Benzyl adenine enhanced stomatal opening in isolated epidermal strips of Commelina benghalensis and Tridax procumhens. The stimulation was maximum at a concentration of 5&#215;10&#8722;5 M BA. But kinetin had no remarkable effect on stomatal opening. The activity of benzyl adenine was observed over a narrow range of concentrations, from 10&#8722;5 M to 10&#8722;4 M. The increase in stomatal aperture was more pronounced in Commelina than in Tridax. Presence of benzyl adenine in the medium prevented the stomatal closure expected from abscisic acid and could reverse considerably the stomatal closure induced earlier by abscisic acid. It is felt that the balance between abscisic acid and cytokinins can possibly control the stomatal aperture effectively

Das, V. S. R.

Raghavendra, A. S.

Rao, I. M.

English

Publications of the IAS Fellows

(1976) 7
REVERSAL OF ABSCISIC ACID INDUCED STOMATAL
CLOSURE BY BENZYL ADENINE
BY V. S. R. DAS, I. M. RAO AND A. S. RAGHAVENDRA*
Department of Botany, Sri Venkateswara University, Tirupati 51702, India
' {Received 10 October K)']^)
SUMMARY
Benzyl adenine enhanced stomatal opening in isolated epidermal strips of Commelina benghalensis
and Tridax procumhens. The stimulation was maximum at a concentration of 5 x io"^ M BA.
But kinetin had no remarkable effect on stomatal opening. The activity of benzyl adenine was
observed over a narrow range of concentrations, from io~ M to io"* M. The increase in
stomatal aperture was more pronounced in Commelina than in Tridax. Presence of benzyl
adenine in the medium prevented the stomatal closure expected from abscisic acid and could
reverse considerably the stomatal closure induced earlier by abscisic acid. It is felt that the
balance between abscisic acid and cytokinins can possibly control the stomatal aperture
effectively. ,- -, ,i: -; . . . , ' ^ ,• • -,,:.• ,,.. ;. , ;-j,;"v A , :' ir's
INTRODUCTION
The control of stomatal aperture and transpiration through the exogenous application
of chemicals has been receiving considerable attention. Abscisic acid (ABA) is a povi'erful
inhibitor of stomatal movement. It significantly reduced the stomatal aperture when
sprayed on to leaves (Mittleheuser and Vansteveninck, 1969, 1971; Jones and Mans-
field, 1970; Talha and Larsen, 1975) and was considered to be as an ideal antitranspirant.
On the other hand, transpiration and stomatal opening were increased by the application
of cytokinins (Livne and Vaadia, 1965; Meidner, 1967; Luke and Freeman, 1967).
Abscisic acid was effective in decreasing the stomatal aperture even in isolated epider-
mal strips. But stomata did not respond to kinetin in epidermal strips (Tucker and
Mansfield, 1971; Horton, 1971). A recent study indicated the significant interaction
betv^'een ABA and kinetin on stomatal opening in barley (Cooper, Digby and Cooper,
1972). In view of the conflicting evidence, we examined in detail the influence of cyto-
kinins, kinetin and benzyl adenine (BA) and their interaction with ABA.
MATERIALS AND METHODS
Lower epidermal strips of about i.o X0.5 cm size were pi^epared from the leaves of
Commelina benghalensis L. and Tridax procumbens L. (Raghavendra and Das, 1972).
The incubation medium for stomatal opening in light was 0.067 ^ phosphate buffer
at pH 7.0 with 10 mM KCl, the various additional compounds were included when
* Present address: Central Plantation Crops Research Institute, Regional Station, Vittal 574243,
Karnataka, India.
Correspondence: Dr A. S. Raghavendra, Central Plantation Crops Research Institute, Regional Station,
Vittal 574243, Karnataka, India.
449 .
45O V. S. R. DAS, I. M. RAO AND A. S. RAGHAVENDRA
required at the given final concentrations and the pH of the incubation medium was
checked and adjusted to 7.0.
The epidermal strips were floated on 20 ml of incubation medium in Petri dishes of
5 cm diameter either illuminated or kept in the dark. The light source was a bank of
incandescent bulbs and the light intensity after passing through the water filter was 12
Klux at the surface of the experimental material. The temperature was maintained at
27 + 2°C. At regular intervals one of the epidermal strips was removed out and examined
under the microscope. The size of stomatal aperture was measured with the help of a
precalibrated occular micrometer. Each time an average of thirty stomata selected at
random was calculated. The experiment was repeated at least thrice on different days.
The average of these is reported.
Abscisic acid was dissolved in a drop of saturated Na2CO3 solution, whereas kinetin
and BA were dissolved in i N NaOH. They were diluted with a few millilitres of distilled
water, pH adjusted to 7.0 and made up to required volume with 0.067 M phosphate
buffer pH 7.0. < ^U^r-.: •-..-•..-:.:• .. ., , f ,:: - • ' : ... f ; ' : I ^ "^ " . S ^ ^ . • ; ' '. ' .
'•• -^'-^'••-•'^ • • ' • ' ' • ' • ' • : ' R E S U L T S ' '' ' '• ' ' ' " '" '
Only benzyl adenine enhanced stomatal opening in epidermal strips of Commelina
benghalensis L. and Tridax procumbens (Table i). Kinetin exerted no remarkable effect.
1. Effect of kinetin and benzyl adenine on stomatal
opening in Commelina benghalensis and Tridax pro-
cumbens
• ' Treatment Stomatal aperture (/jm)
,, C. benghalensis T. procumbens
, Initial* 4.2 2.4
After illumination! '
Control 7 : ; v;. 12.7 • > 8.5
+ Kinetin io~* M - " - ^ "8.6
+ Kinetin 5 X 10 "^ M '' ' 12.9 8.8*
+ Kinetin 10"* M ..', 12.5 . 8.4
+ BA io-= M , . 13-2 8.8
' + B A s x i o - = M - ' 17-6 9.8
, + B A i o - * M 12.8 9.0
* At the start of the experiment.
» , ; ; ; ; ! - :.r;t3^ t At an intensity of 12 Klux for 5 h. -. :-.nr<^. , -. i,. >.-•
The stimulatory effect was more pronounced in case of Commelina, but it was also quite
significant in Tridax. Fig. i shows the critical importance of the concentration of BA
in stimulation of stomatal opening. Since the increase of aperture was maximum in
5 X io~^ M BA. This concentration was used in the following experiments.
Stomata of Commelina benghalensis opened wider in 5 x io~^ M BAbut the stimulation
was partially suppressed by the presence of ABA in the medium (Fig. 2). The stomata
closed when ABA was alone present in the medium, but it was evident that BA maintained
the stomatal opening to a remarkable level even in the presence of ABA. Addition of BA
rapidly reversed the closure induced earlier by ABA. Both the action of ABA as well
as the reversal of it by BA was rapid. . ' . ! , . , : ! , 1 ' - . i : . . J - 'i?
DISCUSSION
The relative insensitivity of stomatal aperture to kinetin and the narrow range of effective
concentrations of BA, clarify the controversies which arise from previous observations.
Reversal of stomatal closure 451
20
18
16
\ I 0
D I
o 8
D
i 6
^ 4
10-6 10- 10
Benzyl adenine ( M )
Fig. I
10-3 2 3
Time(h)
Fig. 2
Fig. I. Stimulation of stomatal opening by benzyl adenine in isolated epidermal strips of
Commdina benghalensis. The epidermal strips were incubated in a light of 12 Klux.
Fig. 2. Reversal of abscisic acid induced stomatal closure by benzyl adenine. The epidermal
strips of Commelina benghalensis were incubated in a light of 12 Klux. Stomata opened as
usual in control sets (•) whereas the inclusion of ABA in the medium (o) closed them.
However ABA induced stomatal closure was rapidly reversed when BA was added after two
hours (A) . The arrow indicates the addition of BA. The course of stomatal opening in
presence of both BA and ABA (A) is also shown.
The failure of stomata in epidermal strips to respond to kinetin is in agreement with the
findings of Tucker and Mansfield (1971). Horton (1971) also could not find any effect
of either BA or kinetin on the stomatal movement in Vicia. The present findings make
it clear that BA can significantly enhance stomatal opening, particularly within a limited
range of concentration. Previous reports have indicated a stimulatory effect of kinetin
on stomatal opening and transpiration in leaves (Livne and Vaadia, 1965; Meidner,
1967; Luke and Freeman, 1967, 1968; Cooper et al., 1972).
The stomata of Commelina benghalensis responded more than those of Tridax pro-
cumbens to the presence of BA in the medium. Such a differential response strikes a
similarity to the earlier findings of Luke and Freeman (1968) and Cooper et al. (1972)
who suggested that the stimulatory effects of kinetin might be confined to graminaceous
genera. The reasons for such differences can be explained only after further detailed
investigations.
There is convincing evidence for the possible control of transpiration in leaves by a
balance between cytokinins and abscisic acid. It is now well established that leaves of
water-stressed plants contain more ABA (Wright, 1969; Milborrow and Noddle, 1970;
Zeevart, 1971; Most, 1971) and less cytokinins (Itai and Vaadia, 1965, 1971). Detailed
investigations with the wilty mutant of tomato also indicated that wilting of plants was
associated with the increase in cytokinin content and decrease in ABA (Tal and Imber,
1970, 1971; Tal, Imber and Itai, 1970).
It seems clear that ABA and BA can cause stomatal opening or closure depending
upon their relative contents. This fact refiiects the key importance of their level in the
leaf in controlling the water loss from the plants.
\ . ACKNOWLEDGMENTS
A.S.R. was in receipt of an U.G.C. Junior Research Fellowship. The generous gifts of
452 V. S. R. D A S , I . M . R A O A N D A. S. R A G H A V E N D R A
ABA from Dr B. V. Milborrow, Milstead Laboratory of Chemical Enzymology, Kent,
U.K. and Messrs P. Hoffman-La Roche, Basel, Switzerland are gratefully acknowledged.
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Reversal of abscisic acid induced stomatal closure by benzyl adenine

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Reversal of abscisic acid induced stomatal closure by benzyl adenine

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