title: Involvement of an extracellular fungus laccase in the flavonoid metabolism in Citrus fruits inoculated with Alternaria alternata.
abstract: Fungi of the genus Alternaria are responsible for substantial pre-harvest losses in Citrus. In this study a degradative metabolism of flavonoids (flavanones, flavones and polymethoxyflavones) was observed when 'Fortune' mandarin, Citrus limon and Citrus paradisi, fruits were inoculated with Alternaria alternata, a pre-harvest pathogenic fungus. Associated to this flavonic metabolism the de novo synthesis of the phytoalexin scoparone was detected. This metabolism of flavonoids is caused by an extracellular fungus laccase. The kinetic characterisation of this enzyme revealed that the activity was induced by Citrus flavonoids and was dependent on flavonoid concentrations. The enzyme exhibited a Km of 1.9 mM using ABTS as substrate with an optimum pH of 3.5 in citrate buffer 100 mM. The enzyme is active between 15 and 45 °C, the optimum temperature being around 35 °C, although 50% of the initial activity is lost after 45 min at 35 °C. The A. alternata laccase was inhibited by 0.5 mM l-cysteine and by caffeic acid. Study of the substrate specificity of this enzyme revealed that Citrus flavonoids are substrates of A. alternata laccase. These results suggest that the laccase enzyme could be involved in the pathogenesis of A. alternata in Citrus.
authors: Departamento de Biología Vegetal (Fisiología Vegetal), Facultad de Biología, Universidad de Murcia, E-30100 Murcia, Spain. Electronic address: l.diazexposito@um.es.
authors: Licinio
authors: L
authors: Díaz
authors: Departamento de Biología Vegetal (Fisiología Vegetal), Facultad de Biología, Universidad de Murcia, E-30100 Murcia, Spain. Electronic address: jadelriro@um.es.
authors: José Antonio
authors: JA
authors: Del Río
authors: Departamento de Bioquímica, Facultad de Veterinaria, Universidad de Murcia, E-30100 Murcia, Spain. Electronic address: mpg@um.es.
authors: Manuela
authors: M
authors: Pérez-Gilabert
authors: Departamento de Biología Vegetal (Fisiología Vegetal), Facultad de Biología, Universidad de Murcia, E-30100 Murcia, Spain. Electronic address: aortuno@um.es.
authors: Ana
authors: A
authors: Ortuño
mesh_terms: D000528:Alternaria / Q000201:enzymology / Q000472:pathogenicity*; D002957:Citrus / Q000378:metabolism / Q000382:microbiology*; D003374:Coumarins / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism*; D005638:Fruit / Q000378:metabolism / Q000382:microbiology*; D042845:Laccase / Q000378:metabolism*; D010935:Plant Diseases / Q000382:microbiology*
chemical_list: D003374:Coumarins; D005419:Flavonoids; D042845:Laccase; C018145:scoparone
keywords: Citrus limon; Citrus paradisi; Flavanones; Flavones; Laccase; ‘Fortune’ mandarin