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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The inhibition of stomatal opening in Commelina communis L. by abscisic acid (ABA) appears to be dependent on the availability of calcium ions. Incubation of epidermal strips in 10"° mol m~^ ABA had little effect in the absence of calcium, and a factorial experiment showed a highly significant calcium x .^BA interaction. The effect of calcium appeared to be on the later stages of the opening process, and might have been the result of an inhibition of potassium uptake by the guard cells. There was no detectable effect of ABA when EGTA was used to chelate calcium from the apoplastic space.The data are consistent with the hypothesis that .\BA increases the permeability of the plasma membranes of the guard cells to calcium. Calcium might then operate as a second messenger to regulate the ionic fluxes that determine guard cell turgor.