The kinetics of zeaxanthin formation is retarded by dicyclohexylcarbodiimide

Abstract

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth --> Zeax) of the reaction sequence seemed to be more affected than the violaxanthin --> Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.

Figure 1

Time course of de-epoxidation in isolated control thylakoids. Thylakoids (50 μg Chl/mL) were incubated for 10 min in the dark and at room temperature in the absence (A) or in the presence (B) of 100 μm DCCD. The relative portions (mol%) of the xanthophyll-cycle pigments Viol (○), Anth (□), and Zeax (•) are plotted. The sum of the three pigments remained constant during the time course of the experiments. The data represent the mean value of three independent experiments. sd was in the range of 5 to 7% of the respective values for Viol and Zeax, and up to 20% of the respective values for Anth.

Figure 2

Time course of de-epoxidation in isolated IML thylakoids. Thylakoids (50 μg Chl/mL) were incubated for 10 min in the dark and at room temperature in the absence (A) or in the presence (B) of 100 μm DCCD. The relative portions of the xanthophyll-cycle pigments Viol (○), Anth (□), and Zeax (•) are plotted. The sum of the three pigments remained constant during the time course of the experiments. The data represent the mean value of three independent experiments. sd was in the range of 2 to 10% of the respective values for Viol and Zeax, and up to 30% of the respective values for Anth.

Figure 3

Time course of de-epoxidation in control (A and C) and IML (B and D) thylakoids. Thylakoids (50 μg Chl/mL) were incubated for 10 min in the dark and at room temperature in the absence (○) or in the presence (•) of 100 μm DCCD. Incubation with DCCD was performed either under HS (A and B) or LS (C and D) conditions. Only the relative portion of Zeax is plotted. The data represent the mean value of three independent experiments. sd was in the range of 3 to 10% of the respective values.

Figure 4

Zeax content in different antenna subcomplexes. Thylakoids (50 μg Chl/mL) were incubated for 10 min in the dark and at room temperature in the absence (white columns) or in the presence (gray and black columns) of 100 μm DCCD. Incubation with DCCD was performed either under LS (gray columns) or HS (black columns) conditions. Different antenna subcomplexes were separated by IEF. The distinct antenna proteins were assigned to the different fractions as in Färber et al. (1997). Only the relative Zeax content of each fraction is plotted. The data represent the mean value of three to five independent experiments. sd is indicated by the respective bars.