Abstract
The conversion of violaxanthin (Vx) to zeaxanthin (Zx) in the de-epoxidation reaction of the xanthophyll cycle plays an important role in the protection of chloroplasts against photooxidative damage. Vx is bound to the antenna proteins of both photosystems. In photosystem II, the formation of Zx is essential for the pH-dependent dissipation of excess light energy as heat. The function of Zx in photosystem I is still unclear. In this work we investigated the de-epoxidation characteristics of light-harvesting complex proteins of photosystem I (LHCI) under in vivo and in vitro conditions. Recombinant LHCI (Lhcal-4) proteins were reconstituted with Vx and lutein, and the convertibility of Vx was studied in an in vitro assay using partially purified Vx de-epoxidase isolated from spinach thylakoids. All four LHCI proteins exhibited unique de-epoxidation characteristics. An almost complete Vx conversion to Zx was observed only in Lhca3, whereas Zx formation in the other LHCI proteins decreased in the order Lhca4 > Lhca1 > Lhca2. Most likely, these differences in Vx de-epoxidation were related to the different accessibility of the respective carotenoid binding sites in the distinct antenna proteins. The results indicate that Vx bound to site V1 and N1 is easily accessible for de-epoxidation, whereas Vx bound to L2 is only partially and/or with the slower kinetics convertible to Zx. The de-epoxidation properties determined for the monomeric recombinant proteins were consistent with those obtained for isolated native LHCI-730 and LHCI-680 in the same in vitro assay and the de-epoxidation state found under in vivo conditions in native LHCIs.
Highlights
Photosynthesis of higher plants is driven by the light-induced electron transport from photosystem II (PSII)1 to photosystem I (PSI)
Recombinant light-harvesting complex proteins of photosystem I (LHCI) (Lhcal-4) proteins were reconstituted with Vx and lutein, and the convertibility of Vx was studied in an in vitro assay using partially purified Vx de-epoxidase isolated from spinach thylakoids
Overexpressed Lhca apoproteins (Lhca1– 4) were reconstituted with pigment mixtures containing only Lut, Vx, Chl a, and Chl b and compared with LHCIs reconstituted with the full complement of thylakoid membrane pigments described previously [10]
Summary
Photosynthesis of higher plants is driven by the light-induced electron transport from photosystem II (PSII) to photosystem I (PSI). The detailed analysis of the npq mutant from Arabidopsis, which is defective in the gene encoding the Vx de-epoxidase (VxDE) and lacks Zx, underlined this proposed function of Zx [22, 23] It is unclear, to what extent this additional function might be related to the conversion of a non-protein-bound pool of Vx molecules in the thylakoid membrane. To what extent this additional function might be related to the conversion of a non-protein-bound pool of Vx molecules in the thylakoid membrane It is well known from early work on the xanthophyll cycle that only a fraction of ϳ60 – 80% of the VAZ pool is convertible to Zx in higher plants [24]. Both the release of Vx from the carotenoid binding site into the lipid phase of the membrane and the rebinding of Zx are likely to be controlled by the pH-regulated conformational changes of antenna proteins [7]
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