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The absorption of radioactive 2,4-dichlorophenoxyacetic acid and the translocation of C14 by bean plants
The Absorption of Radioactive 2,4-Dichlorophenoxyacetic Acid and the Translocation of Cl4 by Bean Plants l* 2 S. C. Fang, E. G. Jaworski, 3 A. V. Logan, V. H. Freed and Joseph S. Butts From the Department
of Agricultural Corvallis, Received
Chemistry, Oregon
January
Oregon State College,
18, 1951
INTRODUCTION
The translocation of plant growth regulators has been studied by the use of 1131as a label (2,3,6,7) in 2-iodo131-3-nitrobenzoic acid and 2,4dichloro-5-iodo131 phenoxyacetic acid. The 2,4-D-51 was found somewhat less toxic when applied to bean plants than was 2,4-dichlorophenoxyacetic acid (2,4-D). The benzoic acid type compound was found to be different -from 2,4-D in that it appeared to react with some constituents in the relatively immature tissues of’the terminal bud somewhat more readily than with those in the more mature parts of the stem (3). The synthesis and mode of action in bean plants of 2,4-D labeled with Cl4 in the carboxyl group has been reported by Holley et al. (1). They concluded that when applied to leaves the main movement of the 2,4-D is down the stems. Some radioactive carbon is lost through CO2 and some is found in the nutrient solution. About one-third of the isolated radioactivity was 2,4-D. More than one-half was bound in such a way that it could not be extracted with ether from an aqueous solution. 1 This research was supported by a grant from the Atomic Energy Commission to the Department of Agricultural Chemistry, Oregon Agricultural Experiment Station, Oregon State College. * Published as Technical Paper No. 656 with the approval of the Director of the Oregon Agricultural Experiment Station. Contribution of the Department of Agricultural Chemistry. 8 Some of the data reported in this paper were taken from a thesis presented by E. G. Jaw&ski to the Faculty of the Graduate School of Oregon State College in partial fulfillment of the Master of Science degree. 249
250
FANG, SAWORSKI, LOGAN, FREED AND BUTTS
It seemed worthwhile to us to synthesize 2,4-D labeled in the methylene group as well as to study the behavior of this compound in bean plants. It was also desired to synthesize a sample of 2,4-D with a high specific activity. The present investigation, employing methylene-labeled 2,4-D, was undertaken to determine the amount of translocation and accumulation of Cl4 vs. (a) time of treatment, (b) age of plant, and (c) the amount of 2,4-D applied. EXPERIMENTAL
METHODS
Xynthesis of a-Methylene-Labeled Cl
0
2,4-D
-O-C’4H~-COOH Cl
Radioactive 2,4-D, labeled in the a-methylene position was synthesized as follows: (Y-labeled methyl iodide was reacted with magnesium in ether to form the Grignard reagent, This was carbonated with CO2 to yield acetic acid. The preceding reactions were carried out in a high vacuum system at 0.1 P pressure. The technique of Tolbert was followed (4). The acetic acid was brominated using liquid bromine and red phosphorus to yield a-bromoacetic acid. The product was then coupled with 2,4dichlorophenol in the presence of sodium hydroxide. The procedure was essentially identical with that of Holley (1). The resulting product was isolated and purified according to Holley (I). Its melting point was found to be 138.9-1338°C. and its mixed melting point 13&139°C. The neutral equivalent was 221. With our counting procedure this gave a product which, when burned and collected as BaC03 and corrected to zero thickness gave approximately 4.2 X lo6 counts/min./mg. of sample. Bean plants (Phaseohs vulgaris, var. Black Valentine) were grown in potted soil under greenhouse conditions throughout the experiment. The plants were treated when the primary leaves were almost fully expanded but the terminal bud was still quite small. 2,4-D in alcohol solution was applied to one primary leaf of each plant along the midrib. All plants after harvesting were dried in vacuum at 59°C. for 24 hr. and then sectioned as indicated. MEASUREMENT
OF RADIOACTIVITY
The sections were individually oxidized using the Van Slyke-Folch combustion solution (5). The carbon dioxide was precipitated as barium carbonate. The barium carbonate was mounted in a thin layer on copper disks which have a diameter of 1 in. The radioactivity was measured by means of a “Tracerlab Autoscaler” with a thin mica window tube (1.5 mg./cm.*). The scale selector was set at 4096 counts which gives 1% error for high activity samples, 5% error for the medium ones and 10% error for the very low ones. The specific activity of all BaCOa samples was corrected to zero thickness. Duplicate determinations were made from each BaC03 sample. In every case an accuracy of 5% or better was realized.
TRANSLOCATION
OF C’” BY BEAN
METHODS
251
PLANTS
OF STUDY
In the first series of study, the bean plants were divided into five groups of six plants each. All groups were treated at the same time with 109 pg. 2,4-D and the groups were then harvested at periods of 2,8,48,96, and 144 hr. The harvested plants were sectioned into treated leaf, untreated leaf, stem, root, and terminal bud. With the exception of the terminal bud, all parts of the plant were individually oxidized and counted. The terminal buds, because of the small size, were pooled. In the second series, twenty-four plants were divided into three groups. Thirtythree pg. of 2,4-D was applied to each plant. The first group was treated when the primary leaves were almost fully expanded and the terminal buds were small.The second and third groups were treated 3 and 6 days later, respectively. All plants were harvested and dried 10 days after treatment. Four plants in each group were pooled and sectioned as indicated. Another three groups of eight plants each were treated with 50 pg., 199 pg., and 150 pg. of 2,4-D on each plant, respectively. All plants were harvested after 7 days of treatment. Samples of different sections were pooled from eight plants in each group. RESULTS
AND
DISCUSSION
The results of the radioactivity measurements are summarized in Tables I, II, and III. Each entry is the average of six plants in the first series and of eight plants in the last two series. The results from the six plants in the first experiment were analyzed statistically to determine the variance between individual plants and the effect of time on the specific activity and amount of accumulation of Cl4 in the various parts. TABLE
I
Results of Radioactivity Measurements on Parts of Bean Plants Which Were Harvested at Diffment Times After Treatment with the Sam Amount Radioactive 2,4-D =
Untreated
leaf
Root
Sterna --
-hr.
i I
Translo-
cation and .ctivity sccumulatior
Terminal
‘ L
e
E m
% 0.89f0.42
% E3 ;:i
, I
3.42f1.28 y; 2; .;g l&2 &.2:70 -
* First internode and hypocotyl. b Standard deviation.
bud
-
--
WUnt8/
min./w.
.-
I Elpecific
I8
= I
-
252
FANG,
JAWORSKI,
LOGAN,
FREED
AND
BUTTS
There were no significant differences in specific activity (counts/ min./mg. of dry plant material) and percentage accumulation among the different parts of the plant at the end of 2 hr. At 8,48, 96, and 144 hr., the specific activity and amount of accumulation of Cl4 in the stem (first internode and hypocotyl) was significantly greater than either the root or the untreated leaf. The rate of accumulation of radioactivity in the untreated leaf did not show a significant change with time. The specific activities of the TABLE
II
Results of Radioactivity Measurements on Parts of Bean Plants at Different Ages After Treutnzed with the Same Amount of Radioactive 2,4-D = 1st
2nd group
grcl"p
Plant aectiom
Tramlospecifiq cation and activity arccumulatior
_-
counls/ min./mg.
Petiole of treated leaf Untreated leaf Petiole, untreated leaf Terminal bud First internode Hypocotyl Main root Branched roots 2nd internode 1st trifoliate
3rd internode 2nd trifoliate 2,PD absorbed and 0’ translocated
Tramlocation and
Specific activity
1 --
127
43.5
3rd group
--
,
counta/ nin./?mJ. 61.8
amcumulation _79
counts/ ?nin./mg. 40.2
?7
0.2
1.5 8.5
0.7
1.4 5.7
0.5 0.3
2.3 2.6
18.4 38.8 22.2 9.2 5.0 -
2.6 18.2 13.1 2.0 3.6 -
14.2 28.6 12.4 5.7 3.5 26.7 6.9 -
0.5 7.9 3.8 0.6 1.4 2.7 1.9 27.2
3.1 19.4 6.4 3.7 1.8 8.9 1.7 3.3 2.7
1.0
55.5
!-
-
1.0
0.1 6.4 2.8 0.7 1.7 1.8 1.4 0.1 0.8 24.5 -
root and the terminal bud were significantly greater at 48, 96, and 144 hr. than they were at 2 and 8 hr. There was no significant difference in specific activity and in amount of accumulation between 2 and 8 hr. or between 48, 96, and 144 hr. In Table II, it is noted that 55.5% of the radioactivity was absorbed and translocated in the first group. Only 27.2% and 24.5% was trans-
TRANSLOCATION
OF
C”
BY
BEAN
253
PLANTS
located in the second and third groups, respectively. It is obvious that the rate of absorption of 2,4-D is greater in younger plants and this is probably one reason why younger plants are more susceptible to 2,4-D treatment than older ones. Most of the radioactivity was found in the petiole of the treated leaf, the first internode, and the hypocotyl. The TABLE III Results of Radtitivity
Measurements 012 Parts of Bean Plants After Treatment with Various Amounts of Radioactive 2,&D
= 50 Irg. pm plant
Plantsections Specific
Ipctivity
Tl.lMl&-
cation and ccumulatic
loo Pg. pm plant
3PeoiftC
TIMdO-
rctivity
cation and rccumulatioI
150 Pg. per plsnt
Specific activity
Translocation and ccumtition
_-
counta/ nnin./wql
27.9 12.2 5.2 25.6 20.3 15.3 12.9 5.2 23.8 17.0
Petiole, treated leaf Petiole, untreated leaf Untreated leaf Terminal bud Fit internode Hypocotyl Main root Branched root 2nd internode Fist trifoliate
5: 0.8 2.8 1.7 7.1 7.1 2.6 4.2 3.2 6.5
:ounts/ rin./ml
58.6 17.2 8.7 57.2 30.1 34.6 13.2 6.7 41.1 23.8
475 0.4 1.6 0.6 4.2 6.0 0.9 2.1 1.8 2.0
cowl&/ zin./?n&
70.5 9.9 4.3 37.2 25.2 11.6 5.6 .4.3 34.2 24.5
4: 0.2 0.6 0.9 2.4 1.0 0.2 0.8 1.5 2.9
_-
Radioactivity applied per plant Radioactivity recovered per plant Radioactivity loss per Plant Radioactivity found in parts of plant except treated leaf
ceunt-s/min.
cou~/min.
counts/min.
20,000
40,000
@woo
11,700
33,100
52,000
8,300
6,900
8,060
4,850
7,560
7,700
-
total in these sections would account for from 70 to 80% of the total radioactivity absorbed. The data in Table III indicate that there was no significant increase in absorption between a 15o_L(g. and a NO-pg. treatment, and slightly less absorption in the group treated with 50 pg. of 2,4-D. These experi-
254
FANG,
JAWORSKI,
LOGAN,
FREED
AND
BUTTS
ments continued for 7 days. In no case were we able to recover all of the radioactivity which was applied to the plant. The loss of radioactivity ranged from 8 to 40% depending on the amount of 2,4-D applied. The amount of radioactivity lost was quite constant, amounting to 7000-8000 counts/min./plant. It has been reported by Holley et al. (1) that some loss of radioactivity occurred, probably through the medium of CY402.To confirm this observation, four bean plants were treated with 100 pg. of 2,4-D (equivalent to 40,000 counts/min. radioactivity) per plant (50 pg. on each leaf) and enclosed in a bell jar under artificial light conditions. Two 100-w. light bulbs were set at about 1 ft. from the plants in opposite directions. The lights were turned on 10 hr./day. The atmosphere in the bell jar was evacuated by aspiration and the gases were passed continuously through an absorption flask containing sodium hydroxide solution at a rate of 9 I./24 hr. In a period of 3 days, 7000 counts/min. of radioactivity, corresponding to 17.5% of the 2,4-D applied had been removed from the atmosphere as C1402. When one considers the data in Table III, it is seen that the radioactivity unaccounted for is of/the same order as reported in experiments in which the C1402was measured. Due to the fact that in one case 100 pg. of 2,4-D was applied to two leaves (50 pg./leaf) under artificial light conditions, and over a 3-day period, while in the experiments reported in Table III varying amounts of 2,4-D were applied under greenhouse conditions to a single leaf over a 7-day period, no direct comparison can be made. It is entirely possible that application to two leaves may increase the absorption considerably, which would account for the increased Cl402 recovery. However, it is believed since conditions were comparable in the 50-pg., the lOO-pg., and the 150-pg. experiments, and since the amount of radioactivity lost per plant was of the same order, that probably absorption was the limiting factor under these conditions and that 50 pg. is approaching the maximum effective amount which could be absorbed. These points are being studied further. SUMMARY
A small amount of radioactivity was found in different parts of bean plants as early as 2 hr. after treatment with radioactive 2,4dichlorophenoxyacetic acid (2,4-D), indicating that it is quickly absorbed and the C” translocated. Only a small amount of Cl4 is translocated to the
TRANSLOCATION
OF C” BY BEAN
PLANTS
255
untreated leaf, root, and terminal bud. The stem (first internode and hypocotyl) is the area where the greatest amount of radioactivity accumulates and increases with time. Young plants will absorb 2,4-D and translocate Cl4 more quickly than will the older plants. The absorption of 2,P-D and translocation of Cl4 is not dependent on the amount applied above 50 pg. when applied to a single leaf. A small amount of 2,4-D was found to be broken down and metabolized in the bean plant with consequent loss of radioactive CO,. REFERENCES 1. HOLLEY, R. W., BOYLE, F. P., AND HAND, D. B. Arch. Biochem. 27, 143 (1950). (1950). 3. MITCHELL, J. W., AND LINDER, P. J., Science 112, 54-5 (1950). 4. TOLBERT, B. M., J. Biol. Chem. 173, 205 (1948). 5. VAN SLYEE, D. D., AND FOLCH, J., J. Biol. Chem. 136, 509 (1940). 6. WOOD, J. W., MITCHELL, J. W., AND IRVING, G. W., JR., Science 106, 337-9 (1947). 7. WOOD, J. W., WOLFE, W. C., AND IRVINQ, G. W., JR., Science 106, 395-7 (1947).
2. LINDEE, P. J., MITCHELL, J. W., AND WOOD, J. W., Science 111,518-519
of Agricultural Corvallis, Received
Chemistry, Oregon
January
Oregon State College,
18, 1951
INTRODUCTION
The translocation of plant growth regulators has been studied by the use of 1131as a label (2,3,6,7) in 2-iodo131-3-nitrobenzoic acid and 2,4dichloro-5-iodo131 phenoxyacetic acid. The 2,4-D-51 was found somewhat less toxic when applied to bean plants than was 2,4-dichlorophenoxyacetic acid (2,4-D). The benzoic acid type compound was found to be different -from 2,4-D in that it appeared to react with some constituents in the relatively immature tissues of’the terminal bud somewhat more readily than with those in the more mature parts of the stem (3). The synthesis and mode of action in bean plants of 2,4-D labeled with Cl4 in the carboxyl group has been reported by Holley et al. (1). They concluded that when applied to leaves the main movement of the 2,4-D is down the stems. Some radioactive carbon is lost through CO2 and some is found in the nutrient solution. About one-third of the isolated radioactivity was 2,4-D. More than one-half was bound in such a way that it could not be extracted with ether from an aqueous solution. 1 This research was supported by a grant from the Atomic Energy Commission to the Department of Agricultural Chemistry, Oregon Agricultural Experiment Station, Oregon State College. * Published as Technical Paper No. 656 with the approval of the Director of the Oregon Agricultural Experiment Station. Contribution of the Department of Agricultural Chemistry. 8 Some of the data reported in this paper were taken from a thesis presented by E. G. Jaw&ski to the Faculty of the Graduate School of Oregon State College in partial fulfillment of the Master of Science degree. 249
250
FANG, SAWORSKI, LOGAN, FREED AND BUTTS
It seemed worthwhile to us to synthesize 2,4-D labeled in the methylene group as well as to study the behavior of this compound in bean plants. It was also desired to synthesize a sample of 2,4-D with a high specific activity. The present investigation, employing methylene-labeled 2,4-D, was undertaken to determine the amount of translocation and accumulation of Cl4 vs. (a) time of treatment, (b) age of plant, and (c) the amount of 2,4-D applied. EXPERIMENTAL
METHODS
Xynthesis of a-Methylene-Labeled Cl
0
2,4-D
-O-C’4H~-COOH Cl
Radioactive 2,4-D, labeled in the a-methylene position was synthesized as follows: (Y-labeled methyl iodide was reacted with magnesium in ether to form the Grignard reagent, This was carbonated with CO2 to yield acetic acid. The preceding reactions were carried out in a high vacuum system at 0.1 P pressure. The technique of Tolbert was followed (4). The acetic acid was brominated using liquid bromine and red phosphorus to yield a-bromoacetic acid. The product was then coupled with 2,4dichlorophenol in the presence of sodium hydroxide. The procedure was essentially identical with that of Holley (1). The resulting product was isolated and purified according to Holley (I). Its melting point was found to be 138.9-1338°C. and its mixed melting point 13&139°C. The neutral equivalent was 221. With our counting procedure this gave a product which, when burned and collected as BaC03 and corrected to zero thickness gave approximately 4.2 X lo6 counts/min./mg. of sample. Bean plants (Phaseohs vulgaris, var. Black Valentine) were grown in potted soil under greenhouse conditions throughout the experiment. The plants were treated when the primary leaves were almost fully expanded but the terminal bud was still quite small. 2,4-D in alcohol solution was applied to one primary leaf of each plant along the midrib. All plants after harvesting were dried in vacuum at 59°C. for 24 hr. and then sectioned as indicated. MEASUREMENT
OF RADIOACTIVITY
The sections were individually oxidized using the Van Slyke-Folch combustion solution (5). The carbon dioxide was precipitated as barium carbonate. The barium carbonate was mounted in a thin layer on copper disks which have a diameter of 1 in. The radioactivity was measured by means of a “Tracerlab Autoscaler” with a thin mica window tube (1.5 mg./cm.*). The scale selector was set at 4096 counts which gives 1% error for high activity samples, 5% error for the medium ones and 10% error for the very low ones. The specific activity of all BaCOa samples was corrected to zero thickness. Duplicate determinations were made from each BaC03 sample. In every case an accuracy of 5% or better was realized.
TRANSLOCATION
OF C’” BY BEAN
METHODS
251
PLANTS
OF STUDY
In the first series of study, the bean plants were divided into five groups of six plants each. All groups were treated at the same time with 109 pg. 2,4-D and the groups were then harvested at periods of 2,8,48,96, and 144 hr. The harvested plants were sectioned into treated leaf, untreated leaf, stem, root, and terminal bud. With the exception of the terminal bud, all parts of the plant were individually oxidized and counted. The terminal buds, because of the small size, were pooled. In the second series, twenty-four plants were divided into three groups. Thirtythree pg. of 2,4-D was applied to each plant. The first group was treated when the primary leaves were almost fully expanded and the terminal buds were small.The second and third groups were treated 3 and 6 days later, respectively. All plants were harvested and dried 10 days after treatment. Four plants in each group were pooled and sectioned as indicated. Another three groups of eight plants each were treated with 50 pg., 199 pg., and 150 pg. of 2,4-D on each plant, respectively. All plants were harvested after 7 days of treatment. Samples of different sections were pooled from eight plants in each group. RESULTS
AND
DISCUSSION
The results of the radioactivity measurements are summarized in Tables I, II, and III. Each entry is the average of six plants in the first series and of eight plants in the last two series. The results from the six plants in the first experiment were analyzed statistically to determine the variance between individual plants and the effect of time on the specific activity and amount of accumulation of Cl4 in the various parts. TABLE
I
Results of Radioactivity Measurements on Parts of Bean Plants Which Were Harvested at Diffment Times After Treatment with the Sam Amount Radioactive 2,4-D =
Untreated
leaf
Root
Sterna --
-hr.
i I
Translo-
cation and .ctivity sccumulatior
Terminal
‘ L
e
E m
% 0.89f0.42
% E3 ;:i
, I
3.42f1.28 y; 2; .;g l&2 &.2:70 -
* First internode and hypocotyl. b Standard deviation.
bud
-
--
WUnt8/
min./w.
.-
I Elpecific
I8
= I
-
252
FANG,
JAWORSKI,
LOGAN,
FREED
AND
BUTTS
There were no significant differences in specific activity (counts/ min./mg. of dry plant material) and percentage accumulation among the different parts of the plant at the end of 2 hr. At 8,48, 96, and 144 hr., the specific activity and amount of accumulation of Cl4 in the stem (first internode and hypocotyl) was significantly greater than either the root or the untreated leaf. The rate of accumulation of radioactivity in the untreated leaf did not show a significant change with time. The specific activities of the TABLE
II
Results of Radioactivity Measurements on Parts of Bean Plants at Different Ages After Treutnzed with the Same Amount of Radioactive 2,4-D = 1st
2nd group
grcl"p
Plant aectiom
Tramlospecifiq cation and activity arccumulatior
_-
counls/ min./mg.
Petiole of treated leaf Untreated leaf Petiole, untreated leaf Terminal bud First internode Hypocotyl Main root Branched roots 2nd internode 1st trifoliate
3rd internode 2nd trifoliate 2,PD absorbed and 0’ translocated
Tramlocation and
Specific activity
1 --
127
43.5
3rd group
--
,
counta/ nin./?mJ. 61.8
amcumulation _79
counts/ ?nin./mg. 40.2
?7
0.2
1.5 8.5
0.7
1.4 5.7
0.5 0.3
2.3 2.6
18.4 38.8 22.2 9.2 5.0 -
2.6 18.2 13.1 2.0 3.6 -
14.2 28.6 12.4 5.7 3.5 26.7 6.9 -
0.5 7.9 3.8 0.6 1.4 2.7 1.9 27.2
3.1 19.4 6.4 3.7 1.8 8.9 1.7 3.3 2.7
1.0
55.5
!-
-
1.0
0.1 6.4 2.8 0.7 1.7 1.8 1.4 0.1 0.8 24.5 -
root and the terminal bud were significantly greater at 48, 96, and 144 hr. than they were at 2 and 8 hr. There was no significant difference in specific activity and in amount of accumulation between 2 and 8 hr. or between 48, 96, and 144 hr. In Table II, it is noted that 55.5% of the radioactivity was absorbed and translocated in the first group. Only 27.2% and 24.5% was trans-
TRANSLOCATION
OF
C”
BY
BEAN
253
PLANTS
located in the second and third groups, respectively. It is obvious that the rate of absorption of 2,4-D is greater in younger plants and this is probably one reason why younger plants are more susceptible to 2,4-D treatment than older ones. Most of the radioactivity was found in the petiole of the treated leaf, the first internode, and the hypocotyl. The TABLE III Results of Radtitivity
Measurements 012 Parts of Bean Plants After Treatment with Various Amounts of Radioactive 2,&D
= 50 Irg. pm plant
Plantsections Specific
Ipctivity
Tl.lMl&-
cation and ccumulatic
loo Pg. pm plant
3PeoiftC
TIMdO-
rctivity
cation and rccumulatioI
150 Pg. per plsnt
Specific activity
Translocation and ccumtition
_-
counta/ nnin./wql
27.9 12.2 5.2 25.6 20.3 15.3 12.9 5.2 23.8 17.0
Petiole, treated leaf Petiole, untreated leaf Untreated leaf Terminal bud Fit internode Hypocotyl Main root Branched root 2nd internode Fist trifoliate
5: 0.8 2.8 1.7 7.1 7.1 2.6 4.2 3.2 6.5
:ounts/ rin./ml
58.6 17.2 8.7 57.2 30.1 34.6 13.2 6.7 41.1 23.8
475 0.4 1.6 0.6 4.2 6.0 0.9 2.1 1.8 2.0
cowl&/ zin./?n&
70.5 9.9 4.3 37.2 25.2 11.6 5.6 .4.3 34.2 24.5
4: 0.2 0.6 0.9 2.4 1.0 0.2 0.8 1.5 2.9
_-
Radioactivity applied per plant Radioactivity recovered per plant Radioactivity loss per Plant Radioactivity found in parts of plant except treated leaf
ceunt-s/min.
cou~/min.
counts/min.
20,000
40,000
@woo
11,700
33,100
52,000
8,300
6,900
8,060
4,850
7,560
7,700
-
total in these sections would account for from 70 to 80% of the total radioactivity absorbed. The data in Table III indicate that there was no significant increase in absorption between a 15o_L(g. and a NO-pg. treatment, and slightly less absorption in the group treated with 50 pg. of 2,4-D. These experi-
254
FANG,
JAWORSKI,
LOGAN,
FREED
AND
BUTTS
ments continued for 7 days. In no case were we able to recover all of the radioactivity which was applied to the plant. The loss of radioactivity ranged from 8 to 40% depending on the amount of 2,4-D applied. The amount of radioactivity lost was quite constant, amounting to 7000-8000 counts/min./plant. It has been reported by Holley et al. (1) that some loss of radioactivity occurred, probably through the medium of CY402.To confirm this observation, four bean plants were treated with 100 pg. of 2,4-D (equivalent to 40,000 counts/min. radioactivity) per plant (50 pg. on each leaf) and enclosed in a bell jar under artificial light conditions. Two 100-w. light bulbs were set at about 1 ft. from the plants in opposite directions. The lights were turned on 10 hr./day. The atmosphere in the bell jar was evacuated by aspiration and the gases were passed continuously through an absorption flask containing sodium hydroxide solution at a rate of 9 I./24 hr. In a period of 3 days, 7000 counts/min. of radioactivity, corresponding to 17.5% of the 2,4-D applied had been removed from the atmosphere as C1402. When one considers the data in Table III, it is seen that the radioactivity unaccounted for is of/the same order as reported in experiments in which the C1402was measured. Due to the fact that in one case 100 pg. of 2,4-D was applied to two leaves (50 pg./leaf) under artificial light conditions, and over a 3-day period, while in the experiments reported in Table III varying amounts of 2,4-D were applied under greenhouse conditions to a single leaf over a 7-day period, no direct comparison can be made. It is entirely possible that application to two leaves may increase the absorption considerably, which would account for the increased Cl402 recovery. However, it is believed since conditions were comparable in the 50-pg., the lOO-pg., and the 150-pg. experiments, and since the amount of radioactivity lost per plant was of the same order, that probably absorption was the limiting factor under these conditions and that 50 pg. is approaching the maximum effective amount which could be absorbed. These points are being studied further. SUMMARY
A small amount of radioactivity was found in different parts of bean plants as early as 2 hr. after treatment with radioactive 2,4dichlorophenoxyacetic acid (2,4-D), indicating that it is quickly absorbed and the C” translocated. Only a small amount of Cl4 is translocated to the
TRANSLOCATION
OF C” BY BEAN
PLANTS
255
untreated leaf, root, and terminal bud. The stem (first internode and hypocotyl) is the area where the greatest amount of radioactivity accumulates and increases with time. Young plants will absorb 2,4-D and translocate Cl4 more quickly than will the older plants. The absorption of 2,P-D and translocation of Cl4 is not dependent on the amount applied above 50 pg. when applied to a single leaf. A small amount of 2,4-D was found to be broken down and metabolized in the bean plant with consequent loss of radioactive CO,. REFERENCES 1. HOLLEY, R. W., BOYLE, F. P., AND HAND, D. B. Arch. Biochem. 27, 143 (1950). (1950). 3. MITCHELL, J. W., AND LINDER, P. J., Science 112, 54-5 (1950). 4. TOLBERT, B. M., J. Biol. Chem. 173, 205 (1948). 5. VAN SLYEE, D. D., AND FOLCH, J., J. Biol. Chem. 136, 509 (1940). 6. WOOD, J. W., MITCHELL, J. W., AND IRVING, G. W., JR., Science 106, 337-9 (1947). 7. WOOD, J. W., WOLFE, W. C., AND IRVINQ, G. W., JR., Science 106, 395-7 (1947).
2. LINDEE, P. J., MITCHELL, J. W., AND WOOD, J. W., Science 111,518-519