Molecular Cloning and Characterization of a meta/para-O-Methyltransferase from Lycoris aurea

Abstract

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 meta O-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.

Identification, molecular characterization and expression of JAZ genes in Lycoris aurea

“…Herb, is an ornamentally and medicinally important perennial herbaceous plant, belonging to the Amaryllidaceae family, from which there were a variety of Amaryllidaceae alkaloids found to exhibit medicinal values [22]. Previous studies of L. aurea have mainly focused on karyotype [23][24][25], physiological analysis [26,27], chemical composition [28][29][30], medicinal usage [31,32], and molecular aspects for gene cloning [33][34][35][36]. For example, several enzymes involved in Amaryllidaceae alkaloids biosynthesis, including cinnamate 4-hydroxylase [34], tyrosine decarboxylase [35] and norbelladine 4'-O-methyltransferase [36] have been identified.…”

“…Previous studies of L. aurea have mainly focused on karyotype [23][24][25], physiological analysis [26,27], chemical composition [28][29][30], medicinal usage [31,32], and molecular aspects for gene cloning [33][34][35][36]. For example, several enzymes involved in Amaryllidaceae alkaloids biosynthesis, including cinnamate 4-hydroxylase [34], tyrosine decarboxylase [35] and norbelladine 4'-O-methyltransferase [36] have been identified. Besides, although the induction effects of exogenous methyl jasmonate (MeJA) treatments on Amaryllidaceae alkaloids accumulation has been reported [37,38], little is known about the possible transcriptional or post-transcriptional regulation mechanism of this biological process in L. aurea.…”

Transcriptome Analysis of Different Tissues Reveals Key Genes Associated With Galanthamine Biosynthesis in Lycoris longituba

“…pseudonarcissus TYDC, with an E-value of zero (Singh and Desgagne-Penix, 2017). Thus, we were able to find seven transcripts with E-values corresponding to genes encoding enzymes involved in Gal biosynthesis (Kilgore et al, 2014;Kilgore et al, 2016;Singh and Desgagne-Penix, 2017;Sun et al, 2018). In the central Gal biosynthetic pathway, sequence reads corresponding to OMT, C4H, and C3H were more abundant than those operating in precursor pathways including PAL, TYDC, and others ( Figure 5A).…”

“…NpN4OMT and LaOMT1 enzymes play key roles in the galanthamine biosynthesis pathway via the methylation of norbelladine to 4'-O-methylnorbelladine (Kilgore et al, 2014;Sun et al, 2018). Consequently, we also examined whether LlOMT exhibits catalytic activities.…”

Recent Progress in Amaryllidaceae Biotechnology

“…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 ].…”