One- and two-photon pump–probe optical spectroscopic measurements reveal the S1 and intramolecular charge transfer states are distinct in fucoxanthin

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S(1), shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S(1)) and chlorophyll (Chl) states, phi(Coupling)(Car S(1)-Chl), correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both Chl fluorescence quenching and phi(Coupling)(Car S(1)-Chl) with the intensity of red-shifted bands in the Chl Q(y) and carotenoid absorption was also observed. These results suggest quenching excitonic Car S(1)-Chl states as origin for the observed effects. Furthermore, real time measurements of the light-dependent down- and up-regulation of the photosynthetic activity and phi(Coupling)(Car S(1)-Chl) in wild-type and mutant (npq1, npq2, npq4, lut2 and WT+PsbS) Arabidopsis thaliana plants reveal that also in vivo the quenching parameter NPQ correlates always linearly with the extent of electronic Car S(1)-Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, phi(Coupling)(Car S(1)-Chl) depends on the presence of PsbS and zeaxanthin (Zea) in an almost identical way as NPQ. In summary, these results provide clear evidence for a very close link between electronic Car S(1)-Chl interactions and the regulation of photosynthesis. These findings support a photophysical mechanism in which short-living, low excitonic carotenoid-chlorophyll states serve as traps and dissipation valves for excess excitation energy.