Cytosolic Alkalinization Mediated by Abscisic Acid Is Necessary, but Not Sufficient, for Abscisic Acid-Induced Gene Expression in Barley Aleurone Protoplasts

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Abstract
We investigated whether intracellular pH (pH(i)) is a causal mediator in abscisic acid (ABA)-induced gene expression. We measured the change in pH(i) by a "null-point" method during stimulation of barley (Hordeum vulgare cv Himalaya) aleurone protoplasts with ABA and found that ABA induces an increase in pH(i) from 7.11 to 7.30 within 45 min after stimulation. This increase is inhibited by plasma membrane H(+)-ATPase inhibitors, which induce a decrease in pH(i), both in the presence and absence of ABA. This ABA-induced pH(i) increase precedes the expression of RAB-16 mRNA, as measured by northern analysis. ABA-induced pH(i) changes can be bypassed or clamped by addition of either the weak acids 5,5-dimethyl-2,4-oxazolidinedione and propionic acid, which decrease the pH(i), or the weak bases methylamine and ammonia, which increase the pH(i). Artificial pH(i) increases or decreases induced by weak bases or weak acids, respectively, do not induce RAB-16 mRNA expression. Clamping of the pH(i) at a high value with methylamine or ammonia treatment affected the ABA-induced increase of RAB-16 mRNA only slightly. However, inhibition of the ABA-induced pH(i) increase with weak acid or proton pump inhibitor treatments strongly inhibited the ABA-induced RAB-16 mRNA expression. We conclude that, although the ABA-induced the pH(i) increase is correlated with and even precedes the induction of RAB-16 mRNA expression and is an essential component of the transduction pathway leading from the hormone to gene expression, it is not sufficient to cause such expression.