Figure 1: The rhc1 mutant plants are defective in stomatal responses to CO₂.

(a–e) Expression of RHC1::GUS in leaves (a), flowers (b), vascular systems (c), stem (d) and guard cells (e). Scale bars, 500 μm (a,b), 100 μm (c,d), 5 μm (e). (f) Subcellular localization of RHC1 in Arabidopsis mesophyll protoplasts. 35S::RHC1–GFP, 35S::SLAC1–GFP and 35S::GFP were transiently expressed in Arabidopsis mesophyll protoplasts, respectively. SLAC1–GFP fusion was used as a control for plasma membrane localization and GFP alone was used as a control for cytosol and nuclear localization. Scale bars, 5 μm. (g) Time courses of stomatal conductance in response to changes in CO₂ concentration in wild-type (WT), rhc1 mutants and a complementation line (comp). Stomatal conductance was measured using a gas-exchange system. The leaves were stabilized in 400 p.p.m. CO₂ before changing to 800 p.p.m. CO₂. Values of stomatal conductance at 400 p.p.m. CO₂ were normalized to 1. (n=5 leaves for rhc1, n=4 for WT and n=3 for the complemented line.) Data are presented as means±s.e.m. (h) High [CO₂]-induced stomatal closing is impaired in rhc1 mutant leaf epidermis. Leaf epidermis were treated with 800 p.p.m. CO₂ for 30 min (n=3 experiments, 60 stomata per condition, genotype blind analyses; *P<0.001, pairwise Student’s t-test). (i) Stomata in rhc1 leaves close in response to abscisic acid (ABA). Leaf epidermis were treated with 0, 1, 10 and 50 μM ABA for 30 min (n=3 experiments, 60 stomata per condition). (j) Stomata in rhc1 leaves close in response to CaCl₂. Leaf epidermis were treated with 0, 2 and 10 mM CaCl₂ for 120 min (n=3 experiments, 60 stomata per condition). Data in h–j are presented as means±s.e.m.