RESUMO
In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.
Assuntos
Di-Inos/metabolismo , Ácidos Graxos/biossíntese , Álcoois Graxos/metabolismo , Solanum lycopersicum/genética , Resistência à Doença/genética , Di-Inos/química , Ácidos Graxos/metabolismo , Álcoois Graxos/química , Regulação da Expressão Gênica de Plantas/genética , Metabolômica , Família Multigênica/genética , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse Fisiológico/genéticaRESUMO
Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.
Assuntos
Bacteroides thetaiotaomicron/metabolismo , Carboidratos da Dieta/metabolismo , Glucosinolatos/metabolismo , Intestinos/microbiologia , Animais , Bacteroides thetaiotaomicron/genética , Bacteroides thetaiotaomicron/patogenicidade , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Camundongos , Óperon , SimbioseRESUMO
Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.
Assuntos
Alcaloides , Anidrases Carbônicas , Modelos Neurológicos , Plantas , Animais , Acetilcolinesterase/metabolismo , Alcaloides/biossíntese , Alcaloides/síntese química , Alcaloides/metabolismo , Alcaloides/farmacologia , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Regulação da Expressão Gênica de Plantas , Neurotransmissores/metabolismo , Plantas/enzimologia , Plantas/genética , Plantas/metabolismo , Sesquiterpenos/síntese química , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Lycopodium/química , Lycopodium/metabolismoRESUMO
Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.
Assuntos
Bactérias , Microbioma Gastrointestinal , Linfócitos T , Animais , Camundongos , Antígenos de Superfície/imunologia , Bactérias/classificação , Bactérias/imunologia , Firmicutes/imunologia , Microbioma Gastrointestinal/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Linfócitos T/imunologia , Simbiose/imunologia , Vida Livre de Germes , Receptores de Antígenos de Linfócitos T/imunologia , Hibridomas/citologia , Hibridomas/imunologia , Separação CelularRESUMO
Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.
Assuntos
Vias Biossintéticas , Colchicina/biossíntese , Engenharia Metabólica , Vias Biossintéticas/genética , Colchicaceae/enzimologia , Colchicaceae/genética , Colchicaceae/metabolismo , Colchicina/química , Colchicina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolômica , Fenilalanina/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Transcriptoma , Tirosina/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Systemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in this process are currently unclear. Here, we report that Arabidopsis thaliana UGT76B1 generated glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants. During infection, Arabidopsis ugt76b1 mutants did not accumulate NHP-Glc and accumulated less glycosylated salicylic acid (SA-Glc) than wild-type plants. The metabolic changes in ugt76b1 plants were accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of the SAR molecules NHP and salicylic acid by UGT76B1 plays an important role in modulating defense responses. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato (Solanum lycopersicum) increased NHP-Glc production and reduced NHP accumulation in local tissue and abolished the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Ácidos Pipecólicos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Imunidade Inata/fisiologia , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/fisiologia , Pseudomonas syringae/patogenicidadeRESUMO
Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer's disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.
Assuntos
Alcaloides/biossíntese , Lycopodium/enzimologia , Lycopodium/metabolismo , Regulon/genética , Alcaloides/química , Vias Biossintéticas , Metabolômica , Oxigenases de Função Mista/metabolismo , Oxirredução , Piperidinas/metabolismo , Sesquiterpenos/química , Transcriptoma/genéticaRESUMO
Microfluidic droplet assays enable single-cell polymerase chain reaction (PCR) and sequencing analyses at unprecedented scales, with most methods encapsulating cells within nanoliter-sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) allows sorting droplets with commercially available fluorescence-activated cell sorter (FACS) machines, making it possible to isolate single cells based on phenotypes of interest for downstream analyses. However, sorting DE droplets with standard cytometers requires small droplets that can pass FACS nozzles. This poses challenges for molecular biology, as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, which can be efficiently alleviated through heat lysis. We further show that the magnitude of inhibition depends on the cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most mouse and human cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL DE droplets with fluorescence quantifiable via FACS. These results open up new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet-based single-cell analysis technologies.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Camundongos , Animais , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Emulsões , Reação em Cadeia da Polimerase/métodos , Microfluídica/métodos , Primers do DNARESUMO
Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete momilactone biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in Nicotiana benthamiana, a heterologous plant host. This required us to solve a central problem: diminishing intermediate and product yields remain a bottleneck for multistep diterpene pathways. We increased intermediate and product titers by rerouting diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement in production versus rice). Pure momilactone B isolated from N. benthamiana inhibited germination and root growth in Arabidopsis thaliana, validating allelopathic activity. We demonstrated the broad utility of this approach by applying it to forskolin, a Hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement in production versus chloroplast expression).
Assuntos
Diterpenos/metabolismo , Lactonas/metabolismo , Plantas/metabolismo , Transdução de Sinais/genética , Alcenos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/genética , Colforsina/farmacologia , Citosol/metabolismo , Diterpenos/farmacologia , Ácido Mevalônico/metabolismo , Oryza/genética , Paclitaxel/biossíntese , Folhas de Planta/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Nicotiana/metabolismoRESUMO
The factors that contribute to the composition of the root microbiome and, in turn, affect plant fitness are not well understood. Recent work has highlighted a major contribution of the soil inoculum in determining the composition of the root microbiome. However, plants are known to conditionally exude a diverse array of unique secondary metabolites, that vary among species and environmental conditions and can interact with the surrounding biota. Here, we explore the role of specialized metabolites in dictating which bacteria reside in the rhizosphere. We employed a reduced synthetic community (SynCom) of Arabidopsis thaliana root-isolated bacteria to detect community shifts that occur in the absence of the secreted small-molecule phytoalexins, flavonoids, and coumarins. We find that lack of coumarin biosynthesis in f6'h1 mutant plant lines causes a shift in the root microbial community specifically under iron deficiency. We demonstrate a potential role for iron-mobilizing coumarins in sculpting the A. thaliana root bacterial community by inhibiting the proliferation of a relatively abundant Pseudomonas species via a redox-mediated mechanism. This work establishes a systematic approach enabling elucidation of specific mechanisms by which plant-derived molecules mediate microbial community composition. Our findings expand on the function of conditionally exuded specialized metabolites and suggest avenues to effectively engineer the rhizosphere with the aim of improving crop growth in iron-limited alkaline soils, which make up a third of the world's arable soils.
Assuntos
Arabidopsis/microbiologia , Cumarínicos/metabolismo , Microbiota , Raízes de Plantas/microbiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fotossíntese , Raízes de Plantas/metabolismo , Pseudomonas/metabolismo , RizosferaRESUMO
Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3ß-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3ß-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.
Assuntos
Azadirachta , Citrus sinensis , Sistema Enzimático do Citocromo P-450 , Genoma de Planta , Limoninas , Proteínas de Plantas , Azadirachta/enzimologia , Azadirachta/genética , Citrus sinensis/enzimologia , Citrus sinensis/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Limoninas/biossíntese , Limoninas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Colchicine (1) is a bioactive plant alkaloid from Colchicum and Gloriosa species that is used as a pharmaceutical treatment for inflammatory diseases, including gouty arthritis and familial Mediterranean fever. The activity of this alkaloid is attributed to its ability to bind tubulin dimers and inhibit microtubule assembly, which not only promotes anti-inflammatory effects, but also makes colchicine a potent mitotic poison. The biochemical origins of colchicine biosynthesis have been investigated for over 50 years, but only recently has the underlying enzymatic machinery become clear. Here, we report the discovery of multiple pathway enzymes from Gloriosa superba that allows for the reconstitution of a complete metabolic route to 1. This includes three enzymes that process a previously established tropolone-containing intermediate into 1 via tailoring of the nitrogen atom. We further demonstrate the total biosynthesis of enantiopure (-)-1 from primary metabolites via heterologous production in a model plant, thus enabling future efforts for the metabolic engineering of this medicinal alkaloid. Additionally, our results provide insight into the timing and tissue specificity for the late stage modifications required in colchicine biosynthesis, which are likely connected to the biological functions for this class of medicinal alkaloids in native producing plants.
Assuntos
Colchicina , Colchicina/biossíntese , Colchicina/química , Colchicum/química , Modelos Moleculares , Estrutura MolecularRESUMO
Phenylpropanoids are a class of abundant building blocks found in plants and derived from phenylalanine and tyrosine. Phenylpropanoid polymerization leads to the second most abundant biopolymer lignin while stereo- and site-selective coupling generates an array of lignan natural products with potent biological activity, including the topoisomerase inhibitor and chemotherapeutic etoposide. A key step in etoposide biosynthesis involves a plant dirigent protein that promotes selective dimerization of coniferyl alcohol, a common phenylpropanoid, to form (+)-pinoresinol, a critical C2 symmetric pathway intermediate. Despite the power of this coupling reaction for the elegant and rapid assembly of the etoposide scaffold, dirigent proteins have not been utilized to generate other complex lignan natural products. Here, we demonstrate that dirigent proteins from Podophyllum hexandrum in combination with a laccase guide the heterocoupling of natural and synthetic coniferyl alcohol analogues for the enantioselective synthesis of pinoresinol analogues. This route for complexity generation is remarkably direct and efficient: three new bonds and four stereocenters are produced from two different achiral monomers in a single step. We anticipate our results will enable biocatalytic routes to difficult-to-access non-natural lignan analogues and etoposide derivatives. Furthermore, these dirigent protein and laccase-promoted reactions of coniferyl alcohol analogues represent new regio- and enantioselective oxidative heterocouplings for which no other chemical methods have been reported.
Assuntos
Produtos Biológicos/síntese química , Proteínas/química , Produtos Biológicos/química , Lignanas/química , Oxirredução , Fenóis/química , Proteínas de Plantas/química , Podophyllum/química , EstereoisomerismoRESUMO
Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.
Assuntos
Amônia/metabolismo , Azospirillum brasilense/metabolismo , Glutamato-Amônia Ligase/metabolismo , Setaria (Planta)/microbiologia , Azospirillum brasilense/genética , Azospirillum brasilense/crescimento & desenvolvimento , Feofitinas/metabolismo , Processamento de Proteína Pós-Traducional , Setaria (Planta)/crescimento & desenvolvimento , SimbioseRESUMO
Thousands of putative biosynthetic genes in Arabidopsis thaliana have no known function, which suggests that there are numerous molecules contributing to plant fitness that have not yet been discovered. Prime among these uncharacterized genes are cytochromes P450 upregulated in response to pathogens. Here we start with a single pathogen-induced P450 (ref. 5), CYP82C2, and use a combination of untargeted metabolomics and coexpression analysis to uncover the complete biosynthetic pathway to 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN), a previously unknown Arabidopsis metabolite. This metabolite harbours cyanogenic functionality that is unprecedented in plants and exceedingly rare in nature; furthermore, the aryl cyanohydrin intermediate in the 4-OH-ICN pathway reveals a latent capacity for cyanogenic glucoside biosynthesis in Arabidopsis. By expressing 4-OH-ICN biosynthetic enzymes in Saccharomyces cerevisiae and Nicotiana benthamiana, we reconstitute the complete pathway in vitro and in vivo and validate the functions of its enzymes. Arabidopsis 4-OH-ICN pathway mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with a role in inducible pathogen defence. Arabidopsis has been the pre-eminent model system for studying the role of small molecules in plant innate immunity; our results uncover a new branch of indole metabolism distinct from the canonical camalexin pathway, and support a role for this pathway in the Arabidopsis defence response. These results establish a more complete framework for understanding how the model plant Arabidopsis uses small molecules in pathogen defence.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Indóis/metabolismo , Nitrilas/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/imunologia , Pseudomonas syringae/imunologia , Pseudomonas syringae/patogenicidade , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosídeos/biossíntese , Imunidade Inata/genética , Imunidade Inata/imunologia , Metabolômica , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Saccharomyces cerevisiae/genética , Metabolismo Secundário , Tiazóis/metabolismo , Nicotiana/genética , Transcriptoma , VirulênciaRESUMO
Systemic acquired resistance (SAR) is a global response in plants induced at the site of infection that leads to long-lasting and broad-spectrum disease resistance at distal, uninfected tissues. Despite the importance of this priming mechanism, the identity and complexity of defense signals that are required to initiate SAR signaling is not well understood. In this paper, we describe a metabolite, N-hydroxy-pipecolic acid (N-OH-Pip) and provide evidence that this mobile molecule plays a role in initiating SAR signal transduction in Arabidopsis thaliana We demonstrate that FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of SAR-associated defense priming, can synthesize N-OH-Pip from pipecolic acid in planta, and exogenously applied N-OH-Pip moves systemically in Arabidopsis and can rescue the SAR-deficiency of fmo1 mutants. We also demonstrate that N-OH-Pip treatment causes systemic changes in the expression of pathogenesis-related genes and metabolic pathways throughout the plant and enhances resistance to a bacterial pathogen. This work provides insight into the chemical nature of a signal for SAR and also suggests that the N-OH-Pip pathway is a promising target for metabolic engineering to enhance disease resistance.
Assuntos
Arabidopsis/imunologia , Resistência à Doença/imunologia , Metabolômica , Ácidos Pipecólicos/metabolismo , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Pseudomonas syringae/patogenicidade , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Transdução de SinaisRESUMO
Iron is an essential but poorly bioavailable nutrient because of its low solubility, especially in alkaline soils. Here, we describe the discovery of a previously undescribed redox-active catecholic metabolite, termed sideretin, which derives from the coumarin fraxetin and is the primary molecule exuded by Arabidopsis thaliana roots in response to iron deficiency. We identified two enzymes that complete the biosynthetic pathway of fraxetin and sideretin. Chemical characterization of fraxetin and sideretin, and biological assays with pathway mutants, suggest that these coumarins are critical for iron nutrition in A. thaliana. Further, we show that sideretin production also occurs in eudicot species only distantly related to A. thaliana. Untargeted metabolomics of the root exudates of various eudicots revealed production of structurally diverse redox-active molecules in response to iron deficiency. Our results indicate that secretion of small-molecule reductants by roots may be a widespread and previously underappreciated component of reduction-based iron uptake.
Assuntos
Arabidopsis/metabolismo , Cumarínicos/metabolismo , Deficiências de Ferro , Oxirredução , Raízes de Plantas/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Cinética , Metabolômica , Mutação , Fenótipo , Filogenia , Plantas Geneticamente Modificadas , Rizosfera , Escopoletina/metabolismo , Solubilidade , TermodinâmicaRESUMO
Brassica crop species are prolific producers of indole-sulfur phytoalexins that are thought to have an important role in plant disease resistance. These molecules are conspicuously absent in the model plant Arabidopsis thaliana, and little is known about the enzymatic steps that assemble the key precursor brassinin. Here, we report the minimum set of biosynthetic genes required to generate cruciferous phytoalexins starting from the well-studied glucosinolate pathway. In vitro biochemical characterization revealed an additional role for the previously described carbon-sulfur lyase SUR1 in processing cysteine-isothiocyanate conjugates, as well as the S-methyltransferase DTCMT that methylates the resulting dithiocarbamate, together completing a pathway to brassinin. Additionally, the ß-glucosidase BABG that is present in Brassica rapa but absent in Arabidopsis was shown to act as a myrosinase and may be a determinant of plants that synthesize phytoalexins from indole glucosinolate. Transient expression of the entire pathway in Nicotiana benthamiana yields brassinin, demonstrating that the biosynthesis of indole-sulfur phytoalexins can be engineered into noncruciferous plants. The identification of these biosynthetic enzymes and the heterologous reconstitution of the indole-sulfur phytoalexin pathway sheds light on an important pathway in an edible plant and opens the door to using metabolic engineering to systematically quantify the impact of cruciferous phytoalexins on plant disease resistance and human health.
Assuntos
Brassica rapa/fisiologia , Resistência à Doença/fisiologia , Glucosinolatos/metabolismo , Indóis/metabolismo , Sesquiterpenos/metabolismo , Tiocarbamatos/metabolismo , Arabidopsis/fisiologia , Liases de Carbono-Enxofre/metabolismo , Engenharia Metabólica/métodos , Metilação , Metiltransferases/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/metabolismo , beta-Glucosidase/metabolismo , FitoalexinasRESUMO
Etoposide is a plant-derived drug used clinically to treat several forms of cancer. Recent shortages of etoposide demonstrate the need for a more dependable production method to replace the semisynthetic method currently in place, which relies on extraction of a precursor natural product from Himalayan mayapple. Here we report milligram-scale production of (-)-deoxypodophyllotoxin, a late-stage biosynthetic precursor to the etoposide aglycone, using an engineered biosynthetic pathway in tobacco. Our strategy relies on engineering the supply of coniferyl alcohol, an endogenous tobacco metabolite and monolignol precursor to the etoposide aglycone. We show that transient expression of 16 genes, encoding both coniferyl alcohol and main etoposide aglycone pathway enzymes from mayapple, in tobacco leaves results in the accumulation of up to 4.3 mg/g dry plant weight (-)-deoxypodophyllotoxin, and enables isolation of high-purity (-)-deoxypodophyllotoxin after chromatography at levels up to 0.71 mg/g dry plant weight. Our work reveals that long (>10 step) pathways can be efficiently transferred from difficult-to-cultivate medicinal plants to a tobacco plant production chassis, and demonstrates mg-scale total biosynthesis for access to valuable precursors of the chemotherapeutic etoposide.