Identificação de marcadores químicos por metabolômica e por rede de moléculas relacionados a resistência constitutiva à Xanthomonas citri subsp. citri em folhas de Citrus spp.
Carregando...
Arquivos
Data
2022-03-14
Autores
Melo, Fernanda Pereira de Souza Rosa de
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Citrus compõem um dos maiores produtos agrícolas de valor agregado no Brasil, movimentando bilhões de dólares por ano, fomentando a economia do país e causando grande impacto social. No entanto, os pomares de Citrus são vulneráveis a patógenos, como por exemplo a bactéria gram-negativa Xanthomonas citri subsp citri (Xcc), agente causador do cancro cítrico. Algumas espécies são consideradas resistentes, todavia, estudos que associam metabólitos especializados à resistência, são escassos. Neste estudo, para identificar marcadores químicos relacionados à resistência, investigamos o metaboloma foliar de 17 variedades comerciais de Citrus com diferentes graus de suscetibilidade e resistência. Preparamos extratos foliares e analisamos por cromatografia líquida de ultra-eficiência acoplada a espectrometria de alta-resolução (UHPLC-HRMS/MS) utilizando um método genérico para se obter uma visão ampla do metaboloma. Variações no fingerprint químico foram investigadas através de análises multivariadas utilizando os dados de LC-MS1. Os dados de MS/MS foram organizados na forma de rede de moléculas evidenciando as famílias estruturais. Os compostos foram anotados na rede por meio de comparação dos espectros experimentais com uma base de espectros in silico de produtos naturais seguido de ponderação taxonômica dos candidatos. Marcadores químicos da resistência foram selecionados a partir de uma rede de moléculas multi-informativa, após a integração dos resultados estatísticos da metabolômica. Isto nos deu uma análise complementar à estatística clássica para a visualização de dados de metabolômica e seleção de marcadores químicos. Seguindo esta abordagem, anotamos 1618 compostos de Citrus, pertencentes a 112 classes químicas normalizadas via ClassyFire. Selecionamos 12 metabólitos como potenciais marcadores químicos de resistência para duas espécies: Citrus reticulata (Ponkan e Satsuma) e Citrus japonica (Kumquat), majoritariamente da classe dos flavonoides, sendo eles: 5,3'-dihidroxi-6,7,8,4'-tetrametoxiflavona, Isoscutelareina 7,8-dimetil éter, 4-O- (beta-D-glucopiranosil) -D-ribitol, Hesperetina 7- (2,6-diramnosilglucosídeo), Ácido 1,4-Dicafeoilquínico, 4,7-Dimetil-6,7-dihidro-5H-2-pirindina, 5,7,3'-trihidroxiflavona, Herclavina, Apigenina 7-O-neohesperidosídeo, 3,7 (11) -Eudesmadien-2-ona, Digicitrina, 2- (1,3-Benzodioxol-5-il) -5,7-di-hidroxi-4H-1-benzopiran-4-ona. Nosso fluxo de trabalho possibilitou verificar variações do metaboloma de várias espécies que puderam ser correlacionadas com a resistência à patógenos. Tais metabólitos podem ser utilizados como marcadores químicos para direcionar a seleção de novas variedades mais resistentes e como alvo de estudos para o desenvolvimento de novos produtos naturais para o controle do cancro cítrico.
Citrus makes up one of the largest value-added agricultural products in Brazil, moving billions of dollars per year, fostering the country's economy and causing great social impact. However, Citrus orchards are vulnerable to pathogens, such as the gram-negative bacterium Xanthomonas citri subsp citri (Xcc), the causative agent of citrus canker. Some species are considered resistant, however, studies associating specialized metabolites with resistance are scarce. In this study, to identify chemical markers related to resistance, we investigated the leaf metabolome of 17 commercial Citrus varieties with different degrees of susceptibility and resistance. We prepared leaf extracts and analyzed them by ultra-high-performance liquid chromatography coupled to high-resolution spectrometry (UHPLC-HRMS/MS) using a generic method to obtain a broad view of the metabolome. Variations in chemical fingerprinting were investigated by multivariate analysis using LC-MS1 data. The MS/MS data were organized in the form of a molecule network highlighting the structural families. Compounds were annotated in the network by comparing the experimental spectra with a base of in silico spectra of natural products followed by taxonomic weighting of candidates. Chemical markers of resistance were selected from a multi-informative molecule network after integration of statistical results from metabolomics. This gave us a complementary analysis to classical statistics for metabolomics data visualization and chemical marker selection. Following this approach, we annotated 1618 Citrus compounds, belonging to 112 chemical classes normalized via ClassyFire. We selected 12 metabolites as potential chemical markers of resistance for two species: Citrus reticulata (Ponkan and Satsuma) and Citrus japonica (Kumquat), mostly from the flavonoid class, being: 5,3'-dihydroxy-6,7,8,4'-tetramethoxyflavone, Isoscutellarein 7,8-dimethyl ether, 4-O- (beta-D-glucopyranosyl)-D-ribitol, Hesperetin 7- (2,6-diramnosylglucoside), 1,4-Dicaffeoylquinic acid, 4,7-Dimethyl-6, 7-dihydro-5H-2-pyrimidine, 5,7,3'-trihydroxyflavone, Herclavine, Apigenin 7-O-neohesperidoside, 3,7 (11)-Eudesmadien-2-one, Digicitrin, 2- (1,3-Benzodioxol-5-yl)-5,7-dihydroxy-4H-1-benzopyran-4-one. Our workflow made it possible to verify variations in the metabolome of several species that could be correlated with pathogen resistance. Such metabolites can be used as chemical markers to direct the selection of new, more resistant varieties and as a target for studies to develop new natural products for the control of citrus canker.
Citrus makes up one of the largest value-added agricultural products in Brazil, moving billions of dollars per year, fostering the country's economy and causing great social impact. However, Citrus orchards are vulnerable to pathogens, such as the gram-negative bacterium Xanthomonas citri subsp citri (Xcc), the causative agent of citrus canker. Some species are considered resistant, however, studies associating specialized metabolites with resistance are scarce. In this study, to identify chemical markers related to resistance, we investigated the leaf metabolome of 17 commercial Citrus varieties with different degrees of susceptibility and resistance. We prepared leaf extracts and analyzed them by ultra-high-performance liquid chromatography coupled to high-resolution spectrometry (UHPLC-HRMS/MS) using a generic method to obtain a broad view of the metabolome. Variations in chemical fingerprinting were investigated by multivariate analysis using LC-MS1 data. The MS/MS data were organized in the form of a molecule network highlighting the structural families. Compounds were annotated in the network by comparing the experimental spectra with a base of in silico spectra of natural products followed by taxonomic weighting of candidates. Chemical markers of resistance were selected from a multi-informative molecule network after integration of statistical results from metabolomics. This gave us a complementary analysis to classical statistics for metabolomics data visualization and chemical marker selection. Following this approach, we annotated 1618 Citrus compounds, belonging to 112 chemical classes normalized via ClassyFire. We selected 12 metabolites as potential chemical markers of resistance for two species: Citrus reticulata (Ponkan and Satsuma) and Citrus japonica (Kumquat), mostly from the flavonoid class, being: 5,3'-dihydroxy-6,7,8,4'-tetramethoxyflavone, Isoscutellarein 7,8-dimethyl ether, 4-O- (beta-D-glucopyranosyl)-D-ribitol, Hesperetin 7- (2,6-diramnosylglucoside), 1,4-Dicaffeoylquinic acid, 4,7-Dimethyl-6, 7-dihydro-5H-2-pyrimidine, 5,7,3'-trihydroxyflavone, Herclavine, Apigenin 7-O-neohesperidoside, 3,7 (11)-Eudesmadien-2-one, Digicitrin, 2- (1,3-Benzodioxol-5-yl)-5,7-dihydroxy-4H-1-benzopyran-4-one. Our workflow made it possible to verify variations in the metabolome of several species that could be correlated with pathogen resistance. Such metabolites can be used as chemical markers to direct the selection of new, more resistant varieties and as a target for studies to develop new natural products for the control of citrus canker.
Descrição
Palavras-chave
Cancro cítrico, Citrus sinensis, Flavonoides, Marcadores químicos, Resistência à patógenos, Noxa, Produtos agricolas