SummaryGuard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K þ -transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO 2 . Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO 2 -free air, depolarized after a switch to air with 700 ll l À1 CO 2 , in a reversible manner. As a result, K þ -fluxes across the plasma membrane changed direction, to favour K þ extrusion and stomatal closure in the presence of CO 2 . Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO 2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO 2 concentration by about 250 ll l À1 , as measured with a miniature CO 2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO 2 concentration.
SummaryStomatal movement is accomplished by changes in the ionic content within guard cells as well as in the cell wall of the surrounding stomatal pore. In this study, the sub-stomatal apoplastic activities of K + , Cl ± , Ca 2+ and H + were continuously monitored by inserting ion-selective micro-electrodes through the open stomata of intact Vicia faba leaves. In light-adapted leaves, the mean activities were 2.59 mM (K + ), 1.26 mM (Cl ± ), 64 mM (Ca 2+ ) and 89 mM (H + ). Stomatal closure was investigated through exposure to abscisic acid (ABA), sudden darkness or both. Feeding the leaves with ABA through the cut petiole initially resulted in peaks after 9±10 min, in which Ca 2+ and H + activities transiently decreased, and Cl ± and K + activities transiently increased. Thereafter, Ca 2+ , H + and Cl ± activities completely recovered, while K + activity approached an elevated level of around 10 mM within 20 min. Similar responses were observed following sudden darkness, with the difference that Cl ± and Ca 2+ activities recovered more slowly. Addition of ABA to dark-adapted leaves evoked responses of Cl ± and Ca 2+ similar to those observed in the light. K + activity, starting from its elevated level, responded to ABA with a transient increase peaking around 16 mM, but then returned to its dark level. During stomatal closure, membrane potential changes in mesophyll cells showed no correlation with the K + kinetics in the sub-stomatal cavity. We thus conclude that the increase in K + activity mainly resulted from K + release by the guard cells, indicating apoplastic compartmentation. Based on the close correlation between Cl ± and Ca 2+ changes, we suggest that anion channels are activated by a rise in cytosolic free Ca 2+ , a process which activates depolarization-activated K + release channels.
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 (NH(3), CO(2)), 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. NH(3), 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, CaCl(2) or sorbitol alkalized the substomatal apoplast. It is suggested that pH may only act as drought signal when accompanied by elevated osmolarity.
To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H(+)- and Ca(2+)-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H(+)- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response.
The apoplastic pH of intact green leaves of Bromus erectus was measured non-invasively by inserting blunt microelectrodes through stomatal openings. After making electrical contact, the recorded signal was stable for hours, yielding a pH of 4.67p0.10. The leaves responded to ' light-off ' with an initial transient acidification and subsequent sustained alkalinization of 0.2-0.3 pH ; ' light-on ' caused the opposite response. Flushing the leaves with 280 nmol NH $ mol −" air within 18p6 s alkalinized the apoplast by 0.22p0.07 pH, followed by a slower pH increase to reach a steady-state alkalinization of 0.53p0.14 after 19p7 min. This pH shift was persistent as long as the NH $ was flushed, and readily returned to its initial value after replacing the NH $ with clean air. The resultant [NH % + ] increase within the apoplast was measured with a NH % + -selective microelectrode. In the presence of 280 nmol NH $ mol −" air, apoplastic NH % + initially increased within 15p10 s to 1.53p0.41 mM, to reach a steady state of 1.62p0.16 mM after 27p7 min. An apoplastic buffer capacity of 6 mM pH −" unit was calculated from the initial changes of pH and [NH % + ], whereas the steady-state values yielded 2.7 mM pH −" . Infiltrated leaves responded to NH % + with concentration-dependent depolarizations, the maxima of which yielded saturation kinetics indicating carrier-mediated NH % + uptake into adjacent cells, as well as a linear component indicating nonspecific transport. We infer that the initial alkalinization is due to rapid conversion of NH $ to NH % + , whereas the slower pH increase would be caused by regulatory processes involving both membrane transport, and (mainly) NH % + assimilation. Possible consequences of the NH $ -induced pH shift for the development of plants growing in polluted areas are discussed.
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