Lycoris longituba is a traditional medicinal plant containing the bioactive compound galanthamine (Gal), a type of Amaryllidaceae alkaloid and can be used to treat Alzheimer’s disease. However, research on its genome or transcriptome and associated genes in the biosynthetic pathway is incomplete. In this study, we estimated the nuclear genome size of this species to be 29.33 Gb by flow cytometry. Then, RNA sequencing of the leaves, roots, and bulbs of L. longituba was carried out. After de novo assembly, 474,589 all-transcripts and 333,440 all-unigenes were finally generated. In addition, the differentially expressed genes (DEGs) were identified, and genes involved in the galanthamine metabolic pathway encoding tyrosine decarboxylase (TYDC), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), p-coumarate 3-hydroxylase (C3H), norbelladine synthase (NBS), norbelladine 4’-O-methyltransferase (OMT), and noroxomaritidine synthase (CYP96T1) were detected and validated by real-time quantitative PCR analysis. One candidate gene, Lycoris longituba O-Methyltransferase (LlOMT), was identified in the proposed galanthamine biosynthetic pathway. Sequence analysis showed that LlOMT is a class I OMT. LlOMT is localized in the cytoplasm, and biochemical analysis indicated that the recombinant LlOMT catalyzes norbelladine to generate 4′-O-methylnorbelladine. The protoplast transformation result showed that the overexpression of LlOMT could increase the Gal content. Our results indicate that LlOMT may play a role in galanthamine biosynthesis in L. longituba. This work provides a useful resource for the metabolic engineering of Amaryllidaceae alkaloids.

The Amaryllidaceae alkaloid galanthamine (Gal) in Lycoris longituba is a secondary metabolite that has been used to treat Alzheimer’s disease. Plant secondary metabolism is affected by methyl jasmonate (MeJA) exposure, although the regulatory mechanisms of MeJA on L. longituba seedlings remains largely unknown. In the present study, 75, 150, and 300 μM MeJA were used as treatments on L. longituba seedlings for 7, 14, 21, and 28 days, while 0 μM MeJA was used as the control (MJ-0). The effect of exogenous MeJA on Gal synthesis in L. longituba was then investigated using transcriptomic sequencing and metabolite profiling via GC-MS and LC-MS analysis. Galanthamine (Gal), lycorine (Lyc), and lycoramine (Lycm) abundances were 2. 71-, 2. 01-, and 2.85-fold higher in 75 μM MeJA (MJ-75) treatment plants compared to MJ-0 treatment plants after 7 days of cultivation. Transcriptomic analysis further showed that MJ-75 treatment significantly induced the expression of norbelladine synthase (NBS) and norbelladine 4′-O-methyltransferase (OMT), which are involved in the Gal biosynthesis pathway. In addition, increased expression was observed in MJ-75 treatment plants for genes in the JA synthesis and JA signaling pathways including those of allene oxide cyclase (AOC), 12-oxo-phytodienoic acid reductase (OPR), jasmonic acid amino acid synthase (JAR), and transcription factor MYC. The L. longituba tyrosine decarboxylase (LlTYDC) enzyme was identified and proposed to be involved in the Gal biosynthetic pathway. Metabolomics results demonstrated that the accumulation of Amaryllidaceae alkaloids, and especially alkaloids in the Gal biosynthesis pathway, could be induced by MJ-75 treatment. Interestingly, metabolites in the JA synthesis pathway were also affected by MeJA treatment. Overall, this multi-omics study suggests that both the JA synthesis/JA signaling and Gal biosynthesis pathways were affected by exogenous MeJA treatment. This comprehensive study of gene expression and metabolite contents can help us better understand the molecular mechanisms underlying MeJA-mediated Gal biosynthesis in L. longituba.

Lycoris species have great ornamental and medicinal values; however, their low regeneration efficiency significantly restricts their commercial production. Exogenous hormone application is an effective way to promote bulblet development, but their effect on Lycoris radiata has not been verified to date. In the present study, we examined the effect of different exogenous hormones on bulblet development in L. radiata, and found that gibberellic acid (GA) significantly inhibited, whereas paclobutrazol (PBZ), abscisic acid (ABA), and ethrel promoted bulblet development, especially PBZ, a GA biosynthesis inhibitor. Furthermore, GA reduced endogenous cytokinin (CK) content, as well as the activities of carbohydrate metabolism enzymes, including sucrose synthase (SUS) and glucose-1-phosphate adenylyltransferase (AGPase), by downregulating the expression levels of LrSUS1, LrSUS2, and genes encoding AGPase large and small subunits. This resulted in the decrease in carbohydrate accumulation in the bulblets, thus hindering their development. PBZ had the opposite effect to GA on carbohydrate metabolism; it decreased endogenous GA₁₅ and GA₂₄, thereby promoting bulblet development. ABA promoted endogenous auxin content and the activities of starch synthesis enzymes, especially soluble starch synthase (SSS) and granule-bound SS (GBSS), through the up-regulation of the expression levels of LrSS1, LrSS2, and LrGBSS1 genes, which could also result in the accumulation of carbohydrates in the bulblets and promote their development. In addition, ethrel application partly promoted bulblet development by promoting endogenous CK content. Although the accumulation of carbohydrates and the activity of starch enzymes were increased by ethrel treatment, we hypothesized that the effect of ethrel on regulating carbohydrate metabolism may be indirect. Our results could provide a basis for improving the propagation efficiency of L. radiata for production, as well as propose some directions for future research.

Alkaloids are a class of nitrogen-containing alkaline organic compounds found in nature, with significant biological activity, and are also important active ingredients in Chinese herbal medicine. Amaryllidaceae plants are rich in alkaloids, among which galanthamine, lycorine, and lycoramine are representative. Since the difficulty and high cost of synthesizing alkaloids have been the major obstacles in industrial production, particularly the molecular mechanism underlying alkaloid biosynthesis is largely unknown. Here, we determined the alkaloid content in Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri, and performed a SWATH-MS (sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in the three Lycoris. A total of 2193 proteins were quantified, of which 720 proteins showed a difference in abundance between Ll and Ls, and 463 proteins showed a difference in abundance between Li and Ls. KEGG enrichment analysis revealed that differentially expressed proteins are distributed in specific biological processes including amino acid metabolism, starch, and sucrose metabolism, implicating a supportive role for Amaryllidaceae alkaloids metabolism in Lycoris. Furthermore, several key genes collectively known as OMT and NMT were identified, which are probably responsible for galanthamine biosynthesis. Interestingly, RNA processing-related proteins were also abundantly detected in alkaloid-rich Ll, suggesting that posttranscriptional regulation such as alternative splicing may contribute to the biosynthesis of Amaryllidaceae alkaloids. Taken together, our SWATH-MS-based proteomic investigation may reveal the differences in alkaloid contents at the protein levels, providing a comprehensive proteome reference for the regulatory metabolism of Amaryllidaceae alkaloids.