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Decarboxylation of alpha-naphthaleneacetic acid by pineapple leaves in sunlight
International Journal of Applied Radiation and hotopu, 1962, Vol. 13, pp. 399-402 Pergamon Press Ltd. Printed in Poland
Decarboxylation of Alpha-Naphthaleneacetic Acid by Pineapple Leaves in Sunlight R. W. LEEPER, D. P. G O W I N G and W. S. S T E W A R T ~ Pineapple Research Institute of Hawaii, Honolulu, Hawaii
PRODUCTIO~ of radioactive carbon dioxide (C140,) has been reported by HOLL~Y et al. ~1) following application on the primary leaf of young red kidney bean plants (Phaseolus vulgaris) of 2,4-dichlorophenoxyacetic acid (2,4-D) labeled in the carboxyl position. W E I N T R A U B et al. (s) showed that Ca40~ was produced f r o m a p p l i c a t i o n of carboxyl-labeled 2,4-D on the terminal bud
of young Black Valentine bean plants in light or in continuous darkness. We have observed evolution of C140~ from carboxyl-labeled 2,4-D, a-naphthaleneacetic acid and fl-naphthoxyacetic acid. The present paper describes the evolution of Ca40~ from carboxyl-labeled a-naphthaleneacetic acid (ANA*) when applied to several types of surfaces and under varying conditions.
METHOD These studies were conducted by application of 1.12 mg of ANA* (2.37 mc/mM) dissolved in 1 ml of 95 per cent ethyl alcohol on a single leaf of a pineapple plant, Ananas comosus (L.) Merr. The ANA* solution was painted, using a rubber policeman, from margin to margin on a 15 cm length of leaf. The treated area was located on green tissue just above the basal white meristematic tissue. Earlier experiments had established that unlabeled a-naphthaleneacetic acid applied in this manner induced
flowering. The treated leaf, still attached to the plant, was immediately inserted into a glass tube and the ends of the tube sealed with sponge-rubber stoppers containing inlet and outlet tubes. The C140~ was trapped in alkali, isolated as barium carbonate, plated upon tared aluminum planchets, and a period of counting was used wherein the observed counts fell within the -_k5 per cent counting error range. The observed activity of all samples was converted to infinite thickness.
RESULTS Figure 1 illustrates the variation in evolution of C140~ from two pineapple plants over a 24 hr period. It is apparent that
sunlight is an important requirement for this evolution and that even low-intensity sunlight caused some destruction of ANA*
t Published with the approval of the Director as Technical Paper No. 286 of the Pineapple Research Institute o f Hawaii, Honolulu, Hawaii. 4: Present address: Los Angeles State and C o u n t y Arboretum, Arcadia, California, U.S.A. 399
400
R. W. Leeper, D. P. Gowing and IV. S. Stewart
on the pineapple leaf~ Collecting the air from about an untreated leaf on the same plant and checking for radioactivity indicated that there were no counts above background. Thus, if there was transport of ANA* from leaf of application to another
dicate that this is usually the period of maxim u m light intensity at the site of the experiments. The unevenness in evolution of C140~ in the period 1-4 p.m. can be attributed to development of intermittent cloudiness.
1200
IOOC
!
¢_ E u~
s
o (.2
40O
I Sunse~
5 Pm
7.17
J-/-,,
I I
1D-,,m,,O
2
3
4
Hours ofter
6
7
8
Actuol
oPpl|cotlon time
14 I~ 6ore 7
,, 16 8
17 9
18 I0
,,, 19 II
20 Noon
21 22 Ipm 2
23 3
;'4 4
FIG. I. Evolution of C1402 from two pineapple plants over a 24 hr period starting 1 hr after application of ANA*.
within the time of this experiment, there was no evolution of C140, from an untreated leaf. Actually, time studies showed there was rapid transport of some ANA* but it seemed to be limited to the very youngest leaves and the last few centimeters of the stem tip. For example, 5 min after application of material with an activity of about 60,000 counts per minute, ANA* with about 200 counts per minute of activity could be isolated from the first centimeter of stem tip. The peak in radioactivity at 11 a.m. is characteristic. Aetinometer measurements in-
Figure 2 illustrates the effect of alternate hours of sunlight and complete darkness upon C140~ output. Application was made in darkness at 5 a.m. and the collecting apparatus wrapped in aluminum foil until 8 a.m. HOurly sampling of the wash solution indicated that no C1~O~ was being evolved while the leaf was in darkness. O n alternate hours after 8 a.m. the aluminum foil cover was removed or replaced. The wash solution was changed on the hour and at 2½ and 15 rain after the hour. In Fig. 2, the direct counts per minute were plotted against time, with no correction
Decarboxylation of a-naphthaleneacetic acid by pineapple leaves in sunlight
after exclusion of sunlight, evolution of C140~ had ceased. To dctcrminc thc effect of tcmperaturc upon C1402 output, a double-wall glass tube was used to enclose the treated leaf. Water at various temperatures was circulated through the interspace and 10 rain samples of aspirated air were taken after the tem-
being made for variation in sampling time. It can be seen that a considerable amount of C140s was present in the 2½ min sampling after shielding from sunlight. It is believed that this represents material evolved during the period of sunlight and "flushed" from the collecting tube during the subsequent sampling period. In any event, 2½ min .
12..
.
.
.
.
/,
.
.'x\~. x\xx
.
.
.
.
.
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.c E
401
b.\N ..--. :.---
!--! ~\x\\\
7~
:?--.
g
.-- .-.-
o L)
5O ~ \ \ \ \ \ \ \ \
b.\\\\\\~
/ II 7
6am
8
I0
II
:
"
i
Noon
2
I
3
Time
FIG. 2.
Effect of sunlight and complete shading u p o n C14Ot evolution.
Direct counts, not corrected for
variation in sampling time.
TxRI~ I. Effect of temperature change upon evolution of C140~ from A N A * on' pineapple leaves Sample
Time collected (p.m.)
1 20-1.30
Air temp. inside glass tube (°F)
Approx.
temp. change
96-104 °
Corrected counts/min 735
--27.5 °
1.40-1.50
72-73 °
2.04-2.14
117-126 °
2.26-2.36
70-75 °
2.53-3.03
88-93 °
Change in counts/rain
--287 448 _L 105
+49 ° 553 --49 °
---339 214
+18 °
-~-189 403
402
R. W. Leeper. D . P . Gowing and IV. S. Stewart TABI.~. 2. Counts per minute from radioactive carbon dioxide evolved from carboxyl-tagged ANA applied to various surfaces
10 a. I1 a. Noon 1 p. 2 p. 3 p.
m. dark m. sunlight dark m. sunlight m. dark m. sunlight
I
Fresh leaf
Steam-killed leaf
582 19,993 688 9211 313 3634
95 5964 347 5670 219 2442
perature within the tube coincided with the temperature of the circulating water. Table 1 contains data obtained in this experiment. The sky was cloudless during the period of samples 1-4, while there was a brief period of cloudiness on sample 5, which would cause the result for that sample to be somewhat lower than if there had been full sunlight. It is interesting to note that with falling temperature there was a decrease in evolution of C140~ and with increasing temperature an increase in evolution. Decarboxylation of ANA* in sunlight can actually occur on m a n y types of surfaces.
Dry leaf Aluminum foil 10 7922 237 (Lost) 398 4336
41 11,081 249 5269 195 2583
In Table 2 comparison is made between fresh pineapple leaf, steam-killed fresh pineapple leaf, dry (dead) pineapple leaf and aluminum foil as surfaces upon which decarboxylation takes place. It is evident that sunlight is an important factor in decarboxylation. It is also evident that decarboxylation is, at least to a large extent, not associated with a biological system. It is our belief this can be an important factor to consider in choosing the time of application or the external conditions to be encountered in experimentation involving materials capable of undergoing decarboxylation.
REFERENCES 1. HOLILY R. W., BOYLE F. P. and HAND D. B. Arch. Biochera. 27, 143 (1950).
2. WEINTRAUB R. L., BROWN J. W., FIELDS M. and ROHAN J. Plant Physiol. 27, 293 (1952).
Decarboxylation of Alpha-Naphthaleneacetic Acid by Pineapple Leaves in Sunlight R. W. LEEPER, D. P. G O W I N G and W. S. S T E W A R T ~ Pineapple Research Institute of Hawaii, Honolulu, Hawaii
PRODUCTIO~ of radioactive carbon dioxide (C140,) has been reported by HOLL~Y et al. ~1) following application on the primary leaf of young red kidney bean plants (Phaseolus vulgaris) of 2,4-dichlorophenoxyacetic acid (2,4-D) labeled in the carboxyl position. W E I N T R A U B et al. (s) showed that Ca40~ was produced f r o m a p p l i c a t i o n of carboxyl-labeled 2,4-D on the terminal bud
of young Black Valentine bean plants in light or in continuous darkness. We have observed evolution of C140~ from carboxyl-labeled 2,4-D, a-naphthaleneacetic acid and fl-naphthoxyacetic acid. The present paper describes the evolution of Ca40~ from carboxyl-labeled a-naphthaleneacetic acid (ANA*) when applied to several types of surfaces and under varying conditions.
METHOD These studies were conducted by application of 1.12 mg of ANA* (2.37 mc/mM) dissolved in 1 ml of 95 per cent ethyl alcohol on a single leaf of a pineapple plant, Ananas comosus (L.) Merr. The ANA* solution was painted, using a rubber policeman, from margin to margin on a 15 cm length of leaf. The treated area was located on green tissue just above the basal white meristematic tissue. Earlier experiments had established that unlabeled a-naphthaleneacetic acid applied in this manner induced
flowering. The treated leaf, still attached to the plant, was immediately inserted into a glass tube and the ends of the tube sealed with sponge-rubber stoppers containing inlet and outlet tubes. The C140~ was trapped in alkali, isolated as barium carbonate, plated upon tared aluminum planchets, and a period of counting was used wherein the observed counts fell within the -_k5 per cent counting error range. The observed activity of all samples was converted to infinite thickness.
RESULTS Figure 1 illustrates the variation in evolution of C140~ from two pineapple plants over a 24 hr period. It is apparent that
sunlight is an important requirement for this evolution and that even low-intensity sunlight caused some destruction of ANA*
t Published with the approval of the Director as Technical Paper No. 286 of the Pineapple Research Institute o f Hawaii, Honolulu, Hawaii. 4: Present address: Los Angeles State and C o u n t y Arboretum, Arcadia, California, U.S.A. 399
400
R. W. Leeper, D. P. Gowing and IV. S. Stewart
on the pineapple leaf~ Collecting the air from about an untreated leaf on the same plant and checking for radioactivity indicated that there were no counts above background. Thus, if there was transport of ANA* from leaf of application to another
dicate that this is usually the period of maxim u m light intensity at the site of the experiments. The unevenness in evolution of C140~ in the period 1-4 p.m. can be attributed to development of intermittent cloudiness.
1200
IOOC
!
¢_ E u~
s
o (.2
40O
I Sunse~
5 Pm
7.17
J-/-,,
I I
1D-,,m,,O
2
3
4
Hours ofter
6
7
8
Actuol
oPpl|cotlon time
14 I~ 6ore 7
,, 16 8
17 9
18 I0
,,, 19 II
20 Noon
21 22 Ipm 2
23 3
;'4 4
FIG. I. Evolution of C1402 from two pineapple plants over a 24 hr period starting 1 hr after application of ANA*.
within the time of this experiment, there was no evolution of C140, from an untreated leaf. Actually, time studies showed there was rapid transport of some ANA* but it seemed to be limited to the very youngest leaves and the last few centimeters of the stem tip. For example, 5 min after application of material with an activity of about 60,000 counts per minute, ANA* with about 200 counts per minute of activity could be isolated from the first centimeter of stem tip. The peak in radioactivity at 11 a.m. is characteristic. Aetinometer measurements in-
Figure 2 illustrates the effect of alternate hours of sunlight and complete darkness upon C140~ output. Application was made in darkness at 5 a.m. and the collecting apparatus wrapped in aluminum foil until 8 a.m. HOurly sampling of the wash solution indicated that no C1~O~ was being evolved while the leaf was in darkness. O n alternate hours after 8 a.m. the aluminum foil cover was removed or replaced. The wash solution was changed on the hour and at 2½ and 15 rain after the hour. In Fig. 2, the direct counts per minute were plotted against time, with no correction
Decarboxylation of a-naphthaleneacetic acid by pineapple leaves in sunlight
after exclusion of sunlight, evolution of C140~ had ceased. To dctcrminc thc effect of tcmperaturc upon C1402 output, a double-wall glass tube was used to enclose the treated leaf. Water at various temperatures was circulated through the interspace and 10 rain samples of aspirated air were taken after the tem-
being made for variation in sampling time. It can be seen that a considerable amount of C140s was present in the 2½ min sampling after shielding from sunlight. It is believed that this represents material evolved during the period of sunlight and "flushed" from the collecting tube during the subsequent sampling period. In any event, 2½ min .
12..
.
.
.
.
/,
.
.'x\~. x\xx
.
.
.
.
.
xx\~
i
IOC
.c E
401
b.\N ..--. :.---
!--! ~\x\\\
7~
:?--.
g
.-- .-.-
o L)
5O ~ \ \ \ \ \ \ \ \
b.\\\\\\~
/ II 7
6am
8
I0
II
:
"
i
Noon
2
I
3
Time
FIG. 2.
Effect of sunlight and complete shading u p o n C14Ot evolution.
Direct counts, not corrected for
variation in sampling time.
TxRI~ I. Effect of temperature change upon evolution of C140~ from A N A * on' pineapple leaves Sample
Time collected (p.m.)
1 20-1.30
Air temp. inside glass tube (°F)
Approx.
temp. change
96-104 °
Corrected counts/min 735
--27.5 °
1.40-1.50
72-73 °
2.04-2.14
117-126 °
2.26-2.36
70-75 °
2.53-3.03
88-93 °
Change in counts/rain
--287 448 _L 105
+49 ° 553 --49 °
---339 214
+18 °
-~-189 403
402
R. W. Leeper. D . P . Gowing and IV. S. Stewart TABI.~. 2. Counts per minute from radioactive carbon dioxide evolved from carboxyl-tagged ANA applied to various surfaces
10 a. I1 a. Noon 1 p. 2 p. 3 p.
m. dark m. sunlight dark m. sunlight m. dark m. sunlight
I
Fresh leaf
Steam-killed leaf
582 19,993 688 9211 313 3634
95 5964 347 5670 219 2442
perature within the tube coincided with the temperature of the circulating water. Table 1 contains data obtained in this experiment. The sky was cloudless during the period of samples 1-4, while there was a brief period of cloudiness on sample 5, which would cause the result for that sample to be somewhat lower than if there had been full sunlight. It is interesting to note that with falling temperature there was a decrease in evolution of C140~ and with increasing temperature an increase in evolution. Decarboxylation of ANA* in sunlight can actually occur on m a n y types of surfaces.
Dry leaf Aluminum foil 10 7922 237 (Lost) 398 4336
41 11,081 249 5269 195 2583
In Table 2 comparison is made between fresh pineapple leaf, steam-killed fresh pineapple leaf, dry (dead) pineapple leaf and aluminum foil as surfaces upon which decarboxylation takes place. It is evident that sunlight is an important factor in decarboxylation. It is also evident that decarboxylation is, at least to a large extent, not associated with a biological system. It is our belief this can be an important factor to consider in choosing the time of application or the external conditions to be encountered in experimentation involving materials capable of undergoing decarboxylation.
REFERENCES 1. HOLILY R. W., BOYLE F. P. and HAND D. B. Arch. Biochera. 27, 143 (1950).
2. WEINTRAUB R. L., BROWN J. W., FIELDS M. and ROHAN J. Plant Physiol. 27, 293 (1952).