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Abstract Leucojum aestivum is known for its ability to biosynthesize alkaloids with therapeutic properties, among which galanthamine used for the treatment of Alzheimer's disease. New sources of this alkaloid are still being explored. In this study, a novel strain PLV of endophytic bacterium Paenibacillus lautus was isolated from in vitro L . aestivum plants. We report the whole genome sequence of that strain and its capacity to produce alkaloids and growth regulators. The effect of elicitation with autoclaved bacteria on the production of alkaloids was examined. Ten alkaloids were identified in bacteria extracts: galanthamine, lycorine, ismine, lycoramine, haemanthamine, tazettine, galanthine, homolycorine, 1,2-dihydrochlidanthine, and hippeastrine. The mean contents of galanthamine and lycorine were 37.51 µg/g of dry weight (DW) and 129.93 µg/g of DW, respectively. Moreover, isolated P . lautus strain synthesized: indole-3-acetic acid, t-zeatin, c-zeatin, kinetin, gibberellin A 1 , abscisic acid, salicylic acid, benzoic acid. In vitro elicitation of cultures with P . lautus increased dry biomass, stimulated galanthamine and lycorine production, contributed to 8,9-desmethylenebis (oxy)-7,9 dimethoxy-crinan biosynthesis, change pigments content, and antioxidant enzymes activities. Our findings for the first time point out that galanthamine can be synthesized by an microorganism. Moreover isolated strain can be used as a new elictor of Amaryllidaceae alkaloids biosynthesis.

Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites with diverse pharmacological properties. Norbelladine is the entry compound in AAs biosynthesis and is produced from the condensation of tyramine and 3,4-dihydroxybenzaldehyde (3,4-DHBA). There are two reported enzymes capable of catalyzing this reaction in-vitro , both with low yield. The first one, norbelladine synthase (NBS), was shown to condense tyramine and 3,4-DHBA, while noroxomaritidine/norcraugsodine reductase (NR), catalyzes a reduction reaction to produce norbelladine. To clarify the mechanisms involved in this controversial step, both NBS and NR homologs were identified from the transcriptome of Narcissus papyraceus and Leucojum aestivum , cloned and expressed in Escherichia coli. Enzymatic assays performed with tyramine and 3,4-DHBA with each enzyme separately or combined, suggested that NBS and NR function together for the condensation of tyramine and 3,4-DHBA into norcraugsodine and further reduction into norbelladine. Using molecular homology modeling and docking studies, we predicted models for the binding of tyramine and 3,4-DHBA to NBS, and of the intermediate norcraugsodine to NR. Moreover, we show that NBS and NR physically interact in yeast and in-planta , that both localize to the cytoplasm and nucleus and are expressed at high levels in bulbs, confirming their colocalization and co-expression thus their ability to work together in the same catalytic route. Finally, their co-expression in yeast led to the production of norbelladine. In all, our study establishes that both NBS and NR participate in the biosynthesis of norbelladine by catalyzing the first key steps associated in the biosynthesis of the Alzheimer’s drug galanthamine.

Light-emitting diodes (LEDs) have emerged as efficient light sources for promoting in vitro plant growth and primary and secondary metabolite biosynthesis. This study investigated the effects of blue, red, and white-red LED lights on plant biomass growth, photosynthetic pigments, soluble sugars, phenolic compounds, the production of Amaryllidaceae alkaloids, and the activities of antioxidant enzymes in Leucojum aestivum L. cultures. A white fluorescent light was used as a control. The plants that were grown under white-red and red light showed the highest fresh biomass increments. The blue light stimulated chlorophyll a, carotenoid, and flavonoid production. The white-red and blue lights were favourable for phenolic acid biosynthesis. Chlorogenic, p-hydroxybenzoic, caffeic, syringic, p-coumaric, ferulic, sinapic, and benzoic acids were identified in plant materials, with ferulic acid dominating. The blue light had a significant beneficial effect both on galanthamine (4.67 µg/g of dry weight (DW)) and lycorine (115 µg/g DW) biosynthesis. Red light treatment increased catalase and superoxide dismutase activities, and high catalase activity was also observed in plants treated with white-red and blue light. This is the first report to provide evidence of the effects of LED light on the biosynthesis of phenolic acid and Amaryllidaceae alkaloids in L. aestivum cultures, which is of pharmacological importance and can propose new strategies for their production.

Summary Amaryllidaceae alkaloids (AAs) are diverse bioactive metabolites with significant pharmaceutical potential, derived from 4′‐O‐methylnorbelladine (4′OM). The biosynthesis of these compounds involves the condensation of tyramine and 3,4‐dihydroxybenzaldehyde by norbelladine synthase (NBS) and/or noroxomaritidine/norcraugsodine reductase (NR), followed by O‐methylation. Cytochrome P450 enzymes, particularly the CYP96T family, introduce further structural diversity through C–C couplings, resulting in lycorine, galanthamine and crinine cores. Despite their importance, the exact biosynthetic pathways remain poorly defined. In this study, we describe key enzymes from Leucojum aestivum (La), providing crucial insight into AA biosynthesis. Transient expression in Nicotiana benthamiana demonstrated that LaNBS and LaNRII catalyse the conversion of tyramine and 3,4‐dihydroxybenzaldehyde to norbelladine, which is subsequently O‐methylated by a norbelladine‐4′‐O‐methyltransferase (LaN4′OMT) in planta. Co‐agroinfiltration of LaNBS, LaNRII, LaN4′OMT and LaCYP96T1 resulted in the production of various phenol‐coupled products, with lycorine as the predominant compound, alongside haemanthamine, crinine/vittatine and norgalanthamine. This study identifies LaCYP96T1 and LaCYP96T2 as the first monocot enzymes capable of catalysing all three regioselective C‐C phenol couplings and also highlights the substrate promiscuity of LaNRII. The findings not only elucidate critical steps in AA biosynthesis but also open new avenues for biotechnological application in producing valuable alkaloids, offering potential for novel drug development. Schematic representation of Amaryllidaceae alkaloid biosynthesis in Leucojum aestivum. Norbelladine synthase (LaNBS) and noroxomaritidine/norcraugsodine reductase (LaNRII) catalyze the condensation of tyramine and 3,4‐dihydroxybenzaldehyde to form norbelladine, which is subsequently O‐methylated by norbelladine‐4′‐O‐methyltransferase (LaN4′OMT). Cytochrome P450 enzymes LaCYP96T1 and LaCYP96T2 mediate C–C phenol couplings, leading to the formation of lycorine, haemanthamine, crinine/vittatine, and norgalanthamine. Transient expression in Nicotiana benthamiana confirms enzymatic activities and alkaloid production, providing new insights into Amaryllidaceae alkaloid biosynthesis and biotechnological applications.

Amaryllidaceae alkaloids are of notable pharmacological relevance. For instance, galanthamine is used in the treatment of Alzheimer’s disease, while other alkaloids (lycorine, crinine, etc.) derived from Amaryllidaceae plants are also of great interest because they exhibit antitumour, antiviral, antibacterial, antifungal, antimalarial, analgesic and cytotoxic properties. Phenolic acids comprise a group of natural bioactive substances that have commercial value in the cosmetic, food and medicinal industries due to their antioxidant, anticancer, anti-inflammatory and cardioprotective potential. In the present study, the effect of temperature (15, 20, 25 and 30 °C) on Amaryllidaceae alkaloid and phenolic acid biosynthesis in Leucojum aestivum in vitro plant cultures was investigated. The highest diversity of alkaloids (i.e., galanthamine, crinan-3-ol, demethylmaritidine, crinine, 11-hydroxyvitattine, lycorine, epiisohaemanthamine, chlidanthine) was noted in plants cultured at 30 °C. By contrast, ismine and tazettine were only present in plants cultured at 15 °C. Temperatures of 20 °C and 30 °C were found to stimulate galanthamine accumulation. The highest lycorine content was noted in plants grown at temperatures of 15 and 30 °C, and it was negatively correlated with the expression of the gene that encodes the cytochrome P450 96T (CYP96T) enzyme which catalyses a key step in the biosynthesis of different types of Amaryllidaceae alkaloids. This observation may reflect temperature-induced shifts in metabolic flux among different branches of Amaryllidaceae alkaloid biosynthesis. The observed stimulating effect of a 15 °C temperature on the chlorogenic, caffeic, p-coumaric, sinapic, ferulic and isoferulic acid content was in line with the highest expression of a gene that encodes the tyrosine decarboxylase (TYDC) enzyme, which is involved in plant stress response mechanisms. At 30 °C, however, the highest content of the caffeic, vanillic, p-coumaric and isoferulic acids was noted.

The Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites, which are known for their biological activities. Although the general chemical reactions in the AA biosynthetic pathway have been proposed, the genes and enzymes of the pathway remain largely unstudied. All AAs are synthesized from a common precursor, norbelladine, by the condensation of tyramine and 3,4-dihydroxybenzaldehyde. The enzyme norbelladine synthase (NBS) which catalyzes the condensation reaction has only been characterized at a molecular level from one species, and the subcellular localizations have not been explored. Hence, the intracellular compartments wherein the AAs are biosynthesized remain unknown. In this study, a first comprehensive transcriptomic analysis of summer snowflake (Leucojum aestivum) was done to identify key genes associated with AA biosynthesis. Fifty orthologous genes were identified and deposited into GenBank. In addition, we identified and further characterized NBS from the transcriptome of L. aestivum and previously reported Narcissus papyraceus. Phylogenetic analysis showed that LaNBS, NpNBS1 and NpNBS2 shared high amino acid identity. The heterologous expression of LaNBS produced a recombinant protein with NBS activity. Bioinformatic prediction and C-terminal GFP tagging in transiently transformed Nicotiana benthamiana showed that LaNBS, NpNBS1 and NpNBS2 were likely localized to the cytosol which suggests that the AA biosynthesis starts in the cytosol. This study provides an Amaryllidaceae transcriptome that will be very helpful to identify genes for characterization studies in AA metabolism in planta or using heterologous systems. In addition, our study will facilitate the bioengineering of AA biosynthetic pathway in plants or in microorganisms.

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (H-1 NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative H-1 NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by H-1 NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L. aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.

Background Leucojum aestivum L. (Amaryllidaceae) is a perennial bulbous species of high pharmaceutical importance, primarily recognized as a natural source of the alkaloid galantamine for Alzheimer’s disease treatment. Iranian L. aestivum populations (LAPs) may contain unique alkaloids with strong acetylcholinesterase (AChE) inhibitory effects, making them a conservation priority. To investigate phytochemical diversity and bioactivity, eight natural populations including LAP1 (Chahar Deh, Astaneh Ashrafieh); LAP2 (Baz Kia Gurab, Lahijan); LAP3 (Sangar); LAP4 (Chomesghal, Ziabar); LAP5 (Pinchah, Astaneh Ashrafieh); LAP6 (Chaboksar); LAP7 (Valiseh, Rudsar); and LAP8 (Vali Abad, Tonkabon) were sampled during the flowering stage. Galantamine, lycorine, total phenolic content (TPC), total flavonoid content (TFC), antioxidant capacity (FRAP and DPPH), and AChE inhibitory activity were quantified in bulbs and leaves. Results The highest AChE inhibition was recorded in bulbs of LAP6 (IC 50 : 95.57 µg/ml) and LAP7 (IC 50 : 102.38 µg/ml), corresponding to elevated galantamine levels (20.31 and 24.92 mg/g dry extract, respectively), which showed a strong positive correlation ( r  = 0.880, p  < 0.01). In contrast, LAP8 contained no detectable galantamine in bulbs but exhibited exceptionally high leaf lycorine content (107.91 mg galanthamine equivalent/g dry extract) and antioxidant capacity (FRAP: 90.71 µmol Fe²⁺/g DW; DPPH, IC₅₀: 95.10 µg/ml), alongside moderate AChE inhibition (IC 50 : 150.80 µg/ml in bulbs, 231.63 µg/ml in leaves). Leaf TPC (8.57–13.39 mg GAE/g DW) consistently exceeded bulb TPC (0.18–7.59 mg GAE/g DW). Cluster analysis grouped populations into three distinct classes: balanced alkaloid levels and antioxidant power ( LAP1 , LAP5 , LAP6 ), lower alkaloid levels ( LAP2 , LAP3 , LAP4 ), and high phenolic content and antioxidant power with relatively elevated alkaloid levels ( LAP7 , LAP8 ). Conclusions These findings demonstrated a strong linkage between alkaloid composition and bioactivity, highlighting the pharmaceutical potential of specific chemotypes and emphasizing the necessity of in situ conservation and ex situ cultivation for sustainable utilization and drug development targeting cholinesterase inhibition and antioxidant activity.

Acetylcholinesterase (AChE) inhibitors are widely used in Alzheimer’s treatment, but they are also crucial for their action on organophosphorus insecticides. The latter exert their toxicity by inhibiting the AChE enzyme in insects, leading to their death. Amaryllidaceae alkaloids have been proven to be potent AChE inhibitors. In the present study methanolic extracts and essential oils being obtained from species of Asteraceae, Lamiaceae, Brassicaceae and Amaryllidaceae were evaluated in vitro for AChE inhibitory activity. Ellman’s colourimetric method, with modifications, was used for AChE activity evaluation. According to the activity level, the tested plant products were divided into three categories. First: plant products with strong activity comparable to that of galanthamine; second: plant products with medium activity, with IC 50 value about 1 mg/ml and the last group with low activity, with IC 50 value greater than 1 mg/ml. Essential oils of Origanum vulgare subsp. hirtum Ietswaart., Satureja pilosa Vel., Monarda fistulosa L., Thymus longedentatus (Degen & Urum.) Ronniger and the methanolic extract of Leucojum aestivum L. showed the most potent activity and were referred to as the first group. Carvacrol was identified as the main component of the most active essential oils. In L. aestivum extract, galanthamine was found as the main alkaloid. The obtained results indicate that essential oils and alkaloid-rich plant extracts possess the strongest AChE inhibitory activity. This gives us a reason to recommend these plant products to be tested for insecticidal activity in the future.

Snowflake genus (Leucojum L.) species currently have wide application as ornamental crops for the landscaping of gardens and parks. Previously, there were conducted several researches aimed at studying the composition of alkaloids as well as prospects for using these herbs as sources for medicines, in particular, containing galanthamine. This review shows systematical data on chemical composition (primarily, galanthamine and lycorine alkaloids) and medical application of two Snowflake species: spring snowflake (Leucojum vernum L.) and summer snowflake (Leucojum aestivum L.). There have also been considered issues concerning the standardization of medicinal plant raw material.