Development of Transcriptomic Resources for Interrogating the Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species

Abstract

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals.

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

“…To identify regulators of the iridoid pathway branch, we set up a screen for transactivators of the G8O promoter (pG8O), the sequence of which had been determined previously (16). Candidate TFs were selected by mining recently generated RNA-Seq data (7,8,26) for TFs that showed coexpression with the iridoid genes upstream of LAMT. Essentially, TF encoding genes that showed expression patterns similar to G8O in the RNA-Seq data generated by the SmartCell consortium (bioinformatics.psb.ugent.be/orcae/ overview/Catro), i.e., JA induced in both cell suspensions and seedlings but not induced by ORCA overexpression in cell suspensions, were selected for further analysis.…”

Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS

“…When we measured extracts of both tissues with LC-MS, leaf tissue was found to contain more TIA metabolites than stem tissues (Table S1), which Góngora-Castillo et al (33) attributed to differences in expression levels of genes involved in TIA biosynthesis between stem and leaf tissue. So far, we have been able to measure cell contents in stem cells because of their physical advantages.…”

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…A. belladonna protein sequences (339 peptides) from GenBank were used to quantify the quality and coverage of the assembly. Transcripts were concatenated into a single pseudomolecule, and expression abundances (fragments per kilobase of transcript per million fragments mapped) were determined in the transcriptome assembly for the 11 single tissue libraries in Table 1 as described by Góngora-Castillo et al (2012).…”

“…The broad utility of alkaloids for humankind has led to considerable interest in understanding the pathways and regulatory mechanisms underlying their synthesis. The development of functional genomics technologies, particularly deep and quantitative transcriptome profiling, has greatly facilitated these efforts (Murata et al, 2008;Liscombe et al, 2009;Desgagné-Penix et al, 2010;Giddings et al, 2011;Farrow et al, 2012;Góngora-Castillo et al, 2012;Winzer et al, 2012).…”