Two-Photon Excitation of Photosynthetic Carotenoids
Photosynthesis
During a typical day plants are exposed to sunlight intensities over several orders of magnitude. Under low light plants use nearly all absorbed energy for the photosynthetic reactions. During high light conditions the photosynthetic apparatus must be protected against excess excitation energy, as it might harm the reaction centers. A quick balance between energy collection on the one hand and energy dissipation on the other hand is crucial for the survival and fitness of natural and gene modified plants.
The photosynthetic pigments (chlorophyll-green, carotenoids-ornage) that absorb the sunlight are located in proteins that are called photosystems. LHCII is the most important light-harvesting pigment-protein complex in the photosynthetic apparatus of plants. It serves as an antenna complex of the photosystem II-supercomplex and transfers all absorbed energy to the reaction centers. It has long been known that for the down-regulation of the photosynthetic activity an increase of the pH-gradient across the thylakoid membrane and the presence of the protein PsbS are needed. Furthermore the carotenoid zeaxanthin (Zea) is simultaneously formed from violaxanthin (Vio) through the enzymatic xanthophyll cycle.
Although many different studies have been undertaken to elucidate the details of this regulation, a complete picture of its mechanisms is still missing. Several different regulation models have been proposed and indeed it cannot be excluded that different mechanisms contribute more or less to plants adaptation to varying light conditions. However, at present even the regulation site and photophysical mechanisms are unresolved, as the models are at least partly contradicting each other.
Photosystem II - Supercomplex with two central PSII proteins, six LHCII proteins and six minor antennas (CP24, CP26, CP29). Absorbed energy (yellow) is transferred via Förster Energy Transfer to the reaction center (violet).
The function of carotenoids in photosynthesis – timeresolved 2-photon fs-spectroscopy
Carotenoids play a crucial role as light-harvesting pigments in photosynthesis and in the regulation of the energy flows in the photosynthetic apparatus. In the Light-harvesting complex II (LHC II), which collects more than 50 % of the light used for photosynthesis on earth, carotenoids contribute about 20-30 % to this huge amount of collected energy. In addition, they seem to play an important role in the regulation of excessive excitation energy under typical conditions of sun light intensities rapidly varying over several orders of magnitude during a day. It is known that the regulation of photosynthetic activity under these circumstances is essential for the survival and fitness of natural and gene modified plants. However, a detailed insight in the underlying mechanisms was very often impossible due the optical forbidden character of the first excited state of carotenoids, Car S1. This is because it has the same Ag- symmetry as the ground state, Car S0 . An elegant approach is the investigation via two-photon excitation because Ag- →Ag- –transitions are generally two-photon allowed. Using this approach we gain important information about the light harvesting mechanisms and the regulation of excessive excitation energy under high-light conditions in the photosynthetic apparatus.
Left: Energy level diagram of the ground and excited states of chlorophyll a. Right: Energy level diagram of the ground and excited states of carotene Middle: The combination of OPE of the chlorophyll ground state and selective two-photon excitation spectroscopy enables the measurement of spectra of the carotenoid dark state within intact pigment-protein complexes and entire plants.
Materials and Methods
To get an insight into the detailed mechanisms of non-photochemical quenching and the contribution of carotenoids we use a simultaneous combination of one- and two-photon excitation (OPE, TPE) on isolated pigment-protein complexes and entire plants of Arabidopsis thaliana. We have developed a two-photon-excitation (TPE) set-up which is combined with a standard PAM fluorescence monitoring system using a confocal arrangement. With the PAM it is not possible to directly excite the carotenoid dark states as they are optically forbidden. But due to different selection rules they can be directly excited by TPE. The dark state of the carotenoids in a living plant or pigment-protein complex is populated selectively via TPE and simultaneous a standard measurement with light and dark adaptation by applying actinic light is performed with the PAM. We are able to determine the different chlorophyll fluorescence parameters after OPE (via the PAM) and TPE (via the new setup) at the same time and on the same spot of a leaf. Thus our measurement allows a deeper insight into the energetic transfer mechanisms between the carotenoid dark state and chlorophylls during light adaptation.
Results
LHCII
In LHCII the electronic Car-Chl interactions correlate always linearly with chlorophyll fluorescence quenching under various experimental conditions. In addition, a linear correlation between both Chl flourescence quenching and the intensity of red-shifted bands in the Chl Qy and Carotenoid absorption can be observed.
LHCII experiments were performed at different pH values. A: Fluorescence after OPE B: Fluorescence after TPE C: Calculated value of coupling between Carotenoids and chlorophylls. At low pH and with high amounts of Zea the coefficient becomes bigger. D-F: Increase in the red shift in the absorption spectra of Zea enriched LHCII corresponds linearly to the regulation status (NPQ).
Arabidopsis thaliana
The electronic Car S1-Chl interactions are invariably and directly correlated to the extent of NPQ, regardless of the current light intensities, adaptation status or growth conditions.They depend on the presence of PsbS and zeaxanthin in an almost identical way as NPQ.
Left: Results of Car-Chl coupling during adaptation for a variety of Arabidopsis thaliana mutants. Right: The Car-Chl interactions are directly correlated to quenching conditions.
Conclusion
All experimental observations and the quenching mechanism can be explained by a model in which the formation of excitonic Car S1–Chl a Qy interactions lead to short-living carotenoid–chlorophyll states that serve as traps and dissipation valves for excess excitation energy. Excitonic interactions generally lead to two new electronic states that are more or less delocalized over the two molecules and have characteristics of both original monomeric states. One state is lower than the energies of the original monomeric states, the other is higher. In a pigment pool, such low lying excitonic states act as local energy trap for the entire pool. Since the excited state lifetime of the original, monomeric Car S1 states is with ~10-30 ps orders of magnitude shorter than the fluorescence lifetime of the original Chl a Qy state (~ 1800 ps) even small excitonic mixing lead to a drastic reduction in the fluorescence lifetime of the involved Chl molecule and consequently to very effective dissipation of excitation energy. These changes in the Chl fluorescence quantum yield and lifetime correspond exactly to the changes in the Chl fluorescence of plants observed during the photosynthetic regulation.
Proposed model: a/b) A quantitative comparison of the chlorophyll fluorescence intensity detected after selective two-photon excitation (TPE) of the carotenoid dark states Car S1, FlTPE, and direct one-photon excitation (OPE) of the chlorophyll states Chl a Qy, FlOPE, allows quantifying the current extent in the electronic interactions, Phi, between these states even during the regulation of in intact plants. When the energy levels of Car S1 and Chl a Qy are similar, increased electronic interactions lead to the formation of excitonic states that are delocalized over both molecules . b/c) The lower and short-lived excitonic Car S1-Chl a Qy state serves as an energy sink and dissipation valve for a large amount of excess excitation energy in the photosynthetic pigment pool, enabling regulation of plant photosynthesis. Molecules and orbitals displayed with VMD.
Literature:
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