Systematic Analysis Of Relationship Between Cell Differentiation And Secondary Metabolism In Taxus Cuspidata Cells

The relationship between cell differentiation and secondary metabolism is a key scientific issue in the process of plan cell culture. In present study, Taxus Cuspidata cells were directly elicited to different cell differentiation level by addition of different combinations of NAA and 6-BA in vitro. Consequently, cell differentiation model was established. High-throughput proteomics, metabolomics and lipidomics analytical protocol combined with bioinformatics strategy were explored to systematically investigate the molecular mechanism of the relationship among cell differentiation, stress response and secondary metabolism involved in the process of Taxus Cuspidata cells culture.In terms of morphological analysis and Taxol detection, Taxus Cuspidata cells displayed significantly different physiological features after being induced by different combinations of NAA and 6-BA for 20 days. In presence of individual 6-BA, Taxol production increased a lot while a large amount of lignin accumulated in cell wall; In the presence of NAA:6-BA=1.0:1.0 and 2.0:2.0, visible treachery elements were appeared in cell wall while Taxol accumulation was promoted. Protein profiles were obtained by using 2-DE combined with MALDI-TOF/MS. 11 differentially expressed protein spots which showed more tight (positive or negative) relations with Taxol production and cell differentiation were indentified by MALDI-TOF/MS. Most of them were cell differentiation related proteins, isoprenoids biosynthesis related proteins and stress response related proteins. Especially, the expressions of protein CDP-FtsH and IPT were positively correlated to Taxol production.Low molecular weight metabolites profile was established by GC-TOF/MS, in which 80 metabolites were identified. Using multivariate analysis PCA, forty-five samples were clustered into four categories, which were coincided with morphological observation results and Taxol production. Most of the potential biomarkers were involved in TCA cycle, amino acid metabolism, polyamines metabolism and piperidine alkaloid biosynthesis.Phospholipids profile was constructed on the basis of LC-MS/MS. Totally 88 kinds of phospholipids were identified, including 9 species of phosphatidylglycerol (PG), 20 species of phosphatidylethanolamine (PE), 18 species of phosphatidylcholine (PC), 10 species of phosphatidylinositol (PI), 10 species of phosphatidylserine (PS), 14 species of phosphatidic acid (PA) and 7 species of Lysophosphatidylcholine (LysoPC). Using PCA analysis for data mining, we discovered that PE, PS and PA were three important lipid groups that were responsible for the discrimination among cells treated by different combinations of NAA and 6-BA. In the presence of individual 6-BA, the interconversion between PE and PS tended to produce PE, meanwhile a lot of PC was converted into PA; In the presence of NAA, the interconversion of PE and PS tended to form PE. The preference of interconversion between PE and PS might be closely related to cell adaptation to new environment. The accumulation of PA in cells elicited by individual 6-BA may positively correlate to stress response.