title: 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.
authors.affiliation: CSIR-National Botanical Research Institute (CSIR-NBRI), Rana Pratap Marg, Lucknow, India.
authors.forename: Sumya
authors.initials: S
authors.lastname: Pathak
authors.forename: Deepika
authors.initials: D
authors.lastname: Lakhwani
authors.forename: Parul
authors.initials: P
authors.lastname: Gupta
authors.forename: Brij Kishore
authors.initials: BK
authors.lastname: Mishra
authors.forename: Sudhir
authors.initials: S
authors.lastname: Shukla
authors.forename: Mehar Hasan
authors.initials: MH
authors.lastname: Asif
authors.forename: Prabodh Kumar
authors.initials: PK
authors.lastname: Trivedi
mesh_terms: D020869:Gene Expression Profiling; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D008780:Methyltransferases / Q000235:genetics; D058977:Molecular Sequence Annotation; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics; D000073718:Pancreatitis-Associated Proteins; D010207:Papaver / Q000235:genetics* / Q000378:metabolism*; D010208:Papaverine / Q000096:biosynthesis* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics
chemical_list: D000073718:Pancreatitis-Associated Proteins; D010940:Plant Proteins; C000612141:REG3A protein, human; D010208:Papaverine; D010088:Oxidoreductases; D008780:Methyltransferases