Transcriptome Profiling of Two Ornamental and Medicinal Papaver Herbs

Abstract

The Papaver spp. (Papaver rhoeas (Corn poppy) and Papaver nudicaule (Iceland poppy)) genera are ornamental and medicinal plants that are used for the isolation of alkaloid drugs. In this study, we generated 700 Mb of transcriptome sequences with the PacBio platform. They were assembled into 120,926 contigs, and 1185 (82.2%) of the benchmarking universal single-copy orthologs (BUSCO) core genes were completely present in our assembled transcriptome. Furthermore, using 128 Gb of Illumina sequences, the transcript expression was assessed at three stages of Papaver plant development (30, 60, and 90 days), from which we identified 137 differentially expressed transcripts. Furthermore, three co-occurrence heat maps are generated from 51 different plant genomes along with the Papaver transcriptome, i.e., secondary metabolite biosynthesis, isoquinoline alkaloid biosynthesis (BIA) pathway, and cytochrome. Sixty-nine transcripts in the BIA pathway along with 22 different alkaloids (quantified with LC-QTOF-MS/MS) were mapped into the BIA KEGG map (map00950). Finally, we identified 39 full-length cytochrome transcripts and compared them with other genomes. Collectively, this transcriptome data, along with the expression and quantitative metabolite profiles, provides an initial recording of secondary metabolites and their expression related to Papaver plant development. Moreover, these profiles could help to further detail the functional characterization of the various secondary metabolite biosynthesis and Papaver plant development associated problems.

Keywords: Papaver nudicaule; Papaver rhoeas; alkaloid biosynthesis; poppy; target metabolome; transcriptome.

Figure 1

Illustration of the transcriptome assembly and functional annotation summary; (a) length distribution of assembled transcripts with and without annotations; (b) distribution of BLAST similarities; and, (c) species distribution from the BLAST outputs (all the images used in this figure are released free of copyright under the Creative Commons CCO license).

Figure 2

Overview of the differential expression patterns obtained from the given conditions: (a) individual sequence library reads mapped to the representative transcriptome; (b) quantitative summary of the differentially expressed transcripts concerning plant developmental stages; (c) a total of 137 differential expressed transcripts from all given pairs of conditions and their selected K-means hierarchical clusters, upon the desired expression patterns; (d) selected clusters from 137 common differentially expressed transcripts; and, (e) secondary metabolite transcripts which are involved in differential expression. Here, the transcripts were grouped into KEGG ortholog IDs, and the average expressions are shown while multiple transcripts belong to same ortholog ID. NW: Papaver nudicaule, RS: Papaver rhoeas.

Figure 3

Overview of secondary metabolite biosynthesis pathways and its coverage. Here the transcripts were linked to the KEGG orthologs (KO), by assessing the sequence similarity between KOs and Papaver transcripts, which was obtained from the ortholog analysis. The coverage of each pathway was calculated and plotted into the percentage scale (0 to 100%). Here, the coverage was calculated by the formula (Coverage per each KEGG map = (Number of KOs present in transcriptome/Total number of KOs) × 100).

Figure 4

The phylogenetic tree reconstructed to understand evolutionary relationship between Papaver and other selected 51 genomes (see Figure 3 for more details) with respect to secondary metabolite transcripts. This tree constructed with 161 single-copy secondary metabolite biosynthesis responsible transcripts, which includes their isoforms. This tree was re-constructed from 1000 bootstrap trees.

Figure 5

Overview of the isoquinoline alkaloid biosynthesis pathway (map00950). Here the transcripts were linked to the KEGG orthologs (KO), by assessing the sequence similarity between KOs and Papaver transcripts, which was obtained from the ortholog analysis. The expression of the heat map is shown in red (transcripts present) and blue (transcripts absent), this information is obtained from the ortholog clusters.

Figure 6

Overview of the isoquinoline alkaloid biosynthesis pathway (KEGG Pathway ID: map00950). The map is marked with the available transcripts and metabolites along with their differential expression patterns using Pathview. The color codes for the transcript differential expression values were in green and red, and for the metabolite, differential quantity values were in blue and yellow color. The sample order is RS (30,60,90), and NW (30,60,90).

Figure 7

The quantification of alkaloids observed from the target metabolite analysis; (a) the total alkaloid content assessed from the Papaver plant developmental stages; (b) The detail expression of individual metabolites with respect to Papaver spp. developmental stages, which used to derive the total metabolite quantifications and these metabolites were mapped to the BIA KEGG reference map (Figure 6).

Figure 8

qRT-PCR validation of twelve transcripts. The M is the marker and the color code for the type is RNA-Seq (Red) and qRT-PCR (Blue) respectively. In the heat-map the relative expression of qRT-PCR and log₂FC values were compared to assesses the similar expression. Here, the 16s rRNA is used as a house keeping in qRT-PCR experiment (bottom row of the gel-image).

Figure 9

Full-length cytochrome existence profile. The expression of the heat map is shown in red (transcripts present) and blue (transcripts absent), this information obtained from the ortholog clusters.

Figure 10

Cytochrome similarity and expression profile: (a) phylogenetic tree for the 39 full-length cytochromes; and (b) the corresponding expression profiles for 39 cytochromes. This tree was re-constructed from 1000 bootstrap trees.