Cell cycle analysis ofTaxus suspension cultures at the single cell level as an indicator of culture heterogeneity

Abstract

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

“…Ketchum and Gibson 1996;Ketchum and Croteau 1998;Yukimune et al 2000;Wu and Lin 2003;Naill and Roberts 2005;Khosroushahi et al 2006;Vongpaseuth and Roberts 2007). This conclusion is further supported by the aforementioned work of Croteau and his associates who succeeded in the isolation of paclitaxel biosynthetic genes of plant origin.…”

Responses of defense signals, biosynthetic gene transcription and taxoid biosynthesis to elicitation by a novel synthetic jasmonate in cell cultures of Taxus chinensis

“…In Taxus cell cultures, various elicitors have been used to stimulate taxoid production (e.g., Huang et al, 2005;Naill and Roberts, 2005;Qian et al, 2004a,b;Tabata, 2004;Wu and Ge, 2004;Ye et al, 2004). Regarding the elicitation mechanism, there are reports on defense responses such as oxidative burst, NO burst and/or JA by cultured cells of Taxus spp.…”

“…Jasmonic acid (JA), methyl jasmonate (MJ) and its derivatives are a family of important signal transducers, and can efficiently stimulate secondary metabolism in plant cells (e.g., Farmer and Ryan, 1990;Gundlach et al, 1992;Naill and Roberts, 2005;. Since the first report regarding the effect of MJ on the accumulation of secondary metabolites in plant cell cultures (Gundlach et al, 1992), more than 100 plant species have been demonstrated to respond to the addition of MJ to the culture medium by accumulating secondary metabolites (Haider et al, 2000).…”

Jasmonic acid elicits oxidative defense and detoxification systems in Cucumis melo L. cells

“…Jasmonic acid, and its derivatives are a family of important signal transducers, that can efficiently stimulate secondary metabolism in plant cells (Gundlach et al, 1992;Naill and Roberts, 2005;. Since the first report regarding the effect of methyl jasmonate (MJ) on the accumulation of secondary metabolites in plant cell cultures (Gundlach et al, 1992), more than 100 plant species have been demonstrated to respond to the addition of MJ to the culture medium by accumulating secondary metabolites (Haider et al, 2000).…”