Stomatal Conductance Measurements

The eight-chamber whole-plant rapid-response gas-exchange measurement device has been described previously (Kollist et al., 2007). This device enables the measurement of eight plants within a 16-min interval. Currently, we used only one plant at a time to get gs data every 2 min. One Arabidopsis plant was inserted into the device, and the high-VPD treatment started when gs had stabilized (i.e. at least 1 h later). Standard conditions during the stabilization were as follows: ambient CO₂, ∼400 µL L⁻¹; light, 150 µmol m⁻² s⁻¹; relative air humidity, ∼70%. Photographs of plants were taken after the experiment, and leaf area was calculated using ImageJ 1.37v (National Institutes of Health). gs was calculated with a custom-written program as described by Kollist et al. (2007).

Pea and tomato plants were measured with a custom-made flow-through four-chamber device, which is suitable for larger and taller plants. The main body of the system consists of four thermostated gas-exchange cuvettes formed by two glass cylinders (i.d., 10.6 cm; height, 15.6 cm) and a thermostated water jacket between them. The cuvettes are placed on a stand composed of two well-fitted glass plates that form a bottom of the gas-exchange cuvette. One of these plates contains perforations for the plant stem and is removable. The other glass plate contains gas input and output ports, a temperature sensor, and a fan to guarantee high turbulence and uniform gas mixing. The fan ensured a high boundary layer conductance of leaves (4,800 mmol m⁻² s⁻¹) and a high heat-exchange coefficient between the leaves and chamber air (36 cal m⁻² s⁻¹ °C⁻¹), minimizing the effect of transpiration on leaf temperature. Modeling gum was used to ensure an air-tight separation of plant shoots within the cuvette from roots in the soil. Chambers are hermetically sealed and operate under slight overpressure of a few millibars to avoid uncontrolled intake of ambient air. Air flow rate through the chamber was 2.5 L min⁻¹. Ambient air passing through a large buffer volume of 25 L was used. The air temperature inside the chambers was measured continuously with thermistors (model -001; RTI Electronics) and was between 24°C and 25°C. Leaf temperature was calculated from the energy balance of leaves based on absorbed light and transpiration (Kollist et al., 2007). All tubing and connections were made of Teflon and stainless steel. For illumination, four 50-W halogen lamps (Kanlux MR16C; Philips) provided PPFD of 500 μmol m⁻² s⁻¹ into each cuvette. Concentrations of CO₂ and water vapor in the reference channel (i.e. air entering the measuring cuvette) and measurement channel (air coming out from the cuvette) were measured with an infrared gas analyzer (Li-7000; Li-Cor), and gs was calculated with a custom-written program as described by Kollist et al. (2007). Standard conditions during the stabilization period in the gas-exchange cuvettes were as follows: ambient CO₂ concentration, ∼400 µL L⁻¹; PPFD, 500 µmol m⁻² s⁻¹; and relative air humidity, ∼70%.

A plant was inserted into one of the four measurement cuvettes and kept under standard conditions for about 1 to 2 h to allow the stabilization of gs. After stabilization, VPD was increased sharply from 1.2 ± 0.1 to 2.2 ± 0.1 (i.e. air humidity was decreased from ∼70% to ∼35%) in all experiments. After 1 h in low-humidity conditions, VPD was decreased to the previous level; thereafter, measurements continued for 40 min. In order to compare the VPD responses of different lines, we calculated the following. (1) The initial rates of stomatal closure/opening, found by fitting a second-order polynomial on the time series of gs values after changes in humidity. The slope of that polynomial at the 4-min time point represents the initial closure/opening rate. (2) The closure/opening half-times obtained by scaling the whole 60-min (closure due to increased VPD) or 40-min (opening due to reduced VPD) stomatal responses to a range from 0% to 100% and by calculating the time when 50% of the stomatal response was achieved. And (3) changes in gs after stimulus application, calculated as the difference gs₄₀ − gs₀, where gs₄₀ is the value of gs after 40 min under high/low VPD and gs₀ is the average of the two last gs values under low VPD/high VPD. These characteristics were not calculated for ost1-3. To induce stomatal opening of ost1-3 plants in Figure 8, we first kept them in low CO₂ (∼50 µL L⁻¹) or high light (white + blue lights, PPFD of ∼500 µmol m⁻² s⁻¹) and applied VPD change after 1.5 to 2 h, when stomata had stabilized. VPD was increased as described above for 30 min and then turned back for another 30 min.

In ABA-spraying experiments, plants were inserted into measurement chambers and, after gs had stabilized, intact plants were sprayed with 5 μm ABA solution (distilled water, 0.012% Silwet L-77 [Duchefa], and 0.05% ethanol). We also performed control experiments, where plants of different genotypes were sprayed with mock solution containing no ABA but 0.012% Silwet L-77 and 0.05% ethanol in distilled water. Supplemental Figure S3 presents the results for Col-0. The volume of solution (ABA or mock) sprayed on one plant was ∼20 µL cm⁻² leaf area. After spraying, gs was measured for 40 min as described previously (Merilo et al., 2015a).