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Plants synthesize specialized metabolites to facilitate environmental and ecological interactions. During evolution, plants diversified in their potential to synthesize these metabolites. Quantitative differences in metabolite levels of natural Arabidopsis (Arabidopsis thaliana) accessions can be employed to unravel the genetic basis for metabolic traits using genome-wide association studies (GWAS). Here, we performed metabolic GWAS on seeds of a panel of 315 A. thaliana natural accessions, including the reference genotypes C24 and Col-0, for polar and semi-polar seed metabolites using untargeted ultra-performance liquid chromatography-mass spectrometry. As a complementary approach, we performed quantitative trait locus (QTL) mapping of near-isogenic introgression lines between C24 and Col-0 for specific seed specialized metabolites. Besides common QTL between seeds and leaves, GWAS revealed seed-specific QTL for specialized metabolites, indicating differences in the genetic architecture of seeds and leaves. In seeds, aliphatic methylsulfinylalkyl and methylthioalkyl glucosinolates associated with the ALKENYL HYDROXYALKYL PRODUCING loci (GS-ALK and GS-OHP) on chromosome 4 containing alkenyl hydroxyalkyl producing 2 (AOP2) and 3 (AOP3) or with the GS-ELONG locus on chromosome 5 containing methylthioalkyl malate synthase (MAM1) and MAM3. We detected two unknown sulfur-containing compounds that were also mapped to these loci. In GWAS, some of the annotated flavonoids (kaempferol 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-rhamnoside) were mapped to transparent testa 7 (AT5G07990), encoding a cytochrome P450 75B1 monooxygenase. Three additional mass signals corresponding to quercetin-containing flavonols were mapped to UGT78D2 (AT5G17050). The association of the loci and associating metabolic features were functionally verified in knockdown mutant lines. By performing GWAS and QTL mapping, we were able to leverage variation of natural populations and parental lines to study seed specialized metabolism. The GWAS data set generated here is a high-quality resource that can be investigated in further studies.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Estudo de Associação Genômica Ampla , Sementes/genética , Mapeamento Cromossômico , Flavonoides , 2-Isopropilmalato Sintase , Proteínas de Arabidopsis/genéticaRESUMO
The diterpenoid paclitaxel (Taxol) is a chemotherapy medication widely used as a first-line treatment against several types of solid cancers. The supply of paclitaxel from natural sources is limited. However, missing knowledge about the genes involved in several specific metabolic steps of paclitaxel biosynthesis has rendered it difficult to engineer the full pathway. In this study, we used a combination of transcriptomics, cell biology, metabolomics, and pathway reconstitution to identify the complete gene set required for the heterologous production of paclitaxel. We identified the missing steps from the current model of paclitaxel biosynthesis and confirmed the activity of most of the missing enzymes via heterologous expression in Nicotiana benthamiana. Notably, we identified a new C4ß-C20 epoxidase that could overcome the first bottleneck of metabolic engineering. We used both previously characterized and newly identified oxomutases/epoxidases, taxane 1ß-hydroxylase, taxane 9α-hydroxylase, taxane 9α-dioxygenase, and phenylalanine-CoA ligase, to successfully biosynthesize the key intermediate baccatin III and to convert baccatin III into paclitaxel in N. benthamiana. In combination, these approaches establish a metabolic route to taxoid biosynthesis and provide insights into the unique chemistry that plants use to generate complex bioactive metabolites.
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Biologia Sintética , Taxoides , Paclitaxel , Oxigenases de Função MistaRESUMO
Plants' ability to chemically modify core structures of specialized metabolites is the main reason why the plant kingdom contains such a wide and rich array of diverse compounds. One of the most important types of chemical modifications of small molecules is the addition of an acyl moiety to produce esters and amides. Large-scale phylogenomics analyses have shown that the enzymes that perform acyl transfer reactions on the myriad small molecules synthesized by plants belong to only a few gene families. This review is focused on describing the biochemistry, evolutionary origins, and chemical ecology implications of one of these families-the BAHD acyltransferases. The growth of advanced metabolomic studies coupled with next-generation sequencing of diverse plant species has confirmed that the BAHD family plays critical roles in modifying nearly all known classes of specialized metabolites. The current and future outlook for research on BAHDs includes expanding their roles in synthetic biology and metabolic engineering.
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Aciltransferases , Plantas , Aciltransferases/genética , Aciltransferases/química , Aciltransferases/metabolismo , Plantas/metabolismo , Evolução Biológica , FilogeniaRESUMO
Heterosis for agronomic traits is a widespread phenomenon that underpins hybrid crop breeding. However, heterosis at the level of cellular metabolites has not yet been fully explored. Some metabolites are highly sought after, like capsaicinoids found in peppers of the Capsicum genus, which confer the characteristic pungent ('hot') flavour of the fruits. We analysed the metabolic profile of the fruit placenta and pericarp of inter- and intra-specific hybrids of two species of Capsicum peppers, C. chinense (cv. Habanero and cv. Biquinho) and C. annuum var. annuum (cv. Jalapeño and cv. Cascadura Ikeda) in complete diallel crosses with reciprocals. The parents and hybrids were grown in a glasshouse and the profile of primary metabolites (sugars, amino acids and organic acids) and capsaicinoids was generated via gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) and ultra-performance liquid chromatography coupled to a mass spectrometer (UPLC-MS), respectively. We found considerable heterotic effects specifically for capsaicinoids accumulation in the fruit placenta of the hybrids, including those derived from non-pungent parents. Furthermore, a large fraction of fruit primary metabolism was influenced by the specific cross combination, with marked parent-of-origin effects, i.e. whether a specific genotype was used as the pistillate or pollen parent. The differences in metabolite levels between the hybrids and their parents provide a snapshot of heterosis for primary and secondary metabolites and may contribute to explain the manifestation of whole-plant heterotic phenotypes.
Assuntos
Capsicum , Capsaicina , Capsicum/química , Capsicum/genética , Cromatografia Líquida , Frutas/química , Frutas/genética , Vigor Híbrido/genética , Melhoramento Vegetal , Espectrometria de Massas em Tandem , VerdurasRESUMO
With the development of large-scale molecular phenotyping platforms, genome-wide association studies have greatly developed, being no longer limited to the analysis of classical agronomic traits, such as yield or flowering time, but also embracing the dissection of the genetic basis of molecular traits. Data generated by omics platforms, however, pose some technical and statistical challenges to the classical methodology and assumptions of an association study. Although genotyping data are subject to strict filtering procedures, and several advanced statistical approaches are now available to adjust for population structure, less attention has been instead devoted to the preparation of omics data prior to GWAS. In the present chapter, we briefly present the methods to acquire profiling data from transcripts, proteins, and small molecules, and discuss the tools and possibilities to clean, normalize, and remove the unwanted variation from large datasets of molecular phenotypic traits prior to their use in GWAS.
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Estudo de Associação Genômica Ampla , Estudo de Associação Genômica Ampla/métodos , FenótipoRESUMO
Plants evolved efficient multifaceted acclimation strategies to cope with low temperatures. Chloroplasts respond to temperature stimuli and participate in temperature sensing and acclimation. However, very little is known about the involvement of chloroplast genes and their expression in plant chilling tolerance. Here we systematically investigated cold acclimation in tobacco seedlings over 2 days of exposure to low temperatures by examining responses in chloroplast genome copy number, transcript accumulation and translation, photosynthesis, cell physiology, and metabolism. Our time-resolved genome-wide investigation of chloroplast gene expression revealed substantial cold-induced translational regulation at both the initiation and elongation levels, in the virtual absence of changes at the transcript level. These cold-triggered dynamics in chloroplast translation are widely distinct from previously described high light-induced effects. Analysis of the gene set responding significantly to the cold stimulus suggested nonessential plastid-encoded subunits of photosynthetic protein complexes as novel players in plant cold acclimation. Functional characterization of one of these cold-responsive chloroplast genes by reverse genetics demonstrated that the encoded protein, the small cytochrome b6f complex subunit PetL, crucially contributes to photosynthetic cold acclimation. Together, our results uncover an important, previously underappreciated role of chloroplast translational regulation in plant cold acclimation.
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Cloroplastos , Complexo de Proteínas do Centro de Reação Fotossintética , Aclimatação/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/genética , Fotossíntese/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , ProteômicaRESUMO
Fruit pungency is caused by the accumulation of capsaicinoids, secondary metabolites whose relation to primary metabolism remains unclear. We have selected ten geographically diverse accessions of Capsicum chinense Jacq with different pungency levels. A detailed metabolic profile was conducted in the fruit placenta and pericarp at 20, 45, and 60 days after anthesis aiming at increasing our understanding of the metabolic changes in these tissues across fruit development and their potential connection to capsaicin metabolism. Overall, despite the variation in fruit pungency among the ten accessions, the composition and metabolite levels in both placenta and pericarp were uniformly stable across accessions. Most of the metabolite variability occurred between the fruit developmental stages rather than among the accessions. Interestingly, different metabolite adjustments in the placenta were observed among pungent and non-pungent accessions, which seem to be related to differences in the genetic background. Furthermore, we observed high coordination between metabolites and capsaicin production in C. chinense fruits, suggesting that pungency in placenta is adjusted with primary metabolism.
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Capsicum , Piper nigrum , Capsaicina/análise , Frutas/química , ReproduçãoRESUMO
Ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) variants currently represent the best tools to tackle the challenges of complexity and lack of comprehensive coverage of the metabolome. UHPLC offers flexible and efficient separation coupled with high-sensitivity detection via HRMS, allowing for the detection and identification of a broad range of metabolites. Here we discuss current common strategies for UHPLC-HRMS-based metabolomics, with a focus on expanding metabolome coverage.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Espectrometria de Mobilidade Iônica/instrumentação , Espectrometria de Mobilidade Iônica/métodos , Espectroscopia de Ressonância Magnética , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Whilst substantial research effort has been placed on understanding the interactions of plant proteins with their molecular partners, relatively few studies in plants - by contrast to work in other organisms - address how these interactions evolve. It is thought that ancestral proteins were more promiscuous than modern proteins and that specificity often evolved following gene duplication and subsequent functional refining. However, ancestral protein resurrection studies have found that some modern proteins have evolved de novo from ancestors lacking those functions. Intriguingly, the new interactions evolved as a consequence of just a few mutations and, as such, acquisition of new functions appears to be neither difficult nor rare, however, only a few of them are incorporated into biological processes before they are lost to subsequent mutations. Here, we detail the approach of ancestral sequence reconstruction (ASR), providing a primer to reconstruct the sequence of an ancestral gene. We will present case studies from a range of different eukaryotes before discussing the few instances where ancestral reconstructions have been used in plants. As ASR is used to dig into the remote evolutionary past, we will also present some alternative genetic approaches to investigate molecular evolution on shorter timescales. We argue that the study of plant secondary metabolism is particularly well suited for ancestral reconstruction studies. Indeed, its ancient evolutionary roots and highly diverse landscape provide an ideal context in which to address the focal issue around the emergence of evolutionary novelties and how this affects the chemical diversification of plant metabolism.
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Feral plants have been known since the inception of modern agriculture, but the genetic changes during what seemed to be a simple reversion of a domesticated form are poorly understood. Recent studies, revealing the changes occurring in weedy rice, show an unexpected degree of differentiation in these feral escapes.
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Oryza , Oryza/genética , Plantas DaninhasRESUMO
The majority of the crops and vegetables of today were domesticated from their wild progenitors within the past 12 000 years. Considerable research effort has been expended on characterizing the genes undergoing positive and negative selection during the processes of crop domestication and improvement. Many studies have also documented how the contents of a handful of metabolites have been altered during human selection, but we are only beginning to unravel the true extent of the metabolic consequences of breeding. We highlight how crop metabolomes have been wittingly or unwittingly shaped by the processes of domestication, and highlight how we can identify new targets for metabolite engineering for the purpose of de novo domestication of crop wild relatives.
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Domesticação , Metaboloma , Produtos Agrícolas/genética , Melhoramento VegetalRESUMO
Our agricultural systems are now in urgent need to secure food for a growing world population. To meet this challenge, we need a better characterization of plant genetic and phenotypic diversity. The combination of genomics, transcriptomics and metabolomics enables a deeper understanding of the mechanisms underlying the complex architecture of many phenotypic traits of agricultural relevance. We review the recent advances in plant genomics to see how these can be integrated with broad molecular profiling approaches to improve our understanding of plant phenotypic variation and inform crop breeding strategies.
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Produtos Agrícolas/genética , Genoma de Planta , Genômica , Metaboloma , Metabolômica , Melhoramento Vegetal/métodos , TranscriptomaRESUMO
For diploid organisms that are highly heterozygous, a phased haploid genome can greatly aid in functional genomic, population genetic and breeding studies. Based on the genome sequencing of 135 single sperm cells of the elite tea cultivar 'Fudingdabai', we herein phased the genome of Camellia sinensis, one of the most popular beverage crops worldwide. High-resolution genetic and recombination maps of Fudingdabai were constructed, which revealed that crossover (CO) positions were frequently located in the 5' and 3' ends of annotated genes, while CO distributions across the genome were random. The low CO frequency in tea can be explained by strong CO interference, and CO simulation revealed the proportion of interference insensitive CO ranged from 5.2% to 11.7%. We furthermore developed a method to infer the relatedness between tea accessions and detected complex kinship and genetic signatures of 106 tea accessions. Among them, 59 accessions were closely related with Fudingdabai and 31 of them were first-degree relatives. We additionally identified genes displaying allele specific expression patterns between the two haplotypes of Fudingdabai and genes displaying significantly differential expression levels between Fudingdabai and other haplotypes. These results lay the foundation for further investigation of genetic and epigenetic factors underpinning the regulation of gene expression and provide insights into the evolution of tea plants as well as a valuable genetic resource for future breeding efforts.
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Camellia sinensis/genética , Troca Genética/genética , Genoma de Planta/genética , Pólen/genética , Alelos , Mapeamento Cromossômico , Genes de Plantas/genética , FilogeniaRESUMO
The presence of various types of structural variants, including transposons, make up the major part of the genomic differences among plant species. Two recent papers, Domínguez et al. and Alonge et al. explore specifically the impact that retrotransposons and other structural variants had on several tomato phenotypes of agricultural importance.
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Solanum lycopersicum , Elementos de DNA Transponíveis/genética , Genoma de Planta/genética , Genômica , Solanum lycopersicum/genética , Retroelementos/genéticaRESUMO
Apple (Malus domestica Borkh) is an important fruit crop cultivated in a broad range of environmental conditions. Apple fruit ripening is a physiological process, whose molecular regulatory network response to different environments is still not sufficiently investigated and this is particularly true of the peel tissue. In this study, the influence of environmental conditions associated with low (20 m) and high (750 m) altitude on peel tissue ripening was assessed by physiological measurements combined with metabolomic and proteomic analyses during apple fruit development and ripening. Although apple fruit ripening was itself not affected by the different environmental conditions, several key color parameters, such as redness and color index, were notably induced by high altitude. Consistent with this observation, increased levels of anthocyanin and other phenolic compounds, including cyanidin-3-O-galactoside, quercetin-3-O-rhamnoside, quercetin-3-O-rutinoside, and chlorogenic acid were identified in the peel of apple grown at high altitude. Moreover, the high-altitude environment was characterized by elevated abundance of various carbohydrates (e.g., arabinose, xylose, and sucrose) but decreased levels of glutamic acid and several related proteins, such as glycine hydroxymethyltransferase and glutamate-glyoxylate aminotransferase. Other processes affected by high altitude were the TCA cycle, the synthesis of oxidative/defense enzymes, and the accumulation of photosynthetic proteins. From the obtained data we were able to construct a metabolite-protein network depicting the impact of altitude on peel ripening. The combined analyses presented here provide new insights into physiological processes linking apple peel ripening with the prevailing environmental conditions.
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Wild teas are valuable genetic resources for studying domestication and breeding. Here we report the assembly of a high-quality chromosome-scale reference genome for an ancient tea tree. The further RNA sequencing of 217 diverse tea accessions clarifies the pedigree of tea cultivars and reveals key contributors in the breeding of Chinese tea. Candidate genes associated with flavonoid biosynthesis are identified by genome-wide association study. Specifically, diverse allelic function of CsANR, CsF3'5'H and CsMYB5 is verified by transient overexpression and enzymatic assays, providing comprehensive insights into the biosynthesis of catechins, the most important bioactive compounds in tea plants. The inconspicuous differentiation between ancient trees and cultivars at both genetic and metabolic levels implies that tea may not have undergone long-term artificial directional selection in terms of flavor-related metabolites. These genomic resources provide evolutionary insight into tea plants and lay the foundation for better understanding the biosynthesis of beneficial natural compounds.
Assuntos
Variação Genética , Genoma de Planta , Melaleuca/genética , Linhagem , Árvores/genética , Alelos , Vias Biossintéticas/genética , Camellia sinensis/genética , Catequina/metabolismo , China , Domesticação , Evolução Molecular , Ácido Gálico/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Estudo de Associação Genômica Ampla , Genômica , Análise de Sequência de RNA , Óleo de MelaleucaRESUMO
While the structures of plant primary metabolic pathways are generally well defined and highly conserved across species, those defining specialized metabolism are less well characterized and more highly variable across species. In this study, we investigated polyphenolic metabolism in the lycopersicum complex by characterizing the underlying biosynthetic and decorative reactions that constitute the metabolic network of polyphenols across eight different species of tomato. For this purpose, GC-MS- and LC-MS-based metabolomics of different tissues of Solanum lycopersicum and wild tomato species were carried out, in concert with the evaluation of cross-hybridized microarray data for MapMan-based transcriptomic analysis, and publicly available RNA-sequencing data for annotation of biosynthetic genes. The combined data were used to compile species-specific metabolic networks of polyphenolic metabolism, allowing the establishment of an entire pan-species biosynthetic framework as well as annotation of the functions of decoration enzymes involved in the formation of metabolic diversity of the flavonoid pathway. The combined results are discussed in the context of the current understanding of tomato flavonol biosynthesis as well as a global view of metabolic shifts during fruit ripening. Our results provide an example as to how large-scale biology approaches can be used for the definition and refinement of large specialized metabolism pathways.
Assuntos
Frutas/metabolismo , Polifenóis/metabolismo , Solanum lycopersicum/metabolismo , Cromatografia Líquida , Flavonoides/metabolismo , Frutas/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Variação Genética , Glicosiltransferases/metabolismo , Solanum lycopersicum/genética , Espectrometria de Massas , Redes e Vias Metabólicas , Metabolômica , Anotação de Sequência Molecular , Especificidade da EspécieRESUMO
Superficial scald is a major physiological disorder in apple fruit that is induced by cold storage and is mainly expressed as brown necrotic patches on peel tissue. However, a global view of the gene-protein-metabolite interactome underlying scald prevention/sensitivity is currently missing. Herein, we have found for the first time that cold storage in an atmosphere enriched with ozone (O3) induced scald symptoms in 'Granny Smith' apple fruits during subsequent ripening at room temperature. In contrast, treatment with the ethylene perception inhibitor 1-methylcyclopropene (1-MCP) reversed this O3-induced scald effect. Amino acids, including branched-chain amino acids, were the most strongly induced metabolites in peel tissue of 1-MCP treated fruits. Proteins involved in oxidative stress and protein trafficking were differentially accumulated prior to and during scald development. Genes involved in photosynthesis, flavonoid biosynthesis and ethylene signaling displayed significant alterations in response to 1-MCP and O3. Analysis of regulatory module networks identified putative transcription factors (TFs) that could be involved in scald. Subsequently, a transcriptional network of the genes-proteins-metabolites and the connected TFs was constructed. This approach enabled identification of several genes coregulated by TFs, notably encoding glutathione S-transferase (GST) protein(s) with distinct signatures following 1-MCP and O3 treatments. Overall, this study is an important contribution to future functional studies and breeding programs for this fruit, aiding to the development of improved apple cultivars to superficial scald.
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The origin of primordial metabolism and its expansion to form the metabolic networks extant today represent excellent systems to study the impact of natural selection and the potential adaptive role of novel compounds. Here we present the current hypotheses made on the origin of life and ancestral metabolism and present the theories and mechanisms by which the large chemical diversity of plants might have emerged along evolution. In particular, we provide a survey of statistical methods that can be used to detect signatures of selection at the gene and population level, and discuss potential and limits of these methods for investigating patterns of molecular adaptation in plant metabolism.