Characterization of the plasma-membrane H+-ATPase from Vicia faba guard cells

Abstract

Stomatal movement is controlled by external and internal signals such as light, phytohormones or cytoplasmic Ca(2+). Using Vicia faba L., we have studied the dose-dependent effect of auxins on the modulation of stomatal opening, mediated through the activity of the plasma-membrane H(+)-ATPase. The patch-clamp technique was used to elucidate the electrical properties of the H(+)-ATPase as effected by growth regulators and seasonal changes. The solute composition of cytoplasmic and extracellular media was selected to record pump currents directly with high resolution. Proton currents through the ATPase were characterized by a voltage-dependent increase in amplitude, positive to the resting potential, reaching a plateau at more depolarized values. Upon changes in extracellular pH, the resting potential of the cell shifted with a non-Nernst potential response (±21 mV), indicating the contribution of a depolarizing ionic conductance other than protons to the permeability of the plasma membrane. The use of selective inhibitors enabled us to identify the currents superimposing the H(+)-pump as carried by Ca(2+). Auxinstimulation of this electroenzyme resulted in a rise in the outwardly directed H(+) current and membrane hyperpolarization, indicating that modulation of the ATPase by the hormone may precede salt accumulation as well as volume and turgor increase. Annual cycles in pump activity (1.5-3.8 μA · cm(-2)) were expressed by a minimum in pump current during January and February. Resting potentials of up to -260 mV and plasmamembrane surface area, on the other hand, did not exhibit seasonal changes. The pump activity per unit surface area was approximately 2- to 3-fold higher in guard cells than in mesophyll cells and thus correlates with their physiological demands.

Stomatal Size, Speed, and Responsiveness Impact on Photosynthesis and Water Use Efficiency

“…By their nature, pH measurements report the net H + flux rather than the true H + -ATPase activity and, therefore, are indirect, subject to the availability of other ions to balance charge during transport Clint and Blatt, 1989;Gradmann et al, 1993). This caveat aside, the rates of H + -ATPase activity recorded directly under voltage clamp (Blatt, 1987(Blatt, , 1988aLohse and Hedrich, 1992), and the corresponding fluxes of K + and Cl 2 (MacRobbie, 1981(MacRobbie, , 1983(MacRobbie, , 1984Clint and Blatt, 1989), have generally proven consistent with the changes in solute content Lettau, 1979, 1980;Blatt, 2000; for a quantitative summary and comparison, see Hills et al, 2012).…”

Roles of ion channels and transporters in guard cell signal transduction

“…Similar to blue light, auxins also cause swelling of guard cells, leading to opening of the stomatal pores, and auxins have been shown to increase ATP-dependent pump currents across the guard cell PM [14,15]. Conversely, abscisic acid (ABA) strongly inhibits light-stimulated stomatal opening.…”

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

“…Under conditions that favor stomatal opening, such as illumination, H'-ATPases in the plasma membrane of guard cells are activated (Assmann et al, 1985;Shimazaki et al, 1986;Serrano et al, 1988;Schwartz et al, 1991;Lohse and Hedrich, 1992), which utilize ATP to pump H+ out of the cytoplasm and build up an H+ gradient across the guard-cell plasma membrane. The resultant electrical gradient drives Kf entry, which is accompanied by C1-influx and malate*-synthesis from starch breakdown.…”

“…ATP was supplied to the cytoplasmic side of the guard cells in most of these investigations, even though it is not known whether addition of ATP was necessary. The ATP concentration supplied varied in different reports, ranging from 1 mM (Schroeder and Hagiwara, 1990) to 2 mM (Schroeder et al, 1987;Schroeder, 1988Schroeder, ,1989Keller et al, 1989;Hedrich et al, 1990;Cosgrove and Hedrich, 1991;Fairley-Grenot and Assmann, 1991, 1992Luan et al, 1993), 4 mM (Schroeder and Hagiwara, 1989;Schroeder and Keller, 1992), and even 9 to 10 mM (Assmann et al, 1985;Marten et al, 1991Marten et al, , 1992Lohse and Hedrich, 1992). On the other hand, ATP was not supplied, or at least not mentioned, in some other experiments (Schroeder et al, 1984;Raschke and Hedrich, 1989;Blatt, 1992).…”