The effects of dithiothreitol on violaxanthin de-epoxidation and absorbance changes in the 500-nm region

Abstract

The effects of dithiothreitol on absorbance changes at 505 and 515 nm in isolated lettuce chloroplasts were investigated. Dithiothreitol inhibited the ascorbate-dependent 505-nm change that is due to the de-epoxidation of violaxanthin to zeaxanthin. Dithiothreitol was effective for both light-induced de-epoxidation at pH 7 and dark de-epoxidation at pH 5. Titration of de-epoxidase activity with dithiothreitol resulted in complete inhibition at about 5 μmoles dithiothreitol per mg chlorophyll. Removal of dithiothreitol restored de-epoxidase activity. These results are consistent with the view that dithiothreitol inhibits violaxanthin de-epoxidation and the corresponding 505-nm change by reducing a disulfide that is required for de-epoxidase activity. Dithiothreitol was effective in resolving absorbance changes due to violaxanthin de-epoxidation and other changes that were superimposed under some conditions. At 515 nm and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), phenazine methosulfate, and ascorbate, dithiothreitol inhibited the large, slow and irreversible change which was due to de-epoxidation but not the fast and reversible so-called 515-nm change. At 505 nm and under similar conditions, dithiothreitol revealed the presence of a slow reversible change in addition to the one from de-epoxidation. Results with dithiothreitol showed that the absorbance change at 505 nm in the presence of DCMU, 2,6-dichlorophenolindophenol and ascorbate was due entirely to de-epoxidation. Similarly, absorbance changes at 515 nm also appeared to be mainly from de-epoxidation but with the presence of a small transient change due to some other components. It is suggested that dithiothreitol may be useful in resolving complex light-induced absorbance changes in other photosynthetic systems as well as in enabling new studies on reversible absorbance changes in the 500-nm region.