Inward K⁺ channel activity underlies stomatal responsiveness to changes in light, VPD, and CO₂ conditions. (A) Rate of light-induced stomatal opening. The consequences of the absence of GCK_in activity on leaf transpiration were then investigated by using a setup allowing to continuously monitor transpirational water loss in an intact plant (10). (Left) Typical recordings of transpiration in a WT or kincless plant during a whole nycthemeral period (8-h/16-h day/night). (Right) Time constants describing the increase in transpiration rate induced by light (derived by fitting the kinetics with monoexponential functions). Data are means ± SE; n = 4. For each of the four plants tested per genotype, the individual time constant integrated values derived from the recording of transpiration rate during at least five successive photoperiods. (B) Responsiveness to a decrease in VPD. An automated growth chamber [Phenopsis robot (28)] was used to impose rapid changes in VPD. (Left) WT or kincless plant transpiration (bottom) during a climatic scenario comprising (top) a dark-light transition (open arrow) 2 h before a sudden decrease in VPD. Data are means ± SE; n = 60. (Right) Kinetics of the increase in transpiration rate due to stomatal aperture readjustment during the first half hour (boxed region in Left) after the change in VPD. (C) Responsiveness to CO₂ availability. (Left) Epidermal peel bioassays. Epidermal strips were peeled at the end of the dark period and incubated in the dark in 30 mM K⁺. Stomatal opening was induced by bubbling CO₂-free air for 3 h. Stomatal aperture was measured just before (control values, two bars on the left) and after (two bars on the right) this 3-h treatment. (Right) In planta stomatal conductance measurement in WT and kincless leaves. Time courses of stomatal conductance in response to changes in CO₂ concentration under dark (Upper) or light (Lower) conditions. Dark and light periods are indicated by black and white boxes under the graphs. The changes in CO₂ concentration in the air flow are indicated (expressed in ppm) in the gray boxes. The decrease in CO₂ concentration in the air flow under dark conditions mimics depletion of internal CO₂ driven by photosynthetic activity (independent of light signal and photosynthesis).