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.
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.
Table 1.
The alkaloids accumulation in different parts and different phase of Macleaya plant.
Table 2.
Summary of Macleaya spp. sequence assembly.
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).
Table 3.
Functional annotation and classification of the unigenes.
Table 4.
The top 10 KEGG pathways of Macleaya spp.
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.
Table 5.
Differential expression Genes(DEGs) statistical result in different organs and species.
Table 6.
The key enzymes of Isoquinoline alkaloid biosynthesis pathway expression in different samples.
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.
Table 7.
Integrated expressions of unigenes and proteins in two databases.
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.