Nonphotochemical Quenching in Plants and Cyanobacteria
The activity of PS II can be efficiently examined by measuring the fluorescence of Chl a, most frequently in form of prompt fluorescence or transient fluorescence measurements by recording Chl fluorescence induction curves imaging the transient development of photochemical and nonphotochemical quenchers in dark-adapted samples after excitation by intense light (Genty et al., 1989, Rohacek, 2002, Stribet and Govindjee, 2011).
Several other techniques of fluorescence detection are used to analyze PSII activity. Delayed fluorescence (DF) is a suitable indicator to measure the recombination fluorescence response at the reaction centers of PSII that can be monitored as a very weak fluorescence signal with characteristic kinetics (Bigler and Schreiber, 1990; Goltsev et al., 2009). Prompt Chl fluorescence is analyzed to obtain the rate constants for charge separation and charge stabilization directly by evaluating time- and wavelength-resolved fluorescence measurements with model-based kinetic analysis (Schatz et al., 1988; Roelofs et al., 1992; Renger et al., 1995; Kramer and Crofts, 1996; Schmitt, 2011). Generally fluorescence is suitable to quantify photochemical or nonphotochemical quenching of excited states.
During evolution, cyanobacteria and plants have developed various mechanisms of acclimation, in particular regulatory pathways for defense to stress induced by unfavorable environmental factors. One important regulation mechanism is nonphotochemical quenching (NPQ) which already starts in the antenna complexes as a regulatory mechanism for EET and excited state accumulation under high light conditions. In such way plants decrease the rate of ROS generation in their leaves, increase the rate of ROS scavenging and accelerate of the repair of damaged cell structures.
The different protection mechanisms operate in markedly different time domains and light intensities. The fastest response is the annihilation process of multiple excitons which are excited in plant antenna complexes, however, such multiexcitons just occur at extreme light intensities or at rare probability.
Light harvesting systems of plants are highly optimized structures measuring coupled domains as the optimum tradeoff between lightharvesting, excitation energy transfer and energy storage to optimize the EET efficiency to the reaction center (Lambrev et al., 2011).
At low light intensities light-harvesting is optimized, whereas at highlight conditions the excess energy needs to be dissipated by photoprotective mechanisms where NPQ is probably the most direct and most efficient mechanism. NPQ starts in the antenna complexes as a regulatory mechanism for excitation energy transfer and excited state accumulation under high light conditions.
The most important mechanisms of NPQ might be quenching of superfluous excitation energy by carotenoids (Cars) and the induction of NPQ processes due to acidification of the thylakoid lumen by formation of a transmembrane pH difference (Niyogi and Truong 2013; Demmig- Adams and Adams, 1996; Demmig-Adams et al., 2014; Ruban et al., 2012). In this context, the large ApH across the thylakoid membrane (with acidic luminal pH) that builds up under extreme light due to the limited capacity of the F()F1-ATPase system triggers NPQ mechanisms (Muller et al., 2001; Szabo et al., 2005).
A regulation of excitation energy funnelling to PS I and PS II in oxygen-evolving organisms occurs via a phenomenon designated “state transitions” which comprises reversible phosphorylation/dephosphoryla- tion of light-harvesting complexes II (Iwai et al., 2010).
Interestingly carotenoids seem to be of optimized chemical structure as dissipation molecules for excess energy. In contrast to the closed porphyrin molecule forming the light-harvesting system, chlorophyll, the carotenoids are long open carbon chains that exhibit a much larger rate constant for internal conversion. All carotenoids with more than ten conjugated C=C bonds have an excited singlet S1 state low enough to accept energy from excited Chl. However, the S1 state cannot be populated by one-photon absorption, but it can be reached upon rapid internal conversion from the S2 state or by direct Dexter-transfer of excitation energy from Chl molecules. In higher plants the dominating NPQ mechanism seems to be the light-induced and pH-dependent xanthophyll cycle (Hartel et al., 1996; Demmig-Adams et al., 1996). Additionally carotenoids effectively reduce the population of 3Chl in antenna systems as well as PS II of plants (Carbonera et al., 2012) and act as direct ROS scavengers. The interaction with singlet oxygen (:AgO2) does not only lead to NPQ, but also to oxidation of carotenoids by formation of species that can act as signal molecules for stress response (Ramel et al., 2012; Schmitt et al., 2014a).
In the violaxanthine cycle of plants and green or brown algae xanthine deepoxidases associate with thylakoid membranes at low pH. In that configuration violaxanthin deepoxygenase converts violaxanthin via antheraxanthin to zeaxanthin. Distinct cycles developed in other organisms as the diadinoxanthin cycle in diatoms (Muller et al., 2001). Zeaxanthin deactivates excited Chl molecules more efficiently than violaxanthin.
For an analysis of the hierarchy of light induced kinetic steps in the PS II by measurement of single flash induced transient quantum yield and modelling with a PS II reaction scheme respecting different NPQ mechanisms by rate equations see (Belyaeva et al., 2008, 2011, 2014, 2015, and references therein).
Also mobile carotenoid binding proteins are found in plants and cyanobacteria which can be pH activated or directly induced by high light due to photoswitchable complexes. The carotenoid binding PsbS subunit of PSII in higher plants acts as a pH activated excess energy quencher after conformational changes by rearranging the PS II complexes. The semi-crystalline ordering and increased fluidity of protein organization in the membrane leads to NPQ (Bergantino et al., 2003; Muller et al., 2001; Niyogi and Truong, 2013; Schmitt et al., 2014a; Goral et al., 2012).
A prominent example of a mobile protein in cyanobacteria is the photo-switchable orange carotenoid protein (OCP) in cyanobacteria (Wilson A. et al., 2008; Wilson et al., 2010; Boulay et al., 2010; Stadnichuk et al., 2013; Maksimov et al., 2014a, 2015). OCP is a directly photoswitchable protein containing 3’-hydroxy-echinenone as cofactor, but also echinenone and canthaxanthin lead to photoconversion and quenching of phycobilisomes (PBS). During its photocycle OCP undergoes a spectral shift by more than 20 nm from the orange OCPO to the photoactive red form OCPR. The structure of the active OCPR form is still unknown, which is likely due to substantial structural flexibility of the OCPR state. OCPR assumes a molten globule-like state with a larger hydrodynamic volume than the inactive OCPO form and attaches quickly to phycobilisomes after photoactivation (Maksimov et al., 2015; 2016). The fluorescence decay curves of phycobilisomes (PBS) interacting with activated OCPR are characterized by short decay components with (170 ps)-1 at strongest NPQ by OCPR. PBS, which are strongly interacting with OCPR, are lacking excitation energy transfer to the terminal emitter of the PBS antennae indicating that OCP quenches mainly the transfer from allophycocyanin in the PBS (Maksimov et al., 2014a). This fact was interpreted as intermolecular interaction between the OCPR and its binding site in the PBS core induced by blue light. Detailed spectroscopic studies and investigations of OCP mutants unraveled most probable H-bonds between two residues, Trp-298 and Tyr-203 and an oxygen localized at the beta-ring of 3’-hydroxyechi- nenone as the most important interaction to stabilize the orange form OCPO (Kirilovsky and Kerfeld, 2013; Leverenz at al., 2014; Maksimov et al., 2015; 2016).
Conclusively, Cars play a pivotal role (for reviews on the key role of Cars in photosynthesis, see Polivka and Sundstrom, 2004, Pogson et al., 2005) for NPQ developed under light stress (for a review, see Ruban et al., 2012) thus effectively reducing the population of 3Chl in antenna systems as well as PS II of plants (Carbonera et al., 2012). Cars, in addition, act as direct ROS scavengers. The interaction between :AgO2 and singlet ground state Cars does not only lead to photophysical quenching, but also to oxidation of Cars by formation of species that can act as signal molecules for stress response (Ramel et al., 2012).
Conformational changes of pigment-protein complexes are typically induced under high light conditions leading to the depletion of excited singlet states by internal conversion and interaction with quenching groups in the protein backbone. Recently, such conformational changes were artificially introduced by freezing of PBS of cyanobacteria and it was shown that this can reduce the fluorescence quantum yield of the PBS by 90% (Maksimov et al., 2013).
Light harvesting complexes containing phycobiliproteins are not prone to triplet formation since phycocyanobilins (linear tetrapyrrols) do not undergo inter-system crossing. However triplet states are the prerequisite for the formation of highly reactive singlet oxygen (1AgO2)-.
As they do not form triplets and 1AgO2, the PBS must not necessarily be quenched by carotenoids at high light conditions. The system just prevents EET to the Chl containing core antenna systems. Decoupling mechanisms seem also to occur that have been extensively studied for PBS and the rod-shaped phycobiliprotein antenna of the cyanobacterium A.marina including the EET processes on a molecular level (Schmitt et al., 2006; Theiss et al., 2008, 2011; Schmitt, 2011). It was found that the phycobiliprotein antenna of A.marina decouples from the PSII under cold stress (Schmitt et al., 2006) therefore reducing the influx of excitation energy into the PS II.
A detailed description of Chl a fluorescence as a reporter of the functional state of the PS II is found in (Strasser et al., 2000; Papageorgiou and Govindjee, 2005).