Possible role of plasma membrane H+-ATPase in the elicitation of phytoalexin and related isoflavone root secretion in chickpea (Cicer arietinum L.) seedlings

Etiolated 3-day old chickpea seedlings constitute a very simple, organized intact-plant system, whose response to elicitors acting at root level is characterized by the excretion rather than the tissue accumulation of phytoalexins (medicarpin and maackiain) and constitutive isoflavones (formononetin and biochanin A). By using this system, it was concluded that seedling incubation in buffered media with high pH values (higher than 6.5) causes a marked induction of isoflavonoid excretion after 24 h. The response was higher for phosphate buffers than for Mes and Pipes buffers, and, with the phosphate buffers, an optimal response was obtained at pH 7.5 (instead of 6.0, 6.5, 7.0 or 8.0), at a concentration of 80 or 120 mM (instead of 40 mM) and potassium salt (instead of sodium salt). Based on the above facts, we tested the hypothesis that the decrease in proton efflux and/or membrane depolarisation caused by plasma membrane H+-ATPase inhibition elicits the phytoalexin response in chickpea. The ATPase inhibitors orthovanadate and N,N'-dicyclohexylcarbodiimide and the ionophores carbonyl cyanide 3-chlorophenylhydrazone and nigericin were all effective elicitors whereas the ionophore valynomycin, which specifically promotes K+ transport and thus should affect the charge but not the pH component of the transmembrane electrochemical gradient, showed little eliciting activity. These data indicate that the inhibition of the plasma membrane H+-ATPase, and more specifically the proton efflux, may be a key step in the signalling pathway leading to the activation of phytoalexin and isoflavone excretion in chickpea seedlings. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.