Stomatal Movement and Sucrose Uptake by Guard Cell Protoplasts of Commelina benghalensis L.

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1 Plant Cell Physiol. 27(8): JSPP 1986 Stomatal Movement and Sucrose Uptake by Guard Cell Protoplasts of Commelina benghalensis L. A. Ramachandra Reddy and V. S. Rama Das Department of Botany, School of Biological and Earth Sciences, Sri Venkateswara University, Tirupati , India Sucrose concentration in guard cells of epidermal strips of Commelina benghalensis increased with stomatal opening. Sucrose uptake patterns were investigated using guard cell protoplasts of C. benghalensis. Sucrose (0.5 miu) uptake into these protoplasts was sensitive to ph, with an optimum at ph 6. Uptake of sucrose into guard cell protoplasts was inhibited by 2,4-dinitrophenol (DNP), diethylstilbestrol (DES) and (/Mrifluoromethoxy)carbonyl cyanide phenylhydrozone (FCCP), while DCMU and o-phenanthroline had no effect on the uptake of sucrose. Fusicoccin (FC) stimulated sucrose influx. The influence of ph and the effect of the metabolic inhibitors on the sucrose uptake into the guard cell protoplasts are consistent with an energy dependent membrane-function. Key words: Commelina benghalensis Guard cell protoplasts Stomatal opening Sucrose. The role of potassium in modulating the turgor potential of guard cells is well established (Imamura 1943, Fischer and Hsiao 1968). Guard cells isolated from epidermal strips were shown to be capable of synthesizing and accumulating malate and this ability has been suggested as the basis for an ingenious system of operation of stomata in leaves (Allaway 1973). Although a positive relationship between malate levels and stomatal opening is well established, the involvement of sugars as important osmotic agents in maintaining the solute potential of guard cells is yet to be clearly defined (Allaway 1981, Outlaw 1982). The role of sucrose as a cytoplasmic osmoticum suggested as early as 1895 by Kohl, now seems to be of greater significance. The present investigation was aimed to determine the role of sucrose in the stomatal movement. Close correlation between the degree of stomatal opening and sucrose levels in guard cells was observed. Materials and Methods Plants of Commelina benghalensis L. were raised from cuttings in 30 cm diameter seed pans. They were grown under natural 12 h photoperiod and average temperatures of 35 C day/20 C night. Third and fourth leaves from the top were used for experiments. The abaxial epidermis was peeled and cut into 10x5 mm strips using a template. The strips were immediately floated on 100 UM CaCl strips were sonicated in 15 ml of 0.05 mm Ca(NO3)2 for 30 s to remove epidermal and mesophyll cells (Durbin and Graniti 1975). The epidermis contained % Abbreviations: DES, diethylstilbestrol; DNP, 2,4-dinitrophenol; FCCP, (/>-trifluoromethoxy)carbonyl cyanide phenylhydrozone; FC. fusicoccin. 1565

2 1566 A. R. Reddy and V. S. Rama Das of guard cells alive as indicated by the uptake of 0.01% (w/v) neutral red strips were placed in 5 cm diameter Petri dishes containing 20 ml of 25 mm MES-Tris buffer, ph 6.2, 25 mm KC1 and 0.1 mm CaCb. The preparations were then illuminated at 500 /j.mol m~ 2 s" 1 ( nm). The illumination was provided by a bank of incandescent lamps. At the end of the incubation period the width of stomatal aperture was determined with the help of a precalibrated ocular micrometer stomata were observed at random in each of at least five strips and the average was calculated. Sucrose content was determined spectrophotometrically according to Jones et al. (1977). Guard cell protoplasts were prepared by digesting the cell walls of epidermal strips (Shimazaki et al. 1982). The strips were incubated in a digesting medium. The enzyme solution for protoplast isolation contained 0.4 M mannitol, 5 mm sodium ascorbate, 2% cellulase Onozuka RS (Yakult Pharmaceutical Industry Co. Japan), 0.5% pectinase, 1 mm CaCl2 and 0.5% BSA at ph 5.5. The contents were incubated for 90 min with constant shaking (40 cycles min- 1 ) at 30 C. Guard cell protoplasts were separated from the strips by passing through 20 fim nylon net. The filtrate was centrifuged at 100 X^ for 5 min and suspended in 0.4 M mannitol and 1 mm CaCl2. The number of guard cells was counted using a hemocytometer. Sucrose uptake by the isolated protoplasts was measured according to the procedure described by Schmitt et al. (1984) with slight modifications. The reaction mixture unless otherwise specified, contained (1 ml) 25 mm MES-KOH, ph 6.0, 0.4 M mannitol, 10 mm KC1, 5 mm CaCl 2, 0.5 mm 14 C-sucrose (containing 0.5,uCi of 14 C-sucrose). Radioactivity in the resuspended protoplasts was counted in a liquid scintillation system (Beckman LS 1800). Results A close positive correlation was noticed between the endogenous sucrose levels in the guard cells and the width of stomatal opening (Fig. 1). There was a remarkable stimulation of stomatal opening in the epidermal tissues with increased sucrose concentration in the incubation medium from 0.1 mm to 0.5 mm (Fig. 2). Sucrose uptake by the isolated guard cell protoplasts was ph dependent. Optimal rates of sucrose uptake were observed at ph 6 (Fig. 3). Sucrose uptake Table 1 Effect of certain chemicals on sucrose influx in the guard cell protoplasts of C. benghalensis Sucrose uptake Experiment /pmoles per 10 2 \ % Value \ protoplasts h" 1 / Control +DES (50 HM) 4-2,4-DNP (100 /M) -(-o-phenanthroline (1 (IM) + DCMU (5/IM) + Fusicoccin (10/IM) + FCCP (10,«M) ± ± ± ± ± ± ± Guard cell protoplasts (6xl0 5 ml~ 1 ) were incubated in 1 ml of 0.4 M mannitol solution consisting of 10 mm KC1, 25 mm MES-KOH (ph 6.0) and 0.5 mm sucrose (containing 0.5 ^Ci of 14 C-sucrose). The inhib tor or promotor was added at the concentrations mentioned in parantheses. The reaction vials were illuminated at 500 //mol m~ 2 s" 1 for 30 min. Radioactivity in the resuspended protoplasts was counted in a liquid scintillation system. Data are means of five experiments ± SE.

3 Sucrose induced stomatal opening 1567 a 5 -i TIME (min) Fig. 1 4 O TIME (min) Fig- 2 Fig. 1 The relationship between the degree of stomatal opening and sucrose levels in epidermal strips of Commelina benghalensis. The epidermal strips were incubated in 5 cm diameter Petridishes containing 25 mm MES-Tris buffer (ph 6.2) consisting of 25 mm KCI, 0.5 min sucrose, 0.1 mm CaCU. The experiments were initiated by illuminating the contents in the Petri dishes at 500 /jmol m~ 2 s -1. Sucrose concentrations were estimated after measuring the width of stomatal aperture. Fifteen to 20 stomata were observed at random in each of at least five strips and the average was calculated. The width of stomatal aperture before incubation was 2.8 /<m. Fig. 2 The course of stomatal opening in epidermal tissue of Commelina benghalensis at different sucrose concentrations. Epidermal strips were incubated in 25 mm MES-Tris buffer which contained 10 mm KCI, and sucrose at concentrations as shown in the figure. The width of stomatal aperture before incubation was 4.5 //m. The results are averages of five determinations. ph Fig. 3 3O 6 O 9 O I2O TIME Fig. 4 Fig. 3 Effect of ph on sucrose uptake by isolated guard cell protoplasts of Commelina benghalensis. Guard cell protoplasts (6 X 10 5 ml" 1 ) were incubated in 0.4 M mannitol solution which contained 10 mm KCI and 0.5 mm sucrose (with 0.5 iic\ of 14 C-sucrose). 25 mm HEPES and citrate mixture was used to adjust different ph of the uptake solution. The results are averages of five determinations. Fig. 4 Time course of stomatal opening in epidermal tissue in presence of FC, FCCP and DES. The epidermal strips were incubated as mentioned in the legend of Fig. 1. After 1 h (indicated by arrow) the effects of FC (10 /UM), FCCP (10 /in) and DES (50 //M) on the degree of stomatal opening were examined. The results are averages of five determinations. The width of stomatal aperture before incubation was 2.8 /im. (min)

4 1568 A. R. Reddy and V. S. Rama Das TIME Fig. 5 (min) TIME Fig. 6 Fig. 5 Effects of different chemicals on the time course of sucrose uptake by isolated guard cell protoplasts. The guard cell protoplasts were incubated in 0.4 M mannitol containing 10 mm KC1, 25 mm MES-KOH, ph 6.0 and 0.5 mm sucrose (0.5 /tci of 14 C-sucrose). After 10 min incubation the effects of various chemicals (at the concentrations as given in Table 1) were monitored. Value for the control experiment was 120 p mol/10 2 protoplasts/h. The results are averages of five determinations. Fig. 6 Effect of ABA on FC induced stomatal opening in epidermal tissue. The epidermal strips were incubated as mentioned in the legend of Fig. 1. ABA (10 /IM) was added as indicated in the figure and the stomatal pore width was monitored. The width of stomatal aperture before incubation was 4.5 fim. The results are averages of five determinations. by guard cell protoplasts was found to be energy dependent (Fig. 4 and 5). DES, a potential inhibitor of ATPase and FCCP, an uncoupler of phosphorylation substantially inhibited the uptake of sucrose by the guard cell protoplasts. DNP, a known inhibitor of cyclic photophosphorylation (Raghavendra and Das 1972) inhibited the sucrose influx into guard cell protoplasts while DCMU and o-phenanthroline had little effect on sucrose uptake (Fig. 5 and Table 1). A potent stimulator of active proton exchange, fusicoccin (Turner 1972), caused substantial increase in sucrose uptake by guard cell protoplasts. in the epidermal tissue was inhibited by ABA (Fig. 6). Discussion (mm) FC stimulated stomatal opening Although a positive relationship between ion exchange and stomatal opening is well established, there has been no general agreement on the significance of carbohydrate metabolism in guard cells. Some of the investigators could notfinda clear relation between the carbohydrate levels of the epidermal tissue and width of stomatal aperture (Pearson 1973, Rutter et al. 1977). But recent reports suggest that changes in carbohydrates in guard cells during the course of stomatal opening are significant (Outlaw and Manchester 1979, Donkin and Martin 1980). Our observations demonstrate that the degree of stomatal opening is related to the levels of sucrose in guard cells (Fig. 1). Sucrose content was higher in guard cells when stomata were wide open than when they were closed. It appears that soluble sugars increase in guard cells when stomata open. However, the source for sucrose in the guard cells is still an enigma. Starch was reported to be a source for malate and sugars in epidermal tissue (Dittrich and

5 Sucrose induced stomatal opening 1569 Raschke 1977). It has been suggested that during stomatal opening the vacuolar ion concentration of guard cells increases so much that the cytosolic osmoticum could not match the same (Allaway 1981). It is hypothesized that something other than inorganic ions is neededto balance the osmotic concentration of the cytoplasm with that of the vacuole. Perhaps the accumulation of sucrose with stomatal opening could suggest sucrose as a cytoplasmic osmoticum. Sucrose could be taken up from the mesophyll cells into the guard cells via the apoplast (Robinson and Preiss 1985). The data on the sucrose uptake patterns in the guard cell protoplasts suggest that sucrose could be transported into the guard cells during stomatal opening. The effects of ph on the sucrose uptake by the guard cell protoplasts, is shown in Fig. 3 which indicates the maximal capacity of guard cell protoplasts was at ph 6. The variations in sucrose uptake by guard cell protoplasts may be due to the buffering capacity of the plasma membranes of guard cell protoplasts when they are directly exposed to the external ph. The physiological significance of this phenomenon in vivo is difficult to explain at present. Sucrose uptake by guard cell protoplasts is strongly energy dependent as evidenced by the inhibition in presence of DNP, FCCP and DES. A 60% inhibition was noticed in the uptake of sucrose in presence of DES (60,UM), a putative ATPase inhibitor. The inhibition of sucrose uptake in presence of FCCP indicates the effect of the uncoupler on the proton gradient-driven transport across the plasmalemma of guard cell protoplasts (Pallaghy and Fischer 1974). The inhibition of sucrose influx by DNP suggests the role of cyclic photophosphorylation in providing energy for membrane transport. The influence of ph and inhibition of sucrose influx by certain metabolic inhibitors strengthen the conclusion that the observed sugar transport reflected membrane function. The lack of inhibition of sucrose uptake by guard cell protoplasts in presence of DCMU or o-phenanthroline indicates the insignificant role of PS II activities in guard cell chloroplasts in providing energy (Rama Das and Raghavendra 1982). However, the presence of light harvesting pigments of both PS I and PS II in guard cell chloroplasts has been a controversial aspect of stomatal physiology (Zeiger 1983). FC is known to stimulate proton efflux from plant cells leading to K+ accumulation (Turner 1972, Travies and Mansfield 1979). The present study has shown that sucrose uptake has been greatly enhanced by FC. The counter effect of ABA on FC stimulated stomatal opening is of particular interest. ABA might affect the proton flux system during stomatal opening which inturn may cause closure of stomata. Finally, we presume that the variations between the apoplastic sucrose concentrations and those in the guard cells may favour sucrose transport into the guard cells by an energy-dependent sucrosyl carrier. A conclusive evidence of this mechanism is likely to constitute one of the most interesting aspects of future stomatal research. This work was supported by a grant from United States Department of Agriculture (FG-IN-576, IN-SEA-171). References Allaway, W. G. (1973) Accumulation of malate in guard cells of Viciafaba during stomatal opening. Planta 110: Allaway, W. G. (1981) Anions in stomatal operation. In Stomatal Physiology. Edited by P. G. Jarvis and T. A. Mansfield p Cambridge University Press, Cambridge. Dittrich, P. and K. Raschke (1977) Uptake and metabolism of carbohydrates by epidermal tissue. Planta 134: Donkin, M. E. and E. S. Martin (1980) Changes in starch and glucose levels of Commelina communis in relation to stomatal movements. Plant. Cell Environ. 3: Durbin, R. D. and A. Graniti (1975) A simple technique of obtaining functionally isolated guard cells in epidermal strips of Viciafaba. Planta 126:

6 1570 A. R. Reddy and V. S. Rama Das Fischer, R. A. and T. C. Hsiao (1968) Stomatal opening in isolated epidermal strips of Viciafaba II. Responses to KC1 concentration and the role of potassium absorption. Plant Physiol. 43: Imamura, S. (1943) Unterschungen Uber den Mechanismus der Turgorschwankungder Spaltoffnungsschliesszellen. Jap.J.Bot. 12: Jones, M. G. K., W. H. Outlaw and O. H. Lowry (1977) Enzymic assay of 10"' to 10~ 14 moles of sucrose in plant tissues. Plant Physiol. 60: Kohl, F. G. (1895) Uber assimilationsenergie und spaltoffnung smechanik. Bolanisches Centralblatt. 64: Outlaw, W. H. (1982) Carbon metabolism in guard cells. In Recent Advances in Phytochemistry. p Edited by L. L. Greasy and G. Hrazdina, Plenum Publishing Co., New York. Outlaw, W. H. and J. Manchester (1979) Guard cell starch concentration quantitatively related to stomatal aperture. Plant Physiol. 55: Pallaghy, C. K. and R. A. Fischer (1974) Metabolic aspects of stomatal opening and ion accumulation by guard cells in Viciafaba. Z. Pflanzenphysiol. 71: Pearson, C. J. (1973) Daily changes in stomatal aperture and in carbohydrates and malate within epidermis and mesophyll leaves of Commelina cyanea and Viciafaba. Aust. J. Biol. Sci. 26: Raghavendra, A. S. and V. S. R. Das (1972) Control of stomatal opening by cyclic photophosphorylation. Curr. Sci. 41: Rama Das, V. S. and A. S. Raghavendra (1982) Stomata: The physiology and biochemistry of their regulation in leaves. Curr. Sci. 51: Robinson, N. and J. Preiss (1985) Biochemical phenomena associated with stomatal function. Physiol. Plant. 64: Rutter.J. C., W. R.Johnston and C. M. Willmer (1977) Free sugars and organic acids in the leaves of various plant species and their compartmentation between the tissues. J. Expt. Bot. 28: Schmitt, M. R., W. D. Hitz, W. Lin and R. T. Giaquinta (1984) Sugar transport into protoplasts isolated from developing soybean cotyledons. Sucrose transport kinetics, selectivity and modelling studies. Plant Physiol. 75: Shimazaki, K., K. Gotow and N. Kondo (1982) Photosynthetic properties of guard cell protoplasts from Viciafaba. Plant Cell Physiol. 23: Travis, A. J. and T. A. Mansfield (1979) Reversal of CO2 responses of stomata by fusicoccin. New Phytol. 83: Turner, N. C. (1972) K + uptake by guard cells stimulated by fusicoccin. Nature 235: Zeiger, E. (1983) The biology of stomatal guard cells. Annu. Rev. Plant Physiol. 34: (Received July 7, 1986; Accepted September 26, 1986)

REVERSAL OF ABSCISIC ACID INDUCED STOMATAL CLOSURE BY BENZYL ADENINE

(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