Fig. 4.

Chloroplasts in guard cells have a central role in the regulation of CO₂- induced stomatal closure via S-type anion channel activation. (A–C) Stomatal aperture in gles1 mutant and WT. The achlorophyllous stomata (SI Appendix, Fig. S2A: type I) in gles1 mutant fail to respond to high [CO₂] (A) and light (B), but show a normal response to ABA (C). Values shown are means ± SE (n = 4 independent experiments with >50 stomata per experiment). Asterisks indicate significant differences (P < 0.05, Student’s t test). (D and E) CO₂ and light responses are impaired in the gles1 mutant. Time course of stomatal conductance in gles1 mutant, gles1/pGLES1:GLES1-GFP, gles1/pGC1:GLES1-GFP, and WT in response to changes in CO₂ concentrations (D) or in light intensity (E). Stomatal conductance was normalized to the average conductance at the last 360 ppm CO₂ data point (D) and the last 0 µmol m⁻²⋅s⁻¹ PAR data point (E). Values shown are means ± SE (n = 5). (F and G) CO₂ activation of S-type anion channels is impaired in gles1 GCPs. Representative current traces (Left) and steady state current–voltage relationships (Right) are shown. CO₂ activates S-type anion channel currents in WT GCPs (F), but not in gles1 GCPs (G). Values shown are means ± SE. Different lowercase letters indicate significant differences at −145 mV (P < 0.05, Tukey-Kramer test). (H) ABA activation of S-type anion channels remains intact in gles1 guard cell protoplasts. Steady state current-voltage relationships of the whole-cell currents recorded in the WT (squares) and gles1 mutant (circles) with (black) or without (white) ABA are shown. Error bars indicate ± SE. Asterisks indicate significant differences (without ABA vs with ABA at −145 mV; P < 0.02, Student’s t test).