CO2-Triggered Chloride Release from Guard Cells in Intact Fava Bean Leaves. Kinetics of the Onset of Stomatal Closure

The influence of CO(2) on Cl(-) release from guard cells was investigated within the intact leaf by monitoring the Cl(-) activity in the apoplastic fluid of guard cells with a Cl(-)-sensitive microelectrode. In illuminated leaves adapted to a CO(2) concentration within the cuvette of 350 microL L(-1), an increase of 250 microL L(-1) CO(2) triggered a transient rise in the apoplastic Cl(-) activity from 3 to 14 mM within 10 min. This Cl(-) response was similar to the Cl(-) efflux evoked by turning off the light, when the substomatal CO(2) was kept constant (CO(2) clamp). Without CO(2) clamp, substomatal CO(2) increased by 120 microL L(-1) upon "light off." The response to an increase in CO(2) within the cuvette from 250 to 500 microL L(-1) in dark-adapted leaves was equivalent to the response to an increase from 350 to 600 microL L(-1) in the light. No Cl(-) efflux was triggered by 2-min CO(2) pulses (150-800 microL L(-1)). After a switch from 350 microL L(-1) to CO(2)-free cuvette air, the guard cells were less sensitive to a rise in CO(2) and to light off, but the sensitivity to both stimuli partially recovered. Changes in CO(2) also caused changes of the guard cell apoplastic voltage, which were generally faster than the observed Cl(-) responses, and which also promptly occurred when CO(2) did not initiate Cl(-) efflux. The comparatively slow activation of Cl(-) efflux by CO(2) indicates that an intermediate effector derived from CO(2) has to accumulate to fully activate plasma membrane anion channels of guard cells.