Comparative Transcriptome Analysis Using High Papaverine Mutant of Papaver somniferum Reveals Pathway and Uncharacterized Steps of Papaverine Biosynthesis

Abstract

The benzylisoquinoline alkaloid papaverine, synthesized in low amount in most of the opium poppy varieties of Papaver somniferum, is used as a vasodilator muscle relaxant and antispasmodic. Papaverine biosynthesis remains controversial as two different routes utilizing either (S)-coclaurine or (S)-reticuline have been proposed with uncharacterized intermediate steps. In an attempt to elucidate papaverine biosynthesis and identify putative genes involved in uncharacterized steps, we carried out comparative transcriptome analysis of high papaverine mutant (pap1) and normal cultivar (BR086) of P. somniferum. This natural mutant synthesizes more than 12-fold papaverine in comparison to BR086. We established more than 238 Mb transcriptome data separately for pap1 and BR086. Assembly of reads generated 127,342 and 106,128 unigenes in pap1 and BR086, respectively. Digital gene expression analysis of transcriptomes revealed 3,336 differentially expressing unigenes. Enhanced expression of (S)-norcoclaurine-6-O-methyltransferase (6OMT), (S)-3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT), norreticuline 7-O-methyltransferase (N7OMT) and down-regulation of reticuline 7-O-methyltransferase (7OMT) in pap1 in comparison to BR086 suggest (S)-coclaurine as the route for papaverine biosynthesis. We also identified several methyltransferases and dehydrogenases with enhanced expression in pap1 in comparison to BR086. Our analysis using natural mutant, pap1, concludes that (S)-coclaurine is the branch-point intermediate and preferred route for papaverine biosynthesis. Differentially expressing methyltransferases and dehydrogenases identified in this study will help in elucidating complete biosynthetic pathway of papaverine. The information generated will be helpful in developing strategies for enhanced biosynthesis of papaverine through biotechnological approaches.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…Another study used a transcriptomics approach to compare high and low papaverine cultivars (Patahnk et al 2013). Similarly, transcripts encoding enzymes in the N-desmethyl pathway were upregulated, whereas transcripts corresponding to 7OMT were downregulated.…”

Comparative analysis of transcription factor gene families from Papaver somniferum: identification of regulatory factors involved in benzylisoquinoline alkaloid biosynthesis

“…This suggested that members of WRKY gene family might play an important role in noscapine biosynthesis. Differential Pathak et al 2013), and expression of contig00139 and contig00217 annotated as WRKY TFs was also observed in these four cultivars only (Fig. 2e).…”

“…through series of enzymatic reactions, (S)-reticuline is synthesized which acts as the central intermediate and is diverted to different branches for the biosynthesis of different types of BIAs. Through various efforts, limited information related to the biosynthetic pathway and enzymes for the morphinans and papaverine biosynthesis has been developed (Facchini and De Luca 2008;Pathak et al 2013). However, with the advancement of technologies and their use in establishing these pathways, pathways have been reconstructed time to time (DellaPenna and O'Connor 2012).…”

“…This study utilized transcriptome datasets of high papaverine mutant (pap1) and normal cultivar (BR086) as well as eight different cultivars of P. somniferum present in public domain Pathak et al 2013) for the identification of members of various TF gene families using Arabidopsis TF database. Different cultivars used in the analysis accumulate highly contrasting levels of specific BIAs Pathak et al 2013) and can provide valuable information for transcription regulation for biosynthesis of specific alkaloid.…”

“…Different cultivars used in the analysis accumulate highly contrasting levels of specific BIAs Pathak et al 2013) and can provide valuable information for transcription regulation for biosynthesis of specific alkaloid. Objective of this study is to gain insight into regulation of secondary metabolism in P. somniferum with a long-term goal to develop genetic engineering strategies for enhanced papaverine biosynthesis in opium poppy as already reported for other secondary plant products.…”