Signal transduction events mediating chitosan stimulation of anthraquinone synthesis in Rubia tinctorum

Abstract

The signalling mechanism by which chitosan increases anthraquinones in Rubia tinctorum L. is largely unknown. We recently showed that the effects of the elicitor require activation of the PLC/PKC cascade. In view of the intrincate network of pathways mediating extracellular stimuli, in this study we investigated whether mitogen activated protein kinase (MAPK), a pathway known to be a PKC target, also participates in chitosan action. Immunoblot analysis revealed a marked stimulation of the MAPK in elicited cells. In presence of the PKC inhibitor calphostin C, lack of activation of MAPK paralleled by a 90% suppression of the chitosan-dependent increase in anthraquinones were observed. Moreover, the elicitor action was decreased 65% by MAPK kinase inhibitor PD 98059. Also, we tested whether the adenylyl cyclase (AC)/cAMP/PKA messenger system plays a role in elicitation. Forskolin, which stimulates AC, and the PKA activators adenosine 3′,5′-cyclic monophosphorothioate-Sp (Sp-cAMPS) or 8-Br-cAMP, were not able to mimic the induction of anthraquinone synthesis in R. tinctorum cells by the elicitor. In addition, the PKA inhibitor adenosine 3′,5′-cyclic monophosphorothiolate-Rp (Rp-cAMPS) did not block chitosan effects. On the other hand, phosphoinositide 3′-OH-kinase (PI3K) inhibitors LY204002 and wortmannin blocked chitosan stimulation of PKC activity and anthraquinone synthesis. These results involve PI3K-mediated activation of PKC and in turn of MAPK in chitosan elicitation.