Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms

The diatom algae, responsible for at least a quarter of the global photosynthetic carbon assimilation in the oceans, are capable of switching on rapid and efficient photoprotection, which helps them cope with the large fluctuations of light intensity in the moving waters. The enhanced dissipation of excess excitation energy becomes visible as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Intact cells of the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum, which show different NPQ induction kinetics under high light illumination, were investigated by picosecond time-resolved fluorescence under dark and NPQ-inducing high light conditions. The fluorescence kinetics revealed that there are two independent sites responsible for NPQ. The first quenching site is located in an FCP antenna system that is functionally detached from both photosystems, while the second quenching site is located in the PSII-attached antenna. Notwithstanding their different npq induction and reversal kinetics, both diatoms showed identical NPQ via both mechanisms in the steady-state. Their fluorescence decays in the dark-adapted states were different, however. A detailed quenching model is proposed for NPQ in diatoms.     

Relaxation of the non-photochemical chlorophyll fluorescence quenching in diatoms: kinetics,components and mechanisms

Diatoms are especially important microorganisms because they constitute the larger group of microalgae. To survive the constant variations of the light environment, diatoms have developed mechanisms aiming at the dissipation of excess energy, such as the xanthophyll cycle and the non-photochemical chlorophyll (Chl) fluorescence quenching. This contribution is dedicated to the relaxation of the latter process when the adverse conditions cease. An original nonlinear regression analysis of the relaxation of non-photochemical Chl fluorescence quenching, qN, in diatoms is presented. It was used to obtain experimental evidence for the existence of three time-resolved components in the diatom Phaeodactylum tricornutum: qNf, qNi and qNs. qNf (s time-scale) and qNs (h time-scale) are exponential in shape. By contrast, qNi (min time-scale) is of sigmoidal nature and is dominant among the three components. The application of metabolic inhibitors (dithiothreitol, ammonium chloride, cadmium and diphenyleneiodonium chloride) allowed the identification of the mechanisms on which each component mostly relies. qNi is linked to the relaxation of the ΔpH gradient and the reversal of the xanthophyll cycle. qNs quantifies the stage of photoinhibition caused by the high light exposure, qNf seems to reflect fast conformational changes within thylakoid membranes in the vicinity of the photosystem II complexes.     

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.     

In diatoms, the transthylakoid proton gradient regulates the photoprotective non-photochemical fluorescence quenching beyond its control on the xanthophyll cycle

In diatoms, the non-photochemical fluorescence quenching (NPQ) regulates photosynthesis during light fluctuations. NPQ is associated with an enzymatic xanthophyll cycle (XC) which is controlled by the light-driven transthylakoid proton gradient (delta pH). In this report, special illumination conditions and chemicals were used to perturb the kinetics of the delta pH build-up, of the XC and of NPQ. We found that the delta pH-related acidification of the lumen is also needed for NPQ to develop by switching the xanthophylls to an 'activated' state, probably via the protonation of light-harvesting antenna proteins. It confirms the NPQ model previously proposed for diatoms.     

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     

Assessing the photoprotective effectiveness of non-photochemical chlorophyll fluorescence quenching: A new approach

The photoprotective nature of non-photochemical quenching (NPQ) has not been effectively quantified and the major reason is the inability to quantitatively separate NPQ that acts directly to prevent photoinhibition of photosystem II (PSII). Here we describe a technique in which we use the values of the PSII yield and qP measured in the dark following illumination. We expressed the quantum yield of PSII (Φ(PSII)) via NPQ as: Φ(PSII)=qP×(Fv/Fo)/(1+Fv/Fo+NPQ). We then tested this theoretical relationship using Arabidopsis thaliana plants that had been exposed to gradually increasing irradiance. The values of qP in the dark immediately after the illumination period (here denoted qPd) were determined using a previously described technique for Fo' calculation: Fo'(calc.)=1/(1/Fo-1/Fm-1/Fm'). We found that in every case the actual Φ(PSII) deviated from theoretical values at the same point that qPd deviated from a value of 1.0. In an increasing series of irradiance levels, WT leaves tolerated 1000μmolm(-2)s(-1) of light before qP(d) declined. Leaves treated with the uncoupler nigericin, leaves of the mutant lacking PsbS protein and leaves overexpressing PsbS showed a qP(d) reduction at 100, 600 and 2000μmolm(-2)s(-1) respectively, each at an increasing value of NPQ. Therefore we suggest that this simple and timely technique will be instrumental for identifying photoprotective NPQ (pNPQ) and that it is more appropriate than the qE component. Its applications should be broad: for example it will be useful in physiology-based studies to define the optimal level of nonphotochemical quenching for plant protection and productivity.     

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.     

Effect of nitric oxide on leaf non-photochemical quenching of fluorescence under heat-stress conditions

Non-photochemical quenching (NPQ) is a photoprotection mechanism in photosynthesis that can be monitored by NPQ of chlorophyll a fluorescence. Comparing NPQ response of Arabidopsis thaliana intact leaves between the wild type and ΔGLB3, which lacks truncated hemoglobin gene, we report here the effects of nitric oxide (NO) on NPQ under stress conditions. Heat stress was found to severely decline NPQ of ΔGLB3. The effect was mimicked by chemical NO donors, and it was completely prevented by the NO scavenger cPTIO. These results suggest that NO is involved in the decline of NPQ which is pronounced under heat stress conditions.     

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.     

On the quenching of the fluorescence yield in photosynthetic systems

A modified matrix model describing transfer of excitation energy in the photosynthetic pigment system is discussed. In addition to the antenna pigments and reaction centers of the simple matrix model, a coupling complex is postulated mediating energy transfer between antenna and reaction centers. The values of the parameters describing the transfer properties of the coupling complex can be chosen in such a way that a number of recent unexplained measurements of fluorescence properties of various purple bacteria can be described. If such coupling complexes are present in oxygen evolving organisms, some of their properties must be different from those of purple bacteria.     

Trapping of the quenched conformation associated with non-photochemical quenching of chlorophyll fluorescence at low temperature

The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25°C and during rapid jumps of the leaf temperature. At 5°C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25°C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (ΔA₅₁₅) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201–4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

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     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

Induction of photochemical and non-photochemical quenching of chlorophyll fluorescence by low concentrations of m-dinitrobenzene

Chlorophyll fluorescence quenching induced by low concentrations of m-dinitrobenzene (DNB) is investigated. In intact spinach chloroplasts DNB causes photochemical and non-photochemical quenching. The two forms of quenching are distinguished by applying the saturation pulse method with a new type of modulation fluorometer. Half-maximal photochemical quenching is observed at about 3 micromolar DNB. It is inhibited by 3-(3,4 dichlorophenyl)-1, 1-dimethylurea (DCMU) and by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Photochemical quenching by DNB leads to suppression of the I-P transient in a fluorescence induction curve. Upon application of saturating continuous light, the increase of fluorescence yield is separated into a photochemical and a thermal part. DNB causes suppression of only the slowest sub-component of the thermal part, in analogy to the action of Hill reagents. Simultaneous measurements of oxygen exchange rate and fluorescence reveal that a part of DNB induced quenching is accompanied by oxygen uptake. Most DNB-induced non-photochemical quenching is prevented by nigericin and, hence, can be considered energy-dependent quenching. The small component persisting in the presence of nigericin is identical to the one observed with methylviologen and other Hill reagents, likely to be due to static quenching by oxidized plastoquinone. The presented data confirm the original finding of Etienne and Lavergne (Biochim Biophys Acta 283: 268–278, 1972) that low concentrations of DNB selectively affect the thermal component of variable fluorescence. However, while these authors interpreted the quenching by a non-photochemical mechanism, the present investigation emphasizes a photochemical mechanism, in analogy to the effect of electron acceptors or mediators.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - DNB m-dinitrobenzene - PGA 3-phosphoglycerate - PMS phenazinemethosulphate - PS I and PS II photosystems I and II     

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.