Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

Lysine decarboxylase converts l ‐lysine to cadaverine as a branching point for the biosynthesis of plant Lys‐derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys‐derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La‐L/ODC) and identified cadaverine‐derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys‐derived alkaloids in the transgenic lines. Moreover, we found several cadaverine‐derived metabolites specifically detected in the transgenic lines compared with the non‐transformed control. Among these, three specific metabolites were identified and confirmed as 5‐aminopentanal, 5‐aminopentanoate and δ‐valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope‐labeled [α‐¹⁵N]‐ or [ε‐¹⁵N]‐l ‐lysine. Our results show similar ¹⁵N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys‐derived alkaloid biosynthesis.     

Codeinone reductase isoforms with differential stability,efficiency and product selectivity in opium poppy

Codeinone reductase (COR) catalyzes the reversible NADPH‐dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3‐O‐demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O‐demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR‐B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1–1.4 and COR‐B) were used to determine sequence–function relationships that influence product selectivity. Biochemical characterization and site‐directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.     

Bioassay‐Guided Isolation of Antifungal Compounds from Disporopsis aspersa (Hua) Engl. ex Diels against Pseudoperonospora cubensis and Phytophthora infestans

Oomycetes are one type of the most highly destructive of the diseases that cause damage to some important crop plants, such as potato late blight, cucumber downy mildew, and grape downy mildew. As main approach of the ongoing search for new botanical fungicide from plant, the secondary metabolites of D. aspersa were investigated. Through efficient bioassay‐guided isolation, two new ( 1 and 2 ) and 12 known compounds ( 3  –  14 ) were isolated, and their structures were determined via extensive NMR, HR‐ESI‐MS, and IR. They were isolated from this genus for the first time except for compounds 11 and 12 . The biological properties of 1  –  14 were evaluated against Pseudoperonospora cubensis and Phytophthora infestans. Compounds 1  –  8 showed potent antifungal activity in vitro. Additionally, compound 3 has preferable control effect on cucumber downy mildew, showing dual effect of protection and treatment in vivo.     

Toxigenic fungi isolated from roquefort cheese

To evaluate the potential for mycotoxin production by fungi contaminating blue-veined cheese, as well as by the ripening fungus,Penicillium roqueforti, the fungal flora of six of local and imported brands was determined. A total of 19 fungi were isolated from the six brands tested. Fourteen of the isolates were toxic to chicken embryos. The toxigenic fungi produced the following mycotoxins:Aspergillus fumigatus, kojic acid;A. versicolor, sterigmatocystin;Penicillium roqueforti, penicillic acid and unidentified toxic metabolites.     

Application of nontargeted metabolite profiling to discover novel markers of quality traits in an advanced population of malting barley

The process of breeding superior varieties for the agricultural industry is lengthy and expensive. Plant metabolites may act as markers of quality traits, potentially expediting the appraisal of experimental lines during breeding. Here, we evaluated the utility of metabolites as markers by assessing metabolic variation influenced by genetic and environmental factors in an advanced breeding setting and in relation to the phenotypic distribution of 20 quality traits. Nontargeted liquid chromatography–mass spectrometry metabolite profiling was performed on barley (Hordeum vulgare L.) grain and malt from 72 advanced malting barley lines grown at two distinct but climatically similar locations, with 2‐row and 6‐row barley as the main genetic factors. 27 420 molecular features were detected, and the metabolite and quality trait profiles were similarly influenced by genotype and environment; however, malt was more influenced by genotype compared with barley. An O2PLS model characterized molecular features and quality traits that covaried, and 1319 features associated with at least one of 20 quality traits. An indiscriminant MS/MS acquisition and novel data analysis method facilitated the identification of metabolites. The analysis described 216 primary and secondary metabolites that correlated with multiple quality traits and included amines, amino acids, alkaloids, polyphenolics and lipids. The mechanisms governing quality trait–metabolite associations were interpreted based on colocalization to genetic markers and their gene annotations. The results of this study support the hypothesis that metabolism and quality traits are co‐influenced by relatively narrow genetic and environmental factors and illustrate the utility of grain metabolites as functional markers of quality traits.     

Three New Ergot Alkaloids from the Fruiting Bodies of Xylaria nigripes (Kl.) Sacc.

Three new ergot alkaloids, xylanigripones A – C ( 1  –  3 ) together with three known compounds, agroclavine ( 4 ), 8,9‐didehydro‐10‐hydroxy‐6,8‐dimethylergolin ( 5 ), and (6S)‐agroclavine N‐oxide ( 6 ) were isolated from the fungus Xylaria nigripes (Kl .) Sacc . Their structures were elucidated by comprehensive spectroscopic analyses and high‐resolution mass spectrometry as well as by comparison with the literature. The absolute configuration was determined by Density Functional Theory (DFT) calculation methods. In addition, all of the compounds were evaluated for bioactivity via a cytotoxicity assay, an acetylcholinesterase inhibition assay and a cholesterol ester transfer protein inhibition assay.     

Genetic and evolution analysis of extrafloral nectary in cotton

Extrafloral nectaries are a defence trait that plays important roles in plant–animal interactions. Gossypium species are characterized by cellular grooves in leaf midribs that secret large amounts of nectar. Here, with a panel of 215 G. arboreum accessions, we compared extrafloral nectaries to nectariless accessions to identify a region of Chr12 that showed strong differentiation and overlapped with signals from GWAS of nectaries. Fine mapping of an F₂ population identified GaNEC1, encoding a PB1 domain‐containing protein, as a positive regulator of nectary formation. An InDel, encoding a five amino acid deletion, together with a nonsynonymous substitution, was predicted to cause 3D structural changes in GaNEC1 protein that could confer the nectariless phenotype. mRNA‐Seq analysis showed that JA‐related genes are up‐regulated and cell wall‐related genes are down‐regulated in the nectary. Silencing of GaNEC1 led to a smaller size of foliar nectary phenotype. Metabolomics analysis identified more than 400 metabolites in nectar, including expected saccharides and amino acids. The identification of GaNEC1 helps establish the network regulating nectary formation and nectar secretion, and has implications for understanding the production of secondary metabolites in nectar. Our results will deepen our understanding of plant–mutualism co‐evolution and interactions, and will enable utilization of a plant defence trait in cotton breeding efforts.     

Five New Alkaloids from the Roots of Sophora flavescens

Five new quinolizidine alkaloids, including three sparteine‐type alkaloids ( 1  –  3 ) and two cytisine‐type alkaloids ( 4 and 5 ), along with four known ones, were isolated from the roots of Sophora flavescens. Their structures were determined by extensive spectroscopic techniques including IR, UV, NMR, and HR‐ESI‐MS. All the compounds were evaluated for their antibacterial activities against Staphylococcus aureus and Escherichia coli.     

Lasiolactols A and B Produced by the Grapevine Fungal Pathogen Lasiodiplodia mediterranea

A strain of Lasiodiplodia mediterranea, a fungus associated with grapevine decline in Sicily, produced several metabolites in liquid medium. Two new dimeric γ‐lactols, lasiolactols A and B ( 1 and 2 ), were characterized as (2S*,3S*,4R*,5R*,2′S*,3′S*,4′R*,5′R*)‐ and (2R*,3S*,4R*,5R*,2′R*,3′S*,4′R*,5′R*)‐(5‐(4‐hydroxymethyl‐3,5‐dimethyl‐tetrahydro‐furan‐2‐yloxy)‐2,4‐dimethyl‐tetrahydro‐furan‐3‐yl]‐methanols by IR, 1D‐ and 2D‐NMR, and HR‐ESI‐MS. Other four metabolites were identified as botryosphaeriodiplodin, (5R)‐5‐hydroxylasiodiplodin, (–)‐(1R,2R)‐jasmonic acid, and (–)‐(3S,4R,5R)‐4‐hydroxymethyl‐3,5‐dimethyldihydro‐2‐furanone ( 3  –  6 , resp.). The absolute configuration (R) at hydroxylated secondary C‐atom C(7) was also established for compound 3 . The compounds 1  –  3 , 5, and 6 , tested for their phytotoxic activities to grapevine cv. Inzolia leaves at different concentrations (0.125, 0.25, 0.5, and 1 mg/ml) were phytotoxic and compound 5 showed the highest toxicity. All metabolites did not show in vitro antifungal activity against four plant pathogens.     

Insecticidal and antifeedant effects of two alkaloids from Cynanchum komarovii against larvae of Plutella xylostella L

A bioassay‐guided fractionation of Cynanchum komarovii crude alkaloid extract led to the isolation of two alkaloids. The isolated alkaloids were identified as 7‐demethoxytylophorine (1) and 6‐hydroxyl‐2,3‐dimethoxy phenanthroindolizidine (2) based on the comparison of their spectroscopic characteristics with the literature data. Insecticidal, antifeedant and growth inhibitory effects of these two alkaloids against the 3rd instar larvae of Plutella xylostella L. (Lepidoptera: Plutellidae) were examined. The results showed that alkaloid 1 was more toxic than alkaloid 2 against the 3rd instar larvae of Plutella xylostella L., but both alkaloids were less toxic than the total alkaloid fraction. For antifeedant activity, alkaloid 1 showed AFC₅₀ of 1.82 mg/ml at 24 h after treatment, alkaloid 2 showed 3.89 mg/ml, while total alkaloids showed 1.56 mg/ml. In dipping toxicity test, alkaloids 1 and 2 produced 93.3% and 63.3% mortality at 72 h after treatment, respectively, while total alkaloids produced 96.7% mortality. The LC₅₀ values for alkaloids 1, 2 and the total alkaloids were 3.54, 9.21 and 2.63 mg/ml, respectively. The development of larvae was also inhibited, and the growth inhibition rates at the concentration of 15.00 mg/ml were 92.8%, 78.2% and 98.6% for alkaloids 1, 2 and total alkaloids, respectively, at 72 h after treatment. Compared with antifeedant and dipping effect, the alkaloids 1, 2 and total alkaloid fraction revealed weak feeding toxicity, and their corrected mortality rates at the concentration of 15.00 mg/ml were 60.0%, 40.0% and 63.3% at 7 days after treatment. The LC₅₀ values for alkaloids 1, 2 and total alkaloids were 12.58, 32.37 and 8.88 mg/ml, respectively, at 7 days after treatment.     

Caterpillars on a phytochemical landscape: The case of alfalfa and the Melissa blue butterfly

Modern metabolomic approaches that generate more comprehensive phytochemical profiles than were previously available are providing new opportunities for understanding plant‐animal interactions. Specifically, we can characterize the phytochemical landscape by asking how a larger number of individual compounds affect herbivores and how compounds covary among plants. Here we use the recent colonization of alfalfa (Medicago sativa) by the Melissa blue butterfly (Lycaeides melissa) to investigate the effects of indivdiual compounds and suites of covarying phytochemicals on caterpillar performance. We find that survival, development time, and adult weight are all associated with variation in nutrition and toxicity, including biomolecules associated with plant cell function as well as putative anti‐herbivore action. The plant‐insect interface is complex, with clusters of covarying compounds in many cases encompassing divergent effects on different aspects of caterpillar performance. Individual compounds with the strongest associations are largely specialized metabolites, including alkaloids, phenolic glycosides, and saponins. The saponins are represented in our data by more than 25 individual compounds with beneficial and detrimental effects on L. melissa caterpillars, which highlights the value of metabolomic data as opposed to approaches that rely on total concentrations within broad defensive classes.     

Four New Diterpenoid Alkaloids from the Roots of Aconitum carmichaelii

Aconitum carmichaelii Debeaux is a widely used traditional Chinese medicine and an important source of clinical drugs, of which the parent and lateral roots are known as ‘Chuanwu’ and ‘Fuzi’, respectively. Four new C₁₉‐diterpenoid alkaloids, carmichasines A – D ( 1 – 4 ), were isolated from the roots of Aconitum carmichaelii, together with twelve known compounds ( 5 – 16 ). Their structures were elucidated via spectroscopic analyses, including HR‐ESI‐MS, IR, and NMR. Carmichasine A ( 1 ) is the first natural C₁₉‐diterpenoid alkaloid possessing a cyano group. Most of the diterpenoid alkaloids isolated were C₁₉‐category, which might provide further clues for understanding the chemotaxonomic significance of this plant. The cytotoxicity of the new compounds was also investigated against several human cancer cell lines, including MCF‐7, HCT116, A549, and 786‐0, and none of them showed considerable cytotoxic activity.     

Targeting novel chemical and constitutive primed metabolites against Plectosphaerella cucumerina

Priming is a physiological state for protection of plants against a broad range of pathogens, and is achieved through stimulation of the plant immune system. Various stimuli, such as beneficial microbes and chemical induction, activate defense priming. In the present study, we demonstrate that impairment of the high‐affinity nitrate transporter 2.1 (encoded by NRT2.1) enables Arabidopsis to respond more quickly and strongly to Plectosphaerella cucumerina attack, leading to enhanced resistance. The Arabidopsis thaliana mutant lin1 (affected in NRT2.1) is a priming mutant that displays constitutive resistance to this necrotroph, with no associated developmental or growth costs. Chemically induced priming by β–aminobutyric acid treatment, the constitutive priming mutant ocp3 and the constitutive priming present in the lin1 mutant result in a common metabolic profile within the same plant–pathogen interactions. The defense priming significantly affects sugar metabolism, cell‐wall remodeling and shikimic acid derivatives levels, and results in specific changes in the amino acid profile and three specific branches of Trp metabolism, particularly accumulation of indole acetic acid, indole‐3–carboxaldehyde and camalexin, but not the indolic glucosinolates. Metabolomic analysis facilitated identification of three metabolites in the priming fingerprint: galacturonic acid, indole‐3–carboxylic acid and hypoxanthine. Treatment of plants with the latter two metabolites by soil drenching induced resistance against P. cucumerina, demonstrating that these compounds are key components of defense priming against this necrotrophic fungus. Here we demonstrate that indole‐3–carboxylic acid induces resistance by promoting papillae deposition and H₂O₂ production, and that this is independent of PR1, VSP2 and PDF1.2 priming.     

A first view on the unsuspected intragenus diversity of N‐glycans in Chlorella microalgae

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health‐promoting food supplements. A mass spectrometry‐based survey of N‐glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N‐glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N‐acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan‐type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4‐O‐methyl‐N‐acetylglucosamine), and substitution of the second N‐acetylglucosamine. Analysis of barcode ITS1–5.8S–ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N‐glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella‐like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N‐glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.     

New Alkaloids and α‐Glucosidase Inhibitory Flavonoids from Ficus hispida

Two new pyrrolidine alkaloids, ficushispimines A ( 1 ) and B ( 2 ), a new ω‐(dimethylamino)caprophenone alkaloid, ficushispimine C ( 3 ), and a new indolizidine alkaloid, ficushispidine ( 4 ), together with the known alkaloid 5 and 11 known isoprenylated flavonoids 6  –  16 , were isolated from the twigs of Ficus hispida. Their structures were elucidated by spectroscopic methods. Isoderrone ( 8 ), 3′‐(3‐methylbut‐2‐en‐1‐yl)biochanin A ( 11 ), myrsininone A ( 12 ), ficusin A ( 13 ), and 4′,5,7‐trihydroxy‐6‐[(1R*,6R*)‐3‐methyl‐6‐(1‐methylethenyl)cyclohex‐2‐en‐1‐yl]isoflavone ( 14 ) showed inhibitory effects on α‐glucosidase in vitro.     

Pyranochromones from Dictyoloma vandellianum A. Juss and Their Cytotoxic Evaluation

One new chromone 3,3‐dimethylallylspatheliachromene methyl ether ( 1 ), as well as five known chromones, 6‐(3‐methylbut‐2‐enyl) allopteroxylin methyl ether ( 2 ), 6‐(3‐methylbut‐2‐enyl) allopteroxylin ( 3 ), 3,3‐dimethylallylspatheliachromene ( 4 ), 5‐O‐methylcneorumchromone K ( 5 ) and spatheliabischromene ( 6 ), two alkaloids, 8‐methoxy‐N‐methylflindersine ( 7 ) and 8‐methoxyflindersine ( 8 ), and two limonoids, limonin diosphenol ( 9 ) and rutaevin ( 10 ), were isolated from Dictyoloma vandellianum A. Juss (Rutaceae). Cytotoxic activities towards tumor cell lines B16‐F10, HepG2, K562 and HL60 and non‐tumor cells PBMC were evaluated for compounds 1  –  6 . Compound 1 was the most active showing IC₅₀ values ranging from 6.26 to 14.82 μg/ml in B16‐F10 and K562 cell lines, respectively, and presented IC₅₀ value of 11.65 μg/ml in PBMC cell line.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen‐fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula–Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen‐fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique – matrix‐assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) – to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8–bis(dimethyl‐amino) naphthalene, DMAN] with a conventional matrix 2,5–dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non‐fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.     

Pyrrole Derivatives and Diterpene Alkaloids from the South China Sea Sponge Agelas nakamurai

Two pairs of new non‐brominated racematic pyrrole derivatives, (±)‐nakamurine D ( 1 ) and (±)‐nakamurine E ( 2 ), two new diterpene alkaloids, isoagelasine C ( 16 ) and isoagelasidine B ( 21 ), together with 13 known pyrrole derivatives ((±)‐ 3  –  15 ), five known diterpene alkaloids ( 17  –  20 , 22 ) were isolated from the South China Sea sponge Agelas nakamurai . The racemic mixtures, compounds 1  –  4 , were resolved into four pairs of enantiomers, (+)‐ 1 and (?)‐ 1 , (+)‐ 2 and (?)‐ 2 , (+)‐ 3 and (?)‐ 3 , and (+)‐ 4 and (?)‐ 4 , by chiral HPLC . The structures and absolute configurations were elucidated on the basis of comprehensive spectroscopic analyses, quantum chemical calculations, quantitative measurements of molar rotations, application of van't Hoff 's principle of optical superposition, and comparison with the literature data. The NMR and MS data of compound 3 are reported for the first time, as the structure was listed in SciFinder Scholar with no associated reference. These non‐brominated pyrrole derivatives were found in this species for the first time. Compound 18 showed valuable cytotoxicities against HL ‐60, K562, and HCT ‐116 cell lines with IC ₅₀ values of 12.4, 16.0, and 19.8 μm , respectively. Compounds 16  –  19 , 21 , and 22 showed potent antifungal activities against Candida albicans with MIC values ranging from 0.59 to 4.69 μg/ml. Compounds 16  –  19 exhibited moderate antibacterial activities against Proteusbacillus vulgaris (MIC values ranging from 9.38 to 18.75 μg/ml).