Transcriptome Analysis and Metabolic Profiling of Lycoris Radiata

Abstract

Lycoris radiata belongs to the Amaryllidaceae family and is a bulbous plant native to South Korea, China, and Japan. Galantamine, a representative alkaloid of Amaryllidaceae plants, including L. radiata, exhibits selective and dominant acetylcholinesterase inhibition. In spite of the economic and officinal importance of L. radiata, the molecular biological and biochemical information on L. radiata is relatively deficient. Therefore, this study provides functional information of L. radiata, describe galantamine biosynthesis in the various organs, and provide transcriptomic and metabolic datasets to support elucidation of galantamine biosynthesis pathway in future studies. The results of studies conducted in duplicate revealed the presence of a total of 325,609 and 404,019 unigenes, acquired from 9,913,869,968 and 10,162,653,038 raw reads, respectively, after trimming the raw reads using CutAdapt, assembly using Trinity package, and clustering using CD-Hit-EST. All of the assembled unigenes were aligned to the public databases, including National Center for Biotechnology Information (NCBI) non-redundant protein (NR) and nucleotide (Nt) database, SWISS-PROT (UniProt) protein sequence data bank, The Arabidopsis Information Resource (TAIR), the Swiss-Prot protein database, Gene Ontology (GO), and Clusters of Orthologous Groups (COG) database to predict potential genes and provide their functional information. Based on our transcriptome data and published literatures, eight full-length cDNA clones encoding LrPAL2, LrPAL3, LrC4H2, LrC3H, LrTYDC2, LrNNR, LrN4OMT, and LrCYP96T genes, involved in galantamine biosynthesis, were identified in L. radiata. In order to investigate galantamine biosynthesis in different plant parts of L. radiata grown in a growth chamber, gene expression levels were measured through quantitative real-time polymerase chain reaction (qRT-PCR) analysis using these identified genes and galantamine levels were quantified by high-performance liquid chromatography (HPLC) analysis. The qRT-PCR data revealed high expression levels of LrNNR, LrN4OMT, and LrCYP96T in the bulbs, and, as expected, we observed higher amounts of galantamine in the bulbs than in the root and leaves. Additionally, a total of 40 hydrophilic metabolites were detected in the different organs using gas-chromatography coupled with time-of-flight mass spectrometry. In particular, a strong positive correlation between galantamine and sucrose, which provides energy for the secondary metabolite biosynthesis, was observed.

Biosynthesis and Biological Activities of Newly Discovered Amaryllidaceae Alkaloids

“…The enzyme responsible for tyramine biosynthesis is the tyrosine decarboxylase (TYDC) ( Figure 2). Two gene transcript variants of TYDC, named TYDC1 and TYDC2, were identified from the transcriptome of different Amaryllidaceae species including N. pseudonarcissus [41], Narcissus papyraceus [42], Lycoris radiata [43], and L. aureus [44].…”

“…Specifically, l-phenylalanine is converted to trans-cinnamic acid by the phenylalanine ammonia-lyase (PAL) ( Figure 2). Several PAL gene transcripts were identified and characterized from different species of Amaryllidaceae [41][42][43][47][48][49]. Interestingly, two main phylogenetic PAL clusters were identified; the first one contained PAL transcripts ubiquitously expressed in Amaryllidaceae whereas the second cluster contained PAL transcripts with expression highest and correlating with organs where AAs accumulated [26].…”

“…Next, trans-cinnamic acid is hydroxylated to form p-coumaric acid by the cinnamate 4-hydroxylase (C4H), a cytochrome P450 of the CYP73 subfamily (cinnamate 4-hydroxylase, C4H) [50,51]. Transcripts encoding C4H were reported from several Amaryllidaceae species [41][42][43]49] including C4H from L. radiate, which was characterized as producing a region-specific 4-hydroxylation of trans-cinnamic acid [49]. The reactions catalyzed by PAL and C4H are also crucial steps in the biosynthesis of many phenylpropanoids such as flavonoids, linins, coumarins and stilbenes.…”

Recent Progress in Amaryllidaceae Biotechnology

“…TYDC is an important enzyme, which catalyzes the production of tyramine from L-tyrosine [ 68 ]. It has been found that two transcript variants of TYDC are present in Amaryllidaceae alkaloid producing plants [ 59 , 65 , 66 , 69 , 70 ]. The phylogenetic analysis showed that Amaryllidaceae TYDCs can be divided into two main clusters: “TYDC1” and “TYDC2” [ 59 ].…”

“…N4OMT has been identified and characterized in many Amaryllidaceae alkaloid producing plants [ 59 ]. The expression analyses showed that N4OMT is highly expressed in bulbs [ 59 , 65 , 70 , 72 ]. Interestingly, the O -methyltransferase found in Lycoris aurea (LauOMT1) was expressed at highest levels in flowers stalks and ovaries, and was demonstrated to have both para ’ and meta ’ O -methylation activities with a strong preference for the meta ’ position methylation [ 73 ].…”