The Investigation of Stomatal Ionic Relations using Guard Cell Protoplasts: I. METHODOLOGY

Clint, G. M. 1985. The investigation of stomatal ionic relationsusing guard cell protoplasts. 1. Methodology.—J exp. Bot.36: 1726–1738. A study was made of the methodology for the production and useof guard cell protoplasts in ion transport studies, with particularemphasis placed on the effects of the composition of the externalmedium on protoplast survival and performance. Addition of externalKCl to media during the production of guard cell protoplastsfrom Commelina communis L. was found to improve viability andto increase K⁺ content and physiological competence of the isolatedprotoplasts. Addition of low levels (20 x 10^–3 mol m^–3)CaCl₂ increased protoplast yield and the maintenance of viabilityin long-term incubation. Ambiguities and uncertainties werefound in the application of methods commonly used for the assessmentof viability of isolated protoplasts. Poor yields (despite highpercentage recoveries) together with difficulties in the assessmentof viability were considered to pose major potential problemsin the use of guard cell protoplasts in ion transport studies. Key words: Guard cell protoplasts, ion transport, Commelina communis     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata     

Vanadate Sensitive ATPase and Phosphatase Activity in Guard Cell Protoplasts of Commelina

Flicker, M. D. and Willmer, C. M. 1986. Vanadate sensitive ATPaseand phosphatase activity in guard cell protoplasts of Commelina.—J.exp. Bot. 38: 642–648. Phosphatase activity was measured in extracts of guard cellprotoplasts of Commelina communis L. using the artificial substratep-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.Ammonium molybdate (Ol mol m^–3) and sodium vanadate (1–0mol m^–3) gave almost complete inhibition of phosphataseactivity at pH 60. ATPase assays were, therefore, conductedin the presence of 0–2 mol m ^–3 molybdate and vanadatewas used as a specific inhibitor of plasmamembrane ATPase activity.Vanadate sensitive ATPase activity showed a pH optimum of 6.6and activity was stimulated by KC1. These properties are characteristicof plasmamembrane proton pumping ATPases in other systems andsuggest that proton extrusion in guard cells could be mediatedby a similar enzyme. The maximum ATPase activity is sufficientto account for all the proton flux observed during the stomatalopening response. Key words: ATPase, Commelina, guard cell protoplasts, phosphatase, vanadate     

Ferricyanide Reduction by Guard Cell Protoplasts

Guard cell protoplasts of Commelina communis L. reduced exogenousferricyanide at pH values lower than 5?0; upon addition of NADH,reduction of ferricyanide by guard cell protoplasts was stimulatedover the pH range 4?0 to 9?0 with two peaks of activity at pH5?0 and between pH 8?0 and pH 9?0. Calcium chloride (1?0 molm^–3) and MgCl2 (1?0 mol m^–3) increased the NADH-stimulatedreduction of ferricyanide. Superoxide dismutase and cyanidehad little effect on the NADH-stimulated reduction of ferricyanideby guard cell protoplasts, but, salicylhydroxamic acid completelyinhibited this activity. The NADH-stimulated reduction of ferricyanidealso occurred in the cell-free supernatant. Horseradish peroxidasedid not reduce ferricyanide in the absence of NADH over a broadrange of pH (4?0 to 9?0). However, in the presence of NADH,horseradish peroxidase reduced ferricyanide over the pH range5?0 to 9?0 with maximal activity at pH 8?0. The NADH-stimulatedreduction of ferricyanide by horseradish peroxidase showed similarproperties to those observed with guard cell protoplasts. Mannitol,superoxide dismutase, and cyanide did not inhibit the NADH-stimulatedreduction of ferricyanide by horseradish peroxidase; SHAM, however,completely inhibited the reduction of ferricyanide by horseradishperoxidase. Catalase inhibited the NADH-stimulated reductionof ferricyanide by horseradish peroxidase by 20%, while absenceof oxygen in the assay medium stimulated this activity over60%. We propose that the reduction of ferricyanide in the presenceof NADH by guard cell protoplasts, can be explained in termsof peroxidase activity associated with the plasma membrane andsecreted to the extracellular medium. However, the capacityof guard cell protoplasts to reduce ferricyanide at acid pHvalues where little peroxidase activity occurs may indicatethe presence of a plasma membrane redox system in guard cellsof C. communis. Key words: Commelina, guard cell protoplasts, ferricyanide reduction, peroxidase, redox system     

Responses of Commelina communis L. Guard Cell Protoplasts to Abscisic Acid

Fitzsimons, P. J. and Weyers, J. D. B. 1987. Responses of Commelinacommunis L. guard cell protoplasts to abscisic acid.—J.exp. Bot. 38: 992–1001. Guard cell protoplasts (GCPs) isolated from the leaf epidermisof Commelina communis L. responded to abscisic acid (ABA) ina manner which was qualitatively and quantitatively similarto that of intact stomata. ABA inhibited swelling of GCPs underlow-CO₂ conditions and swollen GCPs responded to the hormoneby shrinking. Both the absolute volume decrease and the initialrate of shrinking were commensurate with the extent and ratesof solute loss computed for ABA-treated intact, open stomata.This indicates that GCPs represent a suitable experimental systemfor studies of ABA-mediated solute fluxes. A radiotracer equilibrationmethod was developed for the rapid estimation of GCP osmoticvolume changes. Using this technique it was found that, on average,82% of the reduction in solute content caused by ABA treatmentwas due to the loss of K⁺. It is envisaged that electroneutralitymight be maintained during ABA-induced shrinkage of GCPs bynet inward proton movement leading to acidification of the vacuole. Key words: Abscisic acid, Commelina communis L., guard cells, protoplasts     

Studies on the Properties of Carboxylating Enzymes in the Epidermis of Commelina communis

A spectrophotometric assay has been used to measure the activityof PEP carboxylase and RuBP carboxylase in the epidermal andmesophyll tissue of Commelina communis. On both a chlorophylland protein basis the PEP carboxylase activity was always greaterin the epidermis than in the mesophyll, whereas RuBP carboxylaseactivity was always highest in the mesophyll. PEP carboxylaseactivity in epidermal extracts was lost very slowly and itspH optimum was a broad one in the range 7·5–8·0.The K_m values for PEP carboxylase in the epidermis and mesophyllobtained from light- and dark-treated plants were not very differentalthough its V_max was much lower in dark-treated tissue. Thesedata are discussed in relation to the possible role of PEP carboxylasein guard cell metabolism.     

Separation and Purification of Protoplast Types from Commelina communis L. Leaf Epidermis

Guard cell and epidermal/subsidiary cell protoplasts obtainedby enzymic digestion of peeled Commelina communis leaf epidermiswere separated and purified by discontinuous density gradientccntrifugation with media based on Percoll (Pharmacia Fine ChemicalsAB, Uppsala, Sweden). The cell types were recovered over 99.9%pure at yields exceeding 50% efficiency, and mesophyll contaminationcould be virtually eliminated when desired. Osmotic characteristicsof the protoplast types were evaluated and compared to in vivovalues, and the viability of the protoplasts, assessed usinga range of criteria, was found to be high. Purified Commelinaguard cell protoplasts were able to evolve O₂ when illuminated,and this was substantially reduced in the presence of the inhibitorDCMU, indicating that they possess photosystem II activity.Specific advantages of this method of protoplast purification,and the potential uses of separate suspensions of guard cellsand epidermal/subsidiary cells in experiments on stomatal physiologyare discussed. Key words: Commelina communis, Protoplasts, Epidermis     

Nitrate-Sensitive ATPase Activity and Proton Pumping in Guard Cell Protoplasts of Commelina

ATPase activity was measured in crude homogenates of guard cellprotoplasts of Commelina communis L. using a linked enzyme assay.A low level of azide-sensitive ATPase activity was detectedwith a pH optimum of 6.8. This activity was stimulated by 0.01%(v/v) Triton X-100, and the pH optimum shifted to pH 7.4. Nitrate-sensitiveATPase activity was measured in the presence of azide and showeda pH optimum around pH 8.0. Proton pumping activity in a mixedpopulation of vesicles from GCP was monitored using fluorescencequenching of quinacrine. Mg-ATP dependent proton pumping wasobserved at pH 8.0, but not at pH 6.6. The activity at pH 8.0was inhibited by nitrate and DCCD but not vanadate. These dataindicate that activity of the tonoplast proton pump was beingmeasured. There was, however, no evidence for a tonoplast cation(K⁺)/proton antiporter under these assay conditions as potassiumdid not reduce the initial rate of pH gradient formation orincrease the rate of collapse of a pre-formed gradient afterinhibition of the pump. Key words: Tonoplast ATPase, proton pump, guard cell protoplasts, Commelina     

The Investigation of Stomatal Ionic Relations using Guard Cell Protoplasts: II. OSMOTIC RELATIONS OF GUARD CELL PROTOPLASTS IN SHORT AND LONG-TERM INCUBATION

Clint, G. M. 1985. The investigation of stomatal ionic relationsusing guard cell protoplasts. II. Osmotic relations of guardcell protoplasts in short and long-term incubation.—J.exp. Bot 36: 1739–1748 Measurements were made of the volume changes exhibited by isolatedguard cell protoplasts (GCPs) of Commelina communis L, whenexposed to a range of concentrations of external osmotica Inshort-term incubation, GCPs behaved as osmometers and showedrapid volume changes in response to changing external osmoticpressure (₀). In long-term incubation, GCPs prepared and incubatedwith added external KCl showed further slow changes in volume,in a manner suggesting that regulation of volume occurred. Protoplastsprepared and incubated without added external KCl had smallervolumes for a given value of ₀, and their ability to regulatevolume in long-term incubation was reduced or absent. Treatment with fusicoccin caused an increase in both the volumeand the K⁺ content of GCPs. The increase in volume continuingafter the increase in K⁺ content had ceased, in a manner similarto that observed in walled guard cells in epidermal strips. Key words: Guard cell protoplasts, volume regulation, Commelina communis     

Effect of Ferricyanide on Potassium Uptake by Intact Epidermal Tissue and Guard Cell Protoplasts

The effect of ferricyanide on K^$ fluxes in epidermis and inguard cells of Commelina communis L. were studied. Ferricyanideenhanced guard cell protoplasts swelling, which results fromenhanced K^$ uptake. In intact epidermis ferricyanide inhibitedK^$ uptake and consequently, stomatal opening. This was foundin floated and submerged epidermal tissues, indicating thatthe degree of contact with the solution does not affect theresponse to ferricyanide. Investigation of the rate of plasmolysisand de-plasmolysis of guard cells in epidermal tissue revealedthat ferricyanide enhances deplasmolysis, caused by K^$ uptake,only in completely plasmolysed cells, which resemble protoplastsin situ. (Received January 21, 1988; Accepted March 24, 1988)     

Surface Charge Density of Hetero-Fused Plant Protoplasts: an Electrophoretic Study

Electrophoretic mobilities of hetero-fused plant protoplasts,which were obtained by electrofusion of barley mesophyll cellprotoplasts and Rauwolfia serpentina cultured cell protoplasts,and those of the unfused parent protoplasts were measured invarious media of different pH values. At pH 5.2, the zeta potentialof the fused protoplasts was intermediate between those of thebarley and R. serpentina protoplasts and the average surfacecharge density of the fused protoplasts was closer to that ofR. serpentina than to that of barley. The distribution of thesurface charge density of fused protoplast obtained at pH 5.2is discussed in terms of the surface charge densities and thesizes of parent protoplasts. These results revealed that thesurface charge density of fused protoplasts was determined bythe surface charge densities and the ratio of the surface areasof the respective parent protoplasts. (Received December 28, 1989; Accepted August 10, 1990)     

Isolation of Protoplasts from Kappaphycus alvarezii var. tambalang (Rhodophyta) and Secretion of i-Carrageenan Fragments by Cultured Cells

A method for generating protoplasts from the carrageenan-producingred alga Kappaphycus alvarezii was developed. Digestions withcellulase and _k-carrageenase produced only a few cortical cellprotoplasts, while digestions with cellulase and i-carrageenaseonly produced epidermal cell protoplasts. When both carrageenaseswere used in the digestion media with cellulase, protoplastswere released from all cell types and yields ranged from 1·0to 1·2x10⁷ cells g^–1 with sizes from 5 to 200 µmdiameter. Protoplasts were subsequently cultured to study cellwall regeneration. Calcofluor-positive material (probably cellulose)was detected within 6 h after removal of protoplasts from thewall digestion media, whereas, i-carrageenan fragments weredetected in all regenerating protoplast cultures 24 h afterremoval from the digestion media. Protoplasts continued to produceCalcofluorpositive material and secrete carrageenan fragmentsinto culture media for several days. However, cells culturedin media augmented with K⁺ ions stopped secreting carrageenanfragments after 24 h. Cells cultured for 48 h in seawater labelledweakly with an i-carrageenan hybridization probe, but not atall with a corresponding _k-probe. Cells cultured for 48 h, blottedto nylon membranes and probed with anti-carrageenan monoclonalantibodies, showed the presence of gelling carrageenan subunitsin the cell walls. Key words: -Carrageenan, Kappaphycus, protoplasts, Rhodophyta     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

The Regulation of the Starch-Malate Balances during Volume Changes of Guard Cell Protoplasts

The enzymatic activities of phosphoenolpyruvate carboxylase(EC 4.1.1.31 [EC] ), ‘malic enzyme’ (EC 1.1.1.40 [EC] ), phosphofmctokinase(EC 2.7.1.11 [EC] ) and fructosebisphosphatase (EC 3.1.3.11 [EC] ) weremeasured during the swelling and shrinking of isolated and purifiedguard cell protoplasts (Vicia faba) in darkness. The volumeincrease was accompanied by the activation of phosphofructokinaseand a short stimulation of phosphoenolpyruvate carboxylase,at the same time the ‘malic enzyme’ and fructosebisphosphatasewere inhibited. However, during the shrinkage of guard cellprotoplasts these two enzymes were activated in contrast tophosphoenolpyruvate carboxylase and phospho-fructokinase. Becauseof the dramatic increase of phosphoenolpyruvate carboxylaseactivity during the swelling, this enzyme was assumed to actas a trigger for the swelling phase.     

The Membrane Potential of Enzymatically Isolated Nitella expansa Protoplasts as Compared with their Intact Cells

With the enzymatically isolated Nitella protoplasts, sufficientinsertions of micro-electrodes to make a stable measurementof the membrane potential by the conventional method could notbe made because of an ‘elasticity’ of the outermembrane. We developed an effective method in which a micro-electrodecould be inserted after the outer membrane was punctured bypassing an electrical impulse through the micro-electrode. Inthis method, Ca ions play a crucial role in the ‘punching’and ‘healing’ processes of the protoplast membrane. The effects of the cations K⁺, Na⁺, Ca²⁺ and the anions Cl^–,, , on the membrane potentials of Nitella expansa protoplasts were compared with those of intactcells. The membrane potential of protoplasts was less negativethan that of intact cells when concentrations of Na or K, inthe presence of Ca, were below certain levels which increasedwith increasing Ca concentration; and it tended to become identicalto that of intact cells when Na or K concentrations were beyondthose levels. Beyond those levels for K the membrane potentialsof both protoplasts and intact cells typically seemed to bethe Nernst potentials in the presence of 0•1 to 30 molm^–3 Ca²⁺. However, for Na, the difference in potentialsbetween intact cells and protoplasts decreased at much higherconcentrations than for K. Increase of Ca always gave less negativeprotoplast potentials than those in intact cells. Replacementof Ca by Mg did not change the membrane potential of intactcells, although it was deleterious to protoplasts. The cellwall potential of intact cells was also measured by the micro-electrodetechnique and was revealed as a simple Donnan potential, assumingthe fixed negative charge density of 0•8 equivalent perdm³. The membrane potential of intact cells seems to be a significantreflection of the plasmalemma potential which is thought tobe measured directly in their protoplasts in terms of ionicselectivity and concentration dependency of the ion speciesexamined. In addition, increased sensitivity to calcium in protoplastpotentials compared to intact cells is suggested, though themembrane potential of intact cells seems to be largely preservedin their enzymatically isolated protoplasts. Key words: Membrane potential, protoplasts, Nitella expansa, cell wall potential     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

Major Element Composition of Epidermal and Mesophyll Tissues2Commelina Communis L. and Vicia Faba L.: Some Further Considerations of the Role of Ions in Stomatal Functioning

Epidermal and mesophyll tissues of Commelina communis L. andVicia faba L. were analysed by atomic absorption spectrometryfor the major plant inorganic cations and anions (K, Na, Ca,Mg, P, NO₃-N, Cl) when stomata of the leaf were open and closed.Water-soluble and residual levels of the elements were estimatedand a charge balance of the soluble fraction made. The major portion of K, Na, Cl, and P was extracted in the water-solublefraction of the epidermal and mesophyll tissues of both species.In both species the bulk of Ca remained in the insoluble residueof the epidermis whereas in mesophyll tissue it was equallydistributed be-between the two fractions in C. communis butmainly in the insoluble residue in V. faba. Magnesium was predominantlyfound in the water-soluble fraction of V. faba mesophyll tissueand distributed approximately equally between the two fractionsin the epidermal tissue. In C. communis Mg was slightly moreabundant in the water-soluble fraction of both mesophyll andepidermis. In both species no statistically significant differences inthe levels of the elements could be detected between epidermaland mesophyll tissues from leaves with open stomata and thesame tissues from leaves with closed stomata, suggesting thatthere was no major flux of ions between mesophyll and epidermisduring stomatal movements. Regardless of whether the stomata were open or closed, therewere considerably more water-soluble inorganic cations thananions present in all tissues of both species with K being themajor cation and Cl being the major anion. In V.faba and C-communis epidermis there was 49–53 per cent and 56%68per cent excess cation respectively. In the mesophyll tissuethe excess cation was 63–75 per cent and 75%78 per centin V.faba and C. communis respectively. When the partitioning of the levels of the elements betweenepidermis and mesophyll of a leaf is considered, except forNO₃-N in both species and Na in V. faba, 20 per cent or lessof each element was present in the epidermis.     

Osmocytosis and Vacuolar Fragmentation in Guard Cell Protoplasts: Their Relevance to Osmotically-lnduced Volume Changes in Guard Cells

Guard cell protoplasts were prepared from young leaves of peaplants. Under hypertonic conditions they shrink and large numbersof endocytotic (‘osmocytotic’) vacuoles are formedby invagination of the plasma membrane. In thin section theseare indistinguishable from other small vacuoles (‘mini-vacuoles’)which are formed by fragmentation of the large central vacuole.However, the two types of vacuole can be individually recognizedby labelling the central vacuole with neutral red and by performingthe osmotic shrinkage with fluorochromes such as Lucifer Yellow-CHor Cascade Blue present in the extracellular medium. Osmocytoticvacuoles do not fuse with the plasma membrane nor with the mini-vacuolesduring a subsequent swelling phase. After several hours, osmocytosedLucifer Yellow gradually leaks out of the endocytotic vacuoleswhen protoplasts are returned to hypotonic conditions. Thisleakage is not prevented by probenecid at concentrations (20–50mmol m^–3) which do not give rise to pathological changesin protoplast ultrastructure. In order to determine the relevanceof these observations to the situation in planta, intact guardcells in epidermal strips were first allowed to accumulate neutralred in their vacuoles and then subjected to osmotic shrinkagein the presence of external Lucifer Yellow. Osmocytotic vacuoleswere not formed, although the production of mini-vacuoles wasfrequently observed. Key words: Guard cell protoplasts, fluid phase markers, Pisum sativum, probenecid, osmocytosis, shrinkage-swelling cycles     

Calcium Inhibits Ion-Stimulated Stomatal Opening in Epidermal Strips of Commelina communis L.

Inoue, H. and Katoh, Y. 1987. Calcium inhibitsion-stimulatedstomatal opening in epidermal strips of Commelina communis L.—J.exp. Bot. 38: 142–149. Ca²⁺ suppressed both the ion-stimulated stomatal opening andH⁺ extrusion of pre-illuminated epidermal strips isolated fromCommelina communis L. In the absence of Ca²⁺, the rate of H⁺release was 18 nmol H⁺ cm^–2 h^–1 per epidermal stripunit area in 150 mol m^–3 KCL at pH 7?4. Half-maximum inhibitionof stomatal opening was observed with 220 mmol m^–3 ofCa²⁺. The hexavalent dye, ruthenium red, showed concentration-dependentprevention of the inhibition by Ca²⁺ of the ion-stimulated stomatalopening. The effect of ruthenium red was non-competitive, andthe K₁ for the calcium inhibition was found to be 3?6 mmol m^–3.The calcium inhibition of H⁺ extrusion was also prevented byruthenium red. These results suggest that Ca²⁺ inhibits theactivity of electrogenic H⁺ translocating ATPase of the guardcell plasma membrane and leads to the suppression of stomatalopening. Key words: Calcium, Commelina communis, ruthenium red, stomata