Critical influence of Eschscholzia californica cells nutritional state on secondary metabolite production

Abstract

In this study, we investigated the influence of initial internal nutrient concentrations at the time of elicitation on the ability of Eschscholzia californica (EC) cells to produce alkaloids. Three EC cell suspensions cultivated in culture media differing in their PO4(3-) and NO3- contents were sampled daily for 12 days and analyzed for extracellular and intracellular nutrient concentrations. The ability of the cells to produce alkaloids was tested along the three cell suspension cultures. Sampled cells were then further cultured for 7 days in a production medium containing the elicitor and an extraction resin. The alkaloid production of the cells was measured 7 days post-elicitation. In the low-N medium, starch, glucose, and phosphate contents in the biomass was increased by 470, 1624 and 70%, respectively, 10 days after inoculation compared to the control culture in standard B5 medium. Cell concentration was significantly reduced from 10.3 to 8.6 millions cell/mL on this low-N medium compared to the control, nevertheless alkaloid production was multiplied by 39 at day 10 when cells were elicited. Cells grown on the low-N or low-P media accumulated 83% and 188% more carbon, respectively, than control cells at the end of the culture. This intracellular C was mainly stored in the form of starch in low-P medium and both in the form of starch and glucose in the low-N medium. The ability of EC cells to produce alkaloids upon elicitation was shown to be strongly dependent on the initial intracellular C and P content at the time of elicitation. This suggests that reproducibility and productivity during EC cell culture could be enhanced by manipulating the intracellular C and P content at the time of elicitation.

A predictive nutritional model for plant cells and hairy roots

“…Data for E. californica cell suspension culture in Erlenmeyer flask were taken from Lamboursain and Jolicoeur (2005). Cultures were performed in shake flasks on an orbital shaker at 120 rpm using a B5 medium supplemented with 3% glucose (Table II).…”

“…In in vitro culture, Pi and ammonium ions of standard culture media such as B5 and MS are usually quickly depleted and the cell storage pools then support growth for the remaining days or weeks of culture. Subculturing Eschscholtzia californica cells in standard B5, lowPi and low-nitrate B5 media has resulted in cells with significantly different behaviors (Lamboursain and Jolicoeur, 2005). The maximum specific production in total alkaloids after elicitation by chitin was shown to occur at different growth stages such as at stationary phase (6-10 days post-inoculation) for the nitrate-limited medium, and much earlier for the Pi-limited medium at early exponential growth (1-to 3-day-old).…”

“…The model also includes the concept of minimal intracellular concentration in Pi and sugars for growth to proceed allowing description of the lag phase. However, results from Lamboursain and Jolicoeur (2005) suggest that specific growth rate term should also include intracellular pools of nitrogenous compounds. The goal of the present work was thus to evaluate the potential of a structured nutritional model based on intracellular cell contents in Pi, nitrogenous compounds and sugars to describe the growth and nutritional behavior of plant cells grown as suspension cells as well as hairy roots.…”

“…The description of the dynamics for sugars, nitrogen sources and phosphate allows using the model without a priori assumptions on the limiting nutrient. Model parameters were calibrated for E. californica cell suspension and C. roseus and D. carota hairy roots using experimental data taken from previous works (BouchardMarchand, 2000;Lamboursain and Jolicoeur, 2005;Leduc et al, 2006). These experiments are described in the Materials and Methods.…”

Sugar metabolism ofVetiveria zizanioidescells cultured with Amberlite XAD-4. Uptake of radiolabelled sugars, cell wall invertase activity and gene expression

“…Sugars as the main carbon source for non-photosynthetic cells are utilized to maintain most of the biological and physiological activities controlling the synthesis and release of secondary products into the culture media (Lamboursain and Jolicoeur 2005).…”