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Genome characterization of California poppy (Eschscholzia californica cv. "Hitoezaki"), which produces pharmaceutically important benzylisoquinoline alkaloids (BIAs), was carried out using the draft genome sequence. The numbers of tRNA and rRNA genes were close to those of the other plant species tested, whereas the frequency of repetitive sequences was distinct from those species. Comparison of the predicted genes with those of Amborella trichopoda, Nelumbo nucifera, Solanum lycopersicum, and Arabidopsis thaliana, and analyses of gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway indicated that the enzyme genes involved in BIA biosynthesis were highly enriched in the California poppy genome. Further comparative analysis using the genome information of Papaver somniferum and Aquilegia coerulea, both BIA-producing plants, revealed that many genes encoding BIA biosynthetic enzymes, transcription factors, transporters, and candidate proteins, possibly related to BIA biosynthesis, were specifically distributed in these plant species., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active benzylisoquinoline alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Understanding the adaptability of plants to the environment will help us address the challenges posed by the constantly changing climate to sustainable agriculture and industry. The California poppy (Eschscholzia californica Cham.) of Papaveraceae is an ornamentally and medicinally valuable plant species, not only due to its beautiful flower, but also the strong abiotic stress tolerance and production of pharmacologicallly active alkaloids. However, little is known about the mechanisms of its drought tolerance and association with the alkaloids biosynthesis. In this study, significant morphological and physiological changes of E. californica under early and late drought stress were found. Subsequently, the transcriptomic and metabolomic dynamics between normal and drought conditions were independently and jointly analyzed. Transcriptome analysis identified 24,176 differentially expressed genes (DEGs), which were enriched in alkaloid biosynthesis, phytohormone synthesis and signal transduction pathways, amino acid biosynthesis, and reactive oxygen species scavenging, etc. A large number of DEGs encoding transcription factor represent a reservoir for transcriptional regulation of drought stress responses of E. californica. Metabolomic analysis detected 393 drought responsive differentially expressed metabolites (DEMs), predominant in alkaloids and phenolic acids. Consistency between DEGs and DEMs identified by joint analysis revealed critical pathways in response to drought in E. californica and its close association with alkaloid biosynthesis. This study provides new insights into the drought response mechanisms of E. californica. The contribution of alkaloids to drought tolerance is particularly worthy of further in-depth research. [Display omitted] • The first transcriptomic and metabolomic study of drought response in E. californica. • Essential pathways responding to drought were identified in E. californica. • Drought stress promotes biosynthesis of alkaloid , especially isoquinoline alkaloids. • A large number of drought responsive transcription factors were identified. [ABSTRACT FROM AUTHOR]

The effect of iron oxide nanoparticles on medicinal plants biomass biosynthesis and antioxidant defense compounds should be assessed critically to ensure their safe and sustainable use within the agricultural fields and food chains. Current focus has been given to the effects of hematite nanoparticles (α-Fe 2 O 3 NPs, ∼ 27 nm) and ferric ions (Fe3+) at different concentrations [(0, control), 10, 100 and 1000 mg L−1)] on morpho-physiological and biochemical parameters and benzophenanthridine alkaloids content in Eschscholzia californica plants. Upon exposure to α-Fe 2 O 3 NPs, a significant (P < 0.05) inhibition (by 26.6%) of root biomass was observed at the high concentration (1000 mg L−1) over the control. As for the total dry weight of plants, significant (P < 0.01) increase (by 22.7%) was observed at all exposures, except that of 1000 mg L−1 α-Fe 2 O 3 NPs/and Fe+3 treatment which was reduced by 12.8%. The treatment of 100 mg L−1 α-Fe 2 O 3 NPs and Fe3+ caused significant increase in chlorophyll a (29.2% and 40%,), chlorophyll b (59.8% and 49.3%,) and carotenoids (14.3% and 25.4%%,) content when compared to control, respectively. However, accumulation of the lipid peroxidation product, malondialdehyde (MDA), in root and leaf tissues was similar (73.5% vs control) that was induced by 1000 mg L−1 α-Fe 2 O 3 NPs. No significant change in MDA content was observed in leaves and roots of Fe+3 treated plants at all exposure doses. Furthermore, total phenolics and flavonoids content of the leaves and roots exposed to α-Fe 2 O 3 NPs at 100 mg L−1 were significantly (P < 0.01) increased by 41–45%, 27–29%, and 44–48%, respectively, over the control. All obtained extracts revealed strong and significant scavenging effects on DPPH˙ as compared to control, but only exposure to 100 mg L−1 Fe+3 and α-Fe 2 O 3 NPs treatment revealed the highest free radical scavenging/antioxidant activity by 44.2% and 37.65%, respectively. The alkaloid accumulation in leaves exhibited significant (P < 0.05) differences between the control plants and those treated with α-Fe 2 O 3 NPs and Fe+3 of all concentrations. The eshchschollzine and sanguinarine contents showed no significant change in plants treated with Fe+3 concentrations; however, the exposure of high Fe+3 concentration increased (33.4%) the content of chelerythrine compared to control. Foliar applied α-Fe 2 O 3 NPs enhanced the content of californidine (35.4%), eshchschollzine (22.3%), and eshchschollzine (19.1%) compared to control. The observed toxicity was dose-dependent for both α-Fe 2 O 3 NPs and Fe+3. The synthesized endogenous secondary metabolites (e.g., phenolics, flavonoids and benzophenanthridine alkaloids) induce defense mechanism in exposed plants. These results suggest that specific concentrations (10–100 mg L−1) of α-Fe 2 O 3 NPs have extra benefits both as a nanoregulator and nanoelicitor for plant growth promotion and biosynthesis of alkaloids and cellular antioxidants. • Exposure to various doses of α-Fe 2 O 3 NPs and Fe+3 differentially affect root/shoot biomass. • Foliar applied 100 mg L−1 α-Fe 2 O 3 NPs increased photosynthetic pigments content. • The lipid peroxidation product was similarly accumulated in root and leaf tissues of α-Fe 2 O 3 NPs exposed plants. • Hormetic dose-dependent response was found on accumulation of alkaloids in leaves. • Multivariate analyses showed the significance of specific traits for interpreting the observed variance. [ABSTRACT FROM AUTHOR]

Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole alkaloids (MIAs), nicotine alkaloids, and benzylisoquinoline alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis. [ABSTRACT FROM AUTHOR]

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α1β2γ2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α3, α5, and α6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α3β2γ2 and α5β2γ2 GABAA receptors. Interestingly, α1, α3, and α5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine—suspected (S)-reticuline metabolites in the rodent brain. [ABSTRACT FROM AUTHOR]

Genes encoding regulatory factors isolated from Arabidopsis, soybean and corn have been screened to identify those that modulate the expression of genes encoding for enzymes involved in the biosynthesis of morphinan alkaloids in opium poppy ( Papaver somniferum) and benzophenanthridine alkaloids in California poppy ( Eschscholzia californica). In opium poppy, the over-expression of selected regulatory factors increased the levels of PsCOR (codeinone reductase), Ps4′OMT ( S-adenosyl-l-methionine:3′-hydroxy- N-methylcoclaurine 4′- O-methyltransferase) and Ps6OMT [( R, S)-norcoclaurine 6- O-methyltransferase] transcripts by 10- to more than 100-fold. These transcriptional activations translated into an enhancement of alkaloid production in opium poppy of up to at least 10-fold. In California poppy, the transactivation effect of regulatory factor WRKY1 resulted in an increase of up to 60-fold in the level of EcCYP80B1 [( S)- N-methylcoclaurine 3′-hydroxylase] and EcBBE (berberine bridge enzyme) transcripts. As a result, the accumulations of selected alkaloid intermediates were enhanced up to 30-fold. The transactivation effects of other regulatory factors led to the accumulation of the same intermediates. These regulatory factors also led to the production of new alkaloids in California poppy callus culture. [ABSTRACT FROM AUTHOR]

Production of the benzophenanthridine alkaloids in Eschscholtzia californica suspension cell cultures was optimized by adding 0.5 mg methyl jasmonate (MJ) and 0.02 mg salicylic acid (SA)/g FCW after 7 days cultivation. Sanguinarine reached 24 mg/g DCW by such treatment; 10 times higher than in control cell cultures. MJ and SA induced expression of berberine bridge enzyme and 3′-hydroxy-( S)- N-methylcoclaurine-4′- O-methyltransferase, respectively. MJ plus SA induced over-expression of both enzymes. [ABSTRACT FROM AUTHOR]

Cultured cells of Eschscholzia californica respond to a yeast glycoprotein elicitor by producing benzophenanthridine alkaloids, which are excreted into the cell wall and the outer medium. These compounds, preferentially sanguinarine, are efficient phytoalexins because of their ability to intercalate double-stranded DNA (dsDNA), penetrate membranes and inhibit various enzymes containing SH-groups. Externally added sanguinarine is rapidly taken up by intact cells and converted to dihydrosanguinarine, which is substituted intracellularly according to the biosynthetic route. A 29.5 kDa soluble enzyme that catalyses the reduction of sanguinarine and chelerythrine by either NADPH or NADH has been isolated and purified to homogeneity. Benzophenanthridines that accumulate in the outer medium, mainly 10-OH-chelerythrine, chelirubine and macarpine, are converted by the isolated enzyme and by intact cells at much slower rates than sanguinarine. The cellular capacity of uptake and conversion of sanguinarine largely surpasses the rate of alkaloid production. We conclude that the sanguinarine produced by intact cells, after excretion and binding to cell wall elements, is rapidly reabsorbed and reduced to the less toxic dihydrosanguinarine, which then undergoes further biosynthetic reactions. This recycling process would allow the presence of the toxic phytoalexin at the cellular surface without taking the risk of injuring the producing cell. [ABSTRACT FROM AUTHOR]

Eschscholtzia californica preparations are in use as phytopharmaceuticals and as herbal drugs. Studies are described on the metabolism and the toxicological analysis of the Eschscholtzia californica alkaloids californine and protopine in rat urine using gas chromatography–mass spectrometry. The identified metabolites indicated that californine is extensively metabolized by N-demethylation and/or single or double demethylenation with consecutive catechol-O-methylation of one of the hydroxy groups. Protopine, however, only undergoes extensive demethylenation of the 2,3-methylenedioxy group followed by catechol-O-methylation. All phenolic hydroxy metabolites were found to be partly conjugated. The authors’ systematic toxicological analysis procedure using full-scan gas chromatography–mass spectrometry after acid hydrolysis, liquid–liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of californine and protopine in rat urine after a dose which should correspond to that of drug users. Therefore, use of Eschscholtzia californica preparations should also be detectable in human urine by the authors’ systematic toxicological analysis procedure. [Copyright &y& Elsevier]

The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl2 (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4′-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl2 in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites. [ABSTRACT FROM AUTHOR]