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Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6β-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2, exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N-methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N-methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to β-glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Tropane alkaloids (TAs) are widely distributed in the Solanaceae, while some important medicinal tropane alkaloids (mTAs), such as hyoscyamine and scopolamine, are restricted to certain species/tribes in this family. Little is known about the genomic basis and evolution of TAs biosynthesis and specialization in the Solanaceae. Here, we present chromosome-level genomes of two representative mTAs-producing species: Atropa belladonna and Datura stramonium. Our results reveal that the two species employ a conserved biosynthetic pathway to produce mTAs despite being distantly related within the nightshade family. A conserved gene cluster combined with gene duplication underlies the wide distribution of TAs in this family. We also provide evidence that branching genes leading to mTAs likely have evolved in early ancestral Solanaceae species but have been lost in most of the lineages, with A. belladonna and D. stramonium being exceptions. Furthermore, we identify a cytochrome P450 that modifies hyoscyamine into norhyoscyamine. Our results provide a genomic basis for evolutionary insights into the biosynthesis of TAs in the Solanaceae and will be useful for biotechnological production of mTAs via synthetic biology approaches., (© 2023. The Author(s).)

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., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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 6 β -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.

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 K m , V max , and K cat 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 .

Atropa belladonna is one of the most important plant sources for producing pharmaceutical tropane alkaloids (TAs). T1 progeny of transgenic A. belladonna, in which putrescine N-methyltransferase (EC. 2.1.1.53) from Nicotiana tabacum (NtPMT) and hyoscyamine 6β-hydroxylase (EC. 1.14.11.14) from Hyoscyamus niger (HnH6H) were overexpressed, were established to investigate TA biosynthesis and distribution in ripe fruits, leaves, stems, primary roots and secondary roots under field conditions. Both NtPMT and HnH6H were detected at the transcriptional level in transgenic plants, whereas they were not detected in wild-type plants. The transgenes did not influence the root-specific expression patterns of endogenous TA biosynthetic genes in A. belladonna. All four endogenous TA biosynthetic genes (AbPMT, AbTRI, AbCYP80F1 and AbH6H) had the highest/exclusive expression levels in secondary roots, suggesting that TAs were mainly synthesized in secondary roots. T1 progeny of transgenic A. belladonna showed an impressive scopolamine-rich chemotype that greatly improved the pharmaceutical value of A. belladonna. The higher efficiency of hyoscyamine conversion was found in aerial than in underground parts. In aerial parts of transgenic plants, hyoscyamine was totally converted to downstream alkaloids, especially scopolamine. Hyoscyamine, anisodamine and scopolamine were detected in underground parts, but scopolamine and anisodamine were more abundant than hyoscyamine. The exclusively higher levels of anisodamine in roots suggested that it might be difficult for its translocation from root to aerial organs. T1 progeny of transgenic A. belladonna, which produces scopolamine at very high levels (2.94-5.13 mg g(-1)) in field conditions, can provide more valuable plant materials for scopolamine production., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Atropa belladonna is officially deemed as the commercial plant to produce scopolamine in China. In this study we report the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which encode the upstream key enzyme putrescine N-methyltransferase (PMT) and the downstream key enzyme hyoscyamine 6β-hydroxylase (H6H), respectively, in transgenic herbal plants Atropa belladonna. Analysis of gene expression profile indicated that both pmt and h6h were expressed at a higher level in transgenic lines, which would be favorable for biosynthesis of scopolamine. High-performance liquid chromatography result suggested that transgenic lines could produce higher accumulation of scopolamine at different levels compared with wild-type lines. Scopolamine content increased to 7.3-fold in transgenic line D9 compared with control lines. This study not only confirms that co-overexpression of pmt and h6h is an ideal method to improve the biosynthetic capacity of scopolamine but also successfully cultivates the transgenic line D9, which significantly enhanced the scopolamine accumulation. Our research can serve as an alternative choice to provide scopolamine resources for relative industry, which is more competitive than conventional market., (Copyright © Physiologia Plantarum 2011.)

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. [ABSTRACT FROM AUTHOR]

Polyamines, including putrescine, spermidine, and spermine, play critical roles in cell physiology by different forms. As a rate-limiting enzyme that converts ornithine to putrescine, ornithine decarboxylase (ODC, EC 1.1.1.37) has been studied in detail in animals and microorganisms, but its specific functions are poorly understood in plants. In this study, the metabolic and developmental roles of the ODC gene were studied through RNAi-mediated suppression of the ODC gene (AbODC) in A. belladonna. Suppression of AbODC reduced the production of precursors of medicinal tropane alkaloids, including putrescine and N -methylputrescine, as well as hyoscyamine and scopolamine. In AbODC -RNAi roots, the production of putrescine and spermidine in free form was reduced, but in the AbODC -RNAi leaves, the content of free polyamines was not altered. In the roots/leaves of AbODC -RNAi plants, the production of conjugated and bound polyamines was reduced. In addition, suppression of the ODC gene resulted in reduction of polyamines and pollen sterility in AbODC- RNAi flowers. In floral organs, GUS-staining results indicated that AbODC was domainantly expressed in pollen. In summary, ornithine decarboxylase not only plays a key role in regulating the biosynthesis of diverse forms of polyamines and medicinal tropane alkaloids, but also participates in pollen development. [Display omitted] • Suppression of AbODC reduced the yield of tropane alkaloids and its precursors. • Inhibition of AbODC expression suppressed the biosynthesis of various polyamines. • In flower, AbODC was dominantly expressed in pollen. • Suppression of the ODC gene resulted in pollen sterility in AbODC- RNAi plants. [ABSTRACT FROM AUTHOR]

Atropa belladonna is widely utilized for producing anticholinergic tropane alkaloids (TAs). Of them, scopolamine is the most valuable component due to its potent pharmacological effects and minimal side effects. However, the content of scopolamine in planta is extremely low, emphasizing the need to cultivate high-yield varieties of A. belladonna. Here it is reported that the genes encoding the rate-limiting upstream enzyme ornithine decarboxylase from A. belladonna (AbODC) and downstream enzyme hyoscyamine 6 β -hydroxylase from Hyoscyamus niger (HnH6H) were simultaneously overexpressed to genetically modify A. belladonna plants. Metabolite analysis in the AbODC overexpressing plants showed that the levels of hyoscyamine and anisodamine in both the roots and leaves of A. belladonna were significantly increased. However, there was no significant effect on the accumulation of scopolamine. On the other hand, overexpressing HnH6H alone was able to increase the contents of scopolamine to some extent. When AbODC and HnH6H were co-expressed, there was a greater increase in the contents of scopolamine in both roots and leaves, especially in the leaves, where the average content of scopolamine reached 6.67 mg/g dry weight (DW). Moreover, the increasing expression levels of ODC in transgenic plants greatly enhanced the biosynthesis of polyamines, thereby enhancing the cold tolerance trait of A. belladonna plants. These findings highlight the importance of co-overexpressing AbODC and HnH6H in A. belladonna and provide an effective promising approach to developing new varieties of A. belladonna with not only a high yield of scopolamine but also a high tolerance of cold stress. • AbODC overexpression boosts hyoscyamine yield and cold tolerance in A. belladonna. • HnH6H overexpression promotes scopolamine yield in A. belladonna. • Plants co-expressing ODC and H6H show cold tolerance and higher scopolamine yield. [ABSTRACT FROM AUTHOR]

• Glyphosate is used for screening transgenic plants of Atropa belladonna. • Homozygous transgenic plants overexpressing HnH6H and G2-EPSPS are obtained. • Homozygous transgenic plants have high-yield scopolamine and glyphosate resistance. Atropa belladonna , one of the most important medicinal plants in China, is used to produce anticholinergic tropane alkaloids. Weeds severely reduce the agricultural productivity of A. belladonna in fields, and the low content of high-value scopolamine in planta limits its economic value. In this study, homozygous lines of A. belladonna , with glyphosate resistance and high yield of scopolamine, were developed using metabolic engineering technology for the first time. Two homozygous transgenic lines (T2GH03 and T2GH06), harboring G2-EPSPS and HnH6H , were established and tested in the field. Glyphosate resistance assays demonstrated that the two homozygous transgenic lines could resist commercial recommended dose of glyphosate. Alkaloid analysis showed that the transgenic lines produced more scopolamine than wild-type plants. T2GH03 and T2GH06 produced scopolamine at the levels of 5.45 mg/g dry weight (DW) and 7.04 mg/g DW respectively in leaves, and 1.06 mg/g DW to 1.07 mg/g DW respectively in roots. Compared with wild-type plants, the scopolamine contents of homozygous transgenic lines increased by over 10 folds in leaves and nearly 5 folds in roots. To sum up, Atropa belladonna's new varieties with glyphosate resistance and high-yield scopolamine are developed, which are highly valuable for industrial production of scopolamine. [ABSTRACT FROM AUTHOR]