Photochemistry and Photobiology. 1975, Vol. 21, pp. 265-268.
Pergamon Press. Printed in Great Britain
X-RAY SENSITIVITY OF PHOTOSYNTHETIC AND REPRODUCTIVE SYSTEMS IN CHLORELLA M. A. NOKES*and M. SIMICt Laboratory of Radiation Biology, Department of Zoology, The University of Texas at Austin, Austin, Texas 78712, U.S.A.
(Received 22 March 1974; accepted 4 October 1974) Abstract-Some effects of X rays on the green alga Chlorella pyrenoidosa have been investigated by measurements of survival and oxygen-evolving capability. Photosynthetic oxygen evolution at light saturation and endogenous respiration showed the same radiation sensitivity, and these two processes were about 40 times more resistant to X-ray-induced inactivation than survival. Responses of cells receiving doses in excess of 200 krad are in agreement with the existence of two photosystems, photoreaction I1 being apparently more sensitive to X rays than photoreaction I.
The ability of aqueous suspensions of the green alga Chlorella pyrenoidosa to evolve oxygen photosynthetically was investigated as a function of X-ray dose. The reproductive ability, which is expected to be much more radiation-sensitive, was also examined and compared to respiration and photosynthetic oxygen evolution. This investigation provides information on the relative radiation sensitivity of various cell functions and conditions for their selective inactivation. MATERIALS AND METHODS
Algae. The Emerson strain of Chlorella pyrenoidosa was used. The algae were grown in a continuous-culture apparatus (Myers and Clark, 1944) in a modified Knops solution (Myers and Graham, 1971), bubbled in air containing 5% CO, at 2 5 T , and illuminated by four 40 W tungsten lamps. Exact concentrations of algae were obtained from packed-cell volume, which was determined by centrifuging a measured aliquot of cell suspension at 2500g for 1 h in a centrifuge tube with a precision-borecapillary extension (Myers and Graham, 1971). Irradiation. The samples were irradiated with 300 kVp X rays, filtered by the equivalent of 3.25 mrn of A]. The dose rate was 4.6krad/min as determined by Fricke dosimetry (Swallow, 1972)at the position of the sample. 1 0 m 2 aliquots of the algal suspensions from the continuous-culture apparatus were placed in Pyrex test tubes for irradiation. The suspensions were held in darkness and deoxygenated with prepurified grade nitrogen (less than 0.002mol-per cent oxygen) for 10min before and during irradiation. Oxygen was purged from the suspensions in order to eliminate radiation-induced formation of organic peroxides. Continuous stirring of the suspensions ensured uniform distribution of dose to the suspended cells. Survival. Two different techniques were used to assess the effects of X irradiation on the growth and reproduction of Chlorella. The first involved plating the cells on Knops agar (modified Knops solution with 1.8% Difco *Present address: Department of Applied Physics, Stanford University, Stanford, California 94305, U S A . tTo whom all correspondence should be addressed. P \
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Bacto-Agar), exposing the sealed plates to fluorescent light, and counting visible colonies after 10 days of growth. In the second technique, growth was determined by optical absorption of the cell suspensions, the absorption being proportional to the number of algae (or amount of chloroplast) present. A short initial growth period was followed by a period of depressed growth rate after X irradiation. These two periods were followed eventually by normal growth rate with a slope identical to those of unirradiated algae (Redford and Myers, 1951). The ordinate intercept of that latter portion of the growth curve, during which growth proceeded at the normal linear rate (see Fig. 1) was used to calculate the survival. Both methods yielded similar results. All the survival data were obtained using the growth-curve method because of its convenience. Oxygen evolution and consumption. The oxygen concentration in algal suspensions was measured with a Beckman oxygen electrode inserted into the sidearm of a specially constructed glass cuvette (Myers and Graham, 1971). The cuvette was enclosed in a water jacket and maintained at 25.00'2 0.02"C. The suspensions in the cuvette were stirred for homogeneous distribution of oxygen. The cuvette was illuminated by a projection system equipped with an 8 c m water filter during the oxygenevolution measurements. Spectral bands were selected with interference filters (Baird-Atomic, B2 620 and 705), and the intensity was varied by using attenuators and a small change of the projection lamp potential for the lowest intensity. Irradiance was measured with a largearea Cambridge thermopile calibrated against standard lamps. Irradiated cells were centrifuged 10 min at 2500 g, washed and recentrifuged twice, and resuspended in Knops X solution (1.25 g/Y KNO,, l.25g/Y KH2P04, 2.50 g/Y MgS04, adjusted to pH 6.8) at a cell concentration of 0.3-0.5 mm3/mY. This particular operation was introduced to eliminate the inhibitory action of radiolytic products in irradiated suspensions. The cells were held in darkness and bubbled with 5% CO, in air at 25°C prior to injection into the electrode cuvette. When oxygen consumption or evolution, under different conditions of illumination, was measured, a fresh aliquot of cell suspension was pipetted into the cuvette for each intensity or wavelength of illumination used. The oxygen evolution rate, P, was always corrected for dark
265
M. A. NOKESand M. SIMIC
266
processes to 50 per cent of the control value. At doses in excess of 800 krad, the inhibition of these processes took place at an increased rate (see Fig. 2). In an attempt to locate the sites of radiation damage within the photosynthetic process, oxygen evolution rates were measured as a function of exciting light intensity. Figures 3a and 3b show the results of these measurements at 620 and 705 nm, respectively. Under our experimental conditions AaoIA705~ 2 . ~ l l l l l l l i l l l l l l l l l Il I 0 20 40 60 80 I00 120 140 160 I80 200 With light that primarily drives photosystem I (705 nm) the curves (Fig. 3b) showed a monotonic T(h) decrease in the linear, light-limited regions, and in Figure 1. Growth of X-irradiated and unirradiated the saturation levels, as radiation dose increased. Chlorella pyrenoidosa in Knops solution, periodically monitored by light absorption. The irradiation was With light that primarily drives photosystem I1 conducted in deaerated suspensions in darkness at 25°C (620 nm), however, the light-limited regions of the intensity curves were co-linear for cells receiving using 300 kVp X rays. doses of 200, 600,and 1OOOkrad (Fig. 3a), and respiration, R, electrode drift and the concentration of differences in response became apparent only at cells. light saturation. Further differences appear in Figs. 3c and 3d, RESULTS where the normalized photosynthetic activity P / P o The optical growth curves for irradiated (Po represents the oxygen-evolution rate of the Chlorella in anoxic media has a triphasic form. unirradiated control) is plotted as a function of light After a short transient period (1-2 divisions) of intensity. The value of P/Pofor cells receiving exponential growth, the irradiated cells (doses 25 krad was approximately constant at 0.94, regardgreater than 50krad) appeared to cease growth less of the wavelength or intensity of exciting light (also known as delayed death) causing a plateau in used. Cells receiving 200 krad also had a roughly the growth curve (Fig. I). Following the plateau, the constant value of PIPo over the range of intensities growth rate would attain that of unirradiated cells, 30 the level and duration of this plateau being dependent on the remaining fraction of fully viable 25 cells i.e. the dose received. A dose of approximately 18 krad was necessary to reduce survival to 20 50 per cent (Fig. 2). The ratios of oxygen evolution in irradiated cells 15 H to that in the controls as a function of dose are n a 10 shown in Fig. 2. The measurements were made in 620 nm light at saturation intensity (142 W/m’). X5 ray-induced reduction of oxygen evolution and dark respiration followed the same pattern; a dose 0 of about 700 krad was required to reduce those two
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Figure 2. Survival (N/Na), endogenous respiration ( R I R J , and photosynthetic oxygen evolution ( P / P , ) in saturating 620 nrn light for C. pyrenoidosa as a function of X ray dose.
I / I 0 X I00
Figure 3. Photosynthetic oxygen-evolution rates ( P ) and normalized photosynthetic activity ( P / P o )as a function of exciting-light intensity. The values shown in a and c were measured in 620nm light, and those in b and d were obtained in 705 nm light. The rates are expressed in units of chart-recorder divisions per inch, this unit being equivalent to a change of 0.01 nA electrode current in 80 s.
Photosynthesisin X-irradiated Chlorella used. At higher doses, however, this ratio was lower at light saturation than during the lightlimited phase. The light-limited values of P / P owere co-linear in 620 nm light for cells given 200,600, and 1000 krad, while the corresponding values in 705 nm light were separate and distinct, and declined monotonically with increasing radiation dose. These responses indicate different radiation sensitivities for components within the photosynthetic apparatus. DISCUSSION
According to current theories, photosynthesis involves two primary photochemical processes, photoreactions I and 11, which utilize separate pigment systems (Duysens, 1964; Hill and Bendall, 1960). The products of these initial photochemical events undergo a number of enzymatic, temperature-dependent reactions, denoted collectively as the ‘dark reaction’, which result in the evolution of oxygen and the reduction of carbon dioxide. We discuss here the effects of X irradiation on photosynthesis in terms of oxygen evolution only. The rates of the two primary photoreactions are directly proportional to the intensity of illumination. On the other hand, the dark reaction has a maximum capacity, and saturation of this reaction occurs at high light intensities. The rate of oxygen evolution at light saturation is, therefore, determined by the dark reaction. At intensities well below saturation, the rate of oxygen evolution is determined by the photoreaction that proceeds most slowly at that particular wavelength. In 705nm light, the majority of the photons are absorbed by the pigment system which drives photoreaction I, and the rate of the under-driven photoreaction I1 determines the oxygen evolution rate at low intensities. Conversely, photoreaction I is under-driven in 620 nm light, and it becomes the rate-limiting reaction in low-intensity light of this wavelength. Although this concept of a ratelimiting step is not exact, it is useful as a first approximation. One can determine whether treatment of the cells has affected the dark reaction (saturation level), one of the two photoreactions (slope of the linear portion of the rate-vs-intensity curve), or some combination of the three reactions. Consider the response of irradiated and unirradiated Chlorella to different wavelengths and intensities of exciting light. If the radiation damage were distributed uniformly between photoreaction 1, photoreaction 11, and the dark reaction, one would expect the normalized photosynthetic activity P / P o to be independent of the intensity of illumination at a given radiation dose. This behavior was indeed observed in cells irradiated with ultraviolet light (Redford and Myers, 1951), and in cells given X ray doses of less than 200krad. At doses of 600krad or more, however, the values of P/Pn were appreciably lower at light
267
saturation than in the light-limited region. This indicates a higher radiation sensitivity of components of the dark reaction, which determines oxygen evolution rate at saturation, than in the photoreactions. It should be noted that only very small changes in the absorption spectrum of the irradiated algae were observed (less than 3 per cent at 1200 krad) thus the effect cannot be attributed to decreased absorption in the optical range. Since photoreaction I1 is grossly under-driven in 705nm light [the ratio of photons absorbed in pigment system I to those absorbed in pigment system I1 at this wavelength is about 78: 22 (Eley, 1%7)], it is the rate-limiting step at low intensities. Examination of Figs. 3b and 3d shows that the slope of the rate-vs-intensity curve in the lightlimited region and the corresponding values of P/Pndecrease monotonically with increasing radiation dose, indicating appreciable damage to photoreaction 11. On the other hand, photoreaction I is slightly under-driven in 620 nm light [the ratio of photons absorbed in pigment system I to those absorbed in pigment system I1 at 620 nm is about 44: 56 (Eley, 1967)], and one expects damage to photoreaction I to appear in the light-limited region for this type of illumination. However, as seen in Figs. 3a and 3c, the low-intensity regions of the rate-vs-intensity and photosynthetic activity curves were co-linear for cells given doses of 200, 600, and 1000 krad. Even at 1200 krad, where a decrease in the slope of the light-limited portion occurred, the values of P and P / P o at low intensity were higher in 620nm light than the corresponding values in 705 nm light. Thus, no increase in radiation damage to photoreaction I, beyond that at 200 krad, is evident for doses up to 1000 krad. Damage to photoreaction 11, however, can be concealed in 620nm illumination because of the imbalance in photons absorbed by the two pigment systems. In addition, the possibility of exciton transfer between two photounits of system II (Joliot et al., 1968) would allow even further masking of damage; a quantum of excitation energy could be transferred from a damaged unit to an undamaged unit that is still capable of initiating photochemistry. On the basis of the above considerations, we conclude that photoreaction I1 is more sensitive to X-ray-induced inactivation than photoreaction I. Our data also provide additional indirect evidence for the existence of two separate photosystems, because, for one system only, the response would have been independent of the wavelength of illumination. Oxygen evolution in saturating 620 nm light and endogenous respiration showed the same sensitivity to X irradiation (Fig. l), in contrast to results of investigations using ultraviolet radiation, in which respiration was less sensitive than photosynthesis (Redford and Myers, 1951). This can be explained
268
M. A. NOKESand M. SIMIC
in terms of the absorption of radiation within the cell. UV damage will occur preferentially in the highly absorbing photosynthetic apparatus. On the other hand, X rays deposit energy more or less uniformly among the cellular constituents, and X ray damage will be equally extensive in the electron transport chains of either the respiratory system or the photosynthetic dark reaction. Appreciable inhibition of photosynthetic oxygen evolution occurs at doses above 1 Mrad. At these doses, the concentration of damage could be as much as 3 X IO-’M (this is based on a value of W = 35eV, where W represents the average energy required to produce one ion pair, provided n o ion recombination takes place, i.e. each positive hole would produce one damaged site). Enzymes and membranes could be extensively damaged at these high doses (Gray, 1954; Levinson and Garber, 1967). On the other hand, we know very little about the effects of radiation on chlorophyll and the accessory pigments in the chloroplast. It is most likely that disruption of membranes is the major cause of radiation-induced damage to the photosynthetic apparatus. This is substantiated by studies of the effects of X rays on isolated chloroplasts and chloroplast-fragments of different structural state (Perner, Falk and Jacobi, 1%5).
These authors also observe remarkable radiation resistance of the photo-synthetic acitivity in vitro, e.g. for one of the preparations, Olj2= 2 Mrad for the Hill reaction. The similar response of respiration to X irradiation could be based on the similarities (in structure and conductivity) of the chloroplast and the mitochondrion. In the case of the mitochondrion, efficient recombination of electrons and positive holes can take place without significant damage to the electron-transport system. Damage to the membranes, however, leads eventually to inhibition of respiration. The chemistry and mechanisms leading to damage of these biological functions are extremely complex, and considerable effort, involving fastkinetic techniques, will be required to elucidate these ionic and free-radical reactions.
Acknowledgements-The authors thank Dr. E. L. Powers for continuous support and Dr. J. Myers and the staff of the Laboratory of Algal Physiology, University of Texas at Austin, for providing the test organism, essential advice and much of the equipment used in this investigation. The work was supported in part by N.I.H. Grants GM-I 1300 and GM-13557, and by the U.S.Atomic Energy Commission on Contract AT-(40-1)-3408.
REFERENCES
Duysens, L. N. M. (1964) Progr. Biophys. Mol. B i d . 14, 1. Eley, J. H., Jr. (1967) Ph.D. Thesis. University of Texas at Austin. Gray, L. H. (1954) Radiation Res. 1, 189. Hill, R. and F. Bendall (1960) Nature 186, 136. Joliot, P., A. Joliot and B. Kok (1968) Biochim. Biophys. Acta 153, 635. Levinson, H. S. and E. B. Garber (1967) Appl. Microbiol. 15, 431. Myers, J. and L. B. Clark (1944) J. Gen. Physiol. 28, 103. Myers, J and Jo-Ruth Graham (1971) Plant Physiol. 48, 282. Perner, E., S. von Falk and G. Jacobi (1965) 2. Naturforsch. 20b, 1077. Redford, E. L. and J. Myers (1951) J. Cell. Comp. Physibl. 38, 217. Swallow, A. J. (1972) Radiation Chemistry. Wiley, New York.
Pergamon Press. Printed in Great Britain
X-RAY SENSITIVITY OF PHOTOSYNTHETIC AND REPRODUCTIVE SYSTEMS IN CHLORELLA M. A. NOKES*and M. SIMICt Laboratory of Radiation Biology, Department of Zoology, The University of Texas at Austin, Austin, Texas 78712, U.S.A.
(Received 22 March 1974; accepted 4 October 1974) Abstract-Some effects of X rays on the green alga Chlorella pyrenoidosa have been investigated by measurements of survival and oxygen-evolving capability. Photosynthetic oxygen evolution at light saturation and endogenous respiration showed the same radiation sensitivity, and these two processes were about 40 times more resistant to X-ray-induced inactivation than survival. Responses of cells receiving doses in excess of 200 krad are in agreement with the existence of two photosystems, photoreaction I1 being apparently more sensitive to X rays than photoreaction I.
The ability of aqueous suspensions of the green alga Chlorella pyrenoidosa to evolve oxygen photosynthetically was investigated as a function of X-ray dose. The reproductive ability, which is expected to be much more radiation-sensitive, was also examined and compared to respiration and photosynthetic oxygen evolution. This investigation provides information on the relative radiation sensitivity of various cell functions and conditions for their selective inactivation. MATERIALS AND METHODS
Algae. The Emerson strain of Chlorella pyrenoidosa was used. The algae were grown in a continuous-culture apparatus (Myers and Clark, 1944) in a modified Knops solution (Myers and Graham, 1971), bubbled in air containing 5% CO, at 2 5 T , and illuminated by four 40 W tungsten lamps. Exact concentrations of algae were obtained from packed-cell volume, which was determined by centrifuging a measured aliquot of cell suspension at 2500g for 1 h in a centrifuge tube with a precision-borecapillary extension (Myers and Graham, 1971). Irradiation. The samples were irradiated with 300 kVp X rays, filtered by the equivalent of 3.25 mrn of A]. The dose rate was 4.6krad/min as determined by Fricke dosimetry (Swallow, 1972)at the position of the sample. 1 0 m 2 aliquots of the algal suspensions from the continuous-culture apparatus were placed in Pyrex test tubes for irradiation. The suspensions were held in darkness and deoxygenated with prepurified grade nitrogen (less than 0.002mol-per cent oxygen) for 10min before and during irradiation. Oxygen was purged from the suspensions in order to eliminate radiation-induced formation of organic peroxides. Continuous stirring of the suspensions ensured uniform distribution of dose to the suspended cells. Survival. Two different techniques were used to assess the effects of X irradiation on the growth and reproduction of Chlorella. The first involved plating the cells on Knops agar (modified Knops solution with 1.8% Difco *Present address: Department of Applied Physics, Stanford University, Stanford, California 94305, U S A . tTo whom all correspondence should be addressed. P \
i' ?1/4
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Bacto-Agar), exposing the sealed plates to fluorescent light, and counting visible colonies after 10 days of growth. In the second technique, growth was determined by optical absorption of the cell suspensions, the absorption being proportional to the number of algae (or amount of chloroplast) present. A short initial growth period was followed by a period of depressed growth rate after X irradiation. These two periods were followed eventually by normal growth rate with a slope identical to those of unirradiated algae (Redford and Myers, 1951). The ordinate intercept of that latter portion of the growth curve, during which growth proceeded at the normal linear rate (see Fig. 1) was used to calculate the survival. Both methods yielded similar results. All the survival data were obtained using the growth-curve method because of its convenience. Oxygen evolution and consumption. The oxygen concentration in algal suspensions was measured with a Beckman oxygen electrode inserted into the sidearm of a specially constructed glass cuvette (Myers and Graham, 1971). The cuvette was enclosed in a water jacket and maintained at 25.00'2 0.02"C. The suspensions in the cuvette were stirred for homogeneous distribution of oxygen. The cuvette was illuminated by a projection system equipped with an 8 c m water filter during the oxygenevolution measurements. Spectral bands were selected with interference filters (Baird-Atomic, B2 620 and 705), and the intensity was varied by using attenuators and a small change of the projection lamp potential for the lowest intensity. Irradiance was measured with a largearea Cambridge thermopile calibrated against standard lamps. Irradiated cells were centrifuged 10 min at 2500 g, washed and recentrifuged twice, and resuspended in Knops X solution (1.25 g/Y KNO,, l.25g/Y KH2P04, 2.50 g/Y MgS04, adjusted to pH 6.8) at a cell concentration of 0.3-0.5 mm3/mY. This particular operation was introduced to eliminate the inhibitory action of radiolytic products in irradiated suspensions. The cells were held in darkness and bubbled with 5% CO, in air at 25°C prior to injection into the electrode cuvette. When oxygen consumption or evolution, under different conditions of illumination, was measured, a fresh aliquot of cell suspension was pipetted into the cuvette for each intensity or wavelength of illumination used. The oxygen evolution rate, P, was always corrected for dark
265
M. A. NOKESand M. SIMIC
266
processes to 50 per cent of the control value. At doses in excess of 800 krad, the inhibition of these processes took place at an increased rate (see Fig. 2). In an attempt to locate the sites of radiation damage within the photosynthetic process, oxygen evolution rates were measured as a function of exciting light intensity. Figures 3a and 3b show the results of these measurements at 620 and 705 nm, respectively. Under our experimental conditions AaoIA705~ 2 . ~ l l l l l l l i l l l l l l l l l Il I 0 20 40 60 80 I00 120 140 160 I80 200 With light that primarily drives photosystem I (705 nm) the curves (Fig. 3b) showed a monotonic T(h) decrease in the linear, light-limited regions, and in Figure 1. Growth of X-irradiated and unirradiated the saturation levels, as radiation dose increased. Chlorella pyrenoidosa in Knops solution, periodically monitored by light absorption. The irradiation was With light that primarily drives photosystem I1 conducted in deaerated suspensions in darkness at 25°C (620 nm), however, the light-limited regions of the intensity curves were co-linear for cells receiving using 300 kVp X rays. doses of 200, 600,and 1OOOkrad (Fig. 3a), and respiration, R, electrode drift and the concentration of differences in response became apparent only at cells. light saturation. Further differences appear in Figs. 3c and 3d, RESULTS where the normalized photosynthetic activity P / P o The optical growth curves for irradiated (Po represents the oxygen-evolution rate of the Chlorella in anoxic media has a triphasic form. unirradiated control) is plotted as a function of light After a short transient period (1-2 divisions) of intensity. The value of P/Pofor cells receiving exponential growth, the irradiated cells (doses 25 krad was approximately constant at 0.94, regardgreater than 50krad) appeared to cease growth less of the wavelength or intensity of exciting light (also known as delayed death) causing a plateau in used. Cells receiving 200 krad also had a roughly the growth curve (Fig. I). Following the plateau, the constant value of PIPo over the range of intensities growth rate would attain that of unirradiated cells, 30 the level and duration of this plateau being dependent on the remaining fraction of fully viable 25 cells i.e. the dose received. A dose of approximately 18 krad was necessary to reduce survival to 20 50 per cent (Fig. 2). The ratios of oxygen evolution in irradiated cells 15 H to that in the controls as a function of dose are n a 10 shown in Fig. 2. The measurements were made in 620 nm light at saturation intensity (142 W/m’). X5 ray-induced reduction of oxygen evolution and dark respiration followed the same pattern; a dose 0 of about 700 krad was required to reduce those two
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Figure 2. Survival (N/Na), endogenous respiration ( R I R J , and photosynthetic oxygen evolution ( P / P , ) in saturating 620 nrn light for C. pyrenoidosa as a function of X ray dose.
I / I 0 X I00
Figure 3. Photosynthetic oxygen-evolution rates ( P ) and normalized photosynthetic activity ( P / P o )as a function of exciting-light intensity. The values shown in a and c were measured in 620nm light, and those in b and d were obtained in 705 nm light. The rates are expressed in units of chart-recorder divisions per inch, this unit being equivalent to a change of 0.01 nA electrode current in 80 s.
Photosynthesisin X-irradiated Chlorella used. At higher doses, however, this ratio was lower at light saturation than during the lightlimited phase. The light-limited values of P / P owere co-linear in 620 nm light for cells given 200,600, and 1000 krad, while the corresponding values in 705 nm light were separate and distinct, and declined monotonically with increasing radiation dose. These responses indicate different radiation sensitivities for components within the photosynthetic apparatus. DISCUSSION
According to current theories, photosynthesis involves two primary photochemical processes, photoreactions I and 11, which utilize separate pigment systems (Duysens, 1964; Hill and Bendall, 1960). The products of these initial photochemical events undergo a number of enzymatic, temperature-dependent reactions, denoted collectively as the ‘dark reaction’, which result in the evolution of oxygen and the reduction of carbon dioxide. We discuss here the effects of X irradiation on photosynthesis in terms of oxygen evolution only. The rates of the two primary photoreactions are directly proportional to the intensity of illumination. On the other hand, the dark reaction has a maximum capacity, and saturation of this reaction occurs at high light intensities. The rate of oxygen evolution at light saturation is, therefore, determined by the dark reaction. At intensities well below saturation, the rate of oxygen evolution is determined by the photoreaction that proceeds most slowly at that particular wavelength. In 705nm light, the majority of the photons are absorbed by the pigment system which drives photoreaction I, and the rate of the under-driven photoreaction I1 determines the oxygen evolution rate at low intensities. Conversely, photoreaction I is under-driven in 620 nm light, and it becomes the rate-limiting reaction in low-intensity light of this wavelength. Although this concept of a ratelimiting step is not exact, it is useful as a first approximation. One can determine whether treatment of the cells has affected the dark reaction (saturation level), one of the two photoreactions (slope of the linear portion of the rate-vs-intensity curve), or some combination of the three reactions. Consider the response of irradiated and unirradiated Chlorella to different wavelengths and intensities of exciting light. If the radiation damage were distributed uniformly between photoreaction 1, photoreaction 11, and the dark reaction, one would expect the normalized photosynthetic activity P / P o to be independent of the intensity of illumination at a given radiation dose. This behavior was indeed observed in cells irradiated with ultraviolet light (Redford and Myers, 1951), and in cells given X ray doses of less than 200krad. At doses of 600krad or more, however, the values of P/Pn were appreciably lower at light
267
saturation than in the light-limited region. This indicates a higher radiation sensitivity of components of the dark reaction, which determines oxygen evolution rate at saturation, than in the photoreactions. It should be noted that only very small changes in the absorption spectrum of the irradiated algae were observed (less than 3 per cent at 1200 krad) thus the effect cannot be attributed to decreased absorption in the optical range. Since photoreaction I1 is grossly under-driven in 705nm light [the ratio of photons absorbed in pigment system I to those absorbed in pigment system I1 at this wavelength is about 78: 22 (Eley, 1%7)], it is the rate-limiting step at low intensities. Examination of Figs. 3b and 3d shows that the slope of the rate-vs-intensity curve in the lightlimited region and the corresponding values of P/Pndecrease monotonically with increasing radiation dose, indicating appreciable damage to photoreaction 11. On the other hand, photoreaction I is slightly under-driven in 620 nm light [the ratio of photons absorbed in pigment system I to those absorbed in pigment system I1 at 620 nm is about 44: 56 (Eley, 1967)], and one expects damage to photoreaction I to appear in the light-limited region for this type of illumination. However, as seen in Figs. 3a and 3c, the low-intensity regions of the rate-vs-intensity and photosynthetic activity curves were co-linear for cells given doses of 200, 600, and 1000 krad. Even at 1200 krad, where a decrease in the slope of the light-limited portion occurred, the values of P and P / P o at low intensity were higher in 620nm light than the corresponding values in 705 nm light. Thus, no increase in radiation damage to photoreaction I, beyond that at 200 krad, is evident for doses up to 1000 krad. Damage to photoreaction 11, however, can be concealed in 620nm illumination because of the imbalance in photons absorbed by the two pigment systems. In addition, the possibility of exciton transfer between two photounits of system II (Joliot et al., 1968) would allow even further masking of damage; a quantum of excitation energy could be transferred from a damaged unit to an undamaged unit that is still capable of initiating photochemistry. On the basis of the above considerations, we conclude that photoreaction I1 is more sensitive to X-ray-induced inactivation than photoreaction I. Our data also provide additional indirect evidence for the existence of two separate photosystems, because, for one system only, the response would have been independent of the wavelength of illumination. Oxygen evolution in saturating 620 nm light and endogenous respiration showed the same sensitivity to X irradiation (Fig. l), in contrast to results of investigations using ultraviolet radiation, in which respiration was less sensitive than photosynthesis (Redford and Myers, 1951). This can be explained
268
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in terms of the absorption of radiation within the cell. UV damage will occur preferentially in the highly absorbing photosynthetic apparatus. On the other hand, X rays deposit energy more or less uniformly among the cellular constituents, and X ray damage will be equally extensive in the electron transport chains of either the respiratory system or the photosynthetic dark reaction. Appreciable inhibition of photosynthetic oxygen evolution occurs at doses above 1 Mrad. At these doses, the concentration of damage could be as much as 3 X IO-’M (this is based on a value of W = 35eV, where W represents the average energy required to produce one ion pair, provided n o ion recombination takes place, i.e. each positive hole would produce one damaged site). Enzymes and membranes could be extensively damaged at these high doses (Gray, 1954; Levinson and Garber, 1967). On the other hand, we know very little about the effects of radiation on chlorophyll and the accessory pigments in the chloroplast. It is most likely that disruption of membranes is the major cause of radiation-induced damage to the photosynthetic apparatus. This is substantiated by studies of the effects of X rays on isolated chloroplasts and chloroplast-fragments of different structural state (Perner, Falk and Jacobi, 1%5).
These authors also observe remarkable radiation resistance of the photo-synthetic acitivity in vitro, e.g. for one of the preparations, Olj2= 2 Mrad for the Hill reaction. The similar response of respiration to X irradiation could be based on the similarities (in structure and conductivity) of the chloroplast and the mitochondrion. In the case of the mitochondrion, efficient recombination of electrons and positive holes can take place without significant damage to the electron-transport system. Damage to the membranes, however, leads eventually to inhibition of respiration. The chemistry and mechanisms leading to damage of these biological functions are extremely complex, and considerable effort, involving fastkinetic techniques, will be required to elucidate these ionic and free-radical reactions.
Acknowledgements-The authors thank Dr. E. L. Powers for continuous support and Dr. J. Myers and the staff of the Laboratory of Algal Physiology, University of Texas at Austin, for providing the test organism, essential advice and much of the equipment used in this investigation. The work was supported in part by N.I.H. Grants GM-I 1300 and GM-13557, and by the U.S.Atomic Energy Commission on Contract AT-(40-1)-3408.
REFERENCES
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