INVOLVEMENT OF PROTEIN-PHOSPHORYLATION IN THE EARLY STEPS OF TRANSDUCTION OF THE OLIGOGALACTURONIDE SIGNAL IN TOBACCO CELLS

Two structurally unrelated protein kinase inhibitors, staurosporine an d 6-dimethylaminopurine, a substituted purine identified as an inhibit or of a variety of animal protein kinases were able to inhibit the oli gogalacturonide-induced stimulation of the phenylpropanoid pathway in Nicotiana tabacum cells. Extracellular alkalinization and oxidative bu rst, which occur in a few minutes after elicitor addition, were highly susceptible to the inhibitors. When cells were incubated in the prese nce of 500 mu M 6-dimethylaminopurine, elicitor-induced oxidative burs t was completely prevented, although a reduced extracellular alkaliniz ation was still induced by oligogalacturonides. The possible significa nce of this result is discussed in terms of potential involvement of d ifferent protein kinases in the pH and oxidative responses. Two protei n phosphatase inhibitors, cantharidin and calyculin A, were able to in duce extracellular alkalinization and oxidative burst in tobacco cells , confirming the involvement of protein phosphorylation in the two mem brane responses. In the presence of 6-dimethylaminopurine, pH and oxid ative responses induced by phosphatase inhibitors, as well as the olig ogalacturonide-dependent responses, were greatly reduced, showing that the purine very likely acts as an inhibitor of plant protein kinases. In vitro phosphorylation studies indicate that most protein kinases a ctive in a purified plasma membrane fraction are highly susceptible to staurosporine. On the contrary, only a few phosphorylated bands appea r affected by 6-dimethylaminopurine in the same conditions, suggesting a more specific action of this inhibitor. The phosphorylation of two polypeptides, 95 kDa and 20 kDa, which was increased by oligogalacturo nides, was susceptible to staurosporine and 6-dimethylaminopurine. The substituted purine appears as a tool to characterize plasma membrane protein kinases highly susceptible to the inhibitor, which appeared in volved in the oxidative burst.