Mechanism of auxin action: second messengers

Abstract

The study was conducted on maize coleoptile segments from 4-d-old etiolated seedlings. Auxin action was observed by changes in the potential difference between the IAA-treated apical end and the basal end of 20-mm coleoptile segments. It was shown that the bioelectric potential (BEP) changes measured with extracellular electrodes completely coincided with membrane potential (MP) changes in epidermal cells (intracellular measurements). Treatment with IAA or its methyl ester (0.1–10 mg 1^-1) resulted in the BEP becoming negative (depolarization of MP) and was replaced in 5–10 min by an electropositive wave of BEP (hyperpolarization of MP) with an amplitude of 15–20 mV and a duration of 40–50 min. Since IAA action on coleoptile cells in the first 2–5 min was accompanied by a decrease in Ca²⁺ in the incubation medium and the Ca²⁺-channel blockers verapamil (0.1 mM) and nifedipine (0.01 and 0.1 mM) decreased the primary negative amplitude, we concluded that the entrance of Ca²⁺ ions into the cell was one of the primary responses to auxin. It was supposed that the temporary electronegative BEP (MP depolarization) might be caused by a Ca²⁺-induced decrease in plasma membrane H⁺-ATPase activity. IAA could directly open Ca²⁺-channels in the plasma membrane and/or act through the phosphoinositide cycle. In in vitro experiments with microsomal fractions, IAA was shown to decrease the [³³P] radioactivity level in phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, but to increase incorporation in a water-methanol fraction containing inositol polyphosphates. In experiments with microsomes, IAA also increased Ca²⁺-dependent protein kinase activity. The auxin-dependent electropositive response of the BEP (hyperpolarization of MP) was related to plasma membrane H⁺-pump activation and was eliminated by the protonophore 2,4-DNP. It was shown that the auxin-dependent H⁺-pump was of ATPase nature because the IAA effect was abolished by the inhibitors of ATPases — vanadate and DES. We propose that the activity increased due to a rise in H⁺ ions concentration in the cytosol (specifically, in exchange of Ca²⁺ ions through the tonoplast) and IAA activation of protein synthesis. Changes in protein kinase activity, phosphorylation and dephosphorylation of cytoplasmic proteins were detected after a 10-min treatment of the coleoptile segments with IAA. Some protein fractions added to the incubation medium increased the effect of IAA on growth and BEP.