Arabidopsis guard cell CO(2)/HCO(3)(−) response mutant screening by an aequorin-based calcium imaging system

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)(−).