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Diverse networks of specialized metabolites promote plant fitness by mediating beneficial and antagonistic environmental interactions. In maize (Zea mays), constitutive and dynamically formed cocktails of terpenoids, benzoxazinoids, oxylipins, and phenylpropanoids contribute to plant defense and ecological adaptation. Recent research has highlighted the multifunctional nature of many specialized metabolites, serving not only as elaborate chemical defenses that safeguard against biotic and abiotic stress but also as regulators in adaptive developmental processes and microbiome interactions. Great strides have also been made in identifying the modular pathway networks that drive maize chemical diversity. Translating this knowledge into strategies for enhancing stress resilience traits has the potential to address climate-driven yield losses in one of the world's major food, feed, and bioenergy crops.
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Adaptación Fisiológica , Zea mays , Zea mays/microbiología , Zea mays/inmunología , Zea mays/fisiología , Inmunidad de la Planta , Ambiente , Estrés FisiológicoAsunto(s)
Transferasas Alquil y Aril , Zea mays , Zea mays/genética , Terpenos , Proteínas de Plantas/genéticaRESUMEN
Two major groups of specialized metabolites in maize (Zea mays), termed kauralexins and dolabralexins, serve as known or predicted diterpenoid defenses against pathogens, herbivores, and other environmental stressors. To consider the physiological roles of the recently discovered dolabralexin pathway, we examined dolabralexin structural diversity, tissue-specificity, and stress-elicited production in a defined biosynthetic pathway mutant. Metabolomics analyses support a larger number of dolabralexin pathway products than previously known. We identified dolabradienol as a previously undetected pathway metabolite and characterized its enzymatic production. Transcript and metabolite profiling showed that dolabralexin biosynthesis and accumulation predominantly occur in primary roots and show quantitative variation across genetically diverse inbred lines. Generation and analysis of CRISPR-Cas9-derived loss-of-function Kaurene Synthase-Like 4 (Zmksl4) mutants demonstrated dolabralexin production deficiency, thus supporting ZmKSL4 as the diterpene synthase responsible for the conversion of geranylgeranyl pyrophosphate precursors into dolabradiene and downstream pathway products. Zmksl4 mutants further display altered root-to-shoot ratios and root architecture in response to water deficit. Collectively, these results demonstrate dolabralexin biosynthesis via ZmKSL4 as a committed pathway node biochemically separating kauralexin and dolabralexin metabolism, and suggest an interactive role of maize dolabralexins in plant vigor during abiotic stress.
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Diterpenos , Zea mays , Zea mays/metabolismo , Diterpenos/metabolismo , Vías Biosintéticas , Metabolismo de los LípidosRESUMEN
Switchgrass (Panicum virgatum) is a bioenergy model crop valued for its energy efficiency and drought tolerance. The related monocot species rice (Oryza sativa) and maize (Zea mays) deploy species-specific, specialized metabolites as core stress defenses. By contrast, specialized chemical defenses in switchgrass are largely unknown. To investigate specialized metabolic drought responses in switchgrass, we integrated tissue-specific transcriptome and metabolite analyses of the genotypes Alamo and Cave-in-Rock that feature different drought tolerance. The more drought-susceptible Cave-in-Rock featured an earlier onset of transcriptomic changes and significantly more differentially expressed genes in response to drought compared to Alamo. Specialized pathways showed moderate differential expression compared to pronounced transcriptomic alterations in carbohydrate and amino acid metabolism. However, diterpenoid-biosynthetic genes showed drought-inducible expression in Alamo roots, contrasting largely unaltered triterpenoid and phenylpropanoid pathways. Metabolomic analyses identified common and genotype-specific flavonoids and terpenoids. Consistent with transcriptomic alterations, several root diterpenoids showed significant drought-induced accumulation, whereas triterpenoid abundance remained predominantly unchanged. Structural analysis verified select drought-responsive diterpenoids as oxygenated furanoditerpenoids. Drought-dependent transcriptome and metabolite profiles provide the foundation to understand the molecular mechanisms underlying switchgrass drought responses. Accumulation of specialized root diterpenoids and corresponding pathway transcripts supports a role in drought stress tolerance.
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Diterpenos , Oryza , Panicum , Triterpenos , Panicum/metabolismo , Sequías , Transcriptoma/genética , Oryza/genética , Zea mays/genética , Diterpenos/metabolismo , Carbohidratos , Terpenos/metabolismo , Triterpenos/metabolismo , Flavonoides/metabolismo , Aminoácidos/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Flavor is essential to consumer preference of foods and is an increasing focus of plant breeding programs. In fruit crops, identifying genes underlying volatile organic compounds has great promise to accelerate flavor improvement, but polyploidy and heterozygosity in many species have slowed progress. Here we use octoploid cultivated strawberry to demonstrate how genomic heterozygosity, transcriptomic intricacy and fruit metabolomic diversity can be treated as strengths and leveraged to uncover fruit flavor genes and their regulatory elements. Multi-omics datasets were generated including an expression quantitative trait loci map with 196 diverse breeding lines, haplotype-phased genomes of a highly-flavored breeding selection, a genome-wide structural variant map using five haplotypes, and volatile genome-wide association study (GWAS) with > 300 individuals. Overlaying regulatory elements, structural variants and GWAS-linked allele-specific expression of numerous genes to variation in volatile compounds important to flavor. In one example, the functional role of anthranilate synthase alpha subunit 1 in methyl anthranilate biosynthesis was supported via fruit transient gene expression assays. These results demonstrate a framework for flavor gene discovery in fruit crops and a pathway to molecular breeding of cultivars with complex and desirable flavor.
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Fragaria , Compuestos Orgánicos Volátiles , Antranilato Sintasa/metabolismo , Fragaria/genética , Frutas/genética , Estudio de Asociación del Genoma Completo , Fitomejoramiento , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
BACKGROUND: Although the genome for the allotetraploid bioenergy crop switchgrass (Panicum virgatum) has been established, limitations in mutant resources have hampered in planta gene function studies toward crop optimization. Virus-induced gene silencing (VIGS) is a versatile technique for transient genetic studies. Here we report the implementation of foxtail mosaic virus (FoMV)-mediated gene silencing in switchgrass in above- and below-ground tissues and at different developmental stages. RESULTS: The study demonstrated that leaf rub-inoculation is a suitable method for systemic gene silencing in switchgrass. For all three visual marker genes, Magnesium chelatase subunit D (ChlD) and I (ChlI) as well as phytoene desaturase (PDS), phenotypic changes were observed in leaves, albeit at different intensities. Gene silencing efficiency was verified by RT-PCR for all tested genes. Notably, systemic gene silencing was also observed in roots, although silencing efficiency was stronger in leaves (~ 63-94%) as compared to roots (~ 48-78%). Plants at a later developmental stage were moderately less amenable to VIGS than younger plants, but also less perturbed by the viral infection. CONCLUSIONS: Using FoMV-mediated VIGS could be achieved in switchgrass leaves and roots, providing an alternative approach for studying gene functions and physiological traits in this important bioenergy crop.
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Specialized diterpenoid metabolites are important mediators of plant-environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species-specific biosynthetic pathways is required. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25-CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co-expression patterns and the presence of select diterpenoids in switchgrass roots support the occurrence of P450-derived furanoditerpenoids in planta. Integrating molecular dynamics, structural analysis and targeted mutagenesis identified active site determinants that contribute to the distinct catalytic specificities underlying the broad substrate promiscuity of CYP71Z25-CYP71Z29 for native and non-native diterpenoids.
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Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/metabolismo , Diterpenos/metabolismo , Genoma de Planta/genética , Panicum/enzimología , Biocatálisis , Productos Biológicos/química , Productos Biológicos/metabolismo , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/genética , Diterpenos/química , Panicum/química , Panicum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/química , Raíces de Plantas/enzimología , Raíces de Plantas/genéticaRESUMEN
Plants synthesize diverse diterpenoids with numerous functions in organ development and stress resistance. However, the role of diterpenoids in glandular trichome (GT) development and GT-localized biosynthesis in plants remains unknown. Here, the identification of 10 diterpene synthases (diTPSs) revealed the diversity of diterpenoid biosynthesis in Artemisia annua. Protein-protein interactions (PPIs) between AaKSL1 and AaCPS2 in the plastids highlighted their potential functions in modulating metabolic flux to gibberellins (GAs) or ent-isopimara-7,15-diene-derived metabolites (IDMs) through metabolic engineering. A phenotypic analysis of transgenic plants suggested a complex repertoire of diterpenoids in Artemisia annua with important roles in GT formation, artemisinin accumulation and stress resilience. Metabolic engineering of diterpenoids simultaneously increased the artemisinin yield and stress resistance. Transcriptome and metabolic profiling suggested that bioactive GA4 /GA1 promote GT formation. Collectively, these results expand our knowledge of diterpenoids and show the potential of diterpenoids to simultaneously improve both the GT-localized metabolite yield and stress resistance, in planta.
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Artemisia annua , Artemisininas , Artemisia annua/genética , Giberelinas , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , TricomasRESUMEN
Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize (Zea mays) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 (anther ear 2), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.
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Antifúngicos/metabolismo , Diterpenos/metabolismo , Microbiota , Raíces de Plantas/microbiología , Zea mays/metabolismo , Zea mays/microbiología , Bacterias/genética , Bacterias/aislamiento & purificación , RizosferaRESUMEN
Primates, like other mammals, exhibit an annual reproductive pattern that ranges from strictly seasonal breeding to giving birth in all months of the year, but factors mediating this variation are not fully understood. We applied both a categorical description and quantitative measures of the birth peak breadth based on daily observations in zoos to characterise reproductive seasonality in 141 primate species with an average of 941 birth events per species. Absolute day length at the beginning of the mating season in seasonally reproducing species was not correlated between populations from natural habitats and zoos. The mid-point of latitudinal range was a major factor associated with reproductive seasonality, indicating a correlation with photoperiod. Gestation length, annual mean temperature, natural diet and Malagasy origin were other important factors associated with reproductive seasonality. Birth seasons were shorter with increasing latitude of geographical origin, corresponding to the decreasing length of the favourable season. Species with longer gestation periods were less seasonal than species with shorter ones, possibly because shorter gestation periods more easily facilitate the synchronisation of reproductive activity with annual cycles. Habitat conditions with higher mean annual temperature were also linked to less-seasonal reproduction, independently of the latitude effect. Species with a high percentage of leaves in their natural diet were generally non-seasonal, potentially because the availability of mature leaves is comparatively independent of seasons. Malagasy primates were more seasonal in their births than species from other regions. This might be due to the low resting metabolism of Malagasy primates, the comparatively high degree of temporal predictability of Malagasy ecosystems, or historical constraints peculiar to Malagasy primates. Latitudinal range showed a weaker but also significant association with reproductive seasonality. Amongst species with seasonal reproduction in their natural habitats, smaller primate species were more likely than larger species to shift to non-seasonal breeding in captivity. The percentage of species that changed their breeding pattern in zoos was higher in primates (30%) than in previous studies on Carnivora and Ruminantia (13 and 10%, respectively), reflecting a higher concentration of primate species in the tropics. When comparing only species that showed seasonal reproduction in natural habitats at absolute latitudes ≤11.75°, primates did not differ significantly from these two other taxa in the proportion of species that changed to a less-seasonal pattern in zoos. However, in this latitude range, natural populations of primates and Carnivora had a significantly higher proportion of seasonally reproducing species than Ruminantia, suggesting that in spite of their generally more flexible diets, both primates and Carnivora are more exposed to resource fluctuation than ruminants.
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Ecosistema , Reproducción , Animales , Femenino , Fotoperiodo , Embarazo , Primates , Estaciones del AñoRESUMEN
Specialized metabolites constitute key layers of immunity that underlie disease resistance in crops; however, challenges in resolving pathways limit our understanding of the functions and applications of these metabolites. In maize (Zea mays), the inducible accumulation of acidic terpenoids is increasingly considered to be a defence mechanism that contributes to disease resistance. Here, to understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies and targeted mutagenesis. We define ten genes in three zealexin (Zx) gene clusters that encode four sesquiterpene synthases and six cytochrome P450 proteins that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants in which the ability to produce zealexins (ZXs) is blocked exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate promiscuity, creating a biosynthetic hourglass pathway that uses diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes that underlie disease resistance demonstrates a predominant maize defence pathway and informs innovative strategies for transferring chemical immunity between crops.
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Antibacterianos/biosíntesis , Resistencia a la Enfermedad/genética , Inmunidad Innata/genética , Redes y Vías Metabólicas/genética , Zea mays/genética , Resistencia a la Enfermedad/fisiología , Perfilación de la Expresión Génica , Genes de Plantas/genética , Genes de Plantas/fisiología , Metabolómica , Familia de Multigenes/genética , Familia de Multigenes/fisiología , Proteómica , Zea mays/inmunología , Zea mays/metabolismo , Zea mays/microbiologíaRESUMEN
Plants produce a staggering diversity of specialized small molecule metabolites that play vital roles in mediating environmental interactions and stress adaptation. This chemical diversity derives from dynamic biosynthetic pathway networks that are often species-specific and operate under tight spatiotemporal and environmental control. A growing divide between demand and environmental challenges in food and bioenergy crop production has intensified research on these complex metabolite networks and their contribution to crop fitness. High-throughput omics technologies provide access to ever-increasing data resources for investigating plant metabolism. However, the efficiency of using such system-wide data to decode the gene and enzyme functions controlling specialized metabolism has remained limited; due largely to the recalcitrance of many plants to genetic approaches and the lack of 'user-friendly' biochemical tools for studying the diverse enzyme classes involved in specialized metabolism. With emphasis on terpenoid metabolism in the bioenergy crop switchgrass as an example, this review aims to illustrate current advances and challenges in the application of DNA synthesis and synthetic biology tools for accelerating the functional discovery of genes, enzymes and pathways in plant specialized metabolism. These technologies have accelerated knowledge development on the biosynthesis and physiological roles of diverse metabolite networks across many ecologically and economically important plant species and can provide resources for application to precision breeding and natural product metabolic engineering.
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Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent-, (+)- and syn-copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yielded different diterpenoid scaffolds. Additionally, a cytochrome P450 monooxygenase (CYP99A17), which genomically clustered with SiTPS8, catalyzes the C19 hydroxylation of SiTPS8 products to generate the corresponding diterpene alcohols. The presence of syntenic orthologs to about 19% of the S. italica TPSs in related grasses supports a common ancestry of selected pathway branches. Among the identified enzyme products, abietadien-19-ol, syn-pimara-7,15-dien-19-ol and germacrene-d-4-ol were detectable in planta, and gene expression analysis of the biosynthetic TPSs showed distinct and, albeit moderately, inducible expression patterns in response to biotic and abiotic stress. In vitro growth-inhibiting activity of abietadien-19-ol and syn-pimara-7,15-dien-19-ol against Fusarium verticillioides and Fusarium subglutinans may indicate pathogen defensive functions, whereas the low antifungal efficacy of tested sesquiterpenoids supports other bioactivities. Together, these findings expand the known chemical space of monocot terpenoid metabolism to enable further investigations of terpenoid-mediated stress resilience in these agriculturally important species.
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Transferasas Alquil y Aril/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Setaria (Planta)/genética , Genoma de Planta/genética , Familia de Multigenes/genética , Setaria (Planta)/enzimología , Terpenos/metabolismoRESUMEN
Among the myriad specialized metabolites that plants employ to mediate interactions with their environment, diterpenoids form a chemically diverse group with vital biological functions. A few broadly abundant diterpenoids serve as core pathway intermediates in plant general metabolism. The majority of plant diterpenoids, however, function in specialized metabolism as often species-specific chemical defenses against herbivores and microbial diseases, in below-ground allelopathic interactions, as well as abiotic stress responses. Dynamic networks of anti-microbial diterpenoids were first demonstrated in rice (Oryza sativa) over four decades ago, and more recently, unique diterpenoid blends with demonstrated antibiotic bioactivities were also discovered in maize (Zea mays). Enabled by advances in -omics and biochemical approaches, species-specific diterpenoid-diversifying enzymes have been identified in these and other Poaceous species, including wheat (Triticum aestivum) and switchgrass (Panicum virgatum), and are discussed in this article with an emphasis on the critical diterpene synthase and cytochrome P450 monooxygenase families and their products. The continued investigation of the biosynthesis, diversity, and function of terpenoid-mediated crop defenses provides foundational knowledge to enable the development of strategies for improving crop resistance traits in the face of impeding pest, pathogen, and climate pressures impacting global agricultural production.
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Diterpenos , Oryza , Productos Agrícolas , Triticum , Zea maysRESUMEN
The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.
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Sustitución de Aminoácidos , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Enzimas/genética , Oxidorreductasas/genética , Terpenos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vías Biosintéticas , Núcleo Celular/metabolismo , Enzimas/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Oxidorreductasas/metabolismo , Plantas Modificadas Genéticamente , Plastidios/genética , Plastidios/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0176507.].
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Terpenoids comprise tens of thousands of small molecule natural products that are widely distributed across all domains of life. Plants produce by far the largest array of terpenoids with various roles in development and chemical ecology. Driven by selective pressure to adapt to their specific ecological niche, individual species form only a fraction of the myriad plant terpenoids, typically representing unique metabolite blends. Terpene synthase (TPS) enzymes are the gatekeepers in generating terpenoid diversity by catalyzing complex carbocation-driven cyclization, rearrangement, and elimination reactions that enable the transformation of a few acyclic prenyl diphosphate substrates into a vast chemical library of hydrocarbon and, for a few enzymes, oxygenated terpene scaffolds. The seven currently defined clades (a-h) forming the plant TPS family evolved from ancestral triterpene synthase- and prenyl transferase-type enzymes through repeated events of gene duplication and subsequent loss, gain, or fusion of protein domains and further functional diversification. Lineage-specific expansion of these TPS clades led to variable family sizes that may range from a single TPS gene to families of more than 100 members that may further function as part of modular metabolic networks to maximize the number of possible products. Accompanying gene family expansion, the TPS family shows a profound functional plasticity, where minor active site alterations can dramatically impact product outcome, thus enabling the emergence of new functions with minimal investment in evolving new enzymes. This article reviews current knowledge on the functional diversity and molecular evolution of the plant TPS family that underlies the chemical diversity of bioactive terpenoids across the plant kingdom.
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Switchgrass (Panicum virgatum L.), a perennial C4 grass, represents an important species in natural and anthropogenic grasslands of North America. Its resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. However, little is known about the mechanisms of resistance of switchgrass against pathogens and herbivores. Volatile compounds such as terpenes have important activities in plant direct and indirect defense. Here, we show that switchgrass leaves emit blends of monoterpenes and sesquiterpenes upon feeding by the generalist insect herbivore Spodoptera frugiperda (fall armyworm) and in a systemic response to the treatment of roots with defense hormones. Belowground application of methyl jasmonate also induced the release of volatile terpenes from roots. To correlate the emission of terpenes with the expression and activity of their corresponding biosynthetic genes, we identified a gene family of 44 monoterpene and sesquiterpene synthases (mono- and sesqui-TPSs) of the type-a, type-b, type-g, and type-e subfamilies, of which 32 TPSs were found to be functionally active in vitro. The TPS genes are distributed over the K and N subgenomes with clusters occurring on several chromosomes. Synteny analysis revealed syntenic networks for approximately 30-40% of the switchgrass TPS genes in the genomes of Panicum hallii, Setaria italica, and Sorghum bicolor, suggesting shared TPS ancestry in the common progenitor of these grass lineages. Eighteen switchgrass TPS genes were substantially induced upon insect and hormone treatment and the enzymatic products of nine of these genes correlated with compounds of the induced volatile blends. In accordance with the emission of volatiles, TPS gene expression was induced systemically in response to belowground treatment, whereas this response was not observed upon aboveground feeding of S. frugiperda. Our results demonstrate complex above and belowground responses of induced volatile terpene metabolism in switchgrass and provide a framework for more detailed investigations of the function of terpenes in stress resistance in this monocot crop.
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Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.