ENHANCEMENT BY ARTIFICIAL ELECTRON ACCEPTERS OF THYLAKOID LUMEN ACIDIFICATION AND ZEAXANTHIN FORMATION

In chloroplasts the enzyme violaxanthin de-epoxidase is responsible fo r 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 ch aracterized by a narrow pH optimum at 5.2 and is presumed to be activa ted 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 thyl akoids 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. T he xanthophylls were quantified by HPLC. In vitro, when using thylakoi ds that had been illuminated by red light at pH 7.5, artificial electr on accepters (such as methyl viologen (MV) or menadione (MD)) increase d both the light-dependent H+-accumulation within the thylakoid lumen and the transformation of Vio to Zea. In vivo, after infiltration in l eaf 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-con taining 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 inhibitio n was the result of an oxidation of NAD(P)H by MD catalyzed by an unde fined thylakoid enzyme. The decreased level of (reduced) NAD(P)H and t he fact that the reduced MD cannot substitute for NAD(P)H in the epoxi dation reaction caused the diminished Zea epoxidation. The results dem onstrate that under the influence of artificial electron accepters an increased formation of Zea in the thylakoid membrane is strictly corre lated with an increased light-dependent H+ concentration in the thylak oid lumen.