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Plant and Cell Physiology Advance Access published online on December 22, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci245
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received June 1, 2005
Accepted December 12, 2005

Regular Paper

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

Ryouhei Nakano 1, Hiroyuki Ishida 1 *, Amane Makino 1, and Tadahiko Mae 1

1 Department of Applied Plant Science, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan

* To whom correspondence should be addressed.
Hiroyuki Ishida, E-mail: hiroyuki{at}biochem.tohoku.ac.jp


  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 (Ishida et al. (1999) J. Biol. Chem. 274, 5222-5226). 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 a ·OH generating system in vitro on two-dimensional polyacrylamide gel electrophoresis. These results indicate that the direct fragmentation of the LSU by ROS also occurs in an intact leaf.

Keywords: chilling; cucumber (Cucumis sativus L.); photoinhibiton; PSI; reactive oxygen species; Rubisco.
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