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Cadmium Effects on the Energetics of Euglena during the Development of Cadmium Resistance
Cadmium Effects on the Energetics of Euglena during the Development of Cadmium Resistance
J. BONALY, M. MIGINIAC-MAsLOW*), E. BROCHIERO, A. HOARAU*) and]. C. MESTRE Laboratoire de Biologie cellulaire, Faculte de Pharmacie, rue J. B. Clement, 92290 ChatenayMalabry, France, and *) Laboratoire de Photosynthese et Metabolisme, UA 1128, Universite de Paris-Sud, 91405 Orsay, France Received July 1, 1985 . Accepted November 18, 1985
Summary Energy charge values and adenylate contents were determined throughout one culture cycle of Euglena gracilis, and on the 3rd day of the subsequent subcultures. The effects of Cd ions (500/LM) on these parameters were investigated. Energy charge values were decreased in the presence of Cd ions, but progressively recovered during the adaptation of Euglena to Cd. The decrease in energy charge was concomitant with a transient drop in the photosynthetic 14C0 2 fixation and with a gradual decrease in lactate consumption, suggesting that respiration was most affected by Cd. Cd poisoning resulted in increased cell size and protein content. Accordingly, adenylate levels per cell were higher in the presence of Cd. When expressed per protein unit, adenylate levels became lower in Cd - treated Euglena than in control Euglena only 4 days after inoculation. This observation suggested that adenylate metabolism was affected by Cd. The use of energy charge as an energy stress indicator is discussed. Key words: Euglena gracilis, cadmium, energy charge, photosynthesis, respiration.
Introduction Many studies reported toxic effects of Cd ions on animal and plant metabolism (Webb, 1979). Various sites of action have been proposed: oxidative phosphorylation (Sporn et al., 1970; Mustafa and Cross, 1971) and respiratory oxygen consumption (De Filippis et al., 1981 a), photosynthetic electron transport and photophosphorylation (Hampp et al., 1976; Lucero et al., 1976; De Filippis et al., 1981 a) and Calvin cycle stromal enzymes (Wiegel, 1985). Our own results (Duret et al. [in press]) showed an alteration of mitochondrial structure and a decrease in cell respiration activity in Euglena, suggesting an effect on respiratory energetics. Many of the proposed sites have been determined on in vitro systems. In the present study, we tried to get information about toxic effects of Cd ions on energetics of Euglena in conditions as close as possible to an in vivo situation. The determination of the adenylate pool and of energy charge values seemed to be a good approach to this problem, since these parameters have been shown to be differently affected by metabolic stress (nutrient deficiency, Thomas and Dawson, 1977; Matsumoto et al.,
f Plant Physiol. Vol.
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1979; respiratory inhibitors, Matsumoto et al., 1979): cellular adenylate content was decreased in all cases, while energy charge was decreased only when the respiratory pathway was affected. Only one study reported Cd effects on adenylate energy charge (De Filippis et al., 1981 b). It concerned autotrophically grown Euglena cells. Our previous studies on photoheterotrophically grown Euglena gracilis stain Z showed that under our culture conditions this Euglena strain was able to develop a resistance to toxic Cd concentrations (Bonaly et al., 1980). It was therefore of particular interest to study the energetic balance in this strain during the development of Cd resistance. In addition to energy charge and adenylate determinations, measurements of lactate consumption and 14C02 fixation have been made in the conditions of the culture in order to investigate which of the two main energetic processes (respiration or photosynthesis) was the most affected by Cd.
Materials and Methods Algal cultures and determination ofgrowth Axenic cultures of Euglena gracilis strain Z (French CNRS collection) were auto-heterotrophically grown in 150 ml Erlenmeyer flasks under sterile conditions. The growth conditions and the method of cell counting were as previously described (Bonaly et al., 1978). Briefly, cells were grown at 23°C in the light (22.5 Wm -2) in a synthetic medium containing minerals, lactic acid, and vitamins Bl and B12. Cadmium chloride (5 x 10- 4M final concentration) was aseptically added to the medium. Cell counting was carried out daily in a model DI Coulter counter using a 100 JLm aperture. Due to the great number of counted cells, the variability was ca. 1 %. Measurements ofsize Size distribution of Euglena was performed with a cytofluorograf FC 200 (Biophysics) connected to a multichannel distribution analyser (Biophysics, Model 2102). For each sample, (10 5 cells), mean cell size was determined after weighing each histogram. Instrument calibration was done with calibrated polystyrene microspheres (Biophysics). As for cell counting the variability was around 1 %. Determination ofAdenylates To 5 ml of culture suspension were rapidly added 0.5 ml of lN TCA and the mixture was frozen in liquid nitrogen. The frozen extracts were thawed and centrifuged (12,000 xg, 10 min). The pellets were washed once with 2 ml of 0.1 N TCA and centrifuged again. The pooled supernatants were washed 4 times with an equal volume of diethyloxide in order to eliminate TCA. The diethyloxide dissolved in the aqueous phase was eliminated by bubbling air, and the extracts were neutralized with 2M K2 C0 3• Adenylate determinations were made on the extracts by the luciferase method, either directly (ATP), or after enzymatic transformation into ATP (ADP and AMP), as described by Pradet (1967). All the determinations were done in triplicate, with internal standards, on 100 JLI of the extracts. Adenylate energy charge was calculated according to Atkinson (1968): [ATP] + ~ [ADP]i[ATP] + [ADP] + [AMP]. Chlorophyll and protein determinations For chlorophyll determinations, 5 ml of culture suspension was sedimented by centrifugation (12,000 xg, 10 min). The pellets were extracted with 80% aqueous acetone. After centri-
J. Plant Physiol. Vol.
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fugation, the chlorophyll content of the supernatants was determined according to Arnon (1949). The protein content was estimated on the pellets remaining after adenylate extraction. The pellets were dissolved in 2 ml of 2N NaOH and their protein content was measured by the Folin phenol reagent (Lowry et al., 1951).
14C02 incorporation measurements The rate of CO 2 fixation was measured by injecting 5 mM 14C-Iabelled bicarbonate (2 p.Ci/ p.mole) into 5 ml cultures contained in stoppered Erlenmeyer flasks. Fixation was performed by injecting 0.5 ml of pure formic acid, and the unreacted bicarbonate eliminated by aspiration under vacuum into concentrated KOH. 0.3 ml samples were dried in an oven at 80°C and the radioactivity counted in a scintillation counter.
Determination of lactate consumption The determination of lactate in the medium was performed spectrophotometrically according to Hohorst (1963). The basic equation is: L Lactate + NAD+ + hydrazine lacticodehydrogenase. pyruvate hydrazone + NADH H+ + H 0 NADH was spectroH 95 , 2 . photofuetrically measured at 340 nm. Difference between lactate amount on day 0 and lactate amount on day x allowed the calculation of lactate consumption by cells on day x. Results Fig. 1 shows the total levels of adenylates and the energy charge values during one culture cycle of photoheterotrophically grown Euglena cells. In the presence of Cd
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Fig. 1: Changes in total adenylate levels (E) and energy charge values (EC) during one culture cycle of Euglena growing either on control medium (e: energy charge;.: total adenylate) or on a medium containing 500 p.M Cd Ciz (0: energy charge, D: total adenylate). Adenylate levels are expressed per g protein. Each value is the average of 3 separate determinations ± standard deviation (S.D.).
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
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J.
BONALY,
M. MIGINIAC-MAsLOW, E. BROCHIERO, A.
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ions, the energy charge values were sharply decreased during the first 2 days of culture, and finally stabilized at low values ( := 0.5). In the control culture, energy charge values ranged between 0.85 and 0.95. It seemed therefore that the energetic functions of Euglena were impaired by Cd ions. Total adenylate content per g protein increased much less in Cd - treated Euglena than in control cells during the growth cycle. As a matter of fact, it remained nearly constant and significantly lower than in control cells, after a limited but significant
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
Energetics of Euglena and cadmium resistance
18
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increase on day 2 found in 3 independent experiments. At this particular stage, the ad· enylate content per g protein was higher in Cd - treated Euglena than in control Eug· lena. When expressed per 107 cells, and in contrast to the observations of De Filippis et al. (1981 b), the adenylate contents were higher in Cd·treated cells than in control cells throughout the culture cycle (Fig.2). This observation reflects the increase in protein content of Cd-treated cells (Fig. 3) which are also larger than control cells (Fig. 4). On further inoculations of Cd·treated Euglena cells on fresh control or Cd-enriched media, energy charge values measured on the yd day of culture (exponential growth phase, see growth curves in Fig. 5), showed a stepwise recovery of the control values (Fig. 6). The recovery was faster on control medium than on Cd·enriched medium.
J Plant Physiol. Vol.
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J. BONALY, M. MIGINIAC-MASLOW, E. BROCHIEllO, A. HOARAU and J. C. MESTRE
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Fig. 6: Changes in the energy charge values of Cd-treated Euglena cultures, measured on the yd day of each culture cycle, during successive subcultures. C: Euglena growing on control media. - Cd: Euglena grown on 500 I'M CdCb during the first subculture cycle and then returned to a medium without Cd in further subcultures. + Cd: Euglena grown and subcultured on 500 JLM CdCb. Average value of 3 separate determinations ± S.D.
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Fig. 7: Comparison of energy charge values of Euglena cultured on a control medium and of a "Cd-resistant» Euglena strain cultured on a medium containing 1 2 3 4 500 JLM CdCb. Average value of 3 separate determinaDays after inoculation tions ± S.D.
Nevertheless, on Cd-enriched medium, the cells reached control energy charge values on the 6th subculture cycle. It appeared that during the development of resistance to Cd ions, the Euglena recovered normal energetic functions. The comparison of energy charge values of control and Cd-resistant Euglena confirmed this point, as they were identical in both cases (Fig. 7). In order to determine which of the main energetic pathways (respiration or photosynthesis) was the most affected by Cd poisoning under the conditions of the culture, we tested 14C02 incorporation by Euglena maintained under normal growth conditions (22.5 W' m -2 light with lactate as respiratory substrate), and the disappearance of lactate from the culture medium, in the presence or in the absence of Cd. Whether expressed per 106 cells or per mg chlorophyll, CO 2 fixation of Cd-treated cells dropped on the first day of culture and then rapidly recovered (Fig. 8). On the 4th day of culture, CO2 fixation was higher in Cd-treated Euglena than in control
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Energetics of Euglena and cadmium resistance
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Euglena. This observation may be accounted for by the fact that at this stage, the control culture has reached its maximum density and its photosynthetic activity might be limited by availability of light. Nevertheless, maximum activity of the Cd-treated culture was equivalent to the maximal activity of the controls. Then, the Cd effect on the CO 2 fixation activity seems to be transitory. The recovery of photosynthetic activity corresponds to the recovery of a normal chlorophyll content per cell. The CO 2 fixation activity was only consistently lower in Cd-treated Euglena when expressed on a protein unit basis.
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
J. BONALY, M. MIGINIAC-MASLOW, E. BROCHIERO, A. HOARAU and J. C. MEsTRE
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Fig. 9: Lactate consumption per 106 cells by Euglena cultured on a control medium (.) or on a medium containing 8 500 pM CdCh (0). Each point is the average of 4 separate determinations. ±S.D.
In contrast to CO2 fixation activities, lactate consumption was much more steadily affected by Cd throughout the culture cycle (Fig. 9). It then appears that inhibition of photosynthesis contributes to the decrease of energy charge only on the first days of culture: inhibition of respiration seems to be the main factor responsible for decrease in energy charge in Cd-poisoned Euglena.
Discussion Our experiments show that under photo heterotrophic growth conditions, the energetic metabolism of Euglena gracilis strain Z was affected by Cd. Energy charge values were decreased in a similar way as reported by De Filippis et al. (1981 b) for autotrophically grown Euglena. The decrease in the energy charge was correlated with a transient decrease in CO2 fixation activities and a consistent lowering in lactate consumption rates per cell. The maximal CO2 fixation rates per unit chlorophyll were the same in control and Cd-treated Euglena, but occurred later in the growth cycle in the latter case: the maximal efficiency of the photosynthetic apparatus seemed to be identical in both cases. While photosynthesis recovered rapidly, lactate consumption was affected for a much longer time. Therefore respiration seemed to be the major site of Cd inhibition. When Euglena developed a resistance to Cd ions, there was a stepwise recovery in energy charge values. Cd-resistant Euglena strains exhibited identical energy charge values, whether cultivated in the presence of Cd, or not, Bariaud et al. (1985) reported a lower accumulation of Cd in resistant cells than in Cd sensitive cells. However, Cd2 + uptake (in 0.1 mM CdClz) is not negligible (0.36/-tmol (gDW) -Ih -I in resistant cells vs 0.87 /-tmol (gDW) -I h -I in sensitive cells). Therefore, the relief of the depressive effect of Cd on energy charge values may be either due to lower intracellular Cd concentration in Cd-resistant cells and or to a decreased susceptibility of organelles to Cd ions.
J Plant Physiol. Vol. 123. pp. 349-358 (1986)
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In contrast with the results of De Filippis et al (1981 b), total adenylate levels were changed in photo heterotrophically grown Euglena in the presence of Cd. Cd poisoning resulted roughly in a lower adenylate content per unit protein and in a higher adenylate content per cell. Decrease in adenylate content when energy charge is lowered is a rather general phenomenon. It is considered as a regulatory mechanism which avoids extensive changes in energy charge (Chapman and Atkinson, 1973, Matsumoto et aI., 1979; Yoshino and Murakami, 1981, Miginiac-Maslow and Hoarau, 1982).
However, in Cd-treated Euglena, total adenylate content became lower than in control Euglena only 4 days after inoculation, on a protein basis. On the 2nd day of culture the adenylate level was 40 to 50 % higher in the presence of Cd. It seems therefore that on the first days of culture Cd interferes with the regulatory mechanisms generally considered responsible for the decrease in adenylates under stress conditions. When expressed per cell, adenylate content was much higher in the presence of Cd. In spite of the lowered energy charge, the ATP content per cell was also higher, almost twice that of the control cells (results not shown). These results mainly reflect the cell enlargement and the increase in protein content per cell. They point out the possibility of misinterpretation of some results when a cell unit basis is chosen. On the contrary, energy charge seems to be an excellent criterion for energetic stress determination. It is a ratio which reflects the energy-saturation of adenylate pool and it is entirely independent of a quantitative expression basis. Our results indicate that changes in energy charge correlate with impaired energetic functions. Acknowledgements The authors wish to thank Dr. M. L. Champigny for helpful discussions. This work was supported by a grant «Action interdisciplinaire» from the University Paris XI, ORSAY.
References ARNON, D. I.: Copper enzymes in isolated chloroplasts. Plant Physiol. 24, 1-15 (1949). ATKINSON, D. E.: The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7, 4030-4034 (1968). BARIAUD, A., M. BURY, and J. C. MESTRE: Mechanism of Cd2 + resistance in Euglena gracilis. Physiol. Plant. 63, 382-386 (1985). BONALY, J., A. DELCOURT, and J. C. MESTRE: The effects of some metallic ions on the growth of Chlamydomonas variabilis and Euglena gracilis. Mitt. Internat. Verein Limnol. 21, 103 -109 (1978). BONALY, J., A. BARIAUD, S. DURET, and J. C. MEsTRE: Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis. Physiol. Plant. 49, 286-290 (1980). CHAPMAN, A. G. and D. E. ATKINSON: Stabilization of adenylate energy charge by the adenylate deaminase reaction. J. BioI. Chern. 248, 8309-8312 (1973). DE FILIPPIS, L. F., R. HAMPP, and H. ZIEGLER: The effect of zinc, cadmium and mercury on Eug· lena. II Respiration, photosynthesis and photochemical activity. Arch. Microbiol. 128, 407411 (1981 a).
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DE FILIPPIS, L. F., R. HAMpp, and H. ZIEGLER: The effect of sublethal concentrations of zinc, cadmium and mercury on Euglena. Adenylates and energy charge. Z. PflanzenphysioI. 103, 1-7 (1981 b). DURET, S., J. BONALY, A. BARIAUD, A. VANNEREAU, and J. C. MESTRE: Cadmium induced ultrastructural changes in Euglena cells. Environ. Res. 39{1}, 96-103 {1986}. HAMpp, R., K. BEULICH, and H. ZIEGLER: Effect of zinc and cadmium on photosynthetic CO2 fixation and Hill activity of isolated spinach chloroplasts. Z. PflanzenphysioI. 77, 336-344 {1976}. HOHORST, H. J.: L+ lactate determination with lactic dehydrogenase and DPN. In Methods of enzymatic analysis. H. U. Bergmeyer ed. 266-274. Verlag Chemie, Academic Press. N.Y. {1963}. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the folin phenol reagent. J. BioI. Chern. 193, 265-275 {1951}. LUCERO, H. A., C. S. ANDREO, and R. M. VALLEJOS: Sulphydryl groups in photosynthetic energy conservation. III Inhibition of photophosphorylation in spinach chloroplasts by CdCh. Plant Sci. Lett. 6, 309-313 {1976}. MATSUMOTO, S. S., K. O. RA:IVIO, and J. E. SEEGMILLER: Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and energy charge correlation. J. BioI. Chern. 254, 8956-8962 {1979}. MIGINIAc-MAsLOw, M. and A. HOARAU: Variations in the adenylate levels during phosphate depletion in isolated soybean cells and wheat leaf fragments. Z. PflanzenphysioI. 107, 427 -436 {1982}. MUSTAFA, G. M. and C. E. CROSS: Pulmonary alveolar macrophage. Oxidative metabolism of isolated cells and mitochondria and effect of cadmium ion on electron and energy transfer reactions. Biochemistry 16, 4176-4185 {1971}. PRADET, A.: Etude des adenosines 5' mono, di et triphosphates dans les tissus vegetaux. I Dosage enzymatique. PhysioI. Veg. 5, 209-221 {1967}. SPORN, A., I. DINu, and L. STOENESCU: Influence of Cadmium administration on carbonhydrate and cellular energetic metabolism in the rat liver. Rev. Roum. Biochim. 7, 299-305 {1970}. THOMAS, K. C. and P. S. S. DAWSON: Energy charge values of Candida utilis growing under energy-excess and energy-limiting conditions. FEMS Microbiology lett. 1, 347 -349 {1977}. WEBB, M.: The chemistry, biochemistry and biology of cadmium. 465p. Webb M. ed. Elsevier Amsterdam {1979}. WIEGEL, H. J.: The effects of Cd2 + on photosynthetic reactions of mesophyll protoplasts. PhysioI. Plant. 63, 192-200 {1985}. YOSHINO, M. and K. MURAKAMI: In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts. Biochim. Biophys. Acta 672, 16-20 {1981}.
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
J. BONALY, M. MIGINIAC-MAsLOW*), E. BROCHIERO, A. HOARAU*) and]. C. MESTRE Laboratoire de Biologie cellulaire, Faculte de Pharmacie, rue J. B. Clement, 92290 ChatenayMalabry, France, and *) Laboratoire de Photosynthese et Metabolisme, UA 1128, Universite de Paris-Sud, 91405 Orsay, France Received July 1, 1985 . Accepted November 18, 1985
Summary Energy charge values and adenylate contents were determined throughout one culture cycle of Euglena gracilis, and on the 3rd day of the subsequent subcultures. The effects of Cd ions (500/LM) on these parameters were investigated. Energy charge values were decreased in the presence of Cd ions, but progressively recovered during the adaptation of Euglena to Cd. The decrease in energy charge was concomitant with a transient drop in the photosynthetic 14C0 2 fixation and with a gradual decrease in lactate consumption, suggesting that respiration was most affected by Cd. Cd poisoning resulted in increased cell size and protein content. Accordingly, adenylate levels per cell were higher in the presence of Cd. When expressed per protein unit, adenylate levels became lower in Cd - treated Euglena than in control Euglena only 4 days after inoculation. This observation suggested that adenylate metabolism was affected by Cd. The use of energy charge as an energy stress indicator is discussed. Key words: Euglena gracilis, cadmium, energy charge, photosynthesis, respiration.
Introduction Many studies reported toxic effects of Cd ions on animal and plant metabolism (Webb, 1979). Various sites of action have been proposed: oxidative phosphorylation (Sporn et al., 1970; Mustafa and Cross, 1971) and respiratory oxygen consumption (De Filippis et al., 1981 a), photosynthetic electron transport and photophosphorylation (Hampp et al., 1976; Lucero et al., 1976; De Filippis et al., 1981 a) and Calvin cycle stromal enzymes (Wiegel, 1985). Our own results (Duret et al. [in press]) showed an alteration of mitochondrial structure and a decrease in cell respiration activity in Euglena, suggesting an effect on respiratory energetics. Many of the proposed sites have been determined on in vitro systems. In the present study, we tried to get information about toxic effects of Cd ions on energetics of Euglena in conditions as close as possible to an in vivo situation. The determination of the adenylate pool and of energy charge values seemed to be a good approach to this problem, since these parameters have been shown to be differently affected by metabolic stress (nutrient deficiency, Thomas and Dawson, 1977; Matsumoto et al.,
f Plant Physiol. Vol.
123. pp. 349-358 (1986)
350
J. BONALY, M. MIGINIAC-MASLOW, E. BROCHIEllo, A. HOARAU andJ. C. MEsTRE
1979; respiratory inhibitors, Matsumoto et al., 1979): cellular adenylate content was decreased in all cases, while energy charge was decreased only when the respiratory pathway was affected. Only one study reported Cd effects on adenylate energy charge (De Filippis et al., 1981 b). It concerned autotrophically grown Euglena cells. Our previous studies on photoheterotrophically grown Euglena gracilis stain Z showed that under our culture conditions this Euglena strain was able to develop a resistance to toxic Cd concentrations (Bonaly et al., 1980). It was therefore of particular interest to study the energetic balance in this strain during the development of Cd resistance. In addition to energy charge and adenylate determinations, measurements of lactate consumption and 14C02 fixation have been made in the conditions of the culture in order to investigate which of the two main energetic processes (respiration or photosynthesis) was the most affected by Cd.
Materials and Methods Algal cultures and determination ofgrowth Axenic cultures of Euglena gracilis strain Z (French CNRS collection) were auto-heterotrophically grown in 150 ml Erlenmeyer flasks under sterile conditions. The growth conditions and the method of cell counting were as previously described (Bonaly et al., 1978). Briefly, cells were grown at 23°C in the light (22.5 Wm -2) in a synthetic medium containing minerals, lactic acid, and vitamins Bl and B12. Cadmium chloride (5 x 10- 4M final concentration) was aseptically added to the medium. Cell counting was carried out daily in a model DI Coulter counter using a 100 JLm aperture. Due to the great number of counted cells, the variability was ca. 1 %. Measurements ofsize Size distribution of Euglena was performed with a cytofluorograf FC 200 (Biophysics) connected to a multichannel distribution analyser (Biophysics, Model 2102). For each sample, (10 5 cells), mean cell size was determined after weighing each histogram. Instrument calibration was done with calibrated polystyrene microspheres (Biophysics). As for cell counting the variability was around 1 %. Determination ofAdenylates To 5 ml of culture suspension were rapidly added 0.5 ml of lN TCA and the mixture was frozen in liquid nitrogen. The frozen extracts were thawed and centrifuged (12,000 xg, 10 min). The pellets were washed once with 2 ml of 0.1 N TCA and centrifuged again. The pooled supernatants were washed 4 times with an equal volume of diethyloxide in order to eliminate TCA. The diethyloxide dissolved in the aqueous phase was eliminated by bubbling air, and the extracts were neutralized with 2M K2 C0 3• Adenylate determinations were made on the extracts by the luciferase method, either directly (ATP), or after enzymatic transformation into ATP (ADP and AMP), as described by Pradet (1967). All the determinations were done in triplicate, with internal standards, on 100 JLI of the extracts. Adenylate energy charge was calculated according to Atkinson (1968): [ATP] + ~ [ADP]i[ATP] + [ADP] + [AMP]. Chlorophyll and protein determinations For chlorophyll determinations, 5 ml of culture suspension was sedimented by centrifugation (12,000 xg, 10 min). The pellets were extracted with 80% aqueous acetone. After centri-
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
Energetics of Euglena and cadmium resistance
351
fugation, the chlorophyll content of the supernatants was determined according to Arnon (1949). The protein content was estimated on the pellets remaining after adenylate extraction. The pellets were dissolved in 2 ml of 2N NaOH and their protein content was measured by the Folin phenol reagent (Lowry et al., 1951).
14C02 incorporation measurements The rate of CO 2 fixation was measured by injecting 5 mM 14C-Iabelled bicarbonate (2 p.Ci/ p.mole) into 5 ml cultures contained in stoppered Erlenmeyer flasks. Fixation was performed by injecting 0.5 ml of pure formic acid, and the unreacted bicarbonate eliminated by aspiration under vacuum into concentrated KOH. 0.3 ml samples were dried in an oven at 80°C and the radioactivity counted in a scintillation counter.
Determination of lactate consumption The determination of lactate in the medium was performed spectrophotometrically according to Hohorst (1963). The basic equation is: L Lactate + NAD+ + hydrazine lacticodehydrogenase. pyruvate hydrazone + NADH H+ + H 0 NADH was spectroH 95 , 2 . photofuetrically measured at 340 nm. Difference between lactate amount on day 0 and lactate amount on day x allowed the calculation of lactate consumption by cells on day x. Results Fig. 1 shows the total levels of adenylates and the energy charge values during one culture cycle of photoheterotrophically grown Euglena cells. In the presence of Cd
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Fig. 1: Changes in total adenylate levels (E) and energy charge values (EC) during one culture cycle of Euglena growing either on control medium (e: energy charge;.: total adenylate) or on a medium containing 500 p.M Cd Ciz (0: energy charge, D: total adenylate). Adenylate levels are expressed per g protein. Each value is the average of 3 separate determinations ± standard deviation (S.D.).
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
352
J.
BONALY,
M. MIGINIAC-MAsLOW, E. BROCHIERO, A.
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Fig.2: Changes in total adenylate levels per 107 Euglena cells during one culture cycle. .: Euglena cultured on control medium (Ed. 0: Euglena cultured on a medium containing 500 pM CdCh (ET). Each value is the average of 3 separate determinations ± S.D.
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Fig.3: Protein content per 107 Euglena cells during one culture cycle. .&: Eu· glena growing on control medium (C). .6.: Euglena growing on a medium containing 500 pM CdCh (T). Each value is the average of 3 separate determinations ± S.D.
ions, the energy charge values were sharply decreased during the first 2 days of culture, and finally stabilized at low values ( := 0.5). In the control culture, energy charge values ranged between 0.85 and 0.95. It seemed therefore that the energetic functions of Euglena were impaired by Cd ions. Total adenylate content per g protein increased much less in Cd - treated Euglena than in control cells during the growth cycle. As a matter of fact, it remained nearly constant and significantly lower than in control cells, after a limited but significant
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
Energetics of Euglena and cadmium resistance
18
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increase on day 2 found in 3 independent experiments. At this particular stage, the ad· enylate content per g protein was higher in Cd - treated Euglena than in control Eug· lena. When expressed per 107 cells, and in contrast to the observations of De Filippis et al. (1981 b), the adenylate contents were higher in Cd·treated cells than in control cells throughout the culture cycle (Fig.2). This observation reflects the increase in protein content of Cd-treated cells (Fig. 3) which are also larger than control cells (Fig. 4). On further inoculations of Cd·treated Euglena cells on fresh control or Cd-enriched media, energy charge values measured on the yd day of culture (exponential growth phase, see growth curves in Fig. 5), showed a stepwise recovery of the control values (Fig. 6). The recovery was faster on control medium than on Cd·enriched medium.
J Plant Physiol. Vol.
123. pp. 349-358 (1986)
J. BONALY, M. MIGINIAC-MASLOW, E. BROCHIEllO, A. HOARAU and J. C. MESTRE
354
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Fig. 6: Changes in the energy charge values of Cd-treated Euglena cultures, measured on the yd day of each culture cycle, during successive subcultures. C: Euglena growing on control media. - Cd: Euglena grown on 500 I'M CdCb during the first subculture cycle and then returned to a medium without Cd in further subcultures. + Cd: Euglena grown and subcultured on 500 JLM CdCb. Average value of 3 separate determinations ± S.D.
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Nevertheless, on Cd-enriched medium, the cells reached control energy charge values on the 6th subculture cycle. It appeared that during the development of resistance to Cd ions, the Euglena recovered normal energetic functions. The comparison of energy charge values of control and Cd-resistant Euglena confirmed this point, as they were identical in both cases (Fig. 7). In order to determine which of the main energetic pathways (respiration or photosynthesis) was the most affected by Cd poisoning under the conditions of the culture, we tested 14C02 incorporation by Euglena maintained under normal growth conditions (22.5 W' m -2 light with lactate as respiratory substrate), and the disappearance of lactate from the culture medium, in the presence or in the absence of Cd. Whether expressed per 106 cells or per mg chlorophyll, CO 2 fixation of Cd-treated cells dropped on the first day of culture and then rapidly recovered (Fig. 8). On the 4th day of culture, CO2 fixation was higher in Cd-treated Euglena than in control
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
Energetics of Euglena and cadmium resistance
A
355
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E4 8 Fig. 8: CO 2 fixation rates of Euglena cultured on a control medium (.) or on a medium containing 500/LM CdCh (0) during one culture cycle. The rates are expressed either per 106 cells (A) or per mg chlorophyll (B) or per mg protein (C). The values of 2 separate determinations are given.
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Euglena. This observation may be accounted for by the fact that at this stage, the control culture has reached its maximum density and its photosynthetic activity might be limited by availability of light. Nevertheless, maximum activity of the Cd-treated culture was equivalent to the maximal activity of the controls. Then, the Cd effect on the CO 2 fixation activity seems to be transitory. The recovery of photosynthetic activity corresponds to the recovery of a normal chlorophyll content per cell. The CO 2 fixation activity was only consistently lower in Cd-treated Euglena when expressed on a protein unit basis.
J. Plant Physiol. Vol.
123. pp. 349-358 (1986)
J. BONALY, M. MIGINIAC-MASLOW, E. BROCHIERO, A. HOARAU and J. C. MEsTRE
356 \0
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Fig. 9: Lactate consumption per 106 cells by Euglena cultured on a control medium (.) or on a medium containing 8 500 pM CdCh (0). Each point is the average of 4 separate determinations. ±S.D.
In contrast to CO2 fixation activities, lactate consumption was much more steadily affected by Cd throughout the culture cycle (Fig. 9). It then appears that inhibition of photosynthesis contributes to the decrease of energy charge only on the first days of culture: inhibition of respiration seems to be the main factor responsible for decrease in energy charge in Cd-poisoned Euglena.
Discussion Our experiments show that under photo heterotrophic growth conditions, the energetic metabolism of Euglena gracilis strain Z was affected by Cd. Energy charge values were decreased in a similar way as reported by De Filippis et al. (1981 b) for autotrophically grown Euglena. The decrease in the energy charge was correlated with a transient decrease in CO2 fixation activities and a consistent lowering in lactate consumption rates per cell. The maximal CO2 fixation rates per unit chlorophyll were the same in control and Cd-treated Euglena, but occurred later in the growth cycle in the latter case: the maximal efficiency of the photosynthetic apparatus seemed to be identical in both cases. While photosynthesis recovered rapidly, lactate consumption was affected for a much longer time. Therefore respiration seemed to be the major site of Cd inhibition. When Euglena developed a resistance to Cd ions, there was a stepwise recovery in energy charge values. Cd-resistant Euglena strains exhibited identical energy charge values, whether cultivated in the presence of Cd, or not, Bariaud et al. (1985) reported a lower accumulation of Cd in resistant cells than in Cd sensitive cells. However, Cd2 + uptake (in 0.1 mM CdClz) is not negligible (0.36/-tmol (gDW) -Ih -I in resistant cells vs 0.87 /-tmol (gDW) -I h -I in sensitive cells). Therefore, the relief of the depressive effect of Cd on energy charge values may be either due to lower intracellular Cd concentration in Cd-resistant cells and or to a decreased susceptibility of organelles to Cd ions.
J Plant Physiol. Vol. 123. pp. 349-358 (1986)
Energetics of Euglena and cadmium resistance
357
In contrast with the results of De Filippis et al (1981 b), total adenylate levels were changed in photo heterotrophically grown Euglena in the presence of Cd. Cd poisoning resulted roughly in a lower adenylate content per unit protein and in a higher adenylate content per cell. Decrease in adenylate content when energy charge is lowered is a rather general phenomenon. It is considered as a regulatory mechanism which avoids extensive changes in energy charge (Chapman and Atkinson, 1973, Matsumoto et aI., 1979; Yoshino and Murakami, 1981, Miginiac-Maslow and Hoarau, 1982).
However, in Cd-treated Euglena, total adenylate content became lower than in control Euglena only 4 days after inoculation, on a protein basis. On the 2nd day of culture the adenylate level was 40 to 50 % higher in the presence of Cd. It seems therefore that on the first days of culture Cd interferes with the regulatory mechanisms generally considered responsible for the decrease in adenylates under stress conditions. When expressed per cell, adenylate content was much higher in the presence of Cd. In spite of the lowered energy charge, the ATP content per cell was also higher, almost twice that of the control cells (results not shown). These results mainly reflect the cell enlargement and the increase in protein content per cell. They point out the possibility of misinterpretation of some results when a cell unit basis is chosen. On the contrary, energy charge seems to be an excellent criterion for energetic stress determination. It is a ratio which reflects the energy-saturation of adenylate pool and it is entirely independent of a quantitative expression basis. Our results indicate that changes in energy charge correlate with impaired energetic functions. Acknowledgements The authors wish to thank Dr. M. L. Champigny for helpful discussions. This work was supported by a grant «Action interdisciplinaire» from the University Paris XI, ORSAY.
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J. Plant Physiol. Vol.
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