No abstract

It is known that the rolA, rolB, and rolC genes of Agrobacterium rhizogenes T-DNA affect processes of plant development and activate the synthesis of secondary metabolites in transformed plant cells. Although a synergistic activity of the rol genes on root formation is well-documented, little is known about their individual and combined action on secondary metabolism. In the present investigation, we provide evidence indicating that individual rolA, rolB, and rolC genes are capable of increasing biosynthesis of anthraquinones (AQs) in transformed calli of Rubia cordifolia. The stimulatory effect was due to the increased transcription of a key gene of AQ biosynthesis, the isochorismate synthase (ICS) gene. The strongest AQ-stimulating activity was shown for an R. cordifolia culture expressing rolB at high levels, where rolB ensured a 15-fold increase of AQ accumulation compared with the control, non-transformed calli. A tyrosine phosphatase inhibitor abolished the rolB-induced increase of AQ production, thus indicating the involvement of tyrosine (de)phosphorylation in the rolB-mediated AQ stimulation. The rolA- and rolC-expressing cultures produced 2.8- and 4.3-fold higher levels of AQs, respectively, when compared with the control calli. However, the effect of rolA, rolB, and rolC on AQ biosynthesis was not synergistic because rolA and rolC apparently attenuated the stimulatory effect of rolB on AQ biosynthesis. Therefore, the rol-gene-mediated signals that promote root formation and those which activate biosynthesis of secondary metabolites seem to have a point of divergence.

Two new spinochromes, echinamines A (1) and B (2), were isolated from the sea urchin Scaphechinus mirabilis. Compounds 1 and 2 represent the first examples of natural polyhydroxynaphthazarins with a primary amine group. The structures of 1 and 2 were established by analysis of spectroscopic data and synthesis of their dimethyl ethers.

The transformation of Rubia cordifolia L. cells by the 35S- rolB and 35S- rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins. Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca(2+) channel blocker. The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment. Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca(2+)-free medium, whereas growth of the rolB culture was reduced by less than half. We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures. Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca(2+) channel blockers verapamil and LaCl(3), or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine. These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca(2+)-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor-receptor complex via transcriptional activation of defense genes. Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect. These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R. cordifolia.

No abstract