In vivo fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species in an intact leaf of cucumber under chilling-light conditions

Abstract

Previous studies have demonstrated that the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) is site-specifically cleaved by a hydroxyl radical (·OH) generated in the illuminated chloroplast lysates or by an artificial ·OH-generating system. However, it is not known whether such cleavage of the LSU by reactive oxygen species (ROS) actually occurs in an intact leaf. When leaf discs of chilling-sensitive cucumber (Cucumis sativus L.) were illuminated at 4°C, five major fragments of the LSU were observed. This fragmentation was completely inhibited by ROS scavengers, such as n-propyl gallate (for ·OH) and 1,2-dihydroxybenzene- 3,5-disulfonic acid (Tiron) (for superoxide). FeSO4 stimulated this fragmentation, whereas an iron-specific chelator, deferoxamine, suppressed it. Furthermore, such fragments were identical to those generated from the purified Rubisco by an ·OH-generating system in vitro on two-dimensional PAGE. These results indicate that the direct fragmentation of the LSU by reacive oxygen species also occurs in an intact leaf. JSPP © 2006.