Journal of Experimental Botany, Vol. 53, No. 376, pp. 1979±1987, September 2002 DOI: 10.1093/jxb/erf041
Effects of low chronic doses of ionizing radiation on antioxidant enzymes and G6PDH activities in Stipa capillata (Poaceae) R. Zaka1, C. M. Vandecasteele2 and M. T. Misset1,3 1
UMR-CNRS 6553 Ecobio, Equipe Evolution des Populations et des EspeÁces, Universite de Rennes 1, Campus de Beaulieu, F-35042 Rennes Cedex, France 2
Decision Strategy Research Department, SCKCEN Boeretang, 200 B-2400 Mol, Belgium
Received 13 November 2001; Accepted 13 May 2002
Abstract Stipa capillata (Poaceae) seeds were harvested from a control area (displaying a g dose rate of 0.23 mSv h±1) (C plants) and from two contaminated areas (5.4 and 25 mSv h±1) on the Semipalatinsk nuclear test site (SNTS) in Kazakhstan. The plants were grown for 124 d in a greenhouse under controlled conditions and exposed to three different treatments: (0) control; (E) external g irradiation delivered by a sealed 137Cs source with a dose rate of 66 mSv h±1; (E+I) E treatment combined with internal b irradiation due to contamination by 134Cs and 85Sr via root uptake from the soil. The root uptake led to a contamination of 100 Bq g±1 for 85Sr and 5 Bq g±1 for 134Cs (of plant dry weight) as measured at harvest. The activity of SOD, APX, GR, POD, CAT, G6PDH, and MDHAR enzymes was measured in leaves. Under (0) treatment, all enzymes showed similar activities, except POD, which had higher activity in plants originating from contaminated areas. Treatment (E) induced an enhancement of POD, CAT, GR, SOD, and G6PDH activities in plants originating from contaminated areas. Only control plants showed any stimulation of APX activity. Treatment (E+I) had no signi®cant effect on APX, GR, CAT, and POD activities, but MDHAR activity was signi®cantly reduced while SOD and G6PDH activities were signi®cantly increased. The increase occurred in plants from all origins for SOD, with a greater magnitude as a function of their origin, and it occurred only in plants from the more contaminated populations for G6PDH. This suggests that exposure to a low dose rate of ionizing radiation for 3
almost a half century in the original environment of Stipa has led to natural selection of the most adapted genotypes characterized by an ef®cient induction of anti-oxidant enzyme activities, especially SOD and G6PDH, involved in plant protection against reactive oxygen species. Key words: Poaceae.
enzymes,
ionizing
radiation,
Introduction The biological effects of environmental stress and the physiological response of living organisms that allows their survival under stress conditions is a widely studied topic. It is well established that, whatever its nature, stress causes the production of a large amount of highly reactive oxygen species (ROS), including free radicals (H+: hydrogen ion, H.: hydrogen radical, H2O2: hydrogen peroxide, OH.: hydroxyl radical), in living cells. In aerobic organisms, moreover, an enhanced production of other species such as O2.± (superoxide ion) and H2O2 takes place. These latter species are relatively less harmful, but they can enter the `Fenton' reaction catalysed by a metal ion (Fe2+) and generate the highly aggressive OH. radical (Wardman and Candeias, 1996). A large amount of biological damage is due to this radical, which reacts with almost all structural and functional organic molecules, including proteins, lipids and nucleic acids. OH. can cause peroxidation of unsaturated membrane fatty acids, forming peroxyl (ROO.) and alkoxyl (RO.) radicals
To whom correspondence should be addressed. E-mail:
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ã Society for Experimental Biology 2002
Antioxidant
1980 Zaka et al.
(Salter and Hewitt, 1992), resulting in a loss of cellular compartmentation and thus metabolic disturbance. Its oxidative attack on proteins (Wolff and Dean, 1986) can greatly alter their properties and functions. Damage caused to DNA may, in turn, induce mutation and chromosome abnormalities of the meristem cells (Okamoto and Tatara, 1994; Taguchi et al., 1994; Zaka et al., 2002). Living organisms, particularly photosynthetic organisms, are continuously exposed to ROS, but their exposure is signi®cantly enhanced in oxidative conditions. For this reason, they have evolved ef®cient enzymatic and non-enzymatic detoxifying systems to overcome damage due to ROS (Larson, 1988). A large number of studies deal with various oxidative stress factors in plants (drought, salinity, extreme temperatures, atmospheric pollution, UV, and herbicides) and describe how exposed plants adjust their detoxifying enzyme activities (Tsang et al., 1991; Bowler et al., 1991; Sgherri et al., 1993; McKersie et al., 1993; HeÂrouart et al., 1994; Van Camp et al., 1996). Key enzymes considered in these works are namely SOD (superoxide dismutases), CAT (catalases), POD (peroxidases), APX (ascorbate peroxidases), and other enzymes implicated in the Halliwell and Asada cycle (ascorbate±glutathione pathway). Under stress conditions an enhanced activity of almost all of these enzymes is reported and should be related to an increased protein synthesis. However, published works only deal with short-term effects of acute stress factors, and there is little information concerning the effects of ionizing radiation stress on the activity of these enzymes in plants. Knowing that water radiolysis, the predominant effect of ionizing radiation in organisms, induces ROS formation (De Vita et al., 1993), one can assume that plant, bacterial, and animal enzymes that are involved in cell protection against oxidative stress will display similar responses under ionizing radiation stress as under other stress factors. Thus, in this study, an attempt was made to answer the following questions: (i) does chronic radioactive contamination and/or external irradiation signi®cantly modify the activity of oxidative stress defence enzymes? (ii) do plants show the same enzymatic response with respect to ionizing radiation of different biological ef®ciency? and (iii) does chronic exposure of plants to low doses lead to the acquisition of radio-resistance in their progeny? In order to respond to these questions, the activities of key enzymes involved in oxidative stress defence, such as SOD, CAT, POD, APX, MDHAR (monodehydroascorbate reductase), and GR (glutathione reductase) were studied, as well as the activity of one enzyme involved in a speci®c intermediary metabolic pathway, G6PDH (glucose-6phosphate dehydrogenase), in Stipa capillata (Poaceae)
originating from areas of different contamination levels on the SNTS in Kazakhstan. Materials and methods Material Caryopses of Stipa capillata (2n=44; Martinovsky, 1980), a characteristic and predominant perennial grass of Central Asia steppes, were collected within the framework of the European `Environment-Health Program in Kazakhstan' in September 1993 on and near the SNTS where, for 40 years (1949±1989) nuclear tests caused substantial contamination of air, soil and water. In contaminated areas, although many radionuclides can be identi®ed, the most hazardous ones are 90Sr, 137Cs and Pu. Three sites with similar soil characteristics but different external gamma dose rates were chosen (Fig. 1) for the collection of seeds: a control area (C) near Mont Dostar (49° 55¢ N; 76° 20¢ E), located to the west of the SNTS, with 0.23 mSv h±1, and two areas close to each other near Balapan Lake (50° N, 78° 20¢ E) with, respectively, 5.4 and 25 mSv h±1, where plants have been exposed for several decades to a lower (L) and a higher (H) irradiation dose rate. The collected caryopses were stored at 4 °C until used for the experiments. The Stipa caryopses from the three sites (C, L and H) were sown in pots on a sandy soil and cultivated under greenhouse conditions (25 °C, natural photoperiod) at SCK´CEN (Belgian Nuclear Centre, Mol, Belgium). The soil, sampled from the A-horizon of a podzol soil (orthic podzol) developed under pasture land was sieved (2 mm) and characterized in terms of physico-chemical properties (Table 1). Rectangularly shaped darkened containers (20315311 cm3) were ®lled with 3435 g moist (®eld capacity, 20.1%) sandy soil. During the experiment, the soil moisture content was controlled by weighing twice a week, and adjusted with tap-water. During their entire growth, the Stipa plants were submitted to three experimental treatments. The ®rst treatment (E) corresponded to a chronic external g-irradiation from a sealed 137Cs source, leadshielded and delivering 66 mSv h±1 at the level of the plants. The second treatment (E+I) combined the external g-irradiation with a chronic contamination due to 0.7 MBq 134CsCl and 1.2 MBq 85SrCl2 added to the soil (generating the following activities at the beginning of cultivation: 461 and 154.5 Bq g±1 of dry soil, respectively). The third and last treatment (0) was the control corresponding to the local radiation background (0.15 mSv h±1). The material from the ®rst cut (180 d after sowing) was not used for the present study. The leaves of the second cut (124 d after the ®rst one) were promptly frozen in liquid nitrogen, in separated sets corresponding to individual plants, and thereafter stored at ±80 °C prior to enzyme assays. Biochemical assays The proteins from 100 mg of leaves were extracted by grinding frozen material in a mortar in liquid nitrogen and homogenizing them in 1 ml of a buffer solution. The extraction buffer for leaf proteins used for the determination of the activity of most enzymes consisted of 0.1 M TRIS±HCl (pH 7.5), 0.23 M sucrose, 20% PVP (polyvinylpyrrolidone), 4 mM bmercaptoethanol, 1 mM EDTA, 10 mM KCl, and 10 mM MgCl2. 5 mM ascorbic acid was added to the buffer just before use. To extract proteins for the determination of APX and MDHAR, 5% PVP was used and the b-mercaptoethanol was omitted. After homogenization and centrifugation (10 000 g at 4 °C for 20 min), the supernatants were used for enzyme assays. Protein concentration was determined by the dye-binding method (Bradford, 1976) using bovine serum albumin (Sigma) as a standard. APX and GR assays were performed according to Vanaker et al.
Ionizing radiation and plant antoxidant enzymes 1981
Fig. 1. Collecting sites of Stipa capillata seeds on and near the Semipalatinsk (Kazakhstan) nuclear test site (SNTS). Seeds were collected in a control area (C) and in two areas located close to each other near Balapan Lake, characterized by low (L) and relatively high (H) g rates. g rates are given in mSv h-1. (According to C Vandecasteele, Rapport Radiation Biomes, INTAS-KZ-95-3, 1997±1998.)
Table 1. Physico-chemical characteristics of the soil used for Stipa seed cultivation under greenhouse conditions Characteristic
Value -1
Bulk density (kg DW l ) pH H2O KCl Texture (%) < 2 mm 2±50 mm 50±2000 mm Total C (%) Total N (%) Exchangeable cations (NH4Ac, 1 M; cmolc kg-1) K+ Na+ Ca2+ Mg2+
1.40 5.7 5.2 3.54 0.66 93.80 2.15 0.14 2.32 1.13 56.26 3.07
(1998), MDHAR according to Miyake and Asada (1992), SOD according to Giannopolitis and Ries (1977), POD according to Macheix and Quessada (1984), CAT according to Siminis et al. (1994), and G6PDH according to Aoki et al. (1998). All determinations were obtained from triplicate measurements on each of three individual plant samples. The statistical analyses were performed with the STATISTICAâ software package (Statsoft, 1999).
Results After 124 d of growth, (E) and (E+I) plants totalled a 196 mSv external g-dose from the 137Cs sealed source. Moreover, (E+I) plants were subjected to additional external g-irradiation, due to 134Cs and 85Sr in the soil, but this contribution was negligible compared to the dose delivered by the 137Cs source. Root uptake of 134Cs and
1982 Zaka et al. 85
Sr led to a respective internal contamination at harvest of 5 and 100 Bq g±1 of plant dry weight in (E+I) plants. Enzyme assays
The average activities (6SE, n=3) measured in plants originating from the three sites in Kazakhstan are given in Fig. 2A±G. Each histogram represents the mean of nine measurements from triplicate assays on three protein extracts from different plant samples. The statistical analysis was carried out using a mixed model, three-way analysis of variance with two crossed, ®xed effects (`Origin' and `Treatment') and their interactions (Table 2). The 3-way analysis of variance reveals that the response of APX, MDHAR and GR are not in¯uenced by the origin site of the seeds, while SOD, CAT, POD, and G6PDH responses are highly affected by this factor. Except for POD, all enzyme activities are signi®cantly in¯uenced by the treatments. Under control growth conditions (0)
For all enzymes except POD (Fig. 2F), signi®cantly similar activities were obtained in C, L and H plants, while POD displayed 30% higher activity in L and H plants. Under external irradiation (E) No change was observed in any plant origin for MDHAR (Fig. 2B), compared to (0) treatment. For GR (Fig. 2C), the increase of activity in L and H plants was not signi®cant compared to (0) treatment, but it was signi®cant compared to C plants under (E) treatment. In G6PDH (Fig. 2G), however, plants of different origin had similar activities, but compared to their activity observed in (0), L and H plants had signi®cantly higher activities. For SOD (Fig. 2D) and CAT (Fig. 2E), only H plants showed signi®cant increases (respectively, 65% and 50% higher). POD (Fig. 2F) showed signi®cant increase in L and H plants as a function of the dose rate of plant origins. With one exception (APX), all enzyme activities in plants originating from the control site were similar to the (0) treatment, i.e. without stimulation due to the gamma irradiation. By contrast, a more or less signi®cant increase in the activity of most enzymes was measured in plants coming from populations chronically exposed to ionizing radiations (L, H). Indeed, SOD, CAT and POD exhibited the highest activities in H plants whereas the enzymes of the ascorbate±glutathione cycle, APX, MDHAR and GR were little or no (MDHAR) stimulated. Under external and internal irradiation (E+I) In spite of a slight decrease compared to (0) treatment, APX, GR, CAT, and POD showed no signi®cant activity change. MDHAR, furthermore, underwent a signi®cant decrease for plants of all origins. SOD and G6PDH showed a highly signi®cant increase compared to (0) and (E) treatments. The increase in SOD, compared to treatment
(0), involved all plants whatever their origin, with a signi®cantly higher increase in L and H plants (activity35) compared to C plants (activity33). For G6PDH, only H and L plants were stimulated (activity33). Discussion Plants are well known to possess effective enzymatic and non-enzymatic detoxifying systems continuously involved in the cellular protection against ROS coming from both the environment and cell metabolism. The results obtained under (0) treatment show this permanent activity regardless of plant origin. Furthermore, it is supposed that this normal antioxidant activity is largely suf®cient to eliminate the different kinds of ROS, since a 196 mSv external g-dose received during 124 dÐ(E) treatmentÐhad little effect (except for APX activity) on control plants coming from a non-polluted Stipa population (C). In (E+I) conditions, the results were similar for ®ve of the enzymes studied (POD, CAT, APX, GR, and G6PDH). Concerning APX stimulation, a similar response has been observed in wheat growing in a saline environment (Meneguzzo et al., 1998), and in tobacco under UVB (Willekens et al., 1994). According to Karpinski et al. (1997), the APX activity induction in Arabidopsis submitted to oxidative stress conditions such as high light intensity, takes place by an induction of apx1 and apx2 gene transcription. Although these experiments considered the short-term response of the enzyme to an acute stress, one can assume that this induction of the genetic expression of APX can also take place in cells undergoing low chronic g radiation stress. On the other hand, very different results were obtained in plants coming from the two contaminated populations (L and H) with differences with regard to the kind of treatment (E) or (E+I). Thus, data obtained under (E) and then (E+I) treatments will be considered successively. Under (E) conditions, APX, MDHAR and GR were stimulated differently in L and H Stipa compared to plants of the same origin without treatment. Although these enzymes belong to the same metabolic pathway (Halliwell and Asada pathway), they can be diversely involved in the protection against g radiation. These results agree with those of Gupta et al. (1993a, b) on tobacco in which resistance to oxidative stress is due to an over-expression of Cu/ZnSOD and APX activities, while MDHAR and GR activities are not affected. In Arabidopsis thaliana as well, Kubo et al. (1995) have observed that a 1 week exposure of the plants to O3 or SO2 had only a slight effect on the activity of the same enzymes. Considering their results, it can be assumed that, in Stipa under ionizing conditions, the recycling of the oxidized form of ascorbate to the reduced form would involve DHAR (dehydroascorbate reductase) rather than MDHAR. In this case, GR would also be involved in the recycling of the electron donor (GSH), later
Ionizing radiation and plant antoxidant enzymes 1983
Fig. 2. (A-G) Mean foliar enzyme activity values in Stipa (6SE, n=3). (A) APX (mmol oxidized abscorbic acid min±1 mg±1 protein), (B) MDHAR (mmol NADP+ min±1 mg±1 protein), (C) GR (mmol NADP+ min±1 mg±1 protein), (D) SOD (SOD unit mg±1 protein), (E) CAT (mmol reduced H2O2 min±1 mg±1 protein), (F) POD (absorbance variation min±1 mg±1 protein), (G) G6PDH (mmol NADPH min±1 mg±1 protein). (White bars) C-plants, (grey bars) L-plants, (black bars) H-plants grown under the following treatments: (0) control, (E) chronic external g-irradiation and (E+I) combination of external g-irradiation with a chronic contamination due to 137Cs and 85Sr added to the soil. Different letters indicate signi®cant differences (Tukey HSD, P
