Electrogenic activity of plasma membrane H+-ATPase in germinating male gametophyte of petunia and its stimulation by exogenous auxin: Mediatory role of calcium and reactive oxygen species

Abstract

A lipophilic potential-sensitive cationic dye, safranin O was employed to examine the influence of exogenous IAA on plasma membrane electric potential in germinating pollen grains of petunia (Petunia hybrida L.) with the aim of elucidating whether the electrogenic H+-ATPase activity of the plasma membrane is sensitive to this phytohormone. The addition of IAA to pollen grains suspended in a K+-free medium was found to induce significant hyperpolarization of the plasmalemma. This effect was fully blocked by orthovanadate, Ca2+-active reagents (EGTA and verapamil), and by the inhibitor of NADPH oxidase of plasmalemma, diphenyleneiodonium (DPI). It was also strongly inhibited by the presence of K+ at centimolar concentrations in the medium. The hyperpolarizing influence of IAA was mimicked by application of hydrogen peroxide; furthermore, the H2O2-induced shift of the membrane potential was inhibited by the same agents that suppressed the IAA-induced hyperpolarization of the pollen plasmalemma. It is concluded that the IAAinduced hyperpolarization of the plasma membrane in male gametophytes of petunia is caused by the enhanced electrogenic activity of ATP-dependent proton pump in the presence of this phytohormone. It is supposed that the effect of IAA is mediated by the transient increase in cytosolic Ca2+ level and by generation of reactive oxygen species (ROS). Possible mechanisms underlying the mediatory role of calcium and ROS in the auxin signal transduction and the resulting stimulation of electrogenic activity of the plasma membrane H+-ATPase are discussed.