(±)-Hypernumqulins A-H: Eight pairs of unexpected [2+2] cycloaddition sesquiterpenoid alkaloid with 6/6/6/4/10 ring system from Hypericum monogynum L.

(±)-Hypernumqulins A-H (1-8), eight pairs of enantiomeric quinoline alkaloids fused with an isopentenyl and a germacrane-type sesquiterpenoid, featuring an unprecedented skeleton with 6/6/6/4/10 ring system, were isolated from Hypericum monogynum L. under the guidance of molecular networking strategy. Their structures including absolute configuration were elucidated by NMR spectroscopy analysis, X-ray crystallography and quantum chemical calculation. The proposed [2+2] cycloaddition may play a key biogenic step in building the unexpected skeleton. Most of the isolates exhibited cytotoxicity with IC50 values ranging from 2.82 ± 0.03 to 45.25 ± 1.26 µM against MCF-7, A549 or SGC7901 cells. Furthermore, compounds (±)-1 and (-)-1 could induce apoptosis by upregulating the protein expression level of Bax and downregulating of Bcl-2 in MCF-7 cells. These findings provided the first example of germacrane sesquiterpene quinoline alkaloids, and supported the possibilities for the development of new anti-tumor agents.

A Fruit-Expressed MYB Transcription Factor Regulates Anthocyanin Biosynthesis in Atropa belladonna.

Anthocyanins are water-soluble flavonoid pigments that play a crucial role in plant growth and metabolism. They serve as attractants for animals by providing plants with red, blue, and purple pigments, facilitating pollination and seed dispersal. The fruits of solanaceous plants, tomato (Solanum lycopersicum) and eggplant (Solanum melongena), primarily accumulate anthocyanins in the fruit peels, while the ripe fruits of Atropa belladonna (Ab) have a dark purple flesh due to anthocyanin accumulation. In this study, an R2R3-MYB transcription factor (TF), AbMYB1, was identified through association analysis of gene expression and anthocyanin accumulation in different tissues of A. belladonna. Its role in regulating anthocyanin biosynthesis was investigated through gene overexpression and RNA interference (RNAi). Overexpression of AbMYB1 significantly enhanced the expression of anthocyanin biosynthesis genes, such as AbF3H, AbF3'5'H, AbDFR, AbANS, and Ab3GT, leading to increased anthocyanin production. Conversely, RNAi-mediated suppression of AbMYB1 resulted in decreased expression of most anthocyanin biosynthesis genes, as well as reduced anthocyanin contents in A. belladonna. Overall, AbMYB1 was identified as a fruit-expressed R2R3-MYB TF that positively regulated anthocyanin biosynthesis in A. belladonna. This study provides valuable insights into the regulation of anthocyanin biosynthesis in Solanaceae plants, laying the foundation for understanding anthocyanin accumulation especially in the whole fruits of solanaceous plants.

AabHLH113 integrates jasmonic acid and abscisic acid signaling to positively regulate artemisinin biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is in huge market demand due to its efficient antimalarial action, especially after the COVID-19 pandemic. Many researchers have elucidated that phytohormones jasmonic acid (JA) and abscisic acid (ABA) positively regulate artemisinin biosynthesis via types of transcription factors (TFs). However, the crosstalk between JA and ABA in regulating artemisinin biosynthesis remains unclear. Here, we identified a novel ABA- and JA-induced bHLH TF, AabHLH113, which positively regulated artemisinin biosynthesis by directly binding to the promoters of artemisinin biosynthetic genes, DBR2 and ALDH1. The contents of artemisinin and dihydroartemisinic acid increased by 1.71- to 2.06-fold and 1.47- to 2.23-fold, respectively, in AabHLH1113 overexpressed A. annua, whereas they decreased by 14-36% and 26-53%, respectively, in RNAi-AabHLH113 plants. Furthermore, we demonstrated that AabZIP1 and AabHLH112, which, respectively, participate in ABA and JA signaling pathway to regulate artemisinin biosynthesis, directly bind to and activate the promoter of AabHLH113. Collectively, we revealed a complex network in which AabHLH113 plays a key interrelational role to integrate ABA- and JA-mediated regulation of artemisinin biosynthesis.

Comparative analyses of chloroplast genomes from Six Rhodiola species: variable DNA markers identification and phylogenetic relationships within the genus.

BACKGROUND: As a valuable medicinal plant, Rhodiola has a very long history of folk medicine used as an important adaptogen, tonic, and hemostatic. However, our knowledge of the chloroplast genome level of Rhodiola is limited. This drawback has limited studies on the identification, evolution, genetic diversity and other relevant studies on Rhodiola. RESULTS: Six Rhodiola complete chloroplast genomes were determined and compared to another Rhodiola cp genome at the genome scale. The results revealed a cp genome with a typical quadripartite and circular structure that ranged in size from 150,771 to 151,891 base pairs. High similarity of genome organization, gene number, gene order, and GC content were found among the chloroplast genomes of Rhodiola. 186 (R. wallichiana) to 200 (R. gelida) SSRs and 144 pairs of repeats were detected in the 6 Rhodiola cp genomes. Thirteen mutational hotspots for genome divergence were determined and could be used as candidate markers for phylogenetic analyses and Rhodiola species identification. The phylogenetic relationships inferred by members of Rhodiola cluster into two clades: dioecious and hermaphrodite. Our findings are helpful for understanding Rhodiola's taxonomic, phylogenetic, and evolutionary relationships. CONCLUSIONS: Comparative analysis of chloroplast genomes of Rhodiola facilitates medicinal resource conservation, phylogenetic reconstruction and biogeographical research of Rhodiola.

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna.

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs' production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Costunolide protects against alcohol-induced liver injury by regulating gut microbiota, oxidative stress and attenuating inflammation in vivo and in vitro.

Costunolide (cos) derived from the roots of Dolomiaea souliei (Franch.), which belongs to the Dolomiaea genus in the family Compositae, exert the anti-inebriation effect mainly by inhibiting the absorption of alcohol in the gastrointestinal tract. However, the protective effect of cos against alcohol-induced liver injury (ALI) remains obscure. The present study was aimed to evaluate the hepatoprotective effects of cos (silymarin was used as positive control) against ALI and its potential mechanisms. MTT was used to examine the effect of cos on the cell viability of L-02 cells. Plasma was separated from blood that used to test the levels of TNF-α, IL-6 and IL-12, and LPS while serum separated from blood which used to detect the level of ALT and AST. Liver tissues were obtained for histopathological examination and western blot analysis. Fresh mice feces samples were collected for the detection of bacterial composition. Cos exhibited protective effect against alcoholic-induced liver injury by regulating gut microbiota capacities (higher relative abundance of Firmicutes and Actinobacteria while lower in Bacteroidetes and Proteobacteria), adjusting oxidative stress (reduced the activities of MDA and ROS while promoted SOD, GSH and GSH-PX in L-02 cells) and attenuating inflammation (decreased the levels of ALT, AST, LPS, IL-6, IL-12 and TNF-α) via LPS-TLR4-NF-κB p65 signaling pathway, which might be an active therapeutic agent for treatment of ALI.

[Two new triterpeneoids from roots of Dolomiaea souliei].

The present study analyzed the chemical constituents in dried roots of Dolomiaea souliei. Chromatographic methods, such as normal-phase, and reversed-phase column chromatography, TLC, and preparative HPLC, were applied to separate and purify the petroleum ether extract of D. souliei. The structures of the purified constituents were identified by multiple spectroscopic methods including 1 D NMR, 2 D NMR, IR, UV, and HR-ESI-MS. Fourteen triterpenoids were obtained and identified as bauer-8-ene-3,11-dione-7α-ol(1), bauer-8-ene-3-one-7α,11α-diol(2), 3-oxo-11α-hydroxy-urs-12-ene(3), 3-oxour-12-ene-1ß,11α-diol(4), 3ß,11α-dihydroxy-urs-12-ene(5), taraxast-20-ene-3ß,30-diol(6), 28-hydroxy-3-oxo-12-ursene(7), 3ß-hydroxytaraxast-20-ene-30-aldehyde(8), urs-12-ene-2α,3ß,28-triol(9), 20-hydroxylupan-3-one(10), monogynol A(11), obtusalin(12), 3-oxo, 11α-hydroxy-olean-12-ene(13), and isocabralealactone(14). Among them, compounds 1 and 2 were new compounds. Compounds 4-10, 12, and 14 were isolated from this genus for the first time. Compounds 3 and 11 were obtained from D. souliei for the first time.

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6ß-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

[One new coumarin from seeds of Herpetospermum pedunculosum].

This paper aims to investigate the chemical constituents of the seeds of Herpetospermum pedunculosum. One new coumarin and two known lignans were isolated from the ethanolic extract of the seeds of H. pedunculosum with thin layer chromatography(TLC), silica gel column chromatography, Sephedax LH-20 chromatography, Semi-preparative high performance liquid chromatography and recrystallization, etc. Their structures were elucidated as herpetolide H(1), phyllanglaucin B(2), and buddlenol E(3) by analysis of their physicochemical properties and spectral data. Among them, compound 1 was a new compound, and compounds 2 and 3 were isolated from this genus for the first time. In vitro anti-inflammatory activity test showed that herpetolide H had certain NO inhibitory activity for LPS-induced RAW 264.7 cells, with its IC_(50) value of(46.57±3.28) µmol·L~(-1).

Correction of Bcl-x splicing improves responses to imatinib in chronic myeloid leukaemia cells and mouse models.

Imatinib mesylate (IM) resistance has become a major clinical problem for chronic myeloid leukaemia (CML). It is known that Bcl-x splicing is deregulated and is involved in multiple malignant cancer initiation and chemotherapy resistance, including CML. The aim of the present study was to correct the abnormal splicing of Bcl-x in CML and investigate the subsequent malignant phenotype changes, especially response to IM. The aberrant Bcl-x splicing in CML cells was effectively restored using vivo-Morpholino Antisense Oligomer (vMO). CCK-8 cell viability assay and flow cytometry showed that restoring of Bcl-x splicing increases IM-induced growth inhibition and apoptosis of K562 cells. Moreover, a more significant similar phenomenon was observed in imatinib-resistant CML cell lines K562/G01. Finally, establishment of CML xenograft model had also proved that correcting Bcl-x splicing in vivo can also enhance the anti-tumor effect of IM. Our findings suggest that vMO co-operating with IM can effectively increase the sensitivity of CML cells to IM both in vitro and in vivo, and Bcl-x splicing could become good candidates for chemotherapy-sensitized target in IM-resistant CML.

Functional genomics analysis reveals two novel genes required for littorine biosynthesis.

Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood. Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS). UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added. This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

Pharmacokinetics and tissue distribution study of herpetotriol in rats by ultrahigh-performance liquid chromatography with tandem mass spectrometry.

Herpetotriol, a typical lignan in Herpetospermum pedunculosum Wall's seeds that has long been used to treat icterhepatitis and indigestion and other related diseases in Tibet, is of potential hepatoprotection. This study aims to study the pharmacokinetics features of herpetotriol, including the blood drug concentration - time curve and tissue distribution. The ultrahigh-performance liquid chromatography with tandem mass spectrometry method was established to detect herpetotriol concentration in plasma and tissues, and the method showed good linearity from 10 to 2000 ng/mL (r ≥ 0.9972) and sensitivity (≥10 ng/mL). Our blood drug concentration - time curve indicated that herpetotriol was distributed quickly in rats with a Tmax value at about 0.083 h and eliminated rapidly with a clearance rate at 98.13 ± 8.05 and 137.04 ± 9.48 L·h-1·kg-1 with doses of 5 and 2.5 mg/kg, respectively. Although herpetotriol was detectable in all tested tissues, it has a higher concentration in liver than in heart, lung, spleen, and kidney, which is in line with its hepatoprotection. The pharmacokinetics features uncovered by the present study could provide more information for future pharmacological and toxicological study of herpetotriol.

The cold-induced transcription factor bHLH112 promotes artemisinin biosynthesis indirectly via ERF1 in Artemisia annua.

Basic helix-loop-helix (bHLH) proteins are the second largest family of transcription factors (TFs) involved in developmental and physiological processes in plants. In this study, 205 putative bHLH TF genes were identified in the genome of Artemisia annua and expression of 122 of these was determined from transcriptomes used to construct the genetic map of A. annua. Analysis of gene expression association allowed division of the 122 bHLH TFs into five groups. Group V, containing 15 members, was tightly associated with artemisinin biosynthesis genes. Phylogenetic analysis indicated that two bHLH TFs, AabHLH106 and AabHLH112, were clustered with Arabidopsis ICE proteins. AabHLH112 was induced by low temperature, while AabHLH106 was not. We therefore chose AabHLH112 for further examination. AabHLH112 was highly expressed in glandular secretory trichomes, flower buds, and leaves. Dual-luciferase assays demonstrated that AabHLH112 enhanced the promoter activity of artemisinin biosynthesis genes and AaERF1, an AP2/ERF TF that directly and positively regulates artemisinin biosynthesis genes. Yeast one-hybrid assays indicated that AabHLH112 could bind to the AaERF1 promoter, but not to the promoters of artemisinin biosynthesis genes. Overexpression of AabHLH112 significantly up-regulated the expression levels of AaERF1 and artemisinin biosynthesis genes and consequently promoted artemisinin production.

Biochemical characterization reveals the functional divergence of two tropinone reductases from Przewalskia tangutica.

Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine-forming reductase and pseudotropine-forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (Km , Vmax , and Kcat ) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone.

Sweroside Alleviated LPS-Induced Inflammation via SIRT1 Mediating NF-κB and FOXO1 Signaling Pathways in RAW264.7 Cells.

Pterocephalus hookeri was used as a traditional Chinese medicine for the treatment of rheumatoid arthritis. Sweroside was a main iridoid isolated from P. hookeri. The present study aimed to investigate the anti-inflammatory effect mechanism of sweroside. In RAW264.7 cells induced by lipopolysaccharide (LPS), the abnormal proliferation, the NO content increase, and the downregulated Sirtuin1 (SIRT1) expression were observed. Sweroside could alleviate the inflammation by inhibiting cell proliferation through arresting the cell cycle at the G0/G1 phase, by suppressing pro-inflammatory cytokines and by promoting anti-inflammatory cytokines in LPS-induced RAW264.7 cells. Further mechanism research indicated that sweroside could activate the SIRT1, then suppress the nuclear factor-kappa B (NF-κB) and promote the Forkhead transcription factor O1 (FOXO1) signaling pathways. The present study indicated that sweroside may be the main anti-inflammatory constituent of P. hookeri and a promising candidate for anti-inflammation therapy.

[Cloning and expression analysis of calmodulin gene from Pinellia ternate].

According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.

Comparison of two hyoscyamine 6ß-hydroxylases in engineering scopolamine biosynthesis in root cultures of Scopolia lurida.

Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6ß-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.

ARTEMISININ BIOSYNTHESIS PROMOTING KINASE 1 positively regulates artemisinin biosynthesis through phosphorylating AabZIP1.

The plant Artemisia annua produces the anti-malarial compound artemisinin. Although the transcriptional regulation of artemisinin biosynthesis has been extensively studied, its post-translational regulatory mechanisms, especially that of protein phosphorylation, remain unknown. Here, we report that an ABA-responsive kinase (AaAPK1), a member of the SnRK2 family, is involved in regulating artemisinin biosynthesis. The physical interaction of AaAPK1 with AabZIP1 was confirmed by multiple assays, including yeast two-hybrid, bimolecular fluorescence complementation, and pull-down. AaAPK1, mainly expressed in flower buds and leaves, could be induced by ABA, drought, and NaCl treatments. Phos-tag mobility shift assays indicated that AaAPK1 phosphorylated both itself and AabZIP1. As a result, the phosphorylated AaAPK1 significantly enhanced the transactivational activity of AabZIP1 on the artemisinin biosynthesis genes. Substituting the Ser37 with Ala37 of AabZIP1 significantly suppressed its phosphorylation, which inhibited the transactivational activity of AabZIP1. Consistent overexpression of AaAPK1 significantly increased the production of artemisinin, as well as the expression levels of the artemisinin biosynthesis genes. Our study opens a window into the regulatory network underlying artemisinin biosynthesis at the post-translational level. Importantly, and for the first time, we provide evidence for why the kinase gene AaAPK1 is a key candidate for the metabolic engineering of artemisinin biosynthesis.

Bacillamidins A⁻G from a Marine-Derived Bacillus pumilus.

Seven long-chain amides, including five previously undescribed bacillamidins A⁻E (1⁻5) and two previously reported synthetic analogs, bacillamidins F (6) and G (7), were isolated from extracts of the marine-derived Bacillus pumilus strain RJA1515. The structures of the new compounds were established by extensive analysis of 1D and 2D nuclear magnetic resonance (NMR) data as well as high resolution mass spectrometry (HRMS), and the absolute configurations of the stereogenic carbons of 1⁻4 were established by comparison of the calculated and the experimental electronic circular dichroism (ECD) spectra. The cytotoxic and antimicrobial activities of 1⁻7 were evaluated.

[Effects of nitrogen form on accumulation of alkaloids and expression of relative genes in Atropa belladonna].

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value. In this paper, the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, and the expression levels of key enzyme genes PMT, TRⅠ and H6H in secondary metabolism of A. belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50 (NH⁺4/NO⁻3) treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine. The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine, they both increased with the rise of ammonium ratio, reaching the highest at 75/25 (NH⁺4/NO⁻3). The detection of signaling molecule nitric oxide (NO) showed that the NO concentration decreased with the decrease of nitrate proportion. Further detection of gene expression levels of PMT, TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots. When the ratio of ammonium to nitrate was 50/50, PMT, TRⅠ and H6H in leaves and roots had higher expression levels. It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes. 50/50 (NH⁺4/NO⁻3) treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots, promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.