Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.
Atropa belladonna/metabolism* ; Hyoscyamine/analysis* ; Nitrogen/metabolism* ; Nitroprusside ; Scopolamine/analysis* ; Secondary Metabolism ; Sodium Chloride ; Stress, Physiological

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.
Atropa belladonna ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Hyoscyamine ; biosynthesis ; Mixed Function Oxygenases ; Nitrogen ; metabolism ; Scopolamine ; metabolism

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.
Cloning, Molecular ; DNA, Complementary ; Datura ; enzymology ; genetics ; Escherichia coli ; Hyoscyamine ; chemistry ; Mixed Function Oxygenases ; genetics ; Plant Leaves ; enzymology ; Plant Roots ; enzymology ; Recombinant Proteins ; genetics ; Scopolamine Hydrobromide ; chemistry

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 μmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 μmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 μmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.
Acetates ; pharmacology ; Atropa belladonna ; drug effects ; genetics ; metabolism ; Cyclopentanes ; pharmacology ; Gene Expression Regulation, Plant ; Hyoscyamine ; metabolism ; Oxylipins ; pharmacology ; Plant Leaves ; metabolism ; Plant Roots ; metabolism ; Scopolamine ; metabolism

Jimson weed, also known as Datura stramonium, is a member of the Solanaceae family. Jimson weed was used by American Indians for medicinal and religious purposes. All parts of the Jimson weed plant are poisons, containing the alkaloids atropine, hyoscyamine and scopolamine. So, it is caused by these components to make a anticholinergic toxicity within 6h after ingestion. Initial manifestations include dry mucus membrane, blurred, vision, thirst, difficulty swallowing and speaking, and photophobia, and may be followed by hyperthermia, confusion, agitation, combative behavior, hallucination, urinary retention, seizure and coma. We experienced two patients who developed combative behavior and agitation with pupil dilation caused by Jimson weed. They discharged with improvement after supportive for 2days.
Alkaloids ; Animals ; Atropine ; Coma ; Comb and Wattles ; Datura stramonium* ; Deglutition ; Dihydroergotamine ; Eating* ; Fever ; Hallucinations ; Humans ; Hyoscyamine ; Indians, North American ; Membranes ; Mucus ; Photophobia ; Plants ; Poisons ; Pupil ; Scopolamine Hydrobromide ; Seizures ; Solanaceae ; Thirst ; Urinary Retention

Atropa belladonna L. is the officially medicinal plant species and the main commercial source of scopolamine and hyoscyamine in China. In this study, we reported the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which respectively encoded the upstream key enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53) and the downstream key enzyme hyoscyamine 6beta-hydroxylase (H6H; EC 1.14.11.11) in transgenic hair root cultures of Atropa belladonna L. HPLC results suggested that four transgenic hair root lines produced higher content of scopolamine at different levels compared with nontransgenic hair root cultures. And scopolamine content increased to 8.2 fold in transgenic line PH2 compared with that of control line; and the other four transgenic lines showed an increase of scopolamine compared with the control. Two of the transgenic hair root lines produced higher levels of tropane alkaloids, and the content increased to 2.7 fold in transgenic line PH2 compared with the control. The gene expression profile indicated that both PMT and H6H expressed at a different levels in different transgenic hair root lines, which would be helpful for biosynthesis of scopolamine. Our studies suggested that overexpression of A. belladonna endogenous genes PMT and H6H could enhance tropane alkaloid biosynthesis.
Atropa belladonna ; enzymology ; genetics ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Hyoscyamine ; metabolism ; Methyltransferases ; genetics ; metabolism ; Mixed Function Oxygenases ; genetics ; metabolism ; Plant Roots ; enzymology ; genetics ; Plants, Genetically Modified ; enzymology ; genetics ; Plants, Medicinal ; enzymology ; genetics ; Scopolamine Hydrobromide ; metabolism ; Synthetic Biology ; Tropanes ; metabolism