Variability of Isoquinoline Alkaloid Profiles and Anticholinesterase Activities with Binding-Mode Predictions of Glaucium flavum Population.

In this study, phytochemical and biological activity studies supported by docking were carried out on a species of the genus Glaucium, a repository of isoquinoline alkaloids. The GC-MS (Gas Chromatography-Mass Spectrometry) method is used to characterize the isoquinoline alkaloids of Glaucium flavum Crantz. (Papaveraceae). G. flavum was collected from seven different regions of Türkiye (Antalya, Urla-Izmir, Mordogan-Izmir, Mugla, Assos-Canakkale, Karabiga-Canakkale, Giresun) and totally 17 compounds were detected by GC-MS. Glaucine was found to be the major constituent in the sample collected from Mugla, whereas isocorydine was recorded to be the principal alkaloid in other samples. Further fractionation studies on G. flavum collected from Antalya province in Southwestern Türkiye, yielded five major alkaloids (isocorydine 1, dihydrosanguinarine 2, glaucine 3, dehydroglaucine 4, protopine 5) which were characterized by spectroscopic methods. Anticholinesterase activities of the extracts and isolated alkaloids were also tested by in vitro Ellman method. The isolated compounds were also analyzed by a molecular docking technique to determine the binding orientations in the gorge of the active site of acetylcholinesterase (AChE) and a homology model of butyrylcholinesterase (BuChE). This is the first comparative investigation of the phytochemical composition and biodiversity of Glaucium flavum species growing in Türkiye.

Exploring the molecular mechanism of coloration differences in two Meconopsis wilsonii subspecies: australis and orientalis.

Flower color diversity is a key taxonomic trait in Meconopsis species, enhancing their appeal as ornamental flowers. However, knowledge of the molecular mechanisms of flower color formation in Meconopsis species is still limited. M. wilsonii subsp. australis (Australis) and M. wilsonii subsp. orientalis (Orientalis) have a developmental stage presenting red-purple flowers, while Orientalis also presents blue coloration at the full-bloom period, making them an important model for exploring the mechanism of blue flower formation in M. wilsonii. In this study, we collected petals from Australis and Orientalis at different developmental stages to compare the coloration differences between the two species and detect the molecular mechanisms of blue color in Orientalis. We identified that cyanidin was the main anthocyanin in the flowers of both species, and the blue color in Orientalis primarily arises from anthocyanins (Cyanidin-3-O-sambubioside). RNA sequencing analysis was performed to detect the gene expression in the anthocyanin biosynthesis pathway, and the results suggested that gene regulation for anthocyanin biosynthesis may not be the direct reason for blue color formation in Orientalis. In addition, the growth solid of Orientalis was rich in Fe and Mg ions, and a large amount of Fe and Mg ions accumulated in the petals of Orientalis. Combined with the gene functional enrichment results, we found that the purple and red-purple colors of these two species were presented by different glycosylation levels of cyanidin, while the violet color of Orientalis might be the results of metalloanthocyanins by Fe and Mg ions, which also relieved the toxicity caused by the high content of Fe and Mg ions in its cells. The environmental adaptation-related genes were highly expressed of in both species, such as adaptation to desiccation, water deprivation, freezing, etc. Our results revealed the coloration differences between Australis and Orientalis and described the molecular mechanisms of blue coloration in Orientalis. The data in our analysis could enrich the genetic resources for M. wilsonii for further studies.

Diversity of staminal nectariferous appendages in disymmetric and zygomorphic flowers of Fumarioideae (Papaveraceae).

Staminal nectaries show diversity in their position, size, shape, color, and number in Ranunculales. In Papaveraceae, nectaries only appear at the base of stamen in these lineages with disymmetric and zygomorphic flowers. However, the diversity of the staminal nectaries' developmental characteristics and structure is unknown. The diversity of staminal nectaries of Hypecoum erectum, Ichtyoselmis macrantha, Adlumia asiatica, Dactylicapnos torulosa, Corydalis edulis, and Fumaria officinalis (six species belonging to six genera, respectively) in the Fumarioideae was investigated under scanning electron microscopy, light microscopy, and transmission electron microscopy. In all species studied, according to the developmental characteristics of the nectaries, four developmental stages can be divided into initiation, enlargement, differentiation, and maturation, and the number of nectaries can be determined at the stage of initiation (stage 1), and morphological differentiation occurs at the developmental stage 3. The staminal nectaries consist of secretory epidermis, parenchyma tissue, and phloem with some sieve tube elements reaching the secretory parenchyma cells; however, the number of cell layers of parenchyma can vary from 30 to 40 in I. macrantha and D. torulosa, to only 5 to 10 like in F. officinalis. Secretory epidermis cells are larger than secretory parenchyma cells with abundant microchannels on the outer cell wall. There were abundant mitochondria, Golgi bodies, rough endoplasmic reticulum, and plastids in secretory parenchyma cells. Nectar is stored in the intercellular space and exuded to the exterior via microchannels. In A. asiatica, according to the evidence of small secretory cell characteristics such as dense cytoplasm, and numerous mitochondria, together with the filamentous secretions present on the surface of epidermal cells on groove, it can be inferred that the U-shaped sulcate which is located in the white projection formed at the filament of triplets in A. asiatica is nectariferous.

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.

Integration of manganese accumulation, subcellular distribution, chemical forms, and physiological responses to understand manganese tolerance in Macleaya cordata.

Macleaya cordata (Willd.) R. Br. are proposed for the application in phytoremediation of heavy metal-contaminated soil. In this paper, the physiological response, subcellular distribution, chemical form, ultrastructure, and manganese (Mn) absorption characteristics of M. cordata under the stress of 0, 3, 6, 9, 12, and 15 mmol/L manganese concentration were studied by sand culture experiment. The results showed that M. cordata seedlings show high tolerance to Mn stress with a concentration of less than 6 mmol/L, while higher Mn concentration showed a significant toxic effect. A low concentration of Mn (≤ 6 mmol/L) can promote the synthesis of chlorophyll and soluble protein; furthermore, superoxide dismutase and peroxidase activities responded positively. The accumulation of Mn in the inactive metabolic part (cell wall and vacuole) of M. cordata leaves might be one of the main Mn detoxification mechanism. According to the ultrastructure of M. cordata, high-concentration Mn2+ (≥ 12 mmol/L) stress can cause M. cordata cells to be distorted and deformed, black precipitates appeared in the intercellular space, mitochondria decrease, chloroplasts shrink, hungry particles increased, and starch granules decrease. The uptake ability of different tissues for Mn is leaf > root > stem, and transport coefficient decreases with the increase of Mn concentration. Clearly, M. cordata has a certain tolerance to manganese, which has the ecological application potential in Mn-polluted areas.

Chemical profile, acetylcholinesterase, butyrylcholinesterase, and prolyl oligopeptidase inhibitory activity of Glaucium corniculatum subsp. refractum

Abstract Papaveraceae is one of the prominent alkaloid-containing families, and plants of the genus Glaucium (Papaveraceae) are known for their bioactive alkaloids. Glaucium species have been used in traditional medicine in Turkey as an analgesic, narcotic, sedative, and antitussive. In this study, it was planned to evaluate the inhibitory activity of an alkaloidal extract of Glaucium corniculatum subsp. refractum on acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and prolyl oligopeptidase (POP), as well as exploring the chemical profile of the plant by using Gas Chromatography-Mass Spectrometry (GC-MS). The AChE, BuChE and POP inhibition activities of the alkaloidal extract of G. corniculatum subsp. refractum were determined spectrophotometrically. A rapid GC-MS method was used to identify alkaloids that could be responsible for these inhibition activities. In total, eleven alkaloids were identified in the alkaloid extract of the plant by GC-MS. Allocyptopine (52.92%) and protopine (25.38%) were found as the major constituents. The alkaloidal extract of G. corniculatum subsp. refractum showed potent AChE inhibitory activity (IC50:1.25 µg/mL) and BuChE inhibitory activity (IC50: 7.02 µg/mL). The extract also showed a remarkable inhibitory effect on POP with an IC50 value of 123.69 µg/mL. This study presents the first GC-MS investigation and POP inhibitory activity of G. corniculatum subsp. refractum.

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.

Chemical constituents from Macleaya cordata (Willd) R. Br. and their phenotypic functions against a Parkinson's disease patient-derived cell line.

Cytological profiling (CP) assay against a human olfactory neuroshpere-derived (hONS) cell line using a library of traditional Chinese medicinal plant extracts gave indications that the ethanolic extract of Macleaya cordata (Willd) R. Br. elicited strong perturbations to various cellular components. Further chemical investigation of this extract resulted in the isolation of two new benzo[c]phenanthridine alkaloids, (6R)-10-methoxybocconoline (1) and 6-(1-hydroxyethyl)-10-methoxy-5,6-dihydrochelerythrine (2). Their planar structures were elucidated by extensive 1D and 2D NMR studies, together with MS data. The absolute configuration for position C-6 of 1 and relative configurations for position C-6 and C-1' of 2 were assigned by density functional theory (DFT) calculations of ECD and NMR data, respectively. Also isolated were fourteen known metabolites, including ten alkaloids (3-12) and four coumaroyl-containing compounds (13-16). Cytological profiling of the isolates against Parkinson's Disease (PD) patient-derived olfactory cells revealed bocconoline (3) and 6-(1-hydroxyethyl)-5,6-dihydrochelerythrine (4) significantly perturbated the features of cellular organelles including early endosomes, mitochondria and autophagosomes. Given that hONS cells from PD patients model some functional aspects of the disease, the results suggested that these phenotypic profiles may have a role in the mechanisms underlying PD and signified the efficacy of CP in finding potential chemical tools to study the biological pathways in PD.

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.

Identification of drought resistant miRNA in Macleaya cordata by high-throughput sequencing.

Drought is one of the most serious factors affecting crop yields in the world. Macleaya cordata (Willd.) is a draught-tolerant medicinal plant that has been proposed as a pioneer crop to be cultivated in arid areas. However, the exact molecular mechanisms through which M. cordata responds to draught stress remain elusive. In recent years, microRNA (miRNAs) in plants have been associated with stress response. Based on these findings, the current study aimed to shed light on the potential regulatory roles of miRNAs in the draught tolerance of M. cordata by employing high-throughput RNA sequencing and degradation sequencing. Six M. cordata plants were randomly divided into two equal experiment groups, including one draught group and one control group. High-throughput sequencing of the M. cordata samples led to the identification of 895 miRNAs, of which 18 showed significantly different expression levels between the two groups. PsRobot analysis and degradation sequencing predicted the differential miRNAs to target 59 and 36 genes, respectively. Functional analysis showed that 38 of the predicted genes could be implicated in the modulation of stress response. Four miRNAs and eight target genes were selected for quantitative real-time polymerase chain reaction (qRT-PCR) validation. The expression trend of each miRNA analyzed by qRT-PCR was consistent with that determined by sequencing, and was negatively correlated with those of its target genes. The results of our current study supported the involvement of miRNAs in the draught tolerance of M. cordata and could pave the way for further investigation into the related regulatory mechanisms.

Integrative study of subcellular distribution, chemical forms, and physiological responses for understanding manganese tolerance in the herb Macleaya cordata (papaveraceae).

Macleaya cordata is a perennial herb, a candidate phytoremediation plant with high biomass and manganese (Mn) tolerance. To study the mechanism underlying its Mn adaptability, Mn2+ at various concentrations (0, 1000, 5000, 10000, 15000, and 20000 µM) were applied to M. cordata to investigate the subcellular distribution and chemical forms of Mn, as well as the resulting physiological and biochemical changes by pot culture experiment under natural light in a greenhouse. According to our results, Mn level in M. cordata increased with exogenous Mn concentrations; and Mn contents in different tissues exhibited a leaf > root > stem pattern. Meanwhile, biomass and the level of photosynthetic pigments increased at lower Mn concentrations but declined as Mn concentration further ascended. Subcellular distribution analysis revealed that Mn was sequestered in cell wall and vacuole; in addition, it was incorporated into pectates and protein, phosphates, and oxalates. These findings revealed a possible effective strategy for M. cordata to reduce Mn mobility and toxicity. Moreover, a continuous boost in the level of malondialdehyde was observed with Mn gradient; whereas contents of soluble proteins and proline, and the activities of superoxide dismutase and peroxidase were initially increased and then dropped. Altogether, these results indicated that most Mn was trapped in the cell wall and soluble fractions in low toxicity forms such as pectates and protein, phosphates, and oxalates. These strategies, that is functioning cooperatively with the well-coordinated antioxidant defense systems and the non-enzymatic metabolites, confer strong resistance to Mn in M. cordata.

Rapid identification of "mad honey" from Tripterygium wilfordii Hook. f. and Macleaya cordata (Willd) R. Br using UHPLC/Q-TOF-MS.

Cases of honey poisoning have been reported widely, meaning there is a need for methods that detect "mad honey" or honey contaminated with plant-derived toxins to protect human health. In this study, we compared whole flower extracts and honey from Tripterygium wilfordii Hook. f. (TwHf) and Macleaya cordata (Willd) R. Br (McRB) using QuEChERS (quick, easy, cheap, effective, rugged, and safe) and ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). The results revealed several compounds common to whole flowers and honey samples. Triptolide and protopine were selected as potential markers for identifying "mad honeys" from these plants. The developed method can easily detect different honey varieties that were spiked with 5% TwHf and McRB honey samples. Additionally, 90 commercial honey samples were analyzed and determined as free from contamination. The method described in this report could be useful for studies on honey from other poisonous nectar and pollen plants.

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.

Green synthesis of silver nanoparticles using Glaucium corniculatum (L.) Curtis extract and evaluation of its antibacterial activity.

The metal nanoparticles, due to interesting features such as electrical, optical, chemical and magnetic properties, have been investigated repeatedly. Also, the mentioned nanoparticles have specific uses in terms of their antibacterial activity. The biosynthesis method is more appropriate than the chemical method for producing the nanoparticles because it does not need any special facilities; it is also economically affordable. In the current study, the silver nanoparticles (AgNPs) were obtained by using a very simple and low-cost method via Glaucium corniculatum (L.) Curtis plant extract. The characteristics of the AgNPs were investigated using techniques including: X-ray diffraction, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy. The SEM and TEM images showed that the nanoparticles had a spherical shape, and the mean diameter of them was 53.7 and 45 nm, respectively. The results of the disc diffusion test used for measuring the anti-bacterial activity of the synthesised nanoparticles indicated that the formed nanoparticles possessed a suitable anti-bacterial activity.

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.

Several newly discovered Mo-enriched plants with a focus on Macleaya cordata.

Phytoremediation as an alternative strategy has been a widespread attention. The screening of enriched plants and hyperaccumulators is the key of the strategy. So this study examined the status of heavy metal pollution in molybdenum (Mo) mine soils, metal accumulation in plants growing on mine, and their tolerance strategies. The analysis of 14 soils and 27 plant samples in mining area showed that Mo, zinc (Zn), and cadmium (Cd) concentrations exceeded soil safety standards and their levels varied in 27 plant samples. Mo was the heavy pollution with an average total content of 256.1 mg/kg in soils. As Mo-enriched plants, Mo concentrations of Macleaya cordata (Willd.) R. Br. and Morus australis Poir. were 704.4 and 772.4 mg/kg, respectively. M. cordata was selected as the research material, due to its high biomass. Molybdenum significantly decreased the biomass and photosynthesis of M. cordata at high concentration (> 200 µmol/L), but its biomass and photosynthesis reached the maximum after 50 µmol/L Mo treatment, respectively. Analysis of the subcellular distribution and chemical speciation showed that Mo was distributed a certain way in the extracts and that this suggested that it may be present in cell wall and soluble fraction of roots (51.9-63.9%; 26.1-44.7%) or shoots (30.0-44.4%; 47.3-56.0%) and complexed to organic acid, pectate, oxalate, and protein. This might be responsible for the adaptation of M. cordata to Mo stress. Therefore, M. cordata could serve as a potential plant to utilize for the phytoremediation of Mo-contaminated soil.

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.

Tissue-specific metabolite profiling of benzylisoquinoline alkaloids in the root of Macleaya cordata by combining laser microdissection with ultra-high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Tissue-specific metabolite profiling helps to find trace alkaloids masked during organ analysis, which contributes to understanding the alkaloid biosynthetic pathways in vivo and evaluating the quality of medical plants by morphology. As Macleaya cordata contains diverse types of benzylisoquinoline alkaloids (BIAs), the alkaloid metabolite profiling was carried out on various tissues of the root. METHODS: Laser microdissection with fluorescence detection was used to recognize and dissect different tissues from the root of M. cordata. Ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry was applied to analyze the trace alkaloids in tissues. These detected alkaloids were elucidated using their accurate molecular weights, MS/MS data, MS fragmentation patterns and the known biosynthetic pathways of BIAs. Finally, the distribution of alkaloids in dissected tissues and whole sections was mapped. RESULTS: Forty-nine alkaloids were identified from five microdissected tissues, and 24 of them were detected for the first time in M. cordata. Some types of alkaloids occurred specifically in dissected tissues. More alkaloids were detected in the cork and xylem vascular bundles which emit strong fluorescence under fluorescence microscopy. Some of the screened alkaloids were intermediates in sanguinarine and chelerythrine biosynthetic pathways, and others were speculated to be involved in the new branches of biosynthetic pathways. CONCLUSIONS: The integrated method is sensitive, specific and reliable for determining trace alkaloids, which is also a powerful tool for metabolite profiling of tissue-specific BIAs in situ. The present findings should contribute to a better understanding of the biosynthesis of BIAs in M. cordata root and provide scientific evidence for its quality evaluation based on morphological characteristics. Copyright © 2016 John Wiley & Sons, Ltd.