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Extraction and analysis of apoplastic phenolic metabolites in carnation roots and stems (Dianthus caryophyllus L)
Extracción y análisis de metabolitos fenólicos apoplásticos en raíz y tallo de clavel (Dianthus caryophyllus L)
Extração e análise do metabólitos fenólicos apoplásticos na raiz e caule do craveiro (Dianthus caryophyllus L)
Palabras clave:
Apoplastic fluid, Fusarium oxysporum f. sp. dianthi, plant-pathogen, phenolic metabolites (en)fluido apoplástico, Fusarium oxysporum f. sp. dianthi, planta–patógeno, metabolitos fenólicos (es)
Fluido apoplástico, Fusarium oxysporum f sp. dianthi, planta–patógeno, metabólitos fenólicos (pt)
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The present study outlines the conditioning of various parameters for the efficient removal of the apoplastic fraction of carnation enriched in polar compounds, mainly phenolics. Several studies can apply the described workflow to different plant species in the particular or global analysis of those metabolites in this peripheral extracellular space. Hence, using carnation (Dianthus caryophyllus L) roots and stems, we evaluated different infiltration solutions for removing apoplastic metabolites. The best outcome was obtained by using the buffer solution NaH2PO4-Na2HPO4 0.1 M pH 6.5/NaCl 50 mM, since the highest amount of apoplastic phenolic metabolites can be obtained with the slightest contamination of intracellular compounds. The metabolites were separated using HPLC-DAD-ESI-MS, affording chromatographic profiles with reasonable quality parameters based on resolution, selectivity, and number of theoretical plates. It was possible to identify eight differential compounds (one flavone and seven flavonols), whose core moieties consisted of (iso)pratol, kaempferide, (dihydro)kaempferol, quercetin, and myricetin-type flavonoids according to the test organ and the cultivar. We deduced that identified flavonoids are related to phytoanticipin-type metabolites in carnation, such as hydroxy-methoxyflavone, di-o-benzoylquercetin, and kaempferide disalicyloylrhamnoside, which are abundantly present in the resistant cultivar. The conditions described in this work are fundamental for delving into the role of apoplastic phenolic metabolites related to the defense mechanisms of this ornamental plant.
En el presente estudio se describe el acondicionamiento de algunos parámetros con fines de obtención eficiente de extractos apoplásticos enriquecidos en compuestos polares, principalmente fenólicos. Este flujo de trabajo descrito, incluso, puede ser aplicado a diferentes especies vegetales para ser empleado en el análisis particular o global de metabolitos en este espacio extracelular periférico. Para ello, usando raíces y tallos de clavel (Dianthus cariophyllus L), se evaluaron diferentes soluciones de infiltración para la extracción de los metabolitos apoplásticos. El mejor resultado se logró con la disolución amortiguadora NaH2PO4-Na2HPO4 0,1 M pH 6,5/NaCl 50 mM, porque se obtiene la mayor cantidad de metabolitos fenólicos apoplásticos, con la menor contaminación de compuestos intracelulares. Los metabolitos se separaron mediante HPLC-DAD-ESI-MS, obteniendo perfiles cromatográficos con parámetros de calidad razonables basados en resolución, selectividad y número de platos teóricos. Con estas condiciones, fue posible identificar ocho compuestos diferenciales (una flavona y siete flavonoles), cuyas estructuras básicas comprendían flavonoides del tipo (iso)pratol, kaempférido, (dihidro)kaempferol, quercetina y miricetina, según el órgano de prueba y la variedad. Los flavonoides identificados están relacionados con metabolitos de tipo fitoanticipina en el clavel, como hidroxi-metoxiflavona, di-o-benzoilquercetina y kaempférido disaliciloilrhamnósido, abundantemente presentes en la variedad resistente. Las condiciones descritas en este trabajo son fundamentales para profundizar en el papel de los metabolitos fenólicos apoplásticos relacionados con los mecanismos de defensa de esta planta ornamental.
O presente estudo descreve o condicionamento de alguns parâmetros para a obtenção eficiente de extratos apoplásticos enriquecidos em compostos polares, principalmente fenólicos. Este fluxo de trabalho descrito pode até mesmo ser aplicado a diferentes espécies de plantas para serem usadas na análise particular ou global de metabólitos neste espaço extracelular periférico. Para isso, utilizando raízes e caules de craveiro (Dianthus cariophyllus L), diferentes soluções de infiltração foram avaliadas para a extração de metabólitos apoplásticos. O melhor resultado foi obtido com a solução tampão NaH2PO4-Na2HPO4 0.1 M pH 6.5/NaCl 50 mM, pois a maior quantidade de metabólitos fenólicos apoplásticos é extraída. Os metabólitos foram separados por HPLC-DAD-ESI-MS, obtendo-se perfis cromatográficos com parâmetros de qualidade razoáveis baseados na resolução, seletividade e número de pratos teóricos. Nessas condições, foi possível identificar oito compostos diferenciais (uma flavona e sete flavonóis), cujas estruturas básicas compreendem flavonóides do tipo (iso)pratol, kaempferida, (dihidro)kaempferol, quercetina e miricetina, de acordo com o órgão e a variedade de craveiro utilizado. Os flavonóides identificados estão relacionados a metabólitos do tipo fitoanticipinas no craveiro, como hidroxi-metoxiflavona, di-O-benzoilquercetina e kaempférido disaliciloilramnósido, abundantemente ocorridos na cultivar resistente. As condições descritas neste trabalho são essenciais para se aprofundar no papel dos metabólitos fenólicos apoplásticos relacionados aos mecanismos de defesa dessa planta ornamental.
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