Relationship of viability and apoptosis to taxol production in Taxus sp. suspension cultures elicited with methyl jasmonate

Taxus cuspidata P991 in plant cell suspension culture is capable of producing the important anticancer agent Taxol (paclitaxel) and related taxanes. High-level production is obtained by elicitation with methyl jasmonate, but successful elicitation leads to loss of cell viability that cannot be recovered by subculture. Here, we test whether the loss of viability is due to a direct effect of methyl jasmonate. Upon subculture, the reduced viability continued in methyl jasmonate elicited cultures, but not in nonelicited control cultures. The growth reduction in elicited T. cuspidata P991 suspension cultures was evaluated by viability reduction measurements using phenosafranin and fluorescein diacetate. The viability reduction does not appear to be related to apoptosis based on DNA laddering analysis because it occurred very late (at day 35) in the culture period. DNA laddering was also found only after day 28 in T. canadensis C93AD (a Taxol-producing cell line) elicited with methyl jasmonate, implying that apoptosis is not the major death mechanism after elicitation. As compared to Taxol-producing cell lines, the viability of a nonproducing cell line, T. canadensis CO93D, was not severely affected by methyl jasmonate, indicating that methyl jasmonate itself is not the primary factor for viability reduction. Based on Northern analysis of taxadiene synthase mRNA from both elicited and nonelicited T. cuspidata P991, methyl jasmonate directly induces the production of this enzyme, which is the first committed step in the biosynthetic pathway for Taxol. As a result, both viability reduction and growth reduction appear related to a high production level of Taxol (and related taxanes) upon methyl jasmonate elicitation, rather than to the direct effect of methyl jasmonate.     

Manipulation by culture mixing and elicitation of paclitaxel and baccatin III production in Taxus baccata suspension cultures

Summary Experiments were carried out with Taxus baccata cell lines showing different paclitaxel-producing capacities (between 1.74 and 19.91 mgl^−1) when growing in a selected product-formation medium that specifically stimulated the production of taxane to the detriment of cell growth. Through mixing low-, medial- and high-producing lines, it could be observed that paclitaxel productivity in the resulting mixed lines was clearly higher than the mean productivity of the individual lines before mixing. This suggests that culture components generated by high-producing individual lines within the population might induce paclitaxel production. Although the accumulation of paclitaxel and baccatin III was higher when 100 μM methyl jasmonate was added to the subcultures of the mixed lines, the results indicate that exogenously applied methyl jasmonate was not the first factor to stimulate taxane production. The possible effects of methyl jasmonate elicitation and paclitaxel accumulation on cell viability are also considered.     

Effect of the plant peptide regulator, phytosulfokine-alpha, on the growth and Taxol production from Taxus sp. suspension cultures

Phytosulfokine-alpha (PSK-alpha) is a small plant peptide (5 amino acids) that displays characteristics typically associated with animal peptide hormones. PSK-alpha was originally isolated based on its mitogenic activity with plant cultures; it has been reported to increase production of tropane alkaloids from Atropa belladonna, although its general influence on secondary metabolite production is unknown. The studies reported in this article were initiated to evaluate the effects of PSK-alpha supplementation on production of Taxol (paclitaxel) from plant cell cultures of Taxus sp. particularly when methyl jasmonate (MeJA) is added as an elicitor of secondary metabolism. The response to PSK-alpha supplementation was cell line dependent. Taxus cuspidata P93AF showed no statistically significant response to PSK-alpha supplementation while Taxus canadensis C93AD and T. cuspidata PO93X displayed a concentration-dependent response (up to 100 nM PSK-alpha added in first 24 h of culture) with a decrease in initial growth rate, an increase in cell density (dry weight/fresh weight), and increased Taxol production. More remarkably with T. canadensis (C93AD), a very strong synergistic response of PSK-alpha (100 nM) and methyl jasmonate (MeJA, 100 microM) elicitation was observed, resulting in Taxol level of 35.3 +/- 2.1 mg/L or 1.83 +/- 0.02 mg Taxol/g dry cell weight achieved at day 21, a level of approximately 10-fold higher than for either treatment by itself. Although the level of Taxol production achieved is not remarkable, this synergistic treatment was able to partially revive taxane production in cultures that have lost productivity due to extended time (over 10 years) in continuous subculture.     

Instability of anthocyanin accumulation inVitis vinifera L. var. Gamay Fréaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, inVitis vinifera cell cultures. Therefore, four cell line suspensions ofVitis vinifera L. var. Gamay Fréaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73±0.15, 1.45±0.04, 0.77±0.024 and 0.27±0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% forV. vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities tol-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line showed greater potential in enhanced the anthocyanin production.     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.     

A combination of elicitation and precursor feeding leads to increased anthocyanin synthesis in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Both elicitation and precursor feeding are effective strategies for improving secondary metabolite production in plant cell suspension cultures. In this study, cell suspension cultures of Vitis vinifera subjected to methyl jasmonate treatment resulted in a significant increase in levels of anthocyanin production. Moreover, a combination of 5 mg/L phenylalanine and 50 mg/L methyl jasmonate promoted the highest level of anthocyanin biosynthesis, resulting in 4.6- and 3.4-fold increases in anthocyanin content and yield, respectively, over the control. The optimum period for elicitation of anthocyanin synthesis was 4 days following incubation in the presence of elicitors, at the beginning of the exponential growth phase. V. vinifera cell lines of different anthocyanin-producing capabilities responded differently to elicitation and precursor feeding. Anthocyanin production of a low-producing cell line, VV06, could be enhanced with addition of elicitors and precursor feeding. Methyl jasmonate was the only elicitor that increased anthocyanin production of the high-producing cell line VV05, but contributed to moderate enhancement of anthocyanin production compared with VV06. For cell line VV06, synergistic effects were observed for all treatment combinations of methyl jasmonate along with other elicitors and precursors. In addition, 6.1- and 4.6-fold increases in anthocyanin content and yield, respectively, were obtained in the presence of 5 mg/L phenylalanine, 50 mg/L methyl jasmonate, and 1 mg/L dextran. However, none of these treatment combinations exhibited synergistic effects in cell line VV05.     

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

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.     

原位吸附和茉莉酸甲酯联合使用显著提高中国红豆杉细胞云南紫杉烷c的生产

本实验所用的中国红豆杉细胞悬浮培养体系中,云南紫杉烷c(Tc)是主要的次生代谢产物,该化合物有类神经生长因子活性,提高其产量是进一步规模化生产的前提。本研究考察了原位吸附和茉莉酸甲酯(MJA)联合调控提高Tc产量的可能性。在培养的第7天加入浓度为100μmol/L的MJA虽然会使细胞的生物量下降10%～30%,但是单位细胞内Tc含量和Tc产量均有显著提高,分别是对照的3.6和3.3倍。吸附剂XAD-7在不同时间加入对Tc的合成影响显著。在培养的第7天同时加入100μmol/L的MJA和100g/L的XAD-7会使细胞生物量增加,Tc产量显著提高。培养到第21天,Tc产量达477.4mg/L,为对照的6.3倍,为只加MJA的1.9倍,其中94%的Tc被树脂吸附。实验结果表明,在MJA诱导高表达的过程中,吸附剂XAD-7的加入使细胞内代谢产物外泌,浓度降低,减轻产物反馈抑制现象,从而大幅度提高代谢物产量,有较好的生产前景。     

Improved Taxol production by combination of inducing factors in suspension cell culture of Taxus baccata

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.     

Elicitation kinetics of taxane production in suspension cultures of Taxus baccata Pendula

Methyl jasmonate increased taxane production in suspension cultures of Taxus baccata Pendula. Time course changes of taxane production after methyl jasmonate addition were different from normal kinetics without elicitation. Baccatin III and 10-deacetyl baccatin III were detected first and paclitaxel, 10-deacetyl taxol and cephalomanine followed each other in sequence. Paclitaxel was not a dead-end metabolite.     

Novel synthetic jasmonates as highly efficient elicitors for taxoid production by suspension cultures of Taxus chinensis

Suspension cultures of Taxus chinensis were used as a model plant cell system to evaluate novel synthetic jasmonates as elicitors for stimulating the biosynthesis of secondary metabolites. Significant increases in accumulation of taxuyunnanine C (Tc) were observed in the presence of newly synthesized 2-hydroxyethyl jasmonate (HEJA) and trifluoroethyl jasmonate (TFEJA) without their inhibition on cell growth. Addition of 100 microM HEJA or TFEJA on day 7 led to a high Tc content of 44.3 +/- 1.1mg/g or 39.7 +/- 1.1 mg/g (at day 21), while the Tc content was 14.0 +/- 0.1 mg/g and 32.4 +/- 1.6 mg/g for the control and that with addition of 100 microM methyl jasmonate (MJA), respectively. The superior stimulating ability of HEJA and TFEJA over MJA, which was generally considered as the best chemical for eliciting taxoid biosynthesis, suggests that the novel jasmonate analogues may have great potential in application to other cell culture systems for effcient elicitation of plant secondary metabolites.     

前体饲喂、诱导子和光照联合使用对葡萄细胞培养合成花青素的影响

苯丙氨酸前体饲喂分别和环糊精、葡聚糖、茉莉酸甲酯、黑曲霉和直喙镰孢菌提取液五种诱导子联合作用,其中以与茉莉酸甲酯的联合作用对葡萄细胞培养生产花青素的影响最大,可使单位鲜细胞花青素含量提高2.7倍,花青素产量提高3.4倍,实验证明两者在培养后第4天加入效果最好。在30μmol/L苯丙氨酸、218μmol/L茉莉酸甲酯和3000~4000lx光照条件下,不同花青素产量的细胞株都能显著提高花青素产量,但低产株VV06比高产株VV05具有更大的产率提高潜力。该条件下VV05和VV06花青素产量分别达到2975和4090CV/L,是对照组的2.5倍和5.2倍。     

Preparation of single cells from aggregated Taxus suspension cultures for population analysis

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.     

Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.     

Flow cytometric identification of Paclitaxel-accumulating subpopulations

An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC. Paclitaxel accumulation was significantly increased by elicitation with methyl jasmonate (100 microM) on day 7 post-transfer as compared to unelicited cultures. Maximum accumulation was observed by day 12 post-transfer in both total paclitaxel (approximately 0.25 mg/L) and the percentage of paclitaxel-accumulating cells (approximately 95%). A similar trend was observed with isolated protoplasts, although protoplasts accumulated only ca. 40-75% of the paclitaxel present in single cells. In unelicited cell cultures, a small subpopulation (ca. 3-5%) of single cells was shown to accumulate paclitaxel. Although nearly all cells were observed to accumulate paclitaxel in methyl jasmonate-elicited cell cultures, a high degree of cell-to-cell variation was observed in paclitaxel content. The identified subpopulations represent targets for cell sorting, which may be applied to develop higher-accumulating cell lines. The quantification of single cell paclitaxel content is useful for characterizing production variability in cell cultures and can be utilized to develop rational strategies to increase paclitaxel production.     

细胞均一性对葡萄细胞生长和花青素合成的影响

通过色差筛选法建立了一个相对均一的葡萄细胞悬浮系E,其细胞团较小,在长期继代培养过程中花青素合成能力的变异系数为8.7%,重复摇瓶实验的变异系数为5%。以E为实验材料进行的各组前体饲喂、诱导子添加、光照等联合作用实验,其生物量和花青素合成的变异系数均可控制在12%以内,充分说明了培养体系的均一性对维持稳定生产的重要性;黑暗条件下添加30μmol/L苯丙氨酸(Phe)和218μmol/L茉莉酸甲酯(MeJA)可使单位细胞花青素含量达到对照组的5.89倍,花青素产量为对照组的4.30倍,且连续5次继代培养过程中生物量和花青素合成的变异系数均比对照组降低。