Time-resolved fluorescence on oligomers of the main light-harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter-trimer chlorophyll-chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far-red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400 ps fluorescence component appearing in intact leaves of Arabidopsis when non-photochemical quenching (NPQ) is induced. The fluorescence components with high far-red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far-red emitting state is shown to be an emissive Chl-Chl charge transfer state which plays a crucial part in the quenching.

In higher plants, the de-epoxidation of violaxanthin (Vx) to antheraxanthin and zeaxanthin is required for the pH-dependent dissipation of excess light energy as heat and by that process plays an important role in the protection against photo-oxidative damage. The de-epoxidation reaction was investigated in an in vitro system using reconstituted light-harvesting complex II (LHCII) and a thylakoid raw extract enriched in the enzyme Vx de-epoxidase. Reconstitution of LHCII with varying carotenoids was performed to replace lutein and/or neoxanthin, which are bound to the native complex, by Vx. Recombinant LHCII containing either 2 lutein and 1 Vx or 1.6 Vx and 1.1 neoxanthin or 2.8 Vx per monomer were studied. Vx de-epoxidation was inducible for all complexes after the addition of Vx de-epoxidase but to different extents and with different kinetics in each complex. Analysis of the kinetics indicated that the three possible Vx binding sites have at least two, and perhaps three, specific rate constants for de-epoxidation. In particular, Vx bound to one of the two lutein binding sites of the native complex, most likely L1, was not at all or only at a slow rate convertible to Zx. In reisolated LHCII, newly formed Zx almost stoichiometrically replaced the transformed Vx, indicating that LHCII and Vx de-epoxidase stayed in close contact during the de-epoxidation reactions and that no release of carotenoids occurred.

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.

The conversion of violaxanthin to zeaxanthin is essentially required for the pH-regulated dissipation of excess light energy in the antenna of photosystem II. Violaxanthin is bound to each of the antenna proteins of both photosystems. Former studies with recombinant Lhcb1 and different Lhca proteins implied that each antenna protein contributes specifically to violaxanthin conversion related to protein-specific affinities of the different violaxanthin binding sites. We investigated the violaxanthin de-epoxidation in the minor antenna proteins of photosystem II, Lhcb4 -6. Recombinant proteins were reconstituted with different xanthophyll mixtures to study the conversion of violaxanthin at different xanthophyll binding sites in these proteins. The extent and kinetics of violaxanthin de-epoxidation were found to be dependent on the respective protein and, for each protein, also on the binding site of violaxanthin. In particular, violaxanthin bound to Lhcb4 was nearly inconvertible for de-epoxidation, whereas violaxanthin bound to Lhcb5 was fully convertible but with slow kinetics. Lhcb6 exhibited heterogeneous violaxanthin conversion characteristics, which could be assigned to different populations of reconstituted Lhcb6 complexes with respect to violaxanthin binding sites. The results support the proposed different binding affinities of violaxanthin to the three putative violaxanthin binding sites (V1, N1, and L2) in antenna proteins. Under consideration of former studies with Lhcb1 and Lhca proteins, the data imply that violaxanthin bound to the V1 and N1 binding site of antenna proteins is easily accessible for de-epoxidation in all antenna proteins, whereas violaxanthin bound to L2 is either only slowly or not convertible to zeaxanthin, depending on the respective protein.

The enzyme violaxanthin de-epoxidase (VxDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of membrane-bound violaxanthin (Vx) to zeaxanthin. De-epoxidation from the opposite, stroma side of the membrane has been investigated in the npq1 mutant from Arabidopsis thaliana (L.) Heynh. - which lacks VxDE - by adding partially purified VxDE from spinach thylakoids. The accessibility of Vx to the exogenously added enzyme (exoVxDE) and the kinetics of Vx conversion by the exoVxDE in thylakoids from npq1 plants were very similar to the characteristics of Vx conversion by the endogenous enzyme (endoVxDE) in thylakoids from wild-type plants. However, the conversion of Vx by exoVxDE was clearly retarded at lower temperatures when compared with the reaction catalyzed by endoVxDE. Since the exoVxDE - in contrast to the endoVxDE - has no access to the stacked regions of the membrane, where the xanthophylls bound to photosystem II are located, these results support the assumption of pronounced diffusion of xanthophylls within the thylakoid membrane.