Research paperSignal transduction pathway for oxidative burst and taxol production in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysprum
Introduction
Taxol is one of the most effective chemotherapeutic agents against a wide variety of tumors especially ovarian and breast. The very limited supply of the drug from the bark of the Taxus species prompts intense efforts to develop alternative methods for taxol production [18]. Taxus cell suspension cultures could only produce traces of taxanes [12]. To improve the taxol production, various kinds of abiotic and biotic elicitors have been introduced and proved to be very effective for some cultures [6], [24]. The Fusarium oxysprum selected from 7 species was found to be the most effective inducer of taxanes accumulation through our experiments. Suspension-cultured Taxus chinesis var. mairei Y901-L responded to crude elicitors from the fungus Fusarium oxysprum by influencing the general phenylpropanoid pathway and incorporating taxol synthesis. The maximum taxol concentration was 8 fold of control, and increased amount of phenolics was observed in the culture medium. It was found that the active component is oligosaccharide extracted from the cell wall of Fusarium oxysprum.
Plant cells respond to challenge with avirulent pathogens by mounting a multi-component defense response called the hypersensitive response (HR). Characteristics of the HR include an oxidative burst leading to generation of hydrogen peroxide (H2O2), superoxide anion (O2.) and hydroxy-radical (OH.) [8], [14], localized cell death and cell wall cross-linking [15], and synthesis of antimicrobial phytoalexins [4]. The oxidative burst in challenged plant cells resembles that exhibited by human neutrophils [3], producing H2O2 that originates from superoxide generated by a plasma membrane-associated NADPH oxidase [14]. The plasma membrane of plant cells harbours protein components that play an important role in elicitation of the complex response of plants to pathogens [21]. Receptor proteins that bind microbial elicitors may generate signals that are transmitted to the sites of gene expression via different components as ion channels, G-proteins, phospholipase C etc. [20]. While individual members of putative signal chains have been identified in different plants frequently because of homologies of proteins from animal cells whose mode of functioning in the course of signal transduction remains a matter of debate [16]. Many studies over the past years have reported that the pharmacological regents affecting signaling in mammalian cells also influenced the defense response of cultured plant cells [7], [9]. We have developed a Taxus cell suspension culture system that responds to oligosacchride, an active component, from Fusarium oxysprum to study their signal transduction pathway.
Taxus chinensis var. mairei Y901-L suspension culture challenging with oligosaccharide from Fusarium oxysprum resulted in oxidative burst. However, the mechanism of how the cultured cells sense the presence of oligosaccharide and transduct this information to the nucleus to elicit an appropriate response is still unclear. The observed oxidative burst and taxol accumulation raised the question of how signal transfer from outside to inside cells is enlarged in intracellular signal transduction pathway.
In the present work, the potential involvement of Tyr phosphorylation in signal transduction was investigated in the Taxus: oligosaccharide cell culture system. The related position in the signal transduction pathway of Tyr phosphorylation, ion channel activity, G-protein activation and phospholipase C activation were examined. The pathway of signal transduction in cells was confirmed by adding pharmacological inhibitors.
Section snippets
Chemicals
Tyr kinase inhibitors, suramin, amphoterin B, anthracene-9-carboxylate, nifedipine and neomycin were purchased from Sigma. Protein kinase inhibitors, nifedipine and anthracene-9-carboxylic acid were dissolved in DMSO and maintained in 100× concentrated stocks. The other chemicals were dissolved in sterilized distilled water. Control cultures were treated with an equivalent amount of DMSO.
Cell cultures
Taxus chinensis var. mairei (Y901-L) cell suspensions were subcultured every 8–10 d for 5 generations in
Acknowledgements
This work was financially supported by the Natural Science Foundation of China (No. 29976032). Thanks are also given to Prof. An-Ping Zeng for helpful discussions and supply of some reagents.
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