Plant Physiol. 2007 February; 143(2): 1068–1077.
Plant water loss is tightly balanced with water uptake to maintain beneficial water status. The most important control on water transport is the change of stomatal aperture, which governs water diffusion from the leaf interior to the atmosphere, as well as the opposite flow of carbon dioxide (CO2) into the photosynthesizing mesophyll. To balance transpiration and photosynthesis, guard cells may sense and integrate many environmental as well as physiological signals related to photosynthesis, the transpirational demand of the atmosphere, and the plant's current hydraulic status (Buckley, 2005; Roelfsema and Hedrich, 2005). Atmospheric humidity is one of the key environmental signals that stomata need to sense to adjust water loss. The sensing mechanisms involved in the stomatal humidity response are nonetheless still not identified.
Plant Physiol. 2007 February; 143(2): 1068–1077.
Plant Culture and Experimental SetupExperiments were performed on attached leaves of potted Sambucus nigra plants of approximately 50 to 80 cm in size. Plants were drawn from cuttings and cultivated in 40-cm pots in a climatic chamber at a PPFD of 220 μmol m−2 s−1 (16-h light/8-h dark) and a temperature of 20°C. Plants were amply supplied with water and nutrients. Stomatal movements were observed on mature leaves in a gas-exchange chamber designed for simultaneous measurement of CO2-water gas exchange and microscopic observation of stomatal movements under controlled light, humidity, temperature, and CO2 conditions (Kaiser and Kappen, 2001). Temperature, leaf-to-air mole fraction of water vapor (ΔW) and [CO2] in the cuvette were set to 20°C to 22°C, 2 mmol mol−1, and approximately 360 μmol mol−1, respectively. Irradiance (500 μmol m−2 s−1, 16-h light/8-h dark) was provided by a fiber-optic illuminator (Kaltlicht-Fiberleuchte FL-400 with Spezial Fiberoptik 400-F; Walz). After mounting the leaf in the gas-exchange cuvette, the plant was allowed to adjust to measuring conditions for at least 24 h. Leaves were fixed with the adaxial side to a Perspex plate with double-sided transparent adhesive tape (Tesa 56661–2; Tesa) to allow micromanipulation. Subsequently, the plate was mounted inside the cuvette, which allows observation of the lower leaf surface with a long-distance microscope lens (50×) led through the bottom of the gas-exchange cuvette (Kaiser and Grams, 2006). The microscope (Axiovert 25CFL; Zeiss) is mounted on a motorized translation stage, which allows repositioning of samples of selected stomata. Digital images of stomata were recorded with a video camera, digitized, and stored for subsequent measurement of aperture with custom image analysis software. The aperture of oil-treated pores can no longer be measured due to refraction of the oil. In these experiments, the area of the guard cell pair between the anticlinal walls was measured. This measure is linearly related to aperture when pores are open (Kaiser and Kappen, 2001) and can be taken as a surrogate measure of stomatal opening. Pore area or guard cell pair area were converted to circularity (c = width × 100/length) to allow comparison between differently sized stomata. The humidity control by a bypass compensation system was used to perform quick changes in air humidity from ΔW = 2 to approximately 18 mmol mol−1 by switching the humidity of the incoming air to lower humidity. Within approximately 90 s, ΔW arrived at its new steady state (Fig. 3). One hour before increasing ΔW, [CO2] was reduced to approximately 60 to 70 μmol mol−1, which is approximately the CO2 compensation point for C3 plants (von Caemmerer and Farquhar, 1982). This avoids intercellular [CO2] gradients due to locally suppressed CO2 diffusion into the mesophyll. Stomatal responses to a reduction in air humidity were observed before and after the treatment with oil or adhesive foil on subsequent days, always beginning at the same time 4 h after illumination was switched on. Stomatal apertures were observed from at least 30 min before to 1.5 h after reduction of air humidity. Images were taken every 3 to 4 min if apertures changed fast, otherwise at longer intervals of up to 10 min.
Plant Physiol. 2007 February; 143(2): 1068–1077.
Oil Treatment of PoresThe cuvette allows micromanipulation on the lower leaf surface by micropipettes inserted through small holes in the cuvette wall. Micropipettes were drawn from 1.5-mm borosilicate glass capillaries with a pipette puller (L/M-3P-A; List). Tips were ground to a diameter of approximately 5 μm. Mineral oil (M8662; Sigma-Aldrich) was applied to pores by approaching the oil-filled pipette to a pore and gently pressurizing it manually by pressing a rubber ball. In each experiment, six to 20 pores with a spacing of at least 2 mm were selected. Either the observed pore or the observed pore plus the adjacent pores was sealed with oil. In an additional experiment, which was only used for the model parameter estimation, only the adjacent pores were sealed (leaving the observed central pore free). In all experiments, a control sample of the same size as the treatment sample was observed to detect and correct for interday variability, which, however, was found to be small.
Plant Physiol. 2007 February; 143(2): 1068–1077.
Shielding of the Leaf with Perforated Adhesive FoilTo shield the entire leaf except circular areas of 0.8 mm, thin polyethylene plastic foil (20 μm) was punched with a 0.8-mm syringe needle, which was ground squarely and sharpened. The correct size of the holes was confirmed microscopically after attachment to the leaf. Very thin double-sided adhesive tape (Pritt permanent) was used to attach the foil to the leaf, allowing only the epidermis inside the circular holes to transpire. In some cases, the foil was not attached firmly to the epidermis at the edge of the punched hole, leaving a gap between leaf and foil. These stomata were excluded from the experiments. Up to six holes with a distance of at least 10 mm were punched into a foil in one experiment. The foil was at first attached provisionally in its final position to select stomata located inside the holes. The foil was then removed to observe a control response to a decrease in air humidity of selected stomata. Thereafter, the foil was attached firmly in the same position as before to measure the humidity response of the sample of stomata on the next day. In some experiments, the foil was carefully removed afterward and another control response was measured on the next day to test for permanent damage due to experimental treatment.
