Tree Taxus Research Articles

A fungal endophyte induces transcription of genes encoding a redundant fungicide pathway in its host plant.

BackgroundTaxol is an anti-cancer drug harvested from Taxus trees, proposed ecologically to act as a fungicide. Taxus is host to fungal endophytes, defined as organisms that inhabit plants without causing disease. The Taxus endophytes have been shown to synthesize Taxol in vitro, providing Taxus with a second potential biosynthetic route for this protective metabolite. Taxol levels in plants vary 125-fold between individual trees, but the underlying reason has remained unknown.ResultsComparing Taxus trees or branches within a tree, correlations were observed between Taxol content, and quantity of its resident Taxol-producing endophyte, Paraconiothyrium SSM001. Depletion of fungal endophyte in planta by fungicide reduced plant Taxol accumulation. Fungicide treatment of intact plants caused concomitant decreases in transcript and/or protein levels corresponding to two critical genes required for plant Taxol biosynthesis. Taxol showed fungicidal activity against fungal pathogens of conifer wood, the natural habitat of the Taxol-producing endophyte. Consistent with other Taxol-producing endophytes, SSM001 was resistant to Taxol.ConclusionsThese results suggest that the variation in Taxol content between intact Taxus plants and/or tissues is at least in part caused by varying degrees of transcriptional elicitation of plant Taxol biosynthetic genes by its Taxol-producing endophyte. As Taxol is a fungicide, and the endophyte is resistant to Taxol, we discuss how this endophyte strategy may be to prevent colonization by its fungal competitors but at minimal metabolic cost to itself.

Getting to the bottom of Taxol biosynthesis by fungi

Taxol (paclitaxel) is a highly-oxygenated diterpenoid natural product first isolated from the pacific yew tree (Taxus brevifolia). It is one of the most widely used anticancer drugs. Soon after the discovery of its unique mode of action and the resulting high demand, an extensive search was initiated for alternative sources to replace the slow-growing and scarce pacific yew. Thus far, however, Taxol and related compounds have only been found in the genus Taxus, which comprises a small number of slow-growing plants with a broad but generally isolated geographical distribution. In 1993, Stierle and colleagues reported the independent biosynthesis of Taxol in an endophytic fungus isolated from Taxus brevifolia, which resulted in more than 160 subsequent publications and patents addressing the biosynthesis of Taxol and related taxanes by microorganisms. The literature on fungal taxane synthesis contains numerous inconsistencies, prompting us to thoroughly re-examine Taxol biosynthesis in endophytic fungi associated with Taxus spp. Using a combination of phytochemistry, molecular biology and genome sequencing, we were unable to find any evidence for independent taxane biosynthesis in any of the endophytes, including the isolate described in the original publication (Taxomyces andreanae) and several more recent isolates from Taxus trees. Our findings therefore resolve a long-standing mystery concerning the evolution of a complex terpenoid biosynthetic pathway in two distantly-related organisms.

Interactions between Co-Habitating fungi Elicit Synthesis of Taxol from an Endophytic Fungus in Host Taxus Plants

Within a plant, there can exist an ecosystem of pathogens and endophytes, the latter described as bacterial and fungal inhabitants that thrive without causing disease to the host. Interactions between microbial inhabitants represent a novel area of study for natural products research. Here we analyzed the interactions between the fungal endophytes of Taxus (yew) trees. Fungal endophytes of Taxus have been proposed to produce the terpenoid secondary metabolite, Taxol, an anti-cancer drug. It is widely reported that plant extracts stimulate endophytic fungal Taxol production, but the underlying mechanism is not understood. Here, Taxus bark extracts stimulated fungal Taxol production 30-fold compared to a 10-fold induction with wood extracts. However, candidate plant-derived defense compounds (i.e., salicylic acid, benzoic acid) were found to act only as modest elicitors of fungal Taxol production from the endophytic fungus Paraconiothyrium SSM001, consistent with previous studies. We hypothesized the Taxus plant extracts may contain elicitors derived from other microbes inhabiting these tissues. We investigated the effects of co-culturing SSM001 with other fungi observed to inhabit Taxus bark, but not wood. Surprisingly, co-culture of SSM001 with a bark fungus (Alternaria) caused a ∼threefold increase in Taxol production. When SSM001 was pyramided with both the Alternaria endophyte along with another fungus (Phomopsis) observed to inhabit Taxus, there was an ∼eightfold increase in fungal Taxol production from SSM001. These results suggest that resident fungi within a host plant interact with one another to stimulate Taxol biosynthesis, either directly or through their metabolites. More generally, our results suggest that endophyte secondary metabolism should be studied in the context of its native ecosystem.

Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

Background and purpose of the studyTaxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale.MethodsWe investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing.ResultsThe yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1) and 10-deacetyl baccatin III (4.2 mgl-1) were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1) in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1), Baccatin III (5.0 mgl-1), and 10-deacetyl baccatin III (1.45 mgl-1) were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

Adjuvant effect of docetaxel on the immune responses to influenza A H1N1 vaccine in mice.

BackgroundVaccination remains one of the most effective approaches to prevent the spread of infectious diseases. Immune responses to vaccination can be enhanced by inclusion of adjuvant in a vaccine. Paclitaxel extracted from the bark of the Pacific yew tree Taxus brevifola was previously demonstrated to have adjuvant property. Compared to paclitaxel, docetaxel is another member of taxane family, and is more soluble in water and easier to manipulate in medication. To investigate the adjuvant effect of this compound, we measured the immune responses induced by co-administration of a split inactivated influenza H1N1 vaccine antigen with docetaxel.ResultsWhen co-administered with docetaxel, lower dose antigen (equivalent to 10 ng HA) induced similar levels of IgG and IgG isotypes as well as HI titers to those induced by higher dose antigen (equivalent to 100 ng HA). Docetaxel promoted splenocyte responses to H1N1 antigen, ConA and LPS, mRNA expressions of cytokines (IFN-gamma, IL-12, IL-4 and IL-10) and T-bet/GATA-3 by splenocytes. The enhanced immunity was associated with up-expressed microRNAs (miR-155, miR-150 and miR-146a) in docetaxel-stimulated RAW264.7 cells. Docetaxel promoted similar IgE level to but alum promoted significantly higher IgE level than the control.ConclusionDocetaxel has adjuvant effect on the influenza H1N1 vaccine by up-regulation of Th1/Th2 immune responses. Considering its unique vaccine adjuvant property as well as the safe record as an anti-neoplastic agent clinically used in humans during a long period, docetaxel should be further studied for its use in influenza vaccine production.

Chemical Inhibitors Suggest Endophytic Fungal Paclitaxel Is Derived from Both Mevalonate and Non-mevalonate-like Pathways

Taxus trees possess fungal endophytes reported to produce paclitaxel. Inhibitors that block early steps in plant paclitaxel biosynthesis were applied to a paclitaxel-producing fungus to determine whether these steps are shared. The plant paclitaxel backbone is reportedly derived from the non-mevalonate terpenoid pathway, while the side chain is phenylalanine-derived. Evidence that the shikimate pathway contributes to fungal paclitaxel was shown by decreased paclitaxel accumulation following inhibition of phenylalanine ammonia lyase. Expression of another shikimate pathway enzyme, 3-dehydroquinate synthase, coincided with paclitaxel production. The importance of the mevalonate pathway in fungal paclitaxel biosynthesis was shown by inhibition of fungal paclitaxel accumulation using compactin, a specific inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase. Expression of another mevalonate pathway enzyme, 3-hydroxy-3-methyl-glutaryl-CoA synthase, coincided with fungal paclitaxel accumulation. Unexpectedly, results from using fosmidomycin suggested that fungal paclitaxel requires 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), an enzyme in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway normally found in bacteria/plants. Additional lines of evidence support this finding; first, a plant DXR antibody recognized a fungal peptide of the correct size; second, expression of an apparent fungal DXR ortholog correlated to changes in paclitaxel production; finally, BLAST searching identified a gene putatively encoding 1-deoxy-D-xylulose-5-phosphate synthase, the first enzyme in the MEP pathway in Aspergillus.

Nab‐Paclitaxel: a bright new SPARC in taxane therapy of cancer

Nab‐Paclitaxel: a bright new SPARC in taxane therapy of cancer

Rapid and simple methodology for isolation of high quality genomic DNA from coniferous tissues (Taxus baccata)

Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.

Taxol: A complex diterpenoid natural product with an evolutionarily obscure origin

Taxol, a diterpenoid natural product first isolated from Taxus brevifolia, is one of today’s better known anticancer drugs. Despite its clinical efficacy, the difficulty of establishing a secure and cost-effective supply of taxol has limited its use. However, its unique mode of action and efficacy against multiple forms of cancer has ensured continual efforts to achieve total and semisynthesis, as well as biotechnological production methods. Total synthesis is now possible but inefficient, so the production of taxol and related taxoids remains completely dependent on biomass derived from Taxus sp, with cell suspensions and collected plant materials as sources. The key to improving the supply of taxol and other clinically useful taxoids is the detailed elucidation of the taxoid biosynthesis pathway, which has been the subject of intense research. Many genes and enzymes in the Taxus pathway for taxoid biosynthesis have now been identified, although gaps remain. In addition to Taxus sp, taxoids are also synthesized by various endophytic fungi, which often live in association with Taxus trees, thus raising questions about the evolutionary origin of this complex diterpenoid pathway. In the future, it may be possible to improve taxoid synthesis through the genetic modification of Taxus cell cultures, by culturing endophytic fungi or by transferring the entire pathway into a heterologous expression host, such as Saccharomyces cerevisiae.

Do secondary sexual dimorphism and female intolerance to drought influence the sex ratio and extinction risk of Taxus baccata?

Sex ratio and sexual dimorphism were studied in the dioecious tree Taxus baccata. We examined five populations of T. baccata in Poland and Ukraine to identify the differences between male and female individuals. The sex of all individuals, height and diameter, needle length and area, specific leaf area (SLA), the number of stomata rows, stomatal density, and content of carbon and nitrogen were measured to identify the differences between male and female individuals. The relationship between sex ratio and climatic conditions, age and population size were analysed using data collected from the field and the literature. Female trees were shorter than males, but needles of females were longer and had larger area. Although there were no differences among sexes in SLA, nitrogen and carbon concentration, we found a positive correlation between nitrogen concentration and SLA among females. The sex ratio changed with tree height within populations, and taller height classes were biased in favour of males. Regardless of population age, the percentage of females within populations was positively correlated with precipitation. Probably high reproductive effort caused female trees to lose in competition with males, and this loss may also be enhanced by lower drought tolerance in females and could contribute to risk of extinction for T. baccata. The continental geographic range of T. baccata may be restricted by limited occurrence of females, which demand higher water resources than males.

Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidata: How does it divert excess energy from photosynthetic reaction center?

Photosystems must efficiently dissipate absorbed light energy under freezing conditions. To clarify the energy dissipation mechanisms, we examined energy transfer and dissipation dynamics in needles of the evergreen plant Taxus cuspidata by time-resolved fluorescence spectroscopy. In summer and autumn, the energy transfer processes were similar to those reported in other higher plants. However, in winter needles, fluorescence lifetimes became shorter not only in PSII but also in PSI, indicating energy dissipation in winter needles. In addition, almost the same fluorescence spectra were obtained with different excitation wavelengths. In contrast, the fluorescence spectrum showed a large difference due to excitation wavelength in spring needles. The fluorescence spectrum of spring needles in 550-nm excitation showed similar spectra to that of winter needles, however, red-chlorophyll fluorescence was not observed in chlorophyll excitation. These observations suggest that some complexes with some kind of red-shifted carotenoid and red-chlorophyll unlink from the core complex in spring. Seasonal changes of excitation energy dynamics are also discussed in relation to changes in thylakoid stacking.

Antifungal and antibacterial activities of Taxus wallichiana Zucc

Current study was undertaken to evaluate the in vitro antifungal and antibacterial potential of methanol extract and subsequent fractions obtained after partitioning in organic solvents with variable polarity of the aerial parts of the tree Taxus wallichiana Zucc. Traditionally, this plant is often used in folk medicines in Pakistan for treating microbial infections. In order to rationalize the traditional use, methanol extracts of leaf, bark, and heartwood of Taxus wallichiana Zucc. were tested against six bacteria and six fungal strains using the Hole diffusion and macro-dilution methods. All extracts and fractions displayed significant antimicrobial effect. Only three fungal strains, Trichophyton longifusus, Microspoum canis, and Fusarium solani were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. In case of bacterial strains, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. Comparison results were carried out using imipinem, miconazole and amphotericin B as standard antibiotics.

What is the evidence for medicinal value of carnivorous plants?

Throughout history, plants have been used for medicinal purposes. Many of the modern allopathic medicines used today are either directly or indirectly derived from plants. Examples include the cardiotonic and antiarrhythmic agent digoxin (derived from the foxglove—Digitalis purpurea), antimalarial quinine (derived from the bark of the cinchona tree—Cinchona officinalis), the mydriatic (pupil dilatory) agent belladonna (derived from the nightshade Atropa belladonna), aspirin (derived from the bark of the willow tree—Salix alba, etc.), taxol (derived from the yew tree—Taxus brevifolia) and vincristine (derived from the periwinkle—Catharanthus roseus (=Vinca rosea)). Although carnivorous plants have been used medicinally, to date not a single compound derived from carnivorous plants has been widely-accepted in modern allopathic medicine. Pinguicula spp., Dionaea muscipula, Drosera spp., Sarracenia purpurea, and Nepenthes spp. are the carnivorous plants frequently touted as having therapeutic potential. We herein review the existing literature on the medical uses of these plants.

Photosynthetic activity in winter needles of the evergreen tree Taxus cuspidata at low temperatures

Photosystems harvest light energy, yet this energy cannot be efficiently employed for CO(2) assimilation at the below-freezing temperatures to which plants are typically exposed during winter in the temperate and boreal zones. To elucidate the mechanisms whereby this energy is dissipated, I evaluated performance of photosystems in winter needles of the evergreen tree Taxus cuspidata Sieb. et Zucc. Chloroplasts were localized adjacent to plasma membranes in needle cells in summer, whereas they congregated together in the centers of the cells during winter. When winter needles were acclimated to a temperature of 20 degrees C, their chloroplasts gradually dispersed to the edges of the cells, as in the summer. Acclimation-dependent relocalization coincided with changes in CO(2) uptake. Examination of photosystem II fluorescence kinetics in winter needles indicated that the quinone electron acceptor (Q(A)) reduction rate exceeded the Q(A) oxidation rate at low temperatures. The majority of Q(A) remained reduced even when winter needles were subjected to a temperature of -5 degrees C at low irradiance.

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.

Taxol content in the bark of Himalayan Yew in relation to tree age and sex

Taxol content in the bark of Taxus baccata trees growing in a homogenous (uniform) environment at Jageshwar, District Almora in Central Himalaya has been quantified. The average taxol concentration in the bark of sampled trees was 0.0558±0.008% (of dry wt.) and was about 64% higher for male plants (averaged across tree age) in comparison to female trees. Maximum taxol content was recorded in the bark samples collected from trees of >110 yrs age. ANOVA indicates a significant difference in the taxol content of bark from trees of different ages, however, differences were not significant between sexes. Taxol was quantified by HPLC using a standard curve prepared with authentic taxol; the identification of bark taxol was confirmed by UV and mass spectrometry. The total taxol content of the bark of Taxus trees across an age series was found to range between 0.064 to 8.032 g/tree, and a tree of about 100 yrs age can yield 5.74 kg dry bark.

Two novel pseudoalkaloid taxanes from the Chinese yew, Taxus chinensis var. mairei

Two pseudoalkaloid taxane diterpenoids were isolated from seeds of the Chinese yew tree Taxus chinensis var. mairei. Their structures were established as 2α,9α,10β,13α-tetraacetoxy-5α(3′-methylamino-3′-phenyl)-propionyloxy-taxa-4(20),11-diene and 7β,9α,10β,13α-tetraacetoxy-5α(3′-methylamino-3′-phenyl)-propionyloxy-taxa-4(20),11-diene, with the aid of NMR spectral and FAB-MS data analysis.

Microbial Transformation of 10-Deacetyl-7-epitaxol and 1β-Hydroxybaccatin I by Fungi from the Inner Bark of Taxus yunnanensis

Fungi present in the inner bark of the yew tree Taxus yunnanensis were studied for the capability to biotransform natural taxoids. On the basis of screening, three fungi, Microsphaeropsis onychiuri, Mucor sp. and Alternaria alternata, were revealed to be able to selectively hydrolyze and epimerize two taxoids, 10-deacetyl-7-epitaxol (1) and 1β-hydroxybaccatin I (2), previously isolated from the bark of the same yew species. After preparative-scale incubation of 10-deacetyl-7-epitaxol with M. onychiuri and Mucor sp., three metabolites were isolated and characterized as 10-deacetylbaccatin V (3), 10-deacetyltaxol (4), and 10-deacetylbaccatin III (5). Incubation of 1β-hydroxybaccatin I with A. alternata, on the other hand, gave 5-deacetyl-1β-hydroxybaccatin I (6), 13-deacetyl-1β-hydroxybaccatin I (7), and 5,13-dideacetyl-1β-hydroxybaccatin I (8).

Supercritical fluid extraction of paclitaxel and baccatin III from needles of taxus cuspidata

Paclitaxel and baccatin III are extracted from the ground needles of the yew tree Taxus cuspidata using supercritical carbon dioxide with and without the addition of several cosolvents. The pressure and temperature ranges tested were from 100 to 300 bar and from 308 to 343 K, respectively, and the cosolvents were ethylacetate, methanol, dichloromethane, and ethylether. To diminish a variety of waxy and nonpolar compounds the ground needles are treated with n-hexane before the extraction. The highest selectivity of paclitaxel and baccatin III in the extracts during the first four hours of SFE was about 0.262 and 0.644 mass %. respectively. Supercritical fluid extraction using carbon dioxide with and without cosolvents was found to be more selective than the conventional organic solvent extraction.

Taxoids: A New Class of Antimitotic Compounds

Paclitaxel (Taxol®) and docetaxel (Taxotere®) are the first representatives of taxoids , a new class of antitumor compounds. These two taxoids are clinically active against breast, ovarian and lung cancers. Taxoids are highly complex diterpenoids from natural origin. Preclinical and clinical develop-ments have been made possible after a long and sustained chemical effort : paclitaxel is extracted from the barks of the Pacific yew tree Taxus brevifolia whereas docetaxel is prepared by hemisynthesis starting from 10-deacetyl-baccatin ill, a non cytotoxic precursor found in the needles of the European yew tree Taxus baccata . These two drugs are active in various in vitro and in vivo preclinical models (cell lines, cloning of human tumor stem cells, murine grafted tumors , human xenografts). Taxoids constitute anew class of antimitotic agents different from vinca-alkaloids: on the one hand, paclitaxel and docetaxel can be considered as inhibitors of the reaction of depolymerization of microtubules into tubulin ; on the other hand, vinca-a!kaloids inhibit the reaction of polymerization of tubulin into microtubules. An active program of medicinal chemistry is done in various pharmaceutical and academic Institutions with two objectives: knowledge of structure-activity relationships and selection of new candidates for clinical trials. With the taxoid series, a variety of analogs have been prepared for their antitubulin and biological properties . Concerning the tubulin binding, some important structure activity relationships have been proposed. In this review the contribution of each functional group of docetaxel will be discussed following the evolution of antitubulin activity, going from docetaxel to taxoids possessing the minimum requirement of recognition by tubulin . The conformation of docetaxel and analogs will be compared taking into account the contribution and relevance of X-rays, NMR and molecular modelling studies in determining the molecular shape of active and inactive compounds.