DataSheet1_Transcriptome and Metabolome Analyses of Codonopsis convolvulacea Kurz Tuber, Stem, and Leaf Reveal the Presence of Important Metabolites and Key Pathways Controlling Their Biosynthesis.xlsx

Codonopsisconvolvulacea Kurz. var. vinciflora (Kom.) L.T. Shen is a member of Campanulaceae, which is used in traditional Chinese medicine. However, apart from a few Codonopsis species, no detailed knowledge is available on the metabolite composition and respective transcriptome signatures. We performed a combined transcriptome and metabolome analysis of the tuber, stem, and leaf of C. convolvulacea and found 1,144 metabolites and 231,840 unigenes in three experimental groups. The analysis revealed considerable variations in the three tissues. Tubers were rich in amino acids and derivatives, flavonoids, and organic acids, whereas the stems and leaves were rich in alkaloids and flavonoids, respectively. Transcriptome sequencing revealed candidate genes being involved in flavonoid, tryptophan, and alkaloid biosyntheses. In particular, we indicated that the variation in the isoflavone content is linked to the expressions of CHI, CYP73A, C3′H, F3H, CYP75B1, anthocyanidin synthase, and FLS. In a similar way, the levels of indole, L-tyrosine, and tryptamine were also consistent with the expressions of TDC/DDCs in the respective tissues. In addition, the expression levels of ASP5, ARO8, GOT, and AOC3 indicated that L-tryptophan is being converted to downstream metabolites. Overall, our datasets present a useful resource for future research on the uses of this medicinal plant and put forward many research questions.</p

Presentation4_Transcriptome and Metabolome Analyses of Codonopsis convolvulacea Kurz Tuber, Stem, and Leaf Reveal the Presence of Important Metabolites and Key Pathways Controlling Their Biosynthesis.PPTX

Codonopsisconvolvulacea Kurz. var. vinciflora (Kom.) L.T. Shen is a member of Campanulaceae, which is used in traditional Chinese medicine. However, apart from a few Codonopsis species, no detailed knowledge is available on the metabolite composition and respective transcriptome signatures. We performed a combined transcriptome and metabolome analysis of the tuber, stem, and leaf of C. convolvulacea and found 1,144 metabolites and 231,840 unigenes in three experimental groups. The analysis revealed considerable variations in the three tissues. Tubers were rich in amino acids and derivatives, flavonoids, and organic acids, whereas the stems and leaves were rich in alkaloids and flavonoids, respectively. Transcriptome sequencing revealed candidate genes being involved in flavonoid, tryptophan, and alkaloid biosyntheses. In particular, we indicated that the variation in the isoflavone content is linked to the expressions of CHI, CYP73A, C3′H, F3H, CYP75B1, anthocyanidin synthase, and FLS. In a similar way, the levels of indole, L-tyrosine, and tryptamine were also consistent with the expressions of TDC/DDCs in the respective tissues. In addition, the expression levels of ASP5, ARO8, GOT, and AOC3 indicated that L-tryptophan is being converted to downstream metabolites. Overall, our datasets present a useful resource for future research on the uses of this medicinal plant and put forward many research questions.</p

Presentation2_Transcriptome and Metabolome Analyses of Codonopsis convolvulacea Kurz Tuber, Stem, and Leaf Reveal the Presence of Important Metabolites and Key Pathways Controlling Their Biosynthesis.PPTX

Codonopsisconvolvulacea Kurz. var. vinciflora (Kom.) L.T. Shen is a member of Campanulaceae, which is used in traditional Chinese medicine. However, apart from a few Codonopsis species, no detailed knowledge is available on the metabolite composition and respective transcriptome signatures. We performed a combined transcriptome and metabolome analysis of the tuber, stem, and leaf of C. convolvulacea and found 1,144 metabolites and 231,840 unigenes in three experimental groups. The analysis revealed considerable variations in the three tissues. Tubers were rich in amino acids and derivatives, flavonoids, and organic acids, whereas the stems and leaves were rich in alkaloids and flavonoids, respectively. Transcriptome sequencing revealed candidate genes being involved in flavonoid, tryptophan, and alkaloid biosyntheses. In particular, we indicated that the variation in the isoflavone content is linked to the expressions of CHI, CYP73A, C3′H, F3H, CYP75B1, anthocyanidin synthase, and FLS. In a similar way, the levels of indole, L-tyrosine, and tryptamine were also consistent with the expressions of TDC/DDCs in the respective tissues. In addition, the expression levels of ASP5, ARO8, GOT, and AOC3 indicated that L-tryptophan is being converted to downstream metabolites. Overall, our datasets present a useful resource for future research on the uses of this medicinal plant and put forward many research questions.</p

Presentation1_Transcriptome and Metabolome Analyses of Codonopsis convolvulacea Kurz Tuber, Stem, and Leaf Reveal the Presence of Important Metabolites and Key Pathways Controlling Their Biosynthesis.PPTX

Codonopsisconvolvulacea Kurz. var. vinciflora (Kom.) L.T. Shen is a member of Campanulaceae, which is used in traditional Chinese medicine. However, apart from a few Codonopsis species, no detailed knowledge is available on the metabolite composition and respective transcriptome signatures. We performed a combined transcriptome and metabolome analysis of the tuber, stem, and leaf of C. convolvulacea and found 1,144 metabolites and 231,840 unigenes in three experimental groups. The analysis revealed considerable variations in the three tissues. Tubers were rich in amino acids and derivatives, flavonoids, and organic acids, whereas the stems and leaves were rich in alkaloids and flavonoids, respectively. Transcriptome sequencing revealed candidate genes being involved in flavonoid, tryptophan, and alkaloid biosyntheses. In particular, we indicated that the variation in the isoflavone content is linked to the expressions of CHI, CYP73A, C3′H, F3H, CYP75B1, anthocyanidin synthase, and FLS. In a similar way, the levels of indole, L-tyrosine, and tryptamine were also consistent with the expressions of TDC/DDCs in the respective tissues. In addition, the expression levels of ASP5, ARO8, GOT, and AOC3 indicated that L-tryptophan is being converted to downstream metabolites. Overall, our datasets present a useful resource for future research on the uses of this medicinal plant and put forward many research questions.</p

Presentation3_Transcriptome and Metabolome Analyses of Codonopsis convolvulacea Kurz Tuber, Stem, and Leaf Reveal the Presence of Important Metabolites and Key Pathways Controlling Their Biosynthesis.PPTX

Codonopsisconvolvulacea Kurz. var. vinciflora (Kom.) L.T. Shen is a member of Campanulaceae, which is used in traditional Chinese medicine. However, apart from a few Codonopsis species, no detailed knowledge is available on the metabolite composition and respective transcriptome signatures. We performed a combined transcriptome and metabolome analysis of the tuber, stem, and leaf of C. convolvulacea and found 1,144 metabolites and 231,840 unigenes in three experimental groups. The analysis revealed considerable variations in the three tissues. Tubers were rich in amino acids and derivatives, flavonoids, and organic acids, whereas the stems and leaves were rich in alkaloids and flavonoids, respectively. Transcriptome sequencing revealed candidate genes being involved in flavonoid, tryptophan, and alkaloid biosyntheses. In particular, we indicated that the variation in the isoflavone content is linked to the expressions of CHI, CYP73A, C3′H, F3H, CYP75B1, anthocyanidin synthase, and FLS. In a similar way, the levels of indole, L-tyrosine, and tryptamine were also consistent with the expressions of TDC/DDCs in the respective tissues. In addition, the expression levels of ASP5, ARO8, GOT, and AOC3 indicated that L-tryptophan is being converted to downstream metabolites. Overall, our datasets present a useful resource for future research on the uses of this medicinal plant and put forward many research questions.</p

A Phenylpyruvic Acid Reductase Is Required for Biosynthesis of Tropane Alkaloids

Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis

Engineering Tropane Alkaloid Production Based on Metabolic Characterization of Ornithine Decarboxylase in <i>Atropa belladonna</i>

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) such as pharmaceutical hyoscyamine, anisodamine, and scopolamine, remains largely unknown. In this study, we characterized the physiological and metabolic functions of the ODC gene of Atropa belladonna (AbODC) and determined its role in TA production using metabolic engineering approaches. Feeding assays with enzyme inhibitors indicated that ODC, rather than arginine decarboxylase (ADC), plays a major role in TA biosynthesis. Tissue-specific AbODC expression analysis and β-glucuronidase (GUS) staining assays showed that AbODC was highly expressed in secondary roots, especially in the cylinder tissue. Enzymatic assays indicated that AbODC was able to convert ornithine to putrescine, with the highest activity at pH 8.0 and 30 °C. Additionally, AbODC showed higher catalytic efficiency than other plant ODCs, as evident from the Km, Vmax, and Kcat values of AbODC using ornithine as the substrate. In A. belladonna root cultures, suppression of AbODC greatly reduced the production of putrescine, N-methylputrescine, and TAs, whereas overexpression of AbODC significantly increased the biosynthesis of putrescine, N-methylputrescine, hyoscyamine, and anisodamine. Moreover, transgenic A. belladonna plants overexpressing AbODC showed a significantly higher production of hyoscyamine and anisodamine compared with control plants. These findings indicate that AbODC plays a key role in TA biosynthesis and therefore is a valuable candidate for increasing TA production in A. belladonna

Data_Sheet_1_Metabolic Characterization of Hyoscyamus niger Ornithine Decarboxylase.docx

Ornithine decarboxylase (ODC) catalyzes ornithine decarboxylation to yield putrescine, a key precursor of polyamines, and tropane alkaloids (TAs). Here, to investigate in depth the role of ODC in polyamine/TA biosynthesis and to provide a candidate gene for engineering polyamine/TA production, the ODC gene (HnODC) was characterized from Hyoscyamus niger, a TA-producing plant. Our phylogenetic analysis revealed that HnODC was clustered with ODC enzymes of plants. Experimental work showed HnODC highly expressed in H. niger roots and induced by methyl jasmonate (MeJA). In the MeJA treatment, the production of both putrescine and N-methylputrescine were markedly promoted in roots, while contents of putrescine, spermidine, and spermine were all significantly increased in leaves. By contrast, MeJA did not significantly change the production of either hyoscyamine or scopolamine in H. niger plants. Building on these results, the 50-kDa His-tagged HnODC proteins were purified for enzymatic assays. When ornithine was fed to HnODC, the putrescine product was detected by HPLC, indicating HnODC catalyzed ornithine to form putrescine. Finally, we also investigated the enzymatic kinetics of HnODC. Its Km, Vmax, and Kcat values for ornithine were respectively 2.62 ± 0.11 mM, 1.87 ± 0.023 nmol min-1 μg-1 and 1.57 ± 0.015 s-1, at pH 8.0 and at 30°C. The HnODC enzyme displays a much higher catalytic efficiency than most reported plant ODCs, suggesting it may be an ideal candidate gene for engineering polyamine/TA biosynthesis.</p

Establishment of transgenic hairy root cultures of <i>R</i>. <i>crenulata</i> (genomic PCR detection included).

<p>A: bacteria-free seedlings; B: induction of hairy roots; C: subcultured hairy root; D: hairy roots cultured in liquid medium; E: genomic PCR analysis of genes of interest.</p

The full-length cDNA of RcTYDC and its deduced amino acids.

<p>The coding sequence of RcTYDC was shown with capital letters in bold fonts; the untranslated regions were in small letters; and the stop codon was marked with an asterisk.</p