Large Nitrate Permeability

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to CIof NOs> Br> FCII> malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple condudance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of CIand initial malate efflux required for mediation of stomatal closure. l h e large optimize their atmospheric COZ and water vapor exchange by adjusting the aperture of stomatal pores in the leaf epidermis. Stomatal regulation depends on environmental conditions such as humidity, COZ levels, and light intensity. Guard cells surrounding stomata directly control the stomatal aperture. Stomatal opening is mediated by turgor and volume increases of guard cells, which result from ion uptake and intemal conversion of starch to osmotically active malate (Raschke, 1979). Efflux of potassium and anions (mainly chloride and to a small degree malate) from guard cells and malate metabolism produce turgor and volume reduction and the ensuing closure of stomatal pores (Raschke, 1979; MacRobbie, 1981; Outlaw et al., 1981). Anion channels in the plasma membrane of guard cells have been suggested to be central mediators of stomatal closing (Keller et al., 1989; Schroeder and Hagiwara, 1989). Recent studies have shown the presence of at least two different types or modes of anion channel currents in guard cells (Schroeder and Keller, 1992). Both anion channels are likely to be involved in stomatal closing, while showing significantly different activation times, voltage dependencies, and pharmacological properties (Schroeder and Hagiwara, 1989; Hedrich et al., 1990; Marten et al., 1992, 1993; NO,permeability as well as the significant permeabilities of ;I1 anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels. Schroeder and Keller, 1992; Schroeder et al., 1993). Both channels are activated by depolarization but differ in their activation potential by UP to 100 m v and in theb halfactivation times by appro-ately 3 orders of magnitude under identical recording conditions. The rapidly activating anion channels (R type) activate within approximately 50 ms but reclose during prolonged stimulation (hI2 < 1 min) (Hedrich et al., 1990). l%s voltage-