Integration of transcriptome, proteome and metabolism data reveals the alkaloids biosynthesis in Macleaya cordata and Macleaya microcarpa

Abstract

Background: The Macleaya spp., including Macleaya cordata and Macleaya microcarpa, are traditional anti-virus, inflammation eliminating, and insecticide herb medicines for their isoquinoline alkaloids. They are also known as the basis of the popular natural animal food addictive in Europe. However, few studies especially at genomics level were conducted on them. Hence, we performed the Macleaya spp. transcriptome and integrated it with iTRAQ proteome analysis in order to identify potential genes involved in alkaloids biosynthesis.

Methodology and principal findings: We elaborately designed the transcriptome, proteome and metabolism profiling for 10 samples of both species to explore their alkaloids biosynthesis. From the transcriptome data, we obtained 69367 and 78255 unigenes for M. cordata and M. microcarpa, in which about two thirds of them were similar to sequences in public databases. By metabolism profiling, reverse patterns for alkaloids sanguinarine, chelerythrine, protopine, and allocryptopine were observed in different organs of two species. We characterized the expressions of enzymes in alkaloid biosynthesis pathways. We also identified more than 1000 proteins from iTRAQ proteome data. Our results strongly suggest that the root maybe the organ for major alkaloids biosynthesis of Macleaya spp. Except for biosynthesis, the alkaloids storage and transport were also important for their accumulation. The ultrastructure of laticifers by SEM helps us to prove the alkaloids maybe accumulated in the mature roots.

Conclusions/significance: To our knowledge this is the first study to elucidate the genetic makeup of Macleaya spp. This work provides clues to the identification of the potential modulate genes involved in alkaloids biosynthesis in Macleaya spp., and sheds light on researches for non-model medicinal plants by integrating different high-throughput technologies.

Figure 1. Biosynthetic pathway from tyrosine to SA, CHE, PRO and ALL.

The biosynthesis of BIAs starts with the condensation of two tyrosine derivatives, followed by serial reactions to form (S)-reticuline. 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-Nmethylcoclaurine 4′-O-methyltransferase; BBE, berberine bridge enzyme; CFS, cheilanthifoline synthase; STS, stylopine synthase; SMT, (S)-scoulerine 9-O-methyltransferase; TDC, (S)-tetrahydroberberine synthase;TNMT, tetrahydroprotoberberine N-methyltransferase; MSH, methylstylopine hydroxylase; P6H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase.

Figure 2. The strategy overview.

Ten special samples of different time and organs to perform transcriptome, metabolism and proteome analysis were selected to explore their alkaloids biosynthesis. These samples are from Phase I (roots, leaves) and Phase II (roots, leaves and fruit shells) of both M. cordata and M. microcarpa.

Figure 3. Venn diagram shows distribution of similarity search results.

The unigenes annotations of both M. cordata and M. microcarpa were performed by alignment to public databases, including UniProt(Blue), KEGG (the Kyoto Encyclopedia of Genes and Genomes database, Yellow), COGs (Clusters of Orthologous Groups of proteins, Green) and Pfam(Red).

Figure 4. Gene expression cluster for P450 and enzymes in alkaloids pathway. A)

Gene expression analysis of P450. RNA-Seq read counts for representative transcripts (rows), expressed as log2 RPKM, were subjected to hierarchical agglomerative clustering based on their expression pattern across Macleaya spp.(columns). B) BIAs pathway key enzymes differential expression. The expression levels for genes in the pathways and precursor pathways (rows) across the Macleaya spp. assayed tissues (columns). The majority of the genes encoding and precursor pathway enzymes are most highly expressed in the stages of roots and fruits. Gene and pathway names correspond to those used in Fig. 1.

Figure 5. The GO analysis of the iTRAQ identified proteins from different databases.

The results from three databases: the public databases and the assembled M. cordata, M. microcarpa database by BLAST.

Figure 6. Ultrastructure of M. cordata in different developmental stages. A)

The results of optical microscope show the alkaloids distribution in different organs in M. cordata. B) The different stages of roots scan. The radicle stage(D,E), Phase I(F,G) and Phase II(H,I); The ultrastructure is taken by SEM which D,F,H are transverse section and E, G, longitudinal section.

Figure 7. Quantitative RT-PCR validataion.

Four candidate unigenes (BBE, P6H, SAR and TNMT) are shown for involved in the BIAs metabolic pathway show differential expression patterns by RT-PCR in three organs were carried out on cDNA prepared from roots (phases I and II), leaves (phases I and II), and fruits shells (phase II) as described in the Material and Methods (phase I show in blue bars and phase II show in red bars). Expression level is relative to ubiquitin and all results represent the mean (± SD) of three experiments.

Figure 8. The biosynthesis, storage and transport of three main alkaloids (PRO, ALL and SA) in different samples.

The number of plus means the relative abundance and the arrow shows the possible transport direction. All the results are based on the transcriptome and metabolism profiling results.