Integrated mass spectrometry imaging and single-cell transcriptome atlas strategies provide novel insights into taxoid biosynthesis and transport in Taxus mairei stems.

Taxol, which is a widely used important chemotherapeutic agent, was originally isolated from Taxus stem barks. However, little is known about the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems. Here, we used MALDI-IMS analysis to visualize the taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. A single-cell T. mairei stem atlas was created, providing a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused an uneven taxoid distribution across T. mairei stems. We developed a single-cell strategy to screen novel transcription factors (TFs) involved in taxol biosynthesis regulation. Several TF genes, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68, were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. In summary, we generated a single-cell Taxus stem metabolic atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway.

Oxetane Ring Formation in Taxol Biosynthesis Is Catalyzed by a Bifunctional Cytochrome P450 Enzyme.

Taxol is a potent drug used in various cancer treatments. Its complex structure has prompted extensive research into its biosynthesis. However, certain critical steps, such as the formation of the oxetane ring, which is essential for its activity, have remained unclear. Previous proposals suggested that oxetane formation follows the acetylation of taxadien-5α-ol. Here, we proposed that the oxetane ring is formed by cytochrome P450-mediated oxidation events that occur prior to C5 acetylation. To test this hypothesis, we analyzed the genomic and transcriptomic information for Taxus species to identify cytochrome P450 candidates and employed two independent systems, yeast (Saccharomyces cerevisiae) and plant (Nicotiana benthamiana), for their characterization. We revealed that a single enzyme, CYP725A4, catalyzes two successive epoxidation events, leading to the formation of the oxetane ring. We further showed that both taxa-4(5)-11(12)-diene (endotaxadiene) and taxa-4(20)-11(12)-diene (exotaxadiene) are precursors to the key intermediate, taxologenic oxetane, indicating the potential existence of multiple routes in the Taxol pathway. Thus, we unveiled a long-elusive step in Taxol biosynthesis.

Metabolomics analysis of different diameter classes of Taxus chinensis reveals that the resource allocation is related to carbon and nitrogen metabolism.

Taxus chinensis (Taxus cuspidata Sieb. et Zucc.) is a traditional medicinal plant known for its anticancer substance paclitaxel, and its growth age is also an important factor affecting its medicinal value. However, how age affects the physiological and metabolic characteristics and active substances of T. chinensis is still unclear. In this study, carbon and nitrogen accumulation, contents of active substances and changes in primary metabolites in barks and annual leaves of T. chinensis of different diameter classes were investigated by using diameter classes instead of age. The results showed that leaves and barks of small diameter class (D1) had higher content of non-structural carbohydrates and C, which were effective in enhancing defense capacity, while N content was higher in medium (D2) and large diameter classes (D3). Active substances such as paclitaxel, baccatin III and cephalomannine also accumulated significantly in barks of large diameter classes. Moreover, 21 and 25 differential metabolites were identified in leaves and barks of different diameter classes, respectively. The differential metabolites were enhanced the TCA cycle and amino acid biosynthesis, accumulate metabolites such as organic acids, and promote the synthesis and accumulation of active substances such as paclitaxel in the medium and large diameter classes. These results revealed the carbon and nitrogen allocation mechanism of different diameter classes of T. chinensis, and its relationship with medicinal components, providing a guidance for the harvesting and utilization of wild T. chinensis.

Overexpression of BAPT and DBTNBT genes in Taxus baccata in vitro cultures to enhance the biotechnological production of paclitaxel.

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 µm coronatine plus 50 mm of randomly methylated-ß-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.

HPLC/ESI-QTOF-MS/MS based untargeted metabolomics authentication of Taxus × media six tissues.

INTRODUCTION: Taxus media (Taxus × media Rehder) is renowned for its high paclitaxel content, serving as a major source for industrial paclitaxel production. In addition to paclitaxel, T. media contains a diverse range of metabolites, including flavonoids, alkaloids, and terpenoids, which have been shown to possess antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects. However, these compounds have not been thoroughly studied as key metabolites in T. media. OBJECTIVE: The untargeted metabolomics analysis of six T. media tissues provides new insights into the development and utilization of T. media metabolites. METHOD: The extracts from six tissues of T. media were analyzed and subjected to analysis using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) and chemometric techniques. RESULTS: Using a reliable HPLC-Q-TOF-MS/MS method, we identified 312 compounds in six T. media tissues, including 214 previously unreported in T. media. To identify characteristic compounds across different tissues, 34 metabolites were further screened. KEGG metabolic pathway analysis revealed that these compounds primarily occur in the metabolic pathways of terpene glycosides, flavans, and O-methylated flavonoids. CONCLUSION: This study initially utilized an HPLC-QTOF-MS/MS-based metabolomics approach to assess the metabolites in different tissues of T. media, providing a basis for their utilization and management.

Effects of Different Doses of sUV-B Exposure on Taxane Compounds' Metabolism in Taxus wallichiana var. Mairei.

UV-B is an important environmental factor that differentially affects plant growth and secondary metabolites. The effects of supplemental ultraviolet-B (sUV-B) exposure (T1, 1.40 kJ·m-2·day-1; T2, 2.81 kJ·m-2·day-1; and T3, 5.62 kJ·m-2·day-1) on the growth biomass, physiological characteristics, and secondary metabolites were studied. Our results indicated that leaf thickness was significantly (p < 0.05) reduced under T3 relative to the control (natural light exposure, CK); The contents of 6-BA and IAA were significantly reduced (p < 0.05); and the contents of ABA, 10-deacetylbaccatin III, and baccatin III were significantly (p < 0.05) increased under T1 and T2. The paclitaxel content was the highest (0.036 ± 0.0018 mg·g-1) under T3. The cephalomannine content was significantly increased under T1. Hmgr gene expression was upregulated under T1 and T3. The gene expressions of Bapt and Dbtnbt were significantly (p < 0.05) upregulated under sUV-B exposure, and the gene expressions of CoA, Ts, and Dbat were significantly (p < 0.05) downregulated. A correlation analysis showed that the 6-BA content had a significantly (p < 0.05) positive correlation with Dbat gene expression. The IAA content had a significantly (p < 0.05) positive correlation with the gene expression of Hmgr, CoA, Ts, and Dbtnbt. The ABA content had a significantly (p < 0.05) positive correlation with Bapt gene expression. Dbat gene expression had a significantly (p < 0.05) positive correlation with the 10-deacetylbaccatin content. Hmgr gene expression was positively correlated with the contents of baccatin III and cephalomannine. Bapt gene expression had a significantly (p < 0.01) positive correlation with the paclitaxel content. A factor analysis showed that the accumulation of paclitaxel content was promoted under T2, which was helpful in clarifying the accumulation of taxane compounds after sUV-B exposure.

An Integrated Strategy Based on 10-DAB Extraction and In Situ Whole-Cell Biotransformation of Renewable Taxus Needles to Produce Baccatin III.

Baccatin III is a crucial precursor in the biosynthesis pathway of paclitaxel. Its main sources are extraction from Taxus or chemical synthesis using 10-deacetylbaccatin III (10-DAB) as substrate. However, these preparation approaches exhibit serious limitations, including the low content of baccatin III in Taxus and the complicated steps of chemical synthesis. Heterologous expression of 10-deacetylbaccatin III-10-O-acetyltransferase (TcDBAT) in microbial strains for biotransformation of 10-DAB is a promising alternative strategy for baccatin III production. Here, the promotion effects of glycerol supply and slightly acidic conditions with a low-temperature on the catalysis of recombinant TcDBAT strain were clarified using 10-DAB as substrate. Taxus needles is renewable and the content of 10-DAB is relatively high, it can be used as an effective source of the catalytic substrate 10-DAB. Baccatin III was synthesized by integrating the extraction of 10-DAB from renewable Taxus needles and in situ whole-cell catalysis in this study. 40 g/L needles were converted into 20.66 mg/L baccatin III by optimizing and establishing a whole-cell catalytic bioprocess. The method used in this study can shorten the production process of Taxus extraction for baccatin III synthesis and provide a reliable strategy for the efficient production of baccatin III by recombinant strains and the improvement of resource utilization rate of Taxus needles.

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis.

Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7ß,9α,10ß,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1ß-dehydroxybaccatin IV (1 a), and a minor 4ß,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4ß,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4ß,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

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.

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.

Switching cell fate by the actin-auxin oscillator in Taxus: cellular aspects of plant cell fermentation.

KEY MESSAGE: Paclitaxel synthesis in Taxus cells correlates with a cell-fate switch that leads to vacuoles of a glossy appearance and vermiform mitochondria. This switch depends on actin and apoplastic respiratory burst. Plant cell fermentation, the production of valuable products in plant cell culture, has great potential as sustainable alternative to the exploitation of natural resources for compounds of pharmaceutical interest. However, the success of this approach has remained limited, because the cellular aspects of metabolic competence are mostly unknown. The production of the anti-cancer alkaloid Paclitaxel has been, so far, the most successful case for this approach. In the current work, we map cellular aspects of alkaloid synthesis in cells of Taxus chinensis using a combination of live-cell imaging, quantitative physiology, and metabolite analysis. We show evidence that metabolic potency correlates with a differentiation event giving rise to cells with large vacuoles with a tonoplast that is of a glossy appearance, agglomerations of lipophilic compounds, and multivesicular bodies that fuse with the plasma membrane. Cellular features of these glossy cells are bundled actin, more numerous peroxisomes, and vermiform mitochondria. The incidence of glossy cells can be increased by aluminium ions, and this increase is significantly reduced by the actin inhibitor Latrunculin B, and by diphenylene iodonium, a specific inhibitor of the NADPH oxidase Respiratory burst oxidase Homologue (RboH). It is also reduced by the artificial auxin Picloram. This cellular fingerprint matches the implications of a model, where the differentiation into the glossy cell type is regulated by the actin-auxin oscillator that in plant cells acts as dynamic switch between growth and defence.

Characterization of lipid droplets from a Taxus media cell suspension and their potential involvement in trafficking and secretion of paclitaxel.

KEY MESSAGE: Our paper describes the potential roles of lipid droplets of Taxus media cell suspension in the biosynthesis and secretion of paclitaxel and, therefore, highlights their involvement in improving its production. Paclitaxel (PTX) is a highly potent anticancer drug that is mainly produced using Taxus sp. cell suspension cultures. The main purpose of the current study is to characterize cellular LDs from T. media cell suspension with a particular focus on the biological connection of their associated proteins, the caleosins (CLOs), with the biosynthesis and secretion of PTX. A pure LD fraction obtained from T. media cells and characterized in terms of their proteome. Interestingly, the cellular LD in T. media sequester the PTX. This was confirmed in vitro, where about 96% of PTX (C0PTX,aq [mg L-1]) in the aqueous solution was partitioned into the isolated LDs. Furthermore, silencing of CLO-encoding genes in the T. media cells led to a net decrease in the number and size of LDs. This coincided with a significant reduction in expression levels of TXS, DBAT and DBTNBT, key genes in the PTX biosynthesis pathway. Subsequently, the biosynthesis of PTX was declined in cell culture. In contrast, treatment of cells with 13-hydroperoxide C18:3, a substrate of the peroxygenase activity, induced the expression of CLOs, and, therefore, the accumulation of cellular LDs in the T. media cells cultures, thus increasing the PTX secretion. The accumulation of stable LDs is critically important for effective secretion of PTX. This is modulated by the expression of caleosins, a class of LD-associated proteins with a dual role conferring the structural stability of LDs as well as regulating lipidic bioactive metabolites via their enzymatic activity, thus enhancing the biosynthesis of PTX.

TcJAV3-TcWRKY26 Cascade Is a Missing Link in the Jasmonate-Activated Expression of Taxol Biosynthesis Gene DBAT in Taxus chinensis.

Jasmonates (JAs) are the most effective inducers for the biosynthesis of various secondary metabolites. Currently, jasmonate ZIM domain (JAZ) and its interactors, such as MYC2, constitute the main JA signal transduction cascade, and such a cascade fails to directly regulate all the taxol biosynthesis genes, especially the rate-limit gene, DBAT. Another JA signaling branch, JAV and WRKY, would probably fill the gap. Here, TcJAV3 was the closest VQ-motif-containing protein in Taxus chinensis to AtJAV1. Although TcJAV3 was overexpressed in AtJAV1 knockdown mutant, JAVRi17, the enhanced disease resistance to Botrytis cinerea caused by silencing AtJAV1 was completely recovered. The results indicated that TcJAV3 indeed transduced JA signal as AtJAV1. Subsequently, TcWRKY26 was screened out to physically interact with TcJAV3 by using a yeast two-hybrid system. Furthermore, bimolecular fluorescence complementation and luciferase complementary imaging also confirmed that TcJAV3 and TcWRKY26 could form a protein complex in vivo. Our previous reports showed that transient TcWRKY26 overexpression could remarkably increase DBAT expression. Yeast one-hybrid and luciferase activity assays revealed that TcWRKY26 could directly bind with the wa-box of the DBAT promoter to activate downstream reporter genes. All of these results indicated that TcWRKY26 acts as a direct regulator of DBAT, and the TcJAV3−TcWRKY26 complex is actually another JA signal transduction mode that effectively regulates taxol biosynthesis in Taxus. Our results revealed that JAV−WRKY complexes directly regulated DBAT gene in response to JA stimuli, providing a novel model for JA-regulated secondary metabolism. Moreover, JAV could also transduce JA signal and function non-redundantly with JAZ during the regulation of secondary metabolisms.

Tissue-specific study across the stem of Taxus media identifies a phloem-specific TmMYB3 involved in the transcriptional regulation of paclitaxel biosynthesis.

Taxus stem barks can be used for extraction of paclitaxel. However, the composition of taxoids across the whole stem and the stem tissue-specificity of paclitaxel biosynthesis-related enzymes remain largely unknown. We used cultivated Taxus media trees for analyses of the chemical composition and protein of major stem tissues by an integrated metabolomic and proteomic approach, and the role of TmMYB3 in paclitaxel biosynthesis was investigated. The metabolomic landscape analysis showed differences in stem tissue-specific accumulation of metabolites. Phytochemical analysis revealed that there is high accumulation of paclitaxel in the phloem. Ten key enzymes involved in paclitaxel biosynthesis were identified, most of which are predominantly produced in the phloem. The full-length sequence of TmMYB3 and partial promoter sequences of five paclitaxel biosynthesis-related genes were isolated. Several MYB recognition elements were found in the promoters of TBT, DBTNBT and TS. Further in vitro and in vivo investigations indicated that TmMYB3 is involved in paclitaxel biosynthesis by activating the expression of TBT and TS. Differences in the taxoid composition of different stem tissues suggest that the whole stem of T. media has potential for biotechnological applications. Phloem-specific TmMYB3 plays a role in the transcriptional regulation of paclitaxel biosynthesis, and may explain the phloem-specific accumulation of paclitaxel.

Transcriptional reprogramming strategies and miRNA-mediated regulation networks of Taxus media induced into callus cells from tissues.

BACKGROUND: Taxus cells are a potential sustainable and environment-friendly source of taxol, but they have low survival ratios and slow grow rates. Despite these limitations, Taxus callus cells induced through 6 months of culture contain more taxol than their parent tissues. In this work, we utilized 6-month-old Taxus media calli to investigate their regulatory mechanisms of taxol biosynthesis by applying multiomics technologies. Our results provide insights into the adaptation strategies of T. media by transcriptional reprogramming when induced into calli from parent tissues. RESULTS: Seven out of 12 known taxol, most of flavonoid and phenylpropanoid biosynthesis genes were significantly upregulated in callus cells relative to that in the parent tissue, thus indicating that secondary metabolism is significantly strengthened. The expression of genes involved in pathways metabolizing biological materials, such as amino acids and sugars, also dramatically increased because all nutrients are supplied from the medium. The expression level of 94.1% genes involved in photosynthesis significantly decreased. These results reveal that callus cells undergo transcriptional reprogramming and transition into heterotrophs. Interestingly, common defense and immune activities, such as "plant-pathogen interaction" and salicylic acid- and jasmonic acid-signaling transduction, were repressed in calli. Thus, it's an intelligent adaption strategy to use secondary metabolites as a cost-effective defense system. MiRNA- and degradome-sequencing results showed the involvement of a precise regulatory network in the miRNA-mediated transcriptional reprogramming of calli. MiRNAs act as direct regulators to enhance the metabolism of biological substances and repress defense activities. Given that only 17 genes of secondary metabolite biosynthesis were effectively regulated, miRNAs are likely to play intermediate roles in the biosynthesis of secondary metabolites by regulating transcriptional factors (TFs), such as ERF, WRKY, and SPL. CONCLUSION: Our results suggest that increasing the biosynthesis of taxol and other secondary metabolites is an active regulatory measure of calli to adapt to heterotrophic culture, and this alteration mainly involved direct and indirect miRNA-induced transcriptional reprogramming. These results expand our understanding of the relationships among the metabolism of biological substances, the biosynthesis of secondary metabolites, and defense systems. They also provide a series of candidate miRNAs and transcription factors for taxol biosynthesis.

Transfecting Taxus � media Protoplasts to Study Transcription Factors BIS2 and TSAR2 as Activators of Taxane-Related Genes.

Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatment of cancer. Taxus spp. biofactories have been used for taxane production, but the lack of knowledge about the taxane biosynthetic pathway and its molecular regulation hinders their optimal function. The difficulties in introducing foreign genes in Taxus spp. genomes hinder the study of the molecular mechanisms involved in taxane production, and a new approach is required to overcome them. In this study, a reliable, simple and fast method to obtain Taxus � media protoplasts was developed, allowing their manipulation in downstream assays for the study of physiological changes in Taxus spp. cells. Using this method, Taxus protoplasts were transiently transfected for the first time, corroborating their suitability for transfection assays and the study of specific physiological responses. The two assayed transcription factors (BIS2 and TSAR2) had a positive effect on the expression of several taxane-related genes, suggesting their potential use for the improvement of taxane yields. Furthermore, the results indicate that the developed method is suitable for obtaining T. � media protoplasts for transfection with the aim of unraveling regulatory mechanisms in taxane production.

Comprehensive temporal reprogramming ensures dynamicity of transcriptomic profile for adaptive response in Taxus contorta.

Plants respond to the environmental perturbations by triggering the dynamic changes within the transcriptome. The assessment of these oscillations within the transcriptome would offer insights into the ecological adaptation of the plants. We evaluated how the transcriptome of Taxus contorta swings under natural conditions to elucidate its adaptive response. Thus, our study provides new insights into the performance of T. contorta under a changing environment during different seasons. The abundance estimation using the RNAseq approach revealed 6727 differentially expressed genes. Comprehensive reprogramming was observed in Taxol biosynthesis, maintenance of redox homeostasis, and generation of effective shield to UV-B, high light intensity, and temperature. Besides differential expression, the alternative splicing (AS) and single nucleotide variations (SNVs) also confer flexibility to the transcriptome of T. contorta. 1936 differentially expressing transcripts were also found to exhibit Differential Exon Usage (DEU) as well as differential SNVs. LC-MS-based untargeted metabolic analysis revealed 7774 ion features, among which around 334 putatively identified metabolites were differentially regulated. Our results showed that the swing and the oscillations of the transcriptome and metabolome of T. contorta ensure adaptability and better survival under changing environment. In addition, varying patterns of AS and SNVs compliment the adaptation provided by differential expression.

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.

Transcriptome analyses provide insights into the expression pattern and sequence similarity of several taxol biosynthesis-related genes in three Taxus species.

BACKGROUND: Taxol is an efficient anticancer drug; however, the accumulation of taxoids can vary hugely among Taxus species. The mechanism underlying differential accumulation of taxoids is largely unknown. Thus, comparative analysis of the transcriptomes in three Taxus species, including T. media, T. mairei and T. cuspidata, was performed. RESULTS: KEGG enrichment analysis revealed that the diterpenoid biosynthesis and cytochrome P450 pathways were significantly enriched in different comparisons. Differential expressions of these taxol biosynthesis related genes might be a potential explanation for the interspecific differential accumulation of taxol and its derivatives. Besides, the sequences of several MEP pathway-associated genes, such as DXS, DXR, MCT, CMK, MDS, HDS, HDR, IPPI, and GGPPS, were re-assembled based on independent transcriptomes from the three Taxus species. Phylogenetic analysis of these MEP pathway-associated enzymes also showed a high sequence similarity between T. media and T. cuspidata. Moreover, 48 JA-related transcription factor (TF) genes, including 10 MYBs, 5 ERFs, 4 RAPs, 3 VTCs, and 26 other TFs, were analyzed. Differential expression of these JA-related TF genes suggested distinct responses to exogenous JA applications in the three Taxus species. CONCLUSIONS: Our results provide insights into the expression pattern and sequence similarity of several taxol biosynthesis-related genes in three Taxus species. The data give us an opportunity to reveal the mechanism underlying the variations in the taxoid contents and to select the highest-yielding Taxus species.