one can find that this has to be divided into three sections, which is also obvious from the correlation ceofficients. The range 25-75 /tmol· m-3 is represented by the line y = 8.8x + 0.25 (r = 0.99) with Vmax = 3.9±1.7 /tmol Pi'(mg proteint1·h- 1 and Km = 34.7 ± 22.3 /tmol· m-3, that of 75-225 /tmol· m -3 Zn2+ by y = 25.1 x + 0.062 (r = 0.995) with Vrnax = 16.1±1.6/tmol Pi'(mg proteint1·h- 1 and Km = 407± 160/tmol· m- 3 • An evaluation of the range above 225/tmol'm- 3 Zn2+ led to negative kinetic constants. This range will not be considered further. With respect to the stimulation by zinc and the simultaneous influence of magneZ. Pjlanzenphysiol. Rd. 108. S. 457-469. 1982.
464
WERNER VELTRUP
20
2
o
a.en
E
0:.-
"0 E
:J.
10
o
o
100
200
fJM Cu 2+ in th~ test medium
300
Fig. 5: Effect of increasing concentrations of copper upon Zn-stimulated ATPases from the roots of Agrostis tenuis (zinc-ecotype) at 25°C.
sium it can be pointed out that with increasing concentrations of the heavy metal a decrease of the affinity and an increase of the maximum velocity resulted.
Discussion A high dependence of the activity of plant A TPases upon the pH of the test medium was already demonstrated by Hall (1973), Leonard and Hotchkiss (1976), as well as by Stok et al. (1981). This is typical for crop and wild plants (Hill and Hill, 1973; Kuiper and Kuiper, 1979 a, b). Inhibited as well as stimulated fractions of A TPases from the two ecotypes studied showed an optimum in neutral ranges (Fig. 1 and 4). This is in agreement with the results on other ATPases (Hall and Butt, 1969; Kuiper et al., 1974; Kawasaki et al., 1979; Kuiper and Kuiper, 1979 a). A slight shifting of the optimum between the different fractions might depend upon the different soils and the resultant different ion contents of the roots (Tab. 1 and 2). This is also stressed by Kuiper and Kuiper (1979 a, b) in the case of Plantago species. Besides the pH dependence, plant A TPases also show stimulation in the presence of magnesium (Hansson and Kylin, 1969; Leonard and Hodges, 1973; Hendrix and Kennedy, 1977). The investigations of Lindberg et al. (1974), Lindberg (1976), as well as Z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
A TPases from the roots of Agrostis
2.0
1.0
.~
.,
-l-.
. c
C .0;
.. Q
0
a. Q>
$ a..~
465
a.
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0,5
E
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o E .?-
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Q02
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o
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/
0
0
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.
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-'
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100 200 (1M z;l+ in ttu test ITl4qi\!m
300
Fig. 6: Effect of increasing concentrations of zinc upon the activity of Zn-stimulated A TPases from the roots of Agrostis tenuis (zinc-ecotype) at 25°C. The upper part of the figure represents the double reciprocal ~lot of the data in the lower part. +--+ without Mg2+ and 0 - - 0 with 1 mmol· m -3 Mg + in the test medium.
Erdei and Kuiper (1980) showed an optimal activity of the ATPases if the relation between magnesium and A TP was one. In the case of the inhibited A TPases from Agrostis tenuis roots this relation is less than one (Fig. 2)_ With respect to the stimulated fractions (Fig. 4)
z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
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WERNER VELTRUP
must be noted that Mg2+ must be considered as a cofactor for the A TPases studied, as was already stressed by Kylin and Lundborg (1980). With respect to the effect of zinc and copper upon the inhibited ATPases a significantly decreased activity could be found. Similar phenomena were reported for phosphatases. This was shown by Cox and Thurman (1978) who extracted enzymes from the roots of Anthoxantum plants, as well as by Cox and Hutchinson (1980) who studied phosphatases from the roots of Deschampsia plants. The inhibition of the activity might, on the one hand, be explained by the formation of copper-ATP-complexes, which could only be transformed to a small extent according to the comments of Chaberek and Martell (1959), and possibly, on the other hand, by direct interactions between heavy metals and the enzymes (Vallee and Ulmer, 1972; Foy et aI., 1978). The stimulated fractions showed that copper also inhibited, while only zinc led to positive reactions (Figs. 5 and 6). In the investigations of Leonard and Hodges (1973) as well as Perlin and Spanswick (1981) that part of the activity which depends on the presence of zinc was characterized further. Mathys (1975) could demonstrate that the nitrate reductase from the leaves of Silene cucubalus (zinc-ecotype) showed 20 % of the control activity under the influence of 15 p.mol· m -3 Zn 2+, the malate dehydrogenase 60 % under the influence of 1 mmol· m- 3 , and the isocitrate dehydrogenase only 40% of the control activity in the presence of 150 p.mol· m- 3 Zn 2+. Therefore it might be possible that in the case of Agrostis tenuis no tolerance of the enzymes may occur either, and, in consequence, higher concentration ranges should not be relevant from an eco-physiological view. In ecologically important concentration ranges the presence of magnesium is needed, especially in the lower concentration ranges of zinc. Here, a high affinity to zinc could be found because of low Km-values. Furthermore, it could be demonstrated, on account of the lower Pi liberation, that zinc cannot substitute for magnesium. This means that A TPases stimulated by zinc also require magnesium, as do other A TPases (Kylin and Gee, 1970; Lindberg et aI., 1974; Kuiper and Kuiper, 1979 b; Erdei and Kuiper, 1980). Mathys (1973) found in experiments with zinc-ecotypes of Agrostis tenuis that increasing concentrations of zinc led to an increased uptake. The investigations of Turner (1970), Auquier and de Laval (1974), Wainwright and Woolhouse (1975), and Brookes et aI. (1981), demonstrated that grasses do not possess exclusion mechanisms with respect to heavy metals. Considering the discussion concerning the participation of A TPases in ion transport processes (d. Hodges, 1976), the zinc stimulated ATPases might possibly be connected with the zinc transport. This might suggest further aspects of zinc tolerance mechanisms, but more investigations and results are needed.
Z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
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References AUQUIER, P. andJ. DE LEVAL: Teneur en zinc, plomb et manganese, in situ et en culture experimentale, des Festuca des terrains calaminaires Belges. Bull. Soc. roy. Bot. Belg. 107, 375-386 (1974). BAUMEISTER, W.: Dber den EinfluB des Zinks bei Silene inflata Smith. Ber. Deutsch. Bot. Ges. 67, 205-213 (1954). BRADSHAW, A. D.: Populations of Agrostis tenuis resistant to lead and zinc poisoning. Nature 169, 1098 (1952). BROOKES, A., J. C. COLLINS, and D. A. THURMAN: The mechanism of zinc tolerance in grasses. J. Plant Nutr. 3,695-705 (1981). CHABEREK, S. and A. E. MARTELL: Organic sequestering agents. J. Wiley and Sons, Inc., New York,1959. CHRISTIENSEN, H. H. and A. KYLIN: Substrates and modifiers of Mg- and Ca-activated ATPases from wheat roots. I. Congress ofFESPP, pp. 130-131. Edinburgh, Scotland, 1978. Cox, R. M. and T. C. HUTCHINSON: Multiple metal tolerances in the grass Deschampsia cespitosa (L.) Beauv. from the Sudbury smelting area. New Phytol. 84, 631-647 (1980). Cox, R. M. and D. A. THURMAN: Inhibition by zinc of soluble and cell wall acid phosphatases of zinc-tolerant and non-tolerant clones ofAnthoxanthum odoratum. Ibid. 80,17-22 (1978). ERDEI, L. and P. J. C. KUIPER: Substrate-dependent modulation of ATPase activity by Na+ and K+ in roots of Plantago species. Physiol. Plant. 49, 71-77 (1980). ERNST, W.: Okophysiologisch-soziologische Untersuchungen der Schwermetall-Pflanzengesellschaften Mitteleuropas unter EinschluB der Alpen. Abh. Landesmuseum Naturk. Miinster 27, 1-54 (1965). - Okologisch-soziologische Untersuchungen der Schwermetall-Pflanzengesellschaften Siidfrankreichs und des ostlichen Harzvorlandes. Flora B 156, 301-318 (1966). - Schwermetallvegetation der Erde. Fischer Verlag, Stuttgart, 1974. - Physiology of heavy metal resistance in plants. Int. Conf. Heavy Metals in the Environment, II, 121-136. Toronto, Canada, 1975. ERNST, W., W. MATHYS, and P. JANIESCH: Physiologische Grundlagen der Schwermetallresistenz-Enzymaktivitaten und organische Sauren. Forsch.-Ber. Land Nordrhein-Westfalen 2496, 1-38 (1975). FISZ, M.: Wahrscheinlichkeitsrechnung und mathematische Statistik. VEB Deutscher Verlag der Wissenschaften, Berlin, 1970. Foy, C. D., R. L. CHANEY, and M. C. WHITE: The physiology of metal toxicity in plants. Ann. Rev. Plant Physiol. 29, 511-566 (1978). HALL, J. L.: Enzyme localization and ion transport. In: ANDERSON, W. P. (ed.): Ion transport in plants, pp. 11-24. Academic Press, London, New York, 1973. HALL, J. L. and V. S. BUTT: Adenosine triphosphatase activity in cell wall preparations and excised roots of barley. J. Exp. Bot. 20, 751-762 (1969). HANSSON, G. and A. KYLIN: ATPase activities in homogenates from sugar beet roots, relation to Mg2+ and (Na+ + K+)-stimulation. Z. Pflanzenphysiol. 60, 270-275 (1969). HENDRIX, D. L. and R. M. KENNEDY: Adenosine triphosphatase from soybean callus and root cells. Plant Physiol. 59, 264-267 (1977). HILL, B. S. and A. E. HILL: Enzymatic approaches to chloride transport in the Limonium salt gland. In: ANDERSON, W. P. (ed.): Ion transport in plants, pp. 379-384. Academic Press, London, New York, 1973. HODGES, T. K.: ATPases associated with membranes of plant cells. In: LUTTGE, U. and M. G. PITMAN (eds.): Transport in plants. II. Part A Cells, pp. 260-283. Springer-Verlag, Berlin, Heidelberg, New York, 1976. Z. Pjlanzenphysiol. Bd. 108. S. 457-469. 1982.
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