Cyclic AMP-dependent protein phosphorylation in guard cell protoplasts of Vicia faba L

FRIEDRICH, P.; CURVETTO, N.; GIUSTO, N.

BIOCELL

INST HISTOL EMBRIOL-CONICET

Año: 1999 vol. 23 p. 203 - 210

0327-9545

Cyclic AMP dependent phosphorylation of target guard cell proteins could participate in transmission of both environmental and hormonal signals, finally affecting stomatal movement. Changes in protein phosphorylation status induced by cyclic AMP, its analog adenosine-3´, 5´-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), adenylyl cyclase activator forskolin and cAMP-dependent protein kinases (PKA) inhibitors were studied using a cell-free system obtained from disrupted guard cell protoplasts of Vicia faba L., by incubation with [gamma-P-32]-ATP in darkness. Proteins were separated by SDS-PAGE and phosphorylated bands were visualized by autoradiography. Autoradiograms show that micromolar concentrations of exogenous cAMP or Sp-cAMPS noticeably increased phosphorylation level of polypeptides with apparent molecular masses of 42, 50 and 57 kDa, while 100 mu M forskolin induced phosphorylation reactions on these bands and in others with apparent molecular masses of 51, 55, 62 and 64 kDa. The presence of PKA-inhibitors, such as adenosine-3´, 5´-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS), or type II PKA-inhibitor (Walsh-inhibitor) in incubation mixtures, partially or totally inhibited forskolin-induced enhancement of protein phosphorylation level, suggesting the occurrence of a cAMP-dependent protein kinase in guard cells of Vicia faba.