Enhancement by Artificial Electron Acceptors of Thylakoid Lumen Acidification and Zeaxanthin Formation

Abstract

In chloroplasts the enzyme violaxanthin de-epoxidase is responsible for the transformation of violaxanthin (Vio) to zeaxanthin (Zea) in the presence of ascorbate (Asc). The enzyme is mobile within the thylakoid lumen and becomes bound to the membrane in its active state. It is characterized by a narrow pH optimum at 5.2 and is presumed to be activated by the photosynthetic electron transport via the acidification of the lumenal pH of the thylakoid vesicles. Correlations between the pH of the lumen and the formation of Zea were studied using isolated thylakoids and leaf disks from Spinacia oleracea L. The alterations of the lumenal pH were measured by a photometric method using neutral red as indicator. A so far not documented bathochromic spectral shift of the protonated dye in the thylakoid lumen was taken into consideration. The xanthophylls were quantified by HPLC. In vitro , when using thylakoids that had been illuminated by red light at pH 7.5, artificial electron acceptors (such as methyl viologen (MV) or menadione (MD)) increased both the light-dependent H + -accumulation within the thylakoid lumen and the transformation of Vio to Zea. In vivo , after infiltration in leaf disks, MV was also effective. In its presence a 5-min illumination of these disks increased the Zea content by about 100 % in comparison with the MV-free control. Moreover, in isolated preilluminated Zea-containing thylakoids MD strongly inhibited the epoxidation of Zea to Vio, which is catalyzed by a mixed-function oxygenase in the presence of the cosubstrates O 2 and NAD(P)H at pH 7.5 in the dark. This inhibition was the result of an oxidation of NAD(P)H by MD catalyzed by an undefined thylakoid enzyme. The decreased level of (reduced) NAD(P)H and the fact that the reduced MD cannot substitute for NAD(P)H in the epoxidation reaction caused the diminished Zea epoxidation. The results demonstrate that under the influence of artificial electron acceptors an increased formation of Zea in the thylakoid membrane is strictly correlated with an increased light-dependent H + concentration in the thylakoid lumen.