Carbonic Anhydrase Mutants in Zea mays Have Altered Stomatal Responses to Environmental Signals

Stomata regulate transpirational water loss and CO₂ uptake for photosynthesis in response to changing environmental conditions. Research investigating stomatal movement has mostly been conducted in C₃ eudicot species, which have very different CO₂ requirements for photosynthesis relative to C₄ grasses. Carbonic anhydrase (CA) catalyzes the hydration of CO₂, and its activity has been linked to stomatal aperture regulation in eudicots. The number of Ca genes and their evolutionary history differ between monocots and dicots, and many questions remain unanswered about potential neofunctionalization and subfunctionalization of grass Ca paralogs and their roles in photosynthesis and stomatal conductance. To investigate the roles of different Ca genes in maize (Zea mays), we examined stomatal responses in ca1 and ca2 single mutants as well as a ca1ca2 double mutant. The ca1 and ca2 single mutants had 10% and 87% of the CA activity exhibited by the wild type, respectively, while ca1ca2 had less than 5% of wild-type CA activity. The ca mutants had higher stomatal conductance than the wild type and slower stomatal closure in response to increases in CO₂ partial pressure. Contrary to previous reports in eudicots, ca mutants showed slowed stomatal closure in response to the light-dark transition and did not show differences in stomatal density compared with the wild type. These results implicate CA-mediated signaling in the control of stomatal movement but not stomatal development. Drought experiments with ca1ca2 mutant plants suggest a role for CA in water-use efficiency and reveal that Z. mays is not optimized for water-use efficiency under well-watered conditions.