THE EFFECT OF ELEVATED CO2 CONCENTRATIONS ON K-FABA L. GUARD-CELLS( AND ANION CHANNELS OF VICIA)

The effects of elevated CO2 concentrations on stomatal movement, anion -and K+-channel activities were examined in guard cells from epidermal strips of Vicia faba. Membrane voltage was measured using intracellul ar, double-barrelled microelectrodes and ion-channel currents were rec orded under voltage clamp during exposure to media equilibrated with a mbient (350 mu l . l(-1)), 1000 mu l . l(-1) and 10 000 mu l . l(-1) C O2 in 20% O-2 and 80% N-2. The addition of 1000 mu l . l(-1) CO2 to th e bathing solution caused stomata to close with a halftime of approx. 40 min, and with 10 000 mu l . l(-1) CO2 closure occurred with a simil ar time course. Under voltage clamp, exposure to 1000 mu l . l(-1) and 10 000 mu l . l(-1) CO2 resulted in a rapid increase (mean, 1.5 +/- 0 .2-fold, n = 8; range 1.3- to 2.5-fold) in the magnitude of current ca rried by outward-rectifying K+ channels (I-K,I-out). The effect of CO2 on I-K,I-out was essentially complete within 30 s and was independent of clamp voltage, but was associated with 25-40% (mean, 30 +/- 4%) de crease in the halftime for current activation. Exposure to CO2 also re sulted in a four-fold increase in background current near the free-run ning membrane voltage, recorded as the instantaneous current at the st art of depolarising and hyperpolarising voltage steps, and a decrease in the magnitude of current carried by inward-rectifying K+ channels ( I-K,I-in). The effect of CO2 on I-K,I-in was generally slower than on I-K,I-out; it was allied with a transient acceleration of its activati on kinetics during the first 60-120 s of treatment; and it was associa ted with a negative shift in the voltage-sensitivity of gating over a period of 3-5 min. Measurements carried out to isolate the background currents attributable to anion channels (I-Cl), using tetraethylammoni um chloride and CsCl, showed that CO2 also stimulated I-CL and dramati cally altered its relaxation kinetics. Within the timeframe of CO2 act ion at the membrane, no significant effect was observed on cytosolic p H, measured using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-c arboxyflourescein (BCECF) and ratio fluorescence microphotometry. Thes e results are broadly consistent with the pattern of guard-cell respon se to abscisic acid, and indicate that guard cells control both anion and K+ channels to achieve net solute loss in CO2. By contrast with th e effects of abscisic acid, however, the data indicate that CO2 action is not mediated through changes in cytosolic pH and thereby implicate new and, as yet, unidentified pathway(s) for channel regulation in th e guard cells.