| Optimization of nitrogen concentration in medium was previously reported to efficiently improve secondary metabolite biosynthesis in plant cell cultures, as changing nitrate concentration could enhance bioactive compounds production through influencing gene expression. But the influence of ammonium on gene transcription and signal transduction and its regulation had not been known. At present, Taxus spp. cell culture is one of important sources for pharmaceutically active taxol production in industry. However, the productivity of taxoids (including taxol) is still low. Further investigation on the regulation mechanism of secondary metabolite biosynthesis will be helpful for industrial application of plant cell culture and basic researches in the field of biotechnology. By taking Taxus chinensis suspension cells for example, this Ph.D. dissertation was aimed to focus on the effects of medium initial ammonium concentration on bioactive taxuyunnanine C (Tc) accumulation and on the expression of related gene. The signals mediating induction role of changing ammonium concentration on Tc biosynthesis and quantitative relation between intracellular signal level and secondary metabolite accumulation were also studied. This work provided us important information to improve the low yield of bioactive metabolite in plant cell culture, and also is useful for understanding the induction mechanism of low nitrogen on secondary metabolites.At first, effect of medium initial ammonium concentration on Tc biosynthesis in Taxus chinensis cells was investigated When medium initial ammonium was in the range of 0-20 mM, the highest Tc content of 8.07 mg/gDW was achieved at 2 mM initial ammonium, which was about 50% higher than that of control. To provide an insight into the unknown inducible mechanism of initial ammonium, the expression of important genes in taxoid biosynthetic pathway were examined. The quantitative Real Time PCR results showed that the transcription of all taxoid biosynthetic genes were up-regulated when T. chinensis cells was cultivated in medium of 2 mM initial ammonium. Among them, transcription of geranylgeranyl diphosphate synthase (GGPPs) and taxa-4(5),11(12)-diene synthase (TS), key genes in taxoid biosynthesis, achieved maximum at 47-fold and 37-fold compared to control, respectively. Other four taxoid biosynthetic related genes(T5aH, TDAT, T10βH和TaH) were also induced by 3-9 folds. The above results indicated the stimulation of taxoid synthesis by low initial ammonium may be resulted from induced transcription of taxoid biosynthetic genes.However, the cell growth was very poor in the 2 mM initial ammonium medium compared to control (at 20 mM of initial ammonium). To further improve the Tc production, strategy of two stage culture between 2 mM and 20 mM ammonium medium was used. Among them, the maximal Tc production of 154.8 mg/L was obtained in the cells which was cultivated in 2 mM ammonium medium for the first 24 h and then transferred to 20 mM ammonium medium. This two stage culture strategy made the Tc biosynthetic gene transcription maintained at a high level during cultivation period, suggesting that high expression level of biosynthetic related gene may be closely related to higher secondary metabolite production.Based on the stimulation effects of changing initial ammonium concentration on Tc biosynthesis, the unknown signal transduction pathway in low ammonium induction of secondary metabolism was investigated. Choosing the 2 mM initial ammonium as a typical condition, defense signals of H2O2, salicylic acid (SA) and phenylalanine ammonia-lyase (PAL) were detected were examined in cell cultures of T. chinensis. The oxidative burst (induced H2O2 production) was confirmed, and the induction of PAL activity and intracellular SA synthesis were found. Application of diphenylene iodonium (DPI), the H2O2 production inhibitor, and paclobutrazol, the SA biosynthesis inhibitor, inhibited the 2 mM initial ammonium-induced up-regulation of taxoid biosynthetic genes such as GGPPs and TS, as well as induction of Tc accumulation. DPI treatment effectively depressed the 2 mM initial ammonium-stimulated SA accumulation, while paclobutrazol addition didn't affect induced H2O2 production. The above results suggested that both H2O2 and SA signals were involved in 2 mM initial ammonium-induced Tc biosynthesis, and H2O2 was upstream of SA in signal transduction pathway, SA may influence Tc synthesis through mediating the induction of Tc biosynthetic genes expression. To the best of our knowledge, intracellular SA could be a mediatory signal under low nitrogen is proposed for the first time. The obtained information about signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is helpful for further investigation on low nitrogen induction of secondary metabolism.To further study the relation between intracellular signal level and secondary metabolite accumulation in plant cells, endogenous SA level was regulated by applying different concentrations of exogenous SA based on the mediatory role of intracellular SA in Tc biosynthesis, and the quantitative response of Tc accumulation was also investigated. The results showed that intracellular SA increased with increase of exogenous applied SA concentration. The maximal endogenous SA of 97.1μg/gDW was observed at 1 h after addition of 100μM exogenous SA, approximately 20 folds higher than intracellular SA before exogenous SA elicitation. Tc content increased with increase of internal SA, which achieved maximum at 10.3 mg/gDW,2-fold over control upon 100μM exogenous SA. Addition of SA biosynthesis inhibitor, paclobutrazol or (BOC-aminooxy) acetic acid (AOA), with exogenous SA, led to a partial inhibitory effect on endogenous SA accumulation, as well as Tc induction, suggesting Tc biosynthesis was dependent on internal SA level. In order to identify potential quantitative relationships between intracellular SA and Tc biosynthesis, regression analyses were performed for maximum values of intracellular SA and Tc accumulation under different concentrations of exogenous SA and its biosynthesis inhibitor treatments. There was a linear correlation between maximal intracellular SA level and maximal induced Tc biosynthesis (r2=0.8426, p<0.05). As for gene expression, maximal induction of GGPPs transcription showed a good dose-dependent relation with the highest intracellular SA level (r2=0.9574, p<0.05), but this trend was a bit weakly observed in case of TS expression with maximal intracellular SA level (r2=0.8091). The signal-product quantitative relationship observed here could be useful for strategy development based on signal transduction for enhancement secondary metabolite production in plant cell cultures.Collectively, the information of initial ammonium concentration affected Tc biosynthesis in Taxus chinensis cells, especially information on low initial ammonium induced gene transcription and signal transduction pathway, will be useful for the exploitation of the inducible mechanisms of low nitrogen-regulated secondary metabolite biosynthesis and for the objective manipulation of bioactive products production. The strategy based on intracellular signal engineering proposed here will also be helpful for useful metabolites production in other plant cell cultures.
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