Extraction and analysis of apoplastic phenolic metabolites in carnation roots and stems (Dianthus caryo-phyllus L) / Extracción y análisis de metabolitos fenólicos apoplásticos en raíz y tallo de clavel (Dianthus caryophyllus L) / Extração e análise do meta-bólitos fenólicos apoplásticos na raiz e caule do craveiro (Dianthus caryophyllus L)

Abstract 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.

Resumen 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.

Resumo 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-Na2HPO40 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.

Flavonoid biosynthesis in Dianthus caryophyllus L. is early regulated during interaction with Fusarium oxysporum f. sp. dianthi.

Rooted cuttings from two carnation (Dianthus caryophyllus L.) cultivars showing contrasting responses to the vascular wilt caused by Fusarium oxysporum f. sp. dianthi (Fod) were inoculated with this phytopathogen, and some of the biochemical responses associated with flavonoid biosynthesis were investigated in the roots. The resistant cultivar ('Golem') showed a significant increase in the levels of phenolic and flavonoid compounds at 48-96 h post-inoculation (hpi) (α = 0.05). LC-MS-based analysis indicated that the flavonoids mainly included flavanol-type glycosides, especially quercetin and kaempferol aglycones. Quantification of the mRNA levels of genes encoding CHS (Chalcone Synthase), CHI (Chalcone Isomerase), FLS (Flavonol Synthase), and the transcription factor MYB11 by using reverse transcription quantitative polymerase chain reaction (RT-qPCR) indicated that the resistant cultivar exhibited higher expression levels of these genes and, therefore, showed more flavonoid accumulation at 96 hpi. The differences in the temporal regulation of the assessed variables during infection support the idea that the early expression of enzymes of the flavonoid biosynthesis pathway in carnation roots is linked to a resistance response to the hemibiotrophic pathogen Fod race 2. The present experimental approach is the first report describing the molecular mechanisms underlying flavonoid biosynthesis in carnation roots during their interaction with Fod.

Mycelium Dispersion from Fusarium oxysporum f. sp. dianthi Elicits a Reduction of Wilt Severity and Influences Phenolic Profiles of Carnation (Dianthus caryophyllus L.) Roots.

The fungal pathogen Fusarium oxysporum f. sp. dianthi (Fod) is the causal agent of the vascular wilt of carnation (Dianthus caryophyllus L.) and the most prevalent pathogen in the areas where this flower is grown. For this reason, the development of new control strategies against Fod in carnation has been continuously encouraged, in particular those based on the implementation of plant resistance inducers that can trigger defensive responses to reduce the disease incidence, even at lower economical and environmental cost. In the present study, the effect of the soil supplementation of a biotic elicitor (i.e., ultrasound-assisted dispersion obtained from Fod mycelium) on disease severity and phenolic-based profiles of roots over two carnation cultivars was evaluated. Results suggest that the tested biotic elicitor, namely, eFod, substantially reduced the progress of vascular wilting in a susceptible cultivar (i.e., 'Mizuki') after two independent in vivo tests. The LC-MS-derived semi-quantitative levels of phenolic compounds in roots were also affected by eFod, since particular anthranilate derivatives, conjugated benzoic acids, and glycosylated flavonols were upregulated by elicitation after 144 and 240 h post eFod addition. Our findings indicate that the soil-applied eFod has an effect as a resistance inducer, promoting a disease severity reduction and accumulation of particular phenolic-like compounds.

Protein extraction and gel-based separation methods to analyze responses to pathogens in carnation (Dianthus caryophyllus L).

We are currently using a 2-DE-based proteomics approach to study plant responses to pathogenic fungi by using the carnation (Dianthus caryophyllus L)-Fusarium oxysporum f. sp. dianthi pathosystem. It is clear that the protocols for the first stages of a standard proteomics workflow must be optimized to each biological system and objectives of the research. The optimization procedure for the extraction and separation of proteins by 1-DE and 2-DE in the indicated system is reported. This strategy can be extrapolated to other plant-pathogen interaction systems in order to perform an evaluation of the changes in the host protein profile caused by the pathogen and to identify proteins which, at early stages, are involved or implicated in the plant defense response.