Plant Science Letters, 6 (1976) 111--115 III © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

CHARACTERISATION OF ABSCISIC ACID INHIBITION OF STOMATAL OPENING IN ISOLATED EPIDERMAL STRIPS*

A.S. RAGHAVENDRA**, I.M. RAO and V.S.R. DAS

Department of Botany, Sri Venkateswara University, Tirupati 517502, A.P. (India)

(Received July 8th, 1975) (Revision received October 30th, 1975) (Accepted November 28tb, 1975)

SUMMARY

Abscisic acid (ABA) inhibited the light-induced opening of stomata in isolated epidermal strips of Commelina benghalensis. It did not alter stomatal closure in the dark. The ABA-induced inhibition in light was released under conditions conducive for cyclic photophosphorylation and remarkably reversed by ATP in the presence of pyruvate. Cyclic photophosphorylation rates of isolated guard cell chloroplasts were significantly reduced by ABA. It is proposed that the direct effect of ABA on stomatal opening was mediated in two ways: (1) by inhibition of cyclic photophosphorylation activities of guard cell chloroplasts and (2) by blocking organic acid formation in guard cells.

INTRODUCTION

ABA is an effective inhibitor of stomata] movements in intact leaves [1,2] and isolated epidermal strips [3,4]. The action of ABA is rapid [5,6], but how its effects are established is not fully understood. ABA has been proposed to act through an effect on enzymatic hydrolysis of starch [3], because it inhibited amylase activity in barley endosperm [ 7 ]. However, existing know- ledge on the mechanism of stomatal movement indicates three possible ways for a rapid action of ABA: (1) Inhibition of enzymatic starch hydrolysis, (2) Impairment with energy production necessary for stomatal opening, and (3) Acceleration of the closing process of stomata. We did therefore investigate the influence of ABA on the closing as well as the opening movements of stomata. The effect of ABA with special reference to ATP was also examined.

Supported by an U.G.C. Junior Research Fellowship to A.S.R. Present address: Central Plantation Crops Research Institute, Regional Station, Vittal

574243, Karnataka, India. Abbreviations: ABA, abscisic acid; ATP, adenosine triphosphate; DNP, 2,4-dinitrophenol; PS I, Photosystem I.

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MATERIALS AND METHODS

The lower epidermal strips were obtained from the leaves of Commelina benghalensis L. as already described [8]. The strips were kept in 0.067 M phosphate buffer at pH 7.0 until needed. The control medium for studying stomatal opening contained 0.067 M phosphate buffer at pH 7.0 with 10 mM KC1. The control medium for stomatal closure was 0.067 M phosphate buffer pH 7.0 with 2 mM CaC12. The methods for incubation, illumination and recording of stomatal apertures were as before [8,9]. The values expressed are averages of three separate experiments conducted on different days.

Guard cell and mesophyll chloroplasts from leaves of C. benghalensis were isolated in 0.05 M Tris--HC1 pH 7.8 containing 0.35 M NaC1 [9]. The photophosphorylation rates were determined by observing light dependent 32Pi incorporation [ 10].

RESULTS

ABA inhibited stomatal opening in light as shown in Fig. 1A. The most effective concentration of ABA was 10- 4 M. It had no significant effect on stomatal closure (Fig. 1B). Phenazine methosulphate, a catalyst of cyclic photophosphorylation, and o-phenanthroline, an inhibitor of noncyclic photophosphorylation, released the ABA-induced inhibition of the opening. (Table I). On the other hand, ferricyanide and DNP, which block cyclic photophosphorylation, had no effect on stomatal opening in the presence of ABA. ABA could not inhibit the opening in the presence of ATP and pyruvate neither in light nor in darkness. The reversal by pyruvate and ATP was as rapid as the stomatal closure caused by ABA (Fig. 2).

ABA decreased cyclic photophosphorylation activities of isolated guard cell chloroplasts (Table II).

18

16

~= 12 .E

a G( -6

9

2

A

0 j O • •

i i i i i 1 2 3 4 5

B

i i i I i

1 2 3 4 5

Time in hours

Fig.1. Time course o f s tomata l open ing or closure in epidermal str ips o f Commelina benghalensis in a light o f 12 k lux (A) or darkness (B). e, Cont ro l ; A, 10 -6 M ABA and ~, 10 - s M ABA.

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TABLE I

EFFECT OF VARIOUS CATALYSTS AND INHIBITORS OF PHOTOFHOSPHORYLA-- TION ON ABA-INHIBITED STOMATAL OPENING IN LIGHT

The concentrations of chemicals were: ABA, 10 -s M; Phenazine methosulphate, 5 .10 -3 M; Ferricyanide, 10 -2 M; o-phenanthroline, 10 -3 M; and 2,4-dinitrophenol, 10 TM M.

Experimental condit ion Stomatal aperture (#)

Control 10 -s M ABA

Initial a 6.6 6.6 After il lumination for 5 h 12.6 2.4 + Phenazine methosulphate 15.0 7.2 + Ferricyanide 2.4 1.8 + o-phenanthroline 11.4 6.6 + 2,4-Dinitrophenol 2.4 1.8

a At the start of the experiment.

18:

~8 o ~ O -o

• [ : 12 •

°!f t~ ~ O - - - - - - - " O - - -O

I I I I I

1 2 3 4 5

Time in hours

Fig.2. Reversal of ABA-in~ibited opening by ATP and pyruvate. Incubation in light of 12 klux. *, Control; 4, 10-s M ABA + 2.10 -= M pyruvate + 10 -= M ATP; v, 10 -s M ABA; A, Addit ion of 2- 10 -2 M pyruvate + 10 -= M ATP after incubation in 10 -s M ABA for 2 h. Arrow indicates the addit ion of pyruvate + ATP.

DISCUSSION

O u r o b s e r v a t i o n s s h o w t h a t A B A o n l y a f f e c t s t h e o p e n i n g p r o c e s s o f s t o m a t a a n d n o t t h e i r c l o s u r e (F ig . 1). A B A a p p e a r s t o a c t in t w o d i f f e r e n t w a y s . F i r s t l y , i t i m p a i r s A T P p r o d u c t i o n in l i g h t ( T a b l e I I ) . C o n t i n u o u s s u p p l y o f e n e r g y has b e e n p r o p o s e d t o b e e s s e n t i a l f o r t h e s t i m u l a t i o n a n d m a i n t e n a n c e o f s t o m a t a l o p e n i n g in l i g h t [ 9 , 1 1 ] . T h i s e n e r g y a p p a r e n t l y

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TABLE II

P H O T O P H O S P H O R Y L A T I O N A C T I V I T I E S OF I S O L A T E D G U A R D CELL AND M E S O P H Y L L C H L O R O P L A S T S F R O M L E A V E S OF COMMELINA BENGHALENSIS

Trea tmen t Pho tophosphory la t ion (~ moles Pi /mg chl /h)

Guard cell Mesophyll

Cyclic Noncycl ic Cyclic Noncycl ic

Contro l 569 32 418 236 + ABA (10 -s M ) a 143 31 322 222

a Final concen t ra t ion in the assay medium.

comes from cyclic photophosphorylation [8] and PSI in guard cell chloro- plasts [9,12,13]. Secondly, pyruvate was earlier found to be implicated in stomatal opening via its carboxylation and subsequent production of organic acids. ABA blocked the formation of organic acids and thus inhibited pot- assium uptake by ion exchange [14]. This finding is supported by the reversibility of ABA induced inhibition of stomatal opening by pyruvate in the presence of ATP (Fig. 2).

Other workers also noticed the correlation between organic acid production in epidermal strips and stomatal opening [15--17]. The inhibitory action of ABA on organic acid production might also be through its effect on amylase [ 7 ] or by an action at an unknown site in glycolysis, which was observed to play a key role in stomatal opening [18].

The rapidity and the reversibility of the ABA effect indicates its nontoxic nature. The rapid action of ABA has been noticed previously [5,6]. The recovery of stomatal resistance in leaves treated with ABA as observed by Kriedmann et al. [19] does also show the nontoxic nature of ABA.

A C K N O W L E D G E M E N T S

The authors wish to thank Dr. B.V. Milborrow, Milstead Laboratory of Chemical Enzymology, Kent, U.K., and M/S P. Hoffman-La Roche Co., Basel Switzerland for generous gifts of ABA.

R E F E R E N C E S

1 C.J. Mittelheuser and R.F.M. VanSteveninck, Nature, 221 (1969) 281. 2 R.J. Jones and T.A. Mansfield, J. Exp. Bot., 21 (1970) 714. 3 T.A. Mansfield and R.J. Jones, Planta, 101 (1971) 147. 4 R.F. Horton, Can. J. Bot., 49 (1971) 583. 5 C.J. Mittelheuser and R.F.M. VanSteveninck, Planta, 97 (1971) 83. 6 W.R. Cummins, H. Kende and K. Raschke, Planta, 99 (1971) 347. 7 F.T. Addicott and J.L. Lyon, Ann. Rev~ Plant Physiol., 20 (1969) 139.

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8 A.S. Raghavendra and V.S.R. Das, Curt. Sci., 41 (1972) 150. 9 V.S.R. Das and A.S. Raghavendra, in R.L. Bieleski, A.R. Ferguson and M.M. Cre~well

(Eds.), Regulation of Plant Growth, Bulletin No. 12, Royal Society of New Zealand, Wellington, 1974, p. 455.

10 A.S. Raghavendra, Ph.D, Thesis, Sri Venkateswara Univ., Tirnpati, 1975. 11 D.A. Thomas, Aust. J. Biol. Sci., 24 (1971) 689. 12 G.D. Humble and T.C. I-Isiao, Plant Physiol., 46 (1970) 483. 13 N.C. Turner, Nature, 235 (1972) 341. 14 V.S.R. Das and A.S. Raghavendra, Ind. J. Exp. Biol., 12 (1974) 425. 15 J.E. Pallas and B.G. Wright, Plant Physiol., 51 (1973) 588. 16 W.G. Allaway, Planta, 110 (1973) 63. 17 C.J. Pearson, Aust. J. Biol. Sci., 26 (1973) 1035. 18 I. Mouravieff, Physiol. Veg., 9 (1971) 109. 19 P.E. Kriedemann, B.R. Loveys, G.L. Fuller and A.C. Leopold, Plant Physiol.,

49 (1972) 842.