The aggregation dependent correlation between fluorescence quenching and the electronic carotenoid–chlorophyll interactions, ϕCouplingCar S1-Chl, as measured by comparing chlorophyll fluorescence observed after two- and one-photon excitation, has been investigated using native LHC II samples as well as mutants lacking Chl 2 and Chl 13. For native LHC II the same linear correlation between ϕCouplingCar S1-Chl and the fluorescence quenching was observed as previously reported for the pH and Zea-dependent quenching of LHC II [1]. In order to elucidate which carotenoid–chlorophyll pair might dominate this correlation we also investigated the mutants lacking Chl 2 and Chl 13. However, also with these mutants the same linear correlation as for native LHC II was observed. This provides indication that these two chlorophylls play only a minor role for the observed effects. Nevertheless, we also conclude that this does not exclude that their neighboured carotenoids, lutein 1 and neoxanthin, might interact electronically with other chlorophylls close by.

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S1, shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S1) and chlorophyll (Chl) states, φCouplingCar S1−Chl, correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both Chl fluorescence quenching and φCouplingCar S1−Chl with the intensity of red-shifted bands in the Chl Qy and carotenoid absorption was also observed. These results suggest quenching excitonic Car S1−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 φCouplingCar S1−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 S1–Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, φCouplingCar S1−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 S1–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.

The Car S1 → Chl energy transfer efficiency, ΦTransfer, in xanthophyll-cycle mutants of living plants and LHC II was investigated by selective Car S1 two-photon excitation. Before high-light illumination ΦTransfer, of the violaxanthin deficient mutant npq2 is ∼30% smaller than the corresponding value for wild type plants. For the zeaxanthin deficient mutant, npq1, ΦTransfer is ∼30% larger. Wild type Arabidopsis thaliana is the only variant which is capable of a light-dependent decrease of up to 40% and complete recovery to the original ΦTransfer values. In contrast, ΦTransfer remains constant during dark adaptation in both mutants. Surprisingly, changes in ΦTransfer of LHC II preparations were less than 5% only, when substituting violaxanthin by zeaxanthin.