Arabidopsis guard cell CO₂/HCO₃ ^- response mutant screening by an aequorin-based calcium imaging system

The increase in atmospheric CO₂ is causing a number of changes in plant growth such as increases in leaf area and number, branching, plant size and biomass, and growth rate. Despite the importance of stomatal responses to CO₂, little is known about the genetic and molecular mechanisms that mediate stomatal development and movement in response to CO₂ levels. Deciphering the mechanisms that sense changes in CO₂ and/or HCO₃ ^- concentration is critical for unraveling the role of CO₂ in stomatal development movement. In Arabidopsis, CO₂-induced stomatal closure is strongly Ca²⁺-dependent. To further dissect this signaling pathway and identify new components in the CO₂ response pathway, we recorded [Ca²⁺]_cyt changes in mutagenized Arabidopsis leaves and screened for mutants with abnormal guard cell behavior in response to CO₂/HCO₃ ^-.

We observed that 1 mM HCO₃ ^- induces [Ca²⁺]_cys transient changes in guard cells and stomatal closure both in light and darkness. The changes in [Ca²⁺]_cys induced by HCO₃ ^- could be detected by an aequorin-based calcium imaging system. Using this system, we identified a number of Arabidopsis mutants defective in both [Ca²⁺]_cyt changes and the stomatal response to CO₂/HCO₃ ^-.

We provide a sensitive method for isolating stomatal CO₂/HCO₃ ^- response genes that function early in stomatal closure and that have a role in regulating [Ca²⁺]_cyt. This method will be helpful in elucidating the Ca²⁺-dependent regulation of guard cell behavior in response to CO₂/HCO₃ ^-.