ORIGINAL ARTICLE Victor P. Bulgakov Æ M. V. Veselova G. K. Tchernoded Æ K. V. Kiselev Æ S. A. Fedoreyev Yu. N. Zhuravlev Inhibitory effect of the Agrobacterium rhizogenes rolC gene on rabdosiin and rosmarinic acid production in Eritrichium sericeum and Lithospermum erythrorhizon transformed cell cultures Received: 12 October 2004 / Accepted: 21 November 2004 / Published online: 2 February 2005 Ó Springer-Verlag 2005 Abstract Rabdosiin and related caffeic acid metabolites have been proposed as active pharmacological agents demonstrating potent anti-HIV and antiallergic activi- ties. We transformed Eritrichium sericeum and Litho- spermum erythrorhizon seedlings by the rolC gene, which has been recently described as an activator of plant secondary metabolism. Surprisingly, the rolC-trans- formed cell cultures of both plants yielded two- to threefold less levels of rabdosiin and rosmarinic acid (RA) than respective control cultures. This result establishes an interesting precedent when the secondary metabolites are differently regulated by a single gene. We show that the rolC gene affects production of rabdosiin and RA irrespective of the methyl jasmonate (MeJA)- mediated and the Ca 2+ -dependent NADPH oxidase pathways. Cantharidin, an inhibitor of serine/threonine phosphatases, partly diminishes the rolC-gene inhibitory effect that indicates involvement of the rolC-gene-med- iated signal in plant regulatory controls, mediated by protein phosphatases. We also show that the control MeJA-stimulated E. sericeum root culture produces (À)- rabdosiin up to 3.41% dry weight, representing the highest level of this substance for plant cell cultures re- ported so far. Keywords Cantharidin Æ Eritrichium sericeum Æ Lithospermum erythrorhizon Æ Methyl jasmonate Æ rolC gene Æ Rabdosiin Æ Rosmarinic acid Abbreviations CAM: Caffeic acid metabolites Æ DPI: Diphenylene iodonium Æ MeJA: Methyl jasmonate Æ RA: Rosmarinic acid Æ ROS: Reactive oxygen species Introduction The rolC gene of Agrobacterium rhizogenes T-DNA plays an essential role in the development of hairy root disease and its expression in plants causes substantial morphological and biochemical alterations (Spena et al. 1987; Nilsson et al. 1993, 1996). The ability of the rolC gene to alter secondary metabolism in hairy root cul- tures, callus cultures and transgenic plants has recently been revealed (Palazo´n et al. 1998a, b; Bulgakov et al. 1998, 2002, 2003; Bonhomme et al. 2000). The rolC- stimulatory effect on secondary metabolism was dem- onstrated by investigations with different groups of secondary metabolites, such as tropane alkaloids (Bon- homme et al. 2000), pyridine alkaloids (Palazo´n et al. 1998a), indole alkaloids (Palazo´n et al. 1998b), ginse- nosides (Bulgakov et al. 1998) and anthraquinones (Bulgakov et al. 2002, 2003). Some data, however, indicate decreased levels of tropane alkaloids in Hy- oscyamus muticus plants and Duboisia hybrid plants transformed by wild-type A. rhizogenes strains contain- ing the rolC among other T-DNA genes (Sevon et al. 1997; Roig Celma et al. 2001). Such peculiarities of the rolC gene action stimulated investigations aimed to understand how the gene could affect secondary metabolism. The assessment of signal transduction pathways affected by the gene showed that the rolC-gene-mediated signal did not interfere with general plant defense pathways leading to synthesis of phytoalexin-type secondary metabolites. In particular, the Ca 2+ -dependent NADPH oxidase pathway as well as salicylic-acid-mediated and octadecanoid pathways V. P. Bulgakov (&) Æ G. K. Tchernoded Æ K. V. Kiselev Yu. N. Zhuravlev Institute of Biology and Soil Science, Far East Branch of Russian Academy of Sciences, 159 Stoletija Street, Vladivostok, 690022, Russia E-mail: bulgakov@ibss.dvo.ru Fax: +7-4232-310193 M. V. Veselova Æ S. A. Fedoreyev Pacific Institute of Bioorganic Chemistry, Far East Branch of Russian Academy of Sciences, Vladivostok, 690022, Russia Planta (2005) 221: 471–478 DOI 10.1007/s00425-004-1457-5