Hydroxyl Radicals and a Thylakoid-Bound Endopeptidase are Involved in Light- and Oxygen-Induced Proteolysis in Oat Chloroplasts

The hypothesis that light- and oxygen-induced proteolysis inchloroplasts is mediated by active oxygen species was examined.In order to determine whether or not H₂O₂ and/or ^{dot}OH radicalsare involved in these degradative processes we compared thedegradation of proteins in isolated oat chloroplasts exposedto white light at 80 W m^-2 with that in chloroplasts incubatedin darkness in the absence or presence of H₂O₂ or a ^{dot}OH-generatingsystem composed by ascorbic acid, FeCl₃ and H₂O₂ (Asc-Fe-H₂O₂).Light enhanced the rate of degradation of at least 18 polypeptides,while proteolysis was almost negligible in darkness in the abscenceof additives. H₂O₂ had a very small effect. However, Asc-Fe-H₂O₂-treatedchloroplasts in darkness showed a pattern of protein degradationalmost identical to that observed in the light. A thylakoid-boundendopeptidase (EP), the activity of which increased under photooxidativeenvironmental conditions and treatment with an ^{dot}OH-generatingsystem, was partially purified and characterized as a serinetypeprotease. Treatments with inhibitors of serine-type proteaseprevented both light- and Asc- Fe-H₂O₂-induced proteolysis.EP was more active against both soluble and membranous proteinsthat had been pretreated with Asc-Fe-H₂O₂ than against untreatedproteins. It is proposed that a high dose of light irradiationpromotes proteolysis by increasing the formation of ^{dot}OH,which may modify proteins such that they become more susceptibleto EP-catalyzed hydrolysis. ¹Fisiología Vegetal, Dept. de Biología Vegetal,Universidad de Alcalá de Henares, Present address: 28871Alcalá de Henares (Madrid), España.