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Drugs affecting sensitivity to stimuli in the plant Mimosa and the protozoan Spirostomum
Physiology and Behavior, Vol. 9, pp. 869-871. Brain Research Publications Inc., 1972. Printed in U.S.A.
BRIEF COMMUNICATION Drugs Affecting Sensitivity to Stimuli in the Plant Mimosa and the Protozoan Spirostomum PHILIP B. APPLEWHITE
Kline Biology Tower, Biology Department, Yale University New Haven, Connecticut 06520
(Received 15 May 1972)
APPLEWHITE,P. B. Drugs affecting sensitivity to stimuli in the plant Mimosa and the protozoan Spirostomum. PHYSIOL. BEHAV. 9(5) 869-871, 1972.-There is a close similarity in the effects of drugs on the sensitivity to mechanical stimulation in the sensitive plant Mimosa and the protozoan Spirostomum. Certain chemicals also affect the rate of opening to light and closure to dark in Mimosa. The chemicals would appear to affect membrane permeability within the two organisms by similar processes.
Spirostomum
Mimosa
Drugs
Movement
THE EARLIEST studies comparing the action of drugs on plants and animals suggested that in certain instances the underlying physiology of the responses to them was similar [5, 6]. The results were seldom quantitative and less often made direct comparisons of the same kind of phenomemon in both plants and animals. Recently, a direct comparison of habituation behavior to electrical and mechanical stimulation has been reported with the sensitive plant Mimosa and the protozoan Spirostomum [2]. This study dealt with the adaptation of a contraction response to stimulation in the protozoan and rapid leaflet closure in Mimosa and demonstrated rather striking similarities in their behavior. A logical extension would be a comparison of the effect of selected drugs on the initial sensitivity of those organisms to stimulation. For completeness in Mimosa, information can also be obtained on the chemicals' effect upon leaflet opening to light and closure to dark to determine whether they are exerting general, rather than specific, effects on response sensitivity. It has been demonstrated that both the plant growth hormones indole acetic acid (I.A.A.) and gibberellic acid (G.A.) over a period of several hours promote faster opening of Mimosa leaflets to light after closing in the dark and for I.A.A. slower closure to dark after opening to the light [11]. Bose [5, 6] demonstrated many years ago that several chemicals "affected the movement of the petiole (stem) of Mimosa via their effect on the primary pulvinus, the organ controlling movement. No study has been done on changes in the sensitivity of leaflets to touch or
mechanical stimulation, however. Preliminary qualitative studies on Spirostomum indicated strychnine and curare prevented contraction to stimulation [10]. The above chemicals were therefore used in this study on both Mimosa and Spirostomum in addition to the other plant growth hormones abscisic acid (A.A.), kinetin, and the auxin 2, 4-Dichlorophenoxyisobutric acid (P.I.B.A.) [71. The local anesthetic xylocaine; the depressants ethyl alcohol and Librium (chiordiazepoxide); and the stimulants picrotoxin and caffeine were also used to broaden the basis of the study. METHOD The details of the method for stimulating Spirostomum ambiguum have been reported elsewhere [1]. Briefly, a constant intensity mechanical stimulus is given in the form of a solenoid dropping on a slide containing two chambers. Both the control and experimental chambers contain deionized water and the one also containing the chemical to be tested is designated the experimental one. About 30 Spirostomum from our stock cultures are placed in each chamber and a stimulus is given that is sufficient to produce contraction for about 80% of the untreated controls. Photographic recording of the number of protozoa contracting in the experimental and control groups reveals what effect, if any, the added drug has upon their initial response sensitivity. This procedure was repeated 15 times with different protozoa for each chemical. An alternate way of doing this would be to determine what change in 869
870
APPLEWHITE min with Spirostomum (to give adequate uptake time) in measuring changes in stimulus sensitivity. All chemicals were dissolved in distilled water except A. A. which was in 0.3% dimethylsulfoxide, as was its control. Chemicals were tested near their limits of solubility or if already in soluble form at commercial strengths, then diluted accordingly until there was no visible tissue damage. The pH of the controls' water was adjusted, if necessary, to that of the experimental group with the chemical added to eliminate any pH effects. Where an effect was obtained, the chemicals the c o m p o u n d s of interest were supplied with (e.g. sodium bisulfite, sodium chloride, benzyl alcohol, etc.) were also tested at appropriate molarities to verify these were not the active agents. They, in fact, had no effect. To determine if the effects on sensitivity were reversible, b o t h experimental and control groups of Mimosa and Spirostomum were washed twice with distilled water, placed in fresh water for 1 hr, and t h e n retested in the same m a n n e r as before for sensitivity to stimulation. F o r Mimosa t w o additional experiments were performed, each using its o w n set of 24 pinnae pairs as before. F o r one e x p e r i m e n t after the experimental pinnae were exposed as in the sensitivity experiments to the chemical and light for 45 min, all lights were turned out and the time to close c o m p l e t e l y to the dark was recorded under a green safe light for the experimental and control groups. F o r the other, the cut
stimulus intensity was necessary to produce the same population percent-contraction in the experimental as the control group. But, this would have required several successive stimulations which would produce some habituation and would be c o m p o u n d e d with the sensitivity effects. Mimosa pudica was grown from seed and raised as previously described [3]. All experiments were p e r f o r m e d in a growth c h a m b e r at 85% h u m i d i t y , 25°C, and an illumination intensity of 45 m W / c m 2/sec. Individual pinnae (leaves) cut from the plant and containing at least 12 leaflet pairs were floated in distilled water in each c h a m b e r of a quadrant Petri dish, a standard procedure [3]. F o u r such dishes were fastened together, so that two contained 8 control pinnae in water only; and t w o the 8 experimental pinnae, from the same leafs of the same plants to form matched pairs, with the added chemical. The constant stimulus intensity delivery system has been described elsewhere [ 2 ] , but basically is similar to the way in which Spirostomum is stimulated. The difference b e t w e e n experimental and control groups is determined, as with Spirostomum, by the percent difference in initial total leaflet colsure to stimulation. F o r each chemical tested, 2 pinnae from 12 different plants were each used for the experimenta_l and control groups. Each chemical was tested for 1 hr with Mimosa and 30
TABLE 1
DRUGS AND MOVEMENT IN MIMOSA AND SPIROSTOMUM
Mimosa
Mimosa
Stimulus
Mimosa
Spirostomum Stimulus
Closure to
Opening to
Chemical
Sensitivity
Sensitivity
Dark
Light
G.A.
Normal
Normal
Normal
Fast(11 min)
10-3
10-s
A.A.
Normal
Normal
Normal
Normal
10-4
10- s
I.A.A.
Less(75%)
Less(20%)
Slow(19 min)
Fast(15 min)
10-4
10- s
P.I.B.A.
Normal
Normal
Normal
Normal
10- 3
10-4
Kinetin
Normal
Normal
Normal
Normal
10-*
10-4
Ethanol
Normal
Normal
Slow(8 min)
Fast(13 min)
10-1
10-1
Xylocaine
Less(87%)
Less(53%)
Slow(27 min)
Normal
2x10- 2
10-3
Librium
Normal
Normal
Normal
Normal
10- 3
10- s
Tubocurarine
Less(50%)
Less(81%)
Slow(24 min)
Normal
10-4
5x10- s
Strychnine
Less(82%)
Less(70%)
Slow(8 min)
Normal
5x10- 3
5x10 -3
Caffeine
Normal
Normal
Slow(12 min)
Normal
5x10- 3
5x10 -3
Picrotoxin
Less(49%)
Normal
Slow(10 min)
Normal
5x10- 3
10- s
Molarity
Mimosa Spirostomum
*Normal means no significant difference (p>O.05 two-tailed Wilcoxon test) between experimental and control groups: Less, Slow, and Fast mean a significant difference (p
MIMOSA, SPIROSTOMUM AND DRUGS
871
pinnae were put in the dark, where they would not open, with the chemical for 45 min, then the lights were turned on. The time to open fully for both groups was recorded. By the time the controls began to close or open, about 60 min would pass and this closing or opening response would be based on approximately the same amount of time of exposure to the chemical as for the mechanical stimulation responses, allowing direct comparisons to be made. RESULTS AND DISCUSSION The results are presented in Table 1. The Mimosa Stimulus Sensitivity column is computed by subtracting what percentage of the 24 pinnae that received the drug closed to the stimulus from the percentage of control pinnae that did also. The third column for Spirostomum was computed the same way. The fourth and fifth columns are obtained by taking the mean absolute difference between the time of the controls' closure to the dark or opening to the light, respectively, and the experimental's time of response. The reported molarity of the drugs is close to the maximum that could be used. Dose-response curves were also obtained and for the effective drugs were sigmoid curves on a semilog plot. With regard to the chemicals' effects on sensitivity to stimulation, it is apparent there is a high degree of correspondence between such diverse organisms as Mimosa and Spirostomum, the exception being the stimulant picrotoxin, which affects only Mimosa. Not only do drugs that exert effects on animals have similar effects on protozoa and plants but a plant growth hormone also affects a protozoan. Furthermore, with the exception of strychnine, which appears to poison permanently the organisms, all the sensitivity effects are at least 85% reversible after washing; meaning 85% of the pinnae and protozoan respond normally after 1 hr in new media. Of course, some of the drugs used may have no effect simply because they are not taken up by the organisms. The results from the closure to dark and opening to light
experiments, when compared to the effect drugs have on sensitivity, indicate these three processes are somewhat independent of each other, and the chemicals are not simultaneously affecting all three kinds (response to stimulation, light, dark) of plant behavior in identical ways. If Mimosa is made less sensitive to stimulation, it also takes longer to close to dark but the converse is not necessarily true. Furthermore, the time for Mimosa to open to light is unrelated to its touch sensitivity or time to close to dark. The fact these drugs speed up opening to light indicates their action is not just merely one of slowing down leaflet movement. Even with the stimulants, strychnine, caffeine and picrotoxin, sensitivity to stimulation cannot be increased for Mimosa or Spirostomum (nor is there a faster closure to dark, or a slower opening to light). The effects on Spirostomum and Mimosa movement may either be at the level of receptors or effectors. It is not known what the former are for either organism. However, with the exception of I.A.A., the substances which produce a lessened sensitivity to stimulation are known to produce m u s c l e r e l a x a t i o n in vertebrates [8] and since actomyosin-like substances are found in both organisms [2], it seems likely the effectors of movement are involved. Since leaflet movement presumably involves changes in ion flow in the motor cells located in the pulvini of Mimosa [9] or in the compartments adjacent to the contractile myonemes in Spirostomum [4], membrane permeability is changed in some way by these chemicals. The conclusion would have to be that on a biochemical level, plants and animals would not appear that different from one another in their response to drugs. Since a structural nervous system is obviously absent in Mimosa and Spirostomum the correspondence with those possessing one is even more striking. This suggests explanations of drug actions on behavior based solely on neurological concepts (e.g., curare and neuromuscular junctions, picrotoxin and pre-synaptic inhibition) may be inadequate.
REFERENCES 1. Applewhite, P. B. Nonlocal nature of habituation in a rotifer and protozoan. Nature 217: 287-288, 1968. 2. Applewhite, P. B. Behavioral plasticity in the sensitive plant, Mimosa. Behav. Biol. 7: 47-53, 1972. 3. Applewhite, P. B. and F. Gardner. Rapid leaf closure of Mimosa in response to light. Nature 233: 279-287, 1971. 4. Applewhite, P. B. and F. Gardner. Theory of protozoan habituation. Nature New Biology 230: 285-287, 1971. 5. Bose, J. C. Plant Response as a Means of Physiological Invest~'gation. London: Longmans, Green and Co., 1906. 6. Bose, J. C. The Motor Mechanism of Plants. London: Longmans, Green and Co., 1928.
7. Galston, A. W. and P. J. Davies. Control Mechanisms in Plant Development. Englewood Cliffs, New Jersey: Prentice-Hall, 1970. 8. Goodman, L. S. and A. Gilman. The Pharmacological Basis of Therapeutics. New York: Macmillan, 1970. 9. Sibaoka, T. Physiology of rapid movements in higher plants. Ann. Rev. Plant Physiology 20: 165-184,1969. 10. Tartar, V. The Biology of Stentor, New York: Pergamon Press, 1961. 11. Williams, C. N. and V. Raghavan. Effects of light and growth substances on the diurnal movements of the leaflets of Mimosa pudiea. J. Exp. Botany 17: 742-749, 1966.
BRIEF COMMUNICATION Drugs Affecting Sensitivity to Stimuli in the Plant Mimosa and the Protozoan Spirostomum PHILIP B. APPLEWHITE
Kline Biology Tower, Biology Department, Yale University New Haven, Connecticut 06520
(Received 15 May 1972)
APPLEWHITE,P. B. Drugs affecting sensitivity to stimuli in the plant Mimosa and the protozoan Spirostomum. PHYSIOL. BEHAV. 9(5) 869-871, 1972.-There is a close similarity in the effects of drugs on the sensitivity to mechanical stimulation in the sensitive plant Mimosa and the protozoan Spirostomum. Certain chemicals also affect the rate of opening to light and closure to dark in Mimosa. The chemicals would appear to affect membrane permeability within the two organisms by similar processes.
Spirostomum
Mimosa
Drugs
Movement
THE EARLIEST studies comparing the action of drugs on plants and animals suggested that in certain instances the underlying physiology of the responses to them was similar [5, 6]. The results were seldom quantitative and less often made direct comparisons of the same kind of phenomemon in both plants and animals. Recently, a direct comparison of habituation behavior to electrical and mechanical stimulation has been reported with the sensitive plant Mimosa and the protozoan Spirostomum [2]. This study dealt with the adaptation of a contraction response to stimulation in the protozoan and rapid leaflet closure in Mimosa and demonstrated rather striking similarities in their behavior. A logical extension would be a comparison of the effect of selected drugs on the initial sensitivity of those organisms to stimulation. For completeness in Mimosa, information can also be obtained on the chemicals' effect upon leaflet opening to light and closure to dark to determine whether they are exerting general, rather than specific, effects on response sensitivity. It has been demonstrated that both the plant growth hormones indole acetic acid (I.A.A.) and gibberellic acid (G.A.) over a period of several hours promote faster opening of Mimosa leaflets to light after closing in the dark and for I.A.A. slower closure to dark after opening to the light [11]. Bose [5, 6] demonstrated many years ago that several chemicals "affected the movement of the petiole (stem) of Mimosa via their effect on the primary pulvinus, the organ controlling movement. No study has been done on changes in the sensitivity of leaflets to touch or
mechanical stimulation, however. Preliminary qualitative studies on Spirostomum indicated strychnine and curare prevented contraction to stimulation [10]. The above chemicals were therefore used in this study on both Mimosa and Spirostomum in addition to the other plant growth hormones abscisic acid (A.A.), kinetin, and the auxin 2, 4-Dichlorophenoxyisobutric acid (P.I.B.A.) [71. The local anesthetic xylocaine; the depressants ethyl alcohol and Librium (chiordiazepoxide); and the stimulants picrotoxin and caffeine were also used to broaden the basis of the study. METHOD The details of the method for stimulating Spirostomum ambiguum have been reported elsewhere [1]. Briefly, a constant intensity mechanical stimulus is given in the form of a solenoid dropping on a slide containing two chambers. Both the control and experimental chambers contain deionized water and the one also containing the chemical to be tested is designated the experimental one. About 30 Spirostomum from our stock cultures are placed in each chamber and a stimulus is given that is sufficient to produce contraction for about 80% of the untreated controls. Photographic recording of the number of protozoa contracting in the experimental and control groups reveals what effect, if any, the added drug has upon their initial response sensitivity. This procedure was repeated 15 times with different protozoa for each chemical. An alternate way of doing this would be to determine what change in 869
870
APPLEWHITE min with Spirostomum (to give adequate uptake time) in measuring changes in stimulus sensitivity. All chemicals were dissolved in distilled water except A. A. which was in 0.3% dimethylsulfoxide, as was its control. Chemicals were tested near their limits of solubility or if already in soluble form at commercial strengths, then diluted accordingly until there was no visible tissue damage. The pH of the controls' water was adjusted, if necessary, to that of the experimental group with the chemical added to eliminate any pH effects. Where an effect was obtained, the chemicals the c o m p o u n d s of interest were supplied with (e.g. sodium bisulfite, sodium chloride, benzyl alcohol, etc.) were also tested at appropriate molarities to verify these were not the active agents. They, in fact, had no effect. To determine if the effects on sensitivity were reversible, b o t h experimental and control groups of Mimosa and Spirostomum were washed twice with distilled water, placed in fresh water for 1 hr, and t h e n retested in the same m a n n e r as before for sensitivity to stimulation. F o r Mimosa t w o additional experiments were performed, each using its o w n set of 24 pinnae pairs as before. F o r one e x p e r i m e n t after the experimental pinnae were exposed as in the sensitivity experiments to the chemical and light for 45 min, all lights were turned out and the time to close c o m p l e t e l y to the dark was recorded under a green safe light for the experimental and control groups. F o r the other, the cut
stimulus intensity was necessary to produce the same population percent-contraction in the experimental as the control group. But, this would have required several successive stimulations which would produce some habituation and would be c o m p o u n d e d with the sensitivity effects. Mimosa pudica was grown from seed and raised as previously described [3]. All experiments were p e r f o r m e d in a growth c h a m b e r at 85% h u m i d i t y , 25°C, and an illumination intensity of 45 m W / c m 2/sec. Individual pinnae (leaves) cut from the plant and containing at least 12 leaflet pairs were floated in distilled water in each c h a m b e r of a quadrant Petri dish, a standard procedure [3]. F o u r such dishes were fastened together, so that two contained 8 control pinnae in water only; and t w o the 8 experimental pinnae, from the same leafs of the same plants to form matched pairs, with the added chemical. The constant stimulus intensity delivery system has been described elsewhere [ 2 ] , but basically is similar to the way in which Spirostomum is stimulated. The difference b e t w e e n experimental and control groups is determined, as with Spirostomum, by the percent difference in initial total leaflet colsure to stimulation. F o r each chemical tested, 2 pinnae from 12 different plants were each used for the experimenta_l and control groups. Each chemical was tested for 1 hr with Mimosa and 30
TABLE 1
DRUGS AND MOVEMENT IN MIMOSA AND SPIROSTOMUM
Mimosa
Mimosa
Stimulus
Mimosa
Spirostomum Stimulus
Closure to
Opening to
Chemical
Sensitivity
Sensitivity
Dark
Light
G.A.
Normal
Normal
Normal
Fast(11 min)
10-3
10-s
A.A.
Normal
Normal
Normal
Normal
10-4
10- s
I.A.A.
Less(75%)
Less(20%)
Slow(19 min)
Fast(15 min)
10-4
10- s
P.I.B.A.
Normal
Normal
Normal
Normal
10- 3
10-4
Kinetin
Normal
Normal
Normal
Normal
10-*
10-4
Ethanol
Normal
Normal
Slow(8 min)
Fast(13 min)
10-1
10-1
Xylocaine
Less(87%)
Less(53%)
Slow(27 min)
Normal
2x10- 2
10-3
Librium
Normal
Normal
Normal
Normal
10- 3
10- s
Tubocurarine
Less(50%)
Less(81%)
Slow(24 min)
Normal
10-4
5x10- s
Strychnine
Less(82%)
Less(70%)
Slow(8 min)
Normal
5x10- 3
5x10 -3
Caffeine
Normal
Normal
Slow(12 min)
Normal
5x10- 3
5x10 -3
Picrotoxin
Less(49%)
Normal
Slow(10 min)
Normal
5x10- 3
10- s
Molarity
Mimosa Spirostomum
*Normal means no significant difference (p>O.05 two-tailed Wilcoxon test) between experimental and control groups: Less, Slow, and Fast mean a significant difference (p
MIMOSA, SPIROSTOMUM AND DRUGS
871
pinnae were put in the dark, where they would not open, with the chemical for 45 min, then the lights were turned on. The time to open fully for both groups was recorded. By the time the controls began to close or open, about 60 min would pass and this closing or opening response would be based on approximately the same amount of time of exposure to the chemical as for the mechanical stimulation responses, allowing direct comparisons to be made. RESULTS AND DISCUSSION The results are presented in Table 1. The Mimosa Stimulus Sensitivity column is computed by subtracting what percentage of the 24 pinnae that received the drug closed to the stimulus from the percentage of control pinnae that did also. The third column for Spirostomum was computed the same way. The fourth and fifth columns are obtained by taking the mean absolute difference between the time of the controls' closure to the dark or opening to the light, respectively, and the experimental's time of response. The reported molarity of the drugs is close to the maximum that could be used. Dose-response curves were also obtained and for the effective drugs were sigmoid curves on a semilog plot. With regard to the chemicals' effects on sensitivity to stimulation, it is apparent there is a high degree of correspondence between such diverse organisms as Mimosa and Spirostomum, the exception being the stimulant picrotoxin, which affects only Mimosa. Not only do drugs that exert effects on animals have similar effects on protozoa and plants but a plant growth hormone also affects a protozoan. Furthermore, with the exception of strychnine, which appears to poison permanently the organisms, all the sensitivity effects are at least 85% reversible after washing; meaning 85% of the pinnae and protozoan respond normally after 1 hr in new media. Of course, some of the drugs used may have no effect simply because they are not taken up by the organisms. The results from the closure to dark and opening to light
experiments, when compared to the effect drugs have on sensitivity, indicate these three processes are somewhat independent of each other, and the chemicals are not simultaneously affecting all three kinds (response to stimulation, light, dark) of plant behavior in identical ways. If Mimosa is made less sensitive to stimulation, it also takes longer to close to dark but the converse is not necessarily true. Furthermore, the time for Mimosa to open to light is unrelated to its touch sensitivity or time to close to dark. The fact these drugs speed up opening to light indicates their action is not just merely one of slowing down leaflet movement. Even with the stimulants, strychnine, caffeine and picrotoxin, sensitivity to stimulation cannot be increased for Mimosa or Spirostomum (nor is there a faster closure to dark, or a slower opening to light). The effects on Spirostomum and Mimosa movement may either be at the level of receptors or effectors. It is not known what the former are for either organism. However, with the exception of I.A.A., the substances which produce a lessened sensitivity to stimulation are known to produce m u s c l e r e l a x a t i o n in vertebrates [8] and since actomyosin-like substances are found in both organisms [2], it seems likely the effectors of movement are involved. Since leaflet movement presumably involves changes in ion flow in the motor cells located in the pulvini of Mimosa [9] or in the compartments adjacent to the contractile myonemes in Spirostomum [4], membrane permeability is changed in some way by these chemicals. The conclusion would have to be that on a biochemical level, plants and animals would not appear that different from one another in their response to drugs. Since a structural nervous system is obviously absent in Mimosa and Spirostomum the correspondence with those possessing one is even more striking. This suggests explanations of drug actions on behavior based solely on neurological concepts (e.g., curare and neuromuscular junctions, picrotoxin and pre-synaptic inhibition) may be inadequate.
REFERENCES 1. Applewhite, P. B. Nonlocal nature of habituation in a rotifer and protozoan. Nature 217: 287-288, 1968. 2. Applewhite, P. B. Behavioral plasticity in the sensitive plant, Mimosa. Behav. Biol. 7: 47-53, 1972. 3. Applewhite, P. B. and F. Gardner. Rapid leaf closure of Mimosa in response to light. Nature 233: 279-287, 1971. 4. Applewhite, P. B. and F. Gardner. Theory of protozoan habituation. Nature New Biology 230: 285-287, 1971. 5. Bose, J. C. Plant Response as a Means of Physiological Invest~'gation. London: Longmans, Green and Co., 1906. 6. Bose, J. C. The Motor Mechanism of Plants. London: Longmans, Green and Co., 1928.
7. Galston, A. W. and P. J. Davies. Control Mechanisms in Plant Development. Englewood Cliffs, New Jersey: Prentice-Hall, 1970. 8. Goodman, L. S. and A. Gilman. The Pharmacological Basis of Therapeutics. New York: Macmillan, 1970. 9. Sibaoka, T. Physiology of rapid movements in higher plants. Ann. Rev. Plant Physiology 20: 165-184,1969. 10. Tartar, V. The Biology of Stentor, New York: Pergamon Press, 1961. 11. Williams, C. N. and V. Raghavan. Effects of light and growth substances on the diurnal movements of the leaflets of Mimosa pudiea. J. Exp. Botany 17: 742-749, 1966.