THE RESPONSE OF ROOT ACID PHOSPHATASE ACTIVITY TO HEAVY METAL STRESS IN TOLERANT AND NON-TOLERANT CLONES OF TWO GRASS SPECIES

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1 Netv Phytol. (\9S0) 96, THE RESPONSE OF ROOT ACID PHOSPHATASE ACTIVITY TO HEAVY METAL STRESS IN TOLERANT AND NON-TOLERANT CLONES OF TWO GRASS SPECIES S ' ''', " ' i, \..- ' - ' ;. BY R. M. C O X AND T. C. HUTCHINSON Department of Botany and Institute for Environmental Studies^ University of Toronto, Toronto, Ontario, M5S lal, Canada {Accepted 20 February 1980) SUMMARY The inhibition by zinc of phosphatase activity in dialysed root extracts was determined for zinc-tolerant and non-tolerant clones of Anthoxanthum odoratum L. Similarly the inhibition by copper of this enzyme activity in root extracts of copper-tolerant and non-tolerant clones of Deschampsia cespitosa (L.) Beauv. was also determined. It was found for both species that the relationship between the in vitro percentage inhibition of enzymatic activity and the tolerance index of each clone, changed after exposure to sub-lethal concentrations of the respective metal in water culture. The enzyme activity in the root extracts of the tolerant clones was found to be significantly less inhibited by the particular metal, than those of the non-tolerant clones only when the plants were previously exposed to the particular metal in vivo. Since exposure of the roots to metal appeared to induce a positive reaction in tolerant material, it is suggested that physiological and biochemical investigations of metal tolerance may be more informative if plants that have been pre-stressed by a particular metal are used. This should enable a better recognition of adaptive components of metabolism. Examples of such an approach and its implications are discussed. INTRODUCTION Enzymes have been implicated in the enhanced tolerance of certain populations and species to elevated concentrations of heavy metals. Woolhouse (1969) reported differences in the sensitivity of cell wall ATPase activity towards aluminium in edaphic ecotypes of Agrostis tenuis. A copper-tolerant race of this species was found to possess cell wall acid phosphatase activities which were less inhibited in the presence of copper than those from copper-susceptible plants (Wainwright and Woolhouse, 1976). Similarly, Mathys (1975 a) found that carbonic anhydrase activity in Silene cucubalus was stimulated by high amounts of zinc in tolerant populations, but not in non-tolerant populations. Wu, Thurman and Bradshaw (1975) observed differences in the copper inhibition of malate dehydrogenase activity between tolerant and nontolerant strains of Agrostis stolonifera, but only after exposure of the plants to copper. Cox, Thurman and Brett (1976) demonstrated that individuals of Anthoxanthum odoratum, collected on a transect across a zinc-lead mine boundary into an adjacent pasture at Trelogan (Clywd, Wales), showed a significantly different distribution in their acid phosphatase isoenzymes. Differences were noted in substrate affinities (istm's) and in specific activities of root acid phosphatases between zinc-tolerant and non-tolerant clones of this species. Using Anthoxanthum odoratum from the same site. Cox and Thurman (1978) reported that zinc-tolerant and non-tolerant clones possessed X/80/ $0200/0 ^ 1980 The New Phytologist

2 360 R. M. Cox AND T. C. H U T C H I N S O N - isoenzymes of acid phosphatase that varied in their zinc inhibitor constants (^i') and that tolerant clones possessed more isoenzymes with higher Ki' values than did nontolerant clones. It has also been shown that the percentage inhibition of enzymes failed to distinguish between tolerant and non-tolerant ecotypes (Cox et al., 1976). However, differences in activities of various enzymes have been reported after in vivo exposure of other plant species to metals (Wu et al., 1975; Mathys, 1975 b). The above observations prompted the examination of the in vitro inhibition of root acid phosphatase activity of two grasses with known heavy metal tolerant and nontolerant populations; zinc-tolerant and non-tolerant clones of Anthoxanthum odoratum (Cox, 1976) and copper-tolerant and non-tolerant clones of Deschampsia cespitosa. These studies compare the inhibition of enzymatic activity in root extracts before and after in vivo exposure of the tillers to a sub-lethal concentration of the appropriate metal. M A T E R I A L S AND M E T H O D S Plant material Clones of A. odoratum were collected from a zinc-lead mine and from an adjacent pasture at Trelogan, U.K. in January, 1971 and were grown in Vitax soil mix. They are described more fully in Cox et al. (1976). Clones of D. cespitosa were collected in May, 1977 as tillers from around the Coniston nickel-copper smelter at Sudbury, Ontario. The soil on which these plants were growing was highly contaminated with both copper and nickel (Hutchinson and Whitby, 1974). In addition, clones of D. cespitosa were grown from seed collected from plants in an uncontaminated calcareous pasture in Derbyshire, U.K. Methods Clones of A. odoratum of known tolerance to zinc (Cox and Thurman, 1978) and D. cespitosa clones of known tolerance to copper (Cox and Hutchinson, 1979) were used in two separate experiments. Tillers of each clone of A. odoratum were allowed to root in 0-5 g dm~^ calcium nitrate solution alone and also in solutions of calcium nitrate containing 2-5, 5-0 and 7-5 p.p.m. Zn. After 10 days, the longest root of 20 randomly selected tillers of each clone in each zinc concentration was measured to obtain a tolerance index (relative root growth) for the two clones at all zinc exposures prior to excision of the 2-0 cm root tips for extraction. Tillers from six copper-tolerant Coniston clones and six non-tolerant Derbyshire clones of D. cespitosa were allowed to root in the calcium nitrate solution, with and without addition of 0-2 p.p.m. copper. Again, the longest roots of 20 randomly selected tillers were measured to obtain a tolerance index for each clone before excision of the 2-0 cm root tips for extraction. Root extracts were prepared and assayed for acid phosphatase activity as described by Cox et al. (1976). This procedure included dialysis for 15 h against 2 dm^ TrisHCI ph 7-5 buffer. The in vitro percentage inhibition of acid phosphatase activity by zinc and copper was derived from activities in the presence and absence of 0-6 p.p.m. zinc and 0-06 p.p.m. copper respectively. Three replicate determinations were made.

3 Root phosphatase and heavy metals 361 RESULTS The various concentrations of zinc inhibited the root growth of the non-tolerant clone of A. odoratum, but allowed sufficient growth at the highest concentration for the preparation of enzyme extracts. There was a statistically significant negative correlation (P<0-001) between the percentage inhibition of acid phosphatase activity and the tolerance indices of the two clones in each zinc concentration (Fig. 1). u c o E o 5 V. c o Q. O = X = P<O-OOl % Tolerance index for zinc Fig. 1. Relationship between the percentage inhibition of enzyme activity by 0-6 p.p.m. zinc and the tolerance index of zinc-tolerant (O) and non-tolerant (#) clones of Anthoxanthum odoratum, grown in the presence of 2-5, 5-0 and 7-5 p.p.m. zinc in culture medium. Table 1. Tolerance indices and percentage inhibition of root acid phosphatase activity in copper-tolerant and non-tolerant clones of D. cespitosa Clone Percentage tolerance index (0-2 p.p.m. Cu) X % inhibition of Grown in the absence of copper Copper-tolerant enzyme activity (arc sin transformed) by 0-06 p. p.m.copper S.E. clones ±0-3 ±0-7 ± Non-tolerant clones ±0-2 ±1-7 ± ±0-7 Grown in the presence of coppei S.E. ± ±0-3 ±0-2 ±0-1 ± f-tests of differences between non-pretreated roots of copper-tolerant and non-tolerant clones revealed no significant differences. f-tests of differences between copper pre-tieated roots of copper-tolerant and non-tolerant clones showed a significant difference (P< 0-02, t for 5 d.f.).

4 362 R. M. Cox AND T. C. HUTCHINSON Table 1 shows the tolerance indices of the D. cespitosa clones in the absence and presence of 0-2 p.p.m. copper and their subsequent in vitro percentage inhibitions of root acid phosphatase activity by copper. These data demonstrate that after exposure to copper in vivo, tolerant and non-tolerant clones of D. cespitosa exhibit a significant difference (P<0-02) in the degree to which copper inhibits enzyme activity in the extracts. No significant difference was found with tillers not pre-exposed to copper. The relationship between the copper tolerance index and percentage inhibition of enzyme activity is shown in Figures 2(a) and 2(b), for clones grown in the absence and presence of copper respectively. All correlations shown fail to reach the 5 % level of significance. However, there appears to be a clearer negative relationship after exposure to copper. y = I X /-s=-0-3l P-ri.%. / = ^ /'c = P = n.s. O \ = ^5=0-08 /O ' = ,. = P=n.s. // A I L L % Tolerance index for copper Fig. 2. Relationship between the percentage inhibition of enzyme activity by 006 p.p.m. copper and the tolerance index of six copper-tolerant (O) and six non-tolerant (#) clones of Deschampsia cespitosa. Tillers were grown (a) in the absence and (b) in the presence of 0-2 p.p.m. copper. DISCUSSION Work reported by Cox et al. (1976) showed that no differences could be found in the in vitro zinc inhibition of root acid phosphatase activity of zinc-tolerant and nontolerant A. odoratum when the plants were grown in the absence of zinc. Furthermore, there was no relationship between this inhibition and the index of tolerance. In contrast, the current investigation has shown that when tillers were pre-exposed to various concentrations of zinc in water culture there was a highly significant relationship between these two parameters. Likewise, significant differences in the degree of copper inhibition of acid phosphatase activity was found in the two contrasting D. cespitosa populations only after the tillers had been pre-stressed by growth in 0-2 p.p.m. copper. These findings suggest that the inhibition of acid phosphatase activity by these metals may be inducible in the two species. In addition, it seems that a decrease in copper inhibition of the enzyme activity can be more easily achieved with tolerant genotypes. The use of pre-stressed plants to investigate subtle adaptive features of their metabolism may be important. Wu et al. (1975) found differences in malate dehydrogenase activity and in respiration rates between copper-tolerant and non-tolerant clones of Agrostis stolonifera only after in vivo pre-treatment with copper. Mathys

5 Root phosphatase and heavy metals 363 (1975 a, b) was unable to show differences in in vitro inhibition of several leaf enzymes of zinc-tolerant and non-tolerant S. cucubalus until the plants were previously placed under conditions of metal stress. Leaf extracts of zinc-tolerant clones of this species grown in the presence of zinc had higher nitrate reductase activity than those of nontolerant clones. Isocitrate dehydrogenase activity was also increased in the tolerant plants relative to the non-tolerant ones whereas glucose-6-phosphatase activity was decreased in the tolerant and increased in the non-tolerant plants. The above authors recorded enzyme activity in tolerant and non-tolerant genotypes after pre-treatment, but did not measure the effectiveness of the enzymes in the presence of the metal, hence their adaptive significance is obscure. The present study has demonstrated that after in vivo pre-treatment with the particular metal, root acid phosphatase activity of tolerant clones was less inhibited in the presence of the metal than that of non-tolerant clones. This strongly suggests that at least a component of this acid phosphatase activity can be maintained in the presence of the metal in the tolerant plants. This must be of considerable adaptive significance as the low phosphate availability in these heavy metal contaminated sites would necessitate both efficient uptake of phosphorus and its recycling within the plant. The change in the in vitro inhibition of acid phosphatase activity after in vivo exposure is little understood. Possible explanations may include an induced change in the proportion of isoenzymes, with higher inhibitor constants for the particular metal in the root extract (Cox and Thurman, 1978), or the production of a high molecular weight compound that remains after dialysis and reduces the free metal concentration by complex formation (Stokes, Maler and Riordan, 1977). Such a compound, however, would need to have a high complexing capacity and be specific for a particular metal to explain the degree of specificity of metal tolerance (Cox and Hutchinson, 1979). The degree of specificity of the induction needs investigation, especially as it relates to mechanisms of metal tolerances and to co-tolerances. ACKNOWLEDGEMENTS For the Canadian studies we acknowledge the technical assistance of H. Schabelski and financial assistance to T. C. Hutchinson from the Department of Indian and Northern Affairs, Ottawa on their Arctic Land Use Research (ALUR) programme. For the A. odoratum studies, R. M. Cox was a recipient of a U.K. Science Research Council Studentship. We also thank R. L. Jefferies and D. A. Thurman for their helpful criticism of this manuscript. REFERENCES Cox, R. M. (1976). Properties of some enzymes of zinc-tolerant and non-tolerant clones o/anthoxanthum odoratum. Ph.D. Thesis, University of Liverpool. Cox, R. M. & HUTCHINSON, T. C. (1979). Metal co-tolerance in the grass Deschampsia cespitosa. Nature, London, 279, Cox, R. M., THURMAN, D. A. & BRETT, M. J. (1976). Some properties of soluble acid phosphatases of roots of zinc-tolerant and non-tolerant clones of Anthoxanthum odoratum. New Phytologist, 77, Cox, R. M. & THURMAN, D. A. (1978). Inhibition by zinc of soluble and cell vv^all acid phosphatases of zinc-tolerant and non-tolerant clones of Anthoxanthum odoratum. New Phytologist, 80, i7-22. HUTCHINSON, T. C. & WHITBY, L. M. (1974). Heavy-metal pollution in the Sudbury mining and smelting region of Canada. I. Soil and Vegetation Contamination by nickel and copper, and other metals. Environmental Conservation, 1,

6 364 R. M. Cox AND T. C. HUTCHINSON MATHYS, W (1975a). quoted by W. H. O. Ernst (1975). In: Symposium Proceedings of the International Conference on Heavy Metals in the Environment (Ed. by T. C. Hutchinson), Vol. II, Part I, pp Institute for Environmental Studies, University of Toronto, Ontario, Canada. MATHYS, W. (1975 b). Enzymes of heavy metal-resistant and non-resistant populations of Silene cucubalus and their interaction with some heavy metals in vitro and in vivo. Physiologia Plantarum, 33, STOKES, P. M., MALER, T. & RIORDAN, J. (1977). In: Trace Substances in Environmental Health. (Ed. by D. D. Hemphill), Vol. II, pp University of Missouri. WAINWRIGHT, S. J. & WOOLHOUSE, H. W. (1976). Physiological mechanisms of heavy metal tolerance. In: The Ecology of Resource Degradation and Renewal. British Ecological Society Symposium. No. 15 (Ed. by M. J. Chadwick & G. T. Goodman). Blackwells Scientific Publishers, Oxford. WOOLHOUSE, H. W. (1969). Differences in the properties of acid phosphatases of plant roots and their significance in the evolution of edaphic ecotypes. In: Ecological Aspects of Mineral Nutrition of Plants (Ed. by I. H. Rorison). Blackwell, Oxford. Wu, LIN., THURMAN, D. A. & BRADSHAW, A. D. (1975). The uptake of copper and its effects upon respiratory processes of roots of copper-tolerant and non-tolerant clones of Agrostis stolonifera. New Phytologist, 75,

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