Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants

Abstract

BackgroundBenzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts.ResultsIn order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal (www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities.ConclusionsThis study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experiment...

Heterodimeric O‐methyltransferases involved in the biosynthesis of noscapine in opium poppy

“…As with other substrate‐ and regio‐specific OMTs involved in BIA metabolism, a clade of closely related OMT2 orthologs occurs in other BIA‐producing species (Hagel et al ., ) suggesting that the specific (or a related) heterodimeric activity of OMT2:OMT3 or OMT2:6OMT occurs in other plants. Previously, we surveyed seven OMT homodimers encoded by genes expressed in yellow horn poppy ( Glaucium flavum ) roots and detected four enzymes with either relatively specific or broad activities on 1‐benzylisoquinoline and protoberberine substrates (Chang et al ., ).…”

“…An enzyme designated OMT4 from G . flavum exhibited no apparent activity with substrates tested, but is a close homolog of opium poppy OMT2 (Chang et al ., ; Hagel et al ., ), which suggests that OMT4 is functional only when partnered with the product of an OMT paralog. In general, the possible widespread occurrence of heterodimeric OMTs involved in BIA metabolism, and potentially beyond, could expand the diversity of substrate‐ and regio‐specific function beyond a directly proportional relationship with respect to OMT genes and metabolic conversions.…”

Tissue‐specific metabolite profiling of benzylisoquinoline alkaloids in the root of Macleaya cordata by combining laser microdissection with ultra‐high‐performance liquid chromatography/tandem mass spectrometry

“…For example, compounds 3, 17, 27 and 30 were produced by methylation reactions of their respective precursors; 6-hydroxyprotopine and 6-hydroxyallocryptopine were formed by hydroxylation reaction; compounds 10, 26 and 28 were produced by cyclization, and so on. [53][54][55][56][57] According to the common steps, some alkaloids missing in the known pathways were considered to be involved in the biosynthesis of BIAs. Typically, 2-methylmagnocurarine (5) was speculated to be formed by adding a methyl group to the C2 position of magnocurarine (2) under the catalysis of a functional enzyme, as magnocurarine (2) is converted into colletine (14), see the routes labeled in pink in Supplementary Fig.…”

De novo transcriptome sequencing of Rhododendron molle and identification of genes involved in the biosynthesis of secondary metabolites

“…Trinity software and TGI clustering tool (TGICL) were used for the de novo assembly and removed redundant clusters, a total of 100,603 unigenes were generated, with average length of 778 bp, the N50 length of 1,384 bp, and the GC content of 47.7%. 40.88% (41,129) of the assembled unigenes were longer than 500 bp, and 20.96% (20,886) Figure S1b). The highly quali ed sequencing results would be in favor of subsequent functional annotations.…”

“…In recent years, the RNA-Seq approach based on the NGS (next-generation sequencing) technology has been developed and widely used for fast and cost-effective transcriptome characterization of numerous vital medicinal plants like Lilium regale [15], Glycyrrhiza uralensis [16], Eugenia uni ora [17] and Carthamus tinctorius [18]. It also provides an effective way to accelerate discovering novel enzymes involved in the speci c metabolic pathways [19,20]. Recently, the RNA-Seq approach has also been used to analyze the owering and ower color formation mechanism in R. molle [21].…”