Planta (Berl.) 122, 307--310 (1975) 9 by Springer-Verlag 1975
Short Communication The Effect of Vomifoliol on Stomatal Aperture K. L. Stuart and L. B. Coke Departments of Chemistry and of Botany, University of the ~Vest Indies, Mona, Kingston 7, Jamaica Received 11 November; accepted 10 December 1974
Summary. Vomifoliol, a compound related to abscisie acid (ABA) but lacking the 2,4-pentadiene sidechain and so far found in four different plant families, caused as much stomatal closure in epidermal strips of Eichhornia crassipes (Mart.) Solms as ABA at identical concentrations. It appears worthwhile to investigate whether vomifoliol plays a role as an endogenous regulator of stomatal aperture. Vomifoliol has been isolated from Rauwol/ia vomitoria, Apocynaceae (Pouset and Poisson, 1969), three Croton species, Euphorbiaceae (Satish and Bhakuni, 1972; Stuart and Woo-Ming, 1975) and Palicourea alpina (Sw.) I)C, Rubiaceae (Stuart and Woo-Ming, 1975). Blumenol A from Podocarpus blumei, Podocarpaceae (Galbraith and Horn, 1972), has also been identified as vomifoliol (Bhakuni et al., 1974; Stuart and Woo-Ming, 1975). This latter finding defines the stereochemistry of vomifoliol as shown in structure I. Bhakuni et al. (1974) have also reported the occurrence of roseoside in Vinca rosea, Apocynaceae, and established it as the fl-glucoside of vomifoliol. A recent study of endogenous antitranspirants in waterstressed Sorghum plants (Ogunkanmi et al., 1974) reported the effect of trans-farnesol on stomata, but also indicated that further work was necessary to verify that the only active compound was in fact all-trans-farnesol. On the basis of the separation procedure described, it is possible that vomifoliol could be a contaminant of the farnesol fraction, and this should be investigated in the light of our findings. The bioassay method developed by Tucker and Mansfield (1971) for demonstrating the activity of ABA on stomatal closure was modified for our studies. Phosphate buffer was preferred to the citrate buffer recommended by Ogunkanmi et al. (1973) because of its superior stability. The plant Eichhornia crassipes (Mart.) Solms (water hyacinth) was used as it promised certain advantages. As a floating aquatic plant which has been growing in a permanent fresh-water pond at the Botany Department, University of the West Indies, Mona, Jamaica, it is not subject to severe water deficit, one of the principal factors which increases internal levels of ABA and reduces stomatal responsiveness. Epidermal strips were taken from the abaxial surface of the youngest fully expanded leaf on a non-flowering shoot of Eichhornia crassipes. All strips required for a group of assays were obtained from a single leaf as this was more than 100 cm2 in area. Since the plants are vegetatively propagated, genetic constancy in the material is assured. The pores are simple in profile, large, and aligned in near parallel rows which are convenient for measurement. Test solutions of 10-8 M ABA, purchased from ICN-K~K Laboratories~ Inc., Plainview,
308
K.L. Stuart and L. B. Coke
N.Y., USA (batch No. 8594A)1, and of vomifoliol, isolated from Palicourea alpina by a combination of counter-current and chromatographic techniques (Stuart and Woo-Ming, 1975), were prepared by dissolving crystalline material in not more than 0.1 ml 98% ethanol and making up to 5 ml with 0.10 M NaNO8 in 0.01 M KH~PO4 buffer at pH 5.7. Serial dilutions from the stock solution provided concentrations ranging from 10-4 to 10-11M. A number of epidermal strips, 20-30 mm~, were obtained, floated in the pH 5.7 btfffer, illuminated by cool-white fluorescent lamps at 1800 lx, and aerated with a stream of COs-flee air, 20-30 cm3/ rain at 274-1 ~ for 2.5 h before incubation. At intervals of 10 rain, batches of three strips were transferred into 4.5 ml of ~est solutions of varying concentrations in glass vials. These were subjected to illumination and aeration conditions identical to those stated above, and incubation lasted 2 h. For each vial, the strips were then transferred to glass slides in a drop of test solution and the widths of 25 stomatal pores on each strip measured at 480foldmagnification with the aid of an eye-piece micrometer. A series of control treatments, consisting of the buffer solutions and identical ethanol concentrations as present in the test solutions, were performed concurrently with the test treatments. A total of six complete experiments were performed, and in some cases variations were made to aeration rates and light intensity. The reported data are for results obtained in conditions which seem optimal. Stomatal aperture showed a log-linear response to both ABA and vomifoliol (Fig. 1). The range of sensitivity between 10-4 and 10-1~ M is slightly better than that reported by Tucker and Mansfield (1971) for a Commelina epidermal strip assay at p H 6.7 and by Ogunkanmi et al. (1973) for piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer at p i t 6.8, and is comparable to the sensitivity obtained b y the latter authors with citrate buffer (pH 5.5). Regression of stomatal aperture on concentration calculated over the entire range of results (for ABA, y : 0 . 1 9 3 x @ 3.3496; for vomifoliol, y : 0 . 3 0 2 4 x ~ 2.4645) showed highly significant ( p : 0 . 0 1 ) linear responses for both substances. Comparison of the slopes of the two lines showed no significant difference at T=O.05. Stomatal aperture in Eichhornia was therefore equally sensitive to ABA and to vomifoliol. These results could be of considerable value if this effect of vomifoliol was not also accompanied by the other known effects of ABA such as senescence and abscission (Wareing and Rybaek, 1970) and further experiments using synthetic (4-)-vomifoliol (Weiss et al., 1973) are planned. One of these is a time-course study similar to that reported for ABA b y Cummins et al. (1971) since it is conceivable that the 2-h incubation period was sufficient for vomifoliol to be conver~ed to ABA. A n y biogenetic relationship between vomifoliol and ABA must await biosynthetic studies, but it is of interest t h a t conversion of vomifoliol to ABA has been achieved in vitro (Galbraith and Horn, 1973). Other naturally occurring compounds, like cis.trans-xanthoxin (Taylor and Burden, 1970), which seems to have limited distribution, and some synthetic models (Sondheimer and Walton, 1970; MeWha et al., 1973) have been demonstrated to possess varying levels of ABA activity, but vomifoliol is the first compound to our knowledge which has a modified 2,4-pentadiene side chain and has activity equal to that displayed by ABA. Our findings should also arouse interest in the possible biological activity of compounds such as blumenol B (Galbraith and Horn, 1973), 3-oxo-~-ionol (Aasen et al., 1971) and 4-(1-hydroxy-4-keto-2,6,6-trimethyl-2-cyelohexen-l-yl)-3-buten2-one (Roberts et al., 1968). 1 (4-)-ABA.
Vomifolioh Effect on Stomata
309
I.
6-
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6-
--.r
s
4
4
3
3
2
2
I
I
'~~CONTROL
t-.-w a.
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0
o -I tl -II-I0-9-8 -7-6 -5-4 -I0-9-8-7- 6 -5 -4 I.OGIo CONCENTRATION[M] LOGIo CONCENTRATION[M] ABA VOMIFOLIOL '
i
,
,
i
,
i
Fig. 1. The effect of ABA (9 and vomifoliol ( . ) on stomatal aperture in epidermal strips of Eichhornia vrassipes in 0.1 M NAN03+0.01 M KH2POa buffer at pH 5.7. Vertical lines = standard errors
We thank Mr. Rex Woo-Ming for the isolation work which made samples of vomifoliol available for these experiments. References Aasen, A.J., Kimland, B., Enzell, C.R.: Tobacco chemistry. 7. Structure and synthesis of 3-oxo-cc-ionol a new tobacco constituent. Acta chem. scand. 25, 1481-1482 (1971) Bhakuni, D.S., Joshi, P.P., Uprcty, H., Kapil, R. S. : Roseoside-a C13 glycoside from Vinca rosea. Phytochemistry 18, 2541-2543 (1974) Cummins, W.R., Kende, H., Raschke, K. : Specificity and reversibility of the rapid stomatal response to abscisic acid. Planta (Berl.) 99, 347-351 (1971) Galbraith, M.N., Horn, D.H.S.: Structures of the natural products blumenols A, B and C. J.C.S. Chem. Comm. 1972, 113-114 Galbraith, M.N., Horn, D.H.S.: Stereochcmistry of the blumenols. Conversion of blumenol A into (S)-(+)-abscisie acid. J.C.S. Chem. Comm. 1973, 566-567 1KeWha, J.A., Philipson, J.J., Hillman, J.R., Wilkins, M.B.: Molecular requirements for abscisic acid activity in two bioassay systems. Planta (Berl.) 109, 327-336 (1973) Ogunkanmi, A.B., Tucker, I).J., Mansfield, T.A.: An improved bioassay for abseisic acid and other antitranspirants. New Phytol. 72, 277-282 (1973) Ogunkanmi, A.B., Wellburn, A.R., Mansfield, T.A. : Detection and preliminary identification of endogenous antitranspirants in water-stressed Sorghum plants. Planta (Berl.) 117, 293-302 (1974) Pouset, J.-L., Poisson, J.: Vomifolioh alcool terpenique isole des feuilles du Rauwolfia vomitoria Mz. Tetrahedron Letters 1969, 1173-1174 21 Planta(Berl.), Vol. 122
310
K.L. Stuart and L. B. Coke
Roberts, D.L., Heckman, R.A., Hege, B.P., Bellin, S.A.: Synthesis of (RS)-abscisie acid. J. org. Chem. 83, 3566-3569 (1968) Satish, S., Bhakuni, D.S.: Constituents of Indian and other plants. Phytochemistry 11, 2888-2890 (1972) Sondheimer, E., Walton, D.C.: Structure-activity correlations with compounds related to abscisie acid. Plant Physiol. 45, 244-248 (1970) Stuart, K.L., Woo-Ming, R.B.: Vomifoliol in Croton and Palicourea species. Phytochemistry 14, in press (1975) Taylor, H.F., Burden, R.S.: Xanthoxin, a new naturally occurring plant growth inhibitor. Nature (Lond.) 227, 302-304 (1970) Tucker, D.J., Mansfield, T.A.: A simple bioassay for detecting "antitranspirant" activity of naturally occurring compounds such as abscisie acid. Planta (Berl.) 98, 157-163 (1971) Wareing, P.F., Ryback, G.: Abscisic acid: A newly discovered growth-regulating substance in plants. Endeavour 29, 84-88 (1970) Weiss, G., Koreeda, M., Nakanishi, K.: Stereochemistry of theaspirone and the blumenols J.C.S. Chem. Comm. 1978, 565-566
Short Communication The Effect of Vomifoliol on Stomatal Aperture K. L. Stuart and L. B. Coke Departments of Chemistry and of Botany, University of the ~Vest Indies, Mona, Kingston 7, Jamaica Received 11 November; accepted 10 December 1974
Summary. Vomifoliol, a compound related to abscisie acid (ABA) but lacking the 2,4-pentadiene sidechain and so far found in four different plant families, caused as much stomatal closure in epidermal strips of Eichhornia crassipes (Mart.) Solms as ABA at identical concentrations. It appears worthwhile to investigate whether vomifoliol plays a role as an endogenous regulator of stomatal aperture. Vomifoliol has been isolated from Rauwol/ia vomitoria, Apocynaceae (Pouset and Poisson, 1969), three Croton species, Euphorbiaceae (Satish and Bhakuni, 1972; Stuart and Woo-Ming, 1975) and Palicourea alpina (Sw.) I)C, Rubiaceae (Stuart and Woo-Ming, 1975). Blumenol A from Podocarpus blumei, Podocarpaceae (Galbraith and Horn, 1972), has also been identified as vomifoliol (Bhakuni et al., 1974; Stuart and Woo-Ming, 1975). This latter finding defines the stereochemistry of vomifoliol as shown in structure I. Bhakuni et al. (1974) have also reported the occurrence of roseoside in Vinca rosea, Apocynaceae, and established it as the fl-glucoside of vomifoliol. A recent study of endogenous antitranspirants in waterstressed Sorghum plants (Ogunkanmi et al., 1974) reported the effect of trans-farnesol on stomata, but also indicated that further work was necessary to verify that the only active compound was in fact all-trans-farnesol. On the basis of the separation procedure described, it is possible that vomifoliol could be a contaminant of the farnesol fraction, and this should be investigated in the light of our findings. The bioassay method developed by Tucker and Mansfield (1971) for demonstrating the activity of ABA on stomatal closure was modified for our studies. Phosphate buffer was preferred to the citrate buffer recommended by Ogunkanmi et al. (1973) because of its superior stability. The plant Eichhornia crassipes (Mart.) Solms (water hyacinth) was used as it promised certain advantages. As a floating aquatic plant which has been growing in a permanent fresh-water pond at the Botany Department, University of the West Indies, Mona, Jamaica, it is not subject to severe water deficit, one of the principal factors which increases internal levels of ABA and reduces stomatal responsiveness. Epidermal strips were taken from the abaxial surface of the youngest fully expanded leaf on a non-flowering shoot of Eichhornia crassipes. All strips required for a group of assays were obtained from a single leaf as this was more than 100 cm2 in area. Since the plants are vegetatively propagated, genetic constancy in the material is assured. The pores are simple in profile, large, and aligned in near parallel rows which are convenient for measurement. Test solutions of 10-8 M ABA, purchased from ICN-K~K Laboratories~ Inc., Plainview,
308
K.L. Stuart and L. B. Coke
N.Y., USA (batch No. 8594A)1, and of vomifoliol, isolated from Palicourea alpina by a combination of counter-current and chromatographic techniques (Stuart and Woo-Ming, 1975), were prepared by dissolving crystalline material in not more than 0.1 ml 98% ethanol and making up to 5 ml with 0.10 M NaNO8 in 0.01 M KH~PO4 buffer at pH 5.7. Serial dilutions from the stock solution provided concentrations ranging from 10-4 to 10-11M. A number of epidermal strips, 20-30 mm~, were obtained, floated in the pH 5.7 btfffer, illuminated by cool-white fluorescent lamps at 1800 lx, and aerated with a stream of COs-flee air, 20-30 cm3/ rain at 274-1 ~ for 2.5 h before incubation. At intervals of 10 rain, batches of three strips were transferred into 4.5 ml of ~est solutions of varying concentrations in glass vials. These were subjected to illumination and aeration conditions identical to those stated above, and incubation lasted 2 h. For each vial, the strips were then transferred to glass slides in a drop of test solution and the widths of 25 stomatal pores on each strip measured at 480foldmagnification with the aid of an eye-piece micrometer. A series of control treatments, consisting of the buffer solutions and identical ethanol concentrations as present in the test solutions, were performed concurrently with the test treatments. A total of six complete experiments were performed, and in some cases variations were made to aeration rates and light intensity. The reported data are for results obtained in conditions which seem optimal. Stomatal aperture showed a log-linear response to both ABA and vomifoliol (Fig. 1). The range of sensitivity between 10-4 and 10-1~ M is slightly better than that reported by Tucker and Mansfield (1971) for a Commelina epidermal strip assay at p H 6.7 and by Ogunkanmi et al. (1973) for piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer at p i t 6.8, and is comparable to the sensitivity obtained b y the latter authors with citrate buffer (pH 5.5). Regression of stomatal aperture on concentration calculated over the entire range of results (for ABA, y : 0 . 1 9 3 x @ 3.3496; for vomifoliol, y : 0 . 3 0 2 4 x ~ 2.4645) showed highly significant ( p : 0 . 0 1 ) linear responses for both substances. Comparison of the slopes of the two lines showed no significant difference at T=O.05. Stomatal aperture in Eichhornia was therefore equally sensitive to ABA and to vomifoliol. These results could be of considerable value if this effect of vomifoliol was not also accompanied by the other known effects of ABA such as senescence and abscission (Wareing and Rybaek, 1970) and further experiments using synthetic (4-)-vomifoliol (Weiss et al., 1973) are planned. One of these is a time-course study similar to that reported for ABA b y Cummins et al. (1971) since it is conceivable that the 2-h incubation period was sufficient for vomifoliol to be conver~ed to ABA. A n y biogenetic relationship between vomifoliol and ABA must await biosynthetic studies, but it is of interest t h a t conversion of vomifoliol to ABA has been achieved in vitro (Galbraith and Horn, 1973). Other naturally occurring compounds, like cis.trans-xanthoxin (Taylor and Burden, 1970), which seems to have limited distribution, and some synthetic models (Sondheimer and Walton, 1970; MeWha et al., 1973) have been demonstrated to possess varying levels of ABA activity, but vomifoliol is the first compound to our knowledge which has a modified 2,4-pentadiene side chain and has activity equal to that displayed by ABA. Our findings should also arouse interest in the possible biological activity of compounds such as blumenol B (Galbraith and Horn, 1973), 3-oxo-~-ionol (Aasen et al., 1971) and 4-(1-hydroxy-4-keto-2,6,6-trimethyl-2-cyelohexen-l-yl)-3-buten2-one (Roberts et al., 1968). 1 (4-)-ABA.
Vomifolioh Effect on Stomata
309
I.
6-
,.:a., 5 W
6-
--.r
s
4
4
3
3
2
2
I
I
'~~CONTROL
t-.-w a.
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0
o -I tl -II-I0-9-8 -7-6 -5-4 -I0-9-8-7- 6 -5 -4 I.OGIo CONCENTRATION[M] LOGIo CONCENTRATION[M] ABA VOMIFOLIOL '
i
,
,
i
,
i
Fig. 1. The effect of ABA (9 and vomifoliol ( . ) on stomatal aperture in epidermal strips of Eichhornia vrassipes in 0.1 M NAN03+0.01 M KH2POa buffer at pH 5.7. Vertical lines = standard errors
We thank Mr. Rex Woo-Ming for the isolation work which made samples of vomifoliol available for these experiments. References Aasen, A.J., Kimland, B., Enzell, C.R.: Tobacco chemistry. 7. Structure and synthesis of 3-oxo-cc-ionol a new tobacco constituent. Acta chem. scand. 25, 1481-1482 (1971) Bhakuni, D.S., Joshi, P.P., Uprcty, H., Kapil, R. S. : Roseoside-a C13 glycoside from Vinca rosea. Phytochemistry 18, 2541-2543 (1974) Cummins, W.R., Kende, H., Raschke, K. : Specificity and reversibility of the rapid stomatal response to abscisic acid. Planta (Berl.) 99, 347-351 (1971) Galbraith, M.N., Horn, D.H.S.: Structures of the natural products blumenols A, B and C. J.C.S. Chem. Comm. 1972, 113-114 Galbraith, M.N., Horn, D.H.S.: Stereochcmistry of the blumenols. Conversion of blumenol A into (S)-(+)-abscisie acid. J.C.S. Chem. Comm. 1973, 566-567 1KeWha, J.A., Philipson, J.J., Hillman, J.R., Wilkins, M.B.: Molecular requirements for abscisic acid activity in two bioassay systems. Planta (Berl.) 109, 327-336 (1973) Ogunkanmi, A.B., Tucker, I).J., Mansfield, T.A.: An improved bioassay for abseisic acid and other antitranspirants. New Phytol. 72, 277-282 (1973) Ogunkanmi, A.B., Wellburn, A.R., Mansfield, T.A. : Detection and preliminary identification of endogenous antitranspirants in water-stressed Sorghum plants. Planta (Berl.) 117, 293-302 (1974) Pouset, J.-L., Poisson, J.: Vomifolioh alcool terpenique isole des feuilles du Rauwolfia vomitoria Mz. Tetrahedron Letters 1969, 1173-1174 21 Planta(Berl.), Vol. 122
310
K.L. Stuart and L. B. Coke
Roberts, D.L., Heckman, R.A., Hege, B.P., Bellin, S.A.: Synthesis of (RS)-abscisie acid. J. org. Chem. 83, 3566-3569 (1968) Satish, S., Bhakuni, D.S.: Constituents of Indian and other plants. Phytochemistry 11, 2888-2890 (1972) Sondheimer, E., Walton, D.C.: Structure-activity correlations with compounds related to abscisie acid. Plant Physiol. 45, 244-248 (1970) Stuart, K.L., Woo-Ming, R.B.: Vomifoliol in Croton and Palicourea species. Phytochemistry 14, in press (1975) Taylor, H.F., Burden, R.S.: Xanthoxin, a new naturally occurring plant growth inhibitor. Nature (Lond.) 227, 302-304 (1970) Tucker, D.J., Mansfield, T.A.: A simple bioassay for detecting "antitranspirant" activity of naturally occurring compounds such as abscisie acid. Planta (Berl.) 98, 157-163 (1971) Wareing, P.F., Ryback, G.: Abscisic acid: A newly discovered growth-regulating substance in plants. Endeavour 29, 84-88 (1970) Weiss, G., Koreeda, M., Nakanishi, K.: Stereochemistry of theaspirone and the blumenols J.C.S. Chem. Comm. 1978, 565-566
