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Purine alkaloids are naturally occurring nitrogenous methylated derivatives of purine nucleotide degradation products, having essential roles in medicine, food and various other aspects of our daily lives. They are generated through convergent evolution in different plant species. The pivotal reaction steps within the purine alkaloid metabolic pathways have been largely elucidated, and the convergent evolution of purine alkaloids has been substantiated through bioinformatic, biochemical and other research perspectives within S -adenosyl-ʟ-methionine-dependent N -methyltransferases. Currently, the biological and ecological roles of purine alkaloids, further refinement of the purine alkaloid metabolic pathways and the investigation of purine alkaloid adaptive evolutionary mechanisms continue to attract widespread research interest. The exploration of the purine alkaloid metabolic pathways also enhances our comprehension of the biochemical mechanism, providing insights into inter-species interactions and adaptive evolution and offering potential value in drug development and agricultural applications. Here, we review the progress of research in the distribution, metabolic pathway elucidation and regulation, evolutionary mechanism and ecological roles of purine alkaloids in plants. The opportunities and challenges involved in elucidating the biochemical basis and evolutionary mechanisms of the purine alkaloid metabolic pathways, as well as other research aspects, are also discussed. This article is part of the theme issue 'The evolution of plant meta-bolism'.

Covering: up to June 2024Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites derived from L-tyrosine, exhibiting significant pharmacological properties such as anti-microbial, anti-spasmodic, anti-cancer, cardiovascular protection, and analgesic effects. The industrial production of valuable BIAs relies on extraction from plants; however, challenges concerning their low concentration and efficiency hinder drug development. Hence, alternative approaches, including biosynthesis and chemoenzymatic synthesis, have been explored. Model species like Papaver somniferum and Coptis japonica have played a key role in unraveling the biosynthetic pathways of BIAs; however, many aspects, particularly modified steps like oxidation and methylation, remain unclear. Critical enzymes, e.g. , CYP450s and methyltransferases, play a substantial role in BIA backbone formation and modification, which is essential for understanding the origin and adaptive evolution of these plant specialized metabolites. This review comprehensively analyzes the structural diversity of reported BIAs and their distribution in plant lineages. In addition, the progress in understanding biosynthesis, evolution, and catalytic mechanisms underlying BIA biosynthesis is summarized. Finally, we discuss the progress and challenges in metabolic engineering, providing valuable insights into BIA drug development and the sustainable utilization of BIA-producing plants.

Histolocalization and quantification of secondary metabolites established the occurrence of alkaloids, phenols and acetogenins in various plant organs of A. muricata at different stages of plant growth. Annona muricata L. possesses broad assemblage of ethno-pharmaceutical and therapeutic upsides ascribed to biosynthesis of secondary metabolites. Currently, the bioactivity is characterized by the production of acetogenins (ACGs), and also by the biosynthesis of alkaloids, primarily benzylisoquinolines derived from tyrosine and phenolic compounds. As a result of high variability of metabolite production in A. muricata, the present study evaluated the histochemical analysis of various plant parts at different developmental stages of growth. Presences of phytometabolites were determined using in-situ histochemical localization of alkaloids, phenolic compounds and acetogenins, while its estimation employing spectroscopic quantification techniques to understand structure, development, time-course deposition, content and distribution of secondary phytoconstituents. Study revealed distinct presence of secondary metabolites in cells and tissues of all plant organs in various intensities and patterns. Alkaloids and acetogenins occurred mostly in secretory cavities or idioblast cells while sparsely associated with other cells or tissues. Phenolic compounds emerged widely, with no confined distribution patterns occurring in storage cells and tissues. Quantitative analysis revealed highest accumulation of alkaloids, phenols and acetogenins in mature root-barks, mature unripe fruit rind and mature leaf respectively. Secondary metabolites generally occurred in young stages at low concentrations as compared to matured growth stages of plant parts. Such in-situ histochemical and biochemical approach will help in identifying the cells and tissues responsible for synthesis, storage and biological effects of potential secondary metabolites which could be further used for culturing and enhancing its production with biotechnological aids., Competing Interests: Declarations Competing interests The authors declare that they have no conflicts of interest to disclose. Data availability All data generated or analyzed during this study are included in the published article. In case, one wished to consult further information, it will be made available through the corresponding author on reasonable request., (© 2024. The Author(s).)

Trichoderma longibrachiatum UN32 is an industrially important fungus capable of producing Dendrobine-Type Total alkaloids (DTTAs). Several reports have pointed out that branched-chain amino acid aminotransferases (BCATs) participate in backbone modification or promote the production of secondary metabolites. We previously proposed that cobalt chloride increased DTTAs production in T. longibrachiatum UN32, which was associated with enhanced expression of the gene BCAT, TlBCAT (Genbank accession No. PP465542). Following cloning and characterization, the cDNA of TlBCAT was found to consists of 1191 bp, encoding a protein of 397 amino acid residues. The molecular mass of TlBCAT was about 42 kDa through SDS-PAGE analysis. The predicted pI value was 5.54. Recombinant TlBCAT can catalyze L-leu with a catalytic efficiency of 15.91 mM - 1 S - 1 . In the binding pocket, residues interacting with PLP, including Tyr68, Arg93, Tyr136, and Lys194, are highly conserved. TlBCAT exhibits a broad spectrum of substrate specificity, typical for catalyzing the transamination reaction of various branched-chain and hydrophobic amino acids, with α-ketoglutarate as the amino acceptor. Exogenous branched-chain amino acids (BCAAs) positively affect Trichoderma growth and DTTAs production. These findings offer insights into the physiological significance of BCAAs and present a novel approach for enhancing DTTAs production in Trichoderma mycelium cultures., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The objective of the present manuscript was the evaluation of the toxicity and metabolism of the second generation of pyrovalerone cathinones (α-PHP, α-PHPiP, 4-MePPP and TH-PVP), using an early zebrafish (Danio rerio) larvae as in vivo model. Pyrovalerone cathinones LC 50 were determined after 24, 48, 72 and 96 h of exposure. TH-PVP proved to be the most toxic cathinone, whereas 4-MePPP was the least toxic. During acute exposure to pyrovalerone cathinones the main signs of toxicity exhibited by survivors were pericardial edema, yolk sac in embryos, bradycardia, delay in the hatching, malformations, and larvae without touch response. Afterwards, a short-term non-lethal experiment (24 h) was performed with early zebrafish larvae (72 h) for each of the selected compounds. The produced metabolites were tentatively identified by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and the metabolic pathways were proposed. The results showed that hydroxylation and dihydroxylation can be considered the main metabolic pathways, although depending of the cathinone studied, other metabolites can be found., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In this paper, we examine intraspecific variation in the quantity of alkaloid chemical defence in field collected individuals of the polymorphic ladybird beetle Adalia decempunctata (10-spot ladybird). Like its more widely studied relative Adalia bipunctata (2-spot ladybird), A. decempunctata possesses the alkaloids adaline and adalinine, which are, respectively, the major and minor alkaloids of A. bipunctata. We focused especially on alkaloid concentration in relation to colour pattern morph, sex, and the relationship between female and egg parameters. There was a marked sexual dimorphism in the balance of the two alkaloids, with adaline predominating in females and adalinine predominating in males: in males, on average, over 70% of total alkaloid was adalinine. Females had a lower proportion of adalinine (< 10%) than their eggs (> 15%) and relationships between egg alkaloid and female alkaloid or fecundity were weak or non-existent. Colour pattern morph had a borderline (although not) significant relationship with adaline concentration and total alkaloid concentration, which could be further explored with laboratory reared individuals. The sexual dimorphism in alkaloid content, which seems likely due to differences in synthesis, might be related to their relative costs to the two sexes and might provide insight into the evolution of alkaloid diversity in ladybirds., (© 2024. The Author(s).)

Harmful cyanobacterial blooms will be more intense and frequent in the future, contaminating surface waters with cyanotoxins and posing a threat to communities heavily reliant on surface water usage for crop irrigation. Constructed wetlands (CWs) are proposed to ensure safe crop irrigation, but more research is needed before implementation. The present study operated 28 mesocosms in continuous mode mimicking horizontal sub-surface flow CWs. Mesocosms were fed with synthetic lake water and spiked periodically with two cyanotoxins, microcystin-LR (MC-LR) and cylindrospermopsin (CYN), at environmentally relevant cyanotoxins concentrations (10 μg L -1 ). The influence of various design factors, including plant species, porous media, and seasonality, was explored. The mesocosms achieved maximum MC-LR and CYN mass removal rates of 95 % and 98 %, respectively. CYN removal is reported for the first time in CWs mimicking horizontal sub-surface flow CWs. Planted mesocosms consistently outperformed unplanted mesocosms, with Phragmites australis exhibiting superior cyanotoxin mass removal compared to Juncus effusus. Considering evapotranspiration, J. effusus yielded the least cyanotoxin-concentrated effluent due to the lower water losses in comparison with P. australis. Using the P-kC* model, different scaling-up scenarios for future piloting were calculated and discussed. Additionally, bacterial community structure was analyzed through correlation matrices and differential taxa analyses, offering valuable insights into their removal of cyanotoxins. Nevertheless, attempts to validate microcystin-LR biotransformation via the known mlrA gene degradation pathway were unfruitful, indicating alternative enzymatic degradation pathways occurring in such complex CW systems. Further investigation into the precise molecular mechanisms of removal and the identification of transformation products is needed for the comprehensive understanding of cyanotoxin mitigation in CW. This study points towards the feasibility of horizontal sub-surface flow CWs to be employed to control cyanotoxins in irrigation or recreational waters., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pedro N. Carvalho reports financial support was provided by Independent Research Fund Denmark. Carlos A. Arias reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Bletilla striata has been used in traditional Chinese medicine for thousands of years to treat a variety of health diseases. Currently, metabolic causes of differences in medicinal values are unknown, due to the lack of a large-scale and comprehensive investigation of metabolites in Bletilla species. In order to gain a better understanding of the major chemical constituents responsible for the medicinal value, this study aimed to explore the metabolomic differences among three Bletilla species (Bletilla striata: Bs, Bletilla ochracea: Bo and Bletilla formosana: Bf). There were 258 different metabolites between 'Bo' and 'Bf', the contents of 109 metabolites had higher abundance, while 149 metabolites showed less accumulation. There were 165 different metabolites between the 'Bs' and 'Bf', content of 72 metabolites was increased and content of 93 metabolites was decreased. There were 239 different metabolites between the 'Bs' and 'Bo', content of 145 metabolites was increased and content of 94 metabolites was decreased. In the Bo&#95;vs&#95;Bf, Bs&#95;vs&#95;Bf and Bs&#95;vs&#95;Bo groups, the major differential categories were flavonoids, phenolic acids, organic acids and alkaloids. Moreover, the differential metabolites were clustered into clear and distinct profiles via K-means analysis. In addition, the major differential categories were flavonoids, phenolic acids, organic acids and alkaloids. The 'Flavonoid biosynthesis' (ko00941) and 'Phenylalanine metabolism' (ko00360) pathways were significantly enriched in Bo&#95;vs&#95;Bf, Bs&#95;vs&#95;Bf and Bs&#95;vs&#95;Bo comparisons. These results clarify the metabolomics in different Bletilla species, as well as providing basis for the phamaceutical value of novel species of Bletilla., (© 2024. The Author(s).)

Protoberberine alkaloids and benzophenanthridine alkaloids (BZDAs) are subgroups of benzylisoquinoline alkaloids (BIAs), which represent a diverse class of plant-specialized natural metabolites with many pharmacological properties. Microbial biosynthesis has been allowed for accessibility and scalable production of high-value BIAs. Here, we engineer Saccharomyces cerevisiae to de novo produce a series of protoberberines and BZDAs, including palmatine, berberine, chelerythrine, sanguinarine and chelirubine. An ER compartmentalization strategy is developed to improve vacuole protein berberine bridge enzyme (BBE) activity, resulting in >200% increase on the production of the key intermediate (S)-scoulerine. Another promiscuous vacuole protein dihydrobenzophenanthridine oxidase (DBOX) has been identified to catalyze two-electron oxidation on various tetrahydroprotoberberines at N7-C8 position and dihydrobenzophenanthridine alkaloids. Furthermore, cytosolically expressed DBOX can alleviate the limitation on BBE. This study highlights the potential of microbial cell factories for the biosynthesis of a diverse group of BIAs through engineering of heterologous plant enzymes., (© 2024. The Author(s).)

Background: Konjac corms are known for their alkaloid content, which possesses pharmacological properties. In the primary cultivation areas of konjac, nitrogen deficiency is a common problem that significantly influences alkaloid synthesis. The impact of nitrogen deficiency on the alkaloids in konjac corms remains unclear, further complicated by the transition from mother to daughter corms during their growth cycle., Results: This study examined 21 alkaloids, including eight indole alkaloids, five isoquinoline alkaloids, and eight other types of alkaloids, along with the associated gene expressions throughout the development of Amorphophallus muelleri Blume under varying nitrogen levels. Nitrogen deficiency significantly reduced corm diameter and fresh weight and delayed the transformation process. Under low nitrogen conditions, the content of indole alkaloids and the expression of genes involved in their biosynthesis, such as tryptophan synthase (TRP) and tryptophan decarboxylase (TDC), exhibited a substantial increase in daughter corms, with fold changes of 61.99 and 19.31, respectively. Conversely, in the mother corm, TDC expression was markedly reduced, showing only 0.04 times the expression level observed under 10 N treatment. The patterns of isoquinoline alkaloid accumulation in corms subjected to nitrogen deficiency were notably distinct from those observed for indole alkaloids. The accumulation of isoquinoline alkaloids was significantly higher in mother corms, with expression levels of aspartate aminotransferase (GOT), chorismate mutase (CM), tyrosine aminotransferase (TAT), and pyruvate decarboxylase (PD) being 4.30, 2.89, 921.18, and 191.40 times greater, respectively. Conversely, in daughter corms, the expression levels of GOT and CM in the 0 N treatment were markedly lower (0.01 and 0.83, respectively) compared to the 10 N treatment., Conclusions: The study suggests that under nitrogen deficiency, daughter corms preferentially convert chorismate into tryptophan to synthesize indole alkaloids, while mother corms convert it into tyrosine, boosting the production of isoquinoline alkaloids. This research provides valuable insights into the mechanisms of alkaloid biosynthesis in A. muelleri and can aid in developing nitrogen fertilization strategies and in the extraction and utilization of alkaloids., (© 2024. The Author(s).)

Numerous eukaryotic toxins that accumulate in geophytic plants are valuable in the clinic, yet their biosynthetic pathways have remained elusive. A notable example is the >150 Amaryllidaceae alkaloids (AmAs), including galantamine, an FDA-approved treatment for Alzheimer's disease. We show that while AmAs accumulate to high levels in many daffodil tissues, biosynthesis is localized to nascent, growing tissue at the leaf base. A similar trend is found in the production of steroidal alkaloids (e.g., cyclopamine) in corn lily. This model of active biosynthesis enabled the elucidation of a complete set of biosynthetic genes that can be used to produce AmAs. Taken together, our work sheds light on the developmental and enzymatic logic of diverse alkaloid biosynthesis in daffodils. More broadly, it suggests a paradigm for biosynthesis regulation in monocot geophytes, where plants are protected from herbivory through active charging of newly formed cells with eukaryotic toxins that persist as above-ground tissue develops., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plants defend themselves chemically against herbivory through secondary metabolites and phytohormones. Few studies have investigated how constitutive variation in secondary metabolites contributes to systemic herbivory response. We hypothesized that plants with lower constitutive defenses would induce a stronger phytohormone response to spatially separated herbivory than plants with high constitutive defense. We used growth chamber bioassays to investigate how aboveground herbivory by Colorado potato beetle (Leptinotarsa decemlineata, CPB) and belowground herbivory by northern root-knot nematode (Meloidogyne hapla, RKN) altered phytohormones and glycoalkaloids in roots and shoots of two lines of wild potato (Solanum chacoense). These lines had different constitutive levels of chemical defense, particularly leptine glycoalkaloids, which are only present in aboveground tissues. We also determined how these differences influenced the preference and performance of CPB. The susceptible wild potato line responded to aboveground damage by CPB through induction of jasmonic acid (JA) and OPDA. However, when challenged by both RKN and CPB, the susceptible line retained high levels of JA, but not OPDA. Beetles gained more mass after feeding on the susceptible line compared to the resistant line, but were not affected by nematode presence. Belowground, JA, JA-Isoleucine, and OPDA were higher in the resistant line compared to the susceptible line, and some compounds demonstrated response to local herbivory. In contrast, the susceptible line did not induce phytohormone defenses belowground. These findings allow us to predict that constitutive level of defense may influence the threshold of herbivory that may lead to plant-mediated effects on spatially separated herbivores., (© 2024. The Author(s).)

Tomato (Solanum lycopersicum L.) is rich in nutrients and has been an important target for enhancing the accumulation of various metabolites. Tomato also contains cholesterol-derived molecules, steroidal glycoalkaloids (SGAs), which contribute to pathogen defense but are toxic to humans and considered antinutritional compounds. Previous studies suggest the role of various transcription factors in SGA biosynthesis; however, the role of light and associated regulatory factors has not been studied in tomatoes. Here, we demonstrated that SGA biosynthesis is regulated by light through the ELONGATED HYPOCOTYL 5 homolog, SlHY5, by binding to light-responsive G-boxes present in the promoters of structural and regulatory genes. SlHY5 complemented Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) hy5 mutants at molecular, morphological, and biochemical levels. CRISPR/Cas9-based knockout tomato plants, SlHY5CR, showed downregulation of SGA and phenylpropanoid pathway genes, leading to a significant reduction in SGA (α-tomatine and dehydrotomatine) and flavonol contents, whereas plants overexpressing SlHY5 (SlHY5OX) showed the opposite effect. Enhanced SGA and flavonol levels in SlHY5OX lines provided tolerance against Alternaria solani fungus, while SlHY5CR lines were susceptible to the pathogen. This study advances our understanding of the HY5-dependent light-regulated biosynthesis of SGAs and flavonoids and their role in biotic stress in tomatoes., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Urbanization impacts plant-herbivore interactions, which are crucial for ecosystem functions such as carbon sequestration and nutrient cycling. While some studies have reported reductions in insect herbivory in urban areas (relative to rural or natural forests), this trend is not consistent and the underlying causes for such variation remain unclear. We conducted a continental-scale study on insect herbivory along urbanization gradients for three European tree species: Quercus robur, Tilia cordata, and Fraxinus excelsior, and further investigated their biotic and abiotic correlates to get at mechanisms. To this end, we quantified insect leaf herbivory and foliar secondary metabolites (phenolics, terpenoids, alkaloids) for 176 trees across eight European cities. Additionally, we collected data on microclimate (air temperature) and soil characteristics (pH, carbon, nutrients) to test for abiotic correlates of urbanization effects directly or indirectly (through changes in plant secondary chemistry) linked to herbivory. Our results showed that urbanization was negatively associated with herbivory for Q. robur and F. excelsior, but not for T. cordata. In addition, urbanization was positively associated with secondary metabolite concentrations, but only for Q. robur. Urbanization was positively associated with air temperature for Q. robur and F. excelsior, and negatively with soil nutrients (magnesium) in the case of F. excelsior, but these abiotic variables were not associated with herbivory. Contrary to expectations, we found no evidence for indirect effects of abiotic factors via plant defences on herbivory for either Q. robur or F. excelsior. Additional biotic or abiotic drivers must therefore be accounted for to explain observed urbanization gradients in herbivory and their interspecific variation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that possess diverse pharmaceutical properties and are exclusively accumulated in specific plant genera. The Pictet-Spengler condensation, catalyzed by norcoclaurine synthase (NCS), represents a key enzymatic reaction in the biosynthetic pathway of BIAs. While NCS genes have been identified in several plant families such as Papaveraceae, Berberidaceae, and Ranunculaceae, no NCS genes have been reported in Menispermaceae, which is another genus known to accumulate BIAs. Here, NCSs were isolated and functionally characterized from the Menispermaceae family plant Stephania tetrandra. In vitro enzyme assay identified two functional StNCSs which could catalyze the formation of (S)-norcoclaurine. These functionally characterized genes were then integrated into engineered yeast to enable the production of norcoclaurine. Phylogenetic analysis of the NCS enzymes revealed that the StNCSs predominantly clustered into two clades. The functional StNCSs clustered with known NCSs, highlighting the presence of a specific NCS catalytic domain. This study not only provides additional genetic components for the synthetic biology-based production of BIAs in yeast but also contributes to the understanding of the phylogenetic relationships and structure-function relationship of NCS genes involved in the origin and production of BIAs., (© 2023 Wiley Periodicals LLC.)

Common flue-cured tobacco (Nicotiana tabacum L.) primarily accumulates nicotine, and its flue-cured leaves exhibit a lemon appearance. In contrast, a spontaneous cherry-red variant (CR60) primarily accumulates nornicotine, accompanied by distinctive red dapples on the cured leaves. In this study, suppression of conversion of nicotine to nornicotine by genome editing resulted in decreased nornicotine and N-acyl nornicotines (NacNNs), and the subsequent disappearance of red dapples in CR60. Conversely, overexpression of CYP82E4 increased nornicotine and NacNNs accumulation, inducing a red dapple phenotype in common tobacco. Notably, nicotine conversion triggered significant alterations in leaf total sugars, alkaloids, and nitrogens. Metabolome analyses using 1352 identified compounds indicated nicotine conversion dramatically affected the entire metabolic network and induced unique metabolic responses across diverse genetic backgrounds. Further WGCNA analysis revealed that nicotine conversion caused substantial contents variation of alkaloids, flavonoids and amino acids and derivatives in cured leaves. Overall, this research provides valuable insights into the mechanisms underlying red dapple formation in cherry-red tobacco, elucidating profound influence of nicotine conversion on entire metabolic network., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Millipedes have long been known to produce a diverse array of chemical defense agents that deter predation. These compounds, or their precursors, are stored in high concentration within glands (ozadenes) and are released upon disturbance. The subterclass Colobognatha contains four orders of millipedes, all of which are known to produce terpenoid alkaloids-spare the Siphonophorida that produce terpenes. Although these compounds represent some of the most structurally-intriguing millipede-derived natural products, they are the least studied class of millipede defensive secretions. Here, we describe the chemistry of millipede defensive secretions from three species of Brachycybe: Brachycybe producta, Brachycybe petasata, and Brachycybe rosea. Chemical investigations using mass spectrometry-based metabolomics, chemical synthesis, and 2D NMR led to the identification of five alkaloids, three of which are new to the literature. All identified compounds are monoterpene alkaloids with the new compounds representing indolizidine (i.e. hydrogosodesmine) and quinolizidine alkaloids (i.e. homogosodesmine and homo-hydrogosodesmine). The chemical diversity of these compounds tracks the known species phylogeny of this genus, rather than the geographical proximity of the species. The indolizidines and quinolizidines are produced by non-sympatric sister species, B. producta and B. petasata, while deoxybuzonamine is produced by another set of non-sympatric sister species, B. rosea and Brachycybe lecontii. The fidelity between the chemical diversity and phylogeny strongly suggests that millipedes generate these complex defensive agents de novo and begins to provide insights into the evolution of their biochemical pathways., (© 2024. The Author(s).)

Maize originated in southern Mexico and various hybrid varieties have been bred during domestication. All maize tissues are rich in specialized plant metabolites (SPMs), which allow the plants to resist the stresses of herbivores and pathogens or environmental factors. To date, a total of 95 terpenoids, 91 phenolics, 31 alkaloids, and 6 other types of compounds have been identified from maize. Certain volatile sesquiterpenes released by maize plants attract the natural enemies of maize herbivores and provide an indirect defensive function. Kauralexins and dolabralexins are the most abundant diterpenoids in maize and are known to regulate and stabilize the maize rhizosphere microbial community. Benzoxazinoids and benzoxazolinones are the main alkaloids in maize and are found in maize plants at the highest concentrations at the seedling stage. These two kinds of alkaloids directly resist herbivory and pathogenic infection. Phenolics enhance the cross-links between maize cell walls. Meanwhile, SPMs also regulate plant-plant relationships. In conclusion, SPMs in maize show a large diversity of chemical structures and broad-spectrum biological activities. We use these to provide ideas and information to enable the improvement of maize resistances through breeding and to promote the rapid development of the maize industry., (© 2023 Wiley Periodicals LLC.)

As a part of the important species that form coral reef ecosystems, stony corals have become a potential source of pharmacologically active lead compounds for an increasing number of compounds with novel chemical structures and strong biological activity. In this study, the secondary metabolites and biological activities are reported for Aspergillus terreus C21-1, an epiphytic fungus acquired from Porites pukoensis collected from Xuwen Coral Reef Nature Reserve, China. This strain was cultured in potato dextrose broth (PDB) media and rice media with different salinities based on the OSMAC strategy. The mycelial morphology and high-performance thin layer chromatographic (HPTLC) fingerprints of the fermentation extracts together with bioautography were recorded. Furthermore, an untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA), and feature-based molecular networking (FBMN) to analyze their secondary metabolite variations. The comprehensive results revealed that the metabolite expression in A. terreus C21-1 differed significantly between liquid and solid media. The metabolites produced in liquid medium were more diverse but less numerous compared to those in solid medium. Meanwhile, the mycelial morphology underwent significant changes with increasing salinity under PDB cultivation conditions, especially in PDB with 10% salinity. Untargeted metabolomics revealed significant differences between PDB with 10% salinity and other media, as well as between liquid and solid media. FBMN analysis indicated that alkaloids, which might be produced under high salt stress, contributed largely to the differences. The biological activities results showed that six groups of crude extracts exhibited acetylcholinesterase (AChE) inhibitory activities, along with 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and antibacterial activities. The results of this study showed that the increase in salinity favored the production of unique alkaloid compounds by A. terreus C21-1.

Trichoderma longibrachiatum UN32 is a well-documented mutant strain known to produce dendrobine-type total alkaloids (DTTAs). It was serendipitously observed that the addition of Co 2+ to the medium resulted in a notable enhancement in DTTAs production in the T. longibrachiatum UN32 strain, accompanied by an upregulating effect on the expression of antioxidase-related genes. Hence, the objective of the present work was to ascertain whether ROS (intracellular levels of hydrogen peroxide) induced by Co 2+ treatment has a beneficial or detrimental impact on DTTAs biosynthesis. A comparison of the intracellular levels of hydrogen peroxide (H 2 O 2 ) and DTTAs treated with CoCl 2 and CH 3 COOH revealed that CoCl 2 was the optimal inducer for investigating the relationship between ROS formation and DTTAs production. This was due to the observation that ROS formation was reduced by approximately 4% and DTTAs production was increased by 12.55% in comparison to the CH 3 COOH treatment. The physiological results revealed that the introduction of Co 2+ resulted in the oxidative damage and activation of the expression of intracellular superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Furthermore, it was confirmed that ROS induced by Co 2+ was beneficial to DTTAs production by adding exogenous ROS scavengers. The inclusion of all ROS scavengers, including vitamin C, tocopherol, melatonin, mannitol, and sesamol, resulted in a reduction in ROS accumulation and a concomitant decrease in DTTAs production. Specifically, the addition of melatonin at a concentration of 0.4 mg/L demonstrated significant effects, resulting in a 32.53% (P < 0.01) decrease in ROS accumulation and a 45.22% (P < 0.01) reduction in DTTAs production. Subsequently, the timelines of accumulation of intracellular H 2 O 2 and DTTAs content indicated that ROS are also crucial for normal fermentation without CoCl 2 addition. Specifically, the proper H 2 O 2 dose for DTTAs accumulation is between 8.82 and 18.86 μmol/g. The present study offers the initial experimental evidence indicating that CoCl 2 enhance DTTAs production during the culture of T. longibrachiatum UN32 via leading an increase in intracellular ROS, which is conductive to DTTAs production and can be inhibited by the ROS scavengers. Our results provide insights into the mechanistic study of DTTAs biosynthesis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

For several decades, products derived from marine natural sources (PMN) have been widely identified for several therapeutic applications due to their rich sources of bioactive sub-stances, unique chemical diversity, biocompatibility and excellent biological activity. For the past 15 years, our research team explored several PMNs, especially fungi fibrinolytic compounds (FGFCs). FGFC is an isoindolone alkaloid derived from marine fungi, also known as staplabin analogs or Stachybotrys microspora triprenyl phenol (SMTP). For instance, our previous studies explored different types of FGFCs such as FGFC 1, 2, 3 and 4 from the marine fungi Stachybotrys longispora FG216 derived metabolites. The derivatives of FGFC are potentially employed in several disease treatments, mainly for stroke, cancer, ischemia, acute kidney injury, inflammation, cerebral infarction, thrombolysis and hemorrhagic activities, etc. Due to the increasing use of FGFCs in pharmaceutical and biomedical applications, it is important to understand the fundamental signaling concept of FGFCs. Hence, for the first time, this review collectively summarizes the background, types, mode of action and biological applications of FGFCs and their current endeavors for future therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jeevithan, Diao, Hu, Elango, Wu, Mate Sanchez de Val, Rajendran, Sundaram and Rajamani Sekar.)

Both Dendrobium nobile and D. officinale are widely used medicinal plants in China and their major medicinal components are alkaloids and polysaccharides, respectively. It is still unclear why these two closely related orchids synthesize and accumulate different chemical components. Here, we investigated the molecular mechanisms underlying polysaccharide and alkaloid biosynthesis in D. nobile and D. officinale through transcriptome and metabolomic analysis at different growth stages. A total of 1267 metabolites were identified in the juvenile and mature stages of the two species. D. nobile accumulated a large number of alkaloids, benzenoids/phenylpropanoids, flavonoids, and terpenoids during the transition from juvenile to mature plants. In contrast, D. officinale accumulated a small number of those metabolites and an absence of flavonoids. The correlation analysis of polysaccharide contents with the differentially expressed genes suggested that the differential expression of GH1, GH3, and GH9 might be related to the difference in polysaccharide contents between the two Dendrobium species. Meanwhile, the difference in the biosynthesis of dendrobine, the main component of alkaloids in D. nobile, was involved in the differential expression of HMGCR, DXR, DXS, ISPH and eight CYP450s. These findings provided new insights into understanding the biosynthetic mechanisms of the main medicinal components in Dendrobium species., (© 2024 Scandinavian Plant Physiology Society. Published by John Wiley & Sons Ltd.)

A recent leading-edge study by Jiang et al. identified two enzymes that are responsible for key reactions in the biosynthesis of baccatin III. The authors successfully reconstructed the baccatin III synthesis pathway with a minimal number of synthetic enzymes in tobacco leaves, laying the foundation for industrial-scale sustainable production of the anticancer drug paclitaxel., Competing Interests: Declaration of interests The authors declare no competing financial interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alkaloids are the main medicinal components in Houttuynia cordata. In this study, two accessions 6# and 7# of H. cordata underwent thorough metabolomic analyses to identify and quantify alkaloid phytometabolites. It turned out that the alkaloid types were largely similar between 6# and 7#, and the identified 81 alkaloids could be divided into nine structural classes. However, the content of alkaloids in the two accessions was quite different. According to transcriptome data, a total of 114 differentially expressed genes related to alkaloid metabolism were screened. The alkaloid synthesis pathway of the two varieties was mainly different in the isoquinoline alkaloid biosynthesis and indole alkaloid biosynthesis; four genes A22110063c&#95;transcript&#95;59323, A22110063c&#95;transcript&#95;60118, A22110063c&#95;transcript&#95;51672 and A22110063c&#95;transcript&#95;48784 were highly expressed in 7#, which could be key candidate genes of alkaloid metabolism and warrant further analysis. These results provide a reference for the medicinal application of H. cordata and breeding alkaloid rich varieties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Saccharomyces cerevisiae is commonly used as a microbial cell factory to produce high-value compounds or bulk chemicals due to its genetic operability and suitable intracellular physiological environment. The current biosynthesis pathway for targeted products is primarily rewired in the cytosolic compartment. However, the related precursors, enzymes, and cofactors are frequently distributed in various subcellular compartments, which may limit targeted compounds biosynthesis. To overcome above mentioned limitations, the biosynthesis pathways are localized in different subcellular organelles for product biosynthesis. Subcellular compartmentalization in the production of targeted compounds offers several advantages, mainly relieving competition for precursors from side pathways, improving biosynthesis efficiency in confined spaces, and alleviating the cytotoxicity of certain hydrophobic products. In recent years, subcellular compartmentalization in targeted compound biosynthesis has received extensive attention and has met satisfactory expectations. In this review, we summarize the recent advances in the compartmentalized biosynthesis of the valuable compounds in S. cerevisiae, including terpenoids, sterols, alkaloids, organic acids, and fatty alcohols, etc. Additionally, we describe the characteristics and suitability of different organelles for specific compounds, based on the optimization of pathway reconstruction, cofactor supplementation, and the synthesis of key precursors (metabolites). Finally, we discuss the current challenges and strategies in the field of compartmentalized biosynthesis through subcellular engineering, which will facilitate the production of the complex valuable compounds and offer potential solutions to improve product specificity and productivity in industrial processes., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

This study investigates the metabolic responses of Auricularia cornea when cultured on de-oiled leaves of Cinnamomum longepaniculatum (DeCL), an underutilized waste product. The metabolic profiles of A. cornea cultured with four different quality ratios of DeCL substrate (0 %, 14 %, 28 % and 42 %) were analyzed by UHPLC-MS/MS-based metabolomics. A total of 516 metabolites were identified and classified into 78 categories, with phenols, alkaloids and flavonoids accounting for 26.7 % of the total. In addition, 32 metabolite biomarkers associated with eight major metabolic pathways were identified. This pioneering research provides valuable insights into the utilization of DeCL, and expands our knowledge of the metabolic dynamics underlying the growth of A. cornea on alternative substrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Seven previously undescribed compounds, including four diketomorpholine alkaloids (1‒4), one indole diketopiperazine alkaloid (9), one chromone (10), and one benzoic acid derivative (13), and nine known compounds (5-8, 11, 12, and 14-16) were isolated from two different fungal sources. Nine of these metabolites (1-9) were obtained from a seagrass-derived Aspergillus alabamensis SYSU-6778, while the others were obtained from a mixed culture of A. alabamensis SYSU-6778 and a co-isolated fungus A. fumigatiaffinis SYSU-6786. The chemical structures of the compounds were deduced via spectroscopic techniques (including HRESIMS, 1D and 2D NMR), chemical reactions, and ECD calculations. It is worth noting that compound 10 was identified as a defensive secondary metabolite of strain SYSU-6786, produced through the induction of compound 8 under co-culture conditions. Compounds 3 and 4 possessed a naturally rare isotryptophan core. Moreover, compounds 1 and 2 exhibited potent inhibitory activities against fish pathogenic bacterium Edwardsiella ictalurid, with minimum inhibitory concentration values of 10.0 μg/mL for both compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. none., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Studies suggest steroidal alkaloids contribute to the health properties of tomato-rich diets. Untargeted studies have detected tomato steroidal alkaloid metabolites in plasma, tissues, and urine, but concentrations remain unknown. Here we utilize UHPLC-MS/MS to characterize 31 steroidal alkaloid metabolites representing 10 unique masses, and a validated UHPLC-MS method to quantify them in blood plasma. In a two-week parallel-arm study, piglets (n = 20) were fed diets containing 10 % tomato powder or a macronutrient-matched control. Concentrations averaged to 107.7 nmol/L plasma, comprising of phase I (66 %) and phase II (4.5 %) metabolites. Primary phase I metabolites were hydroxylated isomers. MS/MS fragments (m/z 253, 271, 289) in conjunction with analysis of diet profile provided higher confidence when identifying hydroxylated metabolites. These results are the first to report quantitative levels of steroidal alkaloid metabolites in plasma, contributing to an understanding of physiologically relevant concentrations. This data is useful for contextualizing research on the health benefits of tomatoes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jessica Cooperstone reports financial support was provided by The Ohio State University. Jessica Cooperstone, David Francis reports financial support was provided by USDA. Jessica Cooperstone, David Francis, Sheila Jacobi, Michael Dzakovich reports financial support was provided by The Ohio State University Ohio Agricultural Research and Development Center. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fibraurea tinctoria Lour. (Menispermaceae) is a medicinal plant used by many local tribes in Indonesia and other countries. This species has pharmacological potential, such as antimalaria, antibacterial, antitumor, antioxidant, anticancer, antidiabetic, anti-inflammatory, and hepato-protector. However, the secretory structures and sites of secondary metabolite accumulation still need to be studied. This study aimed to explore the secretory structures and reveal the accumulation sites of the main classes of secondary metabolites in the root, stem, and leaves of F. tinctoria. Histochemical analysis using various specific reagents was performed on freehand sections of the fresh organs to detect alkaloids, phenolics, terpenoids, and lipophilic compounds. Observations of secretory structures were carried out using light microscopy. The result showed that idioblast was the predominant secretory structure in the plant organs and contained all the chemical groups investigated, while laticifer accumulated alkaloids were found only in the stem. Some secondary metabolites were localized in the xylem, intercellular space, and cuticle layer. Crystals were observed in the root, stem, and leaf of this species. These results might be helpful as guide information for extracting the particular plant parts to obtain the desired secondary metabolites. It suggested that leaves are a potential alternative source of medicinal raw material of this species, in addition to the stem. RESEARCH HIGHLIGHTS: The first study on secretory structures of Fibraurea tinctoria. Secondary metabolites in F. tinctoria organs accumulate in specialized and unspecialized structures. The idioblast is the primary secretory structure in the organs of F. tinctoria spreading over the root, stem, and leaves., (© 2024 Wiley Periodicals LLC.)

Background: In the kynurenine pathway, it is reported that the essential amino acid tryptophan forms nicotinic acid (NA, vitamin B 3 ) in biological systems. This pathway is part of the de novo pathway to perform nicotinamide adenine dinucleotide (NAD + ) biosynthesis. Additionally, biosynthesis of NAD + via the Preiss-Handler pathway involves NA and its analogue nicotinamide, both designated as niacin. Previous attempts were successful in converting myosmine (MYO) by organic synthesis to NA, and the assumption was that the alkaloid MYO, which is taken in from food, can be converted into NA by biological oxidation., Result: Incubation of HepG2 cells with MYO yielded NA. Moreover, a significant increase of NAD + compared with the control has been found., Conclusion: Hence, MYO could be assumed to be the hitherto unknown origin of an alternative NA biosynthesis additionally influencing NAD + biosynthesis positively. This novel MYO pathway may open new perspectives to improve knowledge and relevance of NA and NAD + biosynthesis and bioactivation in cells and, moreover, in food staples, food, and diet. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Lyciasalamandra species, like most amphibians, secrete a wide array of compounds from their granular and mucous skin glands, including the internally synthesized samandarine alkaloids, making their skin a complex organ performing a variety of functions. Lyciasalamandra helverseni and L. luschani basoglui are insular endemics of the Dodecanese islands of SE Greece, bearing distinct isolated populations, with well-documented phylogenetic profiles. Here, we employ a metabolomics approach, utilizing UPLC-ESI-HRMS/MS data of the skin secretions sampled from a number of specimens found in the islands of Karpathos, Kasos and Kastellorizo, in an effort to reveal aspects of their chemistry and diversity across populations. The results indicated statistically significant variation between all taxa examined, based on various secreted compounds. The underlying factors of variation highlighted by the multivariate analysis were differences in samandarine and other alkaloid content as well as in animal size. Metabolite annotation, based on dereplication tools and most importantly HRMS and HRMS/MS spectra, yielded a number of known samandarine alkaloids, reported for the first time in the currently studied Lyciasalamandra species. We also present documentation for novel members of the samandarine alkaloid family, as well as preliminary evidence for a possible dietary alkaloid sequestration. This work can set the basis for further research of this often-neglected endemic species of the Salamandridae, as well as the structural investigation of the samandarine alkaloid group., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Eleftherakos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Morus alba L. is a tradition medical and edible plant. It is rich in many important bioactive components. However, there is a dearth of systematic information about the components. Here, the Mori Cortex, Mori Folium, Mori Fructus, and Mori Ramulus were studied. Ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) is used to study primary and secondary metabolites. Eight hundred two metabolites were identified and classified into 10 different categories in total. Correlation analysis, hierarchical clustering analysis, and principal component analysis of metabolites showed that different parts of the sample could be significantly different. In different medicinal parts, alkaloids accounted for 4.0%, 3.6%, 5.1%, and 4.5%; flavonoids accounted for 0.7%, 27.2%, 5.6%, 1.2%; terpenes accounted for 20.1%, 2.1%, 2.6%, 2.5%. Furthermore, the abundance of phenols, phenylpropanoids, and lipids metabolites sequentially accounted for 2.3-4.4%, 0.5-1.8%, and 2.4-5.3%. These results have improved our understanding of metabolites and provided a reference for research on the medicinal and edible value of Morus alba L. In addition, the study reveals the correlation between the components of Traditional Chinese medicine and the basic theory of TCM properties and reinterprets the ancient wisdom in the world's traditional herbs through the perspective of modern science.

Isopentenyl diphosphate isomerase (IDI), a key enzyme in the biosynthetic pathway of diterpenoid alkaloids (DAs), plays an essential regulatory role in the synthesis and accumulation of DAs. In this study, the coding sequence (CDS) of AcIDI1 was isolated from the mother roots of Aconitum carmichaelii Debx. (GeneBank accession number OR915879). Bioinformatics analysis showed that the CDS of AcIDI1 was 894 bp, encoding a protein with 297 amino acids and the putative protein localized in the chloroplast. AcIDI1 exhibited significant homology with sequences encoding IDI in other species, and was most closely related to Aconitum vilmorinianum. Furthermore, the fusion protein has been successfully expressed in Escherichia coli (E. coli), providing a basis for future functional studies of AcIDI1. The expression pattern of AcIDI1 was analyzed by real-time quantitative PCR (qPCR), which demonstrates that AcIDI1 is a tissue-specific gene in the roots of A. carmichaelii and exhibits high expression in both daughter and mother roots. By comparing the expression levels of AcIDI1 in three tissues of the roots of A. carmichaelii at different growth stages, we propose that the mother roots (MRs) are the centers of resources allocation. The roots of A. carmichaelii continuously absorb the energy from external environment, while resources transfer behavior from MRs to both daughter roots (DRs) and axillary buds (ABs) occurs as the plant grows. This study establishes a foundation for applying the IDI gene to regulate the biosynthesis and accumulation of DAs in A. carmichaelii., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Solanum xanthocarpum fruits are used in the treatment of cough, fever, and heart disorders. It possesses antipyretic, hypotensive, antiasthmatic, aphrodisiac and antianaphylactic properties. In the present study, 24 elicitors (both biotic and abiotic) were used to enhance the production of glycoalkaloids in cell cultures of S. xanthocarpum. Four concentrations of elicitors were added into the MS culture medium. The maximum accumulation (5.56-fold higher than control) of demissidine was induced by sodium nitroprusside at 50 mM concentration whereas the highest growth of cell biomass (4.51-fold higher than control) stimulated by systemin at 30 mM concentration. A total of 17 genes of biosynthetic pathways of glycoalkaloids were characterized from the cells of S. xanthocarpum. The greater accumulation of demissidine was confirmed with the expression analysis of 11 key biosynthetic pathway enzymes e.g., acetoacetic-CoA thiolase, 3- hydroxy 3-methyl glutaryl synthase, β-hydroxy β-methylglutaryl CoA reductase, mevalonate kinase, farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, squalene-2,3- epoxide cyclase, cycloartenol synthase, UDP-glucose: solanidine glucosyltransferase and UDP-rhamnose: solanidine rhamno-galactosyl transferase. The maximum expression levels of UDP-rhamnose: solanidine rhamno-galactosyl transferase gene was recorded in this study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato, and eggplant. Although it has been known that SGAs play important roles in defense in plants and "anti-nutritional" effects (e.g., toxicity and bitterness) to humans, many of these molecules have documented anti-cancer, anti-microbial, anti-inflammatory, anti-viral, and anti-pyretic activities. Among these, α-solasonine and α-solamargine isolated from black nightshade (Solanum nigrum) are reported to have potent anti-tumor, anti-proliferative, and anti-inflammatory activities. Notably, α-solasonine and α-solamargine, along with the core steroidal aglycone solasodine, are the most widespread SGAs produced among the Solanum plants. However, it is still unknown how plants synthesize these bioactive steroidal molecules. Through comparative metabolomic-transcriptome-guided approach, biosynthetic logic, combinatorial expression in Nicotiana benthamiana, and functional recombinant enzyme assays, here we report the discovery of 12 enzymes from S. nigrum that converts the starting cholesterol precursor to solasodine aglycone, and the downstream α-solasonine, α-solamargine, and malonyl-solamargine SGA products. We further identified six enzymes from cultivated eggplant that catalyze the production of α-solasonine, α-solamargine, and malonyl-solamargine SGAs from solasodine aglycone via glycosylation and atypical malonylation decorations. Our work provides the gene tool box and platform for engineering the production of high-value, steroidal bioactive molecules in heterologous hosts using synthetic biology., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category. In this study, we identified two specific cytochrome P450 enzymes (CYP80G6 and CYP80Q5) with distinct activities toward (S)-configured and (R)-configured substrates from the herbaceous perennial vine Stephania tetrandra, shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories. Additionally, we characterized two CYP719C enzymes (CYP719C3 and CYP719C4) that catalyzed the formation of the methylenedioxy bridge, an essential pharmacophoric group, on the A- and D-rings, respectively, of aporphine alkaloids. Leveraging the functional characterization of these crucial cytochrome P450 enzymes, we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast (Saccharomyces cerevisiae) for the de novo production of compounds such as (R)-glaziovine, (S)-glaziovine, and magnoflorine. This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

A trade-off between growth and defence against biotic stresses is common in plants. Fungal endophytes of the genus Epichloë may relieve this trade-off in their host grasses since they can simultaneously induce plant growth and produce antiherbivore alkaloids that circumvent the need for host defence. The Epichloë ability to decouple the growth-defence trade-off was evaluated by subjecting ryegrass with and without Epichloë endophytes to an exogenous treatment with gibberellin (GA) followed by a challenge with Rhopalosiphum padi aphids. In agreement with the endophyte-mediated trade-off decoupling hypothesis, the GA-derived promotion of plant growth increased the susceptibility to aphids in endophyte-free plants but did not affect the insect resistance in endophyte-symbiotic plants. In line with the unaltered insect resistance, the GA treatment did not reduce the concentration of Epichloë-derived alkaloids. The Epichloë mycelial biomass was transiently increased by the GA treatment but at the expense of hyphal integrity. The response of the phyllosphere bacterial microbiota to both GA treatment and Epichloë was also evaluated. Only Epichloë, and not the GA treatment, altered the composition of the phyllosphere microbiota and the abundance of certain bacterial taxa. Our findings clearly demonstrate that Epichloë does indeed relieve the plant growth-defence trade-off., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Tylophora indica (Burm f.) Merrill, belong to family Asclepiadaceae, is considered to be a natural remedy with high medicinal benefits. The objective of this work is to assess the metabolomic profile of T. indica leaves enriched in alkaloids, as well as to evaluate the in vitro cytotoxicity of these leaves using the MTT assay on human breast MCF-7 and liver HepG2 cancer cell lines. Dried leaves of T. indica were extracted by sonication, using methanol containing 2 % (v/v) of acetic acid and obtained fraction was characterized by HPTLC and UPLC-MS. The UPLC-MS study yielded a preliminary identification of 32 metabolites, with tylophorine, tylophorine B, tylophorinine, and tylophorinidine being the predominant metabolites. The cytotoxicity of the extract of T. indica was evaluated on HepG2 and MCF-7 cell lines, yielding inhibitory concentration (IC 50 ) values of 75.71 μg/mL and 69.60 μg/mL, respectively. Data suggested that the phytochemical screening clearly showed presence of numerous secondary metabolites with moderate cytotoxic efficacy. In conclusion, the future prospects of T. indica appear promising for the advancement of phytopharmaceutical-based anticancer medications, as well as for the design of contemporary pharmaceuticals in the field of cancer chemotherapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Synthetic cathinones are some of the most prevalent new psychoactive substances (NPSs) globally, with alpha-pyrrolidinoisohexanophenone (α-PiHP) being particularly noted for its widespread use in the United States, Europe, and Taiwan. However, the analysis of isomeric NPSs such as α-PiHP and alpha-pyrrolidinohexiophenone (α-PHP) is challenging owing to similarities in their retention times and mass spectra. This study proposes a dual strategy based on in vitro metabolic experiments and machine learning-based classification modelling for differentiating α-PHP and α-PiHP in urine samples: (1) in vitro metabolic experiments using pooled human liver microsomes and liquid chromatography tandem quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were conducted to identify the key metabolites of α-PHP and α-PiHP from the high-resolution MS/MS spectra. After 5 h incubation, 71.4 % of α-PHP and 64.7 % of α-PiHP remained unmetabolised. Nine phase I metabolites were identified for each compound, including primary β-ketone reduction (M1) metabolites. Comparing the metabolites and retention times confirmed the efficacy of in vitro metabolic experiments for differentiating NPS isomers. Subsequently, analysis of seven real urine samples revealed the presence for various metabolites, including M1, that could be used as suitable detection markers at low concentrations. The aliphatic hydroxylation (M2) metabolite peak counts and metabolite retention times were used to determine α-PiHP use. (2) Classification models for the parent compounds and M1 metabolites were developed using principal component analysis for feature extraction and logistic regression for classification. The training and test sets were devised from the spectra of standard samples or supernatants from in vitro metabolism experiments with different incubation times. Both models had classification accuracies of 100 % and accurately identified α-PiHP and its M1 metabolite in seven real urine samples. The proposed methodology effectively distinguished between such isomers and confirmed their presence at low concentrations. Overall, this study introduces a novel concept that addresses the complexities in analysing isomeric NPSs and suggests a path towards enhancing the accuracy and reliability of NPS detection., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Solanine (SOL) and chaconine (CHA) are glycoalkaloids (GAs) produced mainly by Solanum plants. These plant secondary metabolites affect insect metabolism; thus, they have the potential to be applied as natural plant protection products. However, it is not known which GA concentration induces physiological changes in animals. Therefore, the aim of this study was to perform a quantitative analysis of SOL and CHA in the larvae of Tenebrio molitor using LC‒MS to assess how quickly they are eliminated or metabolised. In this experiment, the beetles were injected with 2 μL of 10 -5  M SOL or CHA solution, which corresponds to a dosage range of 0.12-0.14 ng/mg body mass. Then, 0.5, 1.5, 8, and 24 h after GA application, the haemolymph (H), gut (G), and the remainder of the larval body (FB) were isolated. GAs were detected in all samples tested for 24 h, with the highest percentage of the amount applied in the FB, while the highest concentration was measured in the H sample. The SOL and CHA concentrations decreased in the haemolymph over time, while they did not change in other tissues. CHA had the highest elimination rate immediately after injection, while SOL slightly later. None of the GA hydrolysis products were detected in the tested samples. One possible mechanism of the detoxification of GAs may be oxidation and/or sequestration. They may be excreted by Malpighian tubules, with faeces or with cuticles during moulting. The results presented are significant because they facilitate the interpretation of studies related to the effects of toxic substances on insect metabolism., (© 2024. The Author(s).)

Background: Alkaloids, important secondary metabolites produced by plants, play a crucial role in responding to environmental stress. Heuchera micrantha, a well-known plant used in landscaping, has the ability to purify air, and absorb toxic and radioactive substances, showing strong environmental adaptability. However, there is still limited understanding of the accumulation characteristics and metabolic mechanism of alkaloids in H. micrantha., Results: In this study, four distinct varieties of H. micrantha were used to investigate the accumulation and metabolic traits of alkaloids in its leaves. We conducted a combined analysis of the plant's metabolome and transcriptome. Our analysis identified 44 alkaloids metabolites in the leaves of the four H. micrantha varieties, with 26 showing different levels of accumulation among the groups. The HT and JQ varieties exhibited higher accumulation of differential alkaloid metabolites compared to YH and HY. We annotated the differential alkaloid metabolites to 22 metabolic pathways, including several alkaloid metabolism. Transcriptome data revealed 5064 differentially expressed genes involved in these metabolic pathways. Multivariate analysis showed that four key metabolites (N-hydroxytryptamine, L-tyramine, tryptamine, and 2-phenylethylamine) and three candidate genes (Cluster-15488.116815, Cluster-15488.146268, and Cluster-15488.173297) that merit further investigation., Conclusions: This study provided preliminarily insight into the molecular mechanism of the biosynthesis of alkaloids in H. micrantha. However, further analysis is required to elucidate the specific regulatory mechanisms of the candidate gene involved in the synthesis of key alkaloid metabolites. In summary, our findings provide important information about how alkaloid metabolites build up and the metabolic pathways involved in H. micrantha varieties. This gives us a good starting point for future research on the regulation mechanism, and development, and utilization of alkaloids in H. micrantha., (© 2024. The Author(s).)

Ziziphi Spinosae Semen (ZSS) is the first choice for the treatment of insomnia. This research aimed to reveal the spatial distribution of identifying quality markers of ZSS and to illustrate the metabolite quality characteristics of this herbal medicine. Here, we performed a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in situ to detect and image 33 metabolites in ZSS, including three saponins, six flavonoids, four alkaloids, eight fatty acids, and 12 amino acids. The MALDI images of the metabolites clearly showed the heterogeneous spatial distribution in different regions of ZSS tissues, such as the cotyledon, endosperm, and radicle. The distribution area of two saponins, six flavonoids, and three alkaloids increased significantly after the fried processing of ZSS. Based on the ion images, samples with different processing technologies were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 23 major influencing components exerting higher ion intensities were identified as the potential quality markers of ZSS. Results obtained in the current research demonstrate that the processing of ZSS changes its content and distribution of the medicinal components. The analysis of MALDI-MSI provides a novel MS-based molecular imaging approach to investigate and monitor traditional medicinal plants., (© 2024. The Author(s).)

Daphniphyllum macropodum produces alkaloids that are structurally complex with polycyclic, stereochemically rich carbon skeletons. Understanding how these compounds are formed by the plant may enable exploration of their biological function and bioactivities. We employed multiple metabolomics techniques, including a workflow to annotate compounds in the absence of standards, to compare alkaloid content across plants and tissues. Different alkaloid structural types were found to have distinct distributions between genotypes, between tissues and within tissues. Alkaloid structural types also showed different isotope labelling enrichments that matched their biosynthetic relationships. The work suggests that mevalonate derived 30-carbon alkaloids are formed in the phloem region before their conversion to 22-carbon alkaloids which accumulate in the epidermis. This sets the stage for further investigation into the biosynthetic pathway., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Variations in cultivars and cultivation altitudes have significant impacts on tea flavour compounds however lack of comprehensive understanding. This study provided insights into differential accumulation of crucial flavour compounds in response to cultivars, cultivation altitudes, and processing. Twelve flavonoids (262.4 ∼ 275.4 mg•g -1 ) and 20 amino acids (AAs) (56.5 ∼ 64.8 mg•g -1 ) were comparative analyzed in 'Longjing 43' and 'Qunti' fresh leaves harvested at low (80 m, LA) and high (500 m, HA) altitudes. Additionally, an in-depth correlation unravelling of 31 alkaloids, 25 fatty acids, 31 saccharides, 8 organic acids, and 7 vitamins and flavonoids/AAs during green tea (GT) and black tea (BT) processing was performed. Enhenced flavonoid accumulation alongside higher AAs and saccharides in HA GT promoted a sweet/mellow flavour. Abundant flavonoids, AAs, and saccharides derivates in LA BT gave rise to a sweet aftertaste. The study presents an integrated illustration of major flavour compounds' differential accumulation patterns and their interrelations, providing new insights into the influence of cultivation conditions on tea flavour., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpene indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus., Competing Interests: Conflict of interest statement. Authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Anisodus tanguticus (Maxim.) Pascher (A. tanguticus) is a valuable botanical for extracting tropane alkaloids, which are widely used in the pharmaceutical industry. Implementing appropriate cultivation methods can improve both the quality and yield of A. tanguticus. A two-year field experiment was conducted from 2021 to 2023 using a single-factor randomized complete block design replicated three times. The study examined the effects of different nutrient levels (nitrogen: 0, 75, 150, 225, 300, 375 kg/ha; phosphorus: 0, 600, 750, 900, 1050, 1200 kg/ha; potassium: 0, 75, 112.5, 150, 187.5, 225 kg/ha) on the growth, primary alkaloid contents, and alkaloid yield of A. tanguticus at different growth stages (S-Greening, S-Growing, S-Wilting; T-Greening, T-Growing, and T-Wilting) in both the roots and aboveground portions., Results: Our results demonstrate that nutrient levels significantly affect the growth and alkaloid accumulation in A. tanguticus. High nitrogen levels (375 kg/ha) notably increased both root and aboveground biomass, while phosphorus had a minimal effect, especially on aboveground biomass. For alkaloid content (scopolamine, anisodamine, anisodine, atropine), a moderate nitrogen level (225 kg/ha) was most effective, followed by low potassium (75 kg/ha), with phosphorus showing a limited impact. Increased phosphorus levels led to a decrease in scopolamine content. During the T-Growing period, moderate nitrogen addition (225 kg/ha) yielded the highest alkaloid levels per unit area (205.79 kg/ha). In the T-Wilting period, low potassium (75 kg/ha) and low phosphorus (750 kg/ha) resulted in alkaloid levels of 146.91 kg/ha and 142.18 kg/ha, respectively. This indicates nitrogen has the most substantial effect on alkaloid accumulation, followed by potassium and phosphorus. The Douglas production function analysis suggests focusing on root biomass and the accumulation of scopolamine and atropine in roots to maximize alkaloid yield in A. tanguticus cultivation., Conclusions: Our findings show that the optimum harvesting period for A. tanguticus is the T-Wilting period, and that the optimal nitrogen addition is 225 kg/ha, the optimal potassium addition is 75 kg/ha, and the optimal phosphorus addition is 600 kg/ha or less., (© 2024. The Author(s).)

Harmful cyanobacterial blooms have increased globally, releasing hazardous cyanotoxins that threaten the safety of water resources. Constructed wetlands (CWs) are a nature-based and low-cost solution to purify and remove cyanotoxins from water. However, bio-mechanistic understanding of the biotransformation processes expected to drive cyanotoxin removal in such systems is poor, and primarily focused on bacteria. Thus, the present study aimed at exploring the fungal contribution to microcystin-LR and cylindrospermopsin biodegradation in CWs. Based on CW mesocosms, two experimental approaches were taken: a) amplicon sequencing studies were conducted to investigate the involvement of the fungal community; and b) CW fungal isolates were tested for their microcystin-LR and cylindrospermopsin degradation capabilities. The data uncovered effects of seasonality (spring or summer), cyanotoxin exposure, vegetation (unplanted, Juncus effusus or Phragmites australis) and substratum (sand or gravel) on the fungal community structure. Additionally, the arbuscular mycorrhizal fungus Rhizophagus and the endophyte Myrmecridium showed positive correlations with cyanotoxin removal. Fungal isolates revealed microcystin-LR-removal potentials of approximately 25 % in in vitro biodegradation experiments, while the extracellular chemical fingerprint of the cultures suggested a potential intracellular metabolization. The results from this study may help us understand the fungal contribution to cyanotoxin removal, as well as their ecology in CWs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pedro N Carvalho reports financial support was provided by Independent Research Fund Denmark. Pedro N Carvalho, Carlos A Arias reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Achyranthes bidentata (AR) is a traditional Chinese herb used for the treatment of hypertension and cerebral ischemia, but its pharmacological effects are not known., Aim of Study: We aimed to detect and accurately identify the components and metabolites of AR in the plasma and brain tissue of Sprague Dawley rats., Methods: We employed ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HR-MS) to detect AR components in the plasma and brain tissue of rats. The absorption and metabolites in the plasma and brain tissue of normal control rats and rats that underwent middle cerebral artery occlusion (MCAO) were characterized and compared., Results: A total of 281 compounds, including alkaloids, flavonoids, terpenoids, phenylpropanes, sugars and glycosides, steroids, triterpenes, amino acids, and peptides, was identified in samples of Achyranthes bidentata (TCM-AR). Four types of absorbable prototype components and 48 kinds of metabolites were identified in rats in the normal control plasma group which were given AR (AR plasma group), and five kinds of metabolites were identified in rats of the normal control brain tissue group which were given AR (AR brain group). Three absorbed prototype components and 13 metabolites were identified in the plasma of rats which underwent MCAO and were given AR (MCAO + AR plasma group). Six absorbed prototype components and two metabolites were identified in the brain tissue of rats who underwent MCAO and were administered AR (MCAO + AR brain group). These results showed that, after the oral administration of AR, the number of identified components in plasma was more than that in brain tissue. The number of prototype components in the AR plasma group was higher than that in the MCAO + AR plasma group, which may indicate that metabolite absorption in rats undergoing MCAO was worse. The number of prototype components in the MCAO + AR brain group was higher than that in the AR brain group, indicating that the blood-brain barrier was destroyed after MCAO, resulting in more compounds entering brain tissue., Conclusions: UHPLC-HR-MS was used to rapidly analyze the components and metabolites of AR in the blood and brain of rats under normal and pathologic conditions, and to comprehensively characterize the components of TCM-AR. We also analyzed and compared the absorbable components and metabolites of normal rats under cerebral ischemia-reperfusion injury to explore the potential mechanism of action. This method could be applied to various Chinese herbs and disease models, which could promote TCM modernization.

Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing pxaB within the pyrrolizixenamide biosynthetic pathway by braC does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.

Synthetic cathinones represent one of the largest and most abused new psychoactive substance classes, and have been involved in numerous intoxications and fatalities worldwide. Methcathinone analogues like 3-methylmethcathinone (3-MMC), 3-chloromethcathinone (3-CMC), and 4-CMC currently constitute most of synthetic cathinone seizures in Europe. Documenting their consumption in clinical/forensic casework is therefore essential to tackle this trend. Targeting metabolite markers is a go-to to document consumption in analytical toxicology, and metabolite profiling is crucial to support investigations. We sought to identify 3-CMC, 4-CMC, and 4-bromomethcathinone (4-BMC) human metabolites. The substances were incubated with human hepatocytes; incubates were screened by liquid chromatography-high-resolution tandem mass spectrometry and data were mined with Compound Discoverer (Themo Scientific). 3-CMC-positive blood, urine, and oral fluid and 4-CMC-positive urine and saliva from clinical/forensic casework were analyzed. Analyses were supported by metabolite predictions with GLORYx freeware. Twelve, ten, and ten metabolites were identified for 3-CMC, 4-CMC, and 4-BMC, respectively, with similar transformations occurring for the three cathinones. Major reactions included ketoreduction and N-demethylation. Surprisingly, predominant metabolites were produced by combination of N-demethylation and ω-carboxylation (main metabolite in 3-CMC-positive urine), and combination of β-ketoreduction, oxidative deamination, and O-glucuronidation (main metabolite in 4-CMC-positive urine). These latter metabolites were detected in negative-ionization mode only and their non-conjugated form was not detected after glucuronide hydrolysis; this metabolic pathway was never reported for any methcathinone analogue susceptible to undergo the same transformations. These results support the need for comprehensive screening strategies in metabolite identification studies, to avoid overlooking significant metabolites and major markers of consumption., (© 2024. The Author(s).)