Planta (Berl.) 125, 149--160 (1975) 9 by Springer-Verlag 1975
Stomatal Closure in Response to Xanthoxin and Abscisic Acid* K l a u s R a s c h k e , R i c h a r d D. F i r n * * , a n d M a r g a r e t P i e r c e MSU/ERDA Plant Research Laboratory, East Lansing, Michigan 48824, USA Received 25 April; accepted 16 May 1975 Summary. The stomata of detached leaves of Commelina eommunis L., Hordeum vulgare L., Zea mays L., Vicia 1aba L., Phaseolus vulgaris L. and Xanthium strumarium L. closed when xanthoxin ();AN) was added to the transpiration stream. XAN was approximately half as active as (+)-abscisic acid (ABA) at an equivalent concentration. XAN, like ABA, sensitized stomata of Xanthium strumarium to COs. In contrast to ABA, XAN was ineffective in closing stomata of isolated epidermal strips of C. communis or V. ]aba. This may be because XAN added to the transpiration stream is converted to ABA during passage from the xylem to the epidermis.
Introduction X a n t h o x i n ( X A N ) is a n a t u r a l l y occurring g r o w t h i n h i b i t o r ( F i r n et al., 1972) w h i c h is s t r u c t u r a l l y s i m i l a r to abscisic a c i d (ABA). I n g r o w t h i n h i b i t i o n assays t h e biological a c t i v i t y of X A N is v e r y s i m i l a r t o t h a t of A B A (Taylor a n d B u r d e n , 1972), b u t t h i s a c t i v i t y could be t h e r e s u l t of conversion of X A N to A B A d u r i n g t h e p r o l o n g e d a s s a y periods. I t is k n o w n t h a t exogenous X A N is c o n v e r t e d b y p l a n t tissue to A B A (Taylor a n d B u r d e n , 1973). A m o r e r a p i d a n d specific response to A B A t h a n t h e i n h i b i t i o n of g r o w t h is t h e reversible closure of s t o m a t a (Cummins et al., 1971). A c o m p a r i s o n of t h e effects of A B A a n d X A N on s t o m a t a s h o u l d y i e l d useful i n f o r m a t i o n on t h e biological a c t i v i t y of X A N a n d should also p e r m i t s p e c u l a t i o n as t o w h e t h e r endogenous X A N could p l a y a role in m o d u l a t i n g stomatM aperture.
Materials and Methods Plants. Plants of Commelina communis L. and Vieia ]aba L. (or. Long Pod) were grown in a potting mixture in growth chambers with a daily 16 h light period and an irradiance of 8.5 mW cm -2 from fluorescent tubes (Sylvania Cool White lamps ~R96T12/CW/VHO/135~ supplemented by 40 W incandescent lamps. The day temperature was 27 ~ the night temperature 23~ and the relative humidity 85 %. Plants of Xanthium strumarium L. were grown in a potting mixture in a greenhouse where the natural light period was extended to 20 h/day (by Sylvania Gro-lux fluorescent tubes giving an irradiance of 30 ~zW cm-~) in order to keep this short-day plant in the vegetative state. Temperature maxima were between 23 ~ and 29 ~ and the relative humidity 70-80%. The plants were pruned to 5-6 leaves and did not flower. Zea mays L. (cv. Michigan 500) was grown in growth chambers in a sand and vermiculite mixture, at an irradiance of 40 mW cm -2 (from General Electric lamps H 400DX33-1 and LU400) for 14 h/day. The day temperature was 27 ~ the night temperature was 17~ and the relative humidity 50%. Hordeum vulgare L. (cv. Himalaya) and Phaseolus vulffaris L. (cv. * Abbreviations: ABA ~ Abscisic acid, XAN = xanthoxin. ** Present address: Department of Biology, University of York, York, U. K.
150
K. Rasehke et al.
Black Valentine) were grown in vermiculite and watered with half-strength Hoagland's solution. The plants were grown at 28 ~ during the 16-h day and at 23 ~ during the 8-h dark period.
Commelina communis seeds were a gift from Dr. T. A. Mansfield, University of Lancaster, U . K . The plants of Xanthium strumarium came from a strain originally collected in the vicinity of Chicago and propagated in isolation for several years in California, and more recently in Michigan. Vieia/aba seeds were obtained from Lagomarsina seeds, Sacramento, California, U S A ; Phaseolus vulgaris (Black Valentine) seeds from Vaughan Seed Co., Ovid, Mich., USA. The Washington State University Agronomy Department, Pullman, Wash., USA supplied fruits of Hordeum vulgare "Himalaya", and the Michigan State University Crop and Soil Science Department, East Lansing, Mich., USA supplied the fruits of Zea mays "Michigan 500". Measurement o/ Stomatal Responses in Leaves. Water-jacketed chambers were attached to the upper and lower epidermis of leaves and air with a known CO2 and water content was passed over the leaves at a rate of 501 h -~. The temperature of the water circulating through the water jackets was 22.5 ~. The leaf chambers attached to barley leaves, however, had no temperature control facility. The exposed leaf surface was 2.44 cm 2 in each chamber. In order to reduce formation of endogenous ABA in response to low water potential, all leaf parts not covered by the leaf chambers were trimmed (except in barley), thus reducing the transpiring a~'ea (Rasehke, 1973). Changes in the composition of the air which had passed over each leaf surface were measured separately with a differential infrared gas analyzer (Uras 2; Hartmann & Braun, Frankfurt a.M., Germany). The absolute CO2 content of the air was monitored with an additional infrared gas analyzer. Thermocouples monitored leaf temperatures and the temperature of the condenser which determined the dew point of the air supplied to the leaves. The leaves were illuminated with 30 m W cm -~ of photosynthetically usable light (135 nE cm -2 s-Z). The light was emitted from a water-cooled xenon arc lamp and filtered through an infrared absorbing glass (Coming 4600) and neutral density Plexiglass filters (No. 800; R6hm & Haas, Darmstadt, Germany). I n most experiments the output voltages from the thermocouples and gas analyzers were recorded on magnetic tape. The data were processed and plotted by computer to give time course plots of the exchange of CO~ and water through the upper and lower epidermis, epidermal conduetances and intercellular C02 concentrations ( ~ CO 2 concentration at the sites of evaporation in or on the cell walls of the mesophyll). In some experiments, transpiration and assimilation of C Q were the only parameters recorded. Leaves were usually detached from the plants under water. In the case of X. strumarium the plant tops were submerged for about 2 min (to produce temporary hydropassive stomatal closure) before excision. Solutions of ( =k )-ABA and ( ~ )-XAN were added to the water in which the cut leaves stood, giving a final inhibitor concentration as stated in the results. Measurement of Stomatal Aperture in Epidermal Strips. The first and second fully expanded leaves of Vicia ]aba plants or non-flowering branches of Commelina communis were excised in the morning, out into sections and floated, lower epidermis upwards, on deionized water. The leaf sections were maintained in a stream of COs-free humidified air under General Electric H400 R D X 33-1 mercury-vapor lamps, the light passing through a 5-cm water filter and having an intensity of 8.5 m W em -e. After at least 3 h, epidermal strips were peeled from the leaf sections which by this time had wide open stomata and washed with deionized water before being mounted on a piece of Plexiglass with a central well of 2 mm diameter. The epidermal surface normally in contact with the mesophyll faced the well; the cuticle of the epidermal sample was Covered with a small glass slide. The well was continuously flushed through small channels in the Plexiglass by a stream of O2-saturated buffer (0.01 M citrate buffer, p H 6.3, 0.5 ml min-~). Saturation of the buffer with O~ was required for the maintenance of stomatal opening; simple aeration of the buffer resulted in stomatal closure. Stomatal responses which resulted from the addition of X A N or ABA to the buffer were obselwed with a microscope (Zeiss Planaehromat long working-distance objective, 40 • N.A., with correction collar for cover glasses between 1.1 and 1.5 mm thick). Three stomata were usually selected in an epidermal sample (ca. 3 mm 2) and the aperture changes measured with an eyepiece micrometer or with a ruler on a television monitor which was linked to a television camera mounted
Stomatal Closure by Xanthoxin and ABA Conductance lower epidermis
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Fig. 1. Time courses of stomatal aperture and leaf temperature following an application of ABA or XAN to the irrigation water of detached bean leaves (Phaseolus vulgaris) to give 10-5 M (=k)-ABA or 5 • 10-5 M (-k)-XAI~. Stomatal aperture expressed in terms of eonduetanse of water vapor; data for lower epidermis. Irradianee 60 mW em -2, air 15.7 mg H20 1-1 and 315 {~l CO21-1
on the microscope. Carbon dioxide was added to the solutions by preparing mixtures of C02 and 02 in WSsthoff (Bochum, Germany) mixing pumps and bubbling these gas mixtures through the buffer before it was pumped to the chamber on which the epidermal sample was mounted. Chemicals. Xanthoxin was prepared from violaxanthin by the method of Taylor and Burden (1970). (:k)-kbscisie acid and (• aldehyde were generously donated by Hoffman-La Roche, Nutley, 1~. J., USA.
Results T h e effects of X A N a n d A B A on open s t o m a t a of d e t a c h e d leaves were comp a r e d in Commelina communis, Hordeum vulgate, Zea mays, Vicia /aba, Phaseolus vulgaris a n d Xanthium strumarium. E p i d e r m a l samples w i t h open s t o m a t a could be peeled from C. communis a n d V. ]aba; X A N a n d A B A were a p p l i e d to these i s o l a t e d tissues too. T h e following results were o b t a i n e d .
1. Experiments with Detached Leaves E//eets on Stomata el Phaseolus vulgaris. X A N s u p p l i e d to d e t a c h e d leaves v i a t h e t r a n s p i r a t i o n s t r e a m caused s t o m a t a l closure, t h e response being similar to t h a t i n d u c e d b y A B A (Fig. 1). T h e d a t a in Fig. 1 show t h a t a solution of 5 • 10-sM ( ~ ) - X A N was s l i g h t l y less effective t h a n a solution of 5 • The physiological effects of X A N a n d A B A can be c o m p a r e d b y d e t e r m i n i n g (a) t h e t i m e elapsed b e t w e e n a d d i t i o n of X A N or A B A to t h e i r r i g a t i o n s t r e a m
K. Raschke et al.
152
relative units 6
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Hordeurn vulgore
ABA
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Fig. 2. Time courses of transpiration of detached barley leaves after continuous application or 1 rain pulses of ABA or XAN. At time zero (• or (+)-XAN were added to the irrigation water to give a concentration of 10-5M. For pulse treatments, the irrigation water was changed to deionized water 1 rain after addition of the antitranspirants. Irradianee 19 mW cm-~ from a mercury vapor lamp with a fluorescent layer, air 14 mg H20 1-1 and 320 ~1 COS 1-1
and the time when stomatal conductance was reduced by 5% of the initial value; (b) the rate of stomatal closure once the response was initiated; and (c) the final conductance attained after treatment with X A N or ABA. The results of (b) and(c) are best expressed relative to the conductance at the time of application of the anti-transpirants (Raschke, in press). I n the experiment shown in Fig. 1, the values for the delays were 3.2 and 9.5 min, the relative slopes were 0.104 and 0.099 rain -1, and the relative final conductances were 0.43 and 0.34 for 5 • 10-6M ( + ) - A B A and 5 X 10-SM (-{-)-XAN, respectively. This experiment was repeated four times with similar results. Reversibility o/E/]ects on Stomata o/Hordeum vulgare. I t has previously been reported that the effect of ABA on the stomata of some species is reversible (Cummins et al., 1971; Itorton, 1971). The stomatal closure induced by X A N in barley leaves was reversed when the solution entering the transpiration stream was replaced b y deionized water (Fig. 2). This happened in all of the four re-
Stomatal Closure by Xanthoxin and ABA Conductance lower epidermis 312 .--,, ~ 5 9 9 - - - ~
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Fig. 3. Stomatal sensitization to CO2 following ABA and XAN applications to leaves of Xanthium strumarium to give 2 • 10-5 M (-L)-ABA or 10-5 M (~)-XAN in the transpiration stream. Stomata of control leaves with reduced lamina (Raschke, 1973) did not respond to the indicated changes in the [COs] of air; however, stomata in leaves treated with ABA or X A ~ did respond. Irradianee 42 mW cm-2; temperature of leaf chambers 23~ air 14 mg H20 1-1
p l i c a t i o n s of t h e e x p e r i m e n t . T h e response of b a r l e y leaves to t 0 - S M ( ~ - ) - X A N was a b o u t t h e s a m e as t h e response to 10-~M (=[=)-ABA, i n d i c a t i n g t h a t ( + ) - X A N was a b o u t half as a c t i v e as (~-)-ABA. The E//ects on the Stomata o / Z e a mays. T h e s t o m a t a of d e t a c h e d leaves of Zea mays were s o m e w h a t insensitive to b o t h A B A a n d X A N . I n a n e x p e r i m e n t i n v o l v i n g four leaves p e r t r e a t m e n t t h e a v e r a g e r e l a t i v e slopes of closing of t h e s t o m a t a of t h e lower e p i d e r m i s were 0.039 rain -1 after a p p l i c a t i o n of 2 • 10-sM (• a n d 0.030 rain -1 a f t e r a n a p p l i c a t i o n of 10-~M ( ~ - ) - X A N . T h e final a p e r t u r e s were 0.51 a n d 0.74 of t h e initial vaIues for A B A a n d X A N respectively. T h e conditions for t h e e x p e r i m e n t were: i r r a d i a n c e 41 m W cm-2; t e m p e r a t u r e of
K. Raschke et al.
154
Table i. The effect of the [CO2] in the air on two characteristics of stomatal closure in detached Xanthium strumarium leaves after application of 2 • 10-5 )5 (• or 10-5 )5 (+)-XA~ Two leaves for each treatment. Irradiance: 42 mW em-2, leaf temperature: 23~ air: 14 mg H20 1-1. Data from lower epidermis; similar data obtained from upper epidermis. Parameter computed a
Treatment
[CO2] in the air (~11-1) 0
300
600
900
Delay (rain)
ABA XA~
24.8 48.3
12.8 20.3
9.2 15.8
10.5 21.3
Relative slope (100 min)-1
ABA XAN
1.6 0.7
5.6 2.5
8.1 4.2
8.2 4.8
a As defined in )saterials and )sethods.
Table 2. Relative effectiveness of XAN compared to ABA on some parameters related to stomatal closure Comparisons were made between equal concentration (10-5 )5) of the active enantiomers (Cummins and Sondheimer, 1973) in Xanthium strumarium and between 10-5 )5 (-~)-XAN and 10-5 )5 (=L)-ABA in Commelina communis. The data are ratios and refer to the lower epidermis. Two leaves per treatment. Species
Parameter
Approximate [CO~] in the air (~11-1) 0
300
600
900
X. strumarium
Delay Relative slope
0.53 0.44
0.62 0.45
0.59 0.52
0.50 0.58
C. communis
Delay Relative slope Relative conductance
0.59 0.41 0.72
0.61 0.53 0.67
----
0.44 0.62 0.73
the leaf c h a m b e r 22.5 ~ ; the air c o n t a i n e d 16 m g H 2 0 a n d 300 ~zl CO 21-1. Two other e x p e r i m e n t s c o n d u c t e d u n d e r similar c o n d i t i o n s gave similar results. Stomatal Sensitization to CO2 by X A N in Xanthium strumarium. Open s t o m a t a of X. strumarium are insensitive to CO S b u t t h e y become responsive to CO S w h e n the A B A c o n t e n t of the leaves is increased (Raschke, 1973). X A N can replace A B A i n this response as shown i n Fig. 3. The s t o m a t a of control leaves were insensitive to changes i n [COs] over a range of 0-600 Ezl C02 1-1. Leaves t r e a t e d with A B A or X_AN, however, closed a n d became sensitive to changes i n [COs] as i n d i c a t e d b y the COs-induced closure when the [COs] was raised from 312 to 599 ~l CO s 1-I a n d the s u b s e q u e n t reopening on exposure to COs-free air. Requirement/or COs/or the Response o/Xanthium strumarium to X A N . S t o m a t a of X. strumarium need CO s to be able to respond to A B A (Raschke, i n press). The same is t r u e for the response to X A N (see Table 1). The p h e n o m e n o n appears to s a t u r a t e w i t h respect to [COs]. E q u a l c o n c e n t r a t i o n s of (~-)-ABA a n d ( ~ - ) - X A N were applied, a n d the response to X A N was less t h a n t h a t to ABA.
Stomatal Closure by Xanthoxin and ABA
155
Table 3. The amounts of XAN and ABA taken up by leaves of Xanthium strumarium and Commelina communis during the delay between the addition of the antitranspirants to the transpiration stream of detached leaves and the reduction of the stomatal conductance by 5 % of its original value A correction was applied to compensate for the difference in the XAN and ABA concentrations supplied to C. communis. Experimental conditions as in Table 1. Species
Treatment
X. strumarium
10-5 M (+)-ABA a 10-5 m (-t-)-XAN
C. communis
0.5 • 10-5 M (-t-)-ABAt, 10-5 M (+)-ABA 10-s M (~)-XAN
Amount of ABA or XAN taken up (pmol cm-~) at approximate [002] in the air (~zl1-1) 0
300
600
900
150 300
76 120
42 66
54 104
18 29 c 64
11 18e 49
a Supplied as 2 • 10-~ M (• b Supplied as 10-s M (• e By extrapolation.
Quantitative Comparison o/Stomatal Responses to X A N and A B A . A n evalua t i o n of t h e responses of s t o m a t a of X . strumarium to A B A a n d X A N s h o w e d t h a t ( ~ - ) - A B A was a p p r o x i m a t e l y twice as a c t i v e as ( - ~ ) - X A N (Table 2). I n Commelina communis, X A N was s l i g h t l y less t h a n half as a c t i v e as A B A . T h e a m o u n t of X A N t a k e n u p b y leaves of X . strumarium before s t o m a t a l c o n d u c t a n c e was r e d u c e d b y 5 % was 1.6-2 t i m e s larger t h a n t h a t of A B A (Table 3). I n leaves of C. communis X A N s e e m e d to h a v e been even less effective. I t is k n o w n t h a t t h e r e l a t i v e effectiveness of A B A in closing s t o m a t a decreases w i t h a p o w e r of ~ 1 w i t h increasing c o n c e n t r a t i o n (Raschke, in press). I f a n a p p r o p r i a t e correction is a p p l i e d (using a n e x p o n e n t of 0.67), 2.2-2.7 t i m e s m o r e X A N t h a n A B A has to e n t e r leaves of C. eommunis to p r o d u c e t h e s t a n d a r d closing response (Table 3).
2. Simultaneous Measurements on Epidermal Strips and Detached Leaves A p p l i c a t i o n s of X A N to e p i d e r m a l s t r i p s w i t h open s t o m a t a s u r p r i s i n g l y h a d no effect a l t h o u g h similar s t r i p s r e s p o n d e d t o A B A . Two series of e x p e r i m e n t s were c o n d u c t e d to m e a s u r e t h e responses of s t o m a t a t o X A N a n d A B A in d e t a c h e d leaves a n d e p i d e r m a l s t r i p s from t h e s a m e p l a n t . The y o u n g e s t f u l l y e x p a n d e d leaves were excised from a p l a n t of Commelina communis a n d t h e leaves d i v i d e d into t w o groups. One g r o u p was used to m e a s u r e s t o m a t a l responses in d e t a c h e d leaves w h i l e s i m u l t a n e o u s m e a s u r e m e n t s were m a d e on e p i d e r m a l s t r i p s from t h e o t h e r group. S i m i l a r e x p e r i m e n t s were m a d e on Vicia/aba. Experiments with Commelina communis at Several COs levels. S t o m a t a in epid e r m a l strips d i d n o t r e s p o n d to 10 -8 or 10-5M ( ~ - ) - X A N (18 r e p l i c a t i o n s ) ; indeed, t h e y e v e n o p e n e d f u r t h e r to a b o u t t h e s a m e e x t e n t as s t o m a t a o p e n e d on
K. l~asehke et al.
156
Aperture lower epidermis
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0 15
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AN
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eo
7o
Time (rain)
Fig. 4. Changes in the stomatal aperture in epidermal strips of Commelina communis. The strips were bathed continuously with O~-saturated 0.01 M citrate buffer, pH 6.3, to which (q-)-XAN or (q-)-ABA had been added to give concentrations of 10-sM. Measurements on three individual stomata for each treatment
buffer alone (Fig. 4). In contrast, 10-SM (=J=)-ABA caused stomatal closure within about 30 rain. Further experiments with buffer solutions equilibrated with 03 containing 300 and 1000 ~1 CO s 1-z confirmed these results. I n detached leaves, however, X A N closed stomata in the upper and lower epidermis (Fig. 5) and a high [COs] enhanced the effect. X A N was however less active than ABA in detached leaves. This was observed at 0, 300 and 1000 ~l COs 1-1. Experiments with Vicia ]aba. X A N had no effect on stomatal aperture in epidermal strips although such stomata responded to ABA (Fig. 6). X A N did, however, reduce stomatal aperture when applied to detached leaves of V. /aba (Fig. 7). 3. E//ects o/Abseisic Aldehyde on Stomata Abscisic aldehyde was added to the transpiration stream of detached leaves of Phazeolus vulgaris and applied to epidermal strips of Commelina communis and
Stom~tal Closure by Xanthoxin and ABA Conductance
Commelina commun/s
upper epidermis
%-
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=-D-
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:Fig. 5. Stomatal responses in detached leaves of Commelina communis (same plant as that used for the epidermal strips of Fig. 4) to 10-sM (=k)-ABA or (-k)-XAN. Irradiance 42 mW cm-~; temperature of leaf chambers 23~ air 13.5 mg H~O 1-1 and 0 tzl CO~ 1-1
Vicia /aba. Stomata of bean leaves closed in response to 10-5M (• aldehyde in the same way they responded to 10-sM (• In epidermal samples of C. communis and V./aba, stomata responded to abscisic aldehyde with a higher velocity than to ABA (Fig. 8). The stomata of V./aba responded more uniformly to abscisic aldehyde than to ABA. We conclude that the presence of the aIdehyde group in XAN was not the cause of its inability to close stomata.
Discussion Xanthoxin added to the transpiration stream of detached leaves closed stomata in all six species tested. XAN was in general half as active as ABA. But XAN was without effect on stomatal aperture when applied to epidermal strips of Commelina communis and Vicia /abe, the only species available from which epidermal samples could be peeled. One explanation of these observations would be that XAN has no direct effect on guard cells, but during passage from the xylem to the guard cells some conversion of XAN to ABA occurs; hence the effect if applied to whole Ieaves. Conversion of XAN to ABA by plant tissues has been demonstrated by Taylor and Burden (1973), but how rapidly the conversion became
158
K. Raschke et al. Aperture lower epidermis
Vicia fab~
/Jm 20
15
I0
0 20
ABA
15
I0
0
15
XAN
....
! ..........
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IO
~
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4o
5'o
6'o
7'0
Time (min)
Fig. 6. Changes in stomatal aperture in epidermal strips of Vicia/aba after ABA or XAN application. Details as for Fig. 4
evident was not stated. If X A N is not active per se then the conversion to ABA must in some cases be very rapid. Thus, in Hordeum vulgate > 50% of the X A N reaching the stomata must be converted to ABA in < 5 min (see Fig. 2). On the other hand, endogenous X A N is present in barley leaves (Firn, unpublished results). If exogenous X A N is rapidly converted to ABA it suggests t h a t endogenous X A N must be in a different, well-isolated compartment to exogenously applied XAN. The finding t h a t X A N does influence stomatal aperture in intact leaves raises the question as to whether X A N is an endogenous regulator of stomatal
Conducfance lower epidermis 0.35 f
XAN
~
3
0
Viciofoba
ABA
~
\
o.oI 0.10
0.05
r0
0
i
60
i
90 Time (min)
!
120
Fig. 7. Stomatal responses in detached leaves of Vicia/aba (leaves from the same plant as used in Fig. 6) after application of ABA or XAN to the irrigation water. Irradiance 15.5 mW era-2; temperature of leaf chambers 23~ air 15 mg H20 1-1 and 345 tzl CO21-1 Aperture lower epidermis /lm
Commelina communls
Control
15
5
0
20
I0
AaA o
,~
A.o,d 2~
3'o ~' ,~
_i~ 2~
~o
Time (rain)
Fig. 8. Changes in the stomatal aperture in epidermal strips of Commelina communi# after application of 10-6M (• acid and 10-6M (• aldehyde. Measurements on three individual stomata for each treatment
160
K. Raschke et al.
aperture. Wilting, which causes a large increase in A B A levels within the plant (Hiron and Wright, 1973), does n o t influence X A N levels (Zeevaart, 1974). Hence there is no reason to implicate x a n t h o x i n in the response of plants to water stress. However, X A N levels in m a n y plants are higher t h a n A B A levels (Firn et al., 1972; Zeevaart, 1974), a n d if X A N does n o t normally influence stomatal aperture it m a y be a result of c o m p a r t m e n t a t i o n of X A N . W e do n o t k n o w whether circumstances exist u n d e r which X A N is released from its sequestration and its potential as an antitranspirant is used b y the plant. This work was supported by the Energy Research and Development Administration (formerly U. S. Atomic Energy Commission) under contract AT-(11-1)-1338. We would like to thank Dr. H. F. Taylor for providing an authentic sample of xanthoxin. We greatly appreciated the assistance of Ms. Chu Chen Popiela in measuring stomatal apertures in epidermal strips.
References Cummins, W. R., Kende, H., Raschke, K. : Specificity and reversibility of the rapid stomatal response to abseisie acid. Planta (Berl.) 99, 347-351 (1971) Cummins, W. R., Sondheimer, E. : Activity of the asymmetric isomers of abscisie acid in a rapid bioassay. Planta (Berl.) 111, 365-369 (1973) Firn, R.D., Burden, R. S., Taylor, H. F.: The detection and estimation of the growth inhibitor xanthoxin in plants. Planta (Berl.) 102, 115-126 (1972) ttiron, R. W. P., Wright, S. T. C. : The role of endogenous abscisie acid in the response of plants to stress. J. exp. Bot. 24, 769-781 (1973) ttorton, R. F. : Stomatal opening: the role of abseisie acid. Canad. J. Bot. 49, 583-585 (1971) Raschke, K. : Abscisie acid sensitizes stomata to C02 in leaves of Xanthium strumarium. Proc. 8th Internat. Conf. Plant Growth Substances. Tokyo 1973 (in press) Rasehke, K.: Simultaneous requirement of carbon dioxide and abscisic acid for stomatal closing in Xanthium strumarium L. Planta (Berl.), 125, (1975) Taylor, tI. F., Burden, R. S. : Identification of plant growth inhibitors produced by photolysis of violaxanthin. Phytoehemistry 9, 2217-2223 (1970) Taylor, H.F., Burden, R.S.: Xanthoxin, a recently discovered plant growth inhibitor. Proc. roy. Soe. B 180, 317-346 (1972) Taylor, H. F., Burden, R. S.: Preparation and metabolism of [2--14C]-cls,trans-xanthoxin. J. exp. Bot. 24, 873-880 (1973) Zeevaart, J. A. D,: Levels of (-b)-abscisic acid and xanthoxin in spinach under different environmental conditions. Plant Physiol. 58, 644-648 (1974)
Stomatal Closure in Response to Xanthoxin and Abscisic Acid* K l a u s R a s c h k e , R i c h a r d D. F i r n * * , a n d M a r g a r e t P i e r c e MSU/ERDA Plant Research Laboratory, East Lansing, Michigan 48824, USA Received 25 April; accepted 16 May 1975 Summary. The stomata of detached leaves of Commelina eommunis L., Hordeum vulgare L., Zea mays L., Vicia 1aba L., Phaseolus vulgaris L. and Xanthium strumarium L. closed when xanthoxin ();AN) was added to the transpiration stream. XAN was approximately half as active as (+)-abscisic acid (ABA) at an equivalent concentration. XAN, like ABA, sensitized stomata of Xanthium strumarium to COs. In contrast to ABA, XAN was ineffective in closing stomata of isolated epidermal strips of C. communis or V. ]aba. This may be because XAN added to the transpiration stream is converted to ABA during passage from the xylem to the epidermis.
Introduction X a n t h o x i n ( X A N ) is a n a t u r a l l y occurring g r o w t h i n h i b i t o r ( F i r n et al., 1972) w h i c h is s t r u c t u r a l l y s i m i l a r to abscisic a c i d (ABA). I n g r o w t h i n h i b i t i o n assays t h e biological a c t i v i t y of X A N is v e r y s i m i l a r t o t h a t of A B A (Taylor a n d B u r d e n , 1972), b u t t h i s a c t i v i t y could be t h e r e s u l t of conversion of X A N to A B A d u r i n g t h e p r o l o n g e d a s s a y periods. I t is k n o w n t h a t exogenous X A N is c o n v e r t e d b y p l a n t tissue to A B A (Taylor a n d B u r d e n , 1973). A m o r e r a p i d a n d specific response to A B A t h a n t h e i n h i b i t i o n of g r o w t h is t h e reversible closure of s t o m a t a (Cummins et al., 1971). A c o m p a r i s o n of t h e effects of A B A a n d X A N on s t o m a t a s h o u l d y i e l d useful i n f o r m a t i o n on t h e biological a c t i v i t y of X A N a n d should also p e r m i t s p e c u l a t i o n as t o w h e t h e r endogenous X A N could p l a y a role in m o d u l a t i n g stomatM aperture.
Materials and Methods Plants. Plants of Commelina communis L. and Vieia ]aba L. (or. Long Pod) were grown in a potting mixture in growth chambers with a daily 16 h light period and an irradiance of 8.5 mW cm -2 from fluorescent tubes (Sylvania Cool White lamps ~R96T12/CW/VHO/135~ supplemented by 40 W incandescent lamps. The day temperature was 27 ~ the night temperature 23~ and the relative humidity 85 %. Plants of Xanthium strumarium L. were grown in a potting mixture in a greenhouse where the natural light period was extended to 20 h/day (by Sylvania Gro-lux fluorescent tubes giving an irradiance of 30 ~zW cm-~) in order to keep this short-day plant in the vegetative state. Temperature maxima were between 23 ~ and 29 ~ and the relative humidity 70-80%. The plants were pruned to 5-6 leaves and did not flower. Zea mays L. (cv. Michigan 500) was grown in growth chambers in a sand and vermiculite mixture, at an irradiance of 40 mW cm -2 (from General Electric lamps H 400DX33-1 and LU400) for 14 h/day. The day temperature was 27 ~ the night temperature was 17~ and the relative humidity 50%. Hordeum vulgare L. (cv. Himalaya) and Phaseolus vulffaris L. (cv. * Abbreviations: ABA ~ Abscisic acid, XAN = xanthoxin. ** Present address: Department of Biology, University of York, York, U. K.
150
K. Rasehke et al.
Black Valentine) were grown in vermiculite and watered with half-strength Hoagland's solution. The plants were grown at 28 ~ during the 16-h day and at 23 ~ during the 8-h dark period.
Commelina communis seeds were a gift from Dr. T. A. Mansfield, University of Lancaster, U . K . The plants of Xanthium strumarium came from a strain originally collected in the vicinity of Chicago and propagated in isolation for several years in California, and more recently in Michigan. Vieia/aba seeds were obtained from Lagomarsina seeds, Sacramento, California, U S A ; Phaseolus vulgaris (Black Valentine) seeds from Vaughan Seed Co., Ovid, Mich., USA. The Washington State University Agronomy Department, Pullman, Wash., USA supplied fruits of Hordeum vulgare "Himalaya", and the Michigan State University Crop and Soil Science Department, East Lansing, Mich., USA supplied the fruits of Zea mays "Michigan 500". Measurement o/ Stomatal Responses in Leaves. Water-jacketed chambers were attached to the upper and lower epidermis of leaves and air with a known CO2 and water content was passed over the leaves at a rate of 501 h -~. The temperature of the water circulating through the water jackets was 22.5 ~. The leaf chambers attached to barley leaves, however, had no temperature control facility. The exposed leaf surface was 2.44 cm 2 in each chamber. In order to reduce formation of endogenous ABA in response to low water potential, all leaf parts not covered by the leaf chambers were trimmed (except in barley), thus reducing the transpiring a~'ea (Rasehke, 1973). Changes in the composition of the air which had passed over each leaf surface were measured separately with a differential infrared gas analyzer (Uras 2; Hartmann & Braun, Frankfurt a.M., Germany). The absolute CO2 content of the air was monitored with an additional infrared gas analyzer. Thermocouples monitored leaf temperatures and the temperature of the condenser which determined the dew point of the air supplied to the leaves. The leaves were illuminated with 30 m W cm -~ of photosynthetically usable light (135 nE cm -2 s-Z). The light was emitted from a water-cooled xenon arc lamp and filtered through an infrared absorbing glass (Coming 4600) and neutral density Plexiglass filters (No. 800; R6hm & Haas, Darmstadt, Germany). I n most experiments the output voltages from the thermocouples and gas analyzers were recorded on magnetic tape. The data were processed and plotted by computer to give time course plots of the exchange of CO~ and water through the upper and lower epidermis, epidermal conduetances and intercellular C02 concentrations ( ~ CO 2 concentration at the sites of evaporation in or on the cell walls of the mesophyll). In some experiments, transpiration and assimilation of C Q were the only parameters recorded. Leaves were usually detached from the plants under water. In the case of X. strumarium the plant tops were submerged for about 2 min (to produce temporary hydropassive stomatal closure) before excision. Solutions of ( =k )-ABA and ( ~ )-XAN were added to the water in which the cut leaves stood, giving a final inhibitor concentration as stated in the results. Measurement of Stomatal Aperture in Epidermal Strips. The first and second fully expanded leaves of Vicia ]aba plants or non-flowering branches of Commelina communis were excised in the morning, out into sections and floated, lower epidermis upwards, on deionized water. The leaf sections were maintained in a stream of COs-free humidified air under General Electric H400 R D X 33-1 mercury-vapor lamps, the light passing through a 5-cm water filter and having an intensity of 8.5 m W em -e. After at least 3 h, epidermal strips were peeled from the leaf sections which by this time had wide open stomata and washed with deionized water before being mounted on a piece of Plexiglass with a central well of 2 mm diameter. The epidermal surface normally in contact with the mesophyll faced the well; the cuticle of the epidermal sample was Covered with a small glass slide. The well was continuously flushed through small channels in the Plexiglass by a stream of O2-saturated buffer (0.01 M citrate buffer, p H 6.3, 0.5 ml min-~). Saturation of the buffer with O~ was required for the maintenance of stomatal opening; simple aeration of the buffer resulted in stomatal closure. Stomatal responses which resulted from the addition of X A N or ABA to the buffer were obselwed with a microscope (Zeiss Planaehromat long working-distance objective, 40 • N.A., with correction collar for cover glasses between 1.1 and 1.5 mm thick). Three stomata were usually selected in an epidermal sample (ca. 3 mm 2) and the aperture changes measured with an eyepiece micrometer or with a ruler on a television monitor which was linked to a television camera mounted
Stomatal Closure by Xanthoxin and ABA Conductance lower epidermis
cm s
Phaseolus vulgoris
Leaf temperature
0.4 0.3
oC
|
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temp
+
9
~ m
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Time (min)
Fig. 1. Time courses of stomatal aperture and leaf temperature following an application of ABA or XAN to the irrigation water of detached bean leaves (Phaseolus vulgaris) to give 10-5 M (=k)-ABA or 5 • 10-5 M (-k)-XAI~. Stomatal aperture expressed in terms of eonduetanse of water vapor; data for lower epidermis. Irradianee 60 mW em -2, air 15.7 mg H20 1-1 and 315 {~l CO21-1
on the microscope. Carbon dioxide was added to the solutions by preparing mixtures of C02 and 02 in WSsthoff (Bochum, Germany) mixing pumps and bubbling these gas mixtures through the buffer before it was pumped to the chamber on which the epidermal sample was mounted. Chemicals. Xanthoxin was prepared from violaxanthin by the method of Taylor and Burden (1970). (:k)-kbscisie acid and (• aldehyde were generously donated by Hoffman-La Roche, Nutley, 1~. J., USA.
Results T h e effects of X A N a n d A B A on open s t o m a t a of d e t a c h e d leaves were comp a r e d in Commelina communis, Hordeum vulgate, Zea mays, Vicia /aba, Phaseolus vulgaris a n d Xanthium strumarium. E p i d e r m a l samples w i t h open s t o m a t a could be peeled from C. communis a n d V. ]aba; X A N a n d A B A were a p p l i e d to these i s o l a t e d tissues too. T h e following results were o b t a i n e d .
1. Experiments with Detached Leaves E//eets on Stomata el Phaseolus vulgaris. X A N s u p p l i e d to d e t a c h e d leaves v i a t h e t r a n s p i r a t i o n s t r e a m caused s t o m a t a l closure, t h e response being similar to t h a t i n d u c e d b y A B A (Fig. 1). T h e d a t a in Fig. 1 show t h a t a solution of 5 • 10-sM ( ~ ) - X A N was s l i g h t l y less effective t h a n a solution of 5 • The physiological effects of X A N a n d A B A can be c o m p a r e d b y d e t e r m i n i n g (a) t h e t i m e elapsed b e t w e e n a d d i t i o n of X A N or A B A to t h e i r r i g a t i o n s t r e a m
K. Raschke et al.
152
relative units 6
Transpiration lowerepidermis
Hordeurn vulgore
ABA
~/. 5 ~ f continuous
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20 3'0 go so 6'0 70 6'0 Time (min)
Fig. 2. Time courses of transpiration of detached barley leaves after continuous application or 1 rain pulses of ABA or XAN. At time zero (• or (+)-XAN were added to the irrigation water to give a concentration of 10-5M. For pulse treatments, the irrigation water was changed to deionized water 1 rain after addition of the antitranspirants. Irradianee 19 mW cm-~ from a mercury vapor lamp with a fluorescent layer, air 14 mg H20 1-1 and 320 ~1 COS 1-1
and the time when stomatal conductance was reduced by 5% of the initial value; (b) the rate of stomatal closure once the response was initiated; and (c) the final conductance attained after treatment with X A N or ABA. The results of (b) and(c) are best expressed relative to the conductance at the time of application of the anti-transpirants (Raschke, in press). I n the experiment shown in Fig. 1, the values for the delays were 3.2 and 9.5 min, the relative slopes were 0.104 and 0.099 rain -1, and the relative final conductances were 0.43 and 0.34 for 5 • 10-6M ( + ) - A B A and 5 X 10-SM (-{-)-XAN, respectively. This experiment was repeated four times with similar results. Reversibility o/E/]ects on Stomata o/Hordeum vulgare. I t has previously been reported that the effect of ABA on the stomata of some species is reversible (Cummins et al., 1971; Itorton, 1971). The stomatal closure induced by X A N in barley leaves was reversed when the solution entering the transpiration stream was replaced b y deionized water (Fig. 2). This happened in all of the four re-
Stomatal Closure by Xanthoxin and ABA Conductance lower epidermis 312 .--,, ~ 5 9 9 - - - ~
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Fig. 3. Stomatal sensitization to CO2 following ABA and XAN applications to leaves of Xanthium strumarium to give 2 • 10-5 M (-L)-ABA or 10-5 M (~)-XAN in the transpiration stream. Stomata of control leaves with reduced lamina (Raschke, 1973) did not respond to the indicated changes in the [COs] of air; however, stomata in leaves treated with ABA or X A ~ did respond. Irradianee 42 mW cm-2; temperature of leaf chambers 23~ air 14 mg H20 1-1
p l i c a t i o n s of t h e e x p e r i m e n t . T h e response of b a r l e y leaves to t 0 - S M ( ~ - ) - X A N was a b o u t t h e s a m e as t h e response to 10-~M (=[=)-ABA, i n d i c a t i n g t h a t ( + ) - X A N was a b o u t half as a c t i v e as (~-)-ABA. The E//ects on the Stomata o / Z e a mays. T h e s t o m a t a of d e t a c h e d leaves of Zea mays were s o m e w h a t insensitive to b o t h A B A a n d X A N . I n a n e x p e r i m e n t i n v o l v i n g four leaves p e r t r e a t m e n t t h e a v e r a g e r e l a t i v e slopes of closing of t h e s t o m a t a of t h e lower e p i d e r m i s were 0.039 rain -1 after a p p l i c a t i o n of 2 • 10-sM (• a n d 0.030 rain -1 a f t e r a n a p p l i c a t i o n of 10-~M ( ~ - ) - X A N . T h e final a p e r t u r e s were 0.51 a n d 0.74 of t h e initial vaIues for A B A a n d X A N respectively. T h e conditions for t h e e x p e r i m e n t were: i r r a d i a n c e 41 m W cm-2; t e m p e r a t u r e of
K. Raschke et al.
154
Table i. The effect of the [CO2] in the air on two characteristics of stomatal closure in detached Xanthium strumarium leaves after application of 2 • 10-5 )5 (• or 10-5 )5 (+)-XA~ Two leaves for each treatment. Irradiance: 42 mW em-2, leaf temperature: 23~ air: 14 mg H20 1-1. Data from lower epidermis; similar data obtained from upper epidermis. Parameter computed a
Treatment
[CO2] in the air (~11-1) 0
300
600
900
Delay (rain)
ABA XA~
24.8 48.3
12.8 20.3
9.2 15.8
10.5 21.3
Relative slope (100 min)-1
ABA XAN
1.6 0.7
5.6 2.5
8.1 4.2
8.2 4.8
a As defined in )saterials and )sethods.
Table 2. Relative effectiveness of XAN compared to ABA on some parameters related to stomatal closure Comparisons were made between equal concentration (10-5 )5) of the active enantiomers (Cummins and Sondheimer, 1973) in Xanthium strumarium and between 10-5 )5 (-~)-XAN and 10-5 )5 (=L)-ABA in Commelina communis. The data are ratios and refer to the lower epidermis. Two leaves per treatment. Species
Parameter
Approximate [CO~] in the air (~11-1) 0
300
600
900
X. strumarium
Delay Relative slope
0.53 0.44
0.62 0.45
0.59 0.52
0.50 0.58
C. communis
Delay Relative slope Relative conductance
0.59 0.41 0.72
0.61 0.53 0.67
----
0.44 0.62 0.73
the leaf c h a m b e r 22.5 ~ ; the air c o n t a i n e d 16 m g H 2 0 a n d 300 ~zl CO 21-1. Two other e x p e r i m e n t s c o n d u c t e d u n d e r similar c o n d i t i o n s gave similar results. Stomatal Sensitization to CO2 by X A N in Xanthium strumarium. Open s t o m a t a of X. strumarium are insensitive to CO S b u t t h e y become responsive to CO S w h e n the A B A c o n t e n t of the leaves is increased (Raschke, 1973). X A N can replace A B A i n this response as shown i n Fig. 3. The s t o m a t a of control leaves were insensitive to changes i n [COs] over a range of 0-600 Ezl C02 1-1. Leaves t r e a t e d with A B A or X_AN, however, closed a n d became sensitive to changes i n [COs] as i n d i c a t e d b y the COs-induced closure when the [COs] was raised from 312 to 599 ~l CO s 1-I a n d the s u b s e q u e n t reopening on exposure to COs-free air. Requirement/or COs/or the Response o/Xanthium strumarium to X A N . S t o m a t a of X. strumarium need CO s to be able to respond to A B A (Raschke, i n press). The same is t r u e for the response to X A N (see Table 1). The p h e n o m e n o n appears to s a t u r a t e w i t h respect to [COs]. E q u a l c o n c e n t r a t i o n s of (~-)-ABA a n d ( ~ - ) - X A N were applied, a n d the response to X A N was less t h a n t h a t to ABA.
Stomatal Closure by Xanthoxin and ABA
155
Table 3. The amounts of XAN and ABA taken up by leaves of Xanthium strumarium and Commelina communis during the delay between the addition of the antitranspirants to the transpiration stream of detached leaves and the reduction of the stomatal conductance by 5 % of its original value A correction was applied to compensate for the difference in the XAN and ABA concentrations supplied to C. communis. Experimental conditions as in Table 1. Species
Treatment
X. strumarium
10-5 M (+)-ABA a 10-5 m (-t-)-XAN
C. communis
0.5 • 10-5 M (-t-)-ABAt, 10-5 M (+)-ABA 10-s M (~)-XAN
Amount of ABA or XAN taken up (pmol cm-~) at approximate [002] in the air (~zl1-1) 0
300
600
900
150 300
76 120
42 66
54 104
18 29 c 64
11 18e 49
a Supplied as 2 • 10-~ M (• b Supplied as 10-s M (• e By extrapolation.
Quantitative Comparison o/Stomatal Responses to X A N and A B A . A n evalua t i o n of t h e responses of s t o m a t a of X . strumarium to A B A a n d X A N s h o w e d t h a t ( ~ - ) - A B A was a p p r o x i m a t e l y twice as a c t i v e as ( - ~ ) - X A N (Table 2). I n Commelina communis, X A N was s l i g h t l y less t h a n half as a c t i v e as A B A . T h e a m o u n t of X A N t a k e n u p b y leaves of X . strumarium before s t o m a t a l c o n d u c t a n c e was r e d u c e d b y 5 % was 1.6-2 t i m e s larger t h a n t h a t of A B A (Table 3). I n leaves of C. communis X A N s e e m e d to h a v e been even less effective. I t is k n o w n t h a t t h e r e l a t i v e effectiveness of A B A in closing s t o m a t a decreases w i t h a p o w e r of ~ 1 w i t h increasing c o n c e n t r a t i o n (Raschke, in press). I f a n a p p r o p r i a t e correction is a p p l i e d (using a n e x p o n e n t of 0.67), 2.2-2.7 t i m e s m o r e X A N t h a n A B A has to e n t e r leaves of C. eommunis to p r o d u c e t h e s t a n d a r d closing response (Table 3).
2. Simultaneous Measurements on Epidermal Strips and Detached Leaves A p p l i c a t i o n s of X A N to e p i d e r m a l s t r i p s w i t h open s t o m a t a s u r p r i s i n g l y h a d no effect a l t h o u g h similar s t r i p s r e s p o n d e d t o A B A . Two series of e x p e r i m e n t s were c o n d u c t e d to m e a s u r e t h e responses of s t o m a t a t o X A N a n d A B A in d e t a c h e d leaves a n d e p i d e r m a l s t r i p s from t h e s a m e p l a n t . The y o u n g e s t f u l l y e x p a n d e d leaves were excised from a p l a n t of Commelina communis a n d t h e leaves d i v i d e d into t w o groups. One g r o u p was used to m e a s u r e s t o m a t a l responses in d e t a c h e d leaves w h i l e s i m u l t a n e o u s m e a s u r e m e n t s were m a d e on e p i d e r m a l s t r i p s from t h e o t h e r group. S i m i l a r e x p e r i m e n t s were m a d e on Vicia/aba. Experiments with Commelina communis at Several COs levels. S t o m a t a in epid e r m a l strips d i d n o t r e s p o n d to 10 -8 or 10-5M ( ~ - ) - X A N (18 r e p l i c a t i o n s ) ; indeed, t h e y e v e n o p e n e d f u r t h e r to a b o u t t h e s a m e e x t e n t as s t o m a t a o p e n e d on
K. l~asehke et al.
156
Aperture lower epidermis
Commelino communis
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,o
1
i
eo
7o
Time (rain)
Fig. 4. Changes in the stomatal aperture in epidermal strips of Commelina communis. The strips were bathed continuously with O~-saturated 0.01 M citrate buffer, pH 6.3, to which (q-)-XAN or (q-)-ABA had been added to give concentrations of 10-sM. Measurements on three individual stomata for each treatment
buffer alone (Fig. 4). In contrast, 10-SM (=J=)-ABA caused stomatal closure within about 30 rain. Further experiments with buffer solutions equilibrated with 03 containing 300 and 1000 ~1 CO s 1-z confirmed these results. I n detached leaves, however, X A N closed stomata in the upper and lower epidermis (Fig. 5) and a high [COs] enhanced the effect. X A N was however less active than ABA in detached leaves. This was observed at 0, 300 and 1000 ~l COs 1-1. Experiments with Vicia ]aba. X A N had no effect on stomatal aperture in epidermal strips although such stomata responded to ABA (Fig. 6). X A N did, however, reduce stomatal aperture when applied to detached leaves of V. /aba (Fig. 7). 3. E//ects o/Abseisic Aldehyde on Stomata Abscisic aldehyde was added to the transpiration stream of detached leaves of Phazeolus vulgaris and applied to epidermal strips of Commelina communis and
Stom~tal Closure by Xanthoxin and ABA Conductance
Commelina commun/s
upper epidermis
%-
0.2
=-D-
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157
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t
i
i
i
i
i
t
i
i
i
i
lower epidermis ABA
XAN
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~
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. ,
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o. ~ 0.0
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50
60
70
80
90
I
l
I 0 0 I10
Time (min)
:Fig. 5. Stomatal responses in detached leaves of Commelina communis (same plant as that used for the epidermal strips of Fig. 4) to 10-sM (=k)-ABA or (-k)-XAN. Irradiance 42 mW cm-~; temperature of leaf chambers 23~ air 13.5 mg H~O 1-1 and 0 tzl CO~ 1-1
Vicia /aba. Stomata of bean leaves closed in response to 10-5M (• aldehyde in the same way they responded to 10-sM (• In epidermal samples of C. communis and V./aba, stomata responded to abscisic aldehyde with a higher velocity than to ABA (Fig. 8). The stomata of V./aba responded more uniformly to abscisic aldehyde than to ABA. We conclude that the presence of the aIdehyde group in XAN was not the cause of its inability to close stomata.
Discussion Xanthoxin added to the transpiration stream of detached leaves closed stomata in all six species tested. XAN was in general half as active as ABA. But XAN was without effect on stomatal aperture when applied to epidermal strips of Commelina communis and Vicia /abe, the only species available from which epidermal samples could be peeled. One explanation of these observations would be that XAN has no direct effect on guard cells, but during passage from the xylem to the guard cells some conversion of XAN to ABA occurs; hence the effect if applied to whole Ieaves. Conversion of XAN to ABA by plant tissues has been demonstrated by Taylor and Burden (1973), but how rapidly the conversion became
158
K. Raschke et al. Aperture lower epidermis
Vicia fab~
/Jm 20
15
I0
0 20
ABA
15
I0
0
15
XAN
....
! ..........
../':~.....-~ . :,._.._.~_._._._..~
IO
~
2'o
4o
5'o
6'o
7'0
Time (min)
Fig. 6. Changes in stomatal aperture in epidermal strips of Vicia/aba after ABA or XAN application. Details as for Fig. 4
evident was not stated. If X A N is not active per se then the conversion to ABA must in some cases be very rapid. Thus, in Hordeum vulgate > 50% of the X A N reaching the stomata must be converted to ABA in < 5 min (see Fig. 2). On the other hand, endogenous X A N is present in barley leaves (Firn, unpublished results). If exogenous X A N is rapidly converted to ABA it suggests t h a t endogenous X A N must be in a different, well-isolated compartment to exogenously applied XAN. The finding t h a t X A N does influence stomatal aperture in intact leaves raises the question as to whether X A N is an endogenous regulator of stomatal
Conducfance lower epidermis 0.35 f
XAN
~
3
0
Viciofoba
ABA
~
\
o.oI 0.10
0.05
r0
0
i
60
i
90 Time (min)
!
120
Fig. 7. Stomatal responses in detached leaves of Vicia/aba (leaves from the same plant as used in Fig. 6) after application of ABA or XAN to the irrigation water. Irradiance 15.5 mW era-2; temperature of leaf chambers 23~ air 15 mg H20 1-1 and 345 tzl CO21-1 Aperture lower epidermis /lm
Commelina communls
Control
15
5
0
20
I0
AaA o
,~
A.o,d 2~
3'o ~' ,~
_i~ 2~
~o
Time (rain)
Fig. 8. Changes in the stomatal aperture in epidermal strips of Commelina communi# after application of 10-6M (• acid and 10-6M (• aldehyde. Measurements on three individual stomata for each treatment
160
K. Raschke et al.
aperture. Wilting, which causes a large increase in A B A levels within the plant (Hiron and Wright, 1973), does n o t influence X A N levels (Zeevaart, 1974). Hence there is no reason to implicate x a n t h o x i n in the response of plants to water stress. However, X A N levels in m a n y plants are higher t h a n A B A levels (Firn et al., 1972; Zeevaart, 1974), a n d if X A N does n o t normally influence stomatal aperture it m a y be a result of c o m p a r t m e n t a t i o n of X A N . W e do n o t k n o w whether circumstances exist u n d e r which X A N is released from its sequestration and its potential as an antitranspirant is used b y the plant. This work was supported by the Energy Research and Development Administration (formerly U. S. Atomic Energy Commission) under contract AT-(11-1)-1338. We would like to thank Dr. H. F. Taylor for providing an authentic sample of xanthoxin. We greatly appreciated the assistance of Ms. Chu Chen Popiela in measuring stomatal apertures in epidermal strips.
References Cummins, W. R., Kende, H., Raschke, K. : Specificity and reversibility of the rapid stomatal response to abseisie acid. Planta (Berl.) 99, 347-351 (1971) Cummins, W. R., Sondheimer, E. : Activity of the asymmetric isomers of abscisie acid in a rapid bioassay. Planta (Berl.) 111, 365-369 (1973) Firn, R.D., Burden, R. S., Taylor, H. F.: The detection and estimation of the growth inhibitor xanthoxin in plants. Planta (Berl.) 102, 115-126 (1972) ttiron, R. W. P., Wright, S. T. C. : The role of endogenous abscisie acid in the response of plants to stress. J. exp. Bot. 24, 769-781 (1973) ttorton, R. F. : Stomatal opening: the role of abseisie acid. Canad. J. Bot. 49, 583-585 (1971) Raschke, K. : Abscisie acid sensitizes stomata to C02 in leaves of Xanthium strumarium. Proc. 8th Internat. Conf. Plant Growth Substances. Tokyo 1973 (in press) Rasehke, K.: Simultaneous requirement of carbon dioxide and abscisic acid for stomatal closing in Xanthium strumarium L. Planta (Berl.), 125, (1975) Taylor, tI. F., Burden, R. S. : Identification of plant growth inhibitors produced by photolysis of violaxanthin. Phytoehemistry 9, 2217-2223 (1970) Taylor, H.F., Burden, R.S.: Xanthoxin, a recently discovered plant growth inhibitor. Proc. roy. Soe. B 180, 317-346 (1972) Taylor, H. F., Burden, R. S.: Preparation and metabolism of [2--14C]-cls,trans-xanthoxin. J. exp. Bot. 24, 873-880 (1973) Zeevaart, J. A. D,: Levels of (-b)-abscisic acid and xanthoxin in spinach under different environmental conditions. Plant Physiol. 58, 644-648 (1974)
