Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Determination of malate levels during the swelling of vacuoles isolated from guard-cell protoplasts

A method for the preparation of vacuoles from guard cells ofVicia faba L. is described. Vacuoles were released from guard-cell protoplasts by osmotic shock and purified on a Ficoll gradient. Contamination of the vacuoles was examined by assaying marker enzymes, such as fumarase, glucose-6-phosphate dehydrogenase, phosphofructokinase, acid phosphatase and mannosidase. Potassium ions in the incubation medium caused increases in the volume of the vacuoles by a factor of about 2.6, while the malate level remained unchanged. In contrast, malate synthesis was stimulated during the swelling phase when complete guard-cell protoplasts were exposed to K⁺. The possible role of K⁺ as an efficient osmotic effector is discussed.Abbreviations DEAE diethylaminoethyl - GCP guard-cell protoplast(s) - GCV guard-cell vacuoles(s) - MCP mesophyll cell protoplast(s) - MCV mesophyll cell vacuole(s)     

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     

Modulation of photosynthesis and respiration in guard and mesophyll cell protoplasts by oxygen concentration

Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O₂ mg^?1 chl h^?1, while guard cell protoplasts respired at rates of 200–300 μmoles O₂ mg chl^?1 h^?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m^?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents.     

Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.     

Modulation of the activity of phosphoenolypyruvate carboxylase during potassium-induced swelling of guard-cell protoplasts of Vicia faba L. after light and dark treatments

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) activity was found to be modulated by light and darkness when measured in the presence of K⁺, which had been added to induce swelling of guard-cell protoplasts (GCPs) from Vicia faba L., whereas no modulation was detected in the absence of K⁺ (PEPcase activity remained constant at 1.5±0.15 pmol PEP metabolized · GCP^–1 ·h^–1; subsequently, pmol GCP^–1 ·h^–1 will be used). The activity of PEPCase increased by 100% (from 1.5 to 3 pmol·protoplast^–1·h^–1) in darkness and by 200% (from 1.7 to 5 pmol·protoplast^–1· h^–1) in light and oscillations in activity of these magnitudes were repeated at intervals of 2 min (dark) and 2.5 min (light) for a period of 10 min during K⁺-induced increase in the volume of GCPs. The oscillations were reflected in changes in malate-pool sizes determined in plastids, mitochondria and the supernatant fraction (consisting of the cytosol and the vacuole). Malate probably functioned as a mitochondrial substrate, thus supplying ATP for K⁺ uptake and the swelling of the protoplasts. On the basis of the present paper and previous results (H. Schnabl and B. Michalke 1988, Life Sci. Adv. Plant Physiol. 7, 203–207) involving adenine nucleotidepool sizes in fractionated GCPs, a model is proposed to explain the cause-effect relationship between K⁺, PEPCase, the cytosolic and mitochondrial malate levels and ATP levels during the K⁺-induced increase of GCP volume.Abbreviations GCP dtguard-cell protoplast - PEP phosphoenol-pyruvate - PEPCase PEP carboxylase The authors thank Professor Hermann Schnabl, University of Stuttgart (FRG), for his assistance in applying the graph theory analysis. This work was supported by Deutsche Forschungsgemeinschaft to H.S.     

Stress-related levels of abscisic acid in guard cell protoplasts of Vicia faba L.

Levels of abscisis acid (ABA) were determined in isolated guard cell (GCP) and mesophyll cell (MCP) protoplasts of Vicia faba L. in relation to water stress. Incubation of GCP and MCP in 0.4 M or 0.8 M mannitol resulted in an average increase in the level of free abscisic acid (ABA) in the cells of 34% (GCP) and 38% (MCP) within 15–60 min. It is concluded that guard cell protoplasts form ABA in response to osmotic stress.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - GCP guard cell protoplasts - MCP mesophyll cell protoplasts - MES [2-(N-morpholino)-ethanesulfonic acid] - TLC thin layer chromatography Part 20 in the series, Use of Immunoassay in Plant Science     

Plasma Membrane Redox System in Guard Cell Protoplasts of Pea (Pisum sativum L.)

Guard cell protoplasts (GCP) from leaves of pea (Pisum sativum)were capable of reducing/oxidizing the membrane impermeableelectron carriers, ferricyanide/NADH. The redox activity ofGCP required the presence of both ferricyanide and NADH, althoughsome ferricyanide reduction occurred even in the absence ofNADH. The GCP preferred NADH to NADPH during ferricyanide reductionand the reduction was slow with DCPIP or cytochrome c. A stoichiometryof about 2 existed between moles of ferricyanide reduced andNADH oxidized by GCP. The redox activities of GCP were severaltimes greater than those of mesophyll protoplasts from pea leaves.The ferricyanide reduction or NADH oxidation by GCP was unaffectedby abscisic acid or sodium orthovanadate and fusicoccin indicatingthe non-involvement of plasma membrane ATPase in these redoxreactions.The redox activities were markedly inhibited by chloroquineor 8-hydroxyquinoline. The findings are discussed in relationto the possible regulatory role of a guard cell plasma membraneredox system in stomatal function. Key words: Plasma membrane redox system, mesophyll protoplasts, pea, guard cell protoplasts, stomatal function     

High Mitochondrial Activity but Incomplete Engagement of the Cyanide-Resistant Alternative Pathway in Guard Cell Protoplasts of Pea

The respiratory properties of guard cell protoplasts (GCP) were examined in comparison with those of mesophyll protoplasts (MCP) from the same leaves of pea (Pisum sativum L. cv Arkel). The rates of respiratory O2 uptake by GCP were extremely high (280 [mu]mol mg-1 Chl h-1) and were several times greater than those of MCP. On the other hand, the rates of photosynthetic O2 evolution by GCP were similar to those of MCP. Also on the basis of protoplast volume, the respiratory rates of GCP were higher: more than three times those of MCP. The enzymes of the tricarboxylic acid cycle, per unit protein or unit protoplast volume, had a 2- to 5-fold higher activity in GCP than in MCP, indicating an enrichment of mitochondrial activity in GCP relative to that in MCP. Respiratory inhibitors were used to assess the activity of the cytochrome (cyanide-sensitive) and alternative (cyanide-resistant) pathways in GCP and MCP. The inhibition of respiration by KCN or antimycin A was more in GCP than that in MCP. The marked inhibition of respiratory O2 uptake by salicylhydroxamic acid in the presence of KCN showed the presence of the cyanide-resistant pathway in GCP. The activity of the cyanide-resistant electron transport path constituted only one-third of total respiration in GCP but accounted for two-thirds of respiration in MCP. The alternative pathway was not completely engaged in GCP but reached its full capacity in MCP.     

Development of cyanide-resistant respiration in mitochondria from potato tubers treated with ethanol,acetaldehyde, and acetic acid

Treatment of intact potato (Solanum tuberosum L.) tubers with acetaldehyde, ethanol or acetic-acid vapors led to a respiratory upsurge which was further increased when the volatiles were applied in 100% O₂. Mitochondria from tubers held in 100% O₂ (O₂ control) displayed a substrate state, state 3, and state 4 in respiration, whereas in mitochondria from the volatile-treated tubers the respiratory rate of the different states was virtually indistinguishable. This respiratory pattern was companied by the development of a cyanide-resistant respiration since these mitochondria exhibited resistance to CN and sensitivity to CN+salicylhydroxamic acid. Acetaldehyde-treated potatoes showed a time-course development (up to 36 h) of cyanide resistance and concomitant sensitivity to salicylhydroxamic acid, indicating the onset of synthetic processes leading to the observed changes in mitochondrial respiration.Abbreviations V total respiration rate - V_cyt velocity of O₂ uptake attributable to cytochrome oxidase - V_alt velocity of O₂ uptake attributable to the alternate oxidase - RCR respiratory control ratio - SHAM salicylhydroxamic acid Paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, N.J., USA     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

Measurement of guard-cell respiration rates using Cartesian-diver technique

Dark respiration rates of guard-cell protoplasts of Commelina communis L. were measured over a temperature range (15–30° C) using a Cartesian-diver microrespirometry technique. Measurements were made using a few microliters of suspension medium containing between 400 and 3 700 protoplasts. Respiration rates were approximately linear for at least 1 h at all temperatures. Respiration rates increased rapidly between 20 and 25° C to relatively high levels (6.11·10^-6 mol O₂ h^-1 protoplast^-1=1259 mol O₂ mg^-1 chlorophyll h^-1=22.97 mol O₂ mg^-1 protein h^-1) with no further increases above this temperature. Respiration rates were much lower in protoplasts 15–16 h old than in freshly prepared ones indicating considerable deterioration of their viability over this time period.     

Transport of ascorbate into protoplasts ofNicotiana tabacum Bright Yellow-2 cell line

Summary The uptake of ascorbate into protoplasts isolated from aNicotiana tabacum Bright Yellow-2 (BY-2) cell suspension culture was investigated. Addition of¹⁴C-labelled ascorbate to freshly isolated protoplasts resulted in a time- and substrate-dependent association of radioactive molecules with the protoplasts. The kinetic characterisation of this presumptive uptake revealed kinetics of Michaelis-Menten type with an apparent maximal uptake activity of 24 pmol/min·10⁶ protoplasts and an apparent affinity constant of 139 M. The amount of ascorbate molecules transported into_{N. tabacum} protoplasts decreased when nonlabelled dehydroascorbate or iso-ascorbate were added but was not affected by addition of 5,6-o-cyclohexylidene ascorbate or ascorbate-2-sulfate. These data indicate a carrier-mediated uptake of ascorbate into the protoplasts that shows a high structural specificity. To investigate which redox status of ascorbate is preferentially taken up by theN. tabacum protoplasts, transport was tested in the presence of various compounds that can affect the redox status of ascorbate. Testing uptake in the presence of a reductant, dithiothreitol, resulted in a significant and concentration-dependent inhibition of the amount of ascorbate molecules transported into the protoplasts. On the other hand, ascorbate uptake was significantly stimulated in the presence of the enzyme ascorbate oxidase. Ferricyanide did not affect ascorbate transport. Inhibition studies revealed that ascorbate uptake in the protoplasts is sensitive to addition of sulfhydryl reagents N-ethyl maleimide andp-chloro-mercuribenzenesulfonic acid and to a disruption of the proton gradient by the protonophore carbonylcyanide-3-chlorophenylhydrazone. The uptake of ascorbate was also inhibited by addition of cytochalasin B but not sensitive to addition of phloretin or sulfinpyrazone. Taken together these data indicate the presence of an ascorbate transport system in the plasma membrane ofN. tabacum protoplasts and suggest dehydroascorbate as the preferentially transported redox species. The putative presence of different carriers for reduced and oxidised ascorbate in the plasma membrane is discussed.Abbreviations Asc ascorbate - BY-2 Bright Yellow 2 - CCCP carbonylcyanide-3-chlorophenylhydrazone - DHA dehydroascorbate - DTT dithiothreitol - MS medium Murashige and Skoog medium - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

The chemiosmotic model of stomatal opening revisited

Stomata are light‐activated biological valves in the otherwise gas‐impermeable epidermis of aerial organs of higher plants. Stomata often regulate rates of photosynthesis and transpiration in ways that optimize whole‐plant carbon gain against water loss. Each stoma is flanked by a pair of opposing guard cells. Stomatal opening occurs by light‐activated increases in the turgor pressure of guard cells, which causes them to change shape so that the stomatal pore between them widens. These increases in turgor pressure oppose increases in cellular osmotic pressure that result from uptake of K⁺. K⁺ uptake occurs by a chemiosmotic mechanism in response to light‐activated extrusion of H⁺ outward across the plasma membrane of the guard cell. The initial changes in cellular membrane potential lead to the opening of inward‐rectifying K⁺ channels, after which K⁺ is taken up along its electrochemical gradient. Changes in membrane potential resulting from K⁺ uptake may be balanced by accumulation of Cl^?ions by guard cells and/or by synthesis of malic acid within each cell. Malic acid also acts to buffer increases in cytosolic pH caused by H⁺ extrusion. This review describes how the application of patch‐clamp technology to guard cell protoplasts has enabled investigators to elucidate the mechanisms by which H⁺ is extruded from guard cells, the types of ion channels present in the guard cell plasma membrane, how those ion channels are regulated, and the signal transduction processes that trigger stomatal opening and closing.     

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.     

Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements

Low concentrations of salicylhydroxamic acid (<5 millimolar) stimulate O₂ uptake in intact roots of Pisum sativum. We demonstrate that the hydroxamate-stimulated O₂ uptake does not reside in the mitochondria. We also show that the hydroxamate-stimulated O₂ uptake is due to the activation of a peroxidase catalyzing reduction of O₂. This peroxidase, which can use both NADH and NADPH as a substrate, is stimulated by low concentrations of monophenols, e.g. salicylhydroxamic acid and 2-methoxyphenol. It is inhibited by high (20 millimolar) concentrations of salicylhydroxamic acid, cyanide, and scavengers of the superoxide free radical ion, e.g. ascorbate, gentisic acid, and catechol. In the presence of gentisic acid, O₂ uptake by intact pea roots was no longer stimulated by low concentrations of salicylhydroxamic acid. The consequence of the present finding for in vivo respiration measurements is that the use of low concentrations of salicylhydroxamic acid and uncoupler is reliable only in the presence of a suitable superoxide free radical scavenger which prevents activation of the peroxidase. It also confirms that high concentrations of salicylhydroxamic acid (20-25 millimolar) can be safely used in short-term experiments to assess the activity of the alternative path in intact roots.     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.