Untargeted metabolite profiling the metabolomes of different Taxus species
To explore the comprehensive variations in metabolomes of different Taxus species, an untargeted approach (15 repeats for each group) was applied, identifying 2,246 metabolites from 8,712 ions with a relative standard deviation < 30% (Additional file 1). Similar to the differences in twig morphology, variations in the metabolomes among different Taxus species were also observed (Fig. 1a). For quality checking, total ion chromatograms were generated, suggesting that the sample preparation met the common standards (Additional file 2). To produce an overview of the metabolic variations, a PCA was performed, and the percentages of explained value in the metabolome analysis of PC1 and PC2 were 25.01% and 31.24%, respectively. The PCA data showed three clearly separated sample groups, indicating separations among the three different species (Fig. 1b). Based on their KEGG annotations, 747 metabolites were predicted to be involved in various primary metabolic pathways, including the amino acid-, carbohydrate-, cofactor and vitamin-, energy-, lipid-, nucleotide-, secondary metabolite-, and terpenoid-related pathways (Fig. 1c and Additional file 3).
Clustering of differential accumulated metabolites
All annotated metabolites were clustered to identify the differential accumulated metabolites (DAMs) among three Taxus species (Fig. 2a). All DAMs were grouped into three Clusters: I, II and III. The T. media predominantly accumulated metabolites were grouped into Cluster I (358 metabolites), the T. cuspidata predominantly accumulated metabolites were grouped into Cluster II (220 metabolites), and the T. mairei predominantly accumulated metabolites were grouped into Cluster III (169 metabolites) (Fig. 2b). Our data showed that the DAMs belonging to the ‘secondary metabolites’, ‘lipids’, ‘cofactors and vitamins’, ‘carbohydrate’ and ‘amino acid’ categories were predominantly accumulated in T. media (Fig. 2c). The Cluster I (T. media predominantly accumulated) consisted of 117 secondary metabolites, 91 amino acids, 51 cofactors and vitamins, 48 carbohydrates, 32 lipids, 17 nucleotides and 2 energy-related metabolites; the Cluster II consisted of 80 secondary metabolites, 53 amino acids, 25 cofactors and vitamins, 23 carbohydrates, 18 lipids, 19 nucleotides and 2 energy-related metabolites; and the Cluster III consisted of 71 secondary metabolites, 32 amino acids, 30 cofactors and vitamins, 13 carbohydrates, 11 lipids, 10 nucleotides and 2 energy-related metabolites (Fig. 2c).
To get a comprehensive overview of variations, all DAMs were classified into different known metabolic pathways. In total, 32, 29, and 38 major pathways were enriched in the T. mairei vs T. cuspidata (Additional file 4), T. media vs T. mairei (Additional file 5), and T. media vs T. cuspidata (Additional file 6) comparisons. Interestingly, the largest number of DAMs in each comparison were enriched in the ‘diterpenoid biosynthesis’ pathway.
Variations in the abundance levels of taxoids among three Taxus species
Paclitaxel biosynthesis is an intricate metabolic pathway that involves a number of precursors, intermediates, and derivatives [5, 30]. By searching the metabolite pool, seven precursors from the MEP pathway, nine intermediates and derivatives, two side chain products, and paclitaxel were detected (Fig. 3a). For the MEP pathway, several precursors, such as D-glyceraldehyde 3-phosphate, 1-deoxy-D-xylulose 5-phosphate, and 2-C-methyl-D-erythritol 4-phosphate, were predominantly accumulated in T. mairei. Two precursors, 4-hydroxy-3-methyl-but-2-enyl diphosphate and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate, were significantly accumulated in T. cuspidata. For the intermediate and derivative products, GGPP, Taxa-4(20),11(12)-dien-5α-ol, and Taxa-4(20),11(12)-dien-5α,13α-diol were predominantly accumulated in T. mairei; Taxa-4(20),11(12)-dien-5α cetoxy-10β ol, 10-Deacetyl-2-debenzoylbaccatin III, 10-Deacetylbaccatin III, and Baccatin III were highest in T. mairei and T. media; and 3'-N-Debenzoyl-2'-deoxytaxol, 3'-N-Debenzoyltaxol, and Paclitaxel were predominantly accumulated in T. cuspidata. For the side chain products, β-Phenylalanine was highly accumulated in T. media and β-Phenylalanoyl baccatin III was greatly accumulated in T. mairei (Fig. 3b). The complete biosynthetic pathway, including the elucidated and putative steps, was summarized in Fig. 4. All the taxane precursors that has been determined in our study were highlighted.
Variations in the abundance levels of flavonoids among three Taxus species
For flavonoid biosynthesis pathway, five intermediate products synthesized by chalcone synthase (CHS), six intermediate products synthesized by chalcone isomerase (CHI), five intermediate products synthesized by flavanone 3-hydroxylase (F3H), and four intermediate products synthesized by flavonol synthase (FLS) were identified (Fig. 5a). For the CHS-synthesized flavonoids, pinocembrin chalcone was highly accumulated in T. mairei, isoliquiritigenin, butein and homoeriodictyol chalcone were predominantly accumulated in T. media, and naringenin chalcone was greatly accumulated in both T. media and T. cuspidata. For the CHI-synthesized flavonoids, only pinocembrin was highly accumulated in T. mairei, eriodictyol and butin were largely accumulated in both T. media, and naringenin, pinostrobin and dihydrotricetin were predominantly accumulated in both T. media and T. cuspidata. Most of the F3H-synthesized flavonoids were predominantly accumulated in T. media, except for dihydroquercetin. For the FLS-synthesized flavonoids, 5-deoxyleucopelargonidin, deoxyleucocyanidin, and leucopelargonidin were highly accumulated in T. media, and leucocyanidin was greatly accumulated in T. mairei (Fig. 5b).
Confirmation of the variations in paclitaxel and its derivatives using a targeted approach
To determine more precisely the differences in taxoids among the three Taxus species, a targeted approach was used to measure the concentrations of paclitaxel, 10-DAB III, baccatin III, and 10-DAP (Additional file 7). The untargeted metabolomics analysis indicated that T. cuspidata and T. mairei contained the highest and the lowest levels of paclitaxel, respectively. The direct quantification with an authentic paclitaxel standard showed that T. cuspidata, T. media, and T. mairei contained 1.67 mg.g-1, 1.22 mg.g-1, and 0.66 mg.g-1 of paclitaxel, respectively (Fig. 6a). The order of the paclitaxel contents was in good agreement with the untargeted metabolome results. For other taxoids, the highest levels of baccatin III and 10-DAP were accumulated in T. cuspidata (0.65 mg.g-1 and 0.80 mg.g-1, respectively), and the highest level of 10-DAB III was detected in T. mairei (0.85 mg.g-1) (Fig. 6b-d). To assess variability in taxoid level among different species of the genus Taxus, another three Taxus species, including T. chinensis, T. fuana and T. yunnanensis, have been collected. A more exhaustive profile of taxoids in the genus has been showed in Additional file 8.
Confirmation of the variations in flavonoids using a targeted approach
To determine more precisely the differences in flavonoids among the three Taxus species, a targeted approach was used to measure the concentrations of amentoflavone, ginkgetin, quercetin and luteolin (Additional file 9). Our data showed that amentoflavone highly accumulated in T. cuspidata (0.14 mg.g-1) and lowly accumulated in T. media (0.024 mg.g-1) (Fig. 6e). Interestingly, ginkgetin, quercetin and luteolin were greatly accumulated in T. mairei rather than the other two taxus trees (Fig. 6f-h).
Systematic correlativity analysis identifies a number of metabolites associated with key metabolites of paclitaxel biosynthesis
An analysis of metabolite–metabolite interaction networks contributed to the understanding of functional relationships and the identification of new compounds associated with key metabolites of paclitaxel biosynthesis. In our study, an interaction network based on the differentially accumulated metabolites was constructed. Furthermore, the taxoid-related networks were divided into three clusters surrounding paclitaxel, baccatin III, and 10-DAB III (Additional file 10). The interaction networks suggested that nine classes of metabolites, phenylpropanoids, flavonoids, alkaloids, carboxylic acid derivatives, quinones, glycosides, saccharides, steroids and terpenoids, may also contribute to the variations in taxoid accumulation in different species (Fig. 7). However, the mechanisms underlying the interactions of these potential new metabolites need to be investigated.