| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2006: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Redox regulation in biology is as important as regulation by phosphorylation / de-phosphorylation. The head investigator of this project recently developed the disulfide proteome technique that allows comprehensive analysis of redox regulation of proteins. This study was aimed to apply the technique to clarify new redox regulations in rice seed germination.
First, proteins were extracted from aleurone layer of rice seeds. When NADPH and NADPH-dependent thioredoxin reductase (NTR) were added to the extract, embryo-specific protein (ESP)-2 disappeared. On the other hand, when the experiment was done in the presence of leupeptine, a cysteine-protease inhibitor, degradation of ESP2 did not occur. These results suggested that the endogenous thioredoxin in the aleurone layer activated a cystein protease that digested ESP2. The ESP2 has a high disulfide content (l6S-S/392aa). So thioredoxin seemed to reduce the disulfide bonds to unfold the molecular structure of ESP2. Next, when isolated aleurone layer was incubated in the presence of gibberellic acid and CaCl_2, similar results were obtained as the previous in vitro studies. So, in rice seed germination, endogenous thioredoxin likely activates cysteine protease and concurrently unfolds its substrate, ESP2, to facilitate prompt degradation. It has been reported that a serine-protease, thiocalsin, that digests globulins is synthesized de novo and activated thioredoxhvdependently in the presence of Ca^<2+> in the later stage of seed germination. In our in vivo study, globulins remained intact when ESP2 was digested by the cystein protease. These observations suggest that in seed germination, thioredoxin activates different proteases step by step, and concurrently unfolds the substrate of the respective protease to facilitate degradation. Thus new redox regulation was clarified in the present study.
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