RESUMEN
N-hydroxy pipecolic acid (NHP) plays an important role in plant immunity. In contrast to its biosynthesis, our current knowledge with respect to the transcriptional regulation of the NHP pathway is limited. This study commences with the engineering of Arabidopsis plants that constitutively produce high NHP levels and display enhanced immunity. Label-free proteomics reveals a NAC-type transcription factor (NAC90) that is strongly induced in these plants. We find that NAC90 is a target gene of SAR DEFICIENT 1 (SARD1) and induced by pathogen, salicylic acid (SA), and NHP. NAC90 knockout mutants exhibit constitutive immune activation, earlier senescence, higher levels of NHP and SA, as well as increased expression of NHP and SA biosynthetic genes. In contrast, NAC90 overexpression lines are compromised in disease resistance and accumulated reduced levels of NHP and SA. NAC90 could interact with NAC61 and NAC36 which are also induced by pathogen, SA, and NHP. We next discover that this protein triad directly represses expression of the NHP and SA biosynthetic genes AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1), FLAVIN MONOOXYGENASE 1 (FMO1), and ISOCHORISMATE SYNTHASE 1 (ICS1). Constitutive immune response in nac90 is abolished once blocking NHP biosynthesis in the fmo1 background, signifying that NAC90 negative regulation of immunity is mediated via NHP biosynthesis. Our findings expand the currently documented NHP regulatory network suggesting a model that together with NHP glycosylation, NAC repressors take part in a 'gas-and-brake' transcriptional mechanism to control NHP production and the plant growth and defense trade-off.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Ácidos Pipecólicos , Inmunidad de la Planta , Ácido Salicílico , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/metabolismo , Ácidos Pipecólicos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Inmunidad de la Planta/genética , Ácido Salicílico/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Plantas Modificadas Genéticamente , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Resistencia a la Enfermedad/genética , Proteómica/métodosRESUMEN
Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.
Asunto(s)
Vías Biosintéticas , Alucinógenos , Mescalina , Alucinógenos/metabolismo , Mescalina/metabolismo , Nicotiana/metabolismo , Nicotiana/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMEN
Arabidopsis (Arabidopsis thaliana) seeds expressing the feedback-insensitive form of cystathionine γ-synthase (AtD-CGS), the key gene of methionine (Met) synthesis, under the control of a seed-specific phaseolin promoter (SSE plants) show a significant increase in Met content. This elevation is accompanied by increased levels of other amino acids (AAs), sugars, total protein, and starch, which are important from a nutritional aspect. Here, we investigated the mechanism behind this phenomenon. Gas chromatography-mass spectrometry (GC-MS) analysis of SSE leaves, siliques, and seeds collected at 3 different developmental stages showed high levels of Met, AAs, and sugars compared to the control plants. A feeding experiment with isotope-labeled AAs showed an increased flux of AAs from nonseed tissues toward the developing seeds of SSE. Transcriptome analysis of leaves and seeds displayed changes in the status of methylation-related genes in SSE plants that were further validated by methylation-sensitive enzymes and colorimetric assay. These results suggest that SSE leaves have higher DNA methylation rates than control plants. This occurrence apparently led to accelerated senescence, together with enhanced monomer synthesis, which further resulted in increased transport of monomers from the leaves toward the seeds. The developing seeds of SSE plants, however, show reduced Met levels and methylation rates. The results provide insights into the role of Met in DNA methylation and gene expression and how Met affects the metabolic profile of the plant.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Metionina/metabolismo , Metilación de ADN/genética , Regulación de la Expresión Génica de las Plantas , Semillas/metabolismo , Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves' glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.
Asunto(s)
Cannabinoides , Cannabis , Humanos , Cannabinoides/metabolismo , Cannabis/genética , Flores/metabolismo , Hojas de la Planta/metabolismoRESUMEN
Solanum steroidal glycoalkaloids (SGAs) are renowned defence metabolites exhibiting spectacular structural diversity. Genes and enzymes generating the SGA precursor pathway, SGA scaffold and glycosylated forms have been largely identified. Yet, the majority of downstream metabolic steps creating the vast repertoire of SGAs remain untapped. Here, we discovered that members of the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (2-ODD) family play a prominent role in SGA metabolism, carrying out three distinct backbone-modifying oxidative steps in addition to the three formerly reported pathway reactions. The GLYCOALKALOID METABOLISM34 (GAME34) enzyme catalyses the conversion of core SGAs to habrochaitosides in wild tomato S. habrochaites. Cultivated tomato plants overexpressing GAME34 ectopically accumulate habrochaitosides. These habrochaitoside enriched plants extracts potently inhibit Puccinia spp. spore germination, a significant Solanaceae crops fungal pathogen. Another 2-ODD enzyme, GAME33, acts as a desaturase (via hydroxylation and E/F ring rearrangement) forming unique, yet unreported SGAs. Conversion of bitter α-tomatine to ripe fruit, nonbitter SGAs (e.g. esculeoside A) requires two hydroxylations; while the known GAME31 2-ODD enzyme catalyses hydroxytomatine formation, we find that GAME40 catalyses the penultimate step in the pathway and generates acetoxy-hydroxytomatine towards esculeosides accumulation. Our results highlight the significant contribution of 2-ODD enzymes to the remarkable structural diversity found in plant steroidal specialized metabolism.
Asunto(s)
Alcaloides , Dioxigenasas , Solanum lycopersicum , Solanum tuberosum , Solanum , Alcaloides/metabolismo , Dioxigenasas/genética , Dioxigenasas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Solanum lycopersicum/genética , Solanum/genética , Solanum/metabolismo , Solanum tuberosum/genéticaRESUMEN
Nitrogen (N) fertilization is one of the main inputs to increase crop yield and food production. However, crops utilize only 30-40% of N applied; the remainder is leached into the soil, causing environmental and health damage. In this scenario, the improvement of nitrogen-use efficiency (NUE) will be an essential strategy for sustainable agriculture. Here, we compared two pairs of NUE-contrasting eggplant (Solanum melongena L.) genotypes, employing GC-MS and UPLC-qTOF-MS-based technologies to determine the differential profiles of primary and secondary metabolites in root and shoot tissues, under N starvation as well as at short- and long-term N-limiting resupply. Firstly, differences in the primary metabolism pathways of shoots related to alanine, aspartate and glutamate; starch, sucrose and glycine; serine and threonine; and in secondary metabolites biosynthesis were detected. An integrated analysis between differentially accumulated metabolites and expressed transcripts highlighted a key role of glycine accumulation and the related glyA transcript in the N-use-efficient genotypes to cope with N-limiting stress. Interestingly, a correlation between both sucrose synthase (SUS)- and fructokinase (scrK)-transcript abundances, as well as D-glucose and D-fructose accumulation, appeared useful to distinguish the N-use-efficient genotypes. Furthermore, increased levels of L-aspartate and L-asparagine in the N-use-efficient genotypes at short-term low-N exposure were detected. Granule-bound starch synthase (WAXY) and endoglucanase (E3.2.1.4) downregulation at long-term N stress was observed. Therefore, genes and metabolites related to these pathways could be exploited to improve NUE in eggplant.
RESUMEN
Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.
Asunto(s)
Alcaloides , Solanum lycopersicum , Alcaloides/metabolismo , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Giberelinas/metabolismo , Solanum lycopersicum/metabolismo , Oxilipinas/metabolismoRESUMEN
Plants are unsurpassed biochemists that synthesize a plethora of molecules in response to an ever-changing environment. The majority of these molecules, considered as specialized metabolites, effectively protect the plant against pathogens and herbivores. However, this defense most probably comes at a great expense, leading to reduction of growth (known as the 'growth-defense trade-off'). Plants employ several strategies to reduce the high metabolic costs associated with chemical defense. Production of specialized metabolites is tightly regulated by a network of transcription factors facilitating its fine-tuning in time and space. Multifunctionality of specialized metabolites-their effective recycling system by re-using carbon, nitrogen, and sulfur, thus re-introducing them back to the primary metabolite pool-allows further cost reduction. Spatial separation of biosynthetic enzymes and their substrates, and sequestration of potentially toxic substances and conversion to less toxic metabolite forms are the plant's solutions to avoid the detrimental effects of metabolites they produce as well as to reduce production costs. Constant fitness pressure from herbivores, pathogens, and abiotic stressors leads to honing of specialized metabolite biosynthesis reactions to be timely, efficient, and metabolically cost-effective. In this review, we assess the costs of production of specialized metabolites for chemical defense and the different plant mechanisms to reduce the cost of such metabolic activity in terms of self-toxicity and growth.
Asunto(s)
Herbivoria , PlantasRESUMEN
Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α-tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 resequenced genomes and genotyping a 650-tomato core collection to identify nine bitter-tasting accessions including the 'high tomatine' Peruvian landraces reported in the literature. These 'bitter' accessions contain a deletion in GORKY, a nitrate/peptide family transporter mediating α-tomatine subcellular localization during fruit ripening. GORKY exports α-tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α-tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a notable innovation in the process of tomato fruit domestication and breeding.
Asunto(s)
Frutas/química , Proteínas de Plantas/genética , Solanum lycopersicum/química , Solanum lycopersicum/genética , Gusto , Frutas/genética , Humanos , Solanum lycopersicum/metabolismo , Fitomejoramiento , Proteínas de Plantas/metabolismoRESUMEN
Nitrogen-use efficiency (NUE) is a complex trait of great interest in breeding programs because through its improvement, high crop yields can be maintained whilst N supply is reduced. In this study, we report a transcriptomic analysis of four NUE-contrasting eggplant (Solanum melongena) genotypes following short- and long-term exposure to low N, to identify key genes related to NUE in the roots and shoots. The differentially expressed genes in the high-NUE genotypes are involved in the light-harvesting complex and receptor, a ferredoxin-NADP reductase, a catalase and WRKY33. These genes were then used as bait for a co-expression gene network analysis in order to identify genes with the same trends in expression. This showed that up-regulation of WRKY33 triggered higher expression of a cluster of 21 genes and also of other genes, many of which were related to N-metabolism, that were able to improve both nitrogen uptake efficiency and nitrogen utilization efficiency, the two components of NUE. We also conducted an independent de novo experiment to validate the significantly higher expression of WRKY33 and its gene cluster in the high-NUE genotypes. Finally, examination of an Arabidopsis transgenic 35S::AtWRKY33 overexpression line showed that it had a bigger root system and was more efficient at taking up N from the soil, confirming the pivotal role of WRKY33 for NUE improvement.
Asunto(s)
Nitrógeno , Solanum melongena , Regulación de la Expresión Génica de las Plantas , Nitrógeno/metabolismo , Fitomejoramiento , Solanum melongena/genética , TranscriptomaRESUMEN
Mango fruit exposed to sunlight develops red skin and are more resistant to biotic and abiotic stresses. Here we show that harvested red mango fruit that was exposed to sunlight at the orchard is more resistant than green fruit to Colletotrichum gloeosporioides. LCMS analysis showed high amounts of antifungal compounds, as glycosylated flavonols, glycosylated anthocyanins, and mangiferin in red vs. green mango skin, correlated with higher antioxidant and lower ROS. However, also the green side of red mango fruit that has low levels of flavonoids was resistant, indicated induced resistance. Transcriptomes of red and green fruit inoculated on their red and green sides with C. gloeosporioides were analyzed. Overall, in red fruit skin, 2,187 genes were upregulated in response to C. gloeosporioides. On the green side of red mango, upregulation of 22 transcription factors and 33 signaling-related transcripts indicated induced resistance. The RNA-Seq analysis suggests that resistance of the whole red fruit involved upregulation of ethylene, brassinosteroid, and phenylpropanoid pathways. To conclude, red fruit resistance to fungal pathogen was related to both flavonoid toxicity and primed resistance of fruit that was exposed to light at the orchard.
RESUMEN
Wild tomato species represent a rich gene pool for numerous desirable traits lost during domestication. Here, we exploited an introgression population representing wild desert-adapted species and a domesticated cultivar to establish the genetic basis of gene expression and chemical variation accompanying the transfer of wild-species-associated fruit traits. Transcriptome and metabolome analysis of 580 lines coupled to pathogen sensitivity assays resulted in the identification of genomic loci associated with levels of hundreds of transcripts and metabolites. These associations occurred in hotspots representing coordinated perturbation of metabolic pathways and ripening-related processes. Here, we identify components of the Solanum alkaloid pathway, as well as genes and metabolites involved in pathogen defense and linking fungal resistance with changes in the fruit ripening regulatory network. Our results outline a framework for understanding metabolism and pathogen resistance during tomato fruit ripening and provide insights into key fruit quality traits.
Asunto(s)
Resistencia a la Enfermedad/genética , Metaboloma/genética , Solanum lycopersicum/genética , Transcriptoma/genética , Alcaloides/genética , Domesticación , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/parasitología , Hongos/genética , Hongos/patogenicidad , Regulación de la Expresión Génica de las Plantas/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/microbiología , Redes y Vías Metabólicas/genética , Fenotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Solanum/genética , Solanum/microbiologíaRESUMEN
Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas/genética , Glicosiltransferasas/genética , Proteínas de Plantas/genética , Saponinas/biosíntesis , Spinacia oleracea/metabolismo , Terpenos/metabolismo , Beta vulgaris/genética , Beta vulgaris/metabolismo , Membrana Celular/metabolismo , Pared Celular/metabolismo , Celulosa/metabolismo , Retículo Endoplásmico/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Glucosiltransferasas/metabolismo , Ácido Glucurónico/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Células Vegetales/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Spinacia oleracea/genéticaRESUMEN
Steroidal glycoalkaloids (SGAs) are defense specialized metabolites produced by thousands of Solanum species. These metabolites are remarkable in structural diversity formed following modifications in their core scaffold. In recent years, it became clear that a large portion of this chemical repertoire was acquired through various molecular mechanisms involving 'hijacking' of core metabolism enzymes. This was typically accompanied by gene duplication and divergence and further neofunctionalization as well as modified subcellular localization and evolution of new substrate preferences. In this review, we highlight recent findings in the SGAs biosynthetic pathway and elaborate on similar occurrences in other chemical classes that enabled evolution of specialized metabolic pathways and its underlying structural diversity.
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Solanum lycopersicum , Duplicación de Gen , Redes y Vías MetabólicasRESUMEN
Microbial communities associated with roots confer specific functions to their hosts, thereby modulating plant growth, health, and productivity. Yet, seminal questions remain largely unaddressed including whether and how the rhizosphere microbiome modulates root metabolism and exudation and, consequently, how plants fine tune this complex belowground web of interactions. Here we show that, through a process termed systemically induced root exudation of metabolites (SIREM), different microbial communities induce specific systemic changes in tomato root exudation. For instance, systemic exudation of acylsugars secondary metabolites is triggered by local colonization of bacteria affiliated with the genus Bacillus Moreover, both leaf and systemic root metabolomes and transcriptomes change according to the rhizosphere microbial community structure. Analysis of the systemic root metabolome points to glycosylated azelaic acid as a potential microbiome-induced signaling molecule that is subsequently exuded as free azelaic acid. Our results demonstrate that rhizosphere microbiome assembly drives the SIREM process at the molecular and chemical levels. It highlights a thus-far unexplored long-distance signaling phenomenon that may regulate soil conditioning.
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Bacterias/metabolismo , Microbiota , Exudados de Plantas/metabolismo , Raíces de Plantas/metabolismo , Microbiología del Suelo , Bacterias/clasificación , Bacterias/genética , Bacterias/crecimiento & desarrollo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Raíces de Plantas/microbiología , Plantas/metabolismo , Plantas/microbiología , Rizosfera , Suelo/químicaRESUMEN
The genus Solanum comprises three food crops (potato, tomato, and eggplant), which are consumed on daily basis worldwide and also producers of notorious anti-nutritional steroidal glycoalkaloids (SGAs). Hydroxylated SGAs (i.e. leptinines) serve as precursors for leptines that act as defenses against Colorado Potato Beetle (Leptinotarsa decemlineata Say), an important pest of potato worldwide. However, SGA hydroxylating enzymes remain unknown. Here, we discover that 2-OXOGLUTARATE-DEPENDENT-DIOXYGENASE (2-ODD) enzymes catalyze SGA-hydroxylation across various Solanum species. In contrast to cultivated potato, Solanum chacoense, a widespread wild potato species, has evolved a 2-ODD enzyme leading to the formation of leptinines. Furthermore, we find a related 2-ODD in tomato that catalyzes the hydroxylation of the bitter α-tomatine to hydroxytomatine, the first committed step in the chemical shift towards downstream ripening-associated non-bitter SGAs (e.g. esculeoside A). This 2-ODD enzyme prevents bitterness in ripe tomato fruit consumed today which otherwise would remain unpleasant in taste and more toxic.
Asunto(s)
Dioxigenasas/metabolismo , Frutas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metaboloma , Solanum/metabolismo , Gusto , Alcaloides/química , Alcaloides/metabolismo , Biocatálisis , Genes de Plantas , Hidroxilación , Ácidos Cetoglutáricos/química , Sitios de Carácter Cuantitativo/genética , Solanum/genética , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Esteroides/química , Esteroides/metabolismoRESUMEN
High-throughput RNA sequencing has proven invaluable not only to explore gene expression but also for both gene prediction and genome annotation. However, RNA sequencing, carried out on tens or even hundreds of samples, requires easy and cost-effective sample preparation methods using minute RNA amounts. Here, we present TranSeq, a high-throughput 3'-end sequencing procedure that requires 10- to 20-fold fewer sequence reads than the current transcriptomics procedures. TranSeq significantly reduces costs and allows a greater increase in size of sample sets analyzed in a single experiment. Moreover, in comparison with other 3'-end sequencing methods reported to date, we demonstrate here the reliability and immediate applicability of TranSeq and show that it not only provides accurate transcriptome profiles but also produces precise expression measurements of specific gene family members possessing high sequence similarity. This is difficult to achieve in standard RNA-seq methods, in which sequence reads cover the entire transcript. Furthermore, mapping TranSeq reads to the reference tomato genome facilitated the annotation of new transcripts improving >45% of the existing gene models. Hence, we anticipate that using TranSeq will boost large-scale transcriptome assays and increase the spatial and temporal resolution of gene expression data, in both model and non-model plant species. Moreover, as already performed for tomato (ITAG3.0; www.solgenomics.net), we strongly advocate its integration into current and future genome annotations.
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Secuenciación del Exoma/métodos , Genes de Plantas/genética , Genoma de Planta/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Arabidopsis/genética , Solanum lycopersicum/genética , Análisis de Secuencia de ARN/métodosRESUMEN
Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3ß-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.
Asunto(s)
Alcaloides/biosíntesis , Saponinas/biosíntesis , Solanaceae/química , Alcaloides/química , Regulación de la Expresión Génica de las Plantas/genética , Glicósidos/biosíntesis , Glicósidos/química , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Oxidorreductasas/metabolismo , Extractos Vegetales/química , Plantas Modificadas Genéticamente/metabolismo , Saponinas/química , Saponinas/metabolismo , Solanaceae/metabolismo , Esteroides/química , Tomatina/análogos & derivados , Tomatina/metabolismoRESUMEN
KEY MESSAGE: Exploration with high throughput leaf metabolomics along with functional genomics in wild tomato unreveal potential role of steroidal glyco-alkaloids and phenylpropanoids during early blight resistance. Alternaria solani severely affects tomato (Solanum lycopersicum L.) yield causing early blight (EB) disease in tropical environment. Wild relative, Solanum arcanum Peralta could be a potential source of EB resistance; however, its underlying molecular mechanism largely remains unexplored. Hence, non-targeted metabolomics was applied on resistant and susceptible S. arcanum accessions upon A. solani inoculation to unravel metabolic dynamics during different stages of disease progression. Total 2047 potential metabolite peaks (mass signals) were detected of which 681 and 684 metabolites revealed significant modulation and clear differentiation in resistant and susceptible accessions, respectively. Majority of the EB-triggered metabolic changes were active from steroidal glycol-alkaloid (SGA), lignin and flavonoid biosynthetic pathways. Further, biochemical and gene expression analyses of key enzymes from these pathways positively correlated with phenotypic variation in the S. arcanum accessions indicating their potential role in EB. Additionally, transcription factors regulating lignin biosynthesis were also up-regulated in resistant plants and electrophoretic mobility shift assay revealed sequence-specific binding of rSaWRKY1 with MYB20 promoter. Moreover, transcript accumulation of key genes from phenylpropanoid and SGA pathways along with WRKY and MYB in WRKY1 transgenic tomato lines supported above findings. Overall, this study highlights vital roles of SGAs as phytoalexins and phenylpropanoids along with lignin accumulation unrevealing possible mechanistic basis of EB resistance in wild tomato.