Effects of Ca(2+) channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genes.

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.