Skip to main content
European Commission logo print header

Higher plant photosynthesis: Identification of the chlorophyll fluorescence quencher

Final Report Summary - HIGH-IQ (Higher plant photosynthesis: Identification of the chlorophyll fluorescence quencher)

Final scientific report FP7-People-2007-2-2-ERG: 'Higher plant photosynthesis: Identification of the chlorophyll fluorescence quencher'

The scientific results obtained during the duration of the project were subject to a number of papers and the results are summarized below:

Single-molecule spectroscopy was employed to elucidate the fluorescence spectral heterogeneity and dynamics of individual, immobilized trimeric complexes of the main light-harvesting complex of plants in solution near room temperature. Rapid reversible spectral shifts between various emitting states, each of which was quasi-stable for seconds to tens of seconds, were observed for a fraction of the complexes. Most deviating states were characterized by the appearance of an additional, red-shifted emission band. Reversible shifts of up to 75 nm were detected. By combining modified Redfield theory with a disordered exciton model, fluorescence spectra with peaks between 670 nm and 705 nm could be explained by changes in the realization of the static disorder of the pigment-site energies. Spectral bands beyond this wavelength window suggest the presence of special protein conformations. We attribute the large red shifts to the mixing of an excitonic state with a charge-transfer state in two or more strongly coupled chlorophylls. Spectral bluing is explained by the formation of an energy trap before excitation energy equilibration is completed.

We have developed a simple method to resolve discrete intensity shifts from time-resolved single-molecule fluorescence emission data, where multiples of the standard deviation of the measured intensities is integrated into short time bins. The algorithm has been apply to the intensity traces of trimeric units of the main light-harvesting complex of plants. It has been shown that the amount of information that can be extracted from an intensity time trace increases considerably, thereby enlarging the possibility to reveal new phenomena. The technique is particularly applicable to the analysis of fluorescence intermittency from multichromophoric systems.

We have applied this algorithm to study the time-resolved fluorescence intensity fluctuations from LHCII of plants in different pH environments close to room temperature and under different light conditions. The efficiency of light harvesting, which was represented by complexes typically residing for long periods in strongly fluorescing states, was significantly reduced by decreasing the pH or increasing the incident photon flux. The same environmental changes significantly increased the switching frequency between strongly and weakly fluorescing states. The strong environmental sensitivity suggests that the immediate environment of an LHCII complex can modulate the amount of energy dissipation. We suggest that the dynamic equilibrium between the strongly and weakly fluorescing states can be shifted by environmentally controlling the conformational diffusion on the potential energy surface of LHCII.

Moreover, we have investigated the single-molecule fluorescence intermittency of the LHCII under conditions that mimic efficient utilization of light or light dissipation (qE component of nonphotochemical quenching process), and demonstrate that weakly fluorescing states are stabilized under qE conditions. Thus, qE is explained by biological control over the intrinsic dynamic disorder in the complex – the frequencies of switching establish whether the population of complexes is unquenched or quenched.

Non-photochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in LHCII in the absence of protein–protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In order to gain insight into the changes in conformation of the pigments involved in NPQ we applied resonance Raman spectroscopy on the LHCII gels. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a signi?cant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid dark states (Car S1) performed on LHCII gels shows that the extent of electronic interactions between Car S1 and chlorophyll states correlates linearly with Chl fluorescence quenching, as previously observed for isolated LHCII (aggregated vs trimeric) and whole plants (with vs without NPQ).

However, the role of the carotenoid itself has been challenged recently, studies suggesting that the carotenoid is not at all involved in energy dissipation in LHCII. We addressed this by applying ultrafast transient-absorption spectroscopy on the above mentioned LHCII gels. We show that a carotenoid excited state is populated concomitantly with the decay of the chlorophyll excited state and its transient population reaches its maximum concentration on a timescale comparable with the carotenoid excited state lifetime.

NPQ is triggered by the transmembrane proton gradient (?pH) 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 characterised by a series of positive and negative bands, the best-known of which is ?A535. 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 unique vibrational signature. Using this technique the origin of ?A535 was previously shown to be a sub-population of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin) a portion of NPQ remains and the ?A535 change is blue-shifted to 525 nm (?A525). Using Resonance Raman spectroscopy it is shown that the ?A525 absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted sub-population of violaxanthin molecules formed during NPQ. The presence of the same ?A535 and ?A525 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.

Publications

Krüger, T.P.J. Novoderezhkin V.I. Ilioaia C. and van Grondelle R. (2010) Fluorescence Spectral Dynamics of Single LHCII Trimers- Biophysical Journal 98, 3093-3101

Ilioaia, C., Johnson, M.P. Duffy C.D.P. Pascal, A.A. van Grondelle, R., Robert B. and Ruban A.V. (2011) The origin of absorption changes associated with photoprotective energy dissipation in the absence of zeaxanthin- Journal of Biological Chemistry 286, 91-98

Ilioaia, C., Johnson M. P., Liao, P-N., Pascal, A.A. van Grondelle, R., Walla P.J. Ruban A.V. and Robert B. (2011) Photoprotection in Higher Plants involves a change in Lutein 1 binding site of the Major Light-Harvesting Complexes.-Journal of Biological Chemistry 286, 27247-27254

Krüger, T.P.J. Ilioaia C. and van Grondelle R. (2011) Fluorescence intermittency of the main plant light-harvesting complex: resolving the shifts between intensity levels. - Journal of Physical Chemistry B 115, 5071–5082

Krüger, T.P.J. Ilioaia C., Valkunas, L. and van Grondelle R. (2011) Fluorescence intermittency of the main plant light-harvesting complex: sensitivity to the local environment. -Journal of Physical Chemistry B 115, 5083–50895