Department of Botany, Sri Venkateswara University, Tirupati - 517502, Andhra Pradesh, India

Replacibility of Potassium by Sodium for Stomatal Opening in Epidermal Strips of Cornrnelina benghalensis1

)

A. s. RAGHAVENDRA, 1. M. RAO and V. S. R. DAS

With 4 figures

Received April 4, 1976 . Accepted May 20, 1976

Summary

Sodium enhanced stomatal opening in epidermal strips of Commelina benghalensis, both in light and darkness. Potassium was not needed for stomatal opening when enough sodium ions were already present in the medium. The optimal concentration of sodium required for stomatal opening was nearly double that of potassium. The stimulatory effect of sodium was essentially similar to potassium in all respects. Sodium-stimulated stomatal opening in light was sensitive to the functioning of cyclic photophosphorylation, inhibited by abscisic acid and enhanced by fusicoccin. The results suggested that sodium could meet the requirement of potassium for stomatal opening.

Key words: stomatal opening, stimulatory effect, K, Na, Commelina benghalensis.

Introduction

ILJIN (1959) reported that univalent cations (sodium, potassium and rubidium) stimulated stomatal opening, whereas divalent cations like magnesium, calcium and strontium favoured stomatal closure. The specific role of potassium was revealed from the work of FU]INO (1967) and FISCHER (1968). Recent experiments even established the quantitative relationship between stomatal aperture and guard cell potassium content (SAWHNEY and ZELITCH, 1969; HUMBLE and RASCHKE, 1971; RASCHKE and FELLOWS, 1971; FISCHER, 1972; ALLAWAY and HSIAO, 1973).

But the studies of WILL MER and MANSFIELD (1969) and those of PALLAGHY (1970) suggested that sodium ions stimulate stomatal opening at certain conditions. During our work with isolated epidermal strips, we noticed that at times stomata did not respond to the addition of KCI, as they used to do normally. The possible reason was traced to the presence of Na+ ions already in the medium. Unlike the work on the role of potassium in stomatal opening, the studies with sodium are very few. In view

I) The work was supported by an U.G.C. Junior Research Fellowship to A. S. RAGHA VENDRA.

z. Pflanzenphysiol. Ed. 80. S. 36-42. 1976.

Replacibility of K by Na for stomatal opening 37

of the above fact, the present investigation was initiated to examine the role of sodium in detail on stomatal opening in isolated epidermal strips.

Material and Methods

First, second and third fully expanded leaves were picked from Commelina benghalensis L. grown under 12 h photoperiod. Strips, of about 1.0 X 0.5 cm size were prepared from the lower epidermis of leaves (RAGHAVENDRA and DAs, 1972). The strips were maintained in 0.05 M Tris-HCl buffer, pH 7.0 until used. The incubation medium in control sets was 0.05 M Tris-HCl buffer, pH 7.0. Unless otherwise mentioned, the concentrations of KCl and NaCI in the incubation medium were 20 mM KCl and 40 mM NaCI. When required the test compounds were included in the incubation medium at the given final concentrations. Every time pH of 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. They then either were illuminated or kept in dark. The light source was a bank of incandescent bulbs and the light intensity after passing through the water filter was 12 K lux at the surface of the experimental material. The temperature was 27 ± 2 0c. At regular intervals one of the epidermal strips was removed 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 30 stomata selected at random was taken. The experiment was repeated at least thrice on different days. The average of these is reported.

Results

Table 1 summarizes the effects of both KCl and NaCI on stomatal opening in light as well as in darkness. Sodium also was effective in stimulating stomatal opening even in darkness. However as with potassium, light was enhancing such stimulation.

Stomata readily responded to sodium when added in the complete absence of potassium in the incubation medium (Fig. 1). The enhancement of stomatal opening by the addition of potassium was marked in the absence of sodium (Fig. 2). But the

Table 1: Effect of potassium ;lnd sodium on stomatal opening in light and darkness (Mean ± S. E.).

Experimental condition

Initiala) After five hours Control (Buffer only) + KCl + NaCI

Stomatal aperture (urn)

Light Darkness

0.8 ± 0.06

1.2 ± 0.1 13.2 ± 1.2 12.6 ± 1.8

0.8 ± 0.06

1.0 ± 0.08 9.8 ± 2.2 9.2 ± 1.8

The strips were floated on a control incubation medium of 0.05 M Tris-HCl buffer, pH 7.0. They were then either illuminated at 12 K lux or incubated in darkness at 27 ± 2°C for five hours. a) At the start of the experiment.

Z. Pjlanzenphysiol. Bd. 80. S. 36-42. 1976.

38 A. S. RAGHAVENDRA, 1. M. RAO and V. S. R. DAS

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Fig. 1: The effect of Na+ on stomatal opening. The stomata were allowed to open either in presence (circles) or absence ( closed circles) of N a + ions. The influence of addition of sodium to control medium after allowing the stomata to open for two hours (triangles) is also shown. The arrow indicates the addition of sodium.

addition of potassium did not result any remarkable change when sodium was already present (Fig. 3). However the concentration of sodium (40 mM) required for maximal stomatal opening was above that of potassium (20 mM) (Fig. 4).

Sodium stimulated stomatal opening in light was suppressed by abscisic acid (Table 2). Fusicoccin, ATP and pyruvate were effective in stimulating stomatal aperture

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Fig. 2: Influence of potassium over stomatal opening. The time course of stomatal opening in control (closed circles) and in the presence of K (open circles). Stomata readily responded when K+ was added after 2 hrs to control medium (triangles). The arrow represents the addition of potassium.

Z. Pjlanzenphysiol. Bd. 80. S. 36-42. 1976.

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Replacibility of K by Na for stomatal opening 39

2 3 4- 5

Time (hour~)

Fig. 3: Time course of stomatal opening in presence (open circles) or absence of sodium (closed circles). The addition of K+ (indicated by arrow) did not greatly influence stomatal opening (triangles). The effect in contrast with Fig. 2 can be noted.

both in light and darkness. The stimulation of stomatal opening by sodium was observed only under conditions favourable for cyclic photophosphorylation. Phenazine methosulphate, a catalyst of cyclic photophosphorylation stimulated stomatal opening (Table 3). Ferricyanide, which catalyses non cyclic photophos­phorylation closed down the stomata. O-phenanthroline, an inhibitor of noncyclic photophosphorylation could decrease only slightly stomatal aperture, whereas dinitro-

20~---------------------------'

o 20 40 60 eo '00

Na+ or K+ Concentration (mM)

Fig. 4: Stomatal opening as a function of K+ or Na+ concentration. The stomata were allowed to open in a light of 12 K lux for 5 hrs.

Z. Pjlanzenphysiol. Bd. 80. S. 36-42. 1976.

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

Table 2: Effect of various compounds on sodium stimulated stomatal opening in light and darkness (Mean ± S. E.).

Stomatal aperture (urn) Experimental condition Buffer Buffer + NaCl

Light Darkness Light Darkness

Initiala) 1.2 ± 0.2 1.2 ± 0.2 1.2 ± 0.2 1.2 ± 0.2 After five hours Control 2.4 ± 0.3 1.4 ± 0.3 16.8 ± 2.8 9.2 ± 1.7 + Abscisic acid 1.2 ± 0.6 1.0 ± 0.4 3.2 ± 0.3 2.4 ± 0.6 + Fusicoccin 3.5 ± 0.5 3.5 ± 0.2 18.9 ± 3.6 18.6 ± 4.8 +ATP 2.8 ± 0.4 2.6 ± 0.8 17.2 ± 4.2 14.3 ± 3.4 + Pyruvate 2.8 ± 0.4 2.6 ± 0.2 17.8 ± 4.6 14.6 ± 2.8 + A TP + Pyruvate 3.3 ± 0.5 3.3 ± 0.8 18.6 ± 5.2 15.8 ± 3.8

The concentrations of chemicals were: NaCl, 40 mM; Abscisic acid 10-5 M; Fusicoccin, 10-5 M; A TP, 10-2 M and Pyruvate 2 X 10-2 M. a) At the start of the experiment.

Table 3: Influence of catalysts and inhibitors of photophosphorylation on sodium stimulated stomatal opening in light (Mean ± S. E.).

Experimental condition

Initiala) After five hours control + Phenazine methosulphatc + Ferricyanide + O-phenanthroline + Dinitrophenol

Stomatal aperture (urn)

2.4 ± 0.6 18.8 ± 2.3 20.2 ± 3.5

4.2 ± 0.4 17.4 ± 3.5 3.8 ± 0.6

The epidermal strips were incubated in 0.05 M Tris-HCI buffer pH 7.0 having 40 mM NaCI, in a light of 12 K lux. The concentrations of chemicals used were: Phenazine Methosulphate, 5 X 10-3 M; Ferricyanide, 10-2 M; O-Phenanthroline, 10-3 M and 2,4-Dinitrophenol, 10-4 M. a) At the start of the experiment.

phenol, an inhibitor of cyclic photophosphorylation, significantly inhibited stomatal

opening.

Discussion

The present results suggested that potassium could be replaced effectively by

sodium for stomatal opening. Previous investigations by WILLMER and MANSFIELD

(1969) who observed sodium content in lower epidermal strips to increase

proportionally with the stomatal aperture and PALLAGHY (1970) indicated

z. Pflanunphysiol. Bd. 80. S. 36-42. 1976.

Replacibility of K by Na for stomatal opening 41

stimulation of stomatal opening by Na+ ions. Sodium might have accumulated In

enough quantities to account for turgor pressure in guard cells. Hitherto it was considered that potassium was indispensable for stomatal opening.

However, stomata readily responded to sodium to open wider. In fact stomata did not respond to potassium, after being allowed to open in presence of sodium. This observation also indicates that potassium mainly acts as an agent for increase in osmotic pressure of guard cells, which requirement can be met with sodium ions also.

Studies on K+ deficient sugar beet plants suggest similar situation. GRAHAM and ULRICH (1972) observed that there were two factors for stomatal closure in K+ deficient leaves and one of them was the absolute requirement for K+ for stomatal opening. Severe decreases in leaf diffusion (mainly stomatal) resistance of K+ deficient sugarbeet plants could be obtained only under conditions of low Na+ availability (TERRY and ULRICH, 1973). When sodium was available, such drastic conditions are remarkably masked. Hence TERRY and ULRICH (1973) felt that sodium was able to substitute at least partly the apparent K+ requirement for stomatal opening. The present observations offer a direct evidence for such possible substitution of Na+ for K+ during stomatal opening. Neverthless the greater stimulation of stomatal opening by lower concentrations of potassium, compared to those of sodium, indicated that potassium was preferred to sodium.

The conditions for stimulation of stomatal opening by sodium were exactly similar to those of potassium. Though guard cells are known to take up significant amounts of K+, potassium uptake by guard cells, at least in light, was dependent on the functioning of Photosystem I or cyclic photophosphorylation (HUMBLE and HSIAO, 1970; DAS and RAGHAVENDRA, 1974 a, inhibited by abscisic acid (HORTON and MORAN, 1971; MANSFIELD and JONES, 1971) and stimulated by fusicoccin (SQUIRE and MANSFIELD, 1972, 1974; TURNER, 1972). The stimulation of stomatal opening by sodium, in light, was also seen similarly under conditions favourable for cyclic photophosphorylation (Table 3). The effect of sodium was nullified by abscisic acid and enhanced by fusicoccin (Table 2). The stimulation of stomatal opening by ATP and pyruvate in presence of sodium is also in agreement with the previous findings (DAS and RAGHAVENDRA, 1974 b).

Acknowledgements

The gifts of abscisic acid from Dr. B. V. MILBORROW, Milstead Laboratory of Chemical Enzymology, Kent, U. K. and Hoffmann-La Roche Co., Basel, Switzerland are gratefully acknowledged. The author wish to thank Dr. N. C. TURNER, CSIRO, Canberra for his generous gift of fusicoccin.

References

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Z. PJlanzenphysiol. Bd. 80. S. 36-42. 1976.

42 A. S. RAGHAVENDRA, 1. M. RAO and V. S. R. DAS

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Dr. A. S. RAGHAVENDRA, Central Plantation Crops Research Institute, Regional Station, Vittal 574243, Karnataka, India.

Z. Pflanzenphysiol. Bd. 80. S. 36-42. 1976.