Journal of Experimental Botany, Vol. 53, No. 376, pp. 1979-1987,
September 1, 2002
© 2002 Oxford University Press
Effects of low chronic doses of ionizing radiation on antioxidant enzymes and G6PDH activities in Stipa capillata (Poaceae)
Received 13 November 2001; Accepted 13 May 2002
1 UMR-CNRS 6553 Ecobio, Equipe Evolution des Populations et des Espèces, Université de Rennes 1, Campus de Beaulieu, F-35042 Rennes Cedex, France
2 Decision Strategy Research Department, SCKCEN Boeretang, 200 B-2400 Mol, Belgium
3 To whom correspondence should be addressed. E-mail: misset{at}univ-rennes1.fr
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Abstract |
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Stipa capillata (Poaceae) seeds were harvested from a control area (displaying a
dose rate of 0.23 µSv h–1) (C plants) and from two contaminated areas (5.4 and 25 µSv 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 irradiation delivered by a sealed 137Cs source with a dose rate of 66 µSv h–1; (E+I) E treatment combined with internal ß 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 significant effect on APX, GR, CAT, and POD activities, but MDHAR activity was significantly reduced while SOD and G6PDH activities were significantly 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 almost a half century in the original environment of Stipa has led to natural selection of the most adapted genotypes characterized by an efficient induction of anti-oxidant enzyme activities, especially SOD and G6PDH, involved in plant protection against reactive oxygen species. Key words: Key words: Antioxidant enzymes, ionizing radiation, Poaceae.
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Introduction |
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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 (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 significantly enhanced in oxidative conditions. For this reason, they have evolved efficient 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; Hérouart 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 significantly modify the activity of oxidative stress defence enzymes? (ii) do plants show the same enzymatic response with respect to ionizing radiation of different biological efficiency? 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 specific intermediary metabolic pathway, G6PDH (glucose-6-phosphate dehydrogenase), in Stipa capillata (Poaceae) originating from areas of different contamination levels on the SNTS in Kazakhstan.
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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 identified, 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 µSv h–1, and two areas close to each other near Balapan Lake (50° N, 78° 20' E) with, respectively, 5.4 and 25 µSv 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.
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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 (20x15x11 cm3) were filled with 3435 g moist (field capacity, 20.1%) sandy soil. During the experiment, the soil moisture content was controlled by weighing twice a week, and adjusted with tap-water.
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During their entire growth, the Stipa plants were submitted to three experimental treatments. The first treatment (E) corresponded to a chronic external
-irradiation from a sealed 137Cs source, lead-shielded and delivering 66 µSv h–1 at the level of the plants. The second treatment (E+I) combined the external -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 µSv h–1). The material from the first cut (180 d after sowing) was not used for the present study. The leaves of the second cut (124 d after the first 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 ß-mercaptoethanol, 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 ß-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. (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 STATISTICA® software package (Statsoft, 1999).
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After 124 d of growth, (E) and (E+I) plants totalled a 196 mSv external
-dose from the 137Cs sealed source. Moreover, (E+I) plants were subjected to additional external -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 85Sr 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 (±SE, 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.
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The statistical analysis was carried out using a mixed model, three-way analysis of variance with two crossed, fixed 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 influenced 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 significantly influenced by the treatments.
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Under control growth conditions (0)
For all enzymes except POD (Fig. 2F), significantly 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 significant compared to (0) treatment, but it was significant 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 significantly higher activities. For SOD (Fig. 2D) and CAT (Fig. 2E), only H plants showed significant increases (respectively, 65% and 50% higher). POD (Fig. 2F) showed significant 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 significant 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 significant activity change. MDHAR, furthermore, underwent a significant decrease for plants of all origins. SOD and G6PDH showed a highly significant increase compared to (0) and (E) treatments. The increase in SOD, compared to treatment (0), involved all plants whatever their origin, with a significantly higher increase in L and H plants (activityx5) compared to C plants (activityx3). For G6PDH, only H and L plants were stimulated (activityx3).
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Discussion |
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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 sufficient to eliminate the different kinds of ROS, since a 196 mSv external
-dose received during 124 d—(E) treatment—had 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 five 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 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 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 in the pathway. In these experimental conditions of chronic external irradiation, a slight increase in GR activity in L and H plants was observed, probably due to an enhancement of the transcription rate of encoding genes (Foyer et al., 1991). The hypothesis of GR radio-induction in Stipa is supported by the fact that a similar response was also obtained in these plants for G6PDH. Indeed, this enzyme plays a major role in the ascorbate–glutathione pathway, supplying GR and MDHAR with NADPH. G6PDH activity is also necessary for the thioredoxin–NADPH-dependent system activity (Fridovich, 1983). Both are considered key systems ensuring the protection of the plant cell against oxidative damage.
Besides the weak stimulation of the ascorbate– glutathione pathway enzymes, SOD, POD and CAT were significantly over-expressed, particularly in H plants. These data suggest a more rapid elimination of increased ROS, in particular H2O2 produced by SOD, the key enzyme removing O2– in a dismutation reaction, by way of CAT and POD involvement. The stimulation of SOD activity is possibly due to a positive regulation of SOD genes or of one particular encoding allele, in response to low external chronic irradiation as shown in different biological models (Schiavone and Hassan, 1988; Scandalios, 1993; Inzé and Van Montagu, 1995). It is also important to emphasize that SOD induction seemed to depend on the dose rate in the original environment of the plants, indicating that the acquisition of SOD gene regulation, as well as that observed for POD in the same conditions, is a function of the severity of the ionizing condition.
This over-expression could be compared to that observed in various plant or animal systems submitted to a pre-irradiation leading to an increase in free radical scavenging ability at higher irradiation doses. For example, this adaptation to environmental stress was experienced in rat hepatic cells (Yukawa et al., 1999), and in vascular endothelial bovine cells, where CAT and POD activities were stimulated after exposure to an H2O2 pretreatment (Lu et al., 1993). In the same way, CAT, a thermo-labile enzyme, when exposed to a 14 °C pretreatment inducing an elevation of H2O2 level, led, in maize germination, to a stimulation of cat-3 expression under 4 °C (Prasad et al., 1994). The same treatment generated a similar response for POD and SOD in zucchini (Wang, 1995). This over-expression probably occurs by an efficient regulatory mechanism adjusting, when necessary, enzyme expression by positive regulation of expression of the corresponding genes, which allows better resistance to high doses of oxidant factors after an exposure to lower doses.
However, the plants were not directly subjected to a pre-exposure to low doses of irradiation, and L and H plants have a behaviour different from that observed in C plants under the same treatment. They grew from caryopses collected from populations submitted for more than 40 years to chronic irradiation with variable dose rates. In this case, the differences observed in enzyme activities in L and H plants compared to C plants and (0) treatment have a genetic basis, since they are observed in the progeny of chronically exposed plants and these responses can be qualified as an adaptation to an environment polluted by radionuclides.
Under the combined external and internal chronic irradiation conditions (E+I), in addition to the external irradiation, 85Sr and 134Cs uptake occurs in the plants via the root system. Indeed, the soil used for the cultivation (Table 1) came from an A horizon podzol which has the characteristic of being clayey and poorly silty (Baize and Jabiol, 1995). This type of soil displays a low selective affinity for Cs+ fixation (Lieser and Steinkopff, 1989), and makes Sr2+ root uptake possible because of its low level of organic matter (Veresoglou et al., 1995). The measure of absorbed radioactivity in plants, after their collection for experimental use, indicates a mean concentration of 100 Bq g–1 of dry matter for 85Sr and of 5 Bq g–1 for 134Cs. The higher transfer of Sr2+ compared to Cs+ is in agreement with other works (Buysse et al., 1995; Carini and Lombi, 1997). Thus, this contamination leads to another kind of stress due to the presence of metals within the plant tissues in addition to ß radiations given out by the two radionuclides and, consequently, to another metabolic response. Indeed, in these experimental conditions, significantly higher specific activities of SOD and G6PDH were observed in L and H plants, contrasting with the normal activity of the other enzymes, except for MDHAR, which exhibited a significant decrease in activity in L and H plants as well as in C plants. If it is considered that strontium, easily accumulated in leaves, is mainly adsorbed on cell walls like Ca2+ its antagonist (Menzel et al., 1961; Abbazov et al., 1978), a non-negligible internal chronic electron particle emission occurs in the more or less immediate proximity of the emitting radio-elements. This increases considerably the biological effects of the radio-treatment because of the relatively high biological efficiency (high ionizing capacity) of the ß-emitted particles.
In this case it is not surprising to note a strong stimulation of the genes encoding for the SOD. Scavenging of H2O2 is then performed by the other enzymatic and/or non-enzymatic antioxidant systems. The induction of G6PDH, similar to that of SOD, can be explained in the same way since this enzyme of intermediary metabolism provides NADPH for the efficient functioning of the ascorbate–glutathione pathway and of other H2O2 scavenging systems like flavonoids (Pendharkar and Nair, 1975; Shimoi et al., 1996). This enzyme, not indispensable for survival in normal conditions, is essential in cell defence against oxidative stress (Kletzien et al., 1994; Pandolfi et al., 1995).
Furthermore, caesium, a monovalent cation, more easily absorbed into the cells than the divalent strontium, may have an inhibitory effect on photosynthesis by interacting with chloroplastic enzymes, leading to a limitation of NADPH provided by electron transport in the chloroplasts (van Assche and Clijsters, 1990). In this case the over-expressed G6PDH could compensate for this through the oxidative pentose-phosphate pathway.
What are the mechanisms in Stipa responsible for the adaptive response in oxidative stress conditions? In these experiments, it was noticed that Stipa capillata is well equipped in enzymatic antioxidant systems since control plants (C) apparently endured (E) and (E+I) treatments without drastic modification of their enzyme activities, except for an increase in SOD activity. However, the same treatments led to higher activities in certain detoxifying enzymes in the progeny of plants originating from an ionizing environment. The origin effect is particularly significant for SOD, CAT, POD, and G6PDH activities as shown in Table 2. From an evolutionary point of view, the answer becomes obvious. Indeed, it is generally shown that in natural populations, whatever the nature of the environmental constraints, the best fit individuals are selected through successive generations. In this work, can 50 years of low radioactive contamination level (leading to -irradiation dose rates as low as 4.5 and 25 µSv h–1) be considered as a selective pressure? Does it lead to a modification of the genetic structure of the local Stipa populations?
It can be assumed that the presence of a low level of radioactivity for almost a half century, corresponding to about 20 generations of Stipa, has led to a progressive selection of individuals characterized by the genotypes best adapted to the ionizing environment. This selection has favoured individuals displaying high detoxifying enzyme activities and/or those able to induce them efficiently if necessary, as observed in this paper in L and H plants.
Preliminary research (Zaka, 1995) involved an electrophoretic analysis of nine enzyme systems, including SOD, in the same Stipa populations, growing under temperate greenhouse conditions. Among these populations, a decrease of the genetic diversity as a function of the dose rate occurring in the natural Stipa’s environment was observed. So, in spite of their low values, these dose rates can be considered a factor able to modify the genetic structure of populations. Furthermore, it has recently been shown, on Pisum sativum, that an acute -irradiation with very low to moderate doses (0–10 Gy) induced genetic modifications that persisted beyond the first generation (Zaka et al., 2002).
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Conclusions |
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This work shows, for the first time, that low chronic ionizing radiation has a significant effect on antioxidant enzyme activities that are also involved in acute oxidative stress conditions in plants. This minimizes the direct effects of ionizing radiation on plants, and so provides them with a radio-resistance. It is genetically determined, hence transmissible to the progeny, as seen in L and H plants (SOD, GR, CAT, POD, and G6PDH). The major defence strategy against ROS resulting from the presence of low chronic ionizing radiation in Stipa appears to be the removal of O2.–, probably in order to avoid its reaction with H2O2, which would lead to the formation of the highly aggressive ROS, OH. radical (Fenton reaction). In this context, SOD is the major enzyme involved, especially when plants are exposed to radiation of high biological efficiency. G6PDH, although an intermediary metabolic enzyme, is also affected greatly by low chronic irradiation and contamination. It must be involved in providing non-enzymatic and/or enzymatic protecting pathways other than the ones involved in the ascorbate–glutathione cycle. It is emphasized that the presence of low ionizing radiation rates in Stipa’s original environment for almost a half century has led to a selection of genotypes displaying high antioxidant enzyme activities and/or an important capacity to induce genes coding these enzymes. Further investigation, using molecular biology techniques, are needed to determine the mechanisms involved in enzyme induction under chronic ionizing conditions.
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Acknowledgements |
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This work was financially supported by EU programme INTAS 93-1421. We would like to thank L Gicquiaud for her precious technical help and Dr Michael Gross who kindly corrected our English.
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