Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions.

Lycoris aurea, a medicinal species of the Amaryllidaceae family, is used in the practice of traditional Chinese medicine because of its broad pharmacological activities of Amaryllidaceae alkaloids. Despite the officinal and economic importance of Lycoris species, the secondary mechanism for this species is relatively deficient. In this study, we attempted to characterize the transcriptome profiling of L. aurea seedlings with the methyl jasmonate (MeJA) treatment to uncover the molecular mechanisms regulating plant secondary metabolite pathway. By using short reads sequencing technology (Illumina), two sequencing cDNA libraries prepared from control (Con) and 100 µM MeJA-treated (MJ100) samples were sequenced. A total of 26,809,842 and 25,874,478 clean reads in the Con and MJ100 libraries, respectively, were obtained and assembled into 59,643 unigenes. Among them, 41,585 (69.72%) unigenes were annotated by basic local alignment search tool similarity searches against public sequence databases. These included 55 Gene Ontology (GO) terms, 128Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 25 Clusters of Orthologous Groups (COG) families. Additionally, 4,175 differentially expressed genes (DEGs; false discovery rate ≤ 0.001 and∣log2 Ratio∣≥ 1) with 2,291 up-regulated and 1,884 down-regulated, were found to be affected significantly under MeJA treatment. Subsequently, the DEGs encoding key enzymes involving in the secondary metabolite biosynthetic pathways, transcription factors, and transporter proteins were also analyzed and summarized. Meanwhile, we confirmed the altered expression levels of the unigenes that encode transporters and transcription factors using quantitative real-time PCR (qRT-PCR). With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of L. aurea may be improved. Additionally, the genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of L. aurea.

O-methyltransferases (OMTs) have been demonstrated to play key roles in the biosynthesis of plant secondary metabolites, such as alkaloids, isoprenoids, and phenolic compounds. Here, we isolated and characterized an OMT gene from Lycoris aurea (namely LaOMT1), based on our previous transcriptome sequencing data. Sequence alignment and phylogenetic analysis showed that LaOMT1 belongs to the class I OMT, and shares high identity to other known plant OMTs. Also, LaOMT1 is highly identical in its amino acid sequence to NpN4OMT, a norbelladine 4′-OMT from Narcissus sp. aff. pseudonarcissus involved in the biosynthesis of Amaryllidaceae alkaloids. Biochemical analysis indicated that the recombinant LaOMT1 displayed both para and metaO-methylation activities with caffeic acid and 3,4-dihydroxybenzaldehyde, and showed a strong preference for the meta position. Besides, LaOMT1 also catalyzes the O-methylation of norbelladine to form 4′-O-methylnorbelladine, which has been demonstrated to be a universal precursor of all the primary Amaryllidaceae alkaloid skeletons. The results from quantitative real-time PCR assay indicated that LaOMT1 was ubiquitously expressed in different tissues of L. aurea, and its highest expression level was observed in the ovary. Meanwhile, the largest concentration of lycorine and galanthamine were found in the ovary, whereas the highest level of narciclasine was observed in the bulb. In addition, sodium chloride (NaCl), cold, polyethylene glycol (PEG), sodium nitroprusside (SNP), and methyl jasmonate (MeJA) treatments could significantly increase LaOMT1 transcripts, while abscisic acid (ABA) treatment dramatically decreased the expression level of LaOMT1. Subcellular localization showed that LaOMT1 is mainly localized in cytoplasm and endosome. Our results in this study indicate that LaOMT1 may play a multifunctional role, and lay the foundation for Amaryllidaceae alkaloid biosynthesis in L. aurea.

p-Coumaric acid is a commercially available phenolcarboxylic acid with a great number of important applications in the nutraceutical, pharmaceutical, material and chemical industries. p-Coumaric acid has been biosynthesized in some engineered microbes, but the potential of the plant CYP450-involved biosynthetic route has not investigated in Escherichia coli. In the present study, a novel trans-cinnamic acid 4-hydroxylase (C4H) encoding the LauC4H gene was isolated from Lycoris aurea (L’ Hér.) Herb via rapid amplification of cDNA ends. Then, N-terminal 28 amino acids of LauC4H were characterized, for the subcellular localization, at the endoplasmic reticulum membrane in protoplasts of Arabidopsis thaliana. In E. coli, LauC4H without the N-terminal membrane anchor region was functionally expressed when fused with the redox partner of A. thaliana cytochrome P450 enzyme (CYP450), and was verified to catalyze the trans-cinnamic acid to p-coumaric acid transformation by whole-cell bioconversion, HPLC detection and LC-MS analysis as well. Further, with phenylalanine ammonia-lyase 1 of A. thaliana, p-coumaric acid was de novo biosynthesized from glucose as the sole carbon source via the phenylalanine route in the recombinant E. coli cells. By regulating the level of intracellular NADPH, the production of p-coumaric acid was dramatically improved by 9.18-fold, and achieved with a titer of 156.09 μM in shake flasks. The recombinant cells harboring functional LauC4H afforded a promising chassis for biological production of p-coumaric acid, even other derivatives, via a plant CYP450-involved pathway.

Summary: Lycoris radiata is a main source of Amaryllidaceae alkaloids; however, the low content of these alkaloids in planta remains a limit to their pharmaceutical development and utilization. The accumulation of secondary metabolites can be enhanced in plants inoculated with fungal endophytes. In this study, we analysed the diversity of culturable fungal endophytes in different organs of L . radiata . Then, by analysing the correlation between the detectable rate of each fungal species and the content of each tested alkaloid, we proposed several fungal candidates implicated in the increase of alkaloid accumulation. This was verified by inoculating these candidates to L . radiata plants. Based on the results of two independent experiments conducted in May 2018 and October 2019, the individual inoculation of nine fungal endophytes significantly increased the total content of the tested alkaloids in the entire L . radiata plants. This is the first study in L . radiata to show that fungal endophytes are able to improve the accumulation of various alkaloids. Therefore, our results provide insights into a better understanding of interactions between plants and fungal endophytes and suggest an effective strategy for enhancing the alkaloid content in the cultivation of L . radiata .

Background: Galanthamine, one kind of Amaryllidaceae alkaloid extracted from theLycorisspecies, is used in the treatment of Alzheimer’s disease. In regards to medical and economic importance, the biosynthesis and regulatory mechanism of the secondary metabolites inLycorisremain uninvestigated.Methods: BLAST was used to identify the sequence of tyrosine decarboxylase in the transcriptome ofLycoris aurea(L’Hér) Herb. The enzyme activity of this TYDC was determined by using heterologous expressed protein in theEscherichia colicells. The related productive contents of tyramine were detected using High Performance Liquid Chromatography (HPLC). According to the available micro RNA sequencing profiles and degradome database ofL. aurea, microRNA396 were isolated, which targets toLaTYDC1and RNA Ligase-Mediated-Rapid Amplification of cDNA Ends (RLM-RACE) were used to confirm the cleavage. The expression levels of miR396 andLaTYDC1were measured using a quantitative real-time polymerase chain reaction (qRT-PCR).Results: LaTYDC1 was mainly expressed in root, bulb, leaf and flower fitting the models for galanthamine accumulation. This decarboxylase efficiently catalyzes tyrosine to tyramine conversion. Under methyl jasmonate (MeJA) treatment, the expression ofLaTYDC1and the content of tyramine sharply increase. The use of RLM-RACE confirms that miR396 promotes the degradation ofLaTYDC1mRNA. Under MeJA treatment, the expression of miR396 was suppressed while the expression level ofLaTYDC1sharply increased. Following the increase of the miR396 transcriptional level,LaTYDC1was significantly repressed.Conclusion: LaTYDC1 participates in the biosynthesis of galanthamine, and is regulated by miR396. This finding also provides genetic strategy for improving the yield of galanthamine in the future.