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Background  The increase in atmospheric CO 2  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 CO 2 , little is known about the genetic and molecular mechanisms that mediate stomatal development and movement in response to CO 2  levels. Deciphering the mechanisms that sense changes in CO 2  and/or HCO 3  −  concentration is critical for unraveling the role of CO 2  in stomatal development movement. In  Arabidopsis , CO 2 -induced stomatal closure is strongly Ca 2+ -dependent. To further dissect this signaling pathway and identify new components in the CO 2  response pathway, we recorded [Ca 2+ ] cyt  changes in mutagenized  Arabidopsis  leaves and screened for mutants with abnormal guard cell behavior in response to CO 2 /HCO 3  − .    Results  We observed that 1 mM HCO 3  −  induces [Ca 2+ ] cys  transient changes in guard cells and stomatal closure both in light and darkness. The changes in [Ca 2+ ] cys  induced by HCO 3  −  could be detected by an aequorin-based calcium imaging system. Using this system, we identified a number of  Arabidopsis  mutants defective in both [Ca 2+ ] cyt  changes and the stomatal response to CO 2 /HCO 3  − .    Conclusions  We provide a sensitive method for isolating stomatal CO 2 /HCO 3  −  response genes that function early in stomatal closure and that have a role in regulating [Ca 2+ ] cyt . This method will be helpful in elucidating the Ca 2+ -dependent regulation of guard cell behavior in response to CO 2 /HCO 3  − .

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