ELECTRON TRANSPORT PATHWAY MEDIATED BY CHLOROPLASTIC NAD(P)H DEHYDROGENASE

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Purification of NDHFirst, I tried to isolate chloroplastic NDH from spinach leaves. The content of NDH in the crude extract was very much lower than expected thus I concluded that it would be very difficult to purify enough amount of NDH for enzymological study. Next, I screened more than 200 plant species for high NDH activity using a newly developed assay method with chlorophyll fluorescence. As a results I found that some C4 plants have very high NDH activity. Therefore I purified NDH from a few C4 plant species which could be commercially available. Intact chloroplasts were isolated from leaves, and membrane associated protein complexes were solubilized by detergents. Then NDH was purified through anion exchange and gel filtration chromatographies.Physiological analysisResponses to very high light was investigated using newly generated chloroplast transformants in which NDH K-J or C-K-J subunits were inactivated. Redox states in stroma and the intersystem chain were monitored with P700 redox and chlorophyll fluorescence, respectively. A crucial role of NDH in preventing over-reduction induced after exposure to high light was proposed.