Analysis of the utilization value of different tissues of Taxus×Media based on metabolomics and antioxidant activity.

BACKGROUND: Taxaceae, is a class of dioecious and evergreen plant with substantial economic and ecology value. At present many phytochemical analyses have been performed in Taxus plants. And various biological constituents have been isolated from various Taxus species. However, the difference of compounds and antioxidant capacity of different tissues of T. media is not clear. RESULTS: In the present study, we investigated the metabolites and antioxidant activity of four tissues of T. media, including T. media bark (TB), T. media fresh leaves (TFL), T. media seeds (TS), T. media aril (TA). In total, 808 compounds, covering 11 subclasses, were identified by using UPLC-MS/MS. Paclitaxel, the most popular anticancer compound, was found to accumulate most in TS, followed by TB, TFL and TA in order. Further analysis found that 70 key differential metabolites with VIP > 1.0 and p < 0.05, covering 8 subclasses, were screened as the key differential metabolites in four tissues. The characteristic compounds of TFL mainly included flavonoids and tanninsis. Alkaloids and phenolic acids were major characteristic compounds of TS and TB respectively. Amino acids and derivatives, organic acids, saccharides and lipids were the major characteristic compounds of TA. Additionally, based on FRAP and ABTS method, TS and TFL exhibited higher antioxidant activity than TB and TA. CONCLUSION: There was significant difference in metabolite content among different tissues of T. media. TFL and TS had higher metabolites and antioxidant capacity than other tissues, indicating that TFL and TS were more suitable for the development and utilization of T. media in foods and drinks.

Taxoids-rich extract from Taxus wallichiana alleviates high-fat diet-induced insulin resistance in C57BL/6 mice through inhibition of low-grade inflammation.

The needle powder of Taxus wallichiana is in use for the management of diabetes and inflammation-related complications in the Indian and Chinese Systems of Traditional Medicine but the lack of proper pharmacological intervention has prompted us to investigate the pharmacological mechanism against inflammation-induced insulin resistance in high-fat diet-fed C57BL/6 mice. Hexane (Tw-H), chloroform (Tw-C), and ethyl acetate (Tw-EA) extracts were prepared from a needle of T. wallichiana and its effect on glucose uptake against TNF-α-induced insulin resistance in skeletal muscle cells was studied. Among all, Tw-EA extract has shown promising glucose uptake potential. Tw-EA treatment is also able to decrease the lipid accumulation in adipocytes. Chemical signature of Tw-EA using HPLC showed the presence of taxoids. Efficacy of taxoids-rich extract from T. wallichiana (Tw-EA) was further validated in in vivo system against high-fat diet (HFD)-induced insulin resistance in C57BL/6 mice. Oral treatment of Tw-EA showed significant reduction in blood glucose, pro-inflammatory cytokine production and body weight gain when compared with vehicle-treated HFD-induced insulin resistance in C57BL/6 mice. Histopathology and immunohistochemistry study in skeletal muscle and adipose tissue revealed that oral treatment of Tw-EA is able to reduce the infiltration of inflammatory cells in skeletal muscles, ameliorate the hypertrophy in adipose tissue and upregulate the GLUT4 protein expression. Treatment with Tw-EA significantly up-regulated mRNA expression of insulin signaling pathway (IRS-1, PI3K, AKT, GLUT 4). This study suggested the beneficial effect of taxoids-rich extract from Taxus wallichiana against the inflammation-associated insulin resistance condition.

Understanding of cell death induced by the constituents of Taxus yunnanensis wood.

The ethanol extract from the wood of Taxus Yunnanensis (TY) induced apoptosis in all cancer cell lines tested, which was mainly due to activation of an extrinsic pathway in human colon cancer DLD-1 cells. The extrinsic pathway was activated by the upregulation of the expression levels of Fas and TRAIL/DR5, which led to the activation of caspase-8. Of note, the machinery of this increase in expression was promoted by the upregulation of MIR32a expression, which silenced MIR34a-targeting E2F3 transcription factor. Furthermore, ectopic expression of MIR32a or siR-E2F3 silencing E2F3 increased Fas and TRAIL/DR5 expression. Thus, the extract activated the extrinsic pathway through the MIR34a/E2F3 axis, resulting in the autocrine and paracrine release of TRAIL, and upregulated expression of death receptors Fas and DR5 in the treated DLD-1 cells, which were functionally validated by Fas immunocytochemistry, and using anti-Fas and anti-TRAIL antibodies, respectively. In vivo, TY showed significant anti-tumor effects on xenografted and syngeneic model mice. The extract may also aid in chemoprevention by selectively making marked tumor cells susceptible to the tumor immunosurveillance system.

Endophytic fungus Pseudodidymocyrtis lobariellae KL27 promotes taxol biosynthesis and accumulation in Taxus chinensis.

BACKGROUND: Taxol from Taxus species is a precious drug used for the treatment of cancer and can effectively inhibit the proliferation of cancer cells. However, the growth of Taxus plants is very slow and the content of taxol is quite low. Therefore, it is of great significance to improve the yield of taxol by modern biotechnology without destroying the wild forest resources. Endophytic fungus which symbiosis with their host plants can promote the growth and secondary metabolism of medicinal plants. RESULTS: Here, an endophytic fungus KL27 was isolated from T. chinensis, and identified as Pseudodidymocyrtis lobariellae. The fermentation broth of KL27 (KL27-FB) could significantly promote the accumulation of taxol in needles of T. chinensis, reaching 0.361 ± 0.082 mg/g·DW (dry weight) at 7 days after KL27-FB treatment, which is 3.26-fold increase as compared to the control. The RNA-seq and qRT-PCR showed that KL27-FB could significantly increase the expression of key genes involved in the upstream pathway of terpene synthesis (such as DXS and DXR) and those in the taxol biosynthesis pathway (such as GGPPS, TS, T5OH, TAT, T10OH, T14OH, T2OH, TBT, DBAT and PAM), especially at the early stage of the stimulation. Moreover, the activation of jasmonic acid (JA) biosynthesis and JA signal transduction, and its crosstalk with other hormones, such as gibberellin acid (GA), ethylene (ET) and salicylic acid (SA), explained the elevation of most of the differential expressed genes related to taxol biosynthesis pathway. Moreover, TF (transcriptional factor)-encoding genes, including MYBs, ethylene-responsive transcription factors (ERFs) and basic/helix-loop-helix (bHLH), were detected as differential expressed genes after KL27-FB treatment, further suggested that the regulation of hormone signaling on genes of taxol biosynthesis was mediated by TFs. CONCLUSIONS: Our results indicated that fermentation broth of endophytic fungus KL27-FB could effectively enhance the accumulation of taxol in T. chinensis needles by regulating the phytohormone metabolism and signal transduction and further up-regulating the expression of multiple key genes involved in taxol biosynthesis. This study provides new insight into the regulatory mechanism of how endophytic fungus promotes the production and accumulation of taxol in Taxus sp.

The Taxus genome provides insights into paclitaxel biosynthesis.

The ancient gymnosperm genus Taxus is the exclusive source of the anticancer drug paclitaxel, yet no reference genome sequences are available for comprehensively elucidating the paclitaxel biosynthesis pathway. We have completed a chromosome-level genome of Taxus chinensis var. mairei with a total length of 10.23 gigabases. Taxus shared an ancestral whole-genome duplication with the coniferophyte lineage and underwent distinct transposon evolution. We discovered a unique physical and functional grouping of CYP725As (cytochrome P450) in the Taxus genome for paclitaxel biosynthesis. We also identified a gene cluster for taxadiene biosynthesis, which was formed mainly by gene duplications. This study will facilitate the elucidation of paclitaxel biosynthesis and unleash the biotechnological potential of Taxus.

Comparative metabolomic analysis reveals the variations in taxoids and flavonoids among three Taxus species.

BACKGROUND: Trees of the genus Taxus are highly valuable medicinal plants with multiple pharmacological effects on various cancer treatments. Paclitaxel from Taxus trees is an efficient and widely used anticancer drug, however, the accumulation of taxoids and other active ingredients can vary greatly among Taxus species. In our study, the metabolomes of three Taxus species have been investigated. RESULTS: A total of 2246 metabolites assigned to various primary and secondary metabolic pathways were identified using an untargeted approach. Analysis of differentially accumulated metabolites identified 358 T. media-, 220 T. cuspidata-, and 169 T. mairei-specific accumulated metabolites, respectively. By searching the metabolite pool, 7 MEP pathway precursors, 11 intermediates, side chain products and derivatives of paclitaxel, and paclitaxel itself were detected. Most precursors, initiated intermediates were highly accumulated in T. mairei, and most intermediate products approaching the end point of taxol biosynthesis pathway were primarily accumulated in T. cuspidata and T. media. Our data suggested that there were higher-efficiency pathways to paclitaxel in T. cuspidata and T. media compared with in T. mairei. As an important class of active ingredients in Taxus trees, a majority of flavonoids were predominantly accumulated in T. mairei rather than T. media and T. cuspidata. The variations in several selected taxoids and flavonoids were confirmed using a targeted approach. CONCLUSIONS: Systematic correlativity analysis identifies a number of metabolites associated with paclitaxel biosynthesis, suggesting a potential negative correlation between flavonoid metabolism and taxoid accumulation. Investigation of the variations in taxoids and other active ingredients will provide us with a deeper understanding of the interspecific differential accumulation of taxoids and an opportunity to accelerate the highest-yielding species breeding and resource utilization.

Identification of the antiglycative components of Hong Dou Shan (Taxus chinensis) leaf tea.

The formation of advanced glycation end products (AGEs) may affect life quality and cut lifespan. With the increasingly utilization of new foods and booming development of international trade, large amounts of newly emerging teas have come into our sight. In this study, we evaluated the antiglycative capabilities of five popular herbal teas. Results showed that Chinese Hong Dou Shan (Taxus chinensis; HDS) leaf tea had particularly strong antiglycative activity via scavenging methylglyoxal (MGO) in both chemical (glucose-BSA, fructose-BSA, and MGO-BSA) and cell models (HUVECs). Furthermore, the intracellular AGEs level was alleviated to normal state by the two HDS leaf tea fractions, H-47 and H-57 which were almost as effective as EGCG when added at the same level (50 µg/mL). The effective components against glycation were further separated and tentatively identified as catechin, epicatechin, gallocatechin, epigallocatechin, procyanidin B2, a dihydromyricetin dimer, and a quercetin glucoside by HPLC-DAD and LC-Q-TOF/MS analysis.

Identification of rate-limiting enzymes involved in paclitaxel biosynthesis pathway affected by coronatine and methyl-ß-cyclodextrin in Taxus baccata L. cell suspension cultures.

BACKGROUND: Paclitaxel is a potent antitumor alkaloid widely used for the treatment of several cancer types. This valuable secondary metabolite naturally exists in the inner bark of Taxus species in very low amounts. The small-scale production of paclitaxel in Taxus cell cultures requires utilization of several elicitors. OBJECTIVE: The main objective of this work was to identify key genes that encode rate-limiting enzymes in paclitaxel biosynthesis pathway by investigating the possible relationship between paclitaxel production and a set of 13 involved genes' relative expression in Taxus baccata L. cell suspension cultures affected by coronatine and methyl-ß-cyclodextrin. METHODS: In the present research, the most important key genes were identified using gene expression profiling evaluation and paclitaxel production assessment in Taxus baccata L. cell cultures affected by mentioned elicitors. RESULTS AND CONCLUSION: Gene expression levels were variably increased using methyl-ß-cyclodextrin, and in some cases, a synergistic effect on transcript accumulation was observed when culture medium was supplemented with both elicitors. It was revealed that DBAT, BAPT, and DBTNBT are the most important rate-limiting enzymes in paclitaxel biosynthesis pathway in Taxus baccata L. cell suspension cultures under coronatine and methyl-ß-cyclodextrin elicitation condition. Moreover, PAM was identified as one of the important key genes especially in the absence of ß-phenylalanine. In cell cultures affected by these elicitors, paclitaxel was found largely in the culture media (more than 90%). The secretion of this secondary metabolite suggests a limited feedback inhibition and reduced paclitaxel toxicity for producer cells. It is the result of the ABC gene relative expression level increment under methyl-ß-cyclodextrin elicitation and highly depends on methyl-ß-cyclodextrin's special property (complex formation with hydrophobic compounds). Paclitaxel biosynthesis was obviously increased due to the effect of coronatine and methyl-ß-cyclodextrin elicitation, leading to the production level of 5.62 times higher than that of the untreated cultures. Graphical abstract Rate Limiting Enzymes in Paclitaxel Biosynthesis Pathway: DBAT, BAPT, DBTNBT and PAM.

Chemical Components, Biological Activities, and Toxicological Evaluation of the Fruit (Aril) of Two Precious Plant Species from Genus Taxus.

The fruit (aril) of the endangered genus Taxus plants is an abandoned herbal resource. Traditionally, people enthusiastically focus on its bark, its renewable, tremendous arils fall into the soil with seeds after they are mature. The present research investigated the fruit of two species from the genus Taxus, Taxus chinensis var. mairei, and Taxus media, with regards to their antioxidant and antihyperglycaemic activities, safety, and bioactive constituents. Results showed that T. chinensis var. mairei and T. media both had certain biological activities with T. chinensis var. mairei better in antioxidant activity and T. media better in antihyperglycaemic activity. Correlation analysis revealed that the differences in bioactivities depended on content of their mainly chemical components. The mice acute oral toxicity test indicated that the methanol extracts of the two biotypes of Taxus were safe. And nineteen compounds were tentatively assigned from the two varieties, via tandem mass spectrometry using a LC-ESI-Q-TOF-MS instrument, which included phenols, flavonoids, and terpenes. These results indicate a possible application of Taxus fruit extracts in various fields like in food industry, however, this still needs further investigations.

A suicide attempt by intoxication with Taxus baccata leaves and ultra-fast liquid chromatography-electrospray ionization-tandem mass spectrometry, analysis of patient serum and different plant samples: case report.

BACKGROUND: Taxus (yew) is one of the most frequently reported plants causing potentially fatal outcome when taken incidentally or for suicidal reasons. A fast and reliable method of detection of poisonous compounds or their metabolites is critical in life-saving procedures in cases of yew ingestion. Previously, several chromatographic analytical procedures have been described usually taking longer than one hour of total analysis time. CASE PRESENTATION: In this report we describe a suicide case study and an ad hoc developed fast method of detection and quantitation of 3,5-dimethoxyphenol - the main taxane metabolite in the blood plasma from the patient as well as the determination of major taxine components in the plant material (Taxus baccata). At present, there is no reasonable alternative for mass spectrometry that could match its high sensitivity and accuracy, and Multiple Reaction Monitoring could be adequate and useful mass spectrometry technique in analyzing and identification of plants material compounds that cause severe poisoning in humans. In the reported case, intensive cardiac care together with the astuteness of the treating physicians not only saved the patient's life, but also allowed for his complete recovery and return to work. CONCLUSIONS: The development of ultra fast liquid chromatography tandem mass spectrometry UFLC-MS/MS method provides a fast means to confirm yew alkaloids and their metabolite in various material. The applied analytical procedure allows early detection of main metabolite in patient material as well as comparing to those extracted from the plant. In our study, the taxanes remained undetected, probably due to the time elapsing from the patient admittance and collection of plasma. In cases like those reported in this study, retaining the gastric material should be obligatory to confirm the ingestion of yew. The possibility of using this approach in detection of native taxine compounds in human plasma remains to be verified.

Response and Defense Mechanisms of Taxus chinensis Leaves Under UV-A Radiation are Revealed Using Comparative Proteomics and Metabolomics Analyses.

Taxus chinensis var. mairei is a species endemic to south-eastern China and one of the natural sources for the anticancer medicine paclitaxel. To investigate the molecular response and defense mechanisms of T. chinensis leaves to enhanced ultraviolet-A (UV-A) radiation, gel-free/label-free and gel-based proteomics and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The transmission electron microscopy results indicated damage to the chloroplast under UV-A radiation. Proteomics analyses in leaves and chloroplasts showed that photosynthesis-, glycolysis-, secondary metabolism-, stress-, and protein synthesis-, degradation- and activation-related systems were mainly changed under UV-A radiation. Forty-seven PSII proteins and six PSI proteins were identified as being changed in leaves and chloroplasts under UV-A treatment. This indicated that PSII was more sensitive to UV-A than PSI as the target of UV-A light. Enhanced glycolysis, with four glycolysis-related key enzymes increased, provided precursors for secondary metabolism. The 1-deoxy-d-xylulose-5-phosphate reductoisomerase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase were identified as being significantly increased during UV-A radiation, which resulted in paclitaxel enhancement. Additionally, mRNA expression levels of genes involved in the paclitaxel biosynthetic pathway indicated a down-regulation under UV-A irradiation and up-regulation in dark incubation. These results reveal that a short-term high dose of UV-A radiation could stimulate the plant stress defense system and paclitaxel production.

Metabolic engineering approaches for production of biochemicals in food and medicinal plants.

Historically, plants are a vital source of nutrients and pharmaceuticals. Recent advances in metabolic engineering have made it possible to not only increase the concentration of desired compounds, but also introduce novel biosynthetic pathways to a variety of species, allowing for enhanced nutritional or commercial value. To improve metabolic engineering capabilities, new transformation techniques have been developed to allow for gene specific silencing strategies or stacking of multiple genes within the same region of the chromosome. The 'omics' era has provided a new resource for elucidation of uncharacterized biosynthetic pathways, enabling novel metabolic engineering approaches. These resources are now allowing for advanced metabolic engineering of plant production systems, as well as the synthesis of increasingly complex products in engineered microbial hosts. The status of current metabolic engineering efforts is highlighted for the in vitro production of paclitaxel and the in vivo production of ß-carotene in Golden Rice and other food crops.

Chemical studies on Taxus canadensis.

A series of new taxanes, 1-93, have been isolated, together with 37 known taxoids including Taxol(®) (paclitaxel) and cephalomannine, from the Canadian yew, Taxus canadensis (Taxaceae) in the past 30 years. These new taxoids possess various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, and 6/12 ring systems and six new taxanes with four novel skeletons, i.e., a taxane with a 6/6/8/6 ring system, a taxane with a [3.3.3] propellane skeleton, three taxanes with [3.3.3] [3.4.5] dipropellane sytems, as well as a novel taxane with a unique 5/5/4/6/6/6 hexacyclic skeleton, containing a unique [3.3.2] propellane, were isolated for the first time from natural sources. It should be emphasized that 13-acetyl-9-dihydrobaccatin III, a very useful starting material for the semisynthesis of Taxol(®) and Taxotere(®) , represents the most abundant taxane in the needles of this yew tree. These findings establish the above mentioned yew tree as significantly different from the remaining species. On the other hand, some chemical modifications on the taxanes isolated from this plant were carried out.

Transcriptome analysis of Taxus cuspidata needles based on 454 pyrosequencing.

Taxus species are highly valued as renewable resources for the production of Taxol. Despite the commercial and medicinal importance of Taxus, little genomic information is available for yew species, and Taxol biosynthesis still needs to be fully elucidated. In this study, 454 pyrosequencing technology was employed to produce an expressed sequence tag (EST) from the needles of Taxus cuspidata. In all, 81 148 high-quality reads from the needles of T. cuspidata were produced using Roche GS FLX Titanium. A total of 20,557 unique sequences were obtained, including 12 975 singletons and 7582 contigs. Approximately 14,095 unique sequences were annotated by a similarity search against five public databases. Gene ontology revealed 11,220 unique sequences that could be assigned to 45 vocabularies. In the Kyoto Encyclopedia of Genes and Genomes mapping, 2403 transcripts were established as associated with 3821 biochemical pathways. Enzymes in the plastidial 2-C-methyl-D-erythritol 4-phosphate pathway were well represented. Candidates of the putative genes of Taxol biosynthesis were revealed, including those in the remaining steps. In total, 291 transcripts were identified, representing putative homologues of transcription factors. Furthermore, 753 simple sequence repeat motifs, which are potential molecular markers for genetic application, were identified. These results provide the largest EST collections in TAXUS and will contribute to biosynthetic and biochemical studies that lead to drug improvement.

4-Deacetylbaccatin III: a proposed biosynthetic precursor of paclitaxel from the bark of Taxus wallichiana.

4-Deacetylbaccatin III was isolated and characterized from the bark of Taxus wallichiana Zucc. It was previously identified as an intermediate during the synthesis of paclitaxel and was proposed as a precursor for the biosynthesis of paclitaxel. Existence of 4-deacetylbaccatin III in T. wallichiana is strong evidence for the previously proposed biosynthetic pathway of paclitaxel via 4-deacetylbaccatin III.

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii.

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.

Profiling of yew hair roots from various species using ultra-performance liquid chromatography/electrospray ionization mass spectrometry.

An efficient and sensitive profiling approach to complex yew samples was developed using ultra-performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). The UPLC-based method displayed short analytical time and improved peak capability, as well as high sensitivity. The appropriate in-source collision-induced dissociation (CID) energy was employed to produce informative characteristic ions which could be used for stereochemical and sub-structural assignment of yew constituents. The method was successfully applied in the rapid screening of yew hair roots from various species, and 53 constituents including 47 taxoids were detected from partially purified root extract. Notably, C-7 hydroxytaxane stereoisomers could be identified based on their different fragment ions under the optimal profiling conditions. It was also observed that hair roots from different Taxus species exhibited nearly identical chemical distribution, indicating they had similar metabolic frameworks. Additionally, Taxus root resources also display benign medicinal perspective because they have relatively simple chemical profiles and possess high yields of valuable taxanes such as paclitaxel, cephalomannine, 10-deacetylpaclitaxel and 7-xylosyltaxanes.

Production of paclitaxel and the related taxanes by cell suspension cultures of Taxus species.

Medicinal plants are the most promising source for the development of drugs, and many types of active ingredients from the plant resources have been studied in order to clarify the relationship between the chemical structure and the activity. However, it is not easy to develop drugs from those active compounds, and in many cases, the supply of active compounds can have some problems: 1) limited quantity of active compounds in plant; 2) low plant growth rate; 3) the limited localization of active ingredients in the specific organs; and 4) from the perspective of the conservation of natural resources. Therefore, the stable supply of the compounds commercially is very difficult and contains risk hedge. Plant cell culture is an attractive technology to solve these problems by securing the stable supply of the active compounds without damage to the natural plant resources. Recently, an efficient production process of anticancer drug paclitaxel by Taxus cell suspension cultures was constructed. The established Taxus cell lines produced paclitaxel and related taxanes by specific external stimuli, such as methyl jasmonate. The time-course analysis revealed that there are two regulatory steps existing in the paclitaxel biosynthesis: the taxane-ring formation step that is up-regulated by MeJA, and the acylation step at the C-13 position. By applying the data from the two-stage culture and the high-density culture, a large-scale culture process was developed with a stable paclitaxel production in the range of 140-295 mg L(-1), reaching 295 mg L(-1) at maximum.

Investigations of taxoids by electrospray and atmospheric pressure chemical ionization with tandem mass spectrometry.

The fragmentation behavior of taxoids was studied using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources with multi-stage tandem mass spectrometry. In the positive ion mode taxoids gave prominent [M+Na]+ and [M+K]+ ions with the ESI source, and [M+NH4]+ or [M+H]+ ions with the APCI source. The MS/MS fragmentations of ions produced by APCI and ESI sources were very similar. For both sources, the presence of cinnamoyl or benzoyl groups could be characterized by initial losses of 148 or 122 u, respectively, from molecular adduct ions. However, the elimination of cinnamic acid was relatively difficult for the molecular adduct ions formed by APCI, and was comparable in importance to the loss of acetic acid. The other fragments involved losses of CH2CO, CO, and H2O. The 5/7/6 type taxoids underwent characteristic losses of 58 or 118 u from ions produced by both APCI and ESI sources. The fragmentation behavior was remarkably influenced by substitution locations. The elimination of the C-10 benzoyl group was usually the first fragmentation step, while that of the C-2 benzoyl group was relatively difficult. The acetoxyl group at C-7 was more active than those at C-2, C-9, and C-10, which in turn were more active than that at C-4. These fragmentation rules could facilitate the rapid screening and structural characterization of taxoids in plant extracts by high-performance liquid chromatography/mass spectrometry (HPLC/MS).

Identification of a cDNA clone specific for the taxol synthesis phase of Taxus chinensis cells by mRNA differential display.

In order to identify genes related to Taxol biosynthesis, the mRNA differential display method was employed to compare mRNA populations from suspension cultured Taxus chinensis cells before and after beginning to produce Taxol. From a total display of about 4000 PCR products, 104 were derived from cells during the Taxol synthesis phase but not the non-Taxol synthesis phase. These products were cloned, and one such cDNA clone, named TS1, was confirmed to be specifically expressed in the Taxol synthesis phase by northern blot analysis. The length of the transcript corresponding TS1 was approximately 2.1 kb. DNA sequencing and homology search showed the sequence of TS1 contains a partial open reading frame and has no homologies with other known genes. Hence, this report demonstrated the potential of mRNA differential display for the isolation of genes specific for the period of secondary metabolite production as well as the feasibility of the approach for the identification of genes potentially related to the synthesis of secondary metabolites in higher plants.