RESUMEN
Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and ß-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular compartmentation of FMO(GS-OX1) was also detected by transiently expressing a FMO(GS-OX1)-yellow fluorescent protein (YFP) fusion protein in tobacco leaves. The results showed that FMO(GS-OX1-5) were expressed basically in vascular tissues, especially in phloem cells, like other glucosinolate biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other glucosinolate biosynthetic genes. It is suggested that the spatial expression pattern of FMO(GS-OX1-5) determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. FMO(GS-OX1)-YFP fusion protein analysis identified FMO(GS-OX1) as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalized aliphatic glucosinolate biosynthetic pathway is discussed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Glucosinolatos/biosíntesis , Espacio Intracelular/enzimología , Oxigenasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Espacio Intracelular/genética , Oxigenasas/genética , Transporte de ProteínasRESUMEN
The cancer-preventive activity of cruciferous vegetables is commonly attributed to isothiocyanates resulting from the breakdown of the natural products glucosinolates (GSLs). Sulforaphane, the isothiocyanate derived from 4-methylsulfinylbutyl GSL, is thought to be the major agent conferring cancer-preventive properties, whereas the isothiocyanate of 4-methylthiobutyl GSL does not have the same activity. We report the identification of an Arabidopsis flavin-monooxygenase (FMO) enzyme, FMO(GS-OX1), which catalyzes the conversion of methylthioalkyl GSLs into methylsulfinylalkyl GSLs. This is evidenced by biochemical characterization of the recombinant protein, and analyses of the GSL content in FMO(GS-OX1) overexpression lines and an FMO(GS-OX1) knock-out mutant of Arabidopsis. The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs, with an approximately fivefold increase in 4-methylsulfinylbutyl GSL in seeds. Identification of FMO(GS-OX1) provides a molecular tool for breeding of Brassica vegetable crops with increased levels of this important GSL, which has implications for production of functional foods enriched with the cancer-preventive sulforaphane.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Flavinas/metabolismo , Glucosinolatos/biosíntesis , Oxigenasas de Función Mixta/metabolismo , Arabidopsis/genética , Brassica/enzimología , Brassica/genética , ADN de Plantas/genética , Eliminación de Gen , Oxigenasas de Función Mixta/genética , Mutagénesis Insercional , Oryza/enzimología , Oryza/genética , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The present study reports that specific antibody increased the bactericidal activity of rainbow trout head-kidney macrophages against virulent capsulated Lactococcus garvieae in the absence of complement. The observed increased bactericidal activity appeared to result from increased phagocytosis of capsulated L. garvieae in the presence of specific antibody and may in part explain the protective effect of antibody previously reported against this disease.