Comparative Investigations on the Nitrogen Metabolism of Two Agrostis tenuis Populations from a Metalliferous Waste1)

Flora (1982) 172: 113-124

Comparative Investigations on the Nitrogen Metabolism of Two Agrostis tenuis Populations from a Metalliferous Wastel ) H. J. WEIGEL, A. PRIEBE and H.-J. JAGER Institut fUr Pflanzeniikologie der .Tustus-Liebig-Universitat GieJ.len, FRG

Summary Some aspects of nitrogen metabolism of two Agrostis tenuis populations of different zinc sensitivity originating from a Violetum calaminariae westfalicum at Blankenrode, FRG (51 0 32N 8° 33E) have been investigated. As defined by growth and yield reduction both populations, the natural habitat of which is characterized by soil zinc concentrations of 8,335 mg Zn/kg and 62,138 mg Zn/kg dry soil, respectively, behaved differently towards an experimental zinc treatment of 37,500-75,000 mg Zn/kg dry soil. At the 75,000 mg Zn/kg treatment the population originating from the low zinc soil was zinc sensitive, while no growth retardation could be observed with the population originating from the high zinc soil. Total protein content and the activities of the enzymes glutamate dehydrogenase and malate dehydrogenase were in the same order of magnitude in both populations and were not influenced by any of the zinc treatments. The proportion of non-dialyzable zinc increased with increasing plant zinc concentrations in both populations. Free amino acids and free and bound polyamines did not differ qualitatively between the two populations. However, the sum of the concentrations of all amino acids investigated increased with increasing plant zinc concentrations in the zinc sensitive population, this increase being mainly caused by an increase of the concentrations of the amides glutamine and asparagine. In the zinc tolerant population the concentrations of the free amino acids were lower as compared to the control plants of the zinc sensitive population at soil zinc concentrations (75,000 mg/kg), which were in the same order of magnitude as in the soil of the populations natural habitat. These results provide evidence that the free amino acids investigated do not function as complexing agents for heavy metal tolerant plant species. The significance of the decrease in the concentrations of free polyamines in the zinc sensitive population at high soil zinc concentrations should be elucidated by further investigations.

Introduction Several plant species from metalliferous soils have evolved specific tolerance against those heavy metals which are abundant in their natural habitat (ANTONOVICS et al. 1971; ERNST 1974). Natural populations of these species often differ morphologically and are characterized as edaphic ecotypes (BRADSHAW 1952; HESLOP-HARRISON 1964). While ecological and genetical aspects of heavy metal tolerance have been extensively studied interest has now focussed on the physiological mechanisms possessed by these plants, which enables them to tolerate elevated concentrations of these elements in their environment (WAINWRIGHT & WOOLHOUSE 1975; ERNST 1975, 1977). As tolerance is not based on a differential total uptake of heavy metals, the 1)

Dedicated to Prof. Dr. L.

STEUBING

on the occasion of her 60th birthday.

114

H. J. WEIGEL et al.

resistance seems to involve a special internal metabolism (ERNST 1977). The importance of the cell wall in binding of heavy metals and removing them from the plants metabolism has been shown by several workers (TURNER 1970; MATHYS 1973; ERNST 1977). On the other hand the existence of heavy metal tolerant enzymes had to be denied (MATHYS 1975; ERNST 1976). The occurrence of essentially higher amounts of malic acid in zinc tolerant populations of all plant species investigated compared to zinc intolerant ones, provided evidence for metabolic adaptation of heavy metal tolerant plants (ERNST et al. 1975; MATHYS 1977). Malic acid is thought to chelate zinc and to function p,s a carrier for the transport of zinc across the cytoplasm. In spite of this, metabolic pathways of heavy metal tolerant plants are only poorly characterized, which is mainly true for the nitrogen metabolism. As for example the ability of amino acids to from stable complexes with heavy metals is well known (GURD & WILCOX 1956; SILLEN & MARTELL 1964; HENKIN 1974), it could be possible that nitrogen compounds are also involved in metal detoxification processes possessed by these plants. The objective of the work reported in this paper, therefore, was to investigate aspects of nitrogen metabolism of two Agrostis tenuis populations of different zinc sensitivity. Especially amino acids and free and bound polyamines have not yet been described quantitatively in metal tolerant plants.

Materials and Methods Sampling and preparation of the plant material The study was conducted in a field trial with two different populations of Agro8ti8 tenui8 SIBTH. Seed collections were made at two different sites of the zinc contaminated soil of the "Bleikuhle" of Blankenrode, FRG (51° 32N 8° 33E). The zinc concentrations of the dry soil (HNO a extract) was 8,335.0 ± 194.3 mg Zn/kg and 62,138.0 ± 3,090.0 mg Zn/kg, respectively. Plant populations originating from these soils are designated as S-1 population (low zinc soil) and S-2 population (high zinc soil) throughout this paper. Seeds were germinated on quartz sand in a greenhouse and after 6 weeks individual seedlings were transferred to a 5: 1 mixture of a commercially available artificial soil (Frustosoil N) and quartz sand. The zinc concentration of this soil was 48 mg Zn/kg dry matter. Soil zinc concentrations of 37,500 mg Zn/kg and 75,000 mg Zn/kg dry matter were adjusted by adding ZnO. Plants were grown until maturity in the field during May-August 1979. Zinc analysis Determination of plant and soil zinc concentrations was carried out by atomic absorption srectroscopy according to KLEIN et al. (1979a) using a Pye Unicam SP 90 atomic absorption spectrophotometer. Analysis of free amino acids and free and bound polyamines Extraction of the free amino acids and free (water soluble) polyamines was achieved by homogenization of freeze dried plant material in MeOH/CHCl a/H 2 0 (12: 5: 3, vol/vol) and subsequent centrifugation (BIELESKI & TURNER 1966; JAGER 1975). The resulting pellet was reextracted several times with MeOH/CHCl a/H 2 0. After addition of CHCl a/H2 0 (1 : 1.5, vol/vol) to the supernatant the phases were separated and the water soluble fraction containing free amino acids and free polyamines was dried under vacuum at 35°C. The residue was redissolved in sodium citrate buffer (0.2 M Na, pH 2.2). The methanol insoluble residue containing bound (HCI extractable) polyamines was extracted for 1 h with 12 % HCI at 100°C and centrifuged. The supernatant was

Comparative Investigations on the Nitrogen Metabolism

1I5

dried under vacuum at 55°C and the residue redissolved in sodium citrate buffer (0.2 M Na, pH 2.2.) Quantitative deterrrination of polyamines was carried out by a modification of the methods of BREMER et al. (1971) and JAGER (1975) using an automated amino acid analyzer BC 100 (LKB). For analysis of free amino acids an automated amino acid analyzer 3201 (LKB) was used. Analysis of enzyme activities and protein content Soluble glutamate dehydrogenase (GDH) and malate dehydrogenase (MDH) extracts were made by grinding freeze dried plant material in tris buffer (0.1 M tris/acetate, pH 7.5, 10- 3 M EDTA, I % polyvinylpyrrolidone) at 4°C and centrifuging at 20,000· g for 30 rrin. The enzyme solution was dialyzed against a continous flow of 0.01 M tris/acetate buffer for 16 h. GDH-activity was assayed with oxoglutarate as substrate according to PAHLIeH & JOY (1971). The activity of the MDH (malate formation) was determined as described by HABIG & RACUSEN (1974). Total protein content of the freeze dried plant material was estimated by the Kjeldahl method after extraction with TCA (5%).

Results and Discussions The present investigation was c8rried out with two populations of Agrostis tenuis originating from a soil of different zinc concentrations at the "Bleikuhle" of Blankenrode, FRG (51 ° 32N 8° 33E). The vegetation of this heavy metal contaminated habitat is mainly composed of the species Viola caZaminaria subsp. westtaZica, SiZene cucubalus, Minuartia verna and Festuca ovina. The plant community has been extensively investigated by ERNST (1968, 1974) and can be described as Violetum calaminariae westfalicum. After a 3 month growth period in the field the two Agrostis tenuis populations grown on the control soil (48 mg Znjkg dry matter) did not show significant differences SIBTH.

Table 1. The effect of different soil zinc concentrations (mg Zn/kg dry soil) on biomass production (gram fresh and dry weight) and plant zinc concentration (pg Zn/g dry matter) of two Agrostis tenuis populations (S-l and S-2) differing in zinc tolerance. Results represent the mean of 5 determinations ± standard deviation soil zinc concentration Control 48

37,500

75,000

fresh weight

dry weight

plant zinc population concentration

9.84 ±1.30 11.60 ±2.50

2.05 ±0.26 3.05 ±0.65

6.04 ±1.80 11.49 ±1.71

1.20 ±0.34 2.44 ±0.36

596.6 ±89.0 660.5 ±147.0

S-I

2.88 ±1.30 9.52 ±1.28

0.55 ±0.24 2.16 ±0.29

1,255.6 ±100.5 791.2 ±102.5

8-1

175.0 ±4.90 210.0 ±9.90

S-l (sensitive) 8·2 (tolerant)

S-2

8-2

4

116

H. J.

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et al.

a

b

Fig. 1. The effect of different soil zinc concentrations on the growth of two Agrostis tenuis populations ("Hang" = S-I; "Grund" = S-2) differing in zinc tolerance. Zinc concentrations of 5,000, 15,000, and 30,000 ppm (calculated on the water content of the soil) correspond to 12,500, 37,500 and 75,000 mg Zn/kg dry soil, respectively. Sec also legend of Table 1.

117

Comparative Investigations on the Nitrogen Metabolism

in growth and yield (Table 1, Fig. 1). However, addition of 37,500 mg Zn/kg and mainly 75,000 mg Zn/kg soil resulted in a. significant yield reduction for the population (S-l population) originating from the soil with low zinc concentration (8,335 mg/kg). Additionally yield reduction for this population was accompanied by stunted growth and foliar chlorosis at the 75,000 mg Zn/kg treatment (Fig. 1). Growth and yield of the S-2 population were not affected by any of the zinc treatments (Table 1, Fig. 1). As defined by visual symptoms and biomass production the S-l popula.tion therefore was more sensitive towards a zinc treatment than was the S-2 population. Similarily BRADSHAW et 801. (1965); WAINWRIGHT & WOOLHOUSE (1975); KA.RATAGLIS (1980) found that generally there is a positive correlation between the amount of metal in the soil and the degree of tolerance to this metal of plants growing on that soil. Plant zinc concentrations in the present case increased with increasing soil zinc concentrations, which was mainly true for the S-l population (Table 1). At the 75,000 mg Zn treatment the more sensitive Agrostis tenuis population took up more zinc than did the less sensitive S-2 population. MATHYS (1973) reported similar results with A. tenuis populations of different zinc tolerance, i.e. the tolerant populations had a diminished zinc uptake. However, experiments of TURNER & GREGORY (1967) have shown that the uptake of zinc is the same for both tolerant and non-tolerant populations of Agrostis tenuis. Our results again provide evidence that populations of the same species may differ in metal content under identical uptake conditions (ERNST 1976). These different effects of a long term zinc treatment on growth and yield of the two Agrostis tenuis popUlations could only partly be correlated with zinc caused alterations Table 2. The effect of different soil zinc concentrations (mg Zn/kg dry soil) on protein content (mg/g dry matter), the activities (Units/g dry matter) of the enzymes glutamate dehydrogenase (GDH) and malate dehydrogenase (MDH) and the concentrations of non-dialyzable zinc (I'g Zn!g dry matter) of two Agrostis tenuis populations (S-l and S-2) differing in zinc tolerance. Results represent the mean of 5 determinations ± standard deviation Boil zinc concentration Control 48.0

37,500

75,000

protein

GDH

MDH

non-dialyzable zinc

population

169 ±7.0 lS3 ±21.5

0.85 ±0.18 0.71 ±0.21

225.3 ±9S.4 lS0.3 ±llS.O

26.3 ±11.0 49.0 ±17.0

S-l

179 ±14.0 172 ±19.7

0.71 ±0.14 0.6S ±0.06

lS6.0 ±67.3 95.S ±36.0

Sl.5 ±43.0 133.7 ±lS.3

S-l

lS3 ±24.0 174 ±13.0

1.25 ±0.50 0.59 ±0.10

206.5 ±SS.6 253.7 ±102.4

263.0 ±3S.0 169.5 ±57.0

S-l

S-2

S-2

S-2

ll8

H. J.

WEIGEL

at al.

of the plants metabolism. The activities of the enzymes GDH and MDH, which were chosen representative for two important metabolic pathways in plants, were not affected by any of the zinc treatments in vivo (Table 2). Additionally the activities of both enzymes were in the same order of magnitude in the two populations at any zinc treatment. While the existence of heavy metal resistant enzymes in metal tolerant plant species had to be denied on the basis of results of in vitro investigations (ERNST 1976, 1977), the in vivo behaviour of enzymes of these plants are quite different after a metal treatment compared to the in vitro results. According to ERNST et al. (1975); MATHYS (1975) and ERNST (1976) the in vivo activity of the enzyme MDH remains unchanged in populations of Silene cucUbalus after a zinc treatment, which is in good agreement with the present results (Table 2). On the other hand the activities of the enzymes nitrate reductase and glucose.6-phosphate dehydrogenase are either stimulated or inhibited in zinc tolerant popvlations of the same plant as compared to nontolerant populations (ERNST et al. 1975; ERNST 1977). No difference could be found in the metal tolerance of the glutamate dehydrogenase between tolerant and nontolerant populations in vitro (ERNST 1976), which seems to be true also in vivo (Table2). Although enzyme activities are not affected in vivo our results provide evidence that an intimate contact between zinc and plant proteins might occur (Table 2). As the concentration of non-dialyzable zinc increased with increasing soil and plant zinc concentrations in both populations, increasing amounts of zinc could be bound to high molecular weight proteins. As the activities of the GDH and MDH are not affected by zinc in vivo, the zinc binding sites should not be at these proteins but at other distinct soluble proteins. However, changes in total protein concentrations with increasing plant zinc concentrations could not be observed (Table 2). Changes in enzyme activities, therefore, do not seem to be responsible for the different susceptibility of the Agrostis tenuis populations towards a zinc treatment in vivo. Although no changes in the concentrations of total protein could be observed in the present case (Table 1) the existence of distinct (special) metal binding proteins in higher plants should not be denied (WEIGEL & JAGER 1980; BARTOLF et al. 1980). Further investigations concerning metal binding proteins in metal tolerant plant species are in progress. The possibility of binding of heavy metals to proteins in metal tolerant plant species was also suggested by REILLY et al. (1970) and REILLY (1972), who were able to show that an increased copper content is accompanied by an increase in total nitrogen in the copper tolerant plant Becium homblei. Only recently a special copper binding protein, metallothionein, has been found in roots of Agrostis gigantea tolerant to excess copper (RAUSER & CURVETTO 1980). According to ERNST (1977) special copper and zinc binding proteins have not yet been found in leaves of higher plants. REILLY et al. (1970) were able to demonstrate that copper may also be complexed by amino acids in extracts of leaf tissue of Becium homblei, while no qualitative differences of the total range of amino acids could be found between copper tolerant and non-tolerant populations of the same plants species (REILLY 1972). HOFNER (1970) and GOMAH & DAVIS (1974) were able to show that in crop plants amino acids are able to form complexes with a variety of heavy metals. In spite of these results the amino

119

Comparative Investigations on the Nitrogen Metabolism

Table 3. The effect of different soil zinc concentrations (mg Zn/kg dry soil) on the concentrations of free amino acids (.umoles/g dry matter) of two Agrostis tenuis populations (8-1 and 8-2) differing in zinc tolerance. Results represent the mean of 3 determinations ± standard deviation soil zinc concentration

Control

population

8-1

ABp Asn

2.69 ± 0.30 5.20 ± 11.30 ± 1.84 20.90 ± 1.68 ± 0.24 2.60 ± 3.88 ± 0.18 6.57 ± 4.65 ± 0.60 10.90 ± 9.50 ± 2.00 16.50 ± 2.64 ± 0.32 6.91 ± 0.19 ± 0.02 0.53 ± 3.55 ± 0.30 5.89 ± 0.96 ± 0.08 1.36 ± 0.52 ± 0.04 0.72 ± 1.48 ± 0.20 1.44 ± 0.28 ± 0.02 0.38 ± 0.57 ± 0.04 0.68 ± 5.66 ± 0.94 5.28 ± 0.28 ± 0.02 0.83 ± 0.33 ± 0.01 1.02 ± 0.20 ± 0.01 0.31 ± 5.08 ± 1.00 6.00 ±

Thr

8er GIu GIn Pro GIy Ala Val Ile Leu Tyr Phe Gaba Lys His Arg NH4+

~

55.40

75,000

37,500

48.0

8-1

8-2

93.11

1.20 3.16 0.80 0.84 2.08 2.24 1.38 0.04 1.00 0.24 0.10 0.28 0.01 0.09 1.02 0.04 0.08 0.01 1.42

9.10 ± 2.12 15.40 ± 2.17 4.55 ± 0.42 8.60 ± 1.20 21.50 ± 2.12 10.60 ± 1.25 4.10 ± 0.57 0.45 ± 0.04 8.85 ± 1.98 2.50 ± 0.24 1.70 ± 0.20 2.20 ± 0.15 0.85 ± 0.08 1.25 ± 0.08 7.85 ± 1.18 1.20 ± 0.10 0.75 ± 0.08 0.14 ± 0.01 17.00 ± 1.92 118.54

8-2

8·1

5.46 ± 0.80 3.24 ± 0.42 10.05 ± 1.89 26.10 ± 2.50 2.70 ± 0.24 1.55 ± 0.24 7.64 ± 1.20 2.16 ± 0.30 8.30 ± 1.45 15.90 ± 2.00 1.60 ± 0.40 49.90 ± 4.00 3.06 ± 0.34 3.76 ± 0.34 0.31 ± 0.02 0.51 ± 0.08 6.76 ± 0.84 4.98 ± 0.92 1.45 ± 0.05 1.38 ± 0.42 0.93 ± 0.10 0.79 ± 0.09 3.79 ± 0.32 1.64 ± 0.17 0.46 ± 0.01 0.33 ± 0.02 0.96 ± 0.18 0.65 ± 0.02 6.25 ± 1.84 5.20 ± 0.82 0.98 ± 0.17 0.65 ± 0.12 0.41 ± 0.04 0.42 ± 0.04 0.14 ± 0.01 0.13 ± 0.01 4.04 ± 0.92 14.40 ± 1.89 52.83

145.94

8-2

3.60 ± 0.42 7.26 ± 1.50 1.23 ± 0.28 2.60 ± 0.32 9.32 ± 2.12 4.00 ± 0.60 2.11 ± 0.34 0.22 ± 0.04 2.19 ± 0.95 0.70 ± 0.08 0.39 ± 0.10 1.40 ± 0.25 0.22 ± 0.05 0.48 ± 0.08 1.93 ± 0.30 0.22 ± 0.02 0.17 ± 0.04 0.15 ± 0.02 1.62 ± 0.40 39.85

acid composition of metal tolerant plant species has not been investigated quantitatively up to now. To elucidate a possible role of these nitrogen compounds in metal tolerance we investigated the amino acid composition of the two Agr08tis tenuis populations (Table 3). Moreover, changes in the concentration of free amino acids are highly indicative for stress situations in plants (STEWART & LARHER 1980; JAGER & KLEIN 1980). In Table 3 the concentrations of the free amino acids and the amides glutamine and asparagine of the two Agrostis tenuis populations are shown. The variety of the amino acids as well as their concentrations are in the same range as in other plant species as for example halophytes (PRIEBE & JAGER 1978; Pon & ALBERT 1980). However, differences are obvious between the two Agr08tis tenuis populations originating from soils differing in zinc concentrations. Zinc treatment (75,000 mgjkg) resulted in a decrease of the sum of all amino acids investigated in the S-2 population, which was mainly caused by a decrease in the concentrations of the amides glutamine and asparagine comprising about one third of the concentrations of all amino acids

* 120

H. J.

WEIGEL

et al.

investigated in the control plants. Opposite to this the sum of all amino acids raised about two fold in the 8-1 population after the 75,000 mg Znjkg treatment. This increase again was caused mainly by an increase in the concentrations of the amides glutamine and asparagine together with an increase of free ammonium. According to GIVAN (1979) and JAGER & KLEIN (1980) such an increase in glutamine, asparagine and ammonium reflects a disturbance in protein turnover. In the present case protein synthesis or degradation might have been affected by zinc in the sensitive Agrostis tenuis population, too. The higher concentrations of all amino acids investigated in the control plants of the 8-2 population compared to the 75,000 mg Znjkg treatment and to the control plants of the 8-1 population might reflect a suboptimal zinc supply resulting in a changed protein metabolism and a increase in glutamine, asparagine and other amino acids. For the 8-2 population the 75,000 mg Znjkg treatment, therefore, should results in an optimal zinc supply with low i.e. "normal" concentrations of amino acids and amides. For the 8-1 population this zinc supply is supraoptimal i.e. toxic resulting in reduction of biomass production (Table 1) and elevated concentrations of free amino acids. As the concentration of the free amino acids increased with increasing soil zinc concentrations in the 8-1 population, while at the same time growth and yield of the plants are reduced, it seems unlikely that amino acids play an important role in the detoxification via complexation of heavy metals in metal tolerant plants. This is further confirmed by the finding that the amino acid concentration of the zinc insensitive 8-2 population of Agrostis tenuis is "low" at the 75,000 mg Znjkg treatment. This zinc treatment corresponds to soil zinc concentrations of the plants natural habitat (see Material and Methods) and does not result in restricted growth. Also no information has become available concerning the concentration of polyamines in metal tolerant plant species. Polyamines are metabolically derived from amino acids and since their function is to stabilize macromolecules like DNA and RNA (TABOR & TABOR 1972) and to be involved in homeostatic buffering mechanisms (8MITH & 8INCLAIR 1967) polyamines occur both in free and bound form. Only recently polyamines were shown to be involved in the regulation and induction of metabolic adaptations of plants to extreme environmental conditions as for example crassulaceen acid metabolism (MOREL et al. 1980). Both kinds of polyamines could be found in extracts of the Agrostis tenuis populations investigated in the present case (Table 4). With the exception of free spermidine and spermine the concentrations of free and bound polyamines are in the same order of magnitude in both populations. In the 8-2 population the concentrations of the polyamines are not affected by the zinc treatment with the exception of a decrease of bound putrescine and spermine. In the 8-1 population no free polyamines could be detected in the 75,000 mg Znjkg treated plants, whereas bound polyamines are not affected or slightly increased. Especially for putrescine the high zinc treatment (75,000 mg/kg) may have resulted in a shift of the free to the bound form reflecting a function in the stabilization of macromolecules under these conditions. 8imilarily the concentrations of free polyamines are reduced in differentially salt tolerant plant species under conditions of increasing salinity (PRIEBE

•

'"

-:r

...

f'

I:!j

trace trace trace trace

trace 0.051 ±0.01O trace 0.046 ±0.007

0.080 ±0.004 0_184 ±0.006

trace

0.163 ±0.020

37,500

75,000

spermine

0.079 ±0.004 trace

spermidine

0.136 ±0.018 0.047 ±0.080

putrescine

free

0.158 ±0.009 0.204 ±0.015

Control 48.0

soil zinc concentration

0.248 ±0.024 0.212 ±0.019 0.243 ±0,Ol8 0.225 ±0.027

0.394 ±0.028 0.071 ±0.014

0.225 ±0.024 0.281 ±0.029

spermidine

0.144 ±0.009 0.122 ±0.012

0.179 ±0.016 0.209 ±0.015

putrescine

bound

0.193 ±0.009 0.146 ±0.01l

0.106 ±0.010 0.142 ±0.024

0.173 ±0.013 0.284 ±0.026

spermine

8-2

8-1

8-2

8-1

8-2

8-1

population

of free (water-soluble) and Table 4. The effects of different soil zinc concentrations (mg Zn/kg dry soil) on the concentrations (p.moles/g dry matter) the mean of 3 deterrepresent Results tolerance. zinc in differing 8-2) and (8-1 bound (RCI-extractabl e) polyamines of two Agrostis tenuis populations minations ± standard deviation

J

'"

..... NI .....

t

~

~

i

~

z

i

g

i~.

[...

I

~

~

;: 122

H. J. WEIGE:L et al.

& JAGER 1978), whereas in response to potassium deficiency, ammonium supply and after 802 fumigation free and bound putrescine and spermine accumulate in pea plants (KLEIN et al. 1979b; PRIEBE et al. 1978). The present investigatio n was carried out to extend our informatio n on the ecophysiology of plants differing in tolerance to heavy metals. Our results provide evidence that free amino acids in regard to their ability for the complexati on of heavy metals are not involved in metabolic adaptation s possessed by these plants to avoid heavy metal toxicity. Further investigatio ns are needed to elucidate the fact that free polyamines can no longer be detected in zinc sensitive population s of Agrostis tenuis after a zinc treatment.

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Comparative Investigatidns on the ·Nitrogen Metabolism

123

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& KLEIN, H. (1980): Biochemical and physiological effects of S02 on plants. Angew. Bot. 54: 337- 348.

KARATAGLIS, S. S. (1980): Behaviour of Agrostis tenui8 populations against copper ions in combina. tion with the absence of some macronutrient elements. Ber. Deutsch. Bot. Ges. 93: 417-424. KLEIN, H., JENSCH, U .. E., & JAGER, H.·J. (1979a): Die Schwermetallaufnahme von Maispflanzen aus Zink., Cadmium· und Kupferoxid.kontaminiertem Boden. Angew. Bot. 53: 19- 30. -

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