Comparative Analysis of Chloroplast Genome of Meconopsis (Papaveraceae) Provides Insights into Their Genomic Evolution and Adaptation to High Elevation.

The Meconopsis species are widely distributed in the Qinghai-Tibet Plateau, Himalayas, and Hengduan Mountains in China, and have high medicinal and ornamental value. The high diversity of plant morphology in this genus poses significant challenges for species identification, given their propensity for highland dwelling, which makes it a question worth exploring how they cope with the harsh surroundings. In this study, we recently generated chloroplast (cp) genomes of two Meconopsis species, Meconopsis paniculata (M. paniculata) and M. pinnatifolia, and compared them with those of ten Meconopsis cp genomes to comprehend cp genomic features, their phylogenetic relationships, and what part they might play in plateau adaptation. These cp genomes shared a great deal of similarities in terms of genome size, structure, gene content, GC content, and codon usage patterns. The cp genomes were between 151,864 bp and 154,997 bp in length, and contain 133 predictive genes. Through sequence divergence analysis, we identified three highly variable regions (trnD-psbD, ccsA-ndhD, and ycf1 genes), which could be used as potential markers or DNA barcodes for phylogenetic analysis. Between 22 and 38 SSRs and some long repeat sequences were identified from 12 Meconopsis species. Our phylogenetic analysis confirmed that 12 species of Meconopsis clustered into a monophyletic clade in Papaveraceae, which corroborated their intrageneric relationships. The results indicated that M. pinnatifolia and M. paniculata are sister species in the phylogenetic tree. In addition, the atpA and ycf2 genes were positively selected in high-altitude species. The functions of these two genes might be involved in adaptation to the extreme environment in the cold and low CO2 concentration conditions at the plateau.

Extreme Reconfiguration of Plastid Genomes in Papaveraceae: Rearrangements, Gene Loss, Pseudogenization, IR Expansion, and Repeats.

The plastid genomes (plastomes) of angiosperms are typically highly conserved, with extreme reconfiguration being uncommon, although reports of such events have emerged in some lineages. In this study, we conducted a comprehensive comparison of the complete plastomes from twenty-two species, covering seventeen genera from three subfamilies (Fumarioideae, Hypecooideae, and Papaveroideae) of Papaveraceae. Our results revealed a high level of variability in the plastid genome size of Papaveraceae, ranging from 151,864 bp to 219,144 bp in length, which might be triggered by the expansion of the IR region and a large number of repeat sequences. Moreover, we detected numerous large-scale rearrangements, primarily occurring in the plastomes of Fumarioideae and Hypecooideae. Frequent gene loss or pseudogenization were also observed for ndhs, accD, clpP, infA, rpl2, rpl20, rpl32, rps16, and several tRNA genes, particularly in Fumarioideae and Hypecooideae, which might be associated with the structural variation in their plastomes. Furthermore, we found that the plastomes of Fumarioideae exhibited a higher GC content and more repeat sequences than those of Papaveroideae. Our results showed that Papaveroideae generally displayed a relatively conserved plastome, with the exception of Eomecon chionantha, while Fumarioideae and Hypecooideae typically harbored highly reconfigurable plastomes, showing high variability in the genome size, gene content, and gene order. This study provides insights into the plastome evolution of Papaveraceae and may contribute to the development of effective molecular markers.

From genomics to metabolomics: Deciphering sanguinarine biosynthesis in Dicranostigma leptopodum.

Dicranostigma leptopodum (Maxim) Fedde (DLF) is a renowned medicinal plant in China, known to be rich in alkaloids. However, the unavailability of a reference genome has impeded investigation into its plant metabolism and genetic breeding potential. Here we present a high-quality chromosomal-level genome assembly for DLF, derived using a combination of Nanopore long-read sequencing, Illumina short-read sequencing and Hi-C technologies. Our assembly genome spans a size of 621.81 Mb with an impressive contig N50 of 93.04 Mb. We show that the species-specific whole-genome duplication (WGD) of DLF and Papaver somniferum corresponded to two rounds of WGDs of Papaver setigerum. Furthermore, we integrated comprehensive homology searching, gene family analyses and construction of a gene-to-metabolite network. These efforts led to the discovery of co-expressed transcription factors, including NAC and bZIP, alongside sanguinarine (SAN) pathway genes CYP719 (CFS and SPS). Notably, we identified P6H as a promising gene for enhancing SAN production. By providing the first reference genome for Dicranostigma, our study confirms the genomic underpinning of SAN biosynthesis and establishes a foundation for advancing functional genomic research on Papaveraceae species. Our findings underscore the pivotal role of high-quality genome assemblies in elucidating genetic variations underlying the evolutionary origin of secondary metabolites.

Comparative analysis of the complete chloroplast genome of Papaveraceae to identify rearrangements within the Corydalis chloroplast genome.

Chloroplast genomes are valuable for inferring evolutionary relationships. We report the complete chloroplast genomes of 36 Corydalis spp. and one Fumaria species. We compared these genomes with 22 other taxa and investigated the genome structure, gene content, and evolutionary dynamics of the chloroplast genomes of 58 species, explored the structure, size, repeat sequences, and divergent hotspots of these genomes, conducted phylogenetic analysis, and identified nine types of chloroplast genome structures among Corydalis spp. The ndh gene family suffered inversion and rearrangement or was lost or pseudogenized throughout the chloroplast genomes of various Corydalis species. Analysis of five protein-coding genes revealed simple sequence repeats and repetitive sequences that can be potential molecular markers for species identification. Phylogenetic analysis revealed three subgenera in Corydalis. Subgenera Cremnocapnos and Sophorocapnos represented the Type 2 and 3 genome structures, respectively. Subgenus Corydalis included all types except type 3, suggesting that chloroplast genome structural diversity increased during its differentiation. Despite the explosive diversification of this subgenus, most endemic species collected from the Korean Peninsula shared only one type of genome structure, suggesting recent divergence. These findings will greatly improve our understanding of the chloroplast genome of Corydalis and may help develop effective molecular markers.

Sequence Analysis of the Plastomes of Two Tibetan Medicinal Plants of the Family Papaveraceae.

BACKGROUND: With the rapid development of next-generation sequencing technology, more plants plastomes have been sequenced, further advancing species identification and phylogenetic studies. However, there are a few studies on the genetic and phylogenetic analysis of the plastomes of Dicranostigma lactucoides Hook. f. et Thoms. and Hypecoum leptocarpum Hook. f. et Thoms. METHODS: In this study, we sequenced and analyzed the plastomes of Dicranostigma lactucoides Hook. f. et Thoms. and Hypecoum leptocarpum Hook. f. et Thoms., and conducted a phylogenetic analysis using 13 related species. RESULTS: The results showed that the plastomes of both D. lactucoides and H. leptocarpum had a typical tetrad structure, with sizes of 166,819 bp and 163,282 bp, respectively. We annotated 133 genes for D. lactucoides and 120 genes for H. leptocarpum. A total of 72 and 43 simple repetitive sequences were detected in D. lactucoides and H. leptocarpum, respectively. Codon preference analysis showed that the relative usage frequency of codons and the relative abundance of synonymous codons used were the same for both plastomes. Nucleotide polymorphism analysis identified seven variant loci with high nucleotide diversity (Pi) values, all located in the large single copy (LSC) region. Inverted repeat (IR) boundary analysis revealed differences in gene types and locations on both sides of the boundary, except for the small single copy/inverted repeat a (SSC/IRa) boundary. The phylogenetic analysis showed the species clustered into two major groups, one with five genera (Hypecoum, Corydalis, Papaver, Meconopsis, and Dicranostigma) and the other with two genera (Coreanomecon; and Hylomecon). CONCLUSIONS: Comparative analysis of the plastome genomic characteristics and phylogeny of D. lactucoides and H. leptocarpum laid the foundation for identifying the above two species and the phylogenetic study and comprehensive exploitation of the Papaveraceae.

Plant-microbe hybrid synthesis provides new insights for the efficient use of Macleaya cordata.

Sanguinarine and chelerytrine have antibacterial and anti-inflammatory effects and is the main active ingredients of growth promoters in animals. Currently, Sanguinarine and chelerytrine were extracted from the capsules of the medicinal plant Macleaya cordata. However, the biomass of M. cordata nonmedicinal parts (leaves) accounted for a large proportion and contained a rich presentation of protopine and allocryptopine which are the precursor compounds of sanguinarine and chelerytrine. The aim of this study was to develop a new method for producing sanguinarine and chelerytrine through yeast transformation of protopine and allocryptopine in M. cordata leaves. First, we isolated different genes from Papaver somniferum (PsP6H, PsCPR, PsDBOX), Eschscholtzia californica (EcP6H), Cucumis sativus (CuCPR), Arabidopsis thaliana (AtCPR) and M. cordata (Mc11229, Mc11218, Mc6408, Mc6407, Mc19967, Mc13802). Additionally, some of the gene sequences were codon optimized. Then, we transformed these genes into yeast cells to compare the catalytic efficiency. Second, we used the most efficient strains to biotransform the leaves of M. cordata. Finally, we obtained 85.415 ± 11.887 ng mL-1 sanguinarine and 4.288 ± 1.395 ng mL-1 chelerytrine, which was more than 2-3 times the content in leaves of M. cordata. Overall, we using the nonmedicinal parts of M. cordata and successfully obtained sanguinarine and chelerytrine by the plant-microbial hybrid synthesis method.

The genome of Corydalis reveals the evolution of benzylisoquinoline alkaloid biosynthesis in Ranunculales.

Species belonging to the order Ranunculales have attracted much attention because of their phylogenetic position as a sister group to all other eudicot lineages and their ability to produce unique yet diverse benzylisoquinoline alkaloids (BIAs). The Papaveraceae family in Ranunculales is often used as a model system for studying BIA biosynthesis. Here, we report the chromosome-level genome assembly of Corydalis tomentella, a species of Fumarioideae, one of the two subfamilies of Papaveraceae. Based on comparisons of sequenced Ranunculalean species, we present clear evidence of a shared whole-genome duplication (WGD) event that has occurred before the divergence of Ranunculales but after its divergence from other eudicot lineages. The C. tomentella genome enabled us to integrate isotopic labeling and comparative genomics to reconstruct the BIA biosynthetic pathway for both sanguinarine biosynthesis shared by papaveraceous species and the cavidine biosynthesis that is specific to Corydalis. Also, our comparative analysis revealed that gene duplications, especially tandem gene duplications, underlie the diversification of BIA biosynthetic pathways in Ranunculales. In particular, tandemly duplicated berberine bridge enzyme-like genes appear to be involved in cavidine biosynthesis. In conclusion, our study of the C. tomentella genome provides important insights into the occurrence of WGDs during the early evolution of eudicots, as well as into the evolution of BIA biosynthesis in Ranunculales.

An Integrated-Omics/Chemistry Approach Unravels Enzymatic and Spontaneous Steps to Form Flavoalkaloidal Nudicaulin Pigments in Flowers of Papaver nudicaule L.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions.

Influence of different elicitors on BIA production in Macleaya cordata.

Sanguinarine (SAN) and chelerythrine (CHE) have been widely used as substitutes for antibiotics for decades. For a long time, SAN and CHE have been extracted from mainly Macleaya cordata, a plant species that is a traditional herb in China and belongs to the Papaveraceae family. However, with the sharp increase in demand for SAN and CHE, it is necessary to develop a new method to enhance the supply of raw materials. Here, we used methyl jasmonate (MJ), salicylic acid (SA) and wounding alone and in combination to stimulate aseptic seedlings of M. cordata at 0 h, 24 h, 72 h and 120 h and then compared the differences in metabolic profiles and gene expression. Ultimately, we found that the effect of using MJ alone was the best treatment, with the contents of SAN and CHE increasing by 10- and 14-fold, respectively. However, the increased SAN and CHE contents in response to combined wounding and MJ were less than those for induced by the treatment with MJ alone. Additionally, after MJ treatment, SAN and CHE biosynthetic pathway genes, such as those encoding the protopine 6-hydroxylase and dihydrobenzophenanthridine oxidase enzymes, were highly expressed, which is consistent with the accumulation of SAN and CHE. At the same time, we have also studied the changes in the content of synthetic intermediates of SAN and CHE after elicitor induction. This study is the first systematic research report about using elicitors to increase the SAN and CHE in Macleaya cordata.

Over 100 Million Years of Enzyme Evolution Underpinning the Production of Morphine in the Papaveraceae Family of Flowering Plants.

Phylogenomic analysis of whole genome sequences of five benzylisoquinoline alkaloid (BIA)-producing species from the Ranunculales and Proteales orders of flowering plants revealed the sequence and timing of evolutionary events leading to the diversification of these compounds. (S)-Reticuline is a pivotal intermediate in the synthesis of many BIAs and our analyses revealed parallel evolution between the two orders, which diverged ∼122 million years ago (MYA). Berberine is present in species across the entire Ranunculales, and we found co-evolution of genes essential for production of the protoberberine class. The benzophenanthridine class, which includes the antimicrobial compound sanguinarine, is specific to the Papaveraceae family of Ranunculales, and biosynthetic genes emerged after the split with the Ranunculaceae family ∼110 MYA but before the split of the three Papaveraceae species used in this study at ∼77 MYA. The phthalideisoquinoline noscapine and morphinan class of BIAs are exclusive to the opium poppy lineage. Ks estimation of paralogous pairs indicates that morphine biosynthesis evolved more recently than 18 MYA in the Papaver genus. In the preceding 100 million years gene duplication, neofunctionalization and recruitment of additional enzyme classes, combined with gene clustering, gene fusion, and gene amplification, resulted in emergence of medicinally valuable BIAs including morphine and noscapine.

Comparison of Four Complete Chloroplast Genomes of Medicinal and Ornamental Meconopsis Species: Genome Organization and Species Discrimination.

High-throughput sequencing of chloroplast genomes has been used to gain insight into the evolutionary relationships of plant species. In this study, we sequenced the complete chloroplast genomes of four species in the Meconopsis genus: M. racemosa, M. integrifolia (Maxim.) Franch, M. horridula and M. punicea. These plants grow in the wild and are recognized as having important medicinal and ornamental applications. The sequencing results showed that the size of the Meconopsis chloroplast genome ranges from 151864 to 153816 bp. A total of 127 genes comprising 90 protein-coding genes, 37 tRNA genes and 8 rRNA genes were observed in all four chloroplast genomes. Comparative analysis of the four chloroplast genomes revealed five hotspot regions (matK, rpoC2, petA, ndhF, and ycf1), which could potentially be used as unique molecular markers for species identification. In addition, the ycf1 gene may also be used as an effective molecular marker to distinguish Papaveraceae and determine the evolutionary relationships among plant species in the Papaveraceae family. Futhermore, these four genomes can provide valuable genetic information for other related studies.

The study of transcriptome sequencing for flower coloration in different anthesis stages of alpine ornamental herb (Meconopsis 'Lingholm').

Meconopsis (Papaveraceae) is an interesting alpine herb, mainly distributed in the mountainous area of southwest China and high altitude zone in Tibetan-Himalaya. Different Meconopsis species showed a flower color alteration in different anthesis stages, Meconopsis 'Lingholm' is one of the localized species whose petal color changes from purple to blue during the flowering process. In general, the blue color flower is a rare kind, and usually hard to cultivate artificially. The molecular mechanism of flower color formation and color alteration of alpine flowers were reported by many research workers. To find critical genes that regulate Meconopsis 'Lingholm' color alteration and the mechanism of environmental adaptation, the current study performed transcriptome sequencing by using Meconopsis 'Lingholm' petals from different anthesis stages. There were totally 91,615 unigenes obtained from 31.4 Gb sequencing data, and differentially expressed genes between two consecutive flowering stages were obtained. Bioinformatics studies showed genes regulating petal color alteration were activated. Moreover, the functional analysis showed that Meconopsis 'Lingholm' showed a stress response to mechanical damage, non-biological stimulation and water deficiency in the bud stage, as well as showed a stress response to the cold from cracking stage to blooming stage. Furthermore, RNA-Seq results were verified using nine randomly selected genes by qPCR, which showed same expression trend with sequencing results. During this study, 20 candidate genes identified for further studies, which included five petal color related genes and 15 environmental response genes.

Modulation of benzylisoquinoline alkaloid biosynthesis by overexpression berberine bridge enzyme in Macleaya cordata.

Macleaya cordata produces a variety of benzylisoquinoline alkaloids (BIAs), such as sanguinarine, protopine, and berberine, which are potential anticancer drugs and natural growth promoters. The genes encoding the berberine bridge enzyme (BBE) were isolated from M. cordata and Papaver somniferum, and then the two genes were overexpressed in M. cordata. Through liquid chromatography with triple-quadrupole mass spectrometry analysis, it was determined that McBBE-OX caused higher levels of (S)-norcoclaurine, (S)-coclaurine, (S)-N-cis-methylcoclaurine, (S)-reticuline, (S)-tetrahydrocolumbamine, (S)-tetrahydroberberine, (S)-cheilanthifoline, and (S)-scoulerine than PsBBE-OX, empty vector or control treatments. qRT-PCR analysis demonstrated that the introduced genes in the transgenic lines were all highly expressed. However, the levels of sanguinarine (SAN) and chelerythrine (CHE) in all the transgenic lines were slightly lower than those in the wild-type lines, possibly because the overexpression of McBBE causes feedback-inhibition. This is the first report on the overexpression of potential key genes in M. cordata, and the findings are important for the design of metabolic engineering strategies that target BIAs biosynthesis.

Reconfiguration of the plastid genome in Lamprocapnos spectabilis: IR boundary shifting, inversion, and intraspecific variation.

We generated a complete plastid genome (plastome) sequence for Lamprocapnos spectabilis, providing the first complete plastome from the subfamily Fumarioideae (Papaveraceae). The Lamprocapnos plastome shows large differences in size, structure, gene content, and substitution rates compared with two sequenced Papaveraceae plastomes. We propose a model that explains the major rearrangements observed, involving at least six inverted repeat (IR) boundary shifts and five inversions, generating a number of gene duplications and relocations, as well as a two-fold expansion of the IR and miniaturized small single-copy region. A reduction in the substitution rates for genes transferred from the single-copy regions to the IR was observed. Accelerated substitution rates of plastid accD and clpP were detected in the Lamprocapnos plastome. The accelerated substitution rate for the accD gene was correlated with a large insertion of amino acid repeat (AAR) motifs in the middle region, but the forces driving the higher substitution rate of the clpP gene are unclear. We found a variable number of AARs in Lamprocapnos accD and ycf1 genes within individuals, and the repeats were associated with coiled-coil regions. In addition, comparative analysis of three Papaveraceae plastomes revealed loss of rps15 in Papaver, and functional replacement to the nucleus was identified.

Hairy root induction and benzylisoquinoline alkaloid production in Macleaya cordata.

Sanguinarine is currently widely used to replace antibiotic growth promoters in animal feeding and has demonstrated useful anticancer activity. Currently, the main source of sanguinarine is from an important medicinal plant, Macleaya cordata. To obtain a new source of sanguinarine production, we established hairy root cultures of M. cordata by co-cultivating leaf and stem explants with Agrobacterium rhizogenes. Except the co-cultivation medium, all growth media contained 200 mg/L timentin to eliminate A. rhizogenes. Through comparing the metabolic profiles and gene expression of hairy roots and wild-type roots sampled at five time points, we found that the sanguinarine and dihydrosanguinarine contents of hairy roots were far higher than those of wild-type roots, and we revealed the molecular mechanism that causes these metabolites to increase. Consequently, this study demonstrated that the hairy root system has further potential for bioengineering and sustainable production of sanguinarine on a commercial scale. To the best of our knowledge, this is the first efficient protocol reported for the establishment of hairy root cultures in M. cordata using A. rhizogenes.

Evolutionary diversification of CYC/TB1-like TCP homologs and their recruitment for the control of branching and floral morphology in Papaveraceae (basal eudicots).

Angiosperms possess enormous morphological variation in plant architectures and floral forms. Previous studies in Pentapetalae and monocots have demonstrated the involvement of TCP domain CYCLOIDEA/TEOSINTE BRANCHED1-like (CYC/TB1) genes in the control of floral symmetry and shoot branching. However, how TCP/CYC-like (CYL) genes originated, evolved and functionally diversified remain unclear. We conducted a comparative functional study in Ranunculales, the sister lineage to all other eudicots, between Eschscholzia californica and Cysticapnos vesicaria, two species of Papaveraceae with actinomorphic and zygomorphic flowers, respectively. Phylogenetic analysis indicates that CYL genes in Papaveraceae form two paralogous lineages, PapaCYL1 and PapaCYL2. Papaveraceae CYL genes show highly diversified expression patterns as well as functions. Enhanced branching by silencing of EscaCYL1 suggests that the role of CYC/TB1-like genes in branching control is conserved in Papaveraceae. In contrast to the arrest of stamen development in Pentapetalae, PapaCYL genes promote stamen initiation and growth. In addition, we demonstrate that CyveCYLs are involved in perianth development, specifying sepal and petal identity in Cysticapnos by regulating the B-class floral organ identity genes. Our data also suggest the involvement of CyveCYL genes in the regulation of flower symmetry in Cysticapnos. Our work provides evidence of the importance of TCP/CYC-like genes in the promotion of morphological diversity across angiosperms.

Phylogeography of Eomecon chionantha in subtropical China: the dual roles of the Nanling Mountains as a glacial refugium and a dispersal corridor.

BACKGROUND: Mountains have not only provided refuge for species, but also offered dispersal corridors during the Neogene and Quaternary global climate changes. Compared with a plethora of studies on the refuge role of China's mountain ranges, their dispersal corridor role has received little attention in plant phylogeographic studies. Using phylogeographic data of Eomecon chionantha Hance (Papaveraceae), this study explicitly tested whether the Nanling Mountains, which spans from west to east for more than 1000 km in subtropical China, could have functioned as a dispersal corridor during the late Quaternary in addition to a glacial refugium. RESULTS: Our analyses revealed a range-wide lack of phylogeographic structure in E. chionantha across three kinds of molecular markers [two chloroplast intergenic spacers, nuclear ribosomal internal transcribed spacer (nrITS), and six nuclear microsatellite loci]. Demographic inferences based on chloroplast and nrITS sequences indicated that E. chionantha could have experienced a strong postglacial range expansion between 6000 and 1000 years ago. Species distribution modelling showed that the Nanling Mountains and the eastern Yungui Plateau were the glacial refugia of E. chionantha. Reconstruction of dispersal corridors indicated that the Nanling Mountains also have acted as a corridor of population connectivity for E. chionantha during the late Quaternary. CONCLUSIONS: Our results suggest that the Nanling Mountains may acted dual roles as a dispersal corridor in east-west direction and as a glacial refugium in subtropical China during the late Quaternary. The population connectivity mediated by the mountain range and a strong postglacial range expansion are the most likely reasons for the lack of phylogeographic structure in E. chionantha. The hypothesis of dual roles of the mountain range presented here sheds new insights into the phylogeographic patterns of organisms in subtropical China.

Identification of candidate genes involved in isoquinoline alkaloids biosynthesis in Dactylicapnos scandens by transcriptome analysis.

Dactylicapnos scandens (D. Don) Hutch (Papaveraceae) is a well-known traditional Chinese herb used for treatment of hypertension, inflammation, bleeding and pain for centuries. Although the major bioactive components in this herb are considered as isoquinoline alkaloids (IQAs), little is known about molecular basis of their biosynthesis. Here, we carried out transcriptomic analysis of roots, leaves and stems of D. scandens, and obtained a total of 96,741 unigenes. Based on gene expression and phylogenetic relationship, we proposed the biosynthetic pathways of isocorydine, corydine, glaucine and sinomenine, and identified 67 unigenes encoding enzymes potentially involved in biosynthesis of IQAs in D. scandens. High performance liquid chromatography analysis demonstrated that while isocorydine is the most abundant IQA in D. scandens, the last O-methylation biosynthesis step remains unclear. Further enzyme activity assay, for the first time, characterized a gene encoding O- methyltransferase (DsOMT), which catalyzes O-methylation at C7 of (S)-corytuberine to form isocorydine. We also identified candidate transcription factor genes belonging to WRKY and bHLH families that may be involved in the regulation of IQAs biosynthesis. Taken together, we first provided valuable genetic information for D. scandens, shedding light on candidate genes involved in IQA biosynthesis, which will be critical for further gene functional characterization.

The Genome of Medicinal Plant Macleaya cordata Provides New Insights into Benzylisoquinoline Alkaloids Metabolism.

The overuse of antibiotics in animal agriculture and medicine has caused a series of potential threats to public health. Macleaya cordata is a medicinal plant species from the Papaveraceae family, providing a safe resource for the manufacture of antimicrobial feed additive for livestock. The active constituents from M. cordata are known to include benzylisoquinoline alkaloids (BIAs) such as sanguinarine (SAN) and chelerythrine (CHE), but their metabolic pathways have yet to be studied in this non-model plant. The active biosynthesis of SAN and CHE in M. cordata was first examined and confirmed by feeding 13C-labeled tyrosine. To gain further insights, we de novo sequenced the whole genome of M. cordata, the first to be sequenced from the Papaveraceae family. The M. cordata genome covering 378 Mb encodes 22,328 predicted protein-coding genes with 43.5% being transposable elements. As a member of basal eudicot, M. cordata genome lacks the paleohexaploidy event that occurred in almost all eudicots. From the genomics data, a complete set of 16 metabolic genes for SAN and CHE biosynthesis was retrieved, and 14 of their biochemical activities were validated. These genomics and metabolic data show the conserved BIA metabolic pathways in M. cordata and provide the knowledge foundation for future productions of SAN and CHE by crop improvement or microbial pathway reconstruction.

Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

BACKGROUND: Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. RESULTS: In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal ( www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. CONCLUSIONS: This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.