Mechanisms of non-photochemical chlorophyll fluorescence quenching in higher plants

The excitation energy of pigment molecules in photosynthetic antennae systems is utilised by photochemistry, partly it is thermally dissipated, and partly it is emitted as fluorescence. Changes in the quantum yield of chlorophyll (Chl) fluorescence reflect the changes in quantum yield of photochemical reaction and thermal dissipation of the excitation energy. Decrease of the Chl fluorescence quantum yield is called the Chl fluorescence quenching. The decrease of the quantum yield that is accompanied by photochemical reactions has been termed the photochemical quenching, and the decrease accompanied by thermal dissipation of the excitation energy is called the non-photochemical quenching. This review deals with mechanisms of the non-photochemical quenching.     

Mechanisms of non-photochemical chlorophyll fluorescence quenching in higher plants

The excitation energy of pigment molecules in photosynthetic antennae systems is utilised by photochemistry, partly it is thermally dissipated, and partly it is emitted as fluorescence. Changes in the quantum yield of chlorophyll (Chl) fluorescence reflect the changes in quantum yield of photochemical reaction and thermal dissipation of the excitation energy. Decrease of the Chl fluorescence quantum yield is called the Chl fluorescence quenching. The decrease of the quantum yield that is accompanied by photochemical reactions has been termed the photochemical quenching, and the decrease accompanied by thermal dissipation of the excitation energy is called the non-photochemical quenching. This review deals with mechanisms of the non-photochemical quenching.     

On the relationship between non-photochemical quenching and photoprotection of Photosystem II

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high-light stress in photosynthetic organisms. NPQ is typically characterized by modulated pulse fluorometry and it is often assumed implicitly to be a good proxy for the actual physiological photoprotection capacity of the organism. Using the results of previously published ultrafast fluorescence measurements on intact leaves of w.t. and mutants of Arabidopsis (Holzwarth et al. 2009) we have developed exact relationships for the fluorescence quenching and the corresponding Photosystem II acceptor side photoprotection effects under NPQ conditions. The approach based on the exciton-radical pair equilibrium model assumes that photodamage results from triplet states generated in the reaction center. The derived relationships allow one to distinguish and determine the individual and combined quenching as well as photoprotection contributions of each of the multiple NPQ mechanisms. Our analysis shows inter alia that quenching and photoprotection are not linearly related and that antenna detachment, which can be identified with the so-called qE mechanism, contributes largely to the measured fluorescence quenching but does not correspond to the most efficient photoprotective response. Conditions are formulated which allow simultaneously the maximal photosynthetic electron flow as well as maximal acceptor side photoprotection. It is shown that maximal photoprotection can be achieved if NPQ is regulated in such a way that PSII reaction centers are open under given light conditions. The results are of fundamental importance for a proper interpretation of the physiological relevance of fluorescence-based NPQ data.     

Relationship between xanthophyll cycle and non-photochemical quenching in rice (Oryza sativa L.) plants in response to light stress

Thirty days old rice plants grown under low and moderate light conditions were transferred to full sunlight to observe the extent of photoinhibitory damage and protective mechanism, and the relationship between xanthophyll cycle and nonphotochemical quenching (qN) under changing light environment. Control plants (low, moderate and sun grown) exhibited similar Fv/Fm ratio, indicating similar photosynthetic efficiency prior to light stress. On exposure to the high light treatment, low light grown plants exhibited faster and higher degree of photoinhibition compared to moderate and high light grown plants. Moderate and high light grown plants showed relatively less photoinhibition and also showed higher qN, indicating better capacity of energy dissipation. Increase in qN in moderate light and sun grown plants was accompanied by conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) indicating operation of Z-dependent thermal dissipation. Rice plants fed with ascorbate (AsA), a stimulator of the de-epoxidation state of V to Z, showed higher Fv/Fm ratio and qN than the plants fed with dithiothreitol (DTT) an inhibitor of xanthophyll cycle. This indicated that an increased amount of energy reached PS II reaction centre, due to absence of A and Z formation, thereby causing greater damage to photosynthesis in DTT fed rice plants. The present data confirmed the relationship between qN and Z in dissipating the excess light energy, thereby protecting plants against photodamage.     

A kinetic model of non-photochemical quenching in cyanobacteria

High light poses a threat to oxygenic photosynthetic organisms. Similar to eukaryotes, cyanobacteria evolved a photoprotective mechanism, non-photochemical quenching (NPQ), which dissipates excess absorbed energy as heat. An orange carotenoid protein (OCP) has been implicated as a blue-green light sensor that induces NPQ in cyanobacteria. Discovered in vitro, this process involves a light-induced transformation of the OCP from its dark, orange form (OCP(o)) to a red, active form, however, the mechanisms of NPQ in vivo remain largely unknown. Here we show that the formation of the quenching state in vivo is a multistep process that involves both photoinduced and dark reactions. Our kinetic analysis of the NPQ process reveals that the light induced conversion of OCP(o) to a quenching state (OCP(q)) proceeds via an intermediate, non-quenching state (OCP(i)), and this reaction sequence can be described by a three-state kinetic model. The conversion of OCP(o) to OCP(i) is a photoinduced process with the effective absorption cross section of 4.5 × 10(-3)?2 at 470 nm. The transition from OCP(i) to OCP(q) is a dark reaction, with the first order rate constant of approximately 0.1s(-1) at 25°C and the activation energy of 21 kcal/mol. These characteristics suggest that the reaction rate may be limited by cis-trans proline isomerization of Gln224-Pro225 or Pro225-Pro226, located at a loop near the carotenoid. NPQ decreases the functional absorption cross-section of Photosystem II, suggesting that formation of the quenched centers reduces the flux of absorbed energy from phycobilisomes to the reaction centers by approximately 50%.     

Programmed cell death in plants: distinguishing between different modes

Programmed cell death (PCD) in plants is a crucial componentof development and defence mechanisms. In animals, differenttypes of cell death (apoptosis, autophagy, and necrosis) havebeen distinguished morphologically and discussed in these morphologicalterms. PCD is largely used to describe the processes of apoptosisand autophagy (although some use PCD and apoptosis interchangeably)while necrosis is generally described as a chaotic and uncontrolledmode of death. In plants, the term PCD is widely used to describemost instances of death observed. At present, there is a vastarray of plant cell culture models and developmental systemsbeing studied by different research groups and it is clear fromwhat is described in this mass of literature that, as with animals,there does not appear to be just one type of PCD in plants.It is fundamentally important to be able to distinguish betweendifferent types of cell death for several reasons. For example,it is clear that, in cell culture systems, the window of timein which ‘PCD’ is studied by different groups varieshugely and this can have profound effects on the interpretationof data and complicates attempts to compare different researcher'sdata. In addition, different types of PCD will probably havedifferent regulators and modes of death. For this reason, inplant cell cultures an apoptotic-like PCD (AL-PCD) has beenidentified that is fairly rapid and results in a distinct corpsemorphology which is visible 4–6 h after release of cytochromec and other apoptogenic proteins. This type of morphology, distinctfrom autophagy and from necrosis, has also been observed inexamples of plant development. In this review, our model systemand how it is used to distinguish specifically between AL-PCDand necrosis will be discussed. The different types of PCD observedin plants will also be discussed and the importance of distinguishingbetween different forms of cell death will be highlighted. Key words: Apoptosis, apoptosis-like programmed cell death (AL-PCD), Arabidopsis, autophagy, mitochondria, necrosis, programmed cell death (PCD) Received 5 June 2007; Revised 13 September 2007 Accepted 20 September 2007     

Activation of non-photochemical quenching in thylakoids and leaves

The mechanism of rapidly-relaxing non-photochemical quenching in two plant species,Chenopodium album L. andDigitalis purpurea L., that differ considerably in their capacity for such quenching has been investigated (Johnson G.N. et al. 1993, Plant Cell Environ.16, 673–679). Illumination of leaves of both species in the presence of 2% O₂ balance N₂ led to the formation of zeaxanthin. When thylakoids were isolated from leaves of each species that had been so treated it was found that inD. purpurea non-photochemical quenching was “activated” relative to the control; a higher level of quenching was found for a given trans-thylakoid pH gradient. No such activation of non-photochemical quenching was observed inC. album. Similar conclusions were drawn when comparing quenching in intact leaves. It is concluded that light activation of quenching is a process that cannot readily be induced inC. album. Measurement of the sensitivity of non-photochemical quenching in leaves ofC. album andD. purpurea to dithiothreitol (DTT; a reagent that inhibits formation of zeaxanthin) showed differences between the two species. In both cases, feeding leaves with DTT inhibited the light-induced formation of zeaxanthin. InC. album this was accompanied by complete inhibition of reversible non-photochemical quenching, whereas inD. purpurea this inhibition was only partial. Data are discussed in relation to studies on the mechanism of quenching and the role of zeaxanthin in this process.     

Ecophysiological aspects of biomass production in higher plants

Photosynthesis as a cosmic process of local entropy decrease is the basic phenomenon for life on Earth. Biomass production is energetically understood as a long-term storage of hydrogen. Endogeneously, the hierarchy of energy storage begins with the electric and proton gradient across the thylakoid membrane. At all higher levels of energy-converting system, efficiency is determined by the interactions between the autotrophic and heterotrophic part of the studied biosystem. Specially, the regulatory function of anabolic and catabolic reduction charge is discussed. From the frame of exogeneous factors, the action of calcium in connection with calmodulin is described as a second messenger.Summarizing, the findings are integrated into the energy-overflow concept in which the loss terms act as system stabilizers especially under injuring conditions.Paper presented at the FESPP meeting in Strasbourg (1984).     

Engineering photorespiration in chloroplasts: a novel strategy for increasing biomass production

Photosynthetic carbon metabolism is rate limiting in C3 plants because of a competing process: photorespiration. Photorespiration lowers the energy efficiency of photosynthesis by metabolizing glycolate produced by the oxygenate activity of Rubisco. The chloroplasts of Arabidopsis thaliana have recently been reported to contain a novel respiratory pathway that converts glycolate directly to glycerate and thus increases productivity by improving photosynthesis in transgenic plants. This pathway promises to widen the applicability of the approach to other C3 plants.     

Resolution of components of non-photochemical chlorophyll fluorescence quenching in barley leaves

Non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves has been analysed by monitoring its relaxation in the dark, by applying saturating pulses of light. At least three kinetically distinct phases to qN recovery are observed, which have previously been identified (Quick and Stitt 1989) as being due to high-energy state quenching (fast), excitation energy redistribution due to a state transition (medium) and photoinhibition (slow). However, measurements of chlorophyll fluorescence at 77 K from leaf extracts show that state transitions only occur in low light conditions, whereas the medium component of qN is very large in high light. The source of that part of the medium component not accounted for by a state transition is discussed.Abbreviations ATP adenosine 5-triphosphate - DCMU 3[3,4-dichlorophenyl]-1,1 dimethylurea - pH trans-thylakoid pH gradient - F_o, F_m room-temperature chlorophyll fluorescence yield with all reaction centres open, closed - F_v variable fluorescence = F_m–F_o - LHC II Light harvesting complex II - PS I, PS II Photosystem I, II - P700, P680 primary donor in photosystem I, II - qP photochemical quenching of variable fluorescence - qN non-photochemical quenching of variable fluorescence - qN_e, qN_t, qN_i non-photochemical quenching due to high energy state, state transition, photoinhibition - qN_f, qN_m, qN_s components of qN relaxing fast, medium, slow - q_r quenching of r relative to the dark state - tricine N-tris[hydroxymethyl]methylglycine - r ratio of fluorescence maximum from photosystem II to that from photosystem I at 77 K     

The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves

It has been suggested previously that non-photochemical quenching of chlorophyll fluorescence is associated with a decrease in the rate of photosystem 2 (PS 2) photochemistry. In this study analyses of fluorescence yield changes, induced by flashes in leaves exhibiting different amounts of non-photochemical quenching of fluorescence, are made to determine the effect of non-photochemical excitation energy quenching processes on the rate of PS 2 photochemistry. It is demonstrated that both the high-energy state and the more slowly relaxing components of non-photochemical quenching reduce the rate of PS 2 photochemistry. Flash dosage response curves for fluorescence yield show that non-photochemical quenching processes effectively decrease the relative effective absorption cross-section for PS 2 photochemistry. It is suggested that non-photochemical quenching processes exert an effect on the rate of PS 2 photochemistry by increasing the dissipation of excitation energy by non-radiative processes in the pigment matrices of PS 2, which consequently results in a decrease in the efficiency of delivery of excitation energy for PS 2 photochemistry.     

Site,trigger, quenching mechanism and recovery of non-photochemical quenching in cyanobacteria: recent updates

Femtosecond transient absorption was used to study excitation decay in monomeric and trimeric cyanobacterial Photosystem I (PSI) being prepared in three states: (1) in aqueous solution, (2) deposited and dried on glass surface (either conducting or non-conducting), and (3) deposited on glass (conducting) surface but being in contact with aqueous solvent. The main goal of this contribution was to determine the reason of the acceleration of the excitation decay in dried PSI deposited on the conducting surface relative to PSI in solution observed previously using time-resolved fluorescence (Szewczyk et al., Photysnth Res 132(2):111–126, 2017). We formulated two alternative working hypotheses: (1) the acceleration results from electron injection from PSI to the conducting surface; (2) the acceleration is caused by dehydration and/or crowding of PSI proteins deposited on the glass substrate. Excitation dynamics of PSI in all three types of samples can be described by three main components of subpicosecond, 3–5, and 20–26 ps lifetimes of different relative contributions in solution than in PSI-substrate systems. The presence of similar kinetic components for all the samples indicates intactness of PSI proteins after their deposition onto the substrates. The kinetic traces for all systems with PSI deposited on substrates are almost identical and they decay significantly faster than the kinetic traces of PSI in solution. We conclude that the accelerated excitation decay in PSI-substrate systems is caused mostly by dense packing of proteins.     

Theoretical assessment of alternative mechanisms for non-photochemical quenching of PS II fluorescence in barley leaves

The components of non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves have been quantified by a combination of relaxation kinetics analysis and 77 K fluorescence measurements (Walters RG and Horton P 1991). Analysis of the behaviour of chlorophyll fluorescence parameters and oxygen evolution at low light (when only state transitions — measured as qN_t — are present) and at high light (when only photoinhibition — measured as qN_i — is increasing) showed that the parameter qN_t represents quenching processes located in the antenna and that qN_i measures quenching processes located in the reaction centre but which operate significantly only when those centres are closed. The theoretical predictions of a variety of models describing possible mechanisms for high-energy-state quenching, measured as the residual quenching, qN_e, were then tested against the experimental data for both fluorescence quenching and quantum yield of oxygen evolution. Only one model was found to agree with these data, one in which antennae exist in two states, efficient in either energy transfer or energy dissipation, and in which those photosynthetic units in a dissipative state are unable to exchange energy with non-dissipative units.Abbreviations: F_o, F_m room-temperature chlorophyll fluorescence yield with all centres open, closed - F_v variable fluorescence yield - LHC II light-harvesting chlorophyll-protein complex of PS II - PS I, PS II Photosystem I, II - P700, P680 primary donor in Photosystem I, II - Q_A primary electron acceptor of PS II - P_max maximum quantum yield of oxygen evolution - qN coefficient of non-photochemical quenching of variable fluorescence - qN_e, qN_t, qN_i coefficient of non-photochemical quenching due to high-energy-state, state transition, photoinhibition - qO coefficient of quenching of dark level fluorescence - qP coefficient of photochemical quenching of variable fluorescence - _P intrinsic quantum yield of open PS II reaction centres = _s/qP - _{PS 2} quantum yield of PS = qP × F_v/F_m - _S quantum yield of oxygen evolution = rate of oxygen evolution/light intensity     

A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis--chlorophyll fluorescence quenching by the xanthophylls

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary-an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and beta-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and beta-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and beta-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.     

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina¹⁰⁴) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light. It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of Q_A reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flash During greening in continuous light of IL pea leaves, an inhibitory effect on Q_A photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.     

Far-red fluorescence: a direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching

Time-resolved fluorescence on oligomers of the main light-harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter-trimer chlorophyll-chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far-red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400ps fluorescence component appearing in intact leaves of Arabidopsis when non-photochemical quenching (NPQ) is induced. The fluorescence components with high far-red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far-red emitting state is shown to be an emissive Chl-Chl charge transfer state which plays a crucial part in the quenching.     

The occurrence of rapidly reversible non-photochemical quenching of chlorophyll a fluorescence in cyanobacteria

Cyanobacteria have previously been considered to differ fundamentally from plants and algae in their regulation of light harvesting. We show here that in fact the ecologically important marine prochlorophyte, Prochlorococcus, is capable of forming rapidly reversible non-photochemical quenching of chlorophyll a fluorescence (NPQf or qE) as are freshwater cyanobacteria when they employ the iron stress induced chlorophyll-based antenna, IsiA. For Prochlorococcus, the capacity for NPQf is greater in high light-adapted strains, except during iron starvation which allows for increased quenching in low light-adapted strains. NPQf formation in freshwater cyanobacteria is accompanied by deep Fo quenching which increases with prolonged iron starvation.     

LHC II protein phosphorylation in leaves of Arabidopsis thaliana mutants deficient in non-photochemical quenching

Phosphorylation of the light-harvesting chlorophyll a/b complex II (LHC II) proteins is induced in light via activation of the LHC II kinase by reduction of cytochrome b₆f complex in thylakoid membranes. We have recently shown that, besides this activation, the LHC II kinase can be regulated in vitro by a thioredoxin-like component, and H₂O₂ that inserts an inhibitory loop in the regulation of LHC II protein phosphorylation in the chloroplast. In order to disclose the complex network for LHC II protein phosphorylation in vivo, we studied phosphorylation of LHC II proteins in the leaves of npq1-2 and npq4-1 mutants of Arabidopis thaliana. In comparison to wild-type, these mutants showed reduced non-photochemical quenching and increased excitation pressure of Photosystem II (PS II) under physiological light intensities. Peculiar regulation of LHC II protein phosphorylation was observed in mutant leaves under illumination. The npq4-1 mutant was able to maintain a high amount of phosphorylated LHC II proteins in thylakoid membranes at light intensities that induced inhibition of phosphorylation in wild-type leaves. Light intensity-dependent changes in the level of LHC II protein phosphorylation were smaller in the npq1-2 mutant compared to the wild-type. No significant differences in leaf thickness, dry weight, chlorophyll content, or the amount of LHC II proteins were observed between the two mutant and wild-type lines. We propose that the reduced capacity of the mutant lines to dissipate excess excitation energy induces changes in the production of reactive oxygen species in chloroplasts, which consequently affects the regulation of LHC II protein phosphorylation.     

A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis—chlorophyll fluorescence quenching by the xanthophylls

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary—an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and β-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and β-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and β-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.