The effect of ionizing and ultraviolet radiations on photosynthesis

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

76,

196-203

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The Effect of Ionizing and Ultraviolet Radiations on Photosynthesis L. P. Zill’ From the Biology

Division,

and N. E. Tolbert

Oak Ridge National

La6oratory,2

Received

7, 1957

November

Oak Ridge, Tennessee

The elucidation of the photosynthetic carbon cycle provides us with a segment of plant metabolism which should be particularly suitable for studying the in viva effects of radiation. However, the entrance of carbon dioxide into this cycle is dependent on the integrity of the photolytic and energy-transferring system. The utilization of carbon dioxide by the plant after irradiation could therefore reflect radiation damage to either the carbon cycle, or the energy-storing system. Damage to the mechanism of formation of enzymes necessary to catalyze the process would not be apparent in the present experiments performed immediately after irradiation. Such a study with gamma radiation has been made by Gailey and Tolbert (l), on the development of photosynthesis in etiolated plants. In past studies of inhibition of photosynthesis in algae by ultraviolet light (249, damage was assayed by manometric determination of oxygen evolution. The photosynthetic quotient was found to remain close to unity, but the disposition of assimilated carbon dioxide was not determined. In the present study, rates of photosynthesis, after exposure to ultraviolet light or gamma radiation, were measured by fixation of C1402. The specific effects of radiation on the carbon cycle were determined by measurements of the distribution of Cl4 among the products. METHODS AND MATERIALS Experiments were carried out on 7-day-old greenhouse. Cultures of Chlorella pyrenoidosa previously described (6).

Thatcher wheat plants grown in a (Emerson strain) were grown as

1 Present address: RIAS, Inc., 7212 Bellona Ave., Baltimore 12, Md. 2 Operated by Union Carbide Nuclear Company for the U. S. Atomic Commission. 196

Energy

RADIATION EFFECTS ON PHOTOSYNTHESIS

197

Plants were exposed to gamma radiation from a cobalt-69 source delivering about 1515 r./min. The prototype of this source was described by Ghormley and Hochanadel (7). Ultraviolet radiation was obtained from a low-pressure 15-w. General Electric germicidal lamp with about 90% of emitted energy at 2537 A. Plants were exposed directly in the quartz chamber used for CldOz fixation. The leaves, with their flat surface parallel to the lamp envelope, received 35 ergs/sq. mm./sec. at 5 cm. distance . After both types of irradiation, carbon dioxide fixation was determined by the amount of C’402 fixed in 10 min. from a standard amount of Cl402 in excess of that which a normal plant could fix in this period. The leaves were exposed in a small chamber (85 ml.) for 10 min. at 1000 ft.-candles in an atmosphere of air containing 25 PC. of C”OZ (released by the reaction of lactic acid with NaHVOs prepared from BaC”Oa having a specific activity of 25% C”). The chamber was submerged in a water bath to maintain the temperature at 23°C. At the end of the Cl402 fixation period, the leaves (weighing 96-194 mg.) were cut from the plant, frozen with liquid nitrogen, ground in a chilled mortar, and then extracted with boiling water. An aliquot of this extract was plated out on a glass planchet and counted in a gas-flow proportional counter to determine the total counts of Cl402 in soluble constituents. Two-dimensional paper chromatograms were prepared from another aliquot. The first solvent was water-saturated phenol and the second was butanol-propionic acid-water (2:1.4:1) prepared according to Benson et ~2. (8). Labeled compoundswerelocatedon the chromatogram with No-screen x-ray film, and the spots were counted with an end-window Geiger tube to determine the distribution of 04 among them. Phosphorylated compounds are not well separated with the solvent systems described, nor are glycine and serine always resolved. Therefore, the total counts in the phosphate area and combined counts for glycine and serine are given in the tables. Compounds were identified by R, values, spray tests, and chromatography. For Chlorella, photosynthetic oxygen evolution was determined as described by Holt et al. (4). After one-half hour, 25 PC. of NaHC403 was added to the suspension in the Warburg vessel, and the algae were allowed a 10.min. fixation period, after which they were transferred into boiling 800/o ethanol. The total fixation of Cl402 was then determined on an aliquot of extract, as described for wheat. These extracts were not chromatographed because of the large concentration of salts in Warburg buffer No. 9.

RESULTS AND DISCUSSION

Ultraviolet Radiation E$ect on Photosynthetic Capacity. Figure 1 depicts the decrease of phocarbon dioxide fixation by Thatcher wheat with increasing dose of ultraviolet radiation. The rapid decline in photosynthetic rate by about 75 % was followed by a considerable period in which no further inhibition was observed. This suggests that absorption of the ultraviolet radiation by the leaf protects the more deeply situated cells. Measuretosynthetic

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ZILL

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ments with a General Electric germicidal meter in fact showed a 100 % absorption of the incident radiation in the leaf. Experiments with Ch.!orella suspensions indicated a photosynthetic quotient close to unity, for all degrees of radiation damage. The maintenance of this quotient until photosynthesis was completely destroyed suggests the absence of independent damage to the oxygen-evolving system or the carbon dioxide-fixing system. No residual, ultraviolet-resistant photosynthesis was observed in Chlorella, thereby supporting the attribution of such a process in wheat to internal shielding. Distribution of Products Formed from PO2 after Irradiation. The soluble products of Cl402 fixation in wheat, corresponding to the points in Fig. 1, were separated by paper chromatography. By measuring the radioactivity in these substances, the distribution of CY among the products was determined (Table I). In none of the radiation experiments were new labeled compounds formed or any of those present in the control lost. Ultraviolet irradiation caused an increase in the radioactivity of phosphorylated compounds and a decrease in that of sucrose. This suggests that sucrose synthesis is more sensitive to ultraviolet radiation than the reactions of the photosynthetic carboxylation cycle, perhaps because of the participation of uridine diphosphate glucose in this synthesis (9, 10). This suggestion is supported by Sinsheimer’s demonstration of photochemical instability of the uridylic acids (11). An almost identical sensitivity to ultraviolet light has been observed for uridine diphosphate glucose (12). In contrast, adenosine triphosphate, also in-

0' 0

FIG.

IO ~LTRA~~~LET

20 IRRADIATION TIME (minb

1. The effect of ultraviolet radiation dioxide fixation by Thatcher

(2537 A.) on carbon wheat.

30

RADIATION

EFFECTS

ON

TABLE Percentage

0

Phosphate area Sucrose Glucose Glycine and serine Fructose Alanine Malic acid a Percentage

I

Distribution” of Cl* in Products Thatcher Wheat after Ultraviolet

Compound

of total

34 30 Trace 24 2 1 9 04 activity

199

PHOTOSYNTHESIS

from C”O~ Fizalion Irradiation

Minutes of ultraviolet 5

44 17 Trace 26 Trace Trace 10

exposure 10

47 13 Trace 28 Trace Trace 7

by

30

35 24 Trace 30 Trace Trace 3

on the chromatogram.

volved in photosynthesis carbon dioxide fixation, must receive an extremely large dose of ultraviolet light before its decomposition can be detected (13). When CO2 entered the photosynthetic cycle in ultraviolet-irradiated leaf cells, it did so in a more or less normal fashion. If any step of the carbon cycle had been highly affected, there should have been an increase in the intermediate before the block. The gross decrease in photosynthetic fixation of CO2 must therefore be attributed to a process outside the carbon cycle or perhaps to the destruction of the primary CO2 acceptor, ribulose diphosphate, which was not studied. No significance has been attached to the small increase in CO2 fixation noted after 30 min. ultraviolet irradiation (Fig. 2). This could be attributed to differences in the leaf samples, especially in their thickness. The similar increase in rate observed after large doses of gamma radiation cannot, however, be explained in the same way. Stomata1 closing is a factor to be considered in the case of wheat. The large loss of diverse cellular constituents observed with ChEoreZZucells after ultraviolet irradiation (L. P. Zill and N. E. Tolbert, unpublished) suggests that a similar loss in the guard cells of wheat could occur, causing loss of turgor and stomata1 closing. A brief investigation of the stomatal behavior in wheat by the “rapid-weighing” method of Alvim (14) revealed only a 30-50 % closing of the stomata under our experimental conditions of ultraviolet irradiation. The decrease in transpiration indicative of stomata1 closing occurred not during the longest period of irradiation (30 min.) but lagged behind the irradiation period by lo-20 min. From these observations, we conclude that narrowing of the stomata did not significantly limit the availability of COZ for photosynthesis.

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A disturbance in the photolytic system caused by ultraviolet light cannot be evaluated because of our lack of knowledge about. its normal operation. That it is indeed affected can be seen from the decrease in oxygen evolution (3-5). In addition, the loss of chlorophyll after ultraviolet irradiation (15, 16) implicates the photolytic apparatus in the radiation damage. Whether a quantitative relation exists between this damage and the ability to fix COZ has not been ascertained. Gamma Radiation Effect on Photosynthetic Ability. The effect of increasing the dose of gamma radiation on photosynthetic COZ fixation by wheat and Chlorella is illustrated in Fig. 2. The rate of fixation decreased up to about 100 kr., after which no further decrease occurred. The extremely penetrating nature of gamma radiation precludes internal shielding as an explanation of this residual photosynthesis. The increase in CO2 fixation after 500 kr. by wheat, and 250 kr. by Chlorella also remains unexplained. To determine whether the photosynthetic quotient changes after gamma irradiation, we carried out experiments with Chlorella. The results (Fig. 2) show that in contrast to CO2 fixation, oxygen evolution continued steadily up to 100 kr., but decreased at, 250 kr. These data indicate that the CO2 fixation sequence of photosynthesis is more sensitive to gamma radiation than the photolysis of water.

FIG. 2. The effect of gamma radiation on photosynthesis by Thatcher wheat and Chlorella. A-A Photosynthetic oxygen evolution by Chlorella. O----O Cl402 fixation by Chlorella. O-0 Cl402 fixation by Thatcher wheat.

RADIATION

EFFECTS

ON

TABLE Percentage

Distribution Thatcher

II

of Cl4 in Productsa from CldOz Fixation Wheat after Gamma Irradiation Dose in kr.

Compound

0

Phosphate area Sucrose Glucose Glycine and serine Fructose Alanine Malic acid D Percentage

of total

201

PHOTOSYNTHESIS

34 30 Trace 24 2 1 9 Cl4 activity

25

loo

250

35 23 1 32 1 0 7

38 23 1 30 2 1 5

26 23 Trace 38 1 2 4

by

ml

27 22 Trace 42 1 2 4

on the chromatogram.

Distribution of Cl4 in Photosynthesis Products after Gamma Irradiation. Table II shows the distribution of Cl4 among the products formed by Thatcher wheat after various doses of gamma radiation corresponding to each point in Fig. 2. Shifts in the products of photosynthesis are too small to account for the 80 % over-all decrease in COz fixation. Some increase in the amino acids, glycine and serine, indicates that the photosynthesis cycle is operating normally since these acids are probably formed by a side reaction prior to the COZ fixation step (17). Recovery of Photosynthetic Activity after Irradiation. The reversibility of the damage to photosynthesis by ionizing radiation is shown in Table III. Table IV presents the distribution of the Cr402 fixed among the soluble products formed in lo-min. periods at different times after irradiation. Data in Table III indicate that, after an initial strong inhibition of COz fixation, an essentially normal capacity for COz fixation is regained in 5 hr. and continues for at least 24 hr. The significance of the

Recovery

of Capacity Tie

to Fis 0*02

TABLE III by Thatcher

after gamma irradiation hr.

0 1 5 24

Wheat after Gamma Irradiation5

Per cent fixation*

35 62 103 94

D All leaves irradiated with 100 kr. gamma radiation. continuous light after gamma irradiation. * Cl402 fixed in 10 min. compared with nonirradiated

The leaves were kept in controls.

202

ZILL

Percentage

AND

TOLBERT

TABLE

IV

Distribution of Cl4 in Products from C140t Fixation by Thatcher Wheat after Exposure to 100 kr. of Gamma Radiation Uni;;~dili~d Compound

Phosphate area Sucrose Glucose Glycine and serine Fructose Alanine Malic acid

22 26 1 32 1 1 11

Time after gamma radiation,

hr.

0

1

5

24

21 35 2 27 3 1 6

18 31 3 32 3 1 7

17 48 1 21 4 2 5

19 26 4 34 4 2 7

increase in sucrose and decrease in glycine and serine at 5 hr. after irradiation is unknown. Normal distribution of Cl4 in the products is attained in 24 hr. Target Consideration. If one assumes that the initial decline in ability to fix CY402after gamma radiation is exponential for both Chlorella and Thatcher wheat, then a target volume may be estimated (18). Since the Cl402 fixation curves are made up of two or more components, the 0.37 activity levels can only be approximated, by extrapolation, to be at about 25 kr. If it is assumed that for each roentgen two ion pairs/cu. p are produced in wet tissue, the target volume is approximately 2 X lo+ cu. I(. This value may be correct to within a factor of two. At least the target volume is considerably smaller than the whole chloroplast in the cell. In addition, there is a substantial COz fixation that from target analysis would have to be considered as occurring in much smaller units. It should be noted that the target for oxygen evolution is either multihit or multiunit and is thus different from that for CO2 fixation. ACKNOWLEDGMENTS

We wish to express our sincere appreciation to Miss Dora A. Mondon for her competent assistance throughout this investigation. Thanks are also extended to Dr. R. Rabson for supplying the Chlorella cultures. SUMMARY

1. Fixation of CO2 by ChZoreEZawas destroyed by 5 min. ultraviolet irradiation (35 ergs/sq. mm./sec.). Under the same irradiation, wheat retained about 25% of normal COa fixation because of protection by absorption of radiation in the leaf. Sucrose synthesis seems particularly sensitive to ultraviolet light.

RADIATION EFFECTS ON PHOTOSYNTHESIS

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2. Continued normal oxygen evolution despite decreasedCO2 fixation after gamma irradiation suggests independent action of gamma radiation on the carbon cycle and the photolytic system. 3. The inhibition of carbon dioxide fixation was greatest immediately after gamma irradiation. Normal CO2 fixation was found after a 5-hr. postirradiation exposure to light. REFERENCES 1. GAILEY, F. B., AND TOLBERT, N. E., Arch. Biochem. Biophys. 76, 188 (1958). 2. ARNOLD, W. A., J. Gen. Physiol. 17, 135 (1933). 3. FRENKEL, A., Biol. Bull. 97, 222 (1949). 4. HOLT, A. S., BROOKS, I. A., AND ARNOLD, W. A., J. Gen. Physiol. 34,627 (1950). 5. REDFORD, E. L., AND MYERS, J., J. Cellular Comp. Physiol. 36, 217 (1951). 6. TOLBERT, N. E., AND ZILL, L. P., J. Biol. Chem. 322, 895 (1956). 7. GHORMLEY, J. A., AND HOCHANADEL, C. J., Rev. Sci. Znstr. 32,473 (1951). 8. BENSON, A. A., BASSHAM, J. A., GOODALE, T. C., HAAS, V. A., AND STEPKA, W. A., J. Am. Chem. Sot. 72, 1710 (1950). 9. CARDINI, C. E., LELOIR, L. F., AND CHIRIBOGA, J., J. Biol. Chem. 214, 149 (1955). 10. 11. 12. 13. 14. 15. 16. 17.

LELOIR, L. F., AND CARDINI, C. E., J. Biol. Chem. 214, 157 (1955). SINSHEIMER, R. L., Radiation Research 1, 505 (1954). ZILL, L. P., Federation Proc. 16, 276 (1957). CARTER, C. E., J. Am. Chem. Sot. ‘72, 1835 (1950). ALVIM, P. de T., Am. J. Botany 36, 781 (1949). BAWDEN, F. C., AND KLECZKOWSKI, A., Nature 169, 90 (1952). TANADA, T., AND HENDRICKS, S. B., Am. J. Botany 40, 634 (1953). WEISSBACH, A., AND HORECICER, B. L., in “Amino Acid Metabolism”

(W. B. McElroy and B. Glass, eds.), p. 741. The Johns Hopkins Press, Baltimore, 1955. 18. LEA, D. E., “Action of Radiations on Living Cells,” 2nd ed. Cambridge University Press, London, 1955.