Timeâresolved fluorescence analysis of the recombinant photosystem II antenna complex CP29: Effects of zeaxanthin, pH and phosphorylation
2001
Crimi, Massimo | Dorra, Dieter | Bösinger, Carola S. | Giuffra, Elisabetta | Holzwarth, Alfred R. | Bassi, Roberto
Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, overâexpressed in Escherichiaâcoli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steadyâstate chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a ≈â35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthinâfree reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (τ) as well as relative fluorescence quantum yields (rfqy) of the two longâlived components (τ3 and τ4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of τ3 and in the average lifetime (≈ 2.4âns with zeaxanthin and 3.2–3.4âns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2–5.3) is observed on chlorophyll a fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this proteinâbound zeaxanthin induces a significant quenching effect.
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