(a) Catalytic carbonic anhydrase activity assays show a reduction by 65% in carbonic anhydrase activity of the ca1ca4 double mutant (n = 16) compared to wild-type plants (n = 16). Means ± s.e.m., *P<0.05 compared to wild type, unpaired Student’s t-test. Residual carbonic anhydrase activities were not significantly different between ca1 and ca1ca4 mutant plants (P>0.3, pairwise Student’s t-test). (b) RT-PCR analyses confirmed restoration of βCA1 and βCA4 expression in ca1ca4 leaves transformed with genomic βCA1 or βCA4 constructs. Three independent randomly selected transgenic lines per genomic construct were analyzed. Actin (At2g37620) was used as a control. (c) Complementation line with genomic βCA1 construct exhibits recovery of [CO₂]-regulated stomatal conductance changes (n = 8 leaves for ca1ca4, n = 10 for WT and n = 4 for each complemented line). Means ± s.e.m‥ (d) Complementation line with genomic βCA4 construct exhibits recovery of [CO₂]-regulated stomatal conductance changes (n = 8 leaves for ca1ca4, n = 10 for WT and n = 4 for each complemented line). Experiments in c and d were performed in the same experimental set with the same controls. Means ± s.e.m‥ Supplementary Information Fig. S3 shows four other independent transgenic lines analyzed in parallel.

(a) Time-resolved stomatal conductance analyses in ca1ca4 (n = 4), wild-type (n = 4), ht1-2 (n = 7) and ca1ca4ht1-2 triple mutant (n = 7) leaves in response to the indicated [CO₂] changes, show that HT1 is epistatic to βCA1 and βCA4. (b) RT-PCR analyses show human αCAII expression in randomly selected human αCAII transgenic ca1ca4 plant leaves. (c) Stomatal conductance of guard cell-targeted human αCAII-expressing ca1ca4 lines, ca1ca4 and wild-type plants in response to the indicated [CO₂] changes (n = 4, ± s.e.m.). Three human αCAII-expressing lines were randomly chosen for stomatal response experiments and all showed recovery of CO₂ responsiveness. Fig. S10 shows two other independent transgenic lines analyzed in parallel. (d) Elevated bicarbonate activates S-type anion channel currents in Arabidopsis guard cells. Average current-voltage curves were recorded in wild-type guard cells at ambient conditions (open circles) or with intracellular addition of either 13.5 mM bicarbonate, buffered to 2 mM free CO₂ (filled red triangles) or 6.75 mM bicarbonate, buffered to 1 mM free CO₂ (filled circles). Error bars depict means ± s.e.m‥

(a) Chlorophyll-deficient albino wild-type leaves were generated by watering with the carotenoid biosynthesis inhibitor norflurazon. (b) Chlorophyll-deficiency in norflurazon-treated albino leaf guard cells compared to wild type was analyzed using confocal microscopy. (c) The absence of chlorophyll in albino guard cells was quantified by image analyses of chlorophyll fluorescence intensity (n = 3, 12 stomata/sample). *P<0.001, pairwise Student’s t-test. (d) The stomatal CO₂ response to [CO₂] changes was functional in intact albino leaf epidermes (n = 7 experiments, 50 stomata/sample). Means ± s.e.m‥ (e) The maximum efficiency of photosystem II (PSII)- F_v/F_m in dark-adapted leaves was unaffected in ca1ca4ca6 mutant plants (n = 10, ± s.e.m.). (f, g) No difference was observed between wild type (WT) and ca1ca4ca6 mutant plants with respect to the quantum yield of PSII (Φ_PSII) in leaves pre-adapted (f) at 50 µmol m⁻²s⁻¹ (n = 6) or (g) at 2000 µmol m⁻²s⁻¹(n = 6) photosynthetically active radiation. Means ± s.e.m. (h) Red light (300 µmol m⁻²s⁻¹) -induced CO₂ assimilation of intact leaves was not impaired in ca1ca4ca6 plants (n = 6, ± s.e.m.).

(a) RT-PCR analyses of βCA1 expression in guard cell (GC) and mesophyll cell (MC) protoplasts of two ca1ca4 lines expressing βCA1 driven by the pGC1 promoter. GC, guard cell; MC, mesophyll cell. KAT1, At5g46240, leaf guard cell marker; CBP, At4g33050, mesophyll cell marker. (b) βCA1 expression in guard cells restores CO₂ responsiveness in intact leaves. CO₂-induced stomatal conductance changes of guard cell-targeted line CA1gc#1and ca1ca4 and wild-type plants from the same experimental set (n = 4). Fig. S7 shows CO₂ responses of other independent transgenic lines. Note that the starting stomatal conductance in guard cell-targeted lines was lower than that in wild type, probably because pGC1 drives stronger expression in guard cells22 than the native βCA promoters (Fig. 1b). (c) Stomatal conductance of βCA4 over-expressing lines and wild-type (WT) plants in response to the indicated [CO₂] changes (n = 4, ± s.e.m.). Fig. S8 shows other independent transgenic lines analyzed in parallel. Experiments in (b) and (c) were performed in the same experimental set with the same controls. (d) βCA1 and βCA4 over-expressing lines show improved instantaneous water use efficiency (WUE, µmol CO₂ assimilated per mmol H₂O transpired). n = 5, error bars depict means ± s.e.m‥ P<0.01(**) and P<0.05(*), compared to wild type, pairwise Student’s t-test. (e) Rates of photosynthesis (CO₂ assimilation) at ambient (365 ppm) [CO₂] in wild type and the analyzed βCA1 and βCA4 guard cell over-expressing lines. Error bars depict means ± s.e.m‥

(a) Phylogenetic tree (ClustalX 1.83) of Arabidopsis β-carbonic anhydrases (βCAs) and corresponding average guard cell specific microarray expression data in brackets (Left, 8K AG Genechips21; Right, ATH1 Genechips22). βCA1, βCA4 and βCA6 showed the highest expression values among βCAs within guard cells as depicted in bold. βCA1 (At3g01500), βCA2 (At5g14740), βCA3 (At1g23730), βCA4 (At1g70410), βCA5 (At4g33580) and βCA6 (At1g58180)24. (b) Relative transcript levels (compared to EF-1α, At5g60390) of the six βCA genes in guard cells (GC) and mesophyll cells (MC) (qRT-PCR, n = 3 independent biological replicates, ± s.e.m.). qRT-PCR data confirmed high expression of βCA1, βCA4 and βCA6 in guard cells. GC1, At1g2269022. (c–e) Time-resolved stomatal conductance responses to [CO₂] concentrations in wild-type (WT) and ca1ca4 mutant plants (c, d, n = 7; e, n = 5 leaves). (c) shows normalized responses of those shown in d. * means significant difference in the bracketed points between ca1ca4 and wild-type plants (P<0.05, unpaired Students t-test). For initial rates of stomatal conductance changes in d: for 800 ppm to 100 ppm shift, dConductance/dt = 0.028 ± 0. 005 in wild type and 0.008 ± 0.002 in ca1ca4; in e: for 100 ppm to 800 ppm shift, dConductance/dt = −0.034 ± 0. 004 in wild type and −0.005 ± 0.009 in ca1ca4, mmol H₂O m⁻²s⁻¹, means ± s.e.m., P<0.05, unpaired t-test. (f) Analyses of relative stomatal conductance responses to blue light and light-dark transitions in wild-type (WT) and ca1ca4 mutant plants (n = 4, ± s.e.m.). (g) High [CO₂]-induced stomatal closing is impaired in ca1ca4 mutant leaf epidermes (n = 4 experiments, 80 stomata per condition), in which only guard cells and leaf pavement cells were alive and no mesophyll cells were in the vicinity. Leaf epidermes were treated with 800 ppm CO₂ for 30 min. Data represent means ± s.e.m‥ (genotype blind analyses). * P<0.001, pairwise Student’s t-test. See also Supplementary Information Fig. S2d for a 60 min treatment. (h) Stomata in ca1ca4 leaves close in response to abscisic acid (n = 3 experiments, 30 stomata per experiment and condition). Data represent means ± s.e.m‥