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Stomatal Closure in Response to Acetylsalicylic Acid Treatment
Rama de Bodnica, Colegio de Postgraduados, Chapingo, Mexico, Mexico
Stomatal Closure in Response to Acetylsalicylic Acid Treatment A. LARQUt-SAAVEDRA With 1 figure Received January 5, 1979 . Accepted February 2, 1979
Summary Previous work showed that acetylsalicylic acid (ASA) reduces transpiration rate of beans (Phaseolus vulgaris 1.). A concentration response curve of stomatal closure of epidermal strips of Commelina communis 1., to ASA is now presented. It is found that ASA closes stomata at 10-2-10- 3 M concentrations and that this closure takes place within 13 minutes after the treatment has started.
Key words: transpiration rate, ASA treatment, Commelina communis.
Introduction Several compounds have been shown to close stomata and to reduce transpiration. Abscisic acid (ABA) has been called the ideal antitranspirant because it is involved in the endogenous control of stomata and probably in the drought resistance mechanisms of plants (WRIGHT, 1972; MANSFIELD, 1976; LARQut-SAAVEDRA, 1974, 1976). Furthermore it has ben found to be very efficient in closing stomata when externally applied to leaves (JONES and MANSFIELD, 1972). The presence of cyclic nucleotides in plants has been suspected, but not definitely proved as yet (WELLBURN et aI., 1973; LIN, 1974). Nevertheless, the inhibitory action of ABA is enhanced when cyclic adenosine monophosphate is applied at the same time (BIANCO and BULARD, 1974; LARQUt-SAAVEDRA, unpublished results). Prostaglandins, a series of compounds derived from prostanoic acid, are known to have a profound effect on cyclases in animal tissue (HINMAN, 1972). Thus, it was considered, that, although prostaglandins are not known to occur in plants, they may have a physiological action on plant tissue (LARQUt-SAAVEDRA, 1979). It was therefore intended to prevent its action by using ASA which is a known prostaglandin antagonist. In a previous study ASA was found to reduce the transpiration rate of beans (LARQUt-SAAVEDRA, 1978). Such reduction was determined using detached leaves from bean seedlings fed with the chemical via their petioles. In the present Abbreviations: ASA
=
acetylsalicylic acid; ABA
=
abscisic acid.
Z. PJlanzenphysiol. Bd. 93. S. 371-375. 1979.
372
A. LARQut-SAAvEDRA
work the direct effect of ASA on stomatal aperture using epidermal strips of Com-
melina communis L. was investigated. Materials and Methods Plants of Commelina communis L. (Seeds from T. A. Mansfield, University of Lancaster, U. K.) were grown in a greenhouse (20-30 DC/18-20 DC and 14 h daylight photoperiod) for a 6-week period. The fourth, fifth and sixth leaves, fully expanded, were removed under water and left to float lower side down on distilled water in the light [11,700 lux from 4 daylight fluorescent lamps (20 W) and 2 tungsten bulbes (250 W)] for 3 h to allow the stomata to open. Strips of the lower epidermis were removed and pieces of about 2 X 2 mm were transferred to vials containing the solutions to be tested. Each vial contained a citratecitric acid buffer pH 4.5, 0.2 M (330 milliosmoles) with ASA at different concentrations from zero to 10-2 M. For comparision purposes a solution of 10-4 M ABA was applied simultaneously. The vials were then incubated in a water bath at 23 ± 0.5 DC for 1 h (time selected from the literature and our preliminary experiments) with CO 2 -free air bubbled into each vial at a rate of 100 mUmin for further stomatal opening. The light intensity at the level of the vials was aproximately 11,500 lux. At the end of the period, the epidermal pieces were observed under the microscope and the apertures of stomata recorded, with 10 readings for each piece of epidermis. To study the time required for ASA to induce stomatal closure, epidermal strips with fully open stomata were transfered to concave microscope slides in buffer with or without 10-2 or 10-3 M ASA. Apertures of 10 stomata were recorded initially and at different times thereafter. Results
Effect of different ASA concentrations on stomatal aperture A series of experiments were run to determine whether ASA could affect stomatal aperture of epidermal strips. Abscisic acid (ABA) was used only for comparlSlOn purposes for its well-known effect of closing stomata 10 Commelina (OGUMKANMI et aI., 1973). As the data show in table 1, ASA does dose stomata at 10-2 and 10-3 M concentrations. A 50 % reduction in stomatal aperture is obtained at concentrations between 10- 4 and 10- 5 M. Moreover 10-6 M ASA reduces stomatal aperture significantly by 20 Ofo of the buffer control. More dilute concentrations (10- 7-10- 8 M) tend to open stomata further thant he buffer control.
The time required for ASA to induce stomatal closure Next an experiment was carried out in order to know the time required for high concentrations of ASA to induce dosing of stomata. The graph in figure 1 illustrates the time course effect of 10-2 and 10-3 M ASA on stomatal aperture. In ,the figure each curve represents the mean value of 10 stomata and the suddenness of ASA's effect is clearly seen. At a 10-2 M con centraZ. Pflanzenphysiol. Bd. 93. S. 371-375. 1979.
Stomatal closure in response to ASA Table 1: Stomatal aperture (,urn strips of Commelina communis L Experiment
2
X
of control.
%
10-2 M 0 0 0 0
±
S,E) as affected by acetylsalicylic acid in epidermal ASA Concentration 10-5 M 10-6 M
to- 3 M 10-4 M 0 0 0 0
8.2±0.93 9.5 ±0.37 8.8 36
ASA Concentration 10-8 M 10-1 M 1 2
X
of control.
%
373
25.1 ± 0.38 26.2 ±0.49 25.6 105
25.3 ±0.27 26.2±0.38 25.9 106
13,8 ± 1.17 14.5± 1.75 14.1 58
20.7 ± 0.44 18.5 ± 0.49 19.6 80
Buffer control
ABA 10-4 M 2.0±0.10 3.5±0,10 2.7 11
23.0±0.24 25,6±0.30 24.3 100
20
...•.
• 12 :::I
Q.
a
.. ..
a
8
a E
o
(I)
4
o
5
10
15
20
Incubation
30 period
40
50
60
70
80
90
100
(minutes)
Fig. 1: The time required for ASA at two different concentrations to induce stomatal closure (urn ± SD) of epidermal strips of Commelina communis L
Z. PJlanzenphysiol, Bd. 93. S. 371-375. 1979.
374
A. LARQUE-SAAVEDRA
tion, ASA takes about 13 minutes to close totally the stomata. At 10 minutes, when the response was noted, the average diameter was 3 f-lm. ASA at 10-3 M took 75 minutes to induce stomatal closure, and at 70 minutes after the treatment had started the first closing was observed. The latter result differs radically from the one obtained for the same concentration using the incubating method with vials for the dose response curve. Discussion A clear proof that ASA can close stomata is reported here. Stomatal apertures of
Commelina obtained for the present work were in general terms similar (RASCHKE, et aI., 1975), or greater than those reported by others. (WILLMER and MANSFIELD, 1969; OGUNKANMI et aI., 1973 ; TRAVIS and MANSFIELD, 1977).
It was reported that 10-3 M ASA had an equivalent action to 5 X 10-5 M ABA for transpiration rare in detached bean seedlings (LARQUE-SAAVEDRA, 1978). In the present work I found that a 10-4 M ABA is equivalent to an ASA concentration between 10- 3 and 10-4 M, which supports the previous finding. Using the concave microscope slide to determine the time required for ASA to induce stomatal closure, it was found that the time was different from that of the dose response curve. The 75 minutes closing period for a 10-3 M ASA is longer than the time required· in vials. This might be due to the fact that no CO 2-free air was bubbled through the medium and the temperature and illuminating conditions were altered. The strips remained floating with the upper side in contact with air whilst only the lower side was in contact with the ASA solution; thus it was only through this surface that ASA could enter the cells. Nevertheless the results obtained give a good indication as ,to how ASA might act. ITAI et ai. (1978) have found that ABA takes 9 minutes to close stomata. However, the slope of the curve is not mentioned. In this paper I show that curve for stomatal closure induced by ASA is very steep. Possibly as ASA penetrates into the guard cells, an unknown threshold concentration is required before closure takes place. It is worth mentioning that a preliminary assay to determine if the guard cells were alive after ASA treatment was carried out using. A. J. TRAVIS'S technique for stomatal studies in detached epidermis. Cytoplasmic streaming and neutral red uptake were observed in the guard cells after a 10- 3 M ASA treatment, and not definite damage was noticeable. A series of experiments are being carried out in this laboratory to elucidate the mechanism of action of ASA. The author wishes to thank Mr. A. J. TRAVIS for showing his technique and help, and to Professor T. A. MANSFIELD for reading and correcting the manuscript. The suggestions of Dr. E. M. ENGLEMAN are appreciated as well as the technical assistance of Mrs. T. AGUILAR.
z. Pjlanzenphysiol. Bd. 93. S. 371-375. 1979.
Stomatal closure in response to ASA
375
References BIANCO, J. and C. BULARD: AMP cyclique et germination des semences de laitue «Grand Rapids»: Interactions avec gibberellines et acide abscissique. Z. Pflanzenphysio!. 74, 160167 (1974). HINMAN, J. W.: Prostaglandins. Ann. Rev. Biochem. 41,161-178 (1972). ITAI, c., J. D. B. WEYERS, J. R. HILLMAN, H. MEIDNER, and C. WILLMER: Abscisic acid and guard cells of Commelina communis L. Nature 271, 652-653 (1978). LARQuE-SAA VEDRA, A. and R. L. WAIN: Abscisic acid in relation to drought tolerance in varieties of Zea mays L. Nature 251, 716-717 (1974). - - Studies on plant growth regulating substanc.es. XLII. Abscisic acid as a genetic character related to drought tolerance. Ann. App!. Bio!. 83, 291-299 (1976). LARQUE-SAAVEDRA, A.: Studies on hormonal aspects of plant growth in relation to chemical and environmental treatments. Ph. D. Thesis, University of London, 1975. - The antitranspirant effect of acetylsalicylic acid on Phaseolus vulgaris L. Physio!. Plant. 43, 126-128 (1978). - Studies on the effect of prostaglandins on four plant bioassay systems. Z. Pflanzenphysio!. (1979) in the press. LIN, pp-c.: Cyclic nucleotides in plants In: P. GREENGARD and G. A. ROBINSON (Eds.): Advanc Cyc!. nucleotides Res. Vo!' 4, pp. 439-461. Raven Press, New York, 1974. MANSFIELD, T. A.: Stomatal behaviour. Chemical control of stomatal movements. Phil. Trans. R. Soc. Lond. B. 273, 541-550 (1976). OGUNKANMI, A. B., D. J. TUCKER, and T. A. MANSFIELD: An improved bioassay for abscisic acid and other antitranspirants. New Phytol. 73,277-282 (1973). RASCHKE, K., R. D. FIRN, and M. PIERCE: Stomatal closure in response to xanthoxin and abscisic acid. Planta (Berl.) 125, 149-160 (1975). TRAVIS, A. J. and T. A. MANSFIELD: Studies of malate formation in isolated guard cells. New Phyto!. 78, 541-546 (1977). WELLBURN, A. T., J. P. ASHBY, and F. A. M. WELLBURN: Occurrence and biosynthesis of adenosine 3': 5'-cyclic monophosphate in isolated Avena etioplasts. Biochem. Biophys. Acta 320, 363-371 (1973). WILLMER, C. M. and T. A. MANSFIELD: A critical examination of the use of detached epidermis in studies of stomatal physiology. New Phyto!' 68, 363-375 (1969). WRIGHT, S. T. c.: Physiological and biochemical responses to wilting and other stress conditions. In: A. R. REEs (Ed.): Crop Processes in Controlled Environments. Applied Bot. Series. vol. II, pp. 349-361. Academic Press, 1972. A. LARQUE-SAAVEDRA, Rama de Botanica, Colegio de Postgraduados, Chapingo, Mexico, Mexico
Z. PJlanzenphysiol. Bd. 93. S. 371-375. 1979.
Stomatal Closure in Response to Acetylsalicylic Acid Treatment A. LARQUt-SAAVEDRA With 1 figure Received January 5, 1979 . Accepted February 2, 1979
Summary Previous work showed that acetylsalicylic acid (ASA) reduces transpiration rate of beans (Phaseolus vulgaris 1.). A concentration response curve of stomatal closure of epidermal strips of Commelina communis 1., to ASA is now presented. It is found that ASA closes stomata at 10-2-10- 3 M concentrations and that this closure takes place within 13 minutes after the treatment has started.
Key words: transpiration rate, ASA treatment, Commelina communis.
Introduction Several compounds have been shown to close stomata and to reduce transpiration. Abscisic acid (ABA) has been called the ideal antitranspirant because it is involved in the endogenous control of stomata and probably in the drought resistance mechanisms of plants (WRIGHT, 1972; MANSFIELD, 1976; LARQut-SAAVEDRA, 1974, 1976). Furthermore it has ben found to be very efficient in closing stomata when externally applied to leaves (JONES and MANSFIELD, 1972). The presence of cyclic nucleotides in plants has been suspected, but not definitely proved as yet (WELLBURN et aI., 1973; LIN, 1974). Nevertheless, the inhibitory action of ABA is enhanced when cyclic adenosine monophosphate is applied at the same time (BIANCO and BULARD, 1974; LARQUt-SAAVEDRA, unpublished results). Prostaglandins, a series of compounds derived from prostanoic acid, are known to have a profound effect on cyclases in animal tissue (HINMAN, 1972). Thus, it was considered, that, although prostaglandins are not known to occur in plants, they may have a physiological action on plant tissue (LARQUt-SAAVEDRA, 1979). It was therefore intended to prevent its action by using ASA which is a known prostaglandin antagonist. In a previous study ASA was found to reduce the transpiration rate of beans (LARQUt-SAAVEDRA, 1978). Such reduction was determined using detached leaves from bean seedlings fed with the chemical via their petioles. In the present Abbreviations: ASA
=
acetylsalicylic acid; ABA
=
abscisic acid.
Z. PJlanzenphysiol. Bd. 93. S. 371-375. 1979.
372
A. LARQut-SAAvEDRA
work the direct effect of ASA on stomatal aperture using epidermal strips of Com-
melina communis L. was investigated. Materials and Methods Plants of Commelina communis L. (Seeds from T. A. Mansfield, University of Lancaster, U. K.) were grown in a greenhouse (20-30 DC/18-20 DC and 14 h daylight photoperiod) for a 6-week period. The fourth, fifth and sixth leaves, fully expanded, were removed under water and left to float lower side down on distilled water in the light [11,700 lux from 4 daylight fluorescent lamps (20 W) and 2 tungsten bulbes (250 W)] for 3 h to allow the stomata to open. Strips of the lower epidermis were removed and pieces of about 2 X 2 mm were transferred to vials containing the solutions to be tested. Each vial contained a citratecitric acid buffer pH 4.5, 0.2 M (330 milliosmoles) with ASA at different concentrations from zero to 10-2 M. For comparision purposes a solution of 10-4 M ABA was applied simultaneously. The vials were then incubated in a water bath at 23 ± 0.5 DC for 1 h (time selected from the literature and our preliminary experiments) with CO 2 -free air bubbled into each vial at a rate of 100 mUmin for further stomatal opening. The light intensity at the level of the vials was aproximately 11,500 lux. At the end of the period, the epidermal pieces were observed under the microscope and the apertures of stomata recorded, with 10 readings for each piece of epidermis. To study the time required for ASA to induce stomatal closure, epidermal strips with fully open stomata were transfered to concave microscope slides in buffer with or without 10-2 or 10-3 M ASA. Apertures of 10 stomata were recorded initially and at different times thereafter. Results
Effect of different ASA concentrations on stomatal aperture A series of experiments were run to determine whether ASA could affect stomatal aperture of epidermal strips. Abscisic acid (ABA) was used only for comparlSlOn purposes for its well-known effect of closing stomata 10 Commelina (OGUMKANMI et aI., 1973). As the data show in table 1, ASA does dose stomata at 10-2 and 10-3 M concentrations. A 50 % reduction in stomatal aperture is obtained at concentrations between 10- 4 and 10- 5 M. Moreover 10-6 M ASA reduces stomatal aperture significantly by 20 Ofo of the buffer control. More dilute concentrations (10- 7-10- 8 M) tend to open stomata further thant he buffer control.
The time required for ASA to induce stomatal closure Next an experiment was carried out in order to know the time required for high concentrations of ASA to induce dosing of stomata. The graph in figure 1 illustrates the time course effect of 10-2 and 10-3 M ASA on stomatal aperture. In ,the figure each curve represents the mean value of 10 stomata and the suddenness of ASA's effect is clearly seen. At a 10-2 M con centraZ. Pflanzenphysiol. Bd. 93. S. 371-375. 1979.
Stomatal closure in response to ASA Table 1: Stomatal aperture (,urn strips of Commelina communis L Experiment
2
X
of control.
%
10-2 M 0 0 0 0
±
S,E) as affected by acetylsalicylic acid in epidermal ASA Concentration 10-5 M 10-6 M
to- 3 M 10-4 M 0 0 0 0
8.2±0.93 9.5 ±0.37 8.8 36
ASA Concentration 10-8 M 10-1 M 1 2
X
of control.
%
373
25.1 ± 0.38 26.2 ±0.49 25.6 105
25.3 ±0.27 26.2±0.38 25.9 106
13,8 ± 1.17 14.5± 1.75 14.1 58
20.7 ± 0.44 18.5 ± 0.49 19.6 80
Buffer control
ABA 10-4 M 2.0±0.10 3.5±0,10 2.7 11
23.0±0.24 25,6±0.30 24.3 100
20
...•.
• 12 :::I
Q.
a
.. ..
a
8
a E
o
(I)
4
o
5
10
15
20
Incubation
30 period
40
50
60
70
80
90
100
(minutes)
Fig. 1: The time required for ASA at two different concentrations to induce stomatal closure (urn ± SD) of epidermal strips of Commelina communis L
Z. PJlanzenphysiol, Bd. 93. S. 371-375. 1979.
374
A. LARQUE-SAAVEDRA
tion, ASA takes about 13 minutes to close totally the stomata. At 10 minutes, when the response was noted, the average diameter was 3 f-lm. ASA at 10-3 M took 75 minutes to induce stomatal closure, and at 70 minutes after the treatment had started the first closing was observed. The latter result differs radically from the one obtained for the same concentration using the incubating method with vials for the dose response curve. Discussion A clear proof that ASA can close stomata is reported here. Stomatal apertures of
Commelina obtained for the present work were in general terms similar (RASCHKE, et aI., 1975), or greater than those reported by others. (WILLMER and MANSFIELD, 1969; OGUNKANMI et aI., 1973 ; TRAVIS and MANSFIELD, 1977).
It was reported that 10-3 M ASA had an equivalent action to 5 X 10-5 M ABA for transpiration rare in detached bean seedlings (LARQUE-SAAVEDRA, 1978). In the present work I found that a 10-4 M ABA is equivalent to an ASA concentration between 10- 3 and 10-4 M, which supports the previous finding. Using the concave microscope slide to determine the time required for ASA to induce stomatal closure, it was found that the time was different from that of the dose response curve. The 75 minutes closing period for a 10-3 M ASA is longer than the time required· in vials. This might be due to the fact that no CO 2-free air was bubbled through the medium and the temperature and illuminating conditions were altered. The strips remained floating with the upper side in contact with air whilst only the lower side was in contact with the ASA solution; thus it was only through this surface that ASA could enter the cells. Nevertheless the results obtained give a good indication as ,to how ASA might act. ITAI et ai. (1978) have found that ABA takes 9 minutes to close stomata. However, the slope of the curve is not mentioned. In this paper I show that curve for stomatal closure induced by ASA is very steep. Possibly as ASA penetrates into the guard cells, an unknown threshold concentration is required before closure takes place. It is worth mentioning that a preliminary assay to determine if the guard cells were alive after ASA treatment was carried out using. A. J. TRAVIS'S technique for stomatal studies in detached epidermis. Cytoplasmic streaming and neutral red uptake were observed in the guard cells after a 10- 3 M ASA treatment, and not definite damage was noticeable. A series of experiments are being carried out in this laboratory to elucidate the mechanism of action of ASA. The author wishes to thank Mr. A. J. TRAVIS for showing his technique and help, and to Professor T. A. MANSFIELD for reading and correcting the manuscript. The suggestions of Dr. E. M. ENGLEMAN are appreciated as well as the technical assistance of Mrs. T. AGUILAR.
z. Pjlanzenphysiol. Bd. 93. S. 371-375. 1979.
Stomatal closure in response to ASA
375
References BIANCO, J. and C. BULARD: AMP cyclique et germination des semences de laitue «Grand Rapids»: Interactions avec gibberellines et acide abscissique. Z. Pflanzenphysio!. 74, 160167 (1974). HINMAN, J. W.: Prostaglandins. Ann. Rev. Biochem. 41,161-178 (1972). ITAI, c., J. D. B. WEYERS, J. R. HILLMAN, H. MEIDNER, and C. WILLMER: Abscisic acid and guard cells of Commelina communis L. Nature 271, 652-653 (1978). LARQuE-SAA VEDRA, A. and R. L. WAIN: Abscisic acid in relation to drought tolerance in varieties of Zea mays L. Nature 251, 716-717 (1974). - - Studies on plant growth regulating substanc.es. XLII. Abscisic acid as a genetic character related to drought tolerance. Ann. App!. Bio!. 83, 291-299 (1976). LARQUE-SAAVEDRA, A.: Studies on hormonal aspects of plant growth in relation to chemical and environmental treatments. Ph. D. Thesis, University of London, 1975. - The antitranspirant effect of acetylsalicylic acid on Phaseolus vulgaris L. Physio!. Plant. 43, 126-128 (1978). - Studies on the effect of prostaglandins on four plant bioassay systems. Z. Pflanzenphysio!. (1979) in the press. LIN, pp-c.: Cyclic nucleotides in plants In: P. GREENGARD and G. A. ROBINSON (Eds.): Advanc Cyc!. nucleotides Res. Vo!' 4, pp. 439-461. Raven Press, New York, 1974. MANSFIELD, T. A.: Stomatal behaviour. Chemical control of stomatal movements. Phil. Trans. R. Soc. Lond. B. 273, 541-550 (1976). OGUNKANMI, A. B., D. J. TUCKER, and T. A. MANSFIELD: An improved bioassay for abscisic acid and other antitranspirants. New Phytol. 73,277-282 (1973). RASCHKE, K., R. D. FIRN, and M. PIERCE: Stomatal closure in response to xanthoxin and abscisic acid. Planta (Berl.) 125, 149-160 (1975). TRAVIS, A. J. and T. A. MANSFIELD: Studies of malate formation in isolated guard cells. New Phyto!. 78, 541-546 (1977). WELLBURN, A. T., J. P. ASHBY, and F. A. M. WELLBURN: Occurrence and biosynthesis of adenosine 3': 5'-cyclic monophosphate in isolated Avena etioplasts. Biochem. Biophys. Acta 320, 363-371 (1973). WILLMER, C. M. and T. A. MANSFIELD: A critical examination of the use of detached epidermis in studies of stomatal physiology. New Phyto!' 68, 363-375 (1969). WRIGHT, S. T. c.: Physiological and biochemical responses to wilting and other stress conditions. In: A. R. REEs (Ed.): Crop Processes in Controlled Environments. Applied Bot. Series. vol. II, pp. 349-361. Academic Press, 1972. A. LARQUE-SAAVEDRA, Rama de Botanica, Colegio de Postgraduados, Chapingo, Mexico, Mexico
Z. PJlanzenphysiol. Bd. 93. S. 371-375. 1979.