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Effect of ionizing radiation on the development of photosynthesis in etiolated wheat leaves
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76, 188-195 (1958)
Effect of Ionizing Radiation on the Development of Photosynthesis in Etiolated Wheat Leaves F. B. Gailey’ and N. E. Tolbert From the Biology
Division,
Oak Ridge National
Laboratory,2 Oak Ridge, Tennessee
Received November 7, 1957 INTRODUCTION
It has been shown that in a green plant photosynthesis is relatively insensitive to ionizing radiation (1). The effect of radiation on the development of photosynthesis when etiolated plants are placed in the light has now been investigated. Controlled experiments on the normal course of development have already been reported (2). PROCEDURE Thatcher wheat seedlings were grown for about 7% days in a sand-soil mixture (2:l) in a black room at 23 f 1°C. Seedlings were grown in Pyrex glass Gooch crucibles (high form, 30-ml. capacity with 30-mm. diameter fritted disk), since these containers would fit into the radiation chamber. The crucibles with the seedlings were wrapped in aluminum foil before moving them from the dark growth room to the radiation chamber. The gamma radiation was obtained from the same cobalt-60 source as used before (l), but it was now delivering 1365 r./min. To produce greening, the etiolated seedlings were illuminated with about loo0 ft.-candles white light from a 150-w. reflector spot bulb. The light was filtered through 238 cm. of flowing water to keep the temperature at about 25°C. Seedlings were taken at intervals for chlorophyll analysis or Cr40z fixation experiments. Since etiolated leaves green acropetally, the analysis was standardized by using only a 3-cm. length of the leaf taken 1 cm. from the tip. For each analysis 12-16 such segments were weighed and ground with sand in about 5 ml. of 86% acetone. The extract was filtered into a graduated cylinder, and the residue was washed repeatedly with acetone until all color was removed. Chlorophyll was determined in the Beckman spectrophotometer at 663 and 645 rnp and calculated as described bv Arnon (3). 1 Work done while on leave of absence from Berea College, Berea, Kentucky, and on an Oak Ridge Institute of Nuclear Studies Research Participantship at Oak Ridge National Laboratory. * Operated by Union Carbide Nuclear Company for the U. S. Atomic Energy Commission. 188
RADIATION ON DEVELOPMENT OF PHOTOSYNTHESIS
189
Seedlings examined for their ability to fix PO2 were cut in the same way, and four 3-cm. segments were exposed in a small chamber for 10 min. to 2000 ft.-candles of white light in an atmosphere of 25 pc. of Cl402 (25% Cl4) in 64 ml. air at about 25°C. After 10 min., the leaf sections were plunged into boiling 40% methanol for several minutes, ground in a mortar, and further extracted in boiling water. After centrifugation, an aliquot was counted to calculate total 040~ fixation in the alcohol and water-soluble fractions. Other portions of the extract were analyzed by paper chromatography as in the preceding paper (1). RESULTS
Inhibition
of Greening by Gamma Radiation
Formation of chlorophyll by etiolated Thatcher wheat seedlings after exposure to various levels of gamma radiation is shown in Fig. 1. Wide variations
were found
in the chlorophyll
content
of irradiated
seedlings
and occasionally also of control plants. Since almost 2 hr. was required to give a dose of 150 kr., and only one sample was exposed at a time, large series of parallel tests could not be made. In the control plants, exposure to light caused immediate conversion of protochlorophyll to chlorophyll, followed 2 hr. later by the start of sustained chlorophyll formation. Plants receiving 12.5 kr. responded similarly. After 50 kr. of gamma radiation, the time lag before the beginning of sustained greening was increased to about 4 hr.; after this
loo bw 0
4
S
1’2 16 20 24 30 DURATION OF EXPOSURE ltl LIGHT(hr)
40
4s
FIG. 1. Chlorophyll synthesis by etiolated Thatcher wheat seedlings after exposure to gamma radiation. All exposures to gamma radiation were made immediately before the first exposure to white light at zero time.
190
GAILEY
AND
TOLBERT
time, the rate of chlorophyll formation was nearly normal. By the end of 2 days, the plants that had received 50 kr. were as green as the nonirradiated controls. In etiolated plants which had received 150 kr., the start of sustained chlorophyll formation was delayed for about 10 hr. After this period, the rate of greening was nearly that of the control plants, but the total chlorophyll content never reached the level of that in the controls. In one experiment, etiolated seedlings were exposed to 1220 kr. These plants never greened and died after a few days. The conversion of protochlorophyll, preformed in the dark, to chlorophyll was not prevented by gamma radiation. For example, etiolated plants which had received 800 kr. in the dark contained immediately after exposure to light 21 pg. chlorophyll and 14 pg. protochlorophyll/g. fresh weight. E$ect of Light on Reversal of Inhibition
Produced by Gamma Radiation
After large doses of radiation, etiolated leaves can recover their capacity to turn green and to develop normal photosynthesis. To determine whether light was needed for this recovery, experiments were performed in which gamma radiation of 150 kr. was followed by a dark period of f3-24 hr. When these plants were finally placed in light, about 10 hr. had to pass before rapid greening began. During the dark period there had been no recovery from the effect of the gamma radiation.
HO”R AFTER y IRRADIATION
Chlorophyll development in Thatcher wheat plants after 0 radiation (closed symbols) or 150 kr. (open symbols) of gamma radiation followed by 1 hr. of light and then darkness for 5 hr. (O), 9 hr. (Cl), or 16 hr. (A). FIQ.
2.
RADIATION
ON
DEVELOPMENT
OF
191
PHOTOSYXTHESIS
In another series of experiments, immediately after 150 kr. of gamma radiation, the etiolated plants were exposed to light for 1 hr. and then returned to the dark for 5, 9, or 16 hr. before exposing them to continuous light (Fig. 2). Greening of control plants was prevented by the dark period after the first hour of light, but when they were again placed in light, sust.ained chlorophyll formation began immediately. Similar results have been reported by Smith (4). With gamma-irradiat.ed plants, a lo-hr. induction period was always required before sustained greening began; but after the inititul hour of light, the period was not light dependent. If the irradiated plants had 1 hr. of light and 9 or 16 hr. of darkness, they began synthesizing chlorophyll as soon as they were placed in light. One hour of illumination was sufficient; a much shorter time may have sufficed. If, after radiation, the plants had 1 hr. of light and only 5 hr. of darkness, t.hey did not begin to green for 4 more hours in light, or a total of 10 hr. Effect of Gamma hdiativn
Applied during GTeming
To test the radiation sensitivity of greening after it has started, plants were irradiated after exposure to a few hours of light. Chlorophyll contents of etiolated plants exposed to 50 or 150 kr. after having first received 2, 4, or 6 hr. of light are shown in Table I. During the initial period of light exposure (col. 2), these plants began to form chlorophyll at the normal rate (col. 4). This greening was interrupted by a 50- or 1.Wkr.
dose of gamma
radiation
(~1. I), and a few hours later (col. :3) the TABLE
I
Chlorophyll Concentralion in Etioluted Plants after Gumma Rudialion at Various Periods during Greening
hr. 50 50 xl
150 150 150 150
hr.
hr.
2 3 6 2 2 4 6
6 4 2 8 14 6 4
rg./g.ljrcrh
110 220 390
110 110 220 390
207 208 291 68 348 93 352
weigh1
97 -12 -99 -42 238 -117 -38
a Includes a dark period of 37 min. after 50.kr. exposure or 110 min. for 150 kr. and then the remainder of the time spent in the light. * Calculated from controls.
192
GAILEY
AND
TOLBERT
plants were analyzed for chlorophyll (col. 5). Plants receiving 50 kr. had to wait longer than 4 hr. for chlorophyll formation to resume; plants receiving 150 kr. produced new chlorophyll 14 hr. later but not 8 hr. later (col. 6). Thus when a greening plant was exposed to gamma radiation, chlorophyll formation was arrested for approximately the same period as that observed in etiolated plants not given any prior light treatment. The loss of chlorophyll observed in the light after radiation was probably the result of photodecomposition (5). However, the irradiated plants did begin to green again after a certain number of hours in the light. The production of substantial amounts of chlorophyll before radiation provided no protection against the temporary inhibition of greening by gamma radiation. C1402Fixation during Greening after Gamma Radiation In previous investigations on nonirradiated plants, the rates of CY402 fixation and the nature of Cl4 products of photosynthesis were determined at hourly intervals during the greening process (2). The following sequence of events seemed to occur when etiolated plants were placed in the light: (a) instantaneous conversion of protochlorophyll to chlorophyll; (5) after 2 hr., beginning of sustained chlorophyll synthesis; (c) after 4-5 hr., beginning of photosynthetic CO2 fixation; and (d) after 9-12 hr., an approach of the photosynthetic carbon cycle to the steadystate conditions. Similar analyses were performed on seedling that had received 50 or 150 kr. (Table II). Total Cl402 fixed was calculated on the basis of fresh weight of the leaf material and of the amount of chlorophyll as determined in a duplicate set of seedlings. Plants irradiated with 50 kr. showed, after 4 hr. in the light, neither sustained chlorophyll formation nor CO2 fixation; after 6 hr., COZ fixation was the same as in the control plants. Plants irradiated with 150 kr. did not begin to fix much C1402 until after 10 hr. of light, the same time when rapid new chlorophyll synthesis began. After 12 hr. of light, Cl402 fixation by these plants approached that in nonirradiated plants, after the same light exposure. Relative to the chlorophyll concentration in the irradiated plants, COZ fixation was very fast. The alcohol- and water-soluble C14-labeled products formed by nonirradiated plants in the IO-min. Cl402 fixation periods during the greening process after 4-5 hr. in light were predominately Cs compounds, such as phosphoglyceric acid and alanine (2). Not until the plants were exposed to light for 4-12 hr. did the distribution of Cl4 approach that
RADIATION
ON
DEVELOPMENT
OF
TABLE C”Oz Fixation
in Light
Illumination hr.
2 4 6 8 9 10 12 14 15 16 20 23 24
II
by Gamma-Irradiated
Control cls.lrec./mg. of leaf
2 52 138 342 242 178 273 321 262 237 315 397 372
193
PHOTOSYNTHESIS
Etiolated
Wheat Seedlings
50 kr.
cts. /sec. / PK. of chloro#hyll
22 234 380 662 504 236 407 333 245 240 285 392 285
cls./scc./mg. of leaf
150
cIs.lsec./
cls./scc.fmg. of leaf
83 600
296
215 332
371 462
cfs./sec./ ChiZ&l
‘hlE&ll
9 158
kr.
8
113
20 242
331 1000
208 218 324
406
328
in steady-state photosynthesis. Although the total Cl402 fixation by gamma-irradiated plants was very small until sustained greening began, the Cl4 fixed prior to rapid greening was distributed among the products in the same pattern as found in nonirradiated controls at the same time. This was true, for instance, after 6 and 10 hr. of light in leaves which had had 150 kr. While radiation prolonged greening, it did not prevent conversion of the protochlorophyll, present before radiation, to chlorophyll and the subsequent adaptation to photosynthetic CO2 fixation by this small amount of chlorophyll. DISCUSSION
Photosynthesis in a green plant has been previously shown to be relatively insensitive to gamma radiation, since it is only partially and temporarily inhibited by 100-500 kr. (1). Likewise, the development of photosynthesis in an etiolated plant has been found to be relatively radiation insensitive. A gamma-irradiation dose of 150 kr. delayed the greening of the etiolated plants for about 10 hr. after the beginning of light exposure, but afterward these plants formed chlorophyll and developed normal photosynthetic CO2 fixation. The nature of the delay in sustained greening is unknown. The data indicate the radiation did not prevent development of the CO2 fixation portion of photosynthesis or the conversion of protochlorophyll to chlorophyll. An inhibition of pro-
194
GAILEY AND TOLBERT
tein synthesis would be expected to cause a delay in synthesis of the chloroplast particles. The dosages of radiation used prevented further growth of the plants insofar as no new leaves were formed. The leaves present at the time of radiation increased in size, but it is likely that this change came from cell elongation. The irradiated plants survived for several weeks longer; their aging and death will be reported subsequently. Etiolated leaves contain large leucoplasts, which correspond in size to chloroplasts and develop into chloroplasts in the light. The bulk of the greening occurs through slow growth of particles originally the size of microsomes (6, 7). Our original hypothesis to explain a normal delay in development of the CO* fixation portion of photosynthesis after chlorophyll formation had begun was that these events might be an adaptive phenomenon. In gamma-irradiated plants, a slightly different sequence of events seems to have occurred. First there was the unimpaired conversion of very small amounts of protochlorophyll to chlorophyll, and afterward an adaptation to a trace of photosynthetic Cl402 fixation in about the same period of light needed to develop sustained CO2 fixation in the unirradiated plants. After the delay in sustained greening imposed by the amout of gamma radiation, a final rapid and sustained chlorophyll formation began, which was accompanied simultaneously by increased photosynthetic CO2 fixation. It is possible that the development of the photosynthetic process in the leucoplasts is not affected by gamma radiation, and would account for the normal appearance of a very small amount of photosynthetically active material. The main delay in greening observed after gamma radiation might be an inhibition of chloroplast formation from the smaller particles. SUMMARY
Doses of gamma radiation as high as 150 kr. delayed the greening of etiolated wheat plants in the light by as much as 10 hr. The irradiated plants developed a complete photosynthetic mechanism, as determined by Cl402 incorporation into normal products. The inhibition of greening caused by gamma radiation could be overcome by exposure to white light, but was not changed after 24 hr. in the dark. Conversion of protochlorophyll to chlorophyll was not affected by gamma radiation. The normal lag between the beginning of sustained chlorophyll synthesis and the development of photosynthetic COZ fixation did not occur after gamma radiation.
RADIATION
ON DEVELOPMENT
OF PHOTOSYNTHESIS
195
REFERENCES 1. ZILL, L. P., AND TOLBERT, N. E., Arch. Biochem. Biophys. 76, 196 (1958). 2. TOLBERT, N. E., AND GAILEY, F. B., Plant Physiol. 30, 491 (1956). 3. ARNON, D. I., Plant Physiol. 24, 1 (1949). 4. SMITH, J. H. C., Plant Physiol. 29, 143 (1954). Vol. I. Interscience Publ., Inc., New 5. RABINOWITCH, E. I., “Photosynthesis,” York, 1945. 6. SMILLIE, R. M., Australian J. Biol. Sci. 9, 339 (1956). 7. DE DEKEN-GRENSON, M., Biochim. et Biophys. Acta 14, 203 (1954).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76, 188-195 (1958)
Effect of Ionizing Radiation on the Development of Photosynthesis in Etiolated Wheat Leaves F. B. Gailey’ and N. E. Tolbert From the Biology
Division,
Oak Ridge National
Laboratory,2 Oak Ridge, Tennessee
Received November 7, 1957 INTRODUCTION
It has been shown that in a green plant photosynthesis is relatively insensitive to ionizing radiation (1). The effect of radiation on the development of photosynthesis when etiolated plants are placed in the light has now been investigated. Controlled experiments on the normal course of development have already been reported (2). PROCEDURE Thatcher wheat seedlings were grown for about 7% days in a sand-soil mixture (2:l) in a black room at 23 f 1°C. Seedlings were grown in Pyrex glass Gooch crucibles (high form, 30-ml. capacity with 30-mm. diameter fritted disk), since these containers would fit into the radiation chamber. The crucibles with the seedlings were wrapped in aluminum foil before moving them from the dark growth room to the radiation chamber. The gamma radiation was obtained from the same cobalt-60 source as used before (l), but it was now delivering 1365 r./min. To produce greening, the etiolated seedlings were illuminated with about loo0 ft.-candles white light from a 150-w. reflector spot bulb. The light was filtered through 238 cm. of flowing water to keep the temperature at about 25°C. Seedlings were taken at intervals for chlorophyll analysis or Cr40z fixation experiments. Since etiolated leaves green acropetally, the analysis was standardized by using only a 3-cm. length of the leaf taken 1 cm. from the tip. For each analysis 12-16 such segments were weighed and ground with sand in about 5 ml. of 86% acetone. The extract was filtered into a graduated cylinder, and the residue was washed repeatedly with acetone until all color was removed. Chlorophyll was determined in the Beckman spectrophotometer at 663 and 645 rnp and calculated as described bv Arnon (3). 1 Work done while on leave of absence from Berea College, Berea, Kentucky, and on an Oak Ridge Institute of Nuclear Studies Research Participantship at Oak Ridge National Laboratory. * Operated by Union Carbide Nuclear Company for the U. S. Atomic Energy Commission. 188
RADIATION ON DEVELOPMENT OF PHOTOSYNTHESIS
189
Seedlings examined for their ability to fix PO2 were cut in the same way, and four 3-cm. segments were exposed in a small chamber for 10 min. to 2000 ft.-candles of white light in an atmosphere of 25 pc. of Cl402 (25% Cl4) in 64 ml. air at about 25°C. After 10 min., the leaf sections were plunged into boiling 40% methanol for several minutes, ground in a mortar, and further extracted in boiling water. After centrifugation, an aliquot was counted to calculate total 040~ fixation in the alcohol and water-soluble fractions. Other portions of the extract were analyzed by paper chromatography as in the preceding paper (1). RESULTS
Inhibition
of Greening by Gamma Radiation
Formation of chlorophyll by etiolated Thatcher wheat seedlings after exposure to various levels of gamma radiation is shown in Fig. 1. Wide variations
were found
in the chlorophyll
content
of irradiated
seedlings
and occasionally also of control plants. Since almost 2 hr. was required to give a dose of 150 kr., and only one sample was exposed at a time, large series of parallel tests could not be made. In the control plants, exposure to light caused immediate conversion of protochlorophyll to chlorophyll, followed 2 hr. later by the start of sustained chlorophyll formation. Plants receiving 12.5 kr. responded similarly. After 50 kr. of gamma radiation, the time lag before the beginning of sustained greening was increased to about 4 hr.; after this
loo bw 0
4
S
1’2 16 20 24 30 DURATION OF EXPOSURE ltl LIGHT(hr)
40
4s
FIG. 1. Chlorophyll synthesis by etiolated Thatcher wheat seedlings after exposure to gamma radiation. All exposures to gamma radiation were made immediately before the first exposure to white light at zero time.
190
GAILEY
AND
TOLBERT
time, the rate of chlorophyll formation was nearly normal. By the end of 2 days, the plants that had received 50 kr. were as green as the nonirradiated controls. In etiolated plants which had received 150 kr., the start of sustained chlorophyll formation was delayed for about 10 hr. After this period, the rate of greening was nearly that of the control plants, but the total chlorophyll content never reached the level of that in the controls. In one experiment, etiolated seedlings were exposed to 1220 kr. These plants never greened and died after a few days. The conversion of protochlorophyll, preformed in the dark, to chlorophyll was not prevented by gamma radiation. For example, etiolated plants which had received 800 kr. in the dark contained immediately after exposure to light 21 pg. chlorophyll and 14 pg. protochlorophyll/g. fresh weight. E$ect of Light on Reversal of Inhibition
Produced by Gamma Radiation
After large doses of radiation, etiolated leaves can recover their capacity to turn green and to develop normal photosynthesis. To determine whether light was needed for this recovery, experiments were performed in which gamma radiation of 150 kr. was followed by a dark period of f3-24 hr. When these plants were finally placed in light, about 10 hr. had to pass before rapid greening began. During the dark period there had been no recovery from the effect of the gamma radiation.
HO”R AFTER y IRRADIATION
Chlorophyll development in Thatcher wheat plants after 0 radiation (closed symbols) or 150 kr. (open symbols) of gamma radiation followed by 1 hr. of light and then darkness for 5 hr. (O), 9 hr. (Cl), or 16 hr. (A). FIQ.
2.
RADIATION
ON
DEVELOPMENT
OF
191
PHOTOSYXTHESIS
In another series of experiments, immediately after 150 kr. of gamma radiation, the etiolated plants were exposed to light for 1 hr. and then returned to the dark for 5, 9, or 16 hr. before exposing them to continuous light (Fig. 2). Greening of control plants was prevented by the dark period after the first hour of light, but when they were again placed in light, sust.ained chlorophyll formation began immediately. Similar results have been reported by Smith (4). With gamma-irradiat.ed plants, a lo-hr. induction period was always required before sustained greening began; but after the inititul hour of light, the period was not light dependent. If the irradiated plants had 1 hr. of light and 9 or 16 hr. of darkness, they began synthesizing chlorophyll as soon as they were placed in light. One hour of illumination was sufficient; a much shorter time may have sufficed. If, after radiation, the plants had 1 hr. of light and only 5 hr. of darkness, t.hey did not begin to green for 4 more hours in light, or a total of 10 hr. Effect of Gamma hdiativn
Applied during GTeming
To test the radiation sensitivity of greening after it has started, plants were irradiated after exposure to a few hours of light. Chlorophyll contents of etiolated plants exposed to 50 or 150 kr. after having first received 2, 4, or 6 hr. of light are shown in Table I. During the initial period of light exposure (col. 2), these plants began to form chlorophyll at the normal rate (col. 4). This greening was interrupted by a 50- or 1.Wkr.
dose of gamma
radiation
(~1. I), and a few hours later (col. :3) the TABLE
I
Chlorophyll Concentralion in Etioluted Plants after Gumma Rudialion at Various Periods during Greening
hr. 50 50 xl
150 150 150 150
hr.
hr.
2 3 6 2 2 4 6
6 4 2 8 14 6 4
rg./g.ljrcrh
110 220 390
110 110 220 390
207 208 291 68 348 93 352
weigh1
97 -12 -99 -42 238 -117 -38
a Includes a dark period of 37 min. after 50.kr. exposure or 110 min. for 150 kr. and then the remainder of the time spent in the light. * Calculated from controls.
192
GAILEY
AND
TOLBERT
plants were analyzed for chlorophyll (col. 5). Plants receiving 50 kr. had to wait longer than 4 hr. for chlorophyll formation to resume; plants receiving 150 kr. produced new chlorophyll 14 hr. later but not 8 hr. later (col. 6). Thus when a greening plant was exposed to gamma radiation, chlorophyll formation was arrested for approximately the same period as that observed in etiolated plants not given any prior light treatment. The loss of chlorophyll observed in the light after radiation was probably the result of photodecomposition (5). However, the irradiated plants did begin to green again after a certain number of hours in the light. The production of substantial amounts of chlorophyll before radiation provided no protection against the temporary inhibition of greening by gamma radiation. C1402Fixation during Greening after Gamma Radiation In previous investigations on nonirradiated plants, the rates of CY402 fixation and the nature of Cl4 products of photosynthesis were determined at hourly intervals during the greening process (2). The following sequence of events seemed to occur when etiolated plants were placed in the light: (a) instantaneous conversion of protochlorophyll to chlorophyll; (5) after 2 hr., beginning of sustained chlorophyll synthesis; (c) after 4-5 hr., beginning of photosynthetic CO2 fixation; and (d) after 9-12 hr., an approach of the photosynthetic carbon cycle to the steadystate conditions. Similar analyses were performed on seedling that had received 50 or 150 kr. (Table II). Total Cl402 fixed was calculated on the basis of fresh weight of the leaf material and of the amount of chlorophyll as determined in a duplicate set of seedlings. Plants irradiated with 50 kr. showed, after 4 hr. in the light, neither sustained chlorophyll formation nor CO2 fixation; after 6 hr., COZ fixation was the same as in the control plants. Plants irradiated with 150 kr. did not begin to fix much C1402 until after 10 hr. of light, the same time when rapid new chlorophyll synthesis began. After 12 hr. of light, Cl402 fixation by these plants approached that in nonirradiated plants, after the same light exposure. Relative to the chlorophyll concentration in the irradiated plants, COZ fixation was very fast. The alcohol- and water-soluble C14-labeled products formed by nonirradiated plants in the IO-min. Cl402 fixation periods during the greening process after 4-5 hr. in light were predominately Cs compounds, such as phosphoglyceric acid and alanine (2). Not until the plants were exposed to light for 4-12 hr. did the distribution of Cl4 approach that
RADIATION
ON
DEVELOPMENT
OF
TABLE C”Oz Fixation
in Light
Illumination hr.
2 4 6 8 9 10 12 14 15 16 20 23 24
II
by Gamma-Irradiated
Control cls.lrec./mg. of leaf
2 52 138 342 242 178 273 321 262 237 315 397 372
193
PHOTOSYNTHESIS
Etiolated
Wheat Seedlings
50 kr.
cts. /sec. / PK. of chloro#hyll
22 234 380 662 504 236 407 333 245 240 285 392 285
cls./scc./mg. of leaf
150
cIs.lsec./
cls./scc.fmg. of leaf
83 600
296
215 332
371 462
cfs./sec./ ChiZ&l
‘hlE&ll
9 158
kr.
8
113
20 242
331 1000
208 218 324
406
328
in steady-state photosynthesis. Although the total Cl402 fixation by gamma-irradiated plants was very small until sustained greening began, the Cl4 fixed prior to rapid greening was distributed among the products in the same pattern as found in nonirradiated controls at the same time. This was true, for instance, after 6 and 10 hr. of light in leaves which had had 150 kr. While radiation prolonged greening, it did not prevent conversion of the protochlorophyll, present before radiation, to chlorophyll and the subsequent adaptation to photosynthetic CO2 fixation by this small amount of chlorophyll. DISCUSSION
Photosynthesis in a green plant has been previously shown to be relatively insensitive to gamma radiation, since it is only partially and temporarily inhibited by 100-500 kr. (1). Likewise, the development of photosynthesis in an etiolated plant has been found to be relatively radiation insensitive. A gamma-irradiation dose of 150 kr. delayed the greening of the etiolated plants for about 10 hr. after the beginning of light exposure, but afterward these plants formed chlorophyll and developed normal photosynthetic CO2 fixation. The nature of the delay in sustained greening is unknown. The data indicate the radiation did not prevent development of the CO2 fixation portion of photosynthesis or the conversion of protochlorophyll to chlorophyll. An inhibition of pro-
194
GAILEY AND TOLBERT
tein synthesis would be expected to cause a delay in synthesis of the chloroplast particles. The dosages of radiation used prevented further growth of the plants insofar as no new leaves were formed. The leaves present at the time of radiation increased in size, but it is likely that this change came from cell elongation. The irradiated plants survived for several weeks longer; their aging and death will be reported subsequently. Etiolated leaves contain large leucoplasts, which correspond in size to chloroplasts and develop into chloroplasts in the light. The bulk of the greening occurs through slow growth of particles originally the size of microsomes (6, 7). Our original hypothesis to explain a normal delay in development of the CO* fixation portion of photosynthesis after chlorophyll formation had begun was that these events might be an adaptive phenomenon. In gamma-irradiated plants, a slightly different sequence of events seems to have occurred. First there was the unimpaired conversion of very small amounts of protochlorophyll to chlorophyll, and afterward an adaptation to a trace of photosynthetic Cl402 fixation in about the same period of light needed to develop sustained CO2 fixation in the unirradiated plants. After the delay in sustained greening imposed by the amout of gamma radiation, a final rapid and sustained chlorophyll formation began, which was accompanied simultaneously by increased photosynthetic CO2 fixation. It is possible that the development of the photosynthetic process in the leucoplasts is not affected by gamma radiation, and would account for the normal appearance of a very small amount of photosynthetically active material. The main delay in greening observed after gamma radiation might be an inhibition of chloroplast formation from the smaller particles. SUMMARY
Doses of gamma radiation as high as 150 kr. delayed the greening of etiolated wheat plants in the light by as much as 10 hr. The irradiated plants developed a complete photosynthetic mechanism, as determined by Cl402 incorporation into normal products. The inhibition of greening caused by gamma radiation could be overcome by exposure to white light, but was not changed after 24 hr. in the dark. Conversion of protochlorophyll to chlorophyll was not affected by gamma radiation. The normal lag between the beginning of sustained chlorophyll synthesis and the development of photosynthetic COZ fixation did not occur after gamma radiation.
RADIATION
ON DEVELOPMENT
OF PHOTOSYNTHESIS
195
REFERENCES 1. ZILL, L. P., AND TOLBERT, N. E., Arch. Biochem. Biophys. 76, 196 (1958). 2. TOLBERT, N. E., AND GAILEY, F. B., Plant Physiol. 30, 491 (1956). 3. ARNON, D. I., Plant Physiol. 24, 1 (1949). 4. SMITH, J. H. C., Plant Physiol. 29, 143 (1954). Vol. I. Interscience Publ., Inc., New 5. RABINOWITCH, E. I., “Photosynthesis,” York, 1945. 6. SMILLIE, R. M., Australian J. Biol. Sci. 9, 339 (1956). 7. DE DEKEN-GRENSON, M., Biochim. et Biophys. Acta 14, 203 (1954).