Paclitaxel is a potent anti-cancer drug that is mainly produced through semi-synthesis, which still requires plant materials as precursors. The content of paclitaxel and 10-deacetyl baccatin III (10-DAB) in Taxus yunnanensis has been found to differ from that of other Taxus species, but there is little research on the mechanism underlying the variation in paclitaxel content in T. yunnanensis of different provenances. In this experiment, the contents of taxoids and precursors in twigs between a high paclitaxel-yielding individual (TG) and a low paclitaxel-yielding individual (TD) of T. yunnanensis were compared, and comparative analyses of transcriptomes as well as chloroplast genomes were performed. High-performance liquid chromatography (HPLC) detection showed that 10-DAB and baccatin III contents in TG were 18 and 47 times those in TD, respectively. Transcriptomic analysis results indicated that genes encoding key enzymes in the paclitaxel biosynthesis pathway, such as taxane 10-β-hydroxylase (T10βH), 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT), and debenzoyl paclitaxel N-benzoyl transferase (DBTNBT), exhibited higher expression levels in TG. Additionally, qRT-PCR showed that the relative expression level of T10βH and DBAT in TG were 29 and 13 times those in TD, respectively. In addition, six putative transcription factors were identified that may be involved in paclitaxel biosynthesis from transcriptome data. Comparative analysis of plastid genomes showed that the TD chloroplast contained a duplicate of rps12, leading to a longer plastid genome length in TD relative to TG. Fifteen mutation hotspot regions were identified between the two plastid genomes that can serve as candidate DNA barcodes for identifying high-paclitaxel-yield individuals. This experiment provides insight into the difference in paclitaxel accumulation among different provenances of T. yunnanensis individuals.

Taxus spp. are ancient tree species that have survived from the Quaternary glacier period, and their metabolites, such as taxol, have been used as anticancer drugs globally. Plant-endophytic microbial interaction plays a crucial role in exerting a profound impact on host growth and secondary metabolite synthesis. In this study, high-throughput sequencing was employed to explore endophytic microbial diversity in the roots, stems, and leaves of the Taxus yunnanensis (T. yunnanensis). The analysis revealed some dominant genera of endophytic bacteria, such as Pseudomonas, Neorhizobium, Acidovorax, and Flavobacterium, with Cladosporium, Phyllosticta, Fusarium, and Codinaeopsis as prominent endophytic fungi genera. We isolated 108 endophytic bacteria and 27 endophytic fungi from roots, stems, and leaves. In vitro assays were utilized to screen for endophytic bacteria with growth-promoting capabilities, including IAA production, cellulase, siderophore production, protease and ACC deaminase activity, inorganic phosphate solubilization, and nitrogen fixation. Three promising strains, Kocuria sp. TRI2-1, Micromonospora sp. TSI4-1, and Sphingomonas sp. MG-2, were selected based on their superior growth-promotion characteristics. These strains exhibited preferable plant growth promotion when applied to Arabidopsis thaliana growth. Fermentation broths of these three strains were also found to significantly promote the accumulation of taxanes in T. yunnanensis stem cells, among which strain TSI4-1 demonstrated outstanding increase potentials, with an effective induction of taxol, baccatin III, and 10-DAB contents. After six days of treatment, the contents of these metabolites were 3.28 times, 2.23 times, and 2.17 times the initial amounts, reaching 8720, 331, and 371 ng/g of dry weight of stem cells, respectively. These findings present new insight into the industrialization of taxol production through Taxus stem cell fermentation, thereby promoting the conservation of wild Taxus resources by maximizing their potential economic benefits.

Taxol is an antitumor drug derived from the bark of the Pacific Yew tree that inhibits microtubule disassembly, resulting in cell cycle arrest in late G2 and M phases. Additionally, Taxol increases cellular oxidative stress by generating reactive oxygen species. We hypothesized that the inhibition of specific DNA repair machinery/mechanisms would increase cellular sensitivity to the oxidative stress capacity of Taxol. Initial screening using Chinese hamster ovary (CHO) cell lines demonstrated that base excision repair deficiency, especially PARP deficiency, caused cellular Taxol hypersensitivity. Taxane diterpenes-containing Taxus yunnanensis extract also showed hypertoxicity in PARP deficient cells, which was consistent with other microtubule inhibitors like colcemid, vinblastine, and vincristine. Acute exposure of 50 nM Taxol treatment induced both significant cytotoxicity and M-phase arrest in PARP deficient cells, but caused neither significant cytotoxicity nor late G2-M cell cycle arrest in wild type cells. Acute exposure of 50 nM Taxol treatment induced oxidative stress and DNA damage. The antioxidant Ascorbic acid 2 glucoside partially reduced the cytotoxicity of Taxol in PARP deficient cell lines. Finally, the PARP inhibitor Olaparib increased cytotoxicity of Taxol in wild type CHO cells and two human cancer cell lines. Our study clearly demonstrates that cytotoxicity of Taxol would be enhanced by inhibiting PARP function as an enzyme implicated in DNA repair for oxidative stress.

The first complete chloroplast genome (cpDNA) sequence of Taxus yunnanensis was determined from Illumina HiSeq pair-end sequencing data in this study. The cpDNA is 129,190 bp in length. Like other species of taxus genus, the chloroplast genome of T. yunnanensis has lost one of the large inverted repeats (IRs). The genome contains 116 genes, including 82 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. Further phylogenomic analysis showed that T. yunnanensis closed to T. brevifolia in Lauraceae family.

A 53-year-old woman presented at our hospital due to nasal bleeding and petechiae with profound thrombocytopenia (0.4×109/L). Her platelet count returned to the normal range immediately following a platelet transfusion. In this case, tea brewed from Taxus yunnanensis was the only suspected agent ingested prior to the onset of thrombocytopenia while all other etiologies for thrombocytopenia were excluded. A re-exposure test to Taxus yunnanensis resulted in the recurrence of acute thrombocytopenia. The association of thrombocytopenia with substances other than drugs has so far only been rarely described and to the best of our knowledge, this is the first reported case of thrombocytopenia caused by Taxus yunnanensis.

Taxus yunnanensis (Yew) is known for natural anticancer metabolite paclitaxel (Taxol) and its biosynthesis pathway in yew species still needs to be completely elucidated. In the current study, productions of paclitaxel and 10-DAB III from three different tissues (needle, branch, and root) of T. yunnanensis wild type (WT) and two new cultivars Zhongda-1 (Zd1) and Zhongda-2 (Zd2) were determined, and significant tissue differences in contents of the taxanes were observed among the three experimental lines. The much higher 10-DAB III and lower paclitaxel contents in needle of Zd2 when compared with that of Zd1 indicated the low conversion from 10-DAB III to paclitaxel in the needle of Zd2. In order to uncover the mechanisms of the tissue-specific biosynthesis of the taxanes, transcriptome analysis of cultivar Zd2 was conducted, and the previously reported transcriptome data of Zd1 and WT were used to perform a comparison. The enhancement of TDAT and T10βH side biosynthetic pathway in roots of Zd2 in early taxane synthesis might lead to the biosynthesis of other toxoids, while the preference of T13αH route in the needle and branch of Zd2 was mainly responsible for the tissue-specific reinforced biosynthesis of 10-DAB III and paclitaxel in Zd2. Different from Zd1, the tissue-specific pattern of paclitaxel biosynthesis genes in Zd2 was similar to WT. However, the lower transcript abundance of final steps genes (TBT, DBAT, BAPT, and DBTNBT) of the paclitaxel biosynthesis pathway in Zd2 than in Zd1 might further promote 10-DAB III accumulation in Zd2.

The difference in contents of paclitaxel and 10-deacetylbaccatin III (10-DABIII) in needles between wildtype (WT) and a new cultivar (Zhongdayihao, ZD1) of Taxus yunnanensis was examined. Transcriptome profiling was conducted for different tissues of the ZD1 and WT to illustrate the regulation mechanism of paclitaxel biosynthesis. It was observed that average contents of paclitaxel and 10-DABIII in ZD1 were 4 folds and 32 folds higher than those in WT, respectively. More significant elevations of differential expressed genes (DEGs) from paclitaxel biosynthesis pathway were revealed in ZD1 rather than WT, which should be responsible for the higher contents of paclitaxel and 10-DABIII in the ZD1. Special tissues-dependent expression patterns of paclitaxel biosynthesis DEGs in ZD1 compared to WT were unraveled. The relative higher expressions of paclitaxel biosynthesis genes in needles than other tissues supported the higher content of paclitaxel and 10-DABIII content in needles of ZD1. Attenuation of plant hormone signal transduction pathway led to the lower expression of TFs in ZD1 rather than WT. Besides, the significant negative correlations between differential expressed TFs and DEGs from paclitaxel biosynthesis pathway displayed a possibly negative regulation pattern of these TFs on paclitaxel biosynthesis pathway genes. These results provided new insights into the molecular process of paclitaxel synthesis in Taxus.

Plant constituents could act as chelating/reducing or capping agents for synthesis of silver nanoparticles (AgNPs). The green synthesis of AgNPs has been considered as an environmental friendly and cost-effective alternative to other fabrication methods. The present work described the biosynthesis of AgNPs using callus extracts from Taxus yunnanensis and evaluated their antibacterial activities in vitro and potential cytotoxicity in cancer cells. Callus extracts were able to reduce silver nitrate at 1 mM in 10 min. Transmission electron microscope (TEM) indicated the synthesized AgNPs were spherical with the size range from 6.4 to 27.2 nm. X-ray diffraction (XRD) confirmed the AgNPs were in the form of nanocrystals. Fourier transform infrared spectroscopy (FTIR) suggested phytochemicals in callus extracts were possible reducing and capping agents. The AgNPs exhibited effective inhibitory activity against all tested human pathogen bacteria and the inhibition against Gram-positive bacteria was stronger than that of Gram-negative bacteria. Furthermore, they exhibited stronger cytotoxic activity against human hepatoma SMMC-7721 cells and induced noticeable apoptosis in SMMC-7721 cells, but showed lower cytotoxic against normal human liver cells (HL-7702). Our results suggested that biosynthesized AgNPs could be an alternative measure in the field of antibacterial and anticancer therapeutics.

A rapid, sensitive and reliable method has been developed and validated for the simultaneous determination of seven taxoids including 10-deacetylbaccatin III (10-DAB III), baccatin III, 5-epi-canadensene, taxinine M, 10-deacetyltaxol (10-DAT), cephalomannine and paclitaxel in rat plasma using docetaxel as the internal standard (IS). The plasma samples were pretreated by liquid-liquid extraction with methyl tert-butyl ether. The chromatographic separation was achieved on a C18 column (50 mm × 2.1 mm, 1.8 μm, Waters, USA) with a gradient elution program consisting of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. Detection was performed under the selected reaction monitoring (SRM) scan using an electrospray ionization (ESI) in the positive ion mode. The mass transitions were as follows: m/z 567.4→444.9 for 10-DAB III, m/z 609.0→549.3 for baccatin III, m/z 617.4→496.9 for 5-epi-canadensene, m/z 709.6→649.3 for taxinine M, m/z 834.8→307.9 for 10-DAT, m/z 854.5→285.4 for cephalomannine, m/z 876.8→307.3 for paclitaxel and m/z 830.8→549.6 for IS, respectively. All calibration curves exhibited good linearity (r(2)>0.99) over a wide concentration range for all components. The intra-day and inter-day precisions at three different levels were both less than 14.3% in terms of relative standard deviation (RSD) and the accuracies ranged from -8.3% to 14.8% in terms of relative error (RE). The extraction recoveries of the seven compounds ranged from 62.5% to 100.5%. The developed method was successfully applied to the pharmacokinetic study of the seven taxoids in rat plasma after oral administration of the crude extract of the twigs and leaves of Taxus yunnanensis.

Three hitherto unknown taxane diterpenoids, namely baccatin VIII (1), baccatin IX (2), and baccatin X (3), along with 10 known analogues were isolated from an ethanolic extract of the twigs and leaves of Taxus yunnanensis. The new structures were characterized based on extensive spectroscopic analysis. Compounds 1 and 2 were tested for their in vitro cytotoxicity against five human tumor cell lines, and 1 exhibited inhibitory effects on HL-60 and MCF-7, with IC50 values of 3.44 and 9.67 μM, respectively.