Abstract
To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.
Highlights
To ensure the efficiency of photosynthesis, even under low light conditions, the photochemically active Chl3 of the photosystem II (PSII) reaction center are served by additional antenna Chl bound to light-harvesting complexes (LHCII) [1]
The maximum quantum yield of PSII (Fv/Fm) was defined as ((Fm Ϫ Fo)/Fm), the quantum yield of PSII (⌽PSII) as ((FmЈ Ϫ Fs)/FmЈ), qP as ((FmЈ Ϫ Fs)/(FmЈ Ϫ FoЈ)), and non-photochemical quenching (NPQ) as ((Fm Ϫ FmЈ)/FmЈ). qE-related absorption changes were recorded on whole leaves using an Aminco DW2000 spectrophotometer as described previously [25], and difference spectra were calculated by subtracting the “dark recovery” spectrum (5 min of preillumination at 1000 mol of photons mϪ2 sϪ1 actinic light followed by 5 min of dark relaxation) from the “light” spectrum recorded in the presence of the actinic light following 5 min of preillumination
The origin of the ⌬A525 absorption change that is characteristic for qE in the absence of zeaxanthin was studied by resonance Raman difference spectroscopy using 528.7 nm excitation
Summary
Chlorophyll(s); PSII, photosystem II; LHCII, light-harvesting antenna complex of photosystem II; NPQ, nonphotochemical Chl fluorescence quenching; qE, rapidly reversible component of NPQ; ⌬pH, proton gradient across the thylakoid membrane; Zea, zeaxanthin; Fm, maximum Chl fluorescence in the dark; FmЈ, maximum Chl fluorescence after illumination. The negative absorption changes in the qE difference spectrum below 500 nm were associated with a loss of xanthophyll resonance confirming that they arise from true electronic transitions rather than light scattering [31]. It is known that the energy of an electronic transition within a molecule is strongly dependent on the refractive index of the solvent environment [39, 40] Such a strong red shift as seen during the ⌬A535 change would require a very dramatic change in the solvent environment, possibly due to the arrival of a static dipole or a point charge in the vicinity of the zeaxanthin [41, 42]. We show that the same Raman signature associated with ⌬A535 and ⌬A525 in vivo is observed upon aggregation of LHCII binding, respectively, zeaxanthin or violaxanthin in the peripheral V1 site
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