Study On Ce⁴⁺-Induced Secondary Metabolism Productions Of Catharanthus Roseus Cells In Suspension Cultures

The effect mechanism of rare earth on secondary metabolism productions in suspension cultured Catharanthus roseus was investigated by addition of 1mM Ce⁴⁺ to cultures. The changes of the activated oxygen species, mitogen-activated protein kinase (MAPK), PAL and secondary metabolites were studied in detail. We attempted to explain the mechanism of secondary metabolites production in Ce⁴⁺ induced Catharanthus roseus.The changes and physiological role of H₂O₂ in Catharanthus roseus induced by Ce⁴⁺ were investigated. H₂O₂ burst after addition of Ce⁴⁺. PAL activation and the catharanthine production decreased significantly while CAT, a H₂O₂ scavenger, was added into cultures. Moreover, H₂O₂ generation was earlier than the catharanthine synthesis. We can conclude that H₂O₂ was the up-stream signal in catharanthine synthesis regulation. The production of catharanthine also increased after addition of H₂O₂. These results indicated that H₂O₂ play a key role in PAL activation and catharanthine synthesis. We also found that the concentration of Ce⁴⁺ strongly influenced H₂O₂ concentration and catharanthine production. Meanwhile, there was a saturated effect according to Ce⁴⁺ concentration.Signal transduction pathway that Ce⁴⁺ induced secondary metabolites synthesis in Catharanthus roseus was investigated by the addition of harmacological inhibitors. JNK,ERK and p38 were all activated in secondary metabolites synthesis induced by Ce⁴⁺. However, only ERK and p38 were activated in H₂O₂ generation induced by Ce⁴⁺. H₂O₂ signal was transduced by JNK,ERK and p38 protein kinase pathway to activated the secondary metabolites synthesis.The effects of Ce⁴⁺ on CAT activation and conformation were investigated by uv-visible spectra, fluorescent emission spectra, synchronous fluorescence, ultraviolet differential spectra, circular dichroism, FT-IR. The CAT conformation was related to the Ce⁴⁺ concentration.α-helix andβ-fold in CAT increase and decrease at lower concentration of Ce⁴⁺, respectively. However, the results reversed at higher concentration of Ce⁴⁺. These changes were related with Ce⁴⁺ concentration.Intracellular contents of Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Fe²⁺ and K+ were analyzed by an inductively coupled plasma-optical emission spectrometry and the relationships between ions contents and total alkaloid in cells at different induced time were investigated. The results showed that the secondary metabolism productions synthesis induced by 1mM Ce⁴⁺ was companying the Ca²⁺ influx and K+ efflux.