Acta Phytopathobgica et Entomologica Hungarica 30 (3-4), pp. 215-220 (1995)

Effects of Chemical Stress Caused by Heavy Metals on the Glutathione S-Transferase Enzyme Activity in Maize

(Zea mays L.)

T. KŐMÍVES, A. A. A. AIOUB and G. GULLNER

Plant Protection Institute, Hungarian Academy of Sciences; H-1525 Budapest, P.O.B. 102, Hungary

Treatment of maize seedlings with different concentrations of micronutrient [copper(Il) and zinc(II)] and environmental toxicant heavy metals [cadmium(II), lead(Il), mercury(II), and nickel(II)] in nutrient solution culture led to markedly changed activities of the enzyme glutathione S-transferase (GST, E.C. 2.5.1.! 8.) depending on the heavy metal, its dose, the tissue of the treated plant and the duration of exposure. The observed responses correlated with heavy metal phytotoxicity, as indicated by their time course and dose-dependence.

Environmental pollutant heavy metals such as cadmium, lead and mercury, are powerful inhibitors of important life processes in mammalian and plant tissues (Almar and Dierickx, 1990, Steffens, 1990). Glutathione (gamma-L-glutamyl-L-cysteinyl-glycine, GSH) is known to participate in different plant stress reactions {Smith et al., 1990). GSH and cysteine-rich oligopeptides, mostly the GSH-derived phytoehelatins [(y-L-glutamyl-L-cysteinyl):i-glycine] play significant roles in heavy metal detoxification processes in plants (Grill et al., 1989; Rüegsegger et al., 1990; Meuwly et al., 1995). We reported earlier that the activity of the enzyme glutathione S-transferase (GST, E.C. 2.5.1.18.), which has an important function in detoxification processes (Kőmíves and Dutka, 1989; Gullner et al., 1991), was strongly influenced by cadmium(II) in pea and wheat (Uotila et al., 1994), and by mercury(II) in maize {Kőmíves et al., 1994) seedlings.

As a result of a continuing study of the basic stress response of plants to heavy metal exposure at the enzymatic level (Kőmíves et al., 1994; Uotila et al., 1994), in this paper we present data of a comparative study on the effects of two micronutrient (copper and zinc) and four toxic heavy metals (cadmium, lead, mercury and nickel) on the activ-ity of GST in maize seedlings.

Materials and Methods

Plant material

Maize seeds (Favea hybrid, obtained from Dr. Endre Széli of the Cereal Research Institute, Szeged, Hungary) were germinated in Petri dishes, and after 72 hours (when root lengths were ca. 25 mm) transferred to nutrient solutions containing different con-centrations of heavy metals as described previously (Kőmíves et al., 1994). Plants were

02 SH-1249/95/$ 5.00© 1995, Akadémiai Kiadó, Budapest

216 Kômives et al.: Glutathione S-transferase

125

1.5 4.5 13.5 40 120 360 1080

[Cadmium chloride],/xM Fig. 1. Effects of di('forent cadmium chloride concentrations on the shoot growth rate of maize seedlings after

48 h of exposure. Control value: 19.1 mm/day

grown ¡¡in greenhouse under normal conditions (18-23°C; supplemental light: 160 ¡aE m : s 1 for 8 h per day; relative humidity: 75-80%). Plant shoot lengths were measured daily.

Chemicals

Solvents, acetate (Pb, Ni) and chloride (Cd, Cu, Hg and Zn) salts of heavy metals, enzyme substrates and other chemicals were obtained from Reanal Fine Chemicals (Budapest, Hungary).

Enzyme assay

Preparations of cell-free homogenates from shoot and root tissues, and^measure-ment of GST activities followed previous lines (Uotila et a!., 1995).

Statistical analysis

Results are expressed as the means of three independent experiments. Significance of differences was evaluated by Student's f-test.

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218 Komives el al.: Glutathione S-transferase 217

Table t

ED5I( values characterizing the ability of heavy metals to reduce the shoot growth rate of maize seedlings (Favea hybrid) cultivated in nutrient solution

Heavy metal E D „ ( m M )

Cd(II) 121 Cu(H) 247 Hg( l l ) 13.6 Ni(ll) 265 Pb(II) 194 Zn(II) 9580

Results

Effects of heavy metals on the growth of maize seedlings

Heavy metal toxicity to plants was manifested by a rapid reduction of growth rate, followed by loss of tissue turgor, yellowing of leaves, and finally by necrosis of plant tissue. A typical dose-response curve on the effects of cadmium in the shoots is shown in Fig. 1. Mercury(II) was the most toxic to maize seedlings, fol lowed by cadmium(II) , !ead(II), copper(II), and nickel(II): EDS1) values on shoot growth reduction by these heavy metals are listed in Table I.

Effects of heavy metals on gluthatione S-transferase activity

GST enzyme activities of shoots and roots of untreated maize seedlings were 0.78 ±0 .11 and 1.01 ± 0 . 1 0 ¡imol g 1 fresh weight min respectively. Treatments with heavy metals resulted in significantly altered enzyme activities. As illustrated by data on effects of cadmium in Fig. 2, treatments with low, non-toxic or slightly toxic heavy metal concentrations led to GST enzyme induction in the shoots. The enzyme activity reached a maximum at 48 h, and ultimately declined to the control value. However, enzyme in-duction by high and strongly toxic heavy metal doses occurred less rapidly, and resulted in higher enzyme activities only after longer times of exposure. In the roots, GST induc-tion appeared only at the highest heavy metal concentration, while at lower concentra-tions the activities were reduced. Thus, Cd(II) caused a strong enzyme induction after 48 h in the shoots, with the largest changes talcing place following a 96 h treatment with 360 |llM heavy metal (241% of the control) (Table 2). In contrast, GST levels in the roots of the Cd(II)-treated maize seedling were reduced (by more than 50% following a 24 h treatment with 120pLM heavy metal), and an increase in the enzyme activity occurred only after long (96 h) incubation with the highest cadmium concentration (360 uM) (Fig.

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218 Komives el al.: Glutathione S-transferase 218

2). Treatments with other heavy metals resulted in similar dose-response relationships: characteristic data on changes of GST activity in heavy metal-treated maize plants are listed in Table 2.

Table 2

Effects of heavy metals on the glutathione S-transferase (GST) activity in shoots and roots of maize (Favea hybrid) grown in nutrient culture. Heavy metals were applied at 1/3 (48 h data) and 3-fold (96 li data) rates of

their ED i0 concentrations (see Table 1). For control values see text in Results

Heavy metal

GST control %

Heavy metal Root Shoot Heavy metal

48 h 96 h 48 h 96 li

Cd(Il) 77 157 189 241 Cu(Il) 81 138 175 322 Hg(II) 82 143 242 303 Ni(Il) 95 159 137 178 Pb(II) 74 125 - 144 169 Zn(II) 111 146 196 195

250r 250

200

s 150 « a © u

fe" 100 a

50

Root

LSD

96h ' *

V

24h

•A Tt to o o o

i—l FT) o CO o

o o o M vo QO » H O ©

[Cadmium chloride], juM [Cadmium chloride], ¿tM

Fig. 2. Effects of cadmium chloride on the glutathione S-transferase (GST) activity in shoots and roots of maize seedlings after different exposure periods. For control values see text in Results

Acta Phytopathologica et Entomologica Hungarica 30, 1995

218 Komives el al.: Glutathione S-transferase 219

Discussion

Chemical stress of plants by heavy metals and herbicides may result in enhanced activities of enzymes that participate in the biosynthesis of phytotoxic natural products (Komives and Casida, 1982; Komives and Casida, 1983), thereby promoting plant tissue damage. Increased activities of GST in maize seedlings (Fig. 2) treated with subtoxic or moderately phytotoxic doses of the heavy metals cadmium and mercury, however, repre-sent a different type of response: in this case the plant's detoxication potency is elevated (Komives et a!., 1994; Uotila et al., 1994). In preliminary experiments (Komives and Brunold, unpublished, 1991) cadmium was found to be an inhibitor of GST from shoots and roots of pea and maize in vitro. In vivo, however, Cd(II) caused massive induction of GST in maize (Fig. 2), similarly to effects by herbicides and herbicide safeners (Komives andHatzios, 1991; Hol te ta l . , 1995; Walton and Casida, 1995). In addition to Cd(II) and Hg(II), all the tested heavy metals had the ability to influence levels of GST. The ob-served responses seem to be heavy metal-specific, as indicated by their nearly identical time-course and the similarity of their dose-dependence.

GST isoenzymes play roles in the detoxication of a number of xenobiotics not only as catalysts of GSH conjugation, but also as ligandins (xenobiotic-binding proteins) (Komives and Dutka, 1989). Inhibition of maize and pea GSTs by Cd(II) and Hg(II) (Komives and Brunold, unpublished, 199!) raises the question, whether GST proteins may be involved in heavy metal detoxication in plants as ligandins. Although relatively high cytosolic concentrations and superb stress-inducibilities of GST proteins (Komives and Dutka, 1989) seem to support this hypothesis, further research is needed for its evaluation.

Acknowledgements This work was supported by grants from the Hungarian Scientific Research Fund (OTK.A T6314 and

F6321). The authors thank Prof. Christian Brunold (University of Bern. Switzerland) for helpful discussions.

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Acta Phvtavatholoffica et Entomolosica Hunsarica 30, ¡995