TY - JOUR
AU - Hanstein, Stefan
AB - Abstract The apoplastic pH of the substomatal cavity is an essential determinant of stomatal movement. In detached leaves of Vicia faba substomatal apoplastic pH and its dependence on external (stress) factors was investigated using a non‐invasive approach: pH‐microsensors were inserted into open stomata, and upon contact with the apoplastic fluid, pH was measured continuously, as apoplastic pH was challenged by changed conditions of light, atmosphere (NH3, CO2), and xylem sap (abscisic acid, cyanide, fusicoccin, pH, inorganic salts). Apoplastic pH proved extremely sensitive to infiltration and local flooding, which rapidly increased the apoplastic pH by more than 1.5 pH units. Recovery from infiltration took several hours, during which light effects on the apoplastic pH were strongly impeded. This indicates that pH tests carried out under such conditions may not be representative of the undisturbed leaf. NH3, flushed across the stomata, yielded a rapid apoplastic alkalinization from which an apoplastic buffer capacity of 2–3 mM per pH unit was calculated. Fusicoccin, fed into the xylem sap acidified the apoplast, whereas cyanide alkalized it, thus underscoring the importance of the plasma membrane H+ pump for apoplastic pH regulation. To address the question to what extent pH was a drought signal, the effect of iso‐osmotic pH changes, fed into the xylem through the petiole were tested. It is demonstrated that the apoplastic response remained below 0.1 pH per pH unit imposed, regardless of the buffer capacity. An increase in the osmolarity of the bath solution (harbouring the cut petiole) using KCl, NaCl, CaCl2 or sorbitol alkalized the substomatal apoplast. It is suggested that pH may only act as drought signal when accompanied by elevated osmolarity. Apoplastic pH, pH regulation, stomata, stress, Vicia faba. Introduction A controlled apoplastic ionic milieu is highly important for the regulation of substrate exchange between symplast and apoplast, a process in which pH, mainly because of chemiosmotic reasons, plays a dominant role. Since membrane transporters measure the activities of their transportees when these are closest to the membrane, and the apoplast fluid is only a very thin film, its composition has to be regulated rather tightly. Since the stomata are control stations of gas exchange and vertical transport of water, ions, amino acids, abscisic acid etc., the understanding of events within that space, the knowledge of composition, regulation and dynamics of the apoplastic milieu is of utmost importance. Because the apoplast fluid film is so thin, direct access to it has always been difficult and determinations of the apoplastic ionic mileu had to rely on estimations using techniques of infiltration and extraction (Schjoerring, 1998, and references therein) or fluorescent dyes (Hoffmann et al., 1992; Mühling and Sattelmacher, 1995; Mühling and Läuchli, 2000). Both techniques have yielded valuable information in the past, but may be rather stressful to the leaf in that they alter the primary composition of the apoplastic fluid, which leaves an uncertainty remains with respect to absolute concentrations of its constituents and to the physiological behaviour of the leaf under such conditions. By using a non‐invasive technique, namely the investigation of the apoplastic fluid with ion‐selective microelectrodes carefully inserted into open stomata, it is now possible to detect absolute activities of ions and continuously any changes thereof while challenging the leaf with different conditions (Hanstein and Felle, 1999; Felle et al., 2000; Szyroki et al., 2001). The main focus of this study is the investigation of the pH regulation of a very sensitive leaf area, the substomatal cavity. This was accomplished by adding a variety of substances to the transpiration stream via the cut petiole, by altering the atmospheric composition, by changing light conditions or by directly changing the apoplastic fluid, while constantly monitoring the apoplastic pH. Materials and methods Plants and general conditions Vicia faba was grown at 20 °C under a 13/11 h light/dark regime. Tests were carried out in the morning hours after adapting the leaves for about 1 h to the light conditions in the test chamber. Leaves were cut by the stem with a razor blade and placed immediately in the standard test solution (1 mM KCl, 0.1 mM CaCl2, pH 5 or as given in the figures or figure legends). The leaves were mounted in a two‐chamber cuvette for gas exchange and measurements of ion activity, as described below. Water‐soluble substances were added to the cut end of the petiole in a flow‐through regime, which provided a constant flow of solution to the cut end of the petiole, from where transpiration drove uptake of test substances into the xylem. Electrical: ion‐selective microelectrodes The electrical set‐up for the fabrication and application of ion‐sensitive microelectrodes has been described previously (Hanstein and Felle, 1999; Felle et al., 2000). The preparation of the ion‐selective electrodes for extracellular use differed from those for intracellular use (Felle and Bertl, 1986) in that the tip was about 2 μm in diameter, blunt and heat‐polished. Single pipettes were pulled on a patch‐clamp puller (List Instruments, Darmstadt, Germany) and silanized internally using a 0.2% tributylsilane/chloroform solution. After heat‐stabilizing at 200 °C for 1 h, the cooled pipettes were backfilled with the respective sensor cocktail. To give the sensor in the tip sufficient firmness, the cocktails (Fluka: No. 95297 H+; No. 60398 K+) were dissolved in a mixture of polyvinylchloride and tetrahydrofuran (40 mg ml−1) at a ratio of 30/70 (v/v). After evaporation of the tetrahydrofuran, the remaining firm gel was topped up with the undiluted sensor cocktail followed by the reference solution, which consisted of 100 mM MES/TRIS mixed to pH 6 in 0.5 M KCl. After equilibrating, these electrodes gave stable responses for several weeks, when stored in a dry chamber. The electrodes were connected to high‐impedance amplifiers (FD 223; WP‐Instruments, Sarasota, FL, USA) which simultaneously measured and subtracted the signals coming from the ion‐selective electrode and the voltage reference electrode to obtain the net kinetics of the free ion concentration under investigation. The signals were recorded on a chart recorder (L 2200, Linseis, Germany). For the sake of clarity, only the net traces are shown. The technique and set‐up for measuring apoplastic ion activities has been decribed recently (Hanstein and Felle, 1999; Felle et al., 2000). Briefly, the two‐chamber cuvette allowed a constant perfusion of one side, but left the other side dry. The ground reference electrode was placed in the perfusion part. The access to the leaf apoplast was carried out in the dry chamber (separated by an insulating Plexiglas bar from the wet chamber) by inserting the blunt electrodes carefully (without damaging cells) at an angle of approximately 45° through the open stomata into the cavity below. Contact of the electrodes with the apoplastic water film was signalled by the closing of the electrical circuit. Prior to tests the electrode was left in a stable position until a stable reading without drift was attained (≥1 h). Closing of the stomata due to darkness or ABA treatment in most cases did not displace the electrodes. In cases where a displacement of the electrode tip did occur during stomatal movement, the electrodes were either repositioned or the measurement was terminated. Since the electrical resistance from the ion selective electrodes through the apoplast network to the grounded bath reference was high (1–20 MΩ, depending on the distance from veins), a blunt leaf voltage reference microelectrode was inserted through a neighbouring stoma. The difference from the signals of these electrodes was recorded as the net trace given in the figures. Fumigation The CO2 concentration in the cuvette was set by mixing CO2‐free air with a CO2 standard (5.04% CO2 in synthetic air). The mixing was carried out using flowmeters (Bailey‐Fischer‐Porter; Göttingen, Germany). Teflon pipes (4 mm o.d., 2 mm i.d.; MAGV, Rabenau, Germany) connected the cuvette with the gas‐cylinders (Messer‐Griesheim, Krefeld, Germany). The flow‐through velocity guaranteed a rapid gas exchange. A certified NH3 standard and clean air from commercially available pressure flasks (Messer–Griesheim) were mixed using flow meters and then flushed at a rate of 1.4 l min−1 across the leaf. Illumination was 300 μE m−2 s−1 through a fibre cold‐light source (KL 1500, Leica, Wetzlar, Germany). Infiltration and flooding of the apoplast Local flooding was carried out using a home‐built pressure device (Herrmann and Felle, 1995). The infiltrate was adjusted as close as possible in composition to the apoplastic fluid, as analysed previously (Felle et al., 2000). After inserting the injection capillary through an open stoma, the pH sensitive‐ and the leaf‐voltage reference electrodes were placed in nearby stomata. After making electrical contact and allowing for the respective adjusting time the infiltrate was gently pressed into the substomatal cavity. It was rapidly distributed within the leaf apoplast and within seconds reached the other electrodes. Infiltration of the entire leaf was carried out by connecting the cut petiole to a vacuum pump through tubing. Since the placing of the leaf and the subsequent manipulations to adjust the electrodes following the infiltration took time, the first reliable measurements of pH could not be carried out before about 1 h had passed. Results The apoplastic pH of the substomatal cavity of intact Vicia faba leaves After mounting the entire leaf in the cuvette, and following an adaptation of about 2 h to light and to a reference bath solution of pH 5.0 (MES/TRIS 0.5 mM), KCl 1 mM, and CaCl2 0.1 mM (chamber with cut petiole), the microelectrodes were carefully inserted through the open stomata. After making electrical contact with the apoplastic fluid of the substomatal cavity and another hour of adaptation with occasional repositioning of the electrodes, the apoplastic pH within the substomatal cavity was measured at 4.7 to 5.2 (mean 4.95±0.12 SE; n=43) depending on the experimental conditions (see below). Challenge of the apoplastic pH through manipulation of the xylem sap The xylem is part of the leaf apoplast and the transport of ions and other transportees do not require the crossing of a membrane to reach the apoplast of the mesophyll or the substomatal cavity. It was of interest, therefore, to discover to what extent substances (naturally occurring as well as laboratory chemicals) fed through the cut petiole influenced the pH of the substomatal cavity and thus could have a potential impact on stomatal aperture. Changes in pH: Apart from representing a basic driving force for membrane transport, apoplastic pH is currently being discussed as signal or messenger for a variety of processes, for example, for channel activation (Blatt, 1992) as well as drought (Wilkinson and Davies, 1997). Here the basic question was: will pH changes that occur within the xylem sap be carried to the stomata? As shown in Fig. 1, changes in the bath pH ranging between two and four units, fed through the cut petiole into the xylem, had only a minor impact on the apoplastic pH of the substomatal cavity. In the presence of 1 mM buffer (Fig. 1a), the apoplastic pH changed by 0.094±0.018 SE (n=5) units per pH unit imposed. Somewhat larger pH responses were observed after increasing the buffer concentration to 5 or 10 mM, respectively (Fig. 1b). In the presence of 5 mM buffer a pH change from 4.1 to 6.8 yielded 0.14±0.03 SE (n=3) units per pH imposed, and 0.17±0.05 SE (n=3) units per pH changed. 10−5 M ABA, added in the same way, transiently alkalized the apoplastic pH by 0.4–0.5 units, irrespective of the pH given in the bath (Fig. 2). Fig. 1. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba as challenged by different pH as indicated, added at different buffer concentrations to the chamber harbouring the cut leaf petiole. (a) Buffer concentration 1 mM; (b) buffer concentrations 5 or 10 mM, as indicated. Representative of at least three equivalent kinetics, each. Fig. 1. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba as challenged by different pH as indicated, added at different buffer concentrations to the chamber harbouring the cut leaf petiole. (a) Buffer concentration 1 mM; (b) buffer concentrations 5 or 10 mM, as indicated. Representative of at least three equivalent kinetics, each. Fig. 2. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba leaves as challenged by 10−5 M abscisic acid (ABA), added to the cut leaf petiole at different pH, as indicated. Representative of three equivalent kinetics, each. Fig. 2. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba leaves as challenged by 10−5 M abscisic acid (ABA), added to the cut leaf petiole at different pH, as indicated. Representative of three equivalent kinetics, each. Ionic and osmotic changes: Apart from their potential effects on membrane transporters, cations may exchange for protons bound to cell wall constituents (Grignon and Sentenac, 1991; Felle, 1998), when forced into that space. As such, changes in cation activity may influence the apoplastic pH. When added to the standard bath solution, 20 mM KCl as well as 20 mM NaCl or 20 mM CaCl2, quite unexpectedly, alkalized the substomatal apoplast by about 0.3 units. Whereas KCl and NaCl elicited similar effects in both shape and magnitude (Fig. 3a), CaCl2 produced a larger pH‐increase (Fig. 3b). When Cl− was exchanged for the membrane impermeable gluconate–, the alkalinization by K+ was slightly reduced. Reducing the [K+] nominally to zero (no K+ added), the pH dropped slightly below the value measured in the presence of 1 mM K+. 38 mM d‐sorbitol (osmotically about equivalent to 20 mM KCl) also alkalized the leaf apoplast. A direct comparison of sorbitol and KCl, exchanging 20 mM for sorbitol, revealed that sorbitol was more effective than KCl (Fig. 3b). To test whether K+ indeed had travelled from the cut petiole to the measuring area within the substomatal cavity and to what extent, the changes in apoplastic K+‐activity were monitored using a K+‐selective microelectrode. As shown in Fig. 3a, there was a rapid increase in apoplastic K+‐activity from 1.8 mM to about 15 mM within 15 min following the addition of 20 mM [K+] to the cut petiole (increase of bath [K+] from 1 to 20 mM). Fig. 3. View largeDownload slide Effect of increased osmolarity on the apoplastic pH of substomatal cavities of Vicia faba as measured after adding (a) 20 mM KCl, NaCl, or K‐gluconate (KGlu) to a bath with either 1 mM KCl or no KCl added, and subsequent removal of the salts (–). To test whether the K+ added in fact reached the stomata, K+ activity changes (pK) was tested following the addition of 20 mM KCl. (b) Addition of 20 mM CaCl2 or 38 mM sorbitol to a bath with either 1 mM or 20 mM KCl present. Representative of at least four equivalent kinetics, each. Fig. 3. View largeDownload slide Effect of increased osmolarity on the apoplastic pH of substomatal cavities of Vicia faba as measured after adding (a) 20 mM KCl, NaCl, or K‐gluconate (KGlu) to a bath with either 1 mM KCl or no KCl added, and subsequent removal of the salts (–). To test whether the K+ added in fact reached the stomata, K+ activity changes (pK) was tested following the addition of 20 mM KCl. (b) Addition of 20 mM CaCl2 or 38 mM sorbitol to a bath with either 1 mM or 20 mM KCl present. Representative of at least four equivalent kinetics, each. Fusicoccin: The phytotoxin fusicoccin (FC) is known to stimulate the plasma membrane H+ ATPase which leads to increased H+ extrusion and plasma membrane hyperpolarization (Marrè, 1979). As shown in Fig. 4, FC, fed through the cut petiole into the xylem, indeed rapidly acidified the apoplast by 0.5 pH units within the first 10 min after application and proceeded with a slower acidification thereafter. This experiment served as a test to show that FC had its usual effect and that even large molecules added this way indeed reached the sensitive tip of the pH‐electrode located within the substomatal cavity. Fig. 4. View largeDownload slide Proton pump activity and apoplastic pH. Apoplastic pH of substomatal cavities of Vicia faba as influenced by FC and cyanide, added to the chamber harbouring the cut leaf petiole. Upper curve: acidification of the apoplast following the addition of 5 μM fusicoccin (FC). Middle curve: effect of 1 mM NaCN and subsequent ‘light‐off’ and ‘light‐on’. Lower curve: alkalinization of the apoplast by 10 mM NaCN and effect of ‘light‐off’ and ‘light‐on’ both in the presence of NaCN. Representative of at least three equivalent kinetics, each. Fig. 4. View largeDownload slide Proton pump activity and apoplastic pH. Apoplastic pH of substomatal cavities of Vicia faba as influenced by FC and cyanide, added to the chamber harbouring the cut leaf petiole. Upper curve: acidification of the apoplast following the addition of 5 μM fusicoccin (FC). Middle curve: effect of 1 mM NaCN and subsequent ‘light‐off’ and ‘light‐on’. Lower curve: alkalinization of the apoplast by 10 mM NaCN and effect of ‘light‐off’ and ‘light‐on’ both in the presence of NaCN. Representative of at least three equivalent kinetics, each. Cyanide: Cyanide is being successfully used in transport studies to inhibit mitochondrial electron transport and, subsequently, respiratory ATP production (Slayman et al., 1973; Felle, 1981, 1996). As such it deactivates the plasma membrane proton pump and depolarizes the affected cells for which, in freely accessible cells, 1 mM cyanide is usually sufficient, unless cyanide‐resistant bypasses prevent such action. As Fig. 4 shows, the response to 1 mM NaCN was very weak, quite contrary to the expected alkalinization. Moreover, the responses to ‘light‐off’ and ‘light‐on’ were in the usual range, albeit the initial fast pH peaks were missing (see below), indicating some effect of cyanide. Addition of 10 mM NaCN, however, yielded the expected result and rapidly increased the apoplastic pH by about 0.4 units. ‘Light‐off’, in the presence of cyanide, then caused further alkalinization of 0.8 units which, after ‘light‐on’, temporarily recovered by about 0.3 units. After this, the apoplastic pH levelled off around 6.4 and light/dark effects were not observed any more. After removal of the cyanide the apoplastic pH slowly (within 2 h) returned to normal levels (data not shown). Challenge of the apoplastic pH through the atmosphere Ammonia: Ammonia is emmited from the soil, from senescing plant parts, and in considerable amounts from agricultural sources. NH3 rapidly dissolves in water and binds H+ due to the low apoplastic pH. As Fig. 5a shows, when leaves were flushed with NH3, a rapid alkalinization occured as an immediate result of NH4+ forming (Husted and Schjoerring, 1995). Following this rapid pH change, which is a good measure of the apoplast's capacity to buffer protons (Hanstein and Felle, 1999), a slower pH increase occurred, which agrees with stomatal closure. The entire alkalinization was reversed when NH3 was exchanged for air (data not shown). In order to test whether the alkalinization was caused by pump inhibition, FC was tested. FC added in the presence of NH3 resulted in a stable, but smaller acidification than observed without NH3 (Fig. 4). After exchanging NH3 for air (in the presence of FC) the full FC‐induced acidification was rapidly attained, as compared with the control experiment without NH3 fumigation. Fig. 5. View largeDownload slide Apoplastic pH‐buffering. (a) Fumigation of Vicia faba leaves with 1 ppm NH3 followed by fusicoccin (FC) addition through the cut petiole and subsequent removal of NH3 (Air). (b) Fumigation with different CO2 concentrations, as indicated. Representative of three equivalent kinetics, each. Fig. 5. View largeDownload slide Apoplastic pH‐buffering. (a) Fumigation of Vicia faba leaves with 1 ppm NH3 followed by fusicoccin (FC) addition through the cut petiole and subsequent removal of NH3 (Air). (b) Fumigation with different CO2 concentrations, as indicated. Representative of three equivalent kinetics, each. Carbon dioxide: The atmospheric CO2 concentration of around 370 ppm is in equilibrium with the leaf apoplastic fluid, which is what the cells encounter. CO2 reacts with water, forms HCO3− and thereby acidifies the aqueous phase. Taking the CO2 solubility and the above stated apoplastic pH into account, physiologically relevant changes in the CO2 concentration, e.g. during stomatal movements, would thereby not alter the pH of the apoplast, unless the apoplastic buffer capacity was very low. In order to cover the range of naturally occurring CO2 changes, CO2 was altered abruptly between 0 and 800 ppm. As shown in Fig. 5b, the apoplastic pH responded to these changes, albeit in the inverse manner than expected: a decrease in CO2 from 700 or 800 ppm to nominally CO2‐free air readily decreased the apoplastic pH by 0.2–0.3 units, addition of CO2 reversed this effect. Infiltration and flooding of the apoplast The apoplastic space is only partly filled with fluid. This raises the question if and how the pH is changed when the entire apoplastic space gets filled with an aqueous solution, which with respect to pH, K+, Ca2+ and Cl− is similar and approximately iso‐osmotic to the apoplastic milieu (Felle et al., 2000). As Fig. 6 shows, pressure‐injection of such a solution into individual open stomata yielded an instantaneous massive pH increase by about 1.5 pH units, which returned within 1 h to the pH measured prior to flooding. Vacuum infiltration of the apoplast yielded responses similar to the one shown in the inset to Fig. 6. It is demonstrated that the pH, measured 1 h after the infiltration (X), neither represented the pH of the infiltrate nor the apoplastic pH measured without infiltration. For instance, when the pH of the infiltrate was pH 4.4 (as shown in the inset) then the first measurement 1 h after infiltration yielded a value around pH 6. Within the next 1–2 h the pH dropped only slightly and reached a value around pH 5 after about 5 h. The latter kinetics were influenced by the buffer concentration of the infiltrate. Whereas there was little difference in the final pH reached in the presence of 0.5 or 5 mM MES/TRIS buffer, 20 mM buffer reduced the ability of the apoplast to restore the pH (data not shown). Since vacuum infiltration and the placing of the electrode thereafter was time‐consuming, no information is available as to what happens immediately upon encounter of the apoplast with the infiltrate. It can be assumed though that pH change took place similar to the one directly observed after local flooding. The typical responses to ‘light‐off’ and ‘light‐on’ were strongly impeded in the infiltrated apoplast. As shown in Fig. 7, in untreated leaves, the light‐adapted leaf responded to ‘light‐off’ with an oscillatory behaviour, starting with a short increase in pH to level off roughly 0.3 pH units more alkaline; ‘light‐on’ essentially inverted this response. In the infiltrated leaf, while apoplastic pH was still readjusting (slope in Fig. 6, inset), the initial fast response is missing and the absolute pH changes are less pronounced. Fig. 6. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba, as influenced by local flooding or infiltration with a medium comprising 2 mM KCl and 0.1 mM CaCl2. Flooding was achieved by injecting the fluid into one stoma while measuring the pH in a neighbouring one; infiltration procedure see Materials and methods. ‘X’ marks the moment of infiltration and the pH of the infiltrate. No data were collected in the first hour after infiltration, because the leaf had to be placed in the cuvette and the electrodes placed within the stomata. Kinetics are representative of at least three equivalent experiments, each. Fig. 6. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba, as influenced by local flooding or infiltration with a medium comprising 2 mM KCl and 0.1 mM CaCl2. Flooding was achieved by injecting the fluid into one stoma while measuring the pH in a neighbouring one; infiltration procedure see Materials and methods. ‘X’ marks the moment of infiltration and the pH of the infiltrate. No data were collected in the first hour after infiltration, because the leaf had to be placed in the cuvette and the electrodes placed within the stomata. Kinetics are representative of at least three equivalent experiments, each. Fig. 7. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba, as influenced by different light conditions (‘light‐off’, light‐on'), as indicated. Upper curve: untreated leaf, lower curve: infiltrated leaf. Representative of nine equivalent kinetics upper curve, three equivalent kinetics lower curve. Fig. 7. View largeDownload slide Apoplastic pH of substomatal cavities of Vicia faba, as influenced by different light conditions (‘light‐off’, light‐on'), as indicated. Upper curve: untreated leaf, lower curve: infiltrated leaf. Representative of nine equivalent kinetics upper curve, three equivalent kinetics lower curve. Discussion pH‐regulation in the substomatal cavity The apoplast of the substomatal cavity is a very sensitive area. This is indicated by the massive pH shifts that were caused by the injection of a solution that was as close as possible in its ionic composition to the apoplastic fluid (Felle et al., 2000). Given that the infiltrate and the apoplastic fluid differed only slightly in ion content, the strong responses must be due to a strong reaction of the cells forming the apoplastic space. Two explanations can explain this strong alkalinization. First, the flooding of the apoplast causes an acute lack of oxygen to which all the involved cells respond with rapid cytosolic acidification and pump deactivation. In fact, the pH peak of about 6.4, reached during flooding, fits well with the pH attained during the cyanide experiments (Fig. 4). Secondly, the strong alkalinization may be a sign of mechanical stress. The fluid entering the apoplast strongly interacts through adhesive forces with the cell wall and plasma membrane and might thus stretch‐activate channels which then release ions and organic acids. Since it is unlikely for several reasons that protons alone entered the cells causing apoplastic alkalinization, it is suggested that the alkalinization was essentially caused by organic acids released through anion channels (H Felle, unpublished results). As Fig. 6 shows, pH recovery following flooding was accomplished within the hour, probably due to recovery of the proton pump, while the infiltrate was being absorbed by the tissue. The much slower pH recovery after vacuum infiltration (Fig. 6; inset) of the entire leaf may be due to the larger liquid volume the leaf tissue had to deal with. Both experiments, however, demonstrate the sensitivity of the leaf apoplast to this kind of manipulation. The recovery of the apoplastic pH takes several hours, depending on the extent of infliltration, and so experiments carried out before that period may yield neither the correct apoplastic pH nor any of the other transport parameters investigated. This idea is supported by the observation that the responses to ‘light‐off’ and ‘light‐on’ considerably differed from those obtained in the untreated tissue (Fig. 7). Apoplastic buffering: In a recent study, the apoplastic buffer capacity has been determined in Bromus erectus by fumigating the leaves with NH3, which then dissolved in the apoplastic fluid, thereby forming NH4+ and increasing the pH (Hanstein and Felle, 1999). According to   1 a buffer capacity (β) of 6 to 8 mM H+ per pH unit was calculated. Here it is shown that NH3 fumigation of Vicia leaves yielded a likewise fast response of 0.3±0.18 (n=4) (Fig. 6a) and a change in [NH4+] of 0.75±0.25 mM (n=2; data not shown), from which a buffer capacity of 2–3 mM H+ per pH unit was calculated. This value compares well with the data from Bromus and with 4 mM per pH unit from potato (Oja et al., 1999), but is only about one‐tenth of typical cytoplasmic values (Guern et al., 1991). Since the apoplast represents the basic reservoir for a variety of substances to be transported in and out of adjacent cells and the apoplastic fluid within the leaf is a rather thin film, small ion fluctuations should rapidly perturb the apoplastic pH unless effective physiological (active) buffering takes place. This is indeed the case: both passive and active buffering are shown in the two‐phased pH response to NH3 fumigation (Fig. 5a). The abrupt stop of alkalinization is indicative of passive buffering, whereas the partial inversion of the pH change, followed by a slow further increase in apoplastic pH, shows active buffering. pH levels off after 20 min, which marks the dynamic equilibrium between the constantly forming NH4+, its transport and possibly metabolism. Similar pH kinetics have been reported for Bromus erectus (Hanstein and Felle, 1999). CO2 also readily dissolves in an aqueous phase with the tendency to acidification. In fact, Savchenko et al. demonstrated in potato leaves, that high CO2 concentrations (1% and higher) immediately and quantitatively acidified the apoplast (Savchenko et al., 2000). But, as Fig. 5b shows, changes in the physiological relevant CO2 concentrations range did not shift the apoplastic pH in the expected direction: a decrease in CO2 caused acidification, its increase alkalinization. Under these conditions passive buffering was sufficient to quench CO2‐produced acidification completely and to permit a physiological response in the opposite direction, which might be indicative of CO2‐sensing with respect to stomatal movement. Although a CO2 sensor within the leaf has not been idetified yet, CO2 will act on channel proteins (Brearley et al., 1997) and thus cause shifts in ion activities, which in turn will have an effect on stomatal action. H+ pump related processes: The importance of the H+ pump for the homeostasis of the apoplastic pH is underscored by the responses to FC and to NaCN. Cyanide, which has been shown in numerous studies to strongly deactivate the pump (Sanders and Slayman, 1982; Felle, 1981), at 10 mM massively alkalized the apoplastic pH to about 6.3 (Fig. 4). Taking into account that at the same time cytosolic pH dropped by 0.6 units, an observation which is generally made under anoxia or with cyanide (Sanders and Slayman, 1982; Felle and Bertl, 1986; Felle, 1996; Fox et al., 1995), the pH gradient across the plasma membrane, which is vital for a variety of substrate transport, was practically eliminated. Although there is no evidence, the relative insensitivity of the apoplastic pH in the vicinity of stomata to 1 mM NaCN, which is usually sufficient to deactivate the pump, could arise from the formation of volatile HCN due to low pH within the apoplast, which then resulted in cyanide concentrations too low to be effective at the point of measurement. In that case the apparent insensitivity of the leaf to low cyanide concentrations would not be due to pH regulation. On the other hand, cyanide‐insensitive bypasses could have prevented pump deactivation enough to cause alkalinization. Although the acidifying effect of FC adds to a recognition of the important regulating role of the H+ pump for the apoplastic pH, it was remarkable that FC could not acidify the apoplast to the full extent while NH3 was being flushed (Fig. 5a), and only removal of NH3 brought the full FC response. 1.5 ppm NH3 flushed will produce approximately 1 mM apoplastic NH4+. This will enter the cell and strongly depolarize the plasma membrane through the rapid import of positive charge (Bertl et al., 1984) and thus short‐circuit the pump, which works against FC action. This shows that, despite the strong influence of the H+ pump, the apoplastic pH is also effectively influenced by processes of association and dissociation, by membrane transport and possibly by metabolism. The xylem filter The xylem is an integral part of the apoplast, and as such is interconnected with the apoplast of the mesophyll and substomatal cavity without membrane barriers. Thus, a variety of substances fed through the petiole into the transpiration stream will leak into the mesophyll apoplast according to their concentration gradients and will sooner or later reach the stomata and produce effects there. FC, for instance, a large inert molecule, readily reached the stomata and caused the expected effects. On the other hand, some substances could, while travelling through the xylem, be taken up by the xylem parenchyma cells, bound by fixed charges or altered chemically and hence would not or only to a minor extent reach the substomatal cavities. pH, a drought signal? Figure 1 demonstrates that only a fraction of a pH change fed into the cut petiole reached the substomatal cavity. Interestingly, this was true for buffer concentrations of 1 mM (with a lower buffer capacity than the apoplastic fluid) as well as for 10 mM buffers with a capacity near that of the apoplastic fluid (tested by titration). The data also show that pH changes within the substomatal cavity do not increase proportional with buffer capacity, which is indicative of active buffering. Although the MES/TRIS buffer used may not be representative for the xylem sap, the results clearly show that iso‐osmolar pH changes are not carried to the stomata, which is underlined by the observation that ABA pH‐transients, measured after adding ABA at different pH to the cut petiole, were also not pH‐dependent (Fig. 2). If pH differences had been carried from the petiole to the stomata, due to the pH‐dependent protonation of ABA, the effects should have differed considerably. Xylem sap pH between 5.8 to 6.6 has been reported (Gollan et al., 1992; Wilkinson and Davies, 1977). This is far less acidic than the apoplastic pH in the vicinity of the stomata, which in all the tested leaves has been found to be around 5 or less: Bromus erectus: pH 4.6–4.8 (Hanstein and Felle, 1999), Vicia faba: 4.8–5.1 (Felle et al., 2000), rapeseed: pH 4.9–5.1 (S Hanstein et al., unpublished results), Hordeum vulgare 4.5–4.8 (S Hanstein and H Felle, unpublished results). Given that in Vicia faba this pH difference between the xylem sap and the substomatal apoplastic fluid also exists, it appears that a dynamic pH barrier exists as a result of active pH regulation. It should be interesting to find out where in the leaf this barrier is built up: gradually along the mesophyll or abruptly between xylem and mesophyll. Apart from ABA, pH is being discussed as a drought root‐sourced signal to the leaves (Wilkinson and Davies, 1997; Wilkinson 1999), and it has been demonstrated that apoplastic alkalinization occurs within a tissue with low water potential (Davies and Zhang, 1991). In the light of the data found in this study, the question must be raised, whether drought experienced by the plant in the root could in fact be signalled to the leaves via pH (increase). In this context it is reported that the xylem pH will increase as the soil dries and the roots experience drought stress (Hartung and Radin, 1989; Wilkinson and Davies, 1997). Since it is accepted that ABA will be released from mesophyll anion traps to the apoplast of a leaf as the apoplast pH increases (Hartung et al., 1988), transmission of a pH change from root to shoot could redistribute ABA in the leaf and thus influence stomatal behaviour (Davies and Zhang, 1991; Zhang and Outlaw, 2001). Drought‐induced pH changes in the xylem were reported to be around 0.5 pH units (Wilkinson and Davies, 1997). According to the data from Fig. 1, this means that in Vicia only about 0.07–0.08 pH units would actually reach the substomatal space, apparently not enough to have any influence of physiological relevance on the apoplastic distribution of ABA or on the stomata directly. Whether pH altered in the root could in fact reach the shoot and if so, under what conditions, has not been measured. But it is known that these pH changes are not carried to a significant extent to the stomata, unless they are accompanied by an osmotic shift, which is possible during drought. There can be no doubt that drought induces an increase in apoplastic pH in the affected organs (Davies and Zhang, 1991), but this might correspond with an increase in osmolarity of the respective fluid. This is indicated by the observation that 20 mM KCl, NaCl, CaCl2 or sorbitol of equivalent osmolarity fed into the transpiration stream, quite substantially alkalized the leaf apoplast (Fig. 3). It can be assumed that larger increases in salt concentrations would cause more substantial pH changes. Since high salt concentrations may produce side‐effects, such experiments were not performed. Alkalinization of the apoplast by an increase in salt concentration was not necessarily to be expected, since in the maize root apoplast (cortex, root hair zone) an acidification in the cell wall space following such treatment: a result which fitted well with the idea of the apoplast being an ion exchanger. Such ion exchange processes apparently do not play a dominating role in the Vicia leaf, but might influence the kinetics in a way that the pH changes shown in Fig. 3 might be underestimations. Apoplastic alkalinization might be due to stimulated uptake of the Cl− via a 2H+/Cl− symport. Although such transport takes place, this idea did not prove valid, because salt‐induced alkalinization also occurred in the presence of the membrane impermeable gluconate–, used instead of Cl− as the anion accompanying K+. In order to explain why apoplastic pH increased in response to increased salt concentration, a reduction in H+‐ATPase activity was discussed (Hartung and Radin, 1989), but strong ion differences (Stewart, 1983; Gerendás and Schurr, 1999) could also be responsible. In support of this, Gollan et al. report a surplus of mobile cations in drought‐stressed xylem (Gollan et al., 1992). It was interesting to observe that non‐ionic substances like sorbitol also alkalized the apoplast, even more than KCl of the same osmolarity, which strongly indicates a process of osmotic origin. K+ and Cl− as membrane permeable ions have a lower reflection coefficient than sorbitol and hence the same amount of solubilized K+ and Cl− would osmotically not be as effective as sorbitol. It is therefore suggested that an iso‐osmolar pH change is not the primary drought signal, but that an increased apoplastic pH is an indication of drought. It appears that the drought‐increased concentration of osmotically effective particles increases the pH in the xylem and, subsequently, also in the leaf. According to this pH increase, part of the anion‐trapped ABA may be released which then causes the stomata to close (Wilkinson and Davies, 1997; Hartung et al., 1998). Since ABA itself has a transient effect on apoplastic pH (Fig. 2), a redistribution of ABA by pH cannot be a merely passive process, but must be tightly regulated. The relatively small pH changes measured during stomatal movements, i.e. 0.3 pH units following ‘light‐off’, 0.2 pH units following increase of CO2, 0.4 pH units following 10−5 M ABA are a clear indication of this. Conclusions Substomatal apoplastic pH is obviously rather sensitive to a variety of factors. This is based on two properties: the low passive buffer capacity and the thin aqueous film covering the apoplast forming cells. The consequence must be that already small fluctuations in membrane transport, gas exchange and intercellular exchange of matter will challenge apoplastic pH. 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TI - The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors
JF - Journal of Experimental Botany
DO - 10.1093/jexbot/53.366.73
DA - 2002-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-apoplastic-ph-of-the-substomatal-cavity-of-vicia-faba-leaves-and-lkWs9IQTWq
SP - 73
EP - 82
VL - 53
IS - 366
DP - DeepDyve
ER -