Origin of absorption changes associated with photoprotective energy dissipation in the absence of zeaxanthin

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.

FIGURE 1.

Light-treated-minus-dark recovery absorption difference spectra in Arabidopsis leaves. Wild-type npq1 and L17+DTT leaves were measured immediately after 5 min of illumination at 1000 μmol of photons m⁻² s⁻¹ (light-treated) or following a further 5 min of dark relaxation (dark recovery). Inset, amplitude of NPQ for each sample, ± S.E.

FIGURE 2.

Comparison of the qE-associated Raman spectrum (difference spectrum of light-treated-minus-dark recovery) induced by 528.7 nm excitation in Arabidopsis leaves from wild type and L17 treated with DTT. rel., relative.

FIGURE 3.

Comparison of the ν₁ region of the qE-associated Raman spectrum (difference spectrum of light-treated-minus-dark recovery) induced by 528.7 nm excitation in Arabidopsis leaves from wild type and L17 treated with DTT. The ν₁ region of the Raman spectra induced by 528.7 nm excitation of isolated zeaxanthin, violaxanthin, lutein, and neoxanthin dissolved in pyridine are also shown for comparison. rel., relative.

FIGURE 4.

Comparison of the ν₃ region of the qE-associated Raman spectrum (difference spectrum of light-treated-minus-dark recovery) induced by 528.7 nm excitation in Arabidopsis leaves from wild type and L17 treated with DTT. The ν₃ region of the Raman spectra induced by 528.7 nm excitation of isolated violaxanthin and lutein dissolved in pyridine and the in vivo violaxanthin spectrum induced by 488.0 nm excitation (difference spectrum of wild-type dark adapted-minus-dark recovery leaves) are shown for comparison. rel., relative.

FIGURE 5.

ν₄ region of the qE-associated Raman spectrum (difference spectrum of light-treated-minus-dark recovery) induced by 528.7 nm excitation in Arabidopsis leaves from L17 plants. The ν4 region of the Raman spectra induced by 528.7 nm excitation (difference spectrum of wild-type dark adapted-minus-dark recovered leaves) (trace 1) is presented in comparison with the spectrum of isolated violaxanthin dissolved in lipid micelles (trace 2). rel., relative.

FIGURE 6.

LHCII aggregation-associated Raman spectra (difference spectrum of aggregated-minus-trimeric) for LHCII binding zeaxanthin (dashed line) and violaxanthin (solid line) in the peripheral V1 binding site in the ν₁ (A) and the ν₃ (B) regions. Excitation is at 528.7 nm. rel., relative.