An Ascorbate-induced Absorbance Change in Chloroplasts from Violaxanthin De-epoxidation

Abstract

A new ascorbate-induced chloroplast absorbance change which has the characteristics of a carotenoid shift is described. The absorbance change was light-dependent at pH 7 but not at pH 5. The difference spectra for the light and dark changes were similar, showing a large absorbance peak at 505 nanometers, smaller peaks near 468 and 437 nanometers, and a sharp valley around 483 nanometers. The absorbance change is assigned to violaxanthin de-epoxidation because various conditions affected the absorbance change and violaxanthin de-epoxidation similarly, and the difference spectrum resembled the spectrum of zeaxanthin minus violaxanthin in organic solvent.Nigericin with KCl inhibited the light-dependent change at 505 nanometers. This effect, as well as the dark change at pH 5, indicated that de-epoxidation requires an acidic condition in chloroplasts. The effects of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, 2,6-dichlorophenolindophenol, and phenazine methosulfate indicated that the chloroplast acidification which mediates the 505 nanometers change is derived from hydrogen-ion transport linked to photosystem 1. Thus the 505 nanometers change could serve as an endogenous probe for chloroplast acidification and an indirect indicator of hydrogenion transport. At pH 5, the role of ascorbate appears to be to provide the reducing potential necessary for reductive de-epoxidation of violaxanthin. At pH 7, ascorbate could have an additional effect of stimulating electron transport and hence the hydrogen-ion transport necessary for de-epoxidation.In contrast to leaves and algae, de-epoxidation in chloroplasts was irreversible under the conditions investigated. Under some conditions other absorbance changes which were apparently due to chlorophyll were superimposed on the de-epoxidation change. The relationship of these ascorbate-induced changes to other absorbance changes observed in chloroplasts and green algae remains to be determined.