Specific genes of cytochrome P450 monooxygenases are implicated in biosynthesis of caffeic acid metabolites in rolC-transgenic culture of Eritrichium sericeum.

Expression of rol agrobacterial oncogenes in plant cell cultures is known to result in activation of secondary metabolite biosynthesis. In the present work, we show that rolC can activate expression of key genes of secondary metabolism using the rolC-transgenic culture of Eritrichium sericeum producing caffeic acid metabolites (rosmarinic acid and rabdosiin) as an example. Increased content of rosmarinic acid in the rolC-transformed callus culture resulted from transcriptional activation of members of the CYP98 family of plant cytochrome P450-containing monooxygenase genes. The rolC gene expression led to increased transcript abundance of the CYP98A3 subfamily members, which are closely related homologs of CYP98A6 of Lithospermum erythrorhizon and are known to be key genes in rosmarinic acid biosynthesis. In contrast, expression of other CYP genes, such as CYP98A1 and CYP98A2, which are not implicated in rosmarinic acid biosynthesis, was not activated in the rolC-transformed calluses. These results are indicative of selective effect of rolC on transcription of particular genes implicated in secondary metabolism.

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses.

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.

Increase in anthraquinone content in Rubia cordifolia cells transformed by rol genes does not involve activation of the NADPH oxidase signaling pathway.

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.

Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes.

It has been suggested that the rol genes of Agrobacterium rhizogenes could play an essential role in the activation of secondary metabolite production in plant transformed cultures. This study investigated whether the content of anthraquinone phytoalexins was changed in callus cultures of Rubia cordifolia transgenic for the 35S-rolB and 35S-rolC genes in comparison with a non-transformed callus culture. The anthraquinone content was shown to be significantly increased in transgenic cultures, thus providing further evidence that the rol-gene transformation can be used for the activation of secondary metabolism in plant cells. Methyl jasmonate and salicylic acid strongly increased anthraquinone accumulation in both transgenic and non-transgenic R. cordifolia calluses, whereas ethephon did not. A treatment of the cultures by cantharidin, the protein phosphatase 2A inhibitor, resulted in massive induction of anthraquinone accumulation in the transgenic cultures only. We suggest the involvement of a cantharidin-sensitive protein phosphorylation mechanism in anthraquinone biosynthesis in transgenic cultures.