title: Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?
abstract: Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.
authors.affiliation: Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), PO Box 164, E-30100 Campus University Espinardo (Murcia), Spain.
authors.forename: Federico
authors.initials: F
authors.lastname: Ferreres
authors.forename: Raquel
authors.initials: R
authors.lastname: Figueiredo
authors.forename: Sara
authors.initials: S
authors.lastname: Bettencourt
authors.forename: Inês
authors.initials: I
authors.lastname: Carqueijeiro
authors.forename: Juliana
authors.initials: J
authors.lastname: Oliveira
authors.forename: Angel
authors.initials: A
authors.lastname: Gil-Izquierdo
authors.forename: David M
authors.initials: DM
authors.lastname: Pereira
authors.forename: Patrícia
authors.initials: P
authors.lastname: Valentão
authors.forename: Paula B
authors.initials: PB
authors.lastname: Andrade
authors.forename: Patrícia
authors.initials: P
authors.lastname: Duarte
authors.forename: Alfonso Ros
authors.initials: AR
authors.lastname: Barceló
authors.forename: Mariana
authors.initials: M
authors.lastname: Sottomayor
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism; D030008:Catharanthus / Q000201:enzymology* / Q000528:radiation effects / Q000648:ultrastructure; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008027:Light; D013058:Mass Spectrometry; D058503:Mesophyll Cells / Q000166:cytology / Q000201:enzymology / Q000528:radiation effects / Q000648:ultrastructure; D010084:Oxidation-Reduction / Q000528:radiation effects; D009195:Peroxidase / Q000378:metabolism*; D010636:Phenols / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010936:Plant Extracts; D018515:Plant Leaves / Q000201:enzymology / Q000528:radiation effects / Q000648:ultrastructure; D010946:Plants, Medicinal / Q000201:enzymology* / Q000528:radiation effects / Q000648:ultrastructure; D011523:Protoplasts / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity / Q000528:radiation effects; D013997:Time Factors; D014617:Vacuoles / Q000201:enzymology* / Q000528:radiation effects / Q000648:ultrastructure
chemical_list: D007527:Isoenzymes; D010636:Phenols; D010936:Plant Extracts; D006861:Hydrogen Peroxide; D009195:Peroxidase; D001205:Ascorbic Acid