Plant Ceil Reports

Plant Cell Reports (1984) 3:199-202

© Springer-Verlag t984

Stomatal opening in isolated epidermis of Commelina benghalensis L. heterophasic response to KCI concentration B.V. Murthy, A. S. Raghavendra, and V. S. Rama Das Plant Physiology Division, Rubber Research Institute of India, Kottayam 686 009, India Received November 14, 1983/Revised version received August 17, 1984 - Communicated by R. g~( F. Hardy

ABSTRACT The pattern of stomatal opening in epidermal strips detached from leaves of Commelina benghalensis was examined. Two different phases could be distinguished in the stomatal response to KC1, one at low concentrations of k~C1 (up to 60 m}~) and the other at high KC1 concentrations (above 1OO ~ ) . The stomatal opening at low l(C1 concentrations was stimulated remarkably by light or fusicoccin and was suppressed by abscisic acid. At higher ~C1 concentrations, the stimulation by light or FC as well as the inhibition by ABA was limited. Both phases of stomatal response to KC1 were sensitive to carbonyl cys~ide-m-chlorophenyl hydrazone. The results suggest that illumination or ~C favours selectively stomatal opening only at low KC1 concentrations. The ionic participation in the stomatal opening is similar to the heterophasic upta~ke of ions by plant cells/roots. Abbreviations : FC, fusicoccin; ABA, abscisic acid ; CCCP, carbonyl cyanide-mchlorophenyl hydrazone.

durinE a re-evaluation, Travis and ~ansfield (1979~ observed a concentration of 50 m~ ~C1 was optimal for stomatal movements in C. com~unis. Recently Zeiger (1983) has noticed that during ~C stimulated stomatai opening in C. communis, 30 ~ KC1 was optimal. We have therefore set out to examine in detail the pattern of stomatal response to wide range ef KC1 concentrations in light as well as darkness. The influence of varying the KC1 concentration in the incubation medium on stomatal movement was assessed in the presence of ABA or FC or an uncoupler of photophosphorylation, CCCP. The response of stomata to the above effectors (ABA, FC) is also checked at low (30 ~ ) or high (100 m~) levels of gC1. ~ATERI ALS A ~

~THODS

The p ! ~ t s of Commelina benghalensis, were raised in 30 cm diameter seed pans grown outdoors in a "natural 12 h photoperiod and average temperature of 300C/20°C day and night. Second to 4th leaves from the top were used for experiments. Strips of 10 x 5 mm were prepared from lower epidermis of leaves as described by Raghavendra ( 1 9 8 1 )

I ~TR ODUC TI ON Stomatal movements are the result of changes in turgor potential of guard cells (Allaway and ~,~ilthorpe 1976, Raschke 1979). The increase in turgor of a guard cell is mainly due to the uptake of potassium and/ or anions like chloride or nitrate (Raschke 1979, '~acRobbie and Lettau 1980). Although the role of potassium in modulating the guard cell turgor has been well established, there were variations in the reported optimal concentration of potassium salts for stomatal opening. Stomata of Vicia faba require as little as I mN KC1 for maximal opening (Fischer 1968, Humble and ~siao 1969), while 1OO mN KC1 was required for maximal stomatal opening in epidermal strips of Commelina sps. (Wilson et al. 1978, Raghavendra et al. 1976). However

Strips were placed in 25 ml beakers containing 5 ml of incubation medium (0.05 5~ Ca (~T03)2; 25 mN Tris-HCl buffer, pH 7.3 with varying KCI concentrations (10 m~ 200 n~). Experiments were carried out with Fo.., ABA, CCCP (10 ~M) in the incubation medium with illumi'nation (100 ~ m-2s -I ) provided by a bank of incandescent bulbs and dark conditions, for a 3 h incubation period. The effect of ABA, FC were examined at 2 KC1 concentrations, one at 30 ~ and the other at 100 m~. At the end of the incubation period, the width of stomatal aperture was determined with a precalibrated occular micrometer. Ten stomata were observed in at least 3 strips and the average aperture size was taken. Experiments were repeated tba~ice on different days.

200

Potassium was estimated with an Elico Flame Fhot ouster. RESULTS There were at least two different phases in the stomatal response to ~C1 concentration, one at low concentrations of ~C1 (up to 60 ~ ) and the other at high I{01 concentrations (above 100 ~ Fig. I).

concentration (100 ~ - ?ig. 4 b). However there was no difference in the saturation response t o FC of stomata at 30 or 100 m~ EC1. The pattern of K + uptake by epidermal tissue also exhibited a heterophasic curve similar to that of stomatal aperture (Fig. 5 ).

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Fig. I : Effect of varying LrCI concentrations on stomatal opening in light or darkness. O - Light

• - dark

& - L i g h t activation of stomatal opening, the readings represent the difference in Bm of the width of stomatal aperture in light and darkness. Calculated from the data of Fig. i. Stomatal opening was stimulated by ~ in both light ~id dark conditions at low KC1 concentrations. At higher EC1 concentrations the stimulation by light or FC was limited (Fig. 2). In the presence of ABA, the opening of stomata was completely suppressed at low E01 concentrations and partial opening occurred at high KCI concentrations (Fig. 2). The degree of stomatal opening in presence of CCCP was markedly less than that in its absence. There was no opening in darkness while ~CCP was present (Fig. 3 b). We monitored the width of s t ~ a t a l aperture every 30 rain during the incubation of epidermal strips w i t h or without PC, ABA or CCCP. ~o marked influence of these effectors was noticed on kinetics of opening. The time ce~rse of stomatal opening in presence of CC~P , a powerful uncoupler, is presented as an example (Fig. 3 a). The stimulation of stomatal opening by light could be observed at 100 m~[ or lower KC! levels only (~ig. I). Stomata were more sensitive to ABA at lower (30 aM) KC1 concentration and less sensitive at higher (100 m~[) ~O1 concentration i_ig. 4 a) Stimulatory effect by FC is more at low ~ l concentration (30 m}~:) and less at higher knG1

~ g . 2 : Effect of 10 ~.~ FC or ABA on Stomital opening in relation to ~TCI concentration in light or darkness. D , |-

Light or Dark FC

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Light or Dark ABA

DI SCUS SI ON The results reveal that there are at least two different phases in the response of stomatal opening to ~C1, one at a low range (up to 50 or 60 ~4) and the other phase at higher levels of KC1 (more than 70 ~;~). The stomatal opening in low range of }~C1 is remarkably stimulated by light (Fig. I), while the opening in darkness is facilitated only at high EC1 concentrations. These observations are similar to those of Humble and Hsiao (1969) who found that the light activated opening of stomata in epidermal strips of V i c i a faba, exhibited a specific requirement for potassium at a concentration of I aM. Only at 10 m~ or higher cation levels stomatal opening could be stimulated by sodium, lithium as well as with p o t ~ s i u m or rubidium in either light or darkness. ~ince all of our experiments were conducted at ambient CO2 (about 320) ppm) in equilibrium with the incubation medium, we believe that the observed stomatal responses are not the result of their sensitivity to CO 2. The two phases could be further differentiated by their response to light and other effectors. The stomatal opening at low T~l levels is markedly stimulated by light

201 (Fig. I), and is extremely sensitive to i~, ABA or COOP (Figs. 2 and 3 b). Fig. I also demonstrates that light activation of stomatal opening can be seen only at lower 7~C1 levels. High levels of KC1 have earlier been noticed to mask the effects of light or O02 (Travis and ~ansfield, 1979).

50 to 200 nt~. Our results (Fig. 2) confirm that the inhibition by ABA can be partially relieved by 80 m~ or more KC1 in light or even higher (160 - 200 m}~) ED1 in the dark.

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