Differential expression of methyltransferases (a) and dehydrogenases (b) with their differential expression in <b><i>pap1</i></b> and BR086.

<p>Two columns in each represent <i>pap1</i> and BR086, while each row represents contigs encoding members of these families (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065622#pone.0065622.s014" target="_blank">Table S10</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065622#pone.0065622.s015" target="_blank">S11</a>). Clustering was carried out with log tpm value of each contig in <i>pap1</i> and BR086 transcriptome to visualize differential expression.</p

Sequencing reads, assembly and annotation.

<p>Size distribution of 454 high-quality reads (a) and assembled contigs (b). Black and white bars represents reads and contigs from <i>pap1</i> and BR086 transcriptome data respectively. Annotation of unigenes of <i>pap1</i> (c) and BR086 (d) was carried out against TAIR, NR and CDD databases. Out of all annotated unigenes, 25,343 and 19,225 unigenes from <i>pap1</i> and BR086, respectively, had common BLAST hits to annotated proteins by different databases.</p

Histogram of gene ontology classification.

<p>The results are summarized in three main categories: Biological Process, Cellular Component and Molecular Function. Bars represent the assign percent of unigenes in <i>pap1</i> (white bars) and BR086 (black bars) with BLAST matches in the TAIR9 database to each GO term.</p

Phenotype of fresh latex and specific alkaloid content in <b><i>pap1</i></b><b> and BR086.</b>

<p>Fresh latex of BR086 is brown or red whereas <i>pap1</i> exuded white latex (a). Content of specific alkaloids in latex was measured in <i>pap1</i> and BR086 (b) using High Pressure Liquid Chromatography. *** indicate values that differ significantly from BR086.</p

Combined assembly and annotation.

<p>Size distribution of combined assembled contig length and frequency (a) using high–quality reads from <i>pap1</i> and BR086. Annotation statistics (b) of unigenes of combined assembly queried against TAIR, NR and CDD databases.</p

Summary of 454 sequencing and assembly for <i>pap1</i> mutant and BR086 line of <i>Papaver somnifera.</i>

<p>Summary of 454 sequencing and assembly for <i>pap1</i> mutant and BR086 line of <i>Papaver somnifera.</i></p

The unigenes related to secondary metabolites.

<p>The unigenes related to secondary metabolites.</p

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

<div><p>The benzylisoquinoline alkaloid papaverine, synthesized in low amount in most of the opium poppy varieties of <i>Papaver somniferum</i>, 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 (<i>pap1</i>) and normal cultivar (BR086) of <i>P. somniferum</i>. This natural mutant synthesizes more than 12-fold papaverine in comparison to BR086. We established more than 238 Mb transcriptome data separately for <i>pap1</i> and BR086. Assembly of reads generated 127,342 and 106,128 unigenes in <i>pap1</i> 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 <i>pap1</i> in comparison to BR086 suggest (S)-coclaurine as the route for papaverine biosynthesis. We also identified several methyltransferases and dehydrogenases with enhanced expression in <i>pap1</i> in comparison to BR086. Our analysis using natural mutant, <i>pap1</i>, 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.</p></div

Validation of differentially expressed transcripts in <b><i>pap1</i></b> and BR086.

<p>Real time quantitative PCR of selected differentially regulated methyltransferase (a) and dehydrogenase gene family (b) was carried out using total RNA isolated from peduncle tissue of prehook stage of <i>pap1</i> mutant and BR086. Digital gene expression of genes used for validation through quantitative Real time PCR is provided in lower panel of (a) and (b). Information about selected up-and down-regulated transcripts is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065622#pone.0065622.s005" target="_blank">Table S1</a>.</p

Clusters containing cytochrome P450 with their differential expression in leaf and root.

<p>Two columns represent leaf and root, while each row represents contigs encoding different members of CYP450 gene family (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062714#pone.0062714.s014" target="_blank">Table S12</a>). Clustering was carried out with log₂tpm value of each contig in leaf and root transcriptome to visualize differential expression.</p