The electrical properties of the tonoplast from a large variety of plant materials such as mesophyll cells, storage cells, tumor cells, suspension cultured cells, guard cells, coleoptile cells, and liverwort cells have been investigated using the patch‐clamp technique. Whole‐vacuole recordings were employed to study the dynamics of an ATP‐dependent proton pump by directly measuring the electrogenic currents. The addition of Mg‐ATP induced an inwardly directed current which depolarized the tonoplast (the vacuole becoming positive inside). Furthermore, voltage‐dependent passive ion fluxes were analyzed using whole vacuoles and isolated membrane patches. Whole‐vacuolar currents and single‐channel currents were induced at hyperpolarizing potentials, whereas currents decreased at positive trans‐tonoplast potentials. The electrical properties of the tonoplast of vacuoles from various plant tissues were similar and it was concluded that ion fluxes across the tonoplast follow the same general mechanisms.

Electron probe microanalysis for K and Cl and enzymic determination of malate were performed on epidermal strips of Vicia faba L. which had been incubated with 0. Republic of Germany.We tested the effect of the availability of Cl-on the ionic composition of the guard cells of Viciafaba by incubating epidermal samples on solutions free of Cl-or containing Cl-and then determining the amounts of K+ and CF-absorbed as well as the amounts of malic acid synthesized. We used established methods: electron probe microanalysis for the determination of K+ and Clin the guard cells (6), and enzymic oxidation coupled to the reduction of NAD for the determination of malate in the epidermal samples (4,14).MATERIALS AND METHODS Plants. V.faba L. plants (cv. Long Pod; seeds from Lagomarsino Seeds Inc., Sacramento, Calif.) were grown for 3 to 4 weeks in a growth chamber in soil consisting of 2 parts Bacto potting soil (Michigan Peat, Houston, Texas) and I part Perlite (W. R. Grace and Co.) in pots with a soil depth of about 12 cm. The plants were watered once a week with Hoagland solution (full strength, pH 6.5), and twice a week with deionized H20. The day length was 16 hr light intensity 85 w m-2. The air temperature was 27 C during the light period and 23 C during the dark period. Relative humidity was about 85%, day and night.Preparation and Incubation of Epidermal Samples. The second, third and fourth fully expanded leaves were removed from the plants in the morning, immediately before the beginning of the light period. The leaves were washed in deionized H20. Sections measuring about 5 x 10 mm2 were cut from the intercostal areas of the leaves and floated, upper side down, on deionized H20 in the dark. Then the lower epidermis was peeled in room light (from fluorescent tubes), a process during which most of the epidermal cells are ruptured (3). The epidermal strips were rinsed in deionized H20, rubbed with a dissecting needle (bent at an angle of 1200) to remove adhering mesophyll cells, and collected on deionized H20. From there, the strips were transferred to the incubation media in small plastic beakers. The beakers were placed under an inverted crystallizing dish (150-mm diameter, 75-mm height). Humidified, C02-free air was passed through the inverted dish for 4 hr at 50 1 hr-1 and 21 C. The samples were illuminated by mercury vapor lamps (General Electric H 400 RDX 33-1); the irradiance below the water filter (5 cm deep) was 85 w m-2. The

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The influence of salicylic acid on the growth and stomatal movements ofVicia faba L. was investigated. Whereas shoot length, fresh weight, and transpiration rates, which are directly correlated with stomatal pore widths, were only affected at salicylic acid concentrations higher than 3.5 mM after long-term treatments, guard cells in epidermal peels exhibited a high sensitivity at concentrations as low as 0.001 mM, resulting in stomatal closing. HPLC analysis of methanolic extracts from roots and leaves revealed the presence of free salicylic acid and a metabolite, whose amount increased with time in plants previously incubated with a medium containing salicylic acid. The possible ability ofVicia faba to detoxify the phenolic acid may be one explanation of the discrepancy between the stomatal reaction in epidermal peels directly treated with the phenolic acid and after application through the transpiration stream. The results indicate that, under natural conditions, salicylic acid will not act as an allelopathic compound whose toxic properties severely affect the growth ofVicia faba.

Guard cell and mesophyll cell protoplasts of Vicia faba L. were purified and separated into cytoplasmic and plastid fractions by a selective silicone-oil filtration. Before fractionation, the protoplasts were ruptured by a low speed centrifugation through a narrow-aperture nylon net placed in a plastic vial. This protoplast homogenation and subsequently the silicone-oil fractionation offer the possibility of investigating the comparatmentation of the enzymatic carboxylating (ribulose bisphosphate carboxylase EC 4.1.1.39, phosphoenolpyruvate carboxylase EC 4.1.1.31, NAD(+) and NADP(+) linked malate dehydrogenase EC 1.1.1.37) and decarboxylating pathways of malic (malic enzyme EC 1.1.1.40, phosphoenolpyruvate carboxykinase EC 4.1.1.32, pyruvate orthophosphate dikinase EC 2.7.9.1) which occur during the swelling and shrinking of the guard cell protoplasts. A model is proposed which describes the transport processes of malic acid during the starch-malate balance as correlated to the volume changes of the protoplasts. As the enzymes and their compartmentation in the guard cell protoplasts seem to be consistent with those of crassulacean acid metabolism (CAM) plants, the metabolism of stomata and of CAM cells is compared.