Effects Of Narciclasine On The Growth Of Tobacco Callus And The Programmed Cell Death In Tobacco Bright Yellow-2 Cells

Narciclasine (NCS) is a bioactive substance isolated from the secreted mucilage of Narcissus tazetta bulbs. It can induce apoptosis by activating the death receptor and/or mitochondrial pathways in cancer cells, now NCS is a commonly anticancer agent in animal systems. In plants, the preliminary studies showed that NCS exhibits a wide range of inhibitory effects on plant growth including seed germination, seedling growth, microbody, plastid and chloroplast development of excised radish cotyledons. In this study, tobacco (Nicotiana tabacum cv. Willow leaf) callus and Tobacco Bright Yellow-2 suspension cell cultures (TBY-2) were used to investigate the regulation mechanism of NCS in plant. The main results as followed:(1) NCS could inhibit the gowth of tobacco callus in a dose-and duration-dependent manner,0.1μM NCS had no effect on the biomass of tobacco callus, whereas 2.5μM NCS could reduce the fresh weight of tobacco callus to 18% of the control (no NCS). We also detected the effect of NCS on the damage to tobacco callus. The results showed that 1.0μM and 2.5μM NCS could induce damage to tobacco callus at 12 d after NCS treatment and the damage seems to more pronounced after 18 d treatment. Finally, we focused our attention on the effect of NCS on the respiratory metabolism of tobacco callus. The results demonstrated that 1.0μM and 2.5μM NCS could significantly increase the alternative pathway and activate the glucose-6-phosphate dehydrogenase (G6PDH, a key enzyme of pentose phosphate pathway, PPP) activity, whereas reduced the cytochrome pathway, tricarboxylic acid (TCA) cycle and Embden-Meyerhof-Parnas pathway (EMP). In view of alternative pathway can reduce the production of reactive oxygen species (ROS) and PPP can supply cells with NADPH and carbon skeletons, we concluded that the alternative pathway together with PPP cauld reduce a slowly growth with low enengy in NCS treated cells by regulating the redox state and substrate metabolism.(2) NCS inhibited to growth and induced cell death of TBY-2 suspension cell in a dose-and duration-dependent manner.2.5μM NCS could totally inhibited the growth of TBY-2 suspension cell and also effectively induced PCD. And all the TBY-2 cells were dead after 2.5μM NCS treatment for 120 h, whereas under control condition (no NCS), the dead cells accounted for less than 5% of the total cells throughout the experimental period. NCS treatment induced typical PCD-associated morphological and biochemical changes, namely cell shrinkage, plasmolysis, chromatin condensation and nuclear DNA degradation. These data showed that the cell death of TBY-2 suspension cell induced by NCS was PCD, but not necrosis. To investigate possible signaling events in PCD induced by NCS, we analyzed the production of ROS and the change of mitochondria function during PCD induced by NCS. The results demonstrated that a biphasic behavior burst of hydrogen peroxide (H2O2) was detected in TBY-2 cells in the present of NCS. They occurred at 1 h and 12 h treated with NCS, respectively. It was found that the accumulation of H2O2 in TBY-2 suspension cell in the present of NCS appeared before the damage of cell membrane by testing the TBARS content and electrolyte leakage. It indicated that the accumulation of H2O2 was not the damaged result of PCD and it maybe act as a signal molecular in PCD induced by NCS. Mitochondrial transmembrane potential (MTP) and cell respiration rate had a similar changing pattern which both decrease occurred after a slight increase by 10%. Pre-incubation with antioxidant catalase (CAT) and N-acetyl-L-cysteine (NAC) not only significantly decreased the H2O2 production but also effectively retarded the decrease of MTP and reduced the percentage of cells undergoing PCD after NCS treatment. On the other hand, when exogenous H2O2 was added to NCS-treated cells, the MTP decreased significantly with an enhanced cell death. This result suggested that mitochondrial activities were regulated by redox state of cells; PCD was induced by mitochondrial dysfuction. In conclusion, our results suggest that NCS induces PCD in plant cells; the oxidative stress (accumulation of H2O2) and the MTP loss play important roles during NCS-induced PCD.