Computational insights into diverse aspects of glutathione S-transferase gene family in Papaver somniferum.

Plant glutathione S-transferases are an ancient protein superfamily having antioxidant activity. These proteins are primarily involved in diverse plant functions such as plant growth and development, secondary metabolism, signaling pathways and defense against biotic and abiotic stresses. The current study aimed to comprehensively identify and characterize the GST gene family in the medicinally important crop Papaver somniferum. A total of 93 GST proteins were identified belonging to eight GST classes and found to be majorly localized in the cytoplasm. All GST genes were found on eleven opium chromosomes. Gene duplication analysis showed segmental duplication as a key factor for opium GST gene family expansion under strong purifying selection. Phylogenetic analysis with gymnosperm, angiosperm and bryophyte revealed the evolution of GSTs earlier than their division into separate groups and also prior to the divergence of monocot and dicot. The secondary structure prediction showed the dominance of α-helices indicative of PsomGSTs as structurally stable and elastic proteins. Gene architecture showed the conservation of number of exons across the classes. MEME analysis revealed only a few class specific and many across class conserved motifs. Ser was found to be the active site residue of tau, phi, theta and zeta class and Cys was catalytic residue of DHAR, lambda and GHR class. Promoter analyses identified many cis-acting regulatory elements related to hormonal, cellular, stress and light response functions. Ser was the key phosphorylation site. Only three glycosylation sites were found across the 93 PsomGSTs. 3D structure prediction was also performed and was validated. Interactome analyses revealed the correlation of PsomGSTs with glutathione metabolizing proteins. Gene enrichment analysis and KEGG pathway analyzed the involvement of PsomGSTs in three major pathways i.e. glutathione metabolism, tyrosine metabolism and ascorbate metabolism. The outcome revealed high model quality of PsomGSTs. The results of the current study will be of potential significance to understand the functional and structural importance of the GST gene family in opium, a medicinally important crop.

Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Improved UAV Opium Poppy Detection Using an Updated YOLOv3 Model.

Rapid detection of illicit opium poppy plants using UAV (unmanned aerial vehicle) imagery has become an important means to prevent and combat crimes related to drug cultivation. However, current methods rely on time-consuming visual image interpretation. Here, the You Only Look Once version 3 (YOLOv3) network structure was used to assess the influence that different backbone networks have on the average precision and detection speed of an UAV-derived dataset of poppy imagery, with MobileNetv2 (MN) selected as the most suitable backbone network. A Spatial Pyramid Pooling (SPP) unit was introduced and Generalized Intersection over Union (GIoU) was used to calculate the coordinate loss. The resulting SPP-GIoU-YOLOv3-MN model improved the average precision by 1.62% (from 94.75% to 96.37%) without decreasing speed and achieved an average precision of 96.37%, with a detection speed of 29 FPS using an RTX 2080Ti platform. The sliding window method was used for detection in complete UAV images, which took approximately 2.2 sec/image, approximately 10× faster than visual interpretation. The proposed technique significantly improved the efficiency of poppy detection in UAV images while also maintaining a high detection accuracy. The proposed method is thus suitable for the rapid detection of illicit opium poppy cultivation in residential areas and farmland where UAVs with ordinary visible light cameras can be operated at low altitudes (relative height < 200 m).

Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Short-chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy.

The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.

Benzylisoquinoline alkaloid biosynthesis in opium poppy.

Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.

Opium alkaloids, biosynthesis, pharmacology and association with cancer occurrence.

Papaver somniferum L. (Family: Papaveraceae) is a species well known for its diverse alkaloids (100 different benzylisoquinoline alkaloids (BIAs)). L-tyrosine serves as a precursor of several specific metabolites like BIAs. It has been used as an antitussive and potent analgesic to alleviate mild to extreme pain since ancient times. The extraction of pharmaceutically important alkaloids like morphine and codeine from poppy plant reflects the need for the most suitable and standard methods. Several analytical and extraction techniques have been reported in open literature for morphine, codeine and other important alkaloids which play a vital function in drug development and drug discovery. Many studies suggest that opioids are also responsible for adverse effects or secondary complications like dependence and withdrawal. In recent years, opium consumption and addiction are the most important risk factors. Many evidence-based reviews suggest that opium consumption is directly linked or acts as a risk factor for different cancers. In this review, we highlight significant efforts related to research which have been done over the past 5 decades and the complete information on Papaver somniferum including its phytochemistry, pharmacological actions, biosynthetic pathways and analytical techniques of opium alkaloid extraction and the link between opium consumption and cancer-related updates.

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

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.

Honeybees foraging response in genetically diversified opium poppy.

Studies were carried out on honeybees foraging on plant flowers. Results showed significantly higher foraging response of honeybees (Apis mellifera) in genetically divergent narcotic plant opium poppy (Papaver somniferum). Of the 18 mutants and two locally adapted cultivars of diverse genotypes screened, eight revealed significantly greater foraging response manifesting honeybee's preference towards specific plant morphotypes. The number of flower bloom did not correspond to number of foraging bees in both mutant and cultivar plant types of opium poppy. The genotype specific foraging response of honeybees could be attributed to physico-chemical properties of opium poppy flowers. This could have implications for the development of opium alkaloid fortified honeys for novel pharmaceuticals and isolation of natural spray compounds to attract honeybee pollinators for promoting crossing and sustainable hybridity in crops.

Gene actions for yield and its attributes and their implications in the inheritance pattern over three generations in opium poppy (Papaver somniferum L.).

The gene actions for yield and its attributes and their inheritance pattern based on five parameter model have been explored in four single crosses (NBIHT-5 × NBIHT-6, NBIHT-5 × NBMHT-1, NBMHT-1 × NBIHT-6 and NBMHT-2 × NBMHT-1) obtained using thebaine rich pure lines of opium poppy (Papaver somniferum L.) for three consecutive generations. All the traits showed nonallelic mode of interaction, however, dominance effect (h) was more pronounced for all the traits except thebaine and papaverine. The dominance × dominance (l) effects were predominant over additive × additive (i) for all traits in all the four crosses except for papaverine. The seed and opium yield, and its contributing traits inherited quantitatively. The fixable gene effects (d) and (i) were lower in magnitude than nonfixable (h) and (l) gene effects. The estimates of heterosis were also higher in comparison to the respective parents which suggested preponderance of dominance gene action for controlling most of the traits. The phenotypic coefficient of variation was marginally higher than those of genotypic coefficient of variation for all the traits. The traits thebaine, narcotine, morphine and opium yield had high heritability coupled with high genetic advance. The leaf number, branches per plant and stem diameter showed positive correlation with opium and seed yields. The selection of plants having large number of leaves, branches and capsules with bigger size would be advantageous to enhance the yield potential.

Silencing nature's narcotics: metabolic engineering of the opium poppy.

The opium poppy, Papaver somniferum L., and its narcotic and analgesic alkaloids, have an ancient history of use (and abuse) by humankind. A recent article by Allen and co-workers describes the metabolic engineering of morphine biosynthesis to block morphine formation and accumulate a potentially valuable pathway intermediate, (S)-reticuline. This work highlights the potential for modifying the production of pharmaceuticals in plants, but also raises questions about the complex regulation of biosynthetic pathways.

The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum.

The opium poppy, Papaver somniferum, is one of mankind's oldest medicinal plants. Opium poppy today is the commercial source of the narcotic analgesics morphine and codeine. Along with these two morphinans, opium poppy produces approximately eighty alkaloids belonging to various tetrahydrobenzylisoquinoline-derived classes. It has been known for over a century that morphinan alkaloids accumulate in the latex of opium poppy. With identification of many of the enzymes of alkaloid biosynthesis in this plant, biochemical data suggested involvement of multiple cell types in alkaloid biosynthesis in poppy. Herein the immunolocalization of five enzymes of alkaloid formation in opium poppy is reported: (R,S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase central to the biosynthesis of tetrahydroisoquinoline-derived alkaloids, the berberine bridge enzyme of the sanguinarine pathway, (R,S)-reticuline 7-O-methyltransferase specific to laudanosine formation, and salutaridinol 7-O-acetyltransferase and codeinone reductase, which lead to morphine. In capsule and stem, both O-methyltransferases and the O-acetyltransferase are found predominantly in parenchyma cells within the vascular bundle, and codeinone reductase is localized to laticifers, the site of morphinan alkaloid accumulation. In developing root tip, both O-methyltransferases and the O-acetyltransferase are found in the pericycle of the stele, and the berberine bridge enzyme is localized to parenchyma cells of the root cortex. Laticifers are not found in developing root tip, and, likewise, codeinone reductase was not detected. These results provide cell-specific localization that gives a coherent picture of the spatial distribution of alkaloid biosynthesis in opium poppy.

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.

A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy.

Opium poppy produces a diverse array of pharmaceutical alkaloids, including the narcotic analgesics morphine and codeine. The benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accompany vascular tissues throughout the plant. However, immunofluorescence labeling using affinity-purified antibodies showed that three key enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), involved in the biosynthesis of morphine and the related antimicrobial alkaloid sanguinarine, are restricted to the parietal region of sieve elements adjacent or proximal to laticifers. The localization of laticifers was demonstrated using antibodies specific to the major latex protein (MLP), which is characteristic of the cell type. In situ hybridization showed that CYP80B1, BBE, and COR gene transcripts were found in the companion cell paired with each sieve element, whereas MLP transcripts were restricted to laticifers. The biosynthesis and accumulation of alkaloids in opium poppy involves cell types not implicated previously in plant secondary metabolism and dramatically extends the function of sieve elements beyond the transport of solutes and information macromolecules in plants.