Planta
Planta 139, 167-170 (1978)
9 by Springer-Verlag 1978
Inhibition of Stomatal Opening during Uptake of Carbohydrates by Guard Cells in Isolated Epidermal Tissues P. Dittrich and M. M a y e r Botanisches Institut der Universitfit, Menzinger Str. 67, D-8000 Miinchen 19, Federal Republic of Germany
Abstract. The uptake of glucose and other carbohydrates into the g u a r d cells o f Commelina communis L. was f o u n d to inhibit the opening of the stomata. The concentration o f glucose necessary to achieve a b o u t 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree o f affinity. It is suggested that the uptake o f glucose occurs via a p r o t o n cotransport, which, depolarizing the m e m b r a n e potential, slows d o w n the electrogenic i m p o r t o f potassium ions. The process of stomatal closure, in contrast, appears not to be affected by c a r b o h y d r a t e uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, i m p o r t of glucose or other carbohydrates did not interfere with the regulation of stomatal movements. Key words: C a r b o h y d r a t e s - Stomata - Commelina - Proton-cotransport.
hydrates fed to epidermal tissues were incorporated into malate and starch (Dittrich and Raschke, 1977 a). However, the notion that starch within the guard cells originates f r o m guard-cell photosynthesis had to be dismissed, since Raschke and Dittrich (1977) demonstrated that the Calvin cycle was not functioning in guard cell chloroplasts. Consequently, the carbohydrates that are used in guard cells to power general metabolism and, in particular, the synthesis o f malic acid, have to be imported from other tissues. Dittrich and Raschke (1977b) have shown that the leaf mesophyll serves as a source of carbohydrates for the auxotrophic epidermal tissue, but so far no specific comp o u n d could be singled out and m a r k e d as the m a j o r transport metabolite. Because o f this dependence o f guard cells u p o n imported sugars, the scope of the present investigation was to elucidate the effect of exogenously applied carbohydrates u p o n the response of guard cells in isolated epidermal tissues with respect to opening or closing o f stomata.
Materials and Methods Introduction The m o v e m e n t o f guard cells and the associated opening or closing o f the stomatal pore is an active process, regulated by factors such as water, c a r b o n dioxide, light, or h o r m o n e s , which alter the osmotic potential o f the guard cell. The osmotic pressure in these cells is increased by u p t a k e o f potassium ions, whose positive charge is c o m p e n s a t e d either by i m p o r t of chloride ions or by endogenous synthesis of malic acid (Raschke, 1975). Since guard cells usually contain starch grains in their chloroplasts, this apparently photosynthetically derived c a r b o h y d r a t e was t h o u g h t to be the source o f c a r b o n for the f o r m a t i o n of malic acid. This contention was supported insofar as carbo-
Plants of Commelina communis L. were grown year round in a greenhouse at 23-24~ C during the day and 18~ C during the night. Daylength was extended to 14 h by illumination with mercury vapor lamps. To accelerate and standardize germination, seeds were incubated for 24h in a solution of 2x 10-SM gibberellic acid and kinetin in dry acetone prior to embedding in wet soil. Plants of Viciafaba L. were grown in a greenhouse at temperatures of 18-22~ C during the day and 12-16~ C during the night. No supplementary illumination was provided. Plants of Tulipa gesneriana L. were obtained from the Botanical Garden, Munich. Labeled compounds, D-[U- 14C]glucose' [U- 14C]malatose' and [U-14C]sucrose were purchased from Amersham-Buchler, Braunschweig, W-Germany. Epidermal strips were peeled from leaves, rinsed in distilled water, cut into sections of about 0.25 cm2, and incubated with the mesophyll side down on various solutions. The incubation took place in a microwaterbath with three chambers mounted on the stage of a microscope equipped with a camera. Illumination (15 mW/cm2) was provided by an incandescent lamp
0032-0935/78/0139/0167/$01.00
168
P. Dittrich and M. Mayer: Inhibition of Stomatal Opening
(M.m)
o KC[ 100 mM z~ KCI 100 mM 9
14
-
9
+
whose light was filtered through a 10 cm wide, water-filled glass cuvette. For the sugar incorporation studies epidermal samples of C. communis L., V. faba L , and T. genseriana L. were incubated for 2 h on solutions of 0.1 M KCI supplemented with labeled glucose (0.6 m M ) or sucrose (0.4 mM). The tissues were extracted thereafter three times in boiling 70% aqueous ethanol and subsequently subjected to microradioautography as described previously (Dittrich and Raschke, 1977a), Stomatal apertures were determined by taking photographs of the epidermal samples. The developed film negatives were projected on a screen for measuring the length and width of 20 stomatal pores. All experiments were carried out at 30 ~ C and, except for those where the closure of stomata was investigated, were performed in the light.
glucose 100mM
" -
§ sucrose
" -
* sorbito{ 100 m M
100mM ~____2
~
o
oE
~ /
g2 0
i 0.5
2
3
incubation time (h) Fig. 1. Kinetics of stomatal opening in isolated epidermis of C. eommunis L. Epidermal samples with closed stomata were incubated in the light at 30 ~ C with the mesophyll side down on solutions of 0.1 M KC1 or KC1 supplemented with 0.1 M concentrations of various carbohydrates. Stomatal apertures were recorded photographically
Table 1. The effect of carbohydrates upon stomatal opening in isolated epidermal tissues of C. communis L. Isolated epidermal strips with stomata closed were incubated in the light at 3 0 ~ on 0.1 M KCI (control) and on a solution of 0.1 M KC1 supplemented with 0.1 M of the individual carbohydrates listed. Apertures were recorded photographically over a period of 2 h Supplied carbohydrate
Stomatal aperture in % of 0.1 M KC1 control
Glucose 3-O-Methylglucose 6-Deoxyglucose Fructose Galactose Mannose Sucrose Maltose Trehalose Melibiose Myo-Inositol Sorbitol
30 45 40 35 50 40 100 110 55 80 100 100
glucose]
KC[
100mM
0
*
mM
_ o /
W,
9 30 mM
~_
-
/////~/j_~_-0.5
I
Uptake of Carbohydrates by Guard Cells and Formation of Starch In a previous publication (Dittrich and Raschke, 1977b), it was demonstrated that carbohydrates fed to epidermal tissue give rise to starch and other general metabolic compounds. No unequivocal evidence was presented as to where these conversions take place within the tissue. Thus, epidermal strips of C. communis L., V. faba L. and T. gesneriana L. were incubated on solutions of 14C-labeled glucose, sucrose, and maltose; after extraction of unconverted fed compounds and soluble conversion products, the epidermal strips were subjected to microradioautography in order to localize the site of starch formation. The developed films showed clearly that the insoluble radioactivity, i.e., starch, was restricted to the guard cells. No apparent difference was observed between the feeding experiments with the three plants and the three sugars. This result shows that glucose, sucrose, and maltose are actually taken up and converted by guard cells into starch.
Effect of Uptake of Carbohydrates upon Stomatal Opening in Commelina communis L.
(~tm)
0
Results
-
2o0 m~
2
3
incubation time (h) Fig. 2. Effect of various concentrations of glucose in 0.I M KCI upon the opening of stomata (light, 30 ~ C) in isolated epidermis of C. communis L.
Samples of epidermal tissue of C. communis L. with closed stomata were incubated on solutions of 0.1 M KC1 and for comparison on 0.1 M KC1 supplemented with 0.1 M of either glucose, sucrose or sorbitol in the light. The degree of stomatal opening was recorded and the results are summarized in Figure 1. The timedependent reaction of the guard cells upon 0.1 M KC1 was taken as a control to evaluate the effect of supplemented carbohydrates. Glucose was found to exhibit a pronounced inhibition of stomatal opening (Fig. 1). To consider a possible osmotic effect, sorbitol (0.1 M) was supplied to the epidermis, but not apparent effect upon the movement of the guard cells was observed. Sucrose, was also fed to guard cells. The guard cells showed no alternation of the
P. Dittrich and M. Mayer: Inhibition of Stomatal Opening opening process as compared to 0.1 M KC1; thus sucrose does not interfere with the mechanism of the stomata. Since osmotic effects or availability for metabolic reactions could be ruled out as a basis of action, other carbohydrates were tested with respect to inhibitory properties. The results are compiled in Table 1. Hexoses in general appear to inhibit the opening of stomata to about the same degree as glucose. It seems to be irrelevant whether the sugar taken up can be further metabolized or not, since 6-deoxyglucose and 3-O-methylglucose also had inhibitory properties. Disaccharides, in contrast, exerted no uniform action, with trehalose clearly inhibiting and maltose slightly stimulating the movement of guard cells. Figure 2 contains a compilation of results of experiments, in which different concentrations of glucose in 0.1 M KC1 were tested with respect to the inhibition of stomatal opening. It was found that concentrations of about 30 mM have to be applied to induce a significant effect, while in the range of 0.1 M inhibition appears to be close to saturation. Effect of Glucose on Stomatal Opening in Vicia faba L. and Tutipa gesneriana L. Epidermal tissues of V. faba L. and T. gesneriana L. were incubated on solutions of 0.1 M KC1 with no supplementation or with supplements of 0.1 M glucose, sucrose, or various other sugars. The opening responses of the guard cells of both plants were not affected by the sugars, and the final aperture of the stomata was in no way different from those of the controls. Since opening of stomata in C. communis L. is inhibited by glucose, it was concluded that the inhibition was due to a stimulated closing response; consequently, open stomata should close more rapidly if incubated on glucose. To elucidate this question, epidermal tissues of C. communis L. were harvested from leaves that had been floating on water at 30 ~ C in the light. The epidermis with stomata open (15 I~m) was subsequently incubated in the dark either on distilled water or on water supplemented with either 0.1 M glucose or 0.1 M sucrose. After about 3 0 4 0 min in the dark the stomata had closed, irrespective of whether sugar was present or not. Furthermore, no difference in the rate of stomatal closure between the incubation with glucose and that with sucrose was noted. Discussion
Raschke and Dittrich (1977) reported that guard cells appear to rely on the underlying mesophyll tissue
169 to supply the carbohydrates necessary for metabolic reactions. Thus, feeding carbohydrates to isolated epidermal tissues should improve the energy status of the cells and consequently could lead to a faster opening of the stomata. The variety of responses of guard cells in epidermal tissues of C. communis L. toward fed carbohydrates however ranged from substantial inhibition (hexoses) to no response (sucrose) to a slight stimulation (maltose). While the latter response might actually be in accord with an improvement of the energy supply of the cells, the pronounced inhibition of stomatal opening by glucose was unexpected. Since neither molecular size, osmotic effects nor sequestration of ATP during uptake (3-O-methylglucose is no subject to phosphorylating reactions) appears to be involved in the mode of action, the inhibitory effect might be a consequence of the mechanism of sugar uptake. The majority of transport systems in microorganisms and plants presumably operate via a proton-cotransport system (Kaback, 1974). Thus; galactosides in E. coli and glucose in Neurospora and Chlorella (Slayman and Slayman, 1974; K o m o r and Tanner, 1976) are taken up concomitantly with protons. In higher plants such as Ricinus communis L., experiments by K o m o r et al. (1977) suggested that sucrose from the endosperm enters the cotyledons jointly with protons. Even the loading of sieve tubes with sucrose appears to be powered by proton-cotransport (Malek and Baker, 1977). Raschke and Humble (1973) and Raschke (1977) reported that the opening of stomata is associated with an extrusion of protons from the guard cells. This efflux of H + generates a negative membrane potential (Pallaghy, 1967), which drives the passive uptake of K § If we assume that glucose is taken up into guard cells concomitantly with protons, a depolarization of the membrane potentiaI would result, limiting the uptake of potassium ions and consequently inhibiting the opening of the stomata. Potassium ions and the H+-glucose system would compete for the electromotive force of the membrane potential. In testing the competitiveness, a concentration of 0.1 M glucose was required in the presence of 0.1 M KC1 to bring about 50% inhibition of stomatal opening. Thus, the affinity of the guard cells for the uptake of potassium is about in the same range as the affinity of the carrier system for glucose uptake. Although many parameters of stomata regulation originate in the leaf mesophyll it is unclear what advantage the guard cells of C. communis L. derive from the operation of a proton-glucose cotransport. Glucose was shown to have no impact upon the closing process of stomata. Since we have in general little information about the mechanism of guard cell deflation, the present evidence suggests that, although opening and
170
closing are active processes, the events during opening are not a mere reversion of the closing procedure. The lack of an effect of glucose upon stomata of V. faba L. and tulip does not necessarily imply that basic mechanisms of osmoregulation of guard cells are different. We have to consider that C. communis L. operates stomata in concert with a number of subsidiary cells that are likely to modify the response. In addition to the impact of morphologic features, it cannot be ruled out that active carbohydrate transport systems m a y or may not be subject to induction phenomena in different organisms. We greatly appreciate the careful technical assistance by M. MeuseI and G. Ohst, the photographic work by K. Liedl and the helpful discussion with D. Gradmann.
References Dittrich, P., Raschke, K. : Malate metabolism in isolated epidermis of Comrnelina communis L. in relation to stomatal functioning. Planta 134, 77-81 (1977a) Dittrich, P., Raschke, K,: Uptake and metabolism of carbohydrates by epidermal tissue. Planta 134, 83-90 (1977b) Kaback, H.R. : Transport studies in bacterial membrane vesicles. Science 184, 882-892 (1974)
P. Dinrich and M. Mayer: Inhibition of Stomatal Opening Komor, E., Tanner, W.: The determination of the membrane potential of Chlorella vulgaris. Europ. J. Biochem. 70, 197-204 (1976) Komor, E., Rotter, M., Tanner, W. : A proton-cotransport system in a higher plant: sucrose transport in Ricinus communis. Plant Sci. Lett. 9, 153-162 (1977) Malek, F., Baker, D.A.: Proton co-transport of sugars in phloem loading. Planta 135, 297-299 (1977) Pallaghy, C.K.: Electrophysiological studies in guard cells of tobacco. Planta 80, 147 153 (1968) Raschke, K., Humble, G.D.: No uptake of anions required by opening stomata of Vicia faba: guard cells release hydrogen ions. Planta 115, 47-57 (1973) Raschke, K.: Stomatal action. Ann. Rev. Plant Physiol. 26, 309-340 (1975) Raschke, K., Dittrich, P. : 14C-Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open. Planta 134, 69-75 (1977) Raschke, K. : The stomatal turgor mechanism and its responses to CO 2 and abscisic acid: observations and a hypothesis. In: Regulation of cell membrane activities in plants, pp. 173-183, Marr6, E., Ciferri, O., eds. Amsterdam: Elsevier 1977 Slayman, C.L, Slayman, C.W.: Depolarization of the plasma membrane of Neurospora during active transport of glucose: evidence for a proton-dependent cotransport system. Proc. Natl. Acad. Sci. USA 71, 135-1939 (1974)
Received 14 October; accepted 14 December 1977
Planta 139, 167-170 (1978)
9 by Springer-Verlag 1978
Inhibition of Stomatal Opening during Uptake of Carbohydrates by Guard Cells in Isolated Epidermal Tissues P. Dittrich and M. M a y e r Botanisches Institut der Universitfit, Menzinger Str. 67, D-8000 Miinchen 19, Federal Republic of Germany
Abstract. The uptake of glucose and other carbohydrates into the g u a r d cells o f Commelina communis L. was f o u n d to inhibit the opening of the stomata. The concentration o f glucose necessary to achieve a b o u t 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree o f affinity. It is suggested that the uptake o f glucose occurs via a p r o t o n cotransport, which, depolarizing the m e m b r a n e potential, slows d o w n the electrogenic i m p o r t o f potassium ions. The process of stomatal closure, in contrast, appears not to be affected by c a r b o h y d r a t e uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, i m p o r t of glucose or other carbohydrates did not interfere with the regulation of stomatal movements. Key words: C a r b o h y d r a t e s - Stomata - Commelina - Proton-cotransport.
hydrates fed to epidermal tissues were incorporated into malate and starch (Dittrich and Raschke, 1977 a). However, the notion that starch within the guard cells originates f r o m guard-cell photosynthesis had to be dismissed, since Raschke and Dittrich (1977) demonstrated that the Calvin cycle was not functioning in guard cell chloroplasts. Consequently, the carbohydrates that are used in guard cells to power general metabolism and, in particular, the synthesis o f malic acid, have to be imported from other tissues. Dittrich and Raschke (1977b) have shown that the leaf mesophyll serves as a source of carbohydrates for the auxotrophic epidermal tissue, but so far no specific comp o u n d could be singled out and m a r k e d as the m a j o r transport metabolite. Because o f this dependence o f guard cells u p o n imported sugars, the scope of the present investigation was to elucidate the effect of exogenously applied carbohydrates u p o n the response of guard cells in isolated epidermal tissues with respect to opening or closing o f stomata.
Materials and Methods Introduction The m o v e m e n t o f guard cells and the associated opening or closing o f the stomatal pore is an active process, regulated by factors such as water, c a r b o n dioxide, light, or h o r m o n e s , which alter the osmotic potential o f the guard cell. The osmotic pressure in these cells is increased by u p t a k e o f potassium ions, whose positive charge is c o m p e n s a t e d either by i m p o r t of chloride ions or by endogenous synthesis of malic acid (Raschke, 1975). Since guard cells usually contain starch grains in their chloroplasts, this apparently photosynthetically derived c a r b o h y d r a t e was t h o u g h t to be the source o f c a r b o n for the f o r m a t i o n of malic acid. This contention was supported insofar as carbo-
Plants of Commelina communis L. were grown year round in a greenhouse at 23-24~ C during the day and 18~ C during the night. Daylength was extended to 14 h by illumination with mercury vapor lamps. To accelerate and standardize germination, seeds were incubated for 24h in a solution of 2x 10-SM gibberellic acid and kinetin in dry acetone prior to embedding in wet soil. Plants of Viciafaba L. were grown in a greenhouse at temperatures of 18-22~ C during the day and 12-16~ C during the night. No supplementary illumination was provided. Plants of Tulipa gesneriana L. were obtained from the Botanical Garden, Munich. Labeled compounds, D-[U- 14C]glucose' [U- 14C]malatose' and [U-14C]sucrose were purchased from Amersham-Buchler, Braunschweig, W-Germany. Epidermal strips were peeled from leaves, rinsed in distilled water, cut into sections of about 0.25 cm2, and incubated with the mesophyll side down on various solutions. The incubation took place in a microwaterbath with three chambers mounted on the stage of a microscope equipped with a camera. Illumination (15 mW/cm2) was provided by an incandescent lamp
0032-0935/78/0139/0167/$01.00
168
P. Dittrich and M. Mayer: Inhibition of Stomatal Opening
(M.m)
o KC[ 100 mM z~ KCI 100 mM 9
14
-
9
+
whose light was filtered through a 10 cm wide, water-filled glass cuvette. For the sugar incorporation studies epidermal samples of C. communis L., V. faba L , and T. genseriana L. were incubated for 2 h on solutions of 0.1 M KCI supplemented with labeled glucose (0.6 m M ) or sucrose (0.4 mM). The tissues were extracted thereafter three times in boiling 70% aqueous ethanol and subsequently subjected to microradioautography as described previously (Dittrich and Raschke, 1977a), Stomatal apertures were determined by taking photographs of the epidermal samples. The developed film negatives were projected on a screen for measuring the length and width of 20 stomatal pores. All experiments were carried out at 30 ~ C and, except for those where the closure of stomata was investigated, were performed in the light.
glucose 100mM
" -
§ sucrose
" -
* sorbito{ 100 m M
100mM ~____2
~
o
oE
~ /
g2 0
i 0.5
2
3
incubation time (h) Fig. 1. Kinetics of stomatal opening in isolated epidermis of C. eommunis L. Epidermal samples with closed stomata were incubated in the light at 30 ~ C with the mesophyll side down on solutions of 0.1 M KC1 or KC1 supplemented with 0.1 M concentrations of various carbohydrates. Stomatal apertures were recorded photographically
Table 1. The effect of carbohydrates upon stomatal opening in isolated epidermal tissues of C. communis L. Isolated epidermal strips with stomata closed were incubated in the light at 3 0 ~ on 0.1 M KCI (control) and on a solution of 0.1 M KC1 supplemented with 0.1 M of the individual carbohydrates listed. Apertures were recorded photographically over a period of 2 h Supplied carbohydrate
Stomatal aperture in % of 0.1 M KC1 control
Glucose 3-O-Methylglucose 6-Deoxyglucose Fructose Galactose Mannose Sucrose Maltose Trehalose Melibiose Myo-Inositol Sorbitol
30 45 40 35 50 40 100 110 55 80 100 100
glucose]
KC[
100mM
0
*
mM
_ o /
W,
9 30 mM
~_
-
/////~/j_~_-0.5
I
Uptake of Carbohydrates by Guard Cells and Formation of Starch In a previous publication (Dittrich and Raschke, 1977b), it was demonstrated that carbohydrates fed to epidermal tissue give rise to starch and other general metabolic compounds. No unequivocal evidence was presented as to where these conversions take place within the tissue. Thus, epidermal strips of C. communis L., V. faba L. and T. gesneriana L. were incubated on solutions of 14C-labeled glucose, sucrose, and maltose; after extraction of unconverted fed compounds and soluble conversion products, the epidermal strips were subjected to microradioautography in order to localize the site of starch formation. The developed films showed clearly that the insoluble radioactivity, i.e., starch, was restricted to the guard cells. No apparent difference was observed between the feeding experiments with the three plants and the three sugars. This result shows that glucose, sucrose, and maltose are actually taken up and converted by guard cells into starch.
Effect of Uptake of Carbohydrates upon Stomatal Opening in Commelina communis L.
(~tm)
0
Results
-
2o0 m~
2
3
incubation time (h) Fig. 2. Effect of various concentrations of glucose in 0.I M KCI upon the opening of stomata (light, 30 ~ C) in isolated epidermis of C. communis L.
Samples of epidermal tissue of C. communis L. with closed stomata were incubated on solutions of 0.1 M KC1 and for comparison on 0.1 M KC1 supplemented with 0.1 M of either glucose, sucrose or sorbitol in the light. The degree of stomatal opening was recorded and the results are summarized in Figure 1. The timedependent reaction of the guard cells upon 0.1 M KC1 was taken as a control to evaluate the effect of supplemented carbohydrates. Glucose was found to exhibit a pronounced inhibition of stomatal opening (Fig. 1). To consider a possible osmotic effect, sorbitol (0.1 M) was supplied to the epidermis, but not apparent effect upon the movement of the guard cells was observed. Sucrose, was also fed to guard cells. The guard cells showed no alternation of the
P. Dittrich and M. Mayer: Inhibition of Stomatal Opening opening process as compared to 0.1 M KC1; thus sucrose does not interfere with the mechanism of the stomata. Since osmotic effects or availability for metabolic reactions could be ruled out as a basis of action, other carbohydrates were tested with respect to inhibitory properties. The results are compiled in Table 1. Hexoses in general appear to inhibit the opening of stomata to about the same degree as glucose. It seems to be irrelevant whether the sugar taken up can be further metabolized or not, since 6-deoxyglucose and 3-O-methylglucose also had inhibitory properties. Disaccharides, in contrast, exerted no uniform action, with trehalose clearly inhibiting and maltose slightly stimulating the movement of guard cells. Figure 2 contains a compilation of results of experiments, in which different concentrations of glucose in 0.1 M KC1 were tested with respect to the inhibition of stomatal opening. It was found that concentrations of about 30 mM have to be applied to induce a significant effect, while in the range of 0.1 M inhibition appears to be close to saturation. Effect of Glucose on Stomatal Opening in Vicia faba L. and Tutipa gesneriana L. Epidermal tissues of V. faba L. and T. gesneriana L. were incubated on solutions of 0.1 M KC1 with no supplementation or with supplements of 0.1 M glucose, sucrose, or various other sugars. The opening responses of the guard cells of both plants were not affected by the sugars, and the final aperture of the stomata was in no way different from those of the controls. Since opening of stomata in C. communis L. is inhibited by glucose, it was concluded that the inhibition was due to a stimulated closing response; consequently, open stomata should close more rapidly if incubated on glucose. To elucidate this question, epidermal tissues of C. communis L. were harvested from leaves that had been floating on water at 30 ~ C in the light. The epidermis with stomata open (15 I~m) was subsequently incubated in the dark either on distilled water or on water supplemented with either 0.1 M glucose or 0.1 M sucrose. After about 3 0 4 0 min in the dark the stomata had closed, irrespective of whether sugar was present or not. Furthermore, no difference in the rate of stomatal closure between the incubation with glucose and that with sucrose was noted. Discussion
Raschke and Dittrich (1977) reported that guard cells appear to rely on the underlying mesophyll tissue
169 to supply the carbohydrates necessary for metabolic reactions. Thus, feeding carbohydrates to isolated epidermal tissues should improve the energy status of the cells and consequently could lead to a faster opening of the stomata. The variety of responses of guard cells in epidermal tissues of C. communis L. toward fed carbohydrates however ranged from substantial inhibition (hexoses) to no response (sucrose) to a slight stimulation (maltose). While the latter response might actually be in accord with an improvement of the energy supply of the cells, the pronounced inhibition of stomatal opening by glucose was unexpected. Since neither molecular size, osmotic effects nor sequestration of ATP during uptake (3-O-methylglucose is no subject to phosphorylating reactions) appears to be involved in the mode of action, the inhibitory effect might be a consequence of the mechanism of sugar uptake. The majority of transport systems in microorganisms and plants presumably operate via a proton-cotransport system (Kaback, 1974). Thus; galactosides in E. coli and glucose in Neurospora and Chlorella (Slayman and Slayman, 1974; K o m o r and Tanner, 1976) are taken up concomitantly with protons. In higher plants such as Ricinus communis L., experiments by K o m o r et al. (1977) suggested that sucrose from the endosperm enters the cotyledons jointly with protons. Even the loading of sieve tubes with sucrose appears to be powered by proton-cotransport (Malek and Baker, 1977). Raschke and Humble (1973) and Raschke (1977) reported that the opening of stomata is associated with an extrusion of protons from the guard cells. This efflux of H + generates a negative membrane potential (Pallaghy, 1967), which drives the passive uptake of K § If we assume that glucose is taken up into guard cells concomitantly with protons, a depolarization of the membrane potentiaI would result, limiting the uptake of potassium ions and consequently inhibiting the opening of the stomata. Potassium ions and the H+-glucose system would compete for the electromotive force of the membrane potential. In testing the competitiveness, a concentration of 0.1 M glucose was required in the presence of 0.1 M KC1 to bring about 50% inhibition of stomatal opening. Thus, the affinity of the guard cells for the uptake of potassium is about in the same range as the affinity of the carrier system for glucose uptake. Although many parameters of stomata regulation originate in the leaf mesophyll it is unclear what advantage the guard cells of C. communis L. derive from the operation of a proton-glucose cotransport. Glucose was shown to have no impact upon the closing process of stomata. Since we have in general little information about the mechanism of guard cell deflation, the present evidence suggests that, although opening and
170
closing are active processes, the events during opening are not a mere reversion of the closing procedure. The lack of an effect of glucose upon stomata of V. faba L. and tulip does not necessarily imply that basic mechanisms of osmoregulation of guard cells are different. We have to consider that C. communis L. operates stomata in concert with a number of subsidiary cells that are likely to modify the response. In addition to the impact of morphologic features, it cannot be ruled out that active carbohydrate transport systems m a y or may not be subject to induction phenomena in different organisms. We greatly appreciate the careful technical assistance by M. MeuseI and G. Ohst, the photographic work by K. Liedl and the helpful discussion with D. Gradmann.
References Dittrich, P., Raschke, K. : Malate metabolism in isolated epidermis of Comrnelina communis L. in relation to stomatal functioning. Planta 134, 77-81 (1977a) Dittrich, P., Raschke, K,: Uptake and metabolism of carbohydrates by epidermal tissue. Planta 134, 83-90 (1977b) Kaback, H.R. : Transport studies in bacterial membrane vesicles. Science 184, 882-892 (1974)
P. Dinrich and M. Mayer: Inhibition of Stomatal Opening Komor, E., Tanner, W.: The determination of the membrane potential of Chlorella vulgaris. Europ. J. Biochem. 70, 197-204 (1976) Komor, E., Rotter, M., Tanner, W. : A proton-cotransport system in a higher plant: sucrose transport in Ricinus communis. Plant Sci. Lett. 9, 153-162 (1977) Malek, F., Baker, D.A.: Proton co-transport of sugars in phloem loading. Planta 135, 297-299 (1977) Pallaghy, C.K.: Electrophysiological studies in guard cells of tobacco. Planta 80, 147 153 (1968) Raschke, K., Humble, G.D.: No uptake of anions required by opening stomata of Vicia faba: guard cells release hydrogen ions. Planta 115, 47-57 (1973) Raschke, K.: Stomatal action. Ann. Rev. Plant Physiol. 26, 309-340 (1975) Raschke, K., Dittrich, P. : 14C-Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open. Planta 134, 69-75 (1977) Raschke, K. : The stomatal turgor mechanism and its responses to CO 2 and abscisic acid: observations and a hypothesis. In: Regulation of cell membrane activities in plants, pp. 173-183, Marr6, E., Ciferri, O., eds. Amsterdam: Elsevier 1977 Slayman, C.L, Slayman, C.W.: Depolarization of the plasma membrane of Neurospora during active transport of glucose: evidence for a proton-dependent cotransport system. Proc. Natl. Acad. Sci. USA 71, 135-1939 (1974)
Received 14 October; accepted 14 December 1977
