Identification and characterization of a new species of Taxus - Taxus qinlingensis by multiple taxonomic methods.

BACKGROUND: The taxonomy of Taxus Linn. remains controversial due to its continuous phenotypic variation and unstable topology, thus adversely affecting the formulation of scientific conservation strategies for this genus. Recently, a new ecotype, known as Qinling type, is mainly distributed in the Qinling Mountains and belongs to a monophyletic group. Here, we employed multiple methods including leaf phenotype comparison (leaf shapes and microstructure), DNA barcoding identification (ITS + trnL-trnF + rbcL), and niche analysis to ascertain the taxonomic status of the Qinling type. RESULTS: Multiple comparisons revealed significant differences in the morphological characters (length, width, and length/width ratio) among the Qinling type and other Taxus species. Leaf anatomical analysis indicated that only the Qinling type and T. cuspidata had no papilla under the midvein or tannins in the epicuticle. Phylogenetic analysis of Taxus indicated that the Qinling type belonged to a monophyletic group. Moreover, the Qinling type had formed a relatively independent niche, it was mainly distributed around the Qinling Mountains, Ta-pa Mountains, and Taihang Mountains, situated at an elevation below 1500 m. CONCLUSIONS: Four characters, namely leaf curvature, margin taper, papillation on midvein, and edges were put forward as primary indexes for distinguishing Taxus species. The ecotype Qingling type represented an independent evolutionary lineage and formed a unique ecological niche. Therefore, we suggested that the Qingling type should be treated as a novel species and named it Taxus qinlingensis Y. F. Wen & X. T. Wu, sp. nov.

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.

[Advances in paclitaxel biosynthesis and transcriptional regulation mechanisms].

Paclitaxel, a rare diterpene extracted from the bark of Chinese yew (Taxus chinensis), is renowned for its anti-cancer activity and serves as a primary drug for treating cancers. Due to the exceptionally low content of paclitaxel in the bark, a semi-synthetic method that depletes Chinese yew resources is used in the production of paclitaxel, which, however, fails to meet the escalating clinical demand. In recent years, researchers have achieved significant progress in heterologous biosynthesis and metabolic engineering for the production of paclitaxel. This article comprehensively reviews the advancements in paclitaxel production, encompassing chemical synthesis, heterologous biosynthesis, and cell engineering. It provides an in-depth introduction to the biosynthetic pathway and transcriptional regulation mechanisms of paclitaxel, aiming to provide a valuable reference for further research on paclitaxel biosynthesis.

Genome-Wide Identification and Expression Pattern Analysis of BAHD Acyltransferase Family in Taxus mairei.

BAHD acyltransferases are involved in catalyzing and regulating the secondary metabolism in plants. Despite this, the members of BAHD family and their functions have not been reported in the Taxus species. In this study, a total of 123 TwBAHD acyltransferases from Taxus wallichiana var. mairei genome were identified and divided into six clades based on phylogenetic analysis, of which Clade VI contained a Taxus-specific branch of 52 members potentially involved in taxol biosynthesis. Most TwBAHDs from the same clade shared similar conserved motifs and gene structures. Besides the typical conserved motifs within the BAHD family, the YPLAGR motif was also conserved in multiple clades of T. mairei. Moreover, only one pair of tandem duplicate genes was found on chromosome 1, with a Ka/Ks ratio < 1, indicating that the function of duplicate genes did not differentiate significantly. RNA-seq analysis revealed different expression patterns of TwBAHDs in MeJA induction and tissue-specific expression experiments. Several TwBAHD genes in the Taxus-specific branch were highly expressed in different tissues of T. mairei, suggesting an important role in the taxol pathway. This study provides comprehensive information for the TwBAHD gene family and sets up a basis for its potential functions.

Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III.

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

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.

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.

Computational redesign of taxane-10ß-hydroxylase for de novo biosynthesis of a key paclitaxel intermediate.

Paclitaxel (Taxol®) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10ß-hydroxylase (T10ßH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10ß-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10ßH, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23 mg/L. Ultimately, the titer of T10OH reached 3.89 mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. KEY POINTS: • The T10ßH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. • The expression and activity of T10ßH were improved through protein engineering. • De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89 mg/L.

Insights from multi-omics integration into seed germination of Taxus chinensis var mairei.

The transition from deep dormancy to seed germination is essential for the life cycle of plants, but how this process occurs in the gymnosperm Chinese yew (Taxus chinensis var mairei), the natural source of the anticancer drug paclitaxel, remains unclear. Herein, we analyse the transcriptome, proteome, spatial metabolome, and spatial lipidome of the Chinese yew and present the multi-omics profiles of dormant and germinating seeds. Our results show that abscisic acid and gibberellic acid 12 homoeostasis is closely associated with gene transcription and protein translation, and the balance between these phytohormones thereby determines if seeds remain dormant or germinate. We find that an energy supply of carbohydrates from glycolysis and the TCA cycle feed into the pentose phosphate pathway during seed germination, and energy supplied from lipids are mainly derived from the lipolysis of triacylglycerols. Using mass spectrometry imaging, we demonstrate that the spatial distribution of plant hormones and phospholipids has a remarkable influence on embryo development. We also provide an atlas of the spatial distribution of paclitaxel C in Chinese yew seeds for the first time. The data from this study enable exploration of the germination mechanism of Chinese yew seeds across several omics levels.

Multiple paternally inherited chloroplast capture events associated with Taxus speciation in the Hengduan Mountains.

Mountainous regions provide a multitude of habitats and opportunities for complex speciation scenarios. Hybridization leading to chloroplast capture, which can be revealed by incongruent phylogenetic trees, is one possible outcome. Four allopatric Taxus lineages (three species and an undescribed lineage) from the Hengduan Mountains, southwest China, exhibit conflicting phylogenetic relationships between nuclear and chloroplast phylogenies. Here, we use multi-omic data at the population level to investigate their historical speciation processes. Population genomic analysis based on ddRAD-seq data revealed limited contemporary inter-specific gene flow involving only populations located close to another species. In a historical context, chloroplast and nuclear data (transcriptome) consistently showed conflicting phylogenetic relationships for T. florinii and the Emei type lineage. ILS and chloroplast recombination were excluded as possible causes, and transcriptome and ddRAD-seq data revealed an absence of the mosaic nuclear genomes that characterize hybrid origin scenarios. Therefore, T. florinii appears to have originated when a lineage of T. florinii captured the T. chinensis plastid type, whereas plastid introgression in the opposite direction generated the Emei Type. All four species have distinct ecological niche based on community investigations and ecological niche analyses. We propose that the origins of both species represent very rare examples of chloroplast capture events despite the paternal cpDNA inheritance of gymnosperms. Specifically, allopatrically and/or ecologically diverged parental species experienced a rare secondary contact, subsequent hybridization and reciprocal chloroplast capture, generating two new lineages, each of which acquired a unique ecological niche. These events might have been triggered by orogenic activities of the Hengduan Mountains and an intensification of the Asian monsoon in the late Miocene, and may represent a scenario more common in these mountains than presently known.

miR5298b regulated taxol biosynthesis by acting on TcNPR3, resulting in an alleviation of the strong inhibition of the TcNPR3-TcTGA6 complex in Taxus chinensis.

Taxol, a valuable but rare secondary metabolite of the genus Taxus, is an effective anticancer drug. Understanding the regulation of taxol biosynthesis may provide a means to increase taxol content. The microRNA miR5298b was found to promote the accumulation of taxol and upregulate several taxol biosynthesis genes, including DBAT, TASY, and T5H, as demonstrated by experiments using the overexpression and mimicry of transient leaves. Moreover, miR5298b cleaves the mRNA sequence of TcNPR3, a homolog of the salicylic acid receptor AtNPR3/4. Overexpression and knockdown by RNA interference of TcNPR3 confirmed that it repressed taxol biosynthesis. These results indicate that miR5298b enhances taxol biosynthesis via the cleavage of TcNPR3. Yeast two-hybrid bimolecular fluorescence complementation and pull-down assays revealed that TcTGA6, a TGA transcription factor, physically interacted with TcNPR3. Functional experiments showed that TcTGA6 negatively regulates taxol biosynthesis by directly combining with the TGACG motif in the promoters of TASY, T5H, and T10H. TcNPR3 enhances TcTGA6 inhibition Luciferase assays showed that miR5298b alleviated the repression of the TcNPR3-TcTGA6 complex. In summary, miR5298b can cleave TcNPR3, thereby alleviating the inhibition of the TcNPR3-TcTGA6 complex to upregulate taxol biosynthesis genes.

Polyploidy as a strategy to increase taxane production in yew cell cultures: Obtaining and characterizing a Taxus baccata tetraploid cell line.

Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug.

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.

Mass spectrometry imaging and single-cell transcriptional profiling reveal the tissue-specific regulation of bioactive ingredient biosynthesis in Taxus leaves.

Taxus leaves provide the raw industrial materials for taxol, a natural antineoplastic drug widely used in the treatment of various cancers. However, the precise distribution, biosynthesis, and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown. Matrix-assisted laser desorption/ionization-mass spectrometry imaging analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei, confirming the tissue-specific accumulation of different active metabolites. Single-cell sequencing was used to produce expression profiles of 8846 cells, with a median of 2352 genes per cell. Based on a series of cluster-specific markers, cells were grouped into 15 clusters, suggesting a high degree of cell heterogeneity in T. mairei leaves. Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways. According to the cell-type annotation, most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells; phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells (including the stomatal complex and guard cells); and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells. A number of novel and cell-specific transcription factors involved in secondary metabolite biosynthesis were identified, including MYB17, WRKY12, WRKY31, ERF13, GT_2, and bHLH46. Our research establishes the transcriptional landscape of major cell types in T. mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism.

Investigation of the role of TmMYB16/123 and their targets (TmMTP1/11) in the tolerance of Taxus media to cadmium.

The toxicity and stress caused by heavy metal contamination has become an important constraint to the growth and flourishing of trees. In particular, species belonging to the genus Taxus, which are the only natural source for the anti-tumor medicine paclitaxel, are known to be highly sensitive to environmental changes. To investigate the response of Taxus spp. to heavy metal stress, we analyzed the transcriptomic profiles of Taxus media trees exposed to cadmium (Cd2+). In total, six putative genes from the metal tolerance protein (MTP) family were identified in T. media, including two Cd2+ stress inducible TMP genes (TmMTP1, TmMTP11 and Taxus media). Secondary structure analyses predicted that TmMTP1 and TmMTP11, which are members of the Zn-CDF and Mn-CDF subfamily proteins, respectively, contained six and four classic transmembrane domains, respectively. The introduction of TmMTP1/11 into the ∆ycf1 yeast cadmium-sensitive mutant strain showed that TmMTP1/11 might regulate the accumulation of Cd2+ to yeast cells. To screen the upstream regulators, partial promoter sequences of the TmMTP1/11 genes were isolated using the chromosome walking method. Several myeloblastosis (MYB) recognition elements were identified in the promoters of these genes. Furthermore, two Cd2+-induced R2R3-MYB TFs, TmMYB16 and TmMYB123, were identified. Both in vitro and in vivo assays confirmed that TmMTB16/123 play a role in Cd2+ tolerance by activating and repressing the expression of TmMTP1/11 genes. The present study elucidated new regulatory mechanisms underlying the response to Cd stress and can contribute to the breeding of Taxus species with high environmental adaptability.

Technology Invention and Mechanism Analysis of Rapid Rooting of Taxus × media Rehder Branches Induced by Agrobacterium rhizogenes.

Taxus, a vital source of the anticancer drug paclitaxel, grapples with a pronounced supply-demand gap. Current efforts to alleviate the paclitaxel shortage involve expanding Taxus cultivation through cutting propagation. However, traditional cutting propagation of Taxus is difficult to root and time-consuming. Obtaining the roots with high paclitaxel content will cause tree death and resource destruction, which is not conducive to the development of the Taxus industry. To address this, establishing rapid and efficient stem rooting systems emerges as a key solution for Taxus propagation, facilitating direct and continuous root utilization. In this study, Agrobacterium rhizogenes were induced in the 1-3-year-old branches of Taxus × media Rehder, which has the highest paclitaxel content. The research delves into the rooting efficiency induced by different A. rhizogenes strains, with MSU440 and C58 exhibiting superior effects. Transcriptome and metabolome analyses revealed A. rhizogenes' impact on hormone signal transduction, amino acid metabolism, zeatin synthesis, and secondary metabolite synthesis pathways in roots. LC-MS-targeted quantitative detection showed no significant difference in paclitaxel and baccatin III content between naturally formed and induced roots. These findings underpin the theoretical framework for T. media rapid propagation, contributing to the sustainable advancement of the Taxus industry.

Transcriptome analysis provides insights into light condition effect on paclitaxel biosynthesis in yew saplings.

BACKGROUND: Taxus is a rare gymnosperm plant that is the sole producer of the anticancer drug paclitaxel. The growth and development of Taxus is affected by environmental factors such as light. However, little is known about how light conditions affect growth and metabolic processes, especially paclitaxel biosynthesis. RESULTS: In this study, we applied three different light conditions to Taxus chinensis young saplings and investigated the physiological response and gene expression. Our observations showed that exposure to high light led to oxidative stress, caused photoinhibition, and damaged the photosynthetic systems in T. chinensis. The paclitaxel content in T. chinensis leaves was significantly decreased after the light intensity increased. Transcriptomic analysis revealed that numerous genes involved in paclitaxel biosynthesis and phenylpropanoid metabolic pathways were downregulated under high light. We also analyzed the expression of JA signaling genes, bHLH, MYB, AP2/ERF transcription factors, and the CYP450 families that are potentially related to paclitaxel biosynthesis. We found that several CYP450s, MYB and AP2/ERF genes were induced by high light. These genes may play an important role in tolerance to excessive light or heat stress in T. chinensis. CONCLUSIONS: Our study elucidates the molecular mechanism of the effects of light conditions on the growth and development of T. chinensis and paclitaxel biosynthesis, thus facilitating the artificial regeneration of Taxus and enhancing paclitaxel production.

Five Species of Taxus Karyotype Based on Oligo-FISH for 5S rDNA and (AG3T3)3.

As a relict plant, Taxus is used in a variety of medicinal ingredients, for instance to treat a variety of cancers. Taxus plants are difficult to distinguish from one another due to their similar morphology; indeed, some species of Taxus cytogenetic data still are unclear. Oligo-FISH can rapidly and efficiently provide insight into the genetic composition and karyotype. This is important for understanding the organization and evolution of chromosomes in Taxus species. We analysed five Taxus species using two oligonucleotide probes. (AG3T3)3 signals were distributed at the chromosome ends and the centromere of five species of Taxus. The 5S rDNA signal was displayed on two chromosomes of five species of Taxus. In addition to Taxus wallichiana var. mairei, 5S rDNA signals were found proximal in the remaining four species, which signals a difference in its location. The karyotype formula of Taxus wallichiana was 2n = 2x = 24m, its karyotype asymmetry index was 55.56%, and its arm ratio was 3.0087. Taxus × media's karyotype formula was 2n = 2x = 24m, its karyotype asymmetry index was 55.09%, and its arm ratio was 3.4198. The karyotype formula of Taxus yunnanensis was 2n = 2x = 24m, its karyotype asymmetry index was 55.56%, and its arm ratio was 2.6402. The karyotype formula of Taxus cuspidate was 2n = 2x = 24m, its karyotype asymmetry index was 54.67%, its arm ratio was 3.0135, and two chromosomes exhibited the 5S rDNA signal. The karyotype formula of T. wallichiana var. mairei was 2n= 2x = 22m + 2sm, its karyotype asymmetry index was 54.33%, and its arm ratio was 2.8716. Our results provide the karyotype analysis and physical genetic map of five species of Taxus, which contributes to providing molecular cytogenetics data for Taxus.

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.