Increase in Anthraquinone Content in Rubia cordifolia Cells Transformed by rol Genes Does Not Involve Activation of the NADPH Oxidase Signaling Pathway

V. P. Bulgakov^1*, G. K. Tchernoded¹, N. P. Mischenko², Yu. N. Shkryl³, V. P. Glazunov², S. A. Fedoreyev², and Yu. N. Zhuravlev¹

¹Institute of Biology and Soil Sciences, Far East Branch of the Russian Academy of Sciences, pr. 100-letiya Vladivostoku 159, Vladivostok 690022, Russia; fax: (4232) 31-0193; E-mail: bulgakov@ibss.dvo.ru

²Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, pr. 100-letiya Vladivostoku 159, Vladivostok 690022, Russia; fax: (4232) 31-4050; E-mail: piboc@stl.ru

³Far East State University, ul. Sukhanova 8, Vladivostok 690600, Russia; fax: (4232) 42-9510; E-mail: shkryl@bio.dvgu.ru

^* To whom correspondence should be addressed.

Received August 13, 2002

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It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells. The activator mechanism is still unknown. In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes. It was demonstrated that the transformation of Rubia cordifolia cells by the rolB and rolC genes caused an induction of biosynthesis of anthraquinone-type phytoalexins. Inhibition studies revealed a striking difference between the rolC and rolB transformed cultures in their sensitivity to Ca²⁺ channel blockers and calcium deficiency. The rolC culture displayed lowered resistance to the inhibitors compared to the non-transformed culture, while the rolB culture was more resistant to the treatment. The assumption was made that the oncogenic potential of rol genes is realized through the alteration of calcium balance in the plant cells. Anthraquinone production was not inhibited in the non-transformed and transformed cultures by Ca²⁺ channel blockers, as well as by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor staurosporine. These results indicate that the induction of anthraquinone production in transgenic cultures does not involve the activation of Ca²⁺-dependent NADPH oxidase pathway.

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KEY WORDS: rolB and rolC genes, Agrobacterium rhizogenes, Ca²⁺, NADPH oxidase, Rubia cordifolia, anthraquinone

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