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Although many studies have reported the negative effects of elevated O 3 on plant physiological characteristics, the influence of elevated O 3 on below-ground processes and soil microbial functioning is less studied. In this study, we examined the effects of elevated O 3 on soil properties, soil microbial biomass, as well as microbial community composition using high-throughput sequencing. Throughout one growing season, one-year old seedlings of two important endemic trees in subtropical China: Taxus chinensis (Pilger) Rehd. var. chinensis, and Machilus ichangensis Rehd. Et Wils, were exposed to charcoal-filtered air (CF as control), 100 nl l -1 (E100) or 150 nl l -1 (E150) O 3 -enriched air, in open top chambers (OTCs). We found that only higher O 3 exposure (E150) significantly decreased soil microbial biomass carbon and nitrogen in M. ichangensis, and the contents of organic matter were significantly decreased by E150 in both tree species. Although both levels of O 3 exposure decreased NO 3 -N in T. chinensis, only E150 increased NO 3 -N in M. ichangensis, and there were no effects of O 3 on NH 4 -N. Moreover, elevated O 3 elicited changes in soil microbial community structure and decreased fungal diversity in both M. ichangensis and T. chinensis. However, even though O 3 exposure reduced bacterial diversity in M. ichangensis, no effect of O 3 exposure on bacterial diversity was detected in soil grown with T. chinensis. Our results showed that elevated O 3 altered the abundance of bacteria and fungi in general, and in particular reduced nitrifiers and increased the relative abundance of some fungal taxa capable of denitrification, which may stimulate N 2 O emissions. Overall, our findings indicate that elevated O 3 not only impacts the soil microbial community structure, but may also exert an influence on the functioning of microbial communities., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Numerous studies have documented the negative effects of ozone (O 3 ) on tree species in growing season, however, little is done in non-growing season. Three evergreen tree species, Phoebe bournei (Hemsl.) Yang (P. bournei), Machilus pauhoi Kanehira (M. pauhoi) and Taxus chinensis (Pilger) Rehd (T. chinensis), were exposed to non-filtered air, 100 nmol mol -1 O 3 air (E1) and 150 nmol mol -1 O 3 air (E2) in open-top chambers in subtropical China. In the entire period of experiment, O 3 fumigation decreased net photosynthesis rate (Pn) through stomatal limitation during the transition period from growing to non-growing season (TGN), and through non-stomatal limitation during the period of non-growing season (NGS) in all species tested. Meanwhile, O 3 fumigation reduced and delayed the resilience of Pn in all species tested during the transition period from non-growing to growing season (TNG). O 3 fumigation significantly decreased chlorophyll contents during NGS, whereas no obvious injury symptoms were observed till the end of experiment. O 3 fumigation induced increases in levels of malondialdehyde, superoxide dismutase, total phenolics and reduced ascorbic acid, and changes in four plant endogenous hormones as well in all species tested during NGS. During NGS, E1 and E2 reduced Pn by an average of 80.11% in P. bournei, 94.56% in M. pauhoi and 12.57% in T. chinensis, indicating that the O 3 sensitivity was in an order of M. pauhoi > P. bournei > T. chinensis. Overall, O 3 fumigation inhibited carbon fixation in all species tested during NGS. Furthermore, O 3 -induced physiological activities also consumed the dry matter. All these suggested that elevated O 3 , which is likely to come true during NGS in the future, will adversely affect the accumulation of dry matter and the resilience of Pn during TNG in evergreen tree species, and further inhibit their growth and development in the upcoming growing season., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Taxus spp. are ancient gymnosperms that produce a unique secondary metabolite, namely, taxol, an anticancer drug. JAZ proteins are key regulators of the JA signaling pathway, which control taxol biosynthesis. However, the JAZ proteins of Taxus spp. are poorly studied. In this work, nine JAZ genes from Taxus chinensis were identified using our previous transcriptome data and named as TcJAZ1-TcJAZ9. Of these nine TcJAZ proteins, eight contain Jas and TIFY domains, and the Jas domain of TcJAZ6 is incomplete. Most TcJAZs and PsJAZs are not related to AtJAZs and OsJAZs. Phylogenetic analysis divided all JAZ proteins from Arabidopsis thaliana, Oryza sativa, Picea sitchensis, and T. chinensis into eight subgroups; gymnosperms JAZs were classified into subgroups V-VIII, and angiosperm JAZs were categorized into subgroups I-V. Three motifs of subgroups VI-VIII were identified in gymnosperm JAZs, indicating that gymnosperm JAZ proteins exhibit a different evolutionary process from those of angiosperms. The expression patterns of nine TcJAZs showed that TcJAZ2/3/8 was a key regulator, indicating their important roles in T. chinensis. Results revealed that gymnosperm JAZs differ from angiosperm JAZs in terms of molecular structure. Three novel conserved motifs were found in TcJAZs and PsJAZs. This study provides a basis for research on JA regulatory system in Taxus spp. and for elucidating the significance of JA signaling pathway to land plants., (Copyright © 2017. Published by Elsevier B.V.)

Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert β-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant cell cultures. Cyclodextrins, which are cyclic oligosaccharides, can form inclusion complexes with nonhydrophilic secondary products, thus increasing their excretion from the producer cells to the culture medium. In the present work, using a selected Taxus x media cell line cultured in a two-stage system, the relationship between taxane production and the transcript profiles of several genes involved in taxol metabolism was studied to gain more insight into the mechanism by which these two elicitors regulate the biosynthesis and excretion of taxol and related taxanes. Gene expression was not clearly enhanced by the presence of cyclodextrins in the culture medium and variably induced by methyl jasmonate, but when the culture was supplemented with both elicitors, a synergistic effect on transcript accumulation was observed. The BAPT and DBTNBT genes, which encode the last two transferases involved in the taxol pathway, appeared to control limiting biosynthetic steps. In the cell cultures treated with both elicitors, the produced taxanes were found mainly in the culture medium, which limited retroinhibition processes and taxane toxicity for the producer cells. The expression level of a putative ABC gene was found to have increased, suggesting it played a role in the taxane excretion. Taxol biosynthesis was clearly increased by the joint action of methyl jasmonate and cyclodextrins, reaching production levels 55 times higher than in nonelicited cultures., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Key Message: Methyl jasmonate elicitation of Taxus cultures enhances paclitaxel accumulation, but represses growth by inhibition of cell cycle progression. Growth repression is evident both at the culture level and transcriptional level. Methyl jasmonate (MeJA) elicitation is an effective strategy to induce and enhance synthesis of the anticancer agent paclitaxel (Taxol(®)) in Taxus cell suspension cultures; however, concurrent decreases in growth are often observed, which is problematic for large-scale bioprocessing. Here, increased accumulation of paclitaxel in Taxus cuspidata suspension cultures with MeJA elicitation was accompanied by a concomitant decrease in cell growth, evident within the first 3 days post-elicitation. Both MeJA-elicited and mock-elicited cultures exhibited similar viability with no apoptosis up to day 16 and day 24 of the cell culture period, respectively, suggesting that growth repression is not attributable to cell death. Flow cytometric analyses demonstrated that MeJA perturbed cell cycle progression of asynchronously dividing Taxus cells. MeJA slowed down cell cycle progression, impaired the G1/S transition as observed by an increase in G0/G1 phase cells, and decreased the number of actively dividing cells. Through a combination of deep sequencing and gene expression analyses, the expression status of Taxus cell cycle-associated genes correlated with observations at the culture level. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.

Metabolomic and transcriptomic profiling data were obtained and integrated to elucidate the crucial network controls on taxol and its precursor biosynthesis during the taxane core functionalization within methyl jasmonate (MJ)-induced Taxus chinensis cells. Twelve metabolites were identified using liquid chromatography-electrospray ionization-mass spectrometry. These metabolites contain taxol (paclitaxel), baccatin III (B-III) and its analogs, a group structurally bearing multiple free hydroxyls (TAX), and another group of multiple acyl taxanes (MAT), including taxuyunnanine C (TC) and its analogs. The metabolomic profile showed a higher increase in TAX than in MAT. Particularly, the ratio of B-III and taxol to the total taxane content increased more significantly in TAX than in MAT. The MAT proportion did not significantly change, although they are predominant components in cell cultures compared with TAX. Quantitative real-time polymerase chain reaction (qRT-RCR) was used to determine the transcription level of 20 genes, among which 11 were reported responsible for taxol biosynthesis and 9 were obtained from our previous transcriptomic data. The total expression levels of hydroxylase after 24 h and 6 days were higher than those of acylase. The principal component analysis (PCA) results validated the metabolomic analysis data, indicating that hydroxylation was more crucial than acylation for controlling the flux toward TAX biosynthesis. Furthermore, the PCA contribution comparison showed that two undefined genes of OHX1 and ACX3 might have good potential in TAX upregulation and MAT downregulation. To the best of our knowledge, this study provides the first experimental evidence on the contribution of total hydroxylation to taxane biosynthesis., (© 2013 American Institute of Chemical Engineers.)

Taxol was originally isolated from the yew Taxus brevifolia. Because taxol inhibits the depolymerization of microtubules, the presence of a self-resistance mechanism in Taxus spp. was hypothesized. The cloning of the cDNA for alpha and beta tubulins from Taxus cuspidata and those from the human embryonic kidney cell line HEK293T revealed that the (26)Asp, (359)Arg, and (361)Leu residues in the human beta tubulin, which are important for taxol binding, were replaced with Glu, Trp, and Met in the beta tubulin of T. cuspidata, respectively. The microtubule assembly of the recombinant alpha and beta tubulins was monitored turbidimetrically, and the results clearly demonstrated that the microtubule from T. cuspidata is less sensitive to taxol than that from HEK293T cells. The Taxus microtubule composed of the wild-type alpha tubulin and the beta tubulin with the E26D mutation restored the sensitivity to taxol. We thus postulated that the mutation identified in the beta tubulin of T. cuspidata plays a role in the self-resistance of this species against taxol.

Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

The ability of an aprotic solvent, dimethylsulfoxide (DMSO), to induce taxane synthesis and release from cell suspension culture of Taxus baccata was examined. The results showed that applying DMSO in optimal conditions not only led to enhancement in taxane excretion from the cells but also led to improvement in taxol synthesis. Maximum yields for taxol [3.34 mg/g dry cell weight (DCW)] were achieved by adding 5% DMSO to the culture at the late-exponential phase of cell growth (day 14) and culturing for 21 days, which was 2.26-fold of that for the control (1.48 mg/g DCW). However, adding 5% DMSO did not affect the yield of 10-deacetyl baccatin III and baccatin III. This condition also resulted in maximum extracellular taxane (4.86 mg/L); this value was 2.82-fold higher than that in the control (1.72 mg/L). We demonstrated that the late-exponential phase of cell growth could be the best time to add elicitor for maximizing the yield of taxol. Adding DMSO at earlier times (days 1 and 7) or in other concentrations (1% and 3%) had negative effects on taxane synthesis. Overall results suggest that DMSO has good potential to enhance synthesis and release taxol from cell suspension culture of T. baccata L., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

Objective: To reveal the influence of harvest season and tree age on the content of taxol and 10-DAB III, and provide the basis for the harvest time of Taxus madia., Method: Branches and leaves of the labeled 25 three-year-old plants and 25 five-year-old plants were collected every two months from March 26,2009 to January 26, 2010. Taxol and 10-DAB III content of different age and growth season of Taxus in branches were determined by HPLC., Result: Taxol and 10-DAB III content were significantly different in different harvesting age. The content of five-year-old plants was significantly higher than that of three-year-old plants. Taxol and 10-DAB III contents were significantly different in different harvesting season, and the highest content of taxol and 10-DAB III was 0.56, 0.32 mg x g(-1), respectively, in May., Conclusion: The May is the suitable harvest season for T. madia, but the suitable harvest age need further study which according to the main active component and biomass accumulation.

Of the initial six cell lines originating from explants of Taxus globosa, or Mexican yew (stem internode, leaves and meristematic tissue), three were selected for their microbial and oxidation resistance, two from leaves and the other from stem internode. A study of their behavior, both in terms of cell growth, and of baccatin III and paclitaxel production, was developed in suspension cultures with an initially standardized biomass (fresh weight 0.23 g/L) using modified Gamborg's B5 medium, and an elicitor (methyl jasmonate), on either the first or seventh day of culture, at several levels (0, 0.1, 1, 10, 100 microM). In most of the conditions used, the three cell lines showed growth associated baccatin III production. The cell line from stem internode was the highest producer of baccatin III using 1 microM elicitor, sampling at 10 days (p < or = 0.01, 6.45 mg/L). This same line also had the highest biomass production (6.85 g/L, p < or = 0.01) at 10 days of culture but at the higher elicitor concentration of 10 microM. All three cell lines did not produce paclitaxel under experimental conditions used.

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1microM of l-phenylalanine (6.2gL(-1)). Addition of 1microM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7microg L(-1)), which was not detected in the untreated control culture. Supplementation with 100microM of l-phenylalanine together with 100microM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3microg L(-1) (control untreated culture) to 568.2microg L(-1), the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8microg L(-1)) was observed when the medium was supplemented with 100microM of the precursor in combination with methyl jasmonate. Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.

To achieve a better understanding of taxol metabolism and accumulation in Taxus cell cultures, a T. baccata cell line growing for 20 days in a selected growth medium was treated at the beginning of the experiment with several concentrations of taxol (25, 50, 100 and 200mgL(-1)). Compared with an untreated control, all these taxol concentrations stimulated cell-associated taxol content (up to 32.7 times in the presence of 200mgL(-1) exogenous taxol), although higher concentrations significantly depressed cell viability. DNA laddering analysis revealed that the viability reduction was not related to apoptosis, suggesting that taxol itself was the primary responsible factor. On the basis of RT-PCR expression analysis of genes encoding taxadiene synthase (ts) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) from treated and nontreated T. baccata cell line cultures, it was observed that exogenous taxol clearly induced the mRNA levels of both taxane-related enzymes. Additionally, we found that exogenous taxol caused a considerable increase in taxadiene synthase activity, although in no case did this coincide with the highest levels of taxol observed at the end of the culture. The effect of exogenous taxol on the content of other related taxanes was also considered.

Lipid signaling involved in mechanotransduction processes in response to shear stress in plants remained elusive. To understand the responses of phospholipids in shear stress-induced mechanotransduction, a lipidomic approach was employed to profile phospholipid species of Taxus cuspidata cells under laminar shear stress. A total of 99 phospholipid species were profiled quantitatively, using the LC/ESI/MS(n) procedure. Potential biomarkers were found by the principal component analysis (PCA) as well as partial least squares (PLS) combined with variable influence in the projection (VIP). Phosphatidic acid (PA) and lysophosphatidylcholine (LysoPC) were two important lipid groups that were responsible for the discrimination between shear stress induced and control cells. Further research revealed that shear stress enhanced the activation of phospholipase D (PLD) and phospholipase C (PLC) compared with control cells and consequently increased PA content in shear stress induced T. cuspidata cells. These results demonstrate that phospholipids and related phospholipases play important roles in mechanotransduction of T. cuspidata cells in response to shear stress.

Methyl jasmonate (MeJA) and cerium (Ce(4+)) elicitation share common features of increasing taxol accumulation of Taxus cuspidata cells. Interestingly, Ce(4+) induces programmed cell death (PCD), but this phenomenon is not observed with MeJA elicitation. Here, using a lipidomic approach to measure more than 100 membrane glycerophospholipids of T. cuspidata cells quantitatively, we discovered that lysophosphatidylcholine (LysoPC), phosphatidic acid (PA) and phosphatidylcholine were three potential lipid markers that were responsible for the differences between Ce(4+)-induced cells and MeJA-induced cells. Compared with MeJA elicitation, marked increase of phospholipase D (PLD) activity was observed following Ce(4+) elicitation, suggesting that the PLD activation and high concentrations of PA production might mediate the PCD. Rapid increase of phospholipase A(2) (PLA(2)) activity caused the release of fatty acids and LysoPC following Ce(4+) elicitation, which enhanced endogenous jasmonic acid (JA) accumulation. In contrast, PLA(2) activity was poorly induced following MeJA elicitation. PLA(2) inhibitor suppressed not only JA accumulation but also taxol production, suggesting that the PLA(2) activation mediated Ce(4+)-induced taxol production partially through a JA-dependent signaling pathway. These results demonstrate that differential alternation of glycerolphospholipids caused by phospholipases constitutes an important step in cell death response to Ce(4+) and increasing taxol production.

The influences of salicylic acid (SA) on taxol production and isopentenyl pyrophosphate (IPP) biosynthesis pathways in suspension cultures of Taxus chinensis var. mairei were investigated by adding SA and mevastatin (MVS), a highly specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase in the mevalonate pathway for IPP biosynthesis, into the culture systems. The cell death and taxol production were induced upon the introduction of SA, and 20mg/l was proved to be the optimal SA concentration in terms of the less damage to Taxus cells and marked activation of phenylalanine ammonia lyase (PAL). In the coexistence of SA (20mg/l) and MVS (100 nmol/l), the taxol content (1.626 mg/g dry wt) was higher than that (0.252 mg/g dry wt) of the MVS-treated system but almost equal to that (1.581 mg/g dry wt) of the SA-treated system. It is thus inferred that the activated non-mevalonate pathway should be responsible for the formation of IPP in taxol biosynthesis in the presence of SA.

A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2. Additionally, a cell line of T. chinensis has been transformed with wild-type A. rhizogenes ATCC strain 25818 containing binary vector pCAMBIA1301. The two transgenic T. cuspidata cell lines have been maintained in culture for more than 20 months, and the transgenic T. chinensis cell line for more than 9 months, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernable effect on growth or Taxol production in the transgenic cell lines when compared to the parent control. The methods for transforming non-embryogenic Taxus suspension cultures are described.

Plant cells cultured in bioreactors are strongly influenced by mechanical forces. However, the molecular mechanism of plant cell mechanoreception has maintained unclear. In animal cells, the Arg-Gly-Asp (RGD) motif can be found in proteins of the extracellular matrix. Integrins link the intracellular cytoskeleton of cells with the extracellular matrix by recognizing this RGD motif. Integrin has been demonstrated to function as an apparatus not only for adhesion but also for mechanotransduction. In plant cells, the molecules that mediate the structural continuity between wall and membrane are unknown. Here, we found that synthetic RGD peptide could dramatically reduce the level of phosphorylation of MAPK-like cascades that are activated by shear stress and reduce the alkalinization response, production of reactive oxygen species (ROS) and accumulation of phenolics by Taxus cuspidata cells during shear stress. These results implicate that a RGD recognition system may exist in Taxus cells and play an important role in signal transduction of shear stress. Although the Arabidopsis genome database shows that the plant seems to lack a homologue of animal integrin, plant cells may use other RGD-binding proteins to recognize the RGD motif. The correlative mechanism is discussed.

In order to establish the chemical biological technology for production of valuable secondary metabolites, a novel family of unnatural elicitors derived from the plant activator benzo-1,2,3-thiadiazole-7-carboxylic acid were designed and synthesized. New synthetic elicitors that showed powerful eliciting activities upon taxoid biosynthesis by Taxus chinensis suspension cells were obtained. For example, benzo-1,2,3-thiadiazole-7-carboxylic acid 2-(2-hydroxybenzoxyl)ethyl ester was more effective and resulted in nearly 40% increase in taxuyunnanine C content and production in comparison with methyl jasmonate, which was previously reported as the most powerful chemical elicitor for taxoid biosynthesis. The novel class of elicitors was found to induce plant defense responses, including promotion of H(2)O(2) levels originating from oxidative burst and activation of phenylalanine ammonia lyase. Interestingly the plant defense responses induced corresponded well to the superior stimulating activity in T. chinensis cell cultures. The work indicates that the newly synthesized benzothiadiazoles can act as a new family of elicitors for taxoid biosynthesis in plant cells.

Cell cultures of Taxus canadensis were subjected to exogenously applied ethylene (ET) hormone and methyl jasmonate (MJ) elicitation in factorial design experiments. Levels of extracellular taxanes, including paclitaxel, were used with principal component analysis for fault detection and real-coded genetic algorithms for parameter optimization to construct a culture sub-population induction model. Culture sub-populations were identified by the model as (1) uninduced, (2) induced to unilateral function of the ET-signaling pathway, and (3) induced to cooperation between jasmonic acid (JA)- and ET-signaling pathways. Comprehensive model results suggested greater rates of cellular induction (resulting in exogenous taxane production) by ET gas as opposed to MJ elicitation. However, cellular induction of ET-signaling pathway genes increased the rate of induction of JA-signaling pathway genes by orders of magnitude. In addition, model results showed that induction of genes leading to extracellular production of the simple taxane 10-deacetylbaccatin III was regulated by the unilateral ET-signaling pathway. However, it was suggested that further processing of this simple taxane to complex taxane structures, such as paclitaxel, required further gene induction by the JA-signaling pathway. Thus, production rate constants of exogenous complex taxanes were predicted to be an order of magnitude lower than that for the simple taxane 10-deacetylbaccatin III. The fraction of the cell culture sub-population displaying unilateral ET-signaling pathway gene induction was found inversely proportional to levels of MJ elicitation. When coupled with simple non-growth product models, levels of all extracellular taxanes were effectively predicted using the culture sub-population induction model.

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., ((c) 2006 Wiley Periodicals, Inc.)

Recently novel synthetic jasmonate derivatives were shown to have very powerful stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells (Biotech Bioeng, 86:595; ibid. 86: 809, 2004). To provide an insight into the elicitation mechanism of the newly synthesized elicitors, by taking 2-hydroxyethyl jasmonate (HEJ, at 100 microM) as a typical example, in this work the defense signals were detected and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in cell cultures of T. chinensis. The oxidative burst (induced H2O2 production) was confirmed, and the induction of lipoxygenase (LOX) activity and intracellular jasmonate acid (JA) synthesis was found. The gene transcription of geranylgeranyl diphosphate synthase (GGPPs) and taxa-4(5),11(12)-diene synthase (TS) was up-regulated by HEJ elicitation compared to control. Inhibition of JA biosynthesis by a putative LOX inhibitor, ibuprofen (IBU), effectively depressed the HEJ-induced up-regulation of GGPPs and TS gene transcription levels. In contrast, the inhibition of H2O2 production by membrane NADPH oxidase inhibitor, diphenylene iodonium (DPI), did not affect the transcriptions of those genes. For the HEJ-induced Tc production, it was suppressed with addition of DPI or IBU. The results suggest that both H2O2 and JA signals were involved in HEJ-induced Tc biosynthesis, and JA mediated the induction of GGPPs and TS genes expression, but H2O2 was not essential to activate them. Finally, a signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed., (Copyright 2006 Wiley Periodicals, Inc.)

Two novel 2,6-dichloroisonicotinic acid (INA) derivatives [trifluoroethyl 2,6-dichloroisonicotinate (TFINA) and 2-(2,6-dichloro-pyridine-4-carbonyloxy)-ethyl jasmonate (DPCEJ)] were chemically synthesized and evaluated by bioassay as potential elicitors for inducing the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which stably produces a high level of bioactive taxuyunnanine C (Tc), was taken as a model plant cell system. A significant increase in Tc accumulation was observed in the presence of TFINA or DPCEJ. For example, addition of 100 microM TFINA or DPCEJ on day 7 led to a high Tc content of 21.6 +/- 2.0 or 27.7 +/- 1.0 mg g(-1) (on day 21), while the Tc content was 13.7 +/- 1.0 and 17.1 +/- 0.9 mg g(-1) for the control and that with addition of 100 microM INA, respectively. To the best of our knowledge, this is the first report on the use of synthetic INA derivatives for inducing the biosynthesis of plant secondary metabolites. The results indicate that the newly synthesized INA analogues can act as promising elicitors for secondary metabolism induction in plant cell cultures.

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.

Ce(4+) (Ce(NH(4))(2)(NO(3))(6)) at 1mM induces apoptosis of suspension cultures of Taxus cuspidata cells; however, the underlying signal mechanisms are unknown. We show here that a 46-kDa ERK (extracellular signal-regulated kinase)-like MAPK appears to be down-regulated at 4h, and remains at low levels for up to 48 h. An inhibitor of superoxide anions (O(2)(-)) generation, diphenyl iodonium (DPI) successfully blocks down-regulation of ERK-like MAPK and degradation of DNA. Moreover, a 41-kDa p38-like MAPK activity remains unchanged from 0.5 to 48 h. The p38 inhibitor SB202190 effectively inhibits p38-like MAPK activity, however, SB202190 fails to modify the apoptotic rate at concentrations up to 100 microM. Three nuclease (34-kDa, 22-kDa and 20-kDa) activities are profoundly enhanced in Ce(4+)-induced T. cuspidata cells. They have an optimum pH at 6.8, and are stimulated by Ca(2+)/Mg(2+). Caspase-3 inhibitor, Ac-DEVD-CHO, does not attenuate the 34-kDa nuclease activity, but inhibits the 22-kDa and the 20-kDa nuclease activities. In addition, inhibition of O(2)(-) generation by DPI significantly reduces the three nuclease activities. In conclusion, the present study suggests that down-regulation of ERK-like MAPK, burst of O(2)(-), activation of caspase-3-like and induction of three nucleases as the key signaling events mediating apoptosis in Ce(4+)-induced cultured T. cuspidata cells.

Trifluoroethyl salicylate (TFESA), a novel salicylate derivative, was chemically synthesized and evaluated by bioassay as a potential elicitor for inducing the biosynthesis of plant secondary metabolites. A cell line of Taxus chinensis, which stably produced a high level of bioactive taxuyunnanine C (Tc), was taken as a model plant cell system. The application of TFESA to the cell cultures could significantly induce Tc biosynthesis, although the cell growth was slightly inhibited. More interestingly, Tc production was enhanced more in the presence of TFESA compared with a structure-similar well-known elicitor, salicylic acid (SA). For example, addition of 100 microM TFESA on day 7 led to a high Tc content of 21.9 +/- 0.1 mg g(-1) (at day 21), whereas the Tc content was 14.0 +/- 0.2 and 16.7 +/- 0.3 mg g(-1) for the control and that with addition of 100 microM SA, respectively. The results indicate that the newly synthesized TFESA can act as a powerful elicitor for secondary metabolism induction in plant cell cultures.

A novel fluoro-containing jasmonate derivative was chemically synthesized and evaluated as a potential elicitor with respect to the induction of plant defense responses and the biosynthesis of plant secondary metabolites. A bioactive taxuyunnanine C (Tc)-producing cell line of Taxus chinensis was taken as a model plant cell system. The presence of novel synthesized pentafluoropropyl jasmonate (PFPJA) induced two early and important events in plant defense responses, including an oxidative burst and activation of L-phenylalanine ammonia lyase. In addition, PFPJA was found to significantly increase Tc accumulation, without any inhibition of cell growth. Moreover, Tc accumulation was increased more in the presence of PFPJA compared with methyl jasmonate (MJA) and previously reported trifluoroethyl jasmonate (TFEJA). For example, addition of 100 muM PFPJA on day 7 led to a high Tc content (38.2 +/- 0.3 mg/g) at day 21, while the Tc content was 29.3 +/- 0.3 mg/g and 34.9 +/- 0.9 mg/g with the addition of 100 microM MJA and TFEJA, respectively. Quantitative structure-activity analysis of fluoro-containing jasmonates suggests that the increase in the fluoro-groups introduced into the carboxyl side-chain of MJA resulted in a higher stimulatory activity for Tc biosynthesis, which corresponds well with the markedly increased lipophilicity after fluorine introduction. These results indicate that newly synthesized fluoro-containing PFPJA can act as a powerful chemical inducing signal for secondary metabolism in plant cell cultures.

Methyl jasmonate (MeJA), a methyl ester of jasmonic acid (JA), is a well-established signal molecule in plant defense responses and an effective inducer of secondary metabolite accumulation in plant cell cultures such as the valuable anticancer diterpenoid taxol (paclitaxel) in Taxus spp. This work examines the involvement of nitric oxide (NO) in MeJA-induced plant defense responses and secondary metabolism in Taxus chinensis cell cultures. Exogenously supplied MeJA at 100 microM induced rapid production of NO in the Taxus cell cultures, reaching a maximum within 6 h of MeJA supply. Several other responses occurred concomitantly, including the production of hydrogen peroxide (H2O2), and the increases in intracellular malondialdehyde (MDA) content, lipoxygenase (LOX) and phenylalanine ammonium-lyase (PAL) activities. The MeJA-induced H2O2 production was suppressed by an NO donor, sodium nitroprusside (SNP), but enhanced by NO inhibitors, N (omega)-nitro-L-arginine (L-NNA) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO). In contrast, the MeJA-induced MDA, LOX and PAL were all enhanced by the NO donor but suppressed by the NO inhibitors. The NO inhibitors also suppressed MeJA-induced taxol accumulation. These results are suggestive of a role for NO as a signal element for activating the MeJA-induced defense responses and secondary metabolism activities of plant cells.

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites., ((c) 2005 Wiley Periodicals, Inc.)

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.

There is a dichotomy in the biosynthetic pathway of terpenoid precursor isopentenyl diphosphate (IPP) in higher plant. One is the classical mevalonate pathway in cytosol, and the other is non-mevalonate pathway in plastid. To know the origin of the taxane ring system of taxol in suspension culture of Taxus chinensis, lovastatin and fosmidomycin were used to block the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (DXR) in the mevalonate and non-mevalonate branch respectively of the terpenoid biosynthetic pathway. Methyl jasmonate (MJ) was used to improve the biosynthesis of taxol. Taxol content was determined by HPLC, the transcriptional expression of genes encoding DXR and HMGR were investigated by real time PCR. Taxol production was lowered by about 2/5 and 1/5 by fosmidomycin (200 mmol/L) and fosmidomycin (200 mmol/L)+MJ (100 mmol/L) treatment respectively, and was lowered by about 1/6 and 1/10 by lovastatin (1 mmol/L) and lovastatin (1 mmol/L) + MJ (100 mmol/L) respectively, which means that both mevalonate and non-mevalonate pathway contribute to taxol biosynthesis, and the latter is the main source of IPP. Inhibitors lovastatin and fosmidomycin both promoted the transcriptional expression of hmgr and dxr, which indicated a metabolic cross talk between cytosolic and plastidial pathways of taxol biosynthesis.

Two-liquid-phase plant cell cultures employ the use of a partitioning system to redirect extracellular product into a second phase. After the addition of organic solvent, in order to understand the defense system of Taxus cuspidata cells to organic solvent in two-liquid-phase suspension cultures, we investigated cells' antioxidant metabolism. The results showed that T. cuspidata cells responded to oleic acid with oxidative bursts in both intracellular H2O2 and extracellular O2-* production. Inhibition studies with diphenylene iodonium suggested that the key enzyme responsible for oxidative bursts was primarily NADPH oxidase. Investigation of the relationship between reactive oxygen species (ROS) and defense responses induced by oleic acid indicated that 4% (v/v) oleic acid increased the levels of antioxidant enzymes of superoxide dismutase, ascorbate peroxidase, and catalase and the antioxidant capacity of reduced ascorbate and glutathione. However, when oleic acid content reached a critical value (6% [v/v]), no further increase in antioxidant enzymes and antioxidant capacity was observed, indicating that the defense responses played a role in a certain range of oleic acid content, beyond which the overall ROS scavenging machinery was not induced and the peroxidation of membrane lipids emerged.

Paclitaxel and baccatin III-producing cells of Taxus baccata were immobilized within Ca(2+)-alginate beads. Under established optimum conditions for the biosynthesis of both taxanes, the yields of paclitaxel and baccatin III in shake-flask cultures of free cells increased by factors of up to 3 and 2, respectively, in the corresponding cultures of immobilized cells. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities when both free and immobilized cells were grown in the same optimum conditions in three different bioreactor types (Stirred, Airlift, and Wave) running for 24 days in a batch mode and with the system optimized in each case, there was a considerable increase in the yields of paclitaxel and baccatin III. Among the reactors, the Stirred bioreactor was the most efficient in promoting immobilized cell production of paclitaxel, giving a content of 43.43 mg.L(-1) at 16 days of culture, equivalent to a rate of 2.71 mg.L(-1).day(-1). To our knowledge, the paclitaxel productivity obtained in this study is one of the highest reported so far by academic laboratories for Taxus species cultures in bioreactors.

This work was to characterize the generation of nitric oxide (NO) in Taxus chinensis cells induced by a fungal elicitor extracted from Fusarium oxysporum mycelium and the signal role of NO in the elicitation of plant defense responses and secondary metabolite accumulation. The fungal elicitor at 10-100 microg/ml (carbohydrate equivalent) induced a rapid and dose-dependent NO production in the Taxus cell culture, which exhibited a biphasic time course, reaching the first plateau within 1 h and the second within 12 h of elicitor treatment. The NO donor sodium nitroprusside potentiated elicitor-induced H2O2 production and cell death but had little influence on elicitor-induced membrane K+ efflux and H+ influx (medium alkalinization). NO inhibitors Nomega-nitro-L-arginine and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide partially blocked the elicitor-induced H2O2 production and membrane ion fluxes. Moreover, the NO inhibitors suppressed elicitor-induced activation of phenylalanine ammonium-lyase and accumulation of diterpenoid taxanes (paclitaxel and baccatin III). These results suggest that NO plays a signal role in the elicitor-induced responses and secondary metabolism activities in the Taxus cells.

The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable. To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics. With Taxus subcultures we observe alternating states of high and low productivity. Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one. These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing. High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition. Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment. Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties. To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture. Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures. The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.

To develop more effective abiotic elicitors for cell suspension cultures of T. chinensis to meet the needs for paclitaxel as anti-tumor drug, some fluoro- or hydroxyl-containing groups are introduced to the ester moiety of jasmonic acid by the esterification or acylation with bis(trichloromethyl) carbonate and corresponding alcohol. Some of them are found to be novel and effective elicitors, which can enhance the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate (MJA) in T. chinensis cell cultures.

Novel hydroxyl-containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 microM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl-containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 microM 2,3-dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 +/- 0.5 mg/g (at day 21), whereas the Tc content was 29.2 +/- 0.6 mg/g (on the same day) with addition of 100 microM MJA. Quantitative structure-activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H(2)O(2) production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl-containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures., (Copyright 2004 Wiley Periodicals, Inc.)

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., (Copyright 2004 Wiley Periodicals, Inc.)

This study evaluates the use of a novel mechanical stimulus, ultrasound (US), and a putative chemical elicitor, methyl jasmonate (MJ), combined with in situ solvent extraction (two-phase culture), to enhance taxol production by Taxus chinensis cells in suspension culture. The volumetric taxol yield was increased 1.5- to 1.8-fold with 2 min US treatment once or twice during a 4-week culture period, about 5-fold with 60-120 microM MJ, and 7- to 9-fold by in situ solvent extraction of taxol with dibutyl phthalate (DBP) (11% v/v). The percent of extracellular taxol or taxol release was also significantly increased. The combined use of US (day 5 or 9) and MJ treatment (day 7) resulted in taxol yields 20-50% higher than each of the treatments used alone. The most favorable strategy for taxol production was the application of US or MJ treatment, followed by in situ solvent extraction, giving rise to a taxol yield of 33-35 mg/l, about 17-fold higher than the control, at 1.9 mg/l. It was found that the organic solvent DBP, as well as US and MJ, stimulated the enzyme activity of secondary metabolic pathways, which was partially responsible for the enhanced taxol production.

Conifers have defenses such as the production of phenolic compounds and resins that can be induced by bark beetles and other invading organisms, but the signaling agents involved are unknown. The anatomical effects of methyl jasmonate (MJ), a potent inducer of certain plant defenses, were compared with wounding of the bark of 12-15-year-old trees of five conifer species. Wounding in all species resulted in tissue necrosis and wound periderm development immediately around the wound site. One cm from the wound, swelling of phloem polyphenolic parenchyma cells and phenolic accumulation were observed in Pseudotsuga menziesii (Mirb.) Franco, Picea pungens Engelman, Larix occidentalis Nutt. and Pinus monticola Douglas ex D. Don, but not in Taxus brevifolia Nutt. Traumatic resin ducts were formed in response to wounding in three species of Pinaceae, but not in P. monticola, which formed irregular clusters of cells rather than ducts. Taxus brevifolia did not form resin ducts in response to either wounding or MJ treatment. In the Pinaceae species studied, surface application of 100 mM MJ caused similar anatomical changes to those observed in response to wounding, including phenolic accumulation, cell swelling and traumatic resin duct formation, but it did not induce a wound periderm. Traumatic resin ducts differed in size among the study species, ranging from small in L. occidentalis to very large in P. menziesii. In P. menziesii, P. pungens and L. occidentalis, traumatic resin ducts were more abundant after MJ treatment than after wounding. We conclude that the octadecanoid pathway is likely involved in defense responses in stems of the Pinaceae, but not necessarily in other taxa.

To achieve a better understanding of the metabolism and accumulation of paclitaxel and baccatin III in cell cultures of Taxus, inhibitors of the early steps in the terpenoid pathway were applied to a cell suspension culture of Taxus baccata: fosmidomycin as an inhibitor of the non-mevalonate branch of the pathway, and mevinolin as an inhibitor of the mevalonate branch. Synthesis of both taxanes in the cell suspension was first increased when cultured in the product formation medium supplemented with methyljasmonate (100 microM). The product formation medium was selected after assaying 24 different culture media. When fosmidomycin (200 microM) was added to the product formation medium together with the elicitor, the accumulation of paclitaxel and baccatin III was reduced by up to 3.0 and 1.5 times, respectively, whereas the inhibitory effect of mevinolin (1 microM) was only clearly exerted in the case of paclitaxel. Under the conditions of our experiment, we conclude that in the synthesis of both taxanes, the non-mevalonate pathway is the main source of the universal terpenoid precursor isopentenyl diphosphate (IPP)., (Copyright 2002 Elsevier Science B.V.)

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.

Apoptotic cell death in suspension cultures of Taxus cuspidata induced by exogenous salicylic acid and/or H2O2 was investigated. H2O2 (0.012% v/v) alone changed the permeability of cell membrane while salicylic acid (0.375 mM) not only altered the permeability but also caused nuclei condensation and a small amount of nuclei fragments. The combined use of salicylic acid (0.375 mM) and H2O2 (0.012% v/v) changed the cell membrane permeability more significantly and nuclei fragments occurred in ca. 30% of the cells at 48 h. DNA ladders of 180 bp and oligopolymers, characteristics of the apoptotic cleavage of nuclei DNA, were observed by agar electrophoresis. These results show that exogenous salicylic acid and H2O2 could synergistically induce the apoptotic cell death of suspension cultures of Taxus cuspidata.

Some metabolic inhibitors, mevastatin (MVS), chlorocholine chloride (CCC), sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), were respectively added into the suspension cultures of the yew Taxus chinensis var. mairei at the late phase of cell growth to study isopentenyl pyrophosphate (IPP) biosynthesis. The content of total taxanes decreased in the cases of MVS, NaPP and DLG addition, regardless of the inhibitor concentration, whereas it increased in the case of CCC addition. It was thus presumed that a mevalonate pathway might exist in IPP biosynthesis at the late growth phase of Taxus cells and that some IPP might transfer from the cytoplasm to the plastids. This presumption was also confirmed by analysing the effects of the inhibitors on taxane content. It was further demonstrated that IPP in the biosynthesis of taxol, baccatin III and 10-deacetylbaccatin III might transfer from the cytoplasm to the plastids, whereas the translocation of IPP might be not involved in cephalomannine biosynthesis. Furthermore, in combination with the results of previous studies, it is very probable that different IPP biosynthesis pathways exist during different growth phases in Taxus cells.

Cell suspension cultures of Taxus chinensis, with 20, 40 and 100 mg fungal elicitor l(-1) from Aspergillus niger, underwent rapid cell death after 24 h, which was about 2, 3.7 and 5-fold of that of the control. At the same time, Taxol production was increased, respectively, to about 5, 8 and 3-fold of that of the control. Inhibition of phenolics biosynthesis resulted in a 150% increase in cell death but a 54% decrease in Taxol production compared with 40 mg elicitor l(-1) alone. O2-free N2 inhibited cell death but had little effect on Taxol production as induced by 40 mg fungal elicitor l(-1).

Suspension cultures of Taxus yunnanensis cells were inoculated with cells of different culture ages (12-24 days) at various densities [50-250 g fresh weight (fw)/l], and treated (on day 7) with a mixture of elicitors, including Ag(+), chitosan and methyl jasmonate. The biomass productivity (during the production stage) increased dramatically with inoculum size, but decreased with inoculum age over 16 days. The volumetric yield and productivity of taxol (paclitaxel) also increased with inoculum size, while the specific taxol yield (per cell) was mainly dependent on inoculum age, with an optimum of 20 days, during the early stationary phase. The highest taxol yield and productivity, 39.8 mg/l and 1.9 mg/l per day, respectively, were obtained with a 20-day-old inoculum at 200 g fw/l. Taxol excretion by the cells increased with inoculum age but decreased with inoculum size. The elicitor-induced activities of catalase (CAT) and phenylalanine ammonia-lyase (PAL) also depended mainly on inoculum age; higher PAL activity and lower CAT activity were obtained with an older inoculum, corresponding to a higher taxol yield. The results show that both inoculum size and age are important variables for taxol production, though the latter more profoundly influences elicitor-induced taxol biosynthesis of the cells. Inoculum size and age are also interrelated and should be optimized together in a two-stage culture process.

Addition of taxol into suspension cultures of Taxus cuspidata induced cell apoptosis, which was confirmed by gel electrophoresis of the DNA ladders indicating the progressive delineation of fragmented nuclear DNA (nDNA) into distinct bodies. The additional taxol not only changed the microtubule assembly of cells, but also affected the gene expression. Fourteen cDNA fragments, named as TIGT9-22, were isolated after addition of taxol and their GenBank accession numbers were given as BF704560-BF704573, respectively. Among them, TIGT13 and TIGT21 were apparently homogeneous with apbE and carbamoylphosphate synthetase, respectively. Other cDNA fragments showed no significant analogy with the known sequences in GenBank.

The trivalent ion of a rare earth element, lanthanum, was tested for elicitor-like effects on taxol production in suspension cultures of four different Taxus spp cells. In T. yunnanensis cell cultures, the lanthanum ion at concentrations from 1.15 to 23.0 microM stimulated taxol production. The lanthanum ion also promoted taxol excretion by the T. yunnanensis cells considerably. The maximum stimulation of taxol production was achieved by the addition of 5.8 microM La3+ to the culture during mid-log growth phase, increasing the volumetric taxol yield by nearly threefold, from 2.61+/-0.37 to 9.89+/-1.92 mg l(-1) over a 28 day culture period. At higher concentrations, i.e. 23.1 and 46.2 microM, however, the lanthanum ion caused significant growth inhibition. For the other three Taxus cell lines, namely an embryo and a leave cell of T. chinensis and a stem cell of T. chinensis marv, the addition of lanthanum ion to the culture only had a significant effect on taxol production by the T. chinensis marv stem cells, increasing the volumetric yield by about threefold to 4.69+/-0.76 mg l(-1). These results suggest that lanthanum has elicitor-like effects on secondary metabolite synthesis of plant cell cultures.