Figure 1.
Phenotype of fresh latex and specific alkaloid content in pap1 and BR086.
Fresh latex of BR086 is brown or red whereas pap1 exuded white latex (a). Content of specific alkaloids in latex was measured in pap1 and BR086 (b) using High Pressure Liquid Chromatography. *** indicate values that differ significantly from BR086.
Figure 2.
Sequencing reads, assembly and annotation.
Size distribution of 454 high-quality reads (a) and assembled contigs (b). Black and white bars represents reads and contigs from pap1 and BR086 transcriptome data respectively. Annotation of unigenes of pap1 (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 pap1 and BR086, respectively, had common BLAST hits to annotated proteins by different databases.
Table 1.
Summary of 454 sequencing and assembly for pap1 mutant and BR086 line of Papaver somnifera.
Figure 3.
Histogram of gene ontology classification.
The results are summarized in three main categories: Biological Process, Cellular Component and Molecular Function. Bars represent the assign percent of unigenes in pap1 (white bars) and BR086 (black bars) with BLAST matches in the TAIR9 database to each GO term.
Figure 4.
Combined assembly and annotation.
Size distribution of combined assembled contig length and frequency (a) using high–quality reads from pap1 and BR086. Annotation statistics (b) of unigenes of combined assembly queried against TAIR, NR and CDD databases.
Figure 5.
Gene ontology classification of unigenes from combined assembly of pap1 and BR086.
The results are summarized in three main categories: Biological Process (a), Cellular Component (b) and Molecular Function (c).
Table 2.
The unigenes related to secondary metabolites.
Figure 6.
Proposed biosynthetic pathways (NH - and NCH3-pathways) and expression of genes in pap1 mutant.
Enzymes for which information about cDNAs is not known are shown by broken arrows. Enzymes in red show up-regulation in pap1 in comparison to BR086, whereas in green (7OMT) shows down-regulation. Transcripts with no change in expression are shown in black. Transcripts identified through comparative transcriptome analysis with putative involvement in papaverine are underlined. Abbreviations: TYDC, tyrosine decarboxylase; NCS, norcoclaurine synthase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; CNMT, (S)-coclaurine N-methyltransferase; NMCH, (S)-N-methylcoclaurine 3′-hydroxylase; 3′OHase, 3′-hydroxylase; 4′OMT, (S)-3′-hydroxyN-methylcoclaurine 4′-O-methyltransferase; N7OMT, norreticuline 7-O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; 3′OMT, 3′-O-methyltransferase; deHase, dehydrogenase; LNdeMT, laudanosine N-demethylase.
Figure 7.
Differential expression of methyltransferases (a) and dehydrogenases (b) with their differential expression in pap1 and BR086.
Two columns in each represent pap1 and BR086, while each row represents contigs encoding members of these families (Table S10 and S11). Clustering was carried out with log tpm value of each contig in pap1 and BR086 transcriptome to visualize differential expression.
Figure 8.
Validation of differentially expressed transcripts in pap1 and BR086.
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 pap1 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 Table S1.