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Enhancing the efficiency of photosynthesis represents a promising strategy to improve crop yields, with keeping the steady state of PSII being key to determining the photosynthetic performance. However, the mechanisms whereby the stability of PSII is maintained in oxygenic organisms remain to be explored. Here, we report that the Psb28 protein functions in regulating the homeostasis of PSII under different light conditions in Arabidopsis thaliana. The psb28 mutant is much smaller than the wild-type plants under normal growth light, which is due to its significantly reduced PSII activity. Similar defects were seen under low light and became more pronounced under photoinhibitory light. Notably, the amounts of PSII core complexes and core subunits are specifically decreased in psb28, whereas the abundance of other representative components of photosynthetic complexes remains largely unaltered. Although the PSII activity of psb28 was severely reduced when subjected to high light, its recovery from photoinactivation was not affected. By contrast, the degradation of PSII core protein subunits is dramatically accelerated in the presence of lincomycin. These results indicate that psb28 is defective in the photoprotection of PSII, which is consistent with the observation that the overall NPQ is much lower in psb28 compared to the wild type. Moreover, the Psb28 protein is associated with PSII core complexes and interacts mainly with the CP47 subunit of PSII core. Taken together, these findings reveal an important role for Psb28 in the protection and stabilization of PSII core in response to changes in light environments.
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Proteínas de Arabidopsis , Arabidopsis , Luz , Fotossíntese , Complexo de Proteína do Fotossistema II , Arabidopsis/metabolismo , Arabidopsis/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Lincomicina/farmacologia , MutaçãoRESUMO
Solanaceae (nightshade family) species synthesize a remarkable array of clade- and tissue-specific specialized metabolites. Protective acylsugars, one such class of structurally diverse metabolites, are produced by ACYLSUGAR ACYLTRANSFERASE (ASAT) enzymes from sugars and acyl-coenzyme A esters. Published research has revealed trichome acylsugars composed of glucose and sucrose cores in species across the family. In addition, acylsugars have been analyzed across a small fraction of the >1200 species in the phenotypically megadiverse Solanum genus, with a handful containing inositol and glycosylated inositol cores. The current study sampled several dozen species across subclades of Solanum to get a more detailed view of acylsugar chemodiversity. In depth characterization of acylsugars from the Clade II species brinjal eggplant (Solanum melongena) led to the identification of eight unusual structures with inositol or inositol glycoside cores and hydroxyacyl chains. Liquid chromatography-mass spectrometry analysis of 31 additional species in the Solanum genus revealed striking acylsugar diversity, with some traits restricted to specific clades and species. Acylinositols and inositol-based acyldisaccharides were detected throughout much of the genus. In contrast, acylglucoses and acylsucroses were more restricted in distribution. Analysis of tissue-specific transcriptomes and interspecific acylsugar acetylation differences led to the identification of the brinjal eggplant ASAT 3-LIKE 1 (SmASAT3-L1; SMEL4.1_12g015780) enzyme. This enzyme is distinct from previously characterized acylsugar acetyltransferases, which are in the ASAT4 clade, and appears to be a functionally divergent ASAT3. This study provides a foundation for investigating the evolution and function of diverse Solanum acylsugar structures and harnessing this diversity in breeding and synthetic biology.
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Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. More than 25 acylsugar variants could be produced in a single enzyme assay with four N. acuminata acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites.
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Aciltransferases , Tricomas , Aciltransferases/genética , Aciltransferases/metabolismo , Carboidratos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Açúcares/metabolismo , Biologia Sintética , Nicotiana/genética , Nicotiana/metabolismo , Tricomas/metabolismoRESUMO
Evolutionary dynamics at the population level play a central role in creating the diversity of life on our planet. In this study, we sought to understand the origins of such population-level variation in mating systems and defensive acylsugar chemistry in Solanum habrochaites-a wild tomato species found in diverse Andean habitats in Ecuador and Peru. Using Restriction-site-Associated-DNA-Sequencing (RAD-seq) of 50 S. habrochaites accessions, we identified eight population clusters generated via isolation and hybridization dynamics of 4-6 ancestral populations. Detailed characterization of mating systems of these clusters revealed emergence of multiple self-compatible (SC) groups from progenitor self-incompatible populations in the northern part of the species range. Emergence of these SC groups was also associated with fixation of deleterious alleles inactivating acylsugar acetylation. The Amotape-Huancabamba Zone-a geographical landmark in the Andes with high endemism and isolated microhabitats-was identified as a major driver of differentiation in the northern species range, whereas large geographical distances contributed to population structure and evolution of a novel SC group in the central and southern parts of the range, where the species was also inferred to have originated. Findings presented here highlight the role of the diverse ecogeography of Peru and Ecuador in generating population differentiation, and enhance our understanding of the microevolutionary processes that create biological diversity.
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Fluxo Gênico , Autoincompatibilidade em Angiospermas/genética , Solanum lycopersicum/genética , Solanum/genética , Acetilação , Equador , Solanum lycopersicum/metabolismo , Peru , Filogeografia , Autofertilização , Solanum/metabolismoRESUMO
Correction for 'Fruity, sticky, stinky, spicy, bitter, addictive, and deadly: evolutionary signatures of metabolic complexity in the Solanaceae' by Paul D. Fiesel et al., Nat. Prod. Rep., 2022, 39, 1438-1464, https://doi.org/10.1039/D2NP00003B.
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Covering: 2000-2022Plants collectively synthesize a huge repertoire of metabolites. General metabolites, also referred to as primary metabolites, are conserved across the plant kingdom and are required for processes essential to growth and development. These include amino acids, sugars, lipids, and organic acids. In contrast, specialized metabolites, historically termed secondary metabolites, are structurally diverse, exhibit lineage-specific distribution and provide selective advantage to host species to facilitate reproduction and environmental adaptation. Due to their potent bioactivities, plant specialized metabolites attract considerable attention for use as flavorings, fragrances, pharmaceuticals, and bio-pesticides. The Solanaceae (Nightshade family) consists of approximately 2700 species and includes crops of significant economic, cultural, and scientific importance: these include potato, tomato, pepper, eggplant, tobacco, and petunia. The Solanaceae has emerged as a model family for studying the biochemical evolution of plant specialized metabolism and multiple examples exist of lineage-specific metabolites that influence the senses and physiology of commensal and harmful organisms, including humans. These include, alcohols, phenylpropanoids, and carotenoids that contribute to fruit aroma and color in tomato (fruity), glandular trichome-derived terpenoids and acylsugars that contribute to plant defense (stinky & sticky, respectively), capsaicinoids in chilli-peppers that influence seed dispersal (spicy), and steroidal glycoalkaloids (bitter) from Solanum, nicotine (addictive) from tobacco, as well as tropane alkaloids (deadly) from Deadly Nightshade that deter herbivory. Advances in genomics and metabolomics, coupled with the adoption of comparative phylogenetic approaches, resulted in deeper knowledge of the biosynthesis and evolution of these metabolites. This review highlights recent progress in this area and outlines opportunities for - and challenges of-developing a more comprehensive understanding of Solanaceae metabolism.
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Frutas , Solanum lycopersicum , Herbivoria , Humanos , Solanum lycopersicum/metabolismo , Filogenia , Tricomas/metabolismoRESUMO
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 , Secas , Transcriptoma/genética , Oryza/genética , Zea mays/genética , Diterpenos/metabolismo , Carboidratos , Terpenos/metabolismo , Triterpenos/metabolismo , Flavonoides/metabolismo , Aminoácidos/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The hormone salicylic acid (SA) plays crucial roles in plant defense, stress responses, and in the regulation of plant growth and development. Whereas the biosynthetic pathways and biological functions of SA have been extensively studied, SA catabolism is less well understood. In this study, we report the identification and functional characterization of an FAD/NADH-dependent SA 1-hydroxylase from tomato (Solanum lycopersicum; SlSA1H), which catalyzes the oxidative decarboxylation of SA to catechol. Transcript levels of SlSA1H were highest in stems and its expression was correlated with the formation of the methylated catechol derivatives guaiacol and veratrole. Consistent with a role in SA catabolism, SlSA1H RNAi plants accumulated lower amounts of guaiacol and failed to produce any veratrole. Two O-methyltransferases involved in the conversion of catechol to guaiacol and guaiacol to veratrole were also functionally characterized. Subcellular localization analyses revealed the cytosolic localization of this degradation pathway. Phylogenetic analysis and functional characterization of SA1H homologs from other species indicated that this type of FAD/NADH-dependent SA 1-hydroxylases evolved recently within the Solanaceae family.
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Oxigenases de Função Mista/metabolismo , Ácido Salicílico/metabolismo , Catecóis/metabolismo , Regulação da Expressão Gênica de Plantas , Guaiacol/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Proteína O-Metiltransferase/metabolismoRESUMO
Plant specialized metabolism (SM) enzymes produce lineage-specific metabolites with important ecological, evolutionary, and biotechnological implications. Using Arabidopsis thaliana as a model, we identified distinguishing characteristics of SM and GM (general metabolism, traditionally referred to as primary metabolism) genes through a detailed study of features including duplication pattern, sequence conservation, transcription, protein domain content, and gene network properties. Analysis of multiple sets of benchmark genes revealed that SM genes tend to be tandemly duplicated, coexpressed with their paralogs, narrowly expressed at lower levels, less conserved, and less well connected in gene networks relative to GM genes. Although the values of each of these features significantly differed between SM and GM genes, any single feature was ineffective at predicting SM from GM genes. Using machine learning methods to integrate all features, a prediction model was established with a true positive rate of 87% and a true negative rate of 71%. In addition, 86% of known SM genes not used to create the machine learning model were predicted. We also demonstrated that the model could be further improved when we distinguished between SM, GM, and junction genes responsible for reactions shared by SM and GM pathways, indicating that topological considerations may further improve the SM prediction model. Application of the prediction model led to the identification of 1,220 A. thaliana genes with previously unknown functions, each assigned a confidence measure called an SM score, providing a global estimate of SM gene content in a plant genome.
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Given the potential health benefits (and adverse effects), of polyphenolic and steroidal glycoalkaloids in the diet there is a growing interest in fully elucidating the genetic control of their levels in foodstuffs. Here we carried out profiling of the specialized metabolites in the seeds of the Solanum pennellii introgression lines identifying 338 putative metabolite quantitative trait loci (mQTL) for flavonoids, steroidal glycoalkaloids and further specialized metabolites. Two putative mQTL for flavonols and one for steroidal glycoalkaloids were cross-validated by evaluation of the metabolite content of recombinants harboring smaller introgression in the corresponding QTL interval or by analysis of lines from an independently derived backcross inbred line population. The steroidal glycoalkaloid mQTL was localized to a chromosomal region spanning 14 genes, including a previously defined steroidal glycoalkaloid gene cluster. The flavonoid mQTL was further validated via the use of transient and stable overexpression of the Solyc12g098600 and Solyc12g096870 genes, which encode seed-specific uridine 5'-diphosphate-glycosyltransferases. The results are discussed in the context of our understanding of the accumulation of polyphenols and steroidal glycoalkaloids, and how this knowledge may be incorporated into breeding strategies aimed at improving nutritional aspects of plants as well as in fortifying them against abiotic stress.
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Alcaloides/metabolismo , Flavonóis/metabolismo , Genes de Plantas/genética , Locos de Características Quantitativas/genética , Sementes/metabolismo , Solanum lycopersicum/genética , Mapeamento Cromossômico , Flavonóis/genética , Solanum lycopersicum/metabolismo , Sementes/genéticaRESUMO
Plants make many biologically active, specialized metabolites, which vary in structure, biosynthesis, and the processes they influence. An increasing number of these compounds are documented to protect plants from insects, pathogens, or herbivores or to mediate interactions with beneficial organisms, including pollinators and nitrogen-fixing microbes. Acylsugars, one class of protective compounds, are made in glandular trichomes of plants across the Solanaceae family. While most described acylsugars are acylsucroses, published examples also include acylsugars with hexose cores. The South American fruit crop naranjilla (lulo; Solanum quitoense) produces acylsugars containing a myoinositol core. We identified an enzyme that acetylates triacylinositols, a function homologous to the last step in the acylsucrose biosynthetic pathway of tomato (Solanum lycopersicum). Our analysis reveals parallels between S. lycopersicum acylsucrose and S. quitoense acylinositol biosynthesis, suggesting a common evolutionary origin.
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Vias Biossintéticas , Inositol/biossíntese , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum/genética , Solanum/metabolismo , Tricomas/metabolismo , Acilação , Variação GenéticaRESUMO
The plant pyrethrum (Tanacetum cinerariifolium) synthesizes highly effective natural pesticides known as pyrethrins. Pyrethrins are esters consisting of an irregular monoterpenoid acid and an alcohol derived from jasmonic acid (JA). These alcohols, referred to as rethrolones, can be jasmolone, pyrethrolone, or cinerolone. We recently showed that jasmolone is synthesized from jasmone, a degradation product of JA, in a single hydroxylation step catalyzed by jasmone hydroxylase (TcJMH). TcJMH belongs to the CYP71 clade of the cytochrome P450 oxidoreductase family. Here, we used coexpression analysis, heterologous gene expression, and in vitro biochemical assays to identify the enzyme responsible for conversion of jasmolone to pyrethrolone. A further T cinerariifolium cytochrome P450 family member, CYP82Q3 (designated Pyrethrolone Synthase; TcPYS), appeared to catalyze the direct desaturation of the C1-C2 bond in the pentyl side chain of jasmolone to produce pyrethrolone. TcPYS is highly expressed in the trichomes of the ovaries in pyrethrum flowers, similar to TcJMH and other T cinerariifolium genes involved in JA biosynthesis. Thus, as previously shown for biosynthesis of the monoterpenoid acid moiety of pyrethrins, rethrolones are synthesized in the trichomes. However, the final assembly of pyrethrins occurs in the developing achenes. Our data provide further insight into pyrethrin biosynthesis, which could ultimately be harnessed to produce this natural pesticide in a heterologous system.
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Chrysanthemum cinerariifolium/metabolismo , Piretrinas/metabolismo , Tanacetum/metabolismo , Ciclopentanos/metabolismo , Inseticidas/metabolismo , Oxigenases de Função Mista/metabolismo , Oxilipinas/metabolismoRESUMO
The branched-chain amino acids (BCAAs) Ile, Val, and Leu are essential nutrients that humans and other animals obtain from plants. However, total and relative amounts of plant BCAAs rarely match animal nutritional needs, and improvement requires a better understanding of the mechanistic basis for BCAA homeostasis. We present an in vivo regulatory model of BCAA homeostasis derived from analysis of feedback-resistant Arabidopsis thaliana mutants for the three allosteric committed enzymes in the biosynthetic network: threonine deaminase (also named l-O-methylthreonine resistant 1 [OMR1]), acetohydroxyacid synthase small subunit 2 (AHASS2), and isopropylmalate synthase 1 (IPMS1). In this model, OMR1 exerts primary control on Ile accumulation and functions independently of AHAS and IPMS AHAS and IPMS regulate Val and Leu homeostasis, where AHAS affects total Val+Leu and IPMS controls partitioning between these amino acids. In addition, analysis of feedback-resistant and loss-of-function single and double mutants revealed that each AHAS and IPMS isoenzyme contributes to homeostasis rather than being functionally redundant. The characterized feedback resistance mutations caused increased free BCAA levels in both seedlings and seeds. These results add to our understanding of the basis of in vivo BCAA homeostasis and inform approaches to improve the amount and balance of these essential nutrients in crops.
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Aminoácidos de Cadeia Ramificada/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Aminoácidos de Cadeia Ramificada/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Homeostase , Isoenzimas/genética , Isoenzimas/metabolismo , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologiaRESUMO
Despite our increasingly sophisticated understanding of mechanisms ensuring efficient photosynthesis under laboratory-controlled light conditions, less is known about the regulation of photosynthesis under fluctuating light. This is important because-in nature-photosynthetic organisms experience rapid and extreme changes in sunlight, potentially causing deleterious effects on photosynthetic efficiency and productivity. Here we report that the chloroplast thylakoid lumenal protein MAINTENANCE OF PHOTOSYSTEM II UNDER HIGH LIGHT 2 (MPH2; encoded by At4g02530) is required for growth acclimation of Arabidopsis thaliana plants under controlled photoinhibitory light and fluctuating light environments. Evidence is presented that mph2 mutant light stress susceptibility results from a defect in photosystem II (PSII) repair, and our results are consistent with the hypothesis that MPH2 is involved in disassembling monomeric complexes during regeneration of dimeric functional PSII supercomplexes. Moreover, mph2-and previously characterized PSII repair-defective mutants-exhibited reduced growth under fluctuating light conditions, while PSII photoprotection-impaired mutants did not. These findings suggest that repair is not only required for PSII maintenance under static high-irradiance light conditions but is also a regulatory mechanism facilitating photosynthetic adaptation under fluctuating light environments. This work has implications for improvement of agricultural plant productivity through engineering PSII repair.
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Aclimatação/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Luz , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Tilacoides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Mutação , Complexo de Proteína do Fotossistema II/genética , Tilacoides/genéticaRESUMO
Flowers of Tanacetum cinerariifolium produce a set of compounds known collectively as pyrethrins, which are commercially important pesticides that are strongly toxic to flying insects but not to most vertebrates. A pyrethrin molecule is an ester consisting of either trans-chrysanthemic acid or its modified form, pyrethric acid, and one of three alcohols, jasmolone, pyrethrolone, and cinerolone, that appear to be derived from jasmonic acid. Chrysanthemyl diphosphate synthase (CDS), the first enzyme involved in the synthesis of trans-chrysanthemic acid, was characterized previously and its gene isolated. TcCDS produces free trans-chrysanthemol in addition to trans-chrysanthemyl diphosphate, but the enzymes responsible for the conversion of trans-chrysanthemol to the corresponding aldehyde and then to the acid have not been reported. We used an RNA sequencing-based approach and coexpression correlation analysis to identify several candidate genes encoding putative trans-chrysanthemol and trans-chrysanthemal dehydrogenases. We functionally characterized the proteins encoded by these genes using a combination of in vitro biochemical assays and heterologous expression in planta to demonstrate that TcADH2 encodes an enzyme that oxidizes trans-chrysanthemol to trans-chrysanthemal, while TcALDH1 encodes an enzyme that oxidizes trans-chrysanthemal into trans-chrysanthemic acid. Transient coexpression of TcADH2 and TcALDH1 together with TcCDS in Nicotiana benthamiana leaves results in the production of trans-chrysanthemic acid as well as several other side products. The majority (58%) of trans-chrysanthemic acid was glycosylated or otherwise modified. Overall, these data identify key steps in the biosynthesis of pyrethrins and demonstrate the feasibility of metabolic engineering to produce components of these defense compounds in a heterologous host.
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Vias Biossintéticas/genética , Chrysanthemum cinerariifolium/enzimologia , Chrysanthemum cinerariifolium/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Inseticidas/química , Monoterpenos/metabolismo , Oxirredutases/metabolismo , Piretrinas/química , Chrysanthemum cinerariifolium/genética , Flores/metabolismo , Genes de Plantas , Estudos de Associação Genética , Cinética , Oxirredutases/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Piretrinas/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Terpenos/química , Terpenos/metabolismoRESUMO
Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330--or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)--and Solyc04g012020 (Sl-ASAT2). These enzymes were used--in concert with two previously identified BAHD acyltransferases--to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates. Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.
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Evolução Biológica , Redes e Vias Metabólicas , Solanum lycopersicum/metabolismo , Sacarose/metabolismo , Acil Coenzima A/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/metabolismo , Substituição de Aminoácidos , Aminoácidos/metabolismo , Solanum lycopersicum/enzimologia , Especificidade de Órgãos , Proteínas de Plantas/metabolismo , Polimorfismo Genético , Especificidade por Substrato , Sacarose/química , Tricomas/enzimologiaRESUMO
The pyrethrum plant, Tanacetum cinerariifolium (Asteraceae) synthesizes a class of compounds called pyrethrins that have strong insecticidal properties but are safe to humans. Class I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of three jasmonic-acid derived alcohols. We reconstructed the trans-chrysanthemic acid biosynthetic pathway in tomato fruits, which naturally produce high levels of the tetraterpene pigment lycopene, an isoprenoid which shares a common precursor, dimethylallyl diphosphate (DMAPP), with trans-chrysanthemic acid. trans-Chrysanthemic acid biosynthesis in tomato fruit was achieved by expressing the chrysanthemyl diphosphate synthase gene from T. cinerariifolium, encoding the enzyme that uses DMAPP to make trans-chrysanthemol, under the control of the fruit specific promoter PG, as well as an alcohol dehydrogenease (ADH) gene and aldehyde dehydrogenase (ALDH) gene from a wild tomato species, also under the control of the PG promoter. Tomato fruits expressing all three genes had a concentration of trans-chrysanthemic acid that was about 1.7-fold higher (by weight) than the levels of lycopene present in non-transgenic fruit, while the level of lycopene in the transgenic plants was reduced by 68%. Ninety seven percent of the diverted DMAPP was converted to trans-chrysanthemic acid, but 62% of this acid was further glycosylated. We conclude that the tomato fruit is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.
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Chrysanthemum cinerariifolium/genética , Frutas , Inseticidas/metabolismo , Plantas Geneticamente Modificadas , Piretrinas/metabolismo , Solanum lycopersicum , Frutas/genética , Frutas/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
Glandular trichomes from tomato (Solanum lycopersicum) and other species in the Solanaceae produce and secrete a mixture of O-acylsugars (aliphatic esters of sucrose and glucose) that contribute to insect defense. Despite their phylogenetic distribution and diversity, relatively little is known about how these specialized metabolites are synthesized. Mass spectrometric profiling of acylsugars in the S. lycopersicum x Solanum pennellii introgression lines identified a chromosome 11 locus containing a cluster of BAHD acyltransferases with one gene (named Sl-ASAT3) expressed in tip cells of type I trichomes where acylsugars are made. Sl-ASAT3 was shown to encode an acyl-CoA-dependent acyltransferase that catalyzes the transfer of short (four to five carbons) branched acyl chains to the furanose ring of di-acylsucrose acceptors to produce tri-acylsucroses, which can be further acetylated by Sl-ASAT4 (previously Sl-AT2). Among the wild tomatoes, diversity in furanose ring acyl chains on acylsucroses was most striking in Solanum habrochaites. S. habrochaites accessions from Ecuador and northern Peru produced acylsucroses with short (≤C5) or no acyl chains on the furanose ring. Accessions from central and southern Peru had the ability to add short or long (up to C12) acyl chains to the furanose ring. Multiple ASAT3-like sequences were found in most accessions, and their in vitro activities correlated with observed geographical diversity in acylsugar profiles.
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Aciltransferases/metabolismo , Proteínas de Plantas/metabolismo , Solanum/enzimologia , Aciltransferases/genética , Alelos , Espectrometria de Massas , Proteínas de Plantas/genética , Solanum/genéticaRESUMO
Glandular trichomes of cultivated tomato (Solanum lycopersicum) and many other species throughout the Solanaceae produce and secrete mixtures of sugar esters (acylsugars) on the plant aerial surfaces. In wild and cultivated tomato, these metabolites consist of a sugar backbone, typically glucose or sucrose, and two to five acyl chains esterified to various positions on the sugar core. The aliphatic acyl chains vary in length and branching and are transferred to the sugar by a series of reactions catalyzed by acylsugar acyltransferases. A phenotypic screen of a set of S. lycopersicum M82 × Solanum pennellii LA0716 introgression lines identified a dominant genetic locus on chromosome 5 from the wild relative that affected total acylsugar levels. Genetic mapping revealed that the reduction in acylsugar levels was consistent with the presence and increased expression of two S. pennellii genes (Sopen05g030120 and Sopen05g030130) encoding putative carboxylesterase enzymes of the α/ß-hydrolase superfamily. These two enzymes, named ACYLSUGAR ACYLHYDROLASE1 (ASH1) and ASH2, were shown to remove acyl chains from specific positions of certain types of acylsugars in vitro. A survey of related genes in M82 and LA0716 identified another trichome-expressed ASH gene on chromosome 9 (M82, Solyc09g075710; LA0716, Sopen09g030520) encoding a protein with similar activity. Characterization of the in vitro activities of the SpASH enzymes showed reduced activities with acylsugars produced by LA0716, presumably contributing to the high-level production of acylsugars in the presence of highly expressed SpASH genes.