Quantification and localization of hesperidin and rutin in Citrus sinensis grafted on C. limonia after Xylella fastidiosa infection by HPLC-UV and MALDI imaging mass spectrometry.
A high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed for quantifying hesperidin and rutin levels in leaves and stems of Citrus limonia, with a good linearity over a range of 1.0-80.0 and 1.0-50.0 μg mL(-1) respectively, with r(2)>0.999 for all curves. The limits of detection (LOD) for both flavonoids were 0.6 and 0.5 μg mL(-1), respectively, with quantification (LOQ) being 2.0 and 1.0 μg mL(-1), respectively. The quantification method was applied to Citrus sinensis grafted onto C. limonia with and without CVC (citrus variegated chlorosis) symptoms after Xylella fastidiosa infection. The total content of rutin was low and practically constant in all analyses in comparison with hesperidin, which showed a significant increase in its amount in symptomatic leaves. Scanning electron microscopy studies on leaves with CVC symptoms showed vessel occlusion by biofilm, and a crystallized material was noted. Considering the difficulty in isolating these crystals for analysis, tissue sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to confirm the presence of hesperidin at the site of infection. The images constructed from MS/MS data with a specific diagnostic fragment ion (m/z 483) also showed higher ion intensities for it in infected plants than in healthy ones, mainly in the vessel regions. These data suggest that hesperidin plays a role in the plant-pathogen interaction, probably as a phytoanticipin. This method was also applied to C. sinensis and C. limonia seedlings, and comparison with the graft results showed that the rootstock had an increased hesperidin content ∼3.6 fold greater in the graft stem than in the stem of C. sinensis seedlings. Increase in hesperidin content by rootstock can be related to induced internal defense mechanisms.
Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos, SP, Brazil.
Márcio Santos
MS
Soares
Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos, SP, Brazil.
Danielle Fernandes
DF
da Silva
Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos, SP, Brazil.
Moacir Rossi
MR
Forim
Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos, SP, Brazil. Electronic address: dmfs@ufscar.br.
Maria Fátima das Graças Fernandes
MF
da Silva
Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos, SP, Brazil.
João Batista
JB
Fernandes
Departamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905 São Carlos, SP, Brazil.
Paulo Cezar
PC
Vieira
Núcleo Pesquisas em Produtos Naturais e Sintéticos, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, SP, Brazil.
Denise Brentan
DB
Silva
Núcleo Pesquisas em Produtos Naturais e Sintéticos, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, SP, Brazil.
Norberto Peporine
NP
Lopes
Centro APTA Citros Sylvio Moreira, Instituto Agronômico, CP 04, 13490-970 Cordeirópolis, São Paulo, Brazil.
Sérgio Alves
SA
de Carvalho
Centro APTA Citros Sylvio Moreira, Instituto Agronômico, CP 04, 13490-970 Cordeirópolis, São Paulo, Brazil.
Alessandra Alves
AA
de Souza
Centro APTA Citros Sylvio Moreira, Instituto Agronômico, CP 04, 13490-970 Cordeirópolis, São Paulo, Brazil.
Marcos Antônio
MA
Machado
D002851:Chromatography, High Pressure Liquid; D002957:Citrus / Q000737:chemistry* / Q000235:genetics; D006569:Hesperidin / Q000032:analysis*; D019906:Nuclear Magnetic Resonance, Biomolecular; D018515:Plant Leaves / Q000737:chemistry; D018547:Plant Stems / Q000737:chemistry; D012431:Rutin / Q000032:analysis; D044167:Xylella / Q000187:drug effects / Q000472:pathogenicity*
D012431:Rutin; D006569:Hesperidin
C. sinensis; Citrus limonia; HPLC-UV; Hesperidin; MALDI MSI; Rutaceae; Xylella fastidiosa