Fig 1.
Stomatal conductance in Arabidopsis ecotypes that have demonstrated a low CO2 responsiveness.
The plants were exposed to 0 ppm CO2 for 2 h and then transferred to 1,000 ppm CO2 for 1.5 h at 40% RH. Relative conductance levels (Relative gs) were calculated as (gs at 1,000 ppm CO2)/(gs at 0 ppm CO2); large values represent small responses. Data presented are means ± SE (n = 3). The commonly used model ecotype Col-0 is highlighted in red and three particularly unresponsive ecotypes that were selected for further experiments are shown in yellow.
Fig 2.
The phenotype of Kl-4, Ga-0, and Chi-1, which exhibit low CO2 responsiveness.
(A) Thermal imaging of the three selected ecotypes Kl-4, Ga-0, Chi-1, and the commonly used ecotype Col-0. Plants were subjected to 0 ppm CO2 for 2 h and then 1,000 ppm CO2 for 1 h at 40% RH. The subtractive image on the right shows that the largest temperature changes were exhibited by Col-0. (B) Time courses of stomatal conductance (gs) in response to changes in CO2 concentration in Kl-4, Ga-0, Chi-1, and Col-0. Col-0 is more responsive to changes in CO2 concentration than Kl-4, Ga-0, Chi-1. (C) Sizes of stomatal apertures at low and high CO2 concentrations. Plants were subjected to 0 ppm CO2 for 2 h and then transferred to 700 ppm CO2 for 1 h at 40% RH with 150 μmol m-2 s-1 photosynthetically active radiation. (D) The relative changes in stomatal aperture (relative stomatal aperture) were calculated as (stomatal aperture in 0 ppm CO2)/(stomatal aperture in 700 ppm CO2). Large values represent small responses. Data presented are means ± SE (n = 60) of five independent experiments. Significant differences from Col-0 at p < 0.05 (Student’s t test) are indicated by asterisks.
Fig 3.
Measures of stomatal size, density, and index in Kl-4, Ga-0, Chi-1, and Col-0.
(A) Guard cell lengths, which were used as a measure of stomatal size. (B) Stomatal densities. (C) Stomatal index. Data presented are means ± SE (n = 3). No significant differences were observed between the four ecotypes.
Fig 4.
Stomatal response to light, humidity, and ABA in Kl-4, Ga-0, Chi-1, and Col-0.
(A) The sizes of stomatal apertures under light and dark conditions are shown on the left. The relative changes in stomatal aperture in response to light (relative stomatal aperture) are shown on the right. Plants were subjected to dark conditions for 2 h after illuminated with white light at an intensity of 250 μmol m-2 s-1 for 3 h. The relative changes were calculated as (stomatal aperture in the light)/(stomatal aperture in the dark). (B) The sizes of stomatal apertures in response to high (80%) and low (40%) RH (left) and humidity-dependent changes of stomatal aperture (right). Plants were kept under 80% RH for 3 h and then transferred to 40% RH for 1 h at 350 ppm CO2 and 150 μmol m-2 s-1 photosynthetically active radiation. The relative changes were calculated as (stomatal aperture in 40% RH)/(stomatal aperture in 80% RH). (C) Influence of ABA on stomatal aperture (left) and ABA-dependent changes in stomatal aperture (right). Epidermal peels were floated on a medium for 1 h, then transferred to the same medium with or without 2 μM ABA, and incubated for a further 2 h. Relative changes were calculated as (stomatal aperture with ABA)/(stomatal aperture without ABA). Data presented are means ± SE (n = 60) of five independent experiments. Significant differences from Col-0 at p < 0.05 (Student’s t test) are indicated by asterisks.
Fig 5.
K+, Cl-, Malate2-, and Na+ levels in guard cell protoplasts.
Ion contents were measured in GCPs from each ecotype under light and dark conditions. The GCPs were incubated with or without white light (70 μmol m-2 s-1) for 1 h; this amount of light is sufficient to induce GCP swelling and stomatal opening in intact leaves. Relative ion content changes were calculated as (ion content in the dark)/(ion content in the light). Each part of the figure shows ion content data on the left and relative ion contents on the right. Data are for K+ (A), Cl- (B), Malate2- (C), and Na+ (D). The data presented are means ± SE (n = 5–6). Significant differences from Col-0 at p < 0.05 (Student’s t test) are indicated by asterisks.
Fig 6.
Relative sensitivities to environmental factors among Kl-4, Ga-0, Chi-1, and Col-0.
The diagram summarizes the relative sensitivities of Kl-4, Ga-0, Chi-1, and the reference ecotype Col-0 to CO2, light, humidity, and ABA. Kl-4 and Col-0 show similar levels of sensitivity to changes in RH. Chi-1 and Ga-0 show similar levels of sensitivity to ABA.