Investigation On The Mechanism Of Mammalian Bax-Mediated Programmed Cell Death And Hyperoside Biosynthesis In Transgenic Bax Hypericum Perforatum L. Cells

Bax, a mammalian pro-apoptotic member of Bcl-2 family, inducesapoptosis in animal cells. It is known that Bax can trigger hypersensitiveresponse (HP) and induce programmed cell death (PCD) in model plants.In this study, we utilized the transgenic Bax H. perforatum cells, whichcan express a mouse Bax protein underβ-estradiol-inducible promoter, tostudy the function of Bax in inducing PCD and enhancing hyperosidesynthesis in H. perforatum cells. And at the same time we studied thesignal transduction mechanism during the above-mentioned process. Themain results are summarized as follows:1,Optimizing the culture condition of suspension culture transgenicBax H. perforatum cells.The suspension culture cells from the transgenic Bax H. perforatumcallus. To get the optimum of cell growth and hyperoside synthesis, multiple factors were studied, such as different types and concentrationsof growth regulators (cytokinin and auxin), pH, sucrose concentrations,temperature, and rating speed of the shaker on cells culture. We got thebest suspension culture condition: MS medium with hormones (1.5×10^-5mol·L^-1 NAA+10^-6 mol·L^-1 BA) and 3% sucrose, the medium wasadjusted to pH 5.8 and then sterilized at 121℃for 20 min before use, theculture temperature is 25℃, shaker's rating speed is 100 rpm, and thesuspension culture cells' cycle is 12 days. In order to get the stabletransgenic Bax H. perforatum suspension cells line, we detected andselected the transgenic Bax cells by PCR. As a result, a stable transgenicBax H. perforatum suspension cells line with high hyeroside content anda high growth speed was obtained.2,The research on Bax-mediated PCD in Bax H. perforatum cells.Bax transgenic cells were treated with 17-β-Estradiol (Est, 25μmol·L^-1). And western blotting analysis indicated that mouse Bax can beexpressed. After treating Bax transgenic cells with Est, the medium beginto alkalize about at the 21st h. cell death was observed about at the 24st hin induced Bax transgenic cells via Sytox green staining. And the deathcells were increasing with time going on. Some morphologiccharacteristics such as chromatin condensation and margination, andapoptotic body formation, appeared about from the 60st to 72st h afterinducement. DNA Ladder was detected about at the 48st h after inducement. As to the control, the above-mentioned characteristics can'tbe detected. It indicated that Bax can induce PCD in Bax transgenic H.perforatun cells.To study the mechanism of Bax-mediated PCD in H. perforatun cells,the NO generation in cells was detected. NO burst was observed aftertreatment with Est. The NO concentration begun to increase about at the12st h, reached the first phase peaked at the 25st h. Consequently, itdecreased a little. However, it increased again about at the 33st h, reachedthe second phase peak at the 40st h. In contast to the transgenic cells, thecontrol was without any change on the kinetics of NO concentration. NOspecific scavenger 2-4-carboxypheny-1-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) not only blocked the NO burst, but suppressed thePCD induced by Bax in Bax transgenic cells. These results suggest thatNO is a key signal for the Bax-mediated PCD. The mammalian neurophilNAD(P)H oxidase specific inhibitor, diphenylene iodonium (DPI), canlag the PCD for about 12 hours, but can't suppress the Bax-induced celldeath. In a word, reactive oxygen species (ROS) which accumulated incells via NADPH oxidase's fuction maybe be involved in theBax-mediated PCD, but doesn't act as the key signal.3,Mouse Bax influenced the hyperoside synthesis of Bax H.perforatum cells.Without the Est treatment, the Bax transgenic cells had the same cell growth and hyperoside synthesis as the wild-type cells. While, aftertreated Est, the hyperoside content in Bax transgenic cells stronglyincreased. To investgate wheather Bax intensified the hyperosidesynthesis or not, inducement with Est was carried on at the differenttimes of the cells culture cycle and the corresponding hyperoside contentwas detect at the 12 d, we drew a conclusion, for Bax transgenic cellstreated by Est at the 9 d (the mid-exponential phase of cell growth cycle),the hyperoside content significantly increased, and the cell fresh weightand total hyperoside yield were, respectively, 0.94 and 2.15-fold higherthan the untreated cell cultures. After treatment with Est, not onlyintensified the synthesis of hyperoside, but also the NO burst generated.The pretreated Bax transgenic cells with cPTIO suppressed theBax-induced NO burst competely and the Bax-induced hyperosidebiosynthesis partly. External application of NO via its donor sodiumnitroprusside (SNP) can induce hyperoside generation in Bax transgeniccells without inducing the Bax expression. And this phenomenon can beremoved by cPTIO completely. The above results indicated that theBax-induced hyperoside biosynthesis partially relied on the NO burst. Inaddition, Bax-induced hyperoside biosynthesis was partly suppressed byDPI, too. Generally speaking, the generation of hydrogen peroxide inplant cells is because of the participation of NADPH oxidase. Externalapplication of hydrogen peroxide can induce hyperoside generation without inducing the Bax expression. Catalase has the same function asDPI—partly inhibiting the Bax-induced hyperoside biosynthesis, but hasno influence on hyperoside generation induced by external application ofNO. cPTIO also has no influence on hyperoside generation induced byexternal application of hydrogen peroxide. Therefore, NO and ROS suchas H₂O₂ synthsized because of participation of NADPH oxidase areinvolved in Bax-induced hyperoside biosynthesis by two different signalpathways.