Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

Abstract

Background: Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies.

Results: A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database.

Conclusions: The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.

Figure 1

Biosynthetic pathway from tyrosine to sanguinarine. Biosynthesis of sanguinarine from tyrosine. Enzymes for which cognate cDNAs have been isolated are shown in black. Abbreviations: TYDC, tyrosine/dopa decarboxylase; NCS, norcoclaurine synthase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; CNMT, (S)-coclaurine N-methyltransferase; NMCH, (S)-N-methylcoclaurine 3'-hydroxylase; 4'OMT, (S)-3'-hdroxy-N-methylcoclaurine 4'-O-methyltransferase; BBE, berberine bridge enzyme; CheSyn, cheilanthifoline synthase; StySyn, stylopine synthase; TNMT, tetrahydroprotoberberine N-methyltransferase; MSH, methylstylopine hydroxylase; P6 H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase. StySyn and CheSyn cDNAs were functionally characterized in plant species other than opium poppy.

Figure 2

Clustering of 454 pyrosequencing data annotated as TNMT. Various examples representing assembly of ESTs for TNMT annotated unigenes found in the 454 database. The upper bar corresponds to the translated TNMT protein (Accession number Q108P1_PAPSO). The lower bar represents the unigene found in the 454 database and labelled with the contig number. The white region reflects the TNMT open reading frame. See Additional File 4 for a summary of unigenes shown in this figure.

Figure 3

Number of 454 pyrosequence reads representing gene transcripts corresponding to known benzylisoquinoline alkaloid biosynthetic enzymes . The cDNA library used for 454 pyrosequencing was prepared from opium poppy cell cultures treated with a fungal elicitor for 10 h. Sequence counts include unigenes encoding predicted proteins with > 90% amino acid sequence identity to known opium poppy enzymes except for CheSyn and StySyn, which were compared with known enzymes from Eschscholzia californica. Black bars represent unigenes encoding enzymes involved in the conversion of precursor tyrosine to the central intermediate (S)-reticuline. Red bars refer to unigenes encoding enzymes involved in the formation of sanguinarine, blue bars represent unigenes encoding enzymes involved in the biosynthesis of morphine, and green bars correspond to other enzymes with a role in benzylisoquinoline alkaloid metabolism. Abbreviations are as indicated in Figure 1 and Additional File 1.

Figure 4

Fractionation of the gel containing proteins separated by SDS-PAGE prior to LC-MS/MS . Coomassie stained gel of a total protein extract (10 μg) from opium poppy cell cultures treated with a fungal elicitor for 50 h. Each of the 12 gel slices was treated with trypsin and independently analyzed by LC-MS/MS peptide analysis.

Figure 5

Functional categories of (A) trans cripts represented in the 454 pyrosequence database and (B) peptides identified by LC-MS/MS . (A) GO annotations were assigned for 23,753 contigs and singletons out of a total of 93,723 unigenes in the opium poppy 454 pyrosequencing database. (B) GO annotations were assigned for a total of 1,004 putative opium poppy proteins identified by LC-MS/MS peptide analysis.

Figure 6

Metabolic networks from sucrose to sanguinarine and morphine. Gene transcripts corresponding to enzymes shown in black or red were identified in the 454 pyrosequencing database, whereas those written in grey were not. Enzymes written in red were found among proteins identified by LC-MS/MS peptide analysis. Cognate cDNAs have not been isolated for enzymes shown in blue.