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Fig. 4. A in Chemical diversity of k¯anuka: Inter- and intraspecific variation of foliage terpenes and flavanones of Kunzea (Myrtaceae) in Aotearoa/New Zealand

Fig. 4. A: PCA score plot for digitized 1 H NMR data (8.5–20 ppm) of solvent extracts from AK herbarium voucher leaf extracts from ten New Zealand Kunzea species; B: PC1 loading plot.

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Fig. 3. A in Chemical diversity of k¯anuka: Inter- and intraspecific variation of foliage terpenes and flavanones of Kunzea (Myrtaceae) in Aotearoa/New Zealand

Fig. 3. A: PCA score plot for digitized GC data of solvent extracts from AK herbarium voucher leaf extracts from ten New Zealand Kunzea species; B PC1 loading plot.

Fig. 4 in Volatile metabolic profiling and functional characterization of four terpene synthases reveal terpenoid diversity in different tissues of Chrysanthemum indicum L

Fig. 4. Characterization of CiTPS enzymatic activity. (A) TIC of α-pinene produced in the root of C. indicum, the enzymatic products of CiTPS1 (without an MBP tag), the enzymatic products of CiTPS2 (pET-(-tp)-CiTPS2), and the negative control (GPP + boiled protein). (B) TIC of the corresponding metabolites produced in the root, the enzymatic products of CiTPS3 (without an MBP tag), and the negative control (FPP + boiled protein). (C) TIC of the enzymatic products of CiTPS4 (with GPP and FPP as substrate and without an MBP tag) and the negative control (GPP/FPP + boiled protein). TIC, total ion chromatogram.

Fig. 5 in Volatile metabolic profiling and functional characterization of four terpene synthases reveal terpenoid diversity in different tissues of Chrysanthemum indicum L

Fig. 5. Expression pattern analysis of CiTPSs in different tissues of C. indicum. (A–D) Expression pattern analysis of CiTPS1, CiTPS2, CiTPS3, and CiTPS4 in the root, stem, leaf, flower bud, and flower. Error bars represent the standard deviations between the three biological replicates. Different letters represent significant differences at P <0.05. (E) The corresponding products (volatile terpenoids) produced by CiTPS1, CiTPS2, CiTPS3 or CiTPS4 in C. indicum. n. d.: not detected; trace: 0–1 × 10 4 ng/mg, FW; +, 0.1–10 ng/mg, FW; + +, 10–100 ng/mg, FW; +++,>100 ng/mg, FW.

Fig. 3 in Volatile metabolic profiling and functional characterization of four terpene synthases reveal terpenoid diversity in different tissues of Chrysanthemum indicum L

Fig. 3. (A) Amino acid sequence alignments of CiTPS1, CiTPS2, CiTPS3, and CiTPS4 (unigene 0021,699, 0037,767, 0060,549, and 0062,052, respectively) and six TPSs from other plants. (B) Phylogenetic analysis of four TPSs from C. indicum and some TPSs from other plants. Detailed information, including plant names and GenBank identification numbers, are shown in Supplementary Table S3. Phylogenetic analysis was performed using the maximum likelihood method and the MEGA and ITOL tools (http://itol.embl.de/).

Fig. 1 in Volatile metabolic profiling and functional characterization of four terpene synthases reveal terpenoid diversity in different tissues of Chrysanthemum indicum L

Fig. 1. Volatile terpenoids in different tissues of C. indicum. (A) The representative total ion chromatogram of the root, stem, leaf, flower bud, and flower. (B) Heatmap of monoterpenoids and sesquiterpenoids in different tissues of C. indicum.

Fig. 7 in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 7. Molecular models of Catharanthus CYP72A1 and Camptotheca CYP72A564 and CYP72A565. (A) Backbone overlays of Catharanthus CYP72A1 and Camptotheca CYP72A564 and CYP72A565 models are shown with the alpha-carbon RMSD amongst CYP72A1, CYP72A564 and CYP72A565 depicted from green (0.0 Å) to yellow (3.0 Å) to red (4.5 Å). (B) SRS regions in CYP72A proteins shown with predicted substrate contact residues (gray fill). (C) Identical versus (D) different side chain residues predicted within 4.5 Å of loganin (aqua) docked in Catharanthus CYP72A1 (blue) and loganic acid (gray) docked in Camptotheca CYP72A564 (orange). (E) Identical versus (F) different side chain residues predicted within 4.5 Å of loganin (aqua) docked in Catharanthus CYP72A1 (blue) and loganic acid (gray) docked in Camptotheca CYP72A565 (rose).

Fig. 5 in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 5. Area of loganic acid, loganin and products from in vitro reconstitution assays conducted with full-length Camptotheca His6-tagged CPR1. Integrated areas from LC-MS analyses of purified His6-tagged CYP72A proteins reconstituted with full-length His6-tagged Caa CPR1 are shown for no NADPH (gray) and plus NADPH (gray slashed) reactions.

Fig. 2. CYP72A in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 2. CYP72A multiple sequence alignment. Signal anchor domain fusion of CYP72A565 into CYP72A564 is underlined; SRS regions are underlined in bold; predicted substrate contacts within 4.5 Å of loganic acid/loganin are gray-filled.

Fig. 1 in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 1. Proposed divergence of the TIA pathway between Camptotheca and Catharanthus. After 7-deoxyloganic acid hydroxylase (7DLH) converts 7-deoxyloganic acid to loganic acid, the pathways in these two species diverge. The Catharanthus pathway uses loganic acid methyltransferase (LAMT) to convert loganic acid into loganin and secologanin synthase (SLS) to convert loganin into secologanin. The Camptotheca pathway bypasses LAMT and uses secologanic acid synthase (SLAS) to metabolize loganic acid directly to secologanic acid.

Fig. 8 in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 8. Molecular models of Camptotheca CYP72A564, CYP72A565 and CYP72A730. (A) Backbone overlays of Camptotheca CYP72A730, CYP72A564 and CYP72A565 models are shown with the RMSD variance of CYP72A564 and CYP72A565 from the CYP72A730 backbone depicted in green (0.0 Å), yellow (3.0 Å) and red (4.5 Å). (B) SRS regions in CYP72A proteins shown with predicted substrate contact residues (gray fill). (C) Identical versus (D) different side chain residues predicted within 4.5 Å of loganic acid (gray) docked in Camptotheca CYP72A564 (orange) versus loganic acid (aqua) docked in CYP72A730 (magenta). (E) Identical versus (F) different side chain residues predicted within 4.5 Å of loganic acid (gray) docked in Camptotheca CYP72A565 (rose) versus loganic acid (aqua) docked in CYP72A730 (magenta).

Fig. 4 in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 4. LC-MS analyses of in vitro assays with purified His6-tagged CYP72A proteins reconstituted with His6-tagged CPR proteins. Reactions containing purified His6- tagged CYP72A protein, full-length His6-tagged Caa CPR1 protein (A, B) or full-length His6-tagged Caa CPR2 protein (C, D), and 250 μM loganic acid (A,C) or loganin (B,D), were incubated at 30◦ C and analyzed by LC-MS as described in experimental procedures. Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089); loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246) are given with stacked chromatograms as marked.

Fig. 3 in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 3. LC-MS analyses of in vitro assays with CYP72A proteins expressed in yeast microsomes. 100 μl reactions containing 10 μl CYP72A microsomes isolated from WAT11 with 250 μM loganic acid or loganin and 500 μM NADPH in 100 mM NaPO (pH 7.4) were incubated at 30◦ C and analyzed by LC-MS as 4 described in the experimental procedures. (A) Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089). (B) Extracted ion chromatograms for loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246).

Fig. 6. Type I in P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Fig. 6. Type I binding spectra of SLAS candidates. Substrate-induced Type I binding spectra for (A) CYP72A564, (B) CYP72A565 and from 998 nM (violet) to 1.90 mM (crimson) using loganic acid (top inset) or loganin (bottom inset). (C) Binding isotherms calculated as the difference of the valley (~419 nm) from the peak (~388 nm) are shown for loganic acid () and loganin (). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11. Experiment 3 in Headspace GC-MS analysis of differences in intra- and interspecific Terpene profiles of Picea pungens Engelm. and P. abies (L.) Karst

Fig. 11. Experiment 3, mean GC/MS peak area (±SE) of branches, buds, and needles in blue and Norway spruce. Means with different letters are significantly different within each species (ANOVA, Tukey's HSD).

Fig. 3 in Headspace GC-MS analysis of differences in intra- and interspecific Terpene profiles of Picea pungens Engelm. and P. abies (L.) Karst

Fig. 3. Sample GC traces of blue spruce (B1), Norway spruce (N1), and cedar of Lebanon (C1). Compounds were identified by retention indices, mass spectra (NIST library), and reference comparisons. Identified compounds are: (1) tricyclene, (2) α-pinene, (3) camphene, (4) sabinene, (5) β-pinene, (6) β-myrcene, (7) 3-carene, (8) limonene, (9) β-phellandrene (coeluting with limonene), (10) 1,8-cineole, (11) γ-terpinene, (12) terpinolene, (13) camphor, (14) α-terpineol, and (15) β-caryophyllene.

Fig. 2. Chemical structures for terpenes detected from branch tip samples. Numbering follows Table 1 in Headspace GC-MS analysis of differences in intra- and interspecific Terpene profiles of Picea pungens Engelm. and P. abies (L.) Karst

Fig. 2. Chemical structures for terpenes detected from branch tip samples. Numbering follows Table 1 (elution order).

Fig. 12. Experiment 4 in Headspace GC-MS analysis of differences in intra- and interspecific Terpene profiles of Picea pungens Engelm. and P. abies (L.) Karst

Fig. 12. Experiment 4, PCA of B1, B7, B8, N1, N7, N11, and C1 run on a chiral column (standardized TIC responses). Largest circles show 95% confidence limits for each tree species, small circles show 95% confidence limits for each tree.

Fig. 1 in Headspace GC-MS analysis of differences in intra- and interspecific Terpene profiles of Picea pungens Engelm. and P. abies (L.) Karst

Fig. 1. Mean abundance of Norway volatiles vs time (left) and the logarithm of average abundance of Norway vs AHS oven temperature (right).

Fig. 9. Experiment 2 in Headspace GC-MS analysis of differences in intra- and interspecific Terpene profiles of Picea pungens Engelm. and P. abies (L.) Karst

Fig. 9. Experiment 2, comparison of N1, N7, and N11 GC traces (standardized TIC responses). Different colored sample at right (N1 – blue, N7 – green, and N11 – red) correspond to colored peaks at left (as indicated by arrows). N1 samples had higher percentages of β-pinene, N7 samples had higher percentages of α-pinene and limonene (and β-phellandrene), and N11 had higher percentages of camphene, β-myrcene and 1,8-cineole. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)