Abscisic acid signal transduction in epidermal cells of Pisum sativum L. Argenteum : both dehydrin mRNA accumulation and stomatal responses require protein phosphorylation and dephosphorylation

Abstract

The effects of a number of treatments, both in planta and in vitro, on the accumulation of mRNA encoding dehydrin, an abscisic acid (ABA)-inducible protein, were determined for guard cells and mesophyll cells prepared from leaves of the Argenteum mutant of Pisum sativum L. Guard cells and mesophyll cells treated for 10 d with ABA in planta accumulated dehydrin mRNA. However, after 1 or 3 d treatment, dehydrin mRNA was induced only in guard cells. Wilting induced dehydrin mRNA accumulation in leaves and epidermal cells. Induction of mRNA in epidermal cells was correlated with an increased ABA content after either ABA application or following wilting. Isolated mesophyll and guard cells responded to ABA in vitro by the induction of dehydrin mRNA. However, osmotic stress, imposed by incubation in mannitol, had no effect on ABA and dehydrin mRNA synthesis in mesophyll cells, and only a slight effect on guard cells. Both protein phosphorylation and dephosphorylation were shown to be required for ABA-induced dehydrin gene expression and stomatal movements. Inhibitors of protein kinases (K-252a) and protein phosphatases 1/2A (okadaic acid) and protein phosphatase 2B (cyclosporin A) all inhibited ABA-induced dehydrin mRNA accumulation. They also reduced the inhibitory effects of ABA on stomatal opening, as did the protein kinase activator phorbol myristate acetate (PMA). While K-252a, cyclosporin A and PMA also inhibited ABA-induced stomatal closure, okadaic acid enhanced the ABA effect, indicating the involvement of multiple protein phosphorylation/dephosphorylation steps in ABA signal transduction in guard cells.