Figure 5

Effects of suramin and heparin on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension cultures of C. roseus. Six-day-old cell suspension cultures were pre-treated with suramin (Sur) or heparin (Hep) at the indicated concentrations and were irradiated with UV-B for 5 min. As control one set of cells was irradiated with UV-B alone or left un-irradiated and the crude extracts from all cells were prepared at the indicated times and assayed for the phosphorylation of H IIIS (a) and MBP (b) under standard conditions as described in materials and methods. A second set of cells was similarly treated and the total RNA was isolated at the indicated times and analyzed for the transcript levels of Tdc and Str by RT-PCR (c). The third set of cells were pretreated with the highest concentration of inhibitor previously used followed by 5 min of UV-B irradiation. After treatment, cells were collected after 48 h and catharanthine content was determined by HPLC (d). These experiments were performed in triplicates and repeated at least twice. Error bars represent mean ± SD (n = 3).