Study On Signal Mechanism Of The Enhancement Of Secondary Metabolite Biosynthesis In Ce⁴⁺-induced Taxus Cells

In order to well-understand the growth and production of Taxus cells during the elicitors-induced apotposis and cellular spontaneous apoptosis, we investigated series changes of signal molecules by using high-throughput technologies, signal transduction strategies as well as bioinformatic methods. From the work we gained some valuable results.A lipidomic approach was employed to investigate the changes in membrane glycerophospholipids during the procession of cellular development and apoptosis of two plant cell lines, Taxus cuspidata and Taxus chinensis var. mairei. Analysis of lipids by LC/ESI/MSn and ESI-MS/MS coupled with principal component analysis profiled 7 glycerophospholipid classes and more than 100 glycerophospholipid molecular species, and indicated significant differences in the abundance of glycerophospholipids. Continuous increase of phospholipase D (PLD) activity from 17 day to 22 day led to PA production in apoptotic T. chinensis var. mairei cells suggesting that PLD activation and PA formation mediated spontaneous apoptosis.Markedly decreased levels of total glycerophospholipids were observed at 48 h and 72 h following treatment with cerium (Ce4+). The total glycerophospholipids levels, however, were not significantly altered for methyl jasmonate (MeJA)-induced T.cuspidata cells. Using multivariate analysis, especially orthogonal partial least squares (OPLS), we discovered that LysoPC, PA and PC were three important lipid groups that were responsible for the discrimination between MeJA/Ce3+-induced non-apoptotic cells and Ce4+-induced apoptotic cells.Continuous activation of PLD from 3 to 24 hour led to PA production during Ce4+-triggered apoptotic response, whereas a delayed and attenuated induction was observed during MeJA-triggered non-apoptotic defense response. PA can act as a mediator in two opposite ways, and appropriately high concentrations of PA were important determinants of apoptosis in Ce4+-induced T.cuspidata cells. Rapid increase of phospholipase A2 (PLA2) activity (1-4 h) caused a massive production of LysoPC following Ce4+ elicitation. In contrast, PLA2 activity was poorly induced following MeJA elicitation. PLA2 inhibitor suppressed not only jasmonic acid (JA) accumulation but also taxol production in Ce4+-induced cells, suggesting that PLA2 activation mediated taxol production partially via a JA-dependent signaling pathway. In addition, JA signaling pathway was not linked directly to apoptosis of T. cuspidata.An approximately 46 kDa ERK-like MAP kinase appeared to be activated at 5 minutes in Ce4+-induced T. cuspidata cells. It was possible that ERK-like activation and O2·- burst were separate signal events in early. O2·- burst formed a signal negative regulation to ERK-like activity. The present study suggested that down-regulation of ERK-like MAPK, burst of O2·-, activation of phospholipase and generation of lipid molecules as the key signaling pathways mediated apoptosis and taxol biosynthesis in Ce4+-induced T. cuspidata cells.