Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

Abstract

Summary. The steady-state, current-voltage (I-V) characteristicsof stomatal guard cells from Vicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50 mM K+-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3-1.0 mM NaCN and 0.4 mM salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V~), as large as + 103 mV, which were generally complete within 60-90 sec (mean response half-time, 10.3 -+ 1.7 sec); values for V~ in NaCN plus SHAM were close or positive to -100 mV and well removed from the K + equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 1().8 + 1.9 sec ([ATP]t~0, 1.32 -+ 0.28 mM; lATP],=60-fs0 .... 0.29 -+ 0.40 mM). In respiring cells, the I-V relations were commonly sigmoid about V,, or gently concave to the voltage axis positive to V~. Inward-and outward-rectifying currents were also observed, especially near the voltage extremes (nominally -350 and +50 mV). Short-circuit currents (at V = 0 mV) were typically about 200-500 mAm -2. The principal effect of cyanide early on was to linearize the I-V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50-75%. The resulting difference-current-voltage (dl-V) curves (+cyanide) showed a marked sensitivity to voltages negative from -100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near -300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATPconsuming ion pump. Fitting dl-V curves to reaction kinetic model for the pump J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m 2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K + transport for stomatal movements. Key Wordsstomatal guard cell -(difference-) current-voltage relation 9 conductance-voltage relation 9 H + pump . kinetic carrier model 9 K + transport -Vicia

Synergistic Effect of Light and Fusicoccin on Stomatal Opening

“…We also demonstrate here (Figs. 5B and 6B) that the FC-stimulated current was essentially voltage independent over the range from -100 to +60 mV, as has also been observed for the pump current of intact guard cells of V. faba, in experiments in which pump current was calculated as the difference in current before and after administration of sodium cyanide plus salicylhydroxamic acid (2,3). This voltage independence contrasts sharply with the voltage dependence of K+ and anion channels over the same voltage range (11,20,21).…”

Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways

“…However, addition of cofactors (1 mM ATP, NADH, or both) visibly enhanced photoreduction. These effects were apparent already at concentrations in the micromolar range for ATP and NADH (Supplemental Figure 2) and, therefore, well within the physiological range of these compounds in most organisms (Blatt, 1987;Albe et al, 1990;Bennett et al, 2009). To more precisely determine the affinity of cry for ATP, we directly obtained a binding constant (K d ) for cry2 of 49 mM (Supplemental Figure 3).…”

Is ATP Required for K+ Channel Activation in Vicia Guard Cells?

“…Some studies suggest that 1 to 2 mM ATP may be close to the actual physiological ATP concentration in the guardcell cytoplasm (Blatt, 1987;Michalke and Schnabl, 1987), which is similar to the cytoplasmic ATP leve1 in other plant cells (Roberts, 1984). Schroeder (1988) showed that guard-cell pump current was not affected by variation of ATP from 2 to 10 mM and was still60% of maximal at 1 mM ATP.…”