RESUMEN
Peroxiredoxins are antioxidative enzymes that catalyze the reduction of alkyl hydroperoxides to alcohols and hydrogen peroxide to water. 1-Cys peroxiredoxins (1-Cys Prxs) perform important roles during late seed development in plants. To characterize their biochemical functions in plants, a 1Cys-Prx gene was cloned from a Chinese cabbage cDNA library and designated as "C1C-Prx". Glutamine synthetase (GS) protection and hydrogen peroxide reduction assays indicated that C1C-Prx was functionally active as a peroxidase. Also C1C-Prx prevented the thermal- or chemical-induced aggregation of malate dehydrogenase and insulin. Hydrogen peroxide treatment changed the mobility of C1C-Prx on a two-dimensional gel, which implies overoxidation of the conserved Cys residue. Furthermore, after overoxidation, the chaperone activity of C1C-Prx increased approximately two-fold, but its peroxidase activity decreased to the basal level of the reaction mixture without enzyme. However, according to the structural analysis using far-UV circular dichroism spectra, intrinsic tryptophan fluorescence spectra, and native-PAGE, overoxidation did not lead to a conformational change in C1C-Prx. Therefore, our results suggest that 1-Cys Prxs function not only to relieve mild oxidative stresses but also as molecular chaperones under severe conditions during seed germination and plant development, and that overoxidation controls the switch in function of 1-Cys-Prxs from peroxidases to molecular chaperones.
Asunto(s)
Brassica/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Semillas/fisiología , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Brassica/enzimología , Brassica/genética , Cisteína/química , Flores/enzimología , Flores/genética , Flores/metabolismo , Dosificación de Gen , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Peróxido de Hidrógeno/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Estrés Oxidativo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Latencia en las Plantas/fisiología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Unión Proteica , Proteínas Recombinantes , Semillas/enzimología , Semillas/genética , Alineación de SecuenciaRESUMEN
We have purified a novel antifungal protein from blast fungus (Magnaporthe grisea)-treated rice leaves using consecutive chromatographies on CM-Sepharose ion-change, Affi-gel blue, and HPLC gel filtration columns. We determined the N-terminal peptide sequence of the purified protein and subjected it to the NCBI/BLAST database and found the protein to be a partial fragment of the peroxisomal receptor protein in rice (OsPex5p). After cloning two cDNAs encoding OsPEX5L and OsPEX5S genes that are splice variants of OsPEX5 from a rice leaf cDNA library, we investigated their antifungal properties. The recombinant proteins were expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the OsPEX5L gene was induced by diverse external stresses such as rice blast fungus, fungal elicitor, and other signaling molecules including H(2)O(2), abscisic acid, jasmonic acid, and salicylic acid. These results suggest that the peroxisomal receptor protein, OsPex5p, plays a critical role in the rice defense system against diverse external stresses including fungal pathogenic attack.