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Ca2+ is implicated as a second messenger in the response of stomata to a range of stimuli. However, the mechanism by which stimulus-induced increases in guard cell cytosolic free Ca2+ ([Ca2+]i) are transduced into different physiological responses remains to be explained. Oscillations in [Ca2+]i may provide one way in which this can occur. We used photometric and imaging techniques to examine this hypothesis in guard cells of Commelina communis. External Ca2+ ([Ca2+]e), which causes an increase in [Ca2+]i, was used as a closing stimulus. The total increase in [Ca2+]i was directly related to the concentration of [Ca2+]e, both of which correlated closely with the degree of stomatal closure. Increases were oscillatory in nature, with the pattern of the oscillations dependent on the concentration of [Ca2+]e. At 0.1 mM, [Ca2+]e induced symmetrical oscillations. In contrast, 1.0 mM [Ca2+]e induced asymmetric oscillations. Oscillations were stimulus dependent and modulated by changing [Ca2+]e. Experiments using Ca2+ channel blockers and Mn2+-quenching studies suggested a role for Ca2+ influx during the oscillatory behavior without excluding the possible involvement of Ca2+ release from intracellular stores. These data suggest a mechanism for encoding the information required to distinguish between a number of different Ca2+-mobilizing stimuli in guard cells, using stimulus-specific patterns of oscillations in [Ca2+]i.

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicais, and (b) H202. Both methyl viologen and H 2 0 2 inhibited stomatal opening and promoted stomatal closure. At concentrations 510-5 M, the effects of methyl viologen and H 2 0 2 on stomatal behavior were reversible and were abolished by 2 mM ECTA or 10 PM verapamil. I n addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by ECTA or verapamil, methyl viologen and H 2 0 2 caused an increase in guard cell cytosolic free Ca2+ ([Ca'+l,), which was abolished in the presence of ECTA. Therefore, at low concentrations of methyl viologen and H202, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatai aperture and the associated increases i n [Ca2+],. This suggests that in this concentration range the effects of the treatments are Ca'+-dependent and are mediated by changes in [Ca2+],. I n contrast, at concentrations of methyl viologen and H,O, >10-5 M, ECTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H202 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+Ii and the possible disruption of guard-cell ca'+ homeostasis are discussed in relation t o the processes of Ca'+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

The hypothesis that increases in cytosolic free calcium ([Ca2+]i) are a component of the CO2 signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura‐2 fluorescence ratio photometry to measure changes in guard cell [Ca2+]i in response to challenge with 700 µl l−1 CO2. Elevated CO2 induced increases in guard cell [Ca2+]i which were similar to those previously reported in response to abscisic acid. [Ca2+]i returned to resting values following removal of the CO2 and further application of CO2 resulted in a second increase in [Ca2+]i. This demonstrated that the CO2‐induced increases in [Ca2+]i were stimulus dependent. Removal of extracellular calcium both prevented the CO2‐induced increase in [Ca2+]i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca2+ acts as a second messenger in the CO2 signal transduction pathway and that an increase in [Ca2+]i may be a requirement for the stomatal response to CO2.

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