A guard cell carbonic anhydrase binds and regulates SLAC1 separate from its catalytic activity

Stomata of plant leaves open to enable CO₂ entry for photosynthesis and close when CO₂ in the leaf is elevated. CO₂ is thought to promote stomatal closure in part by activating the SLAC1 anion channel at the guard cell plasma membrane. Carbonic anhydrases (CAs) contribute to this activation, but their contribution as distinct from CO₂-H₂CO₃ catalysis remains controversial. Here we show that the β-carbonic anhydrase CA4 binds selectively with the guard-cell anion channel SLAC1 to enhance channel current. The interaction is CO₂-dependent, but binding is mediated by amino acids distal from the CO₂-binding site of CA4 and is separable from carbonic-anhydrase activity. CA4 mutants impaired in channel binding eliminate the CO₂-sensitivity of SLAC1 in vivo and slow stomatal kinetics with a commensurate loss in water use efficiency. The findings demonstrate that CA4 contributes directly to the CO₂-response mechanics regulating SLAC1 at near-ambient CO₂ in guard cells and to stomatal kinetics in the plant.