Action potential in a plant cell lowers the light requirement for non-photochemical energy-dependent quenching of chlorophyll fluorescence

This study deals with effects of membrane excitation on photosynthesis and cell protection against excessive light, manifested in non-photochemical quenching (NPQ). In Chara corallina cells, NPQ and pericellular pH displayed coordinated spatial patterns along the length of the cell. The NPQ values were lower in H⁺-extruding cell regions (external pH ∼ 6.5) than in high pH regions (pH ∼ 9.5). Generation of an action potential by applying a pulse of electric current caused NPQ to increase within 30-60 s. This effect, manifested as a long-lived drop of maximum chlorophyll fluorescence (F_m′), occurred at lower photosynthetic flux densities (PFD) in the alkaline as compared to acidic cell regions. The light response curve of NPQ shifted, after generation of an action potential, towards lower PFD. The release of NPQ by nigericin and the rapid reversal of action potential-triggered NPQ in darkness indicate its relation to thylakoid ΔpH. Generation of an action potential shortly after darkening converted the chloroplasts into a latent state with the F_m identical to that of unexcited cells. This state transformed to the quenched state after turning on weak light that was insufficient for NPQ prior to membrane excitation of the cells. The ionophore, A23187, shifted NPQ plots similarly to the action potential effect, consistent with a likely role of a rise in the cytosolic Ca²⁺ level in the action potential-induced quenching. The results suggest that a rapid electric signal, across the plasma membrane, might exert long-lived effects on photosynthesis and chlorophyll fluorescence through ion flux-mediated pathways.     

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.     

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 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.     

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.     

A study on the energy-dependent quenching of chlorophyll fluorescence by means of photoacoustic measurements

The mechanism of energy-dependent quenching (q_E) of chlorophyll fluorescence was studied employing photoacoustic measurements of oxygen evolution and heat release. It is shown that concomitant to the formation of q_E the yield of open reaction centers _p decreases indicating that q_E quenching originates from a process being competitive to fluorescence as well as to photochemistry. The analysis of heat release (rate of thermal deactivation) shows: 1. The competitive process is not given by a still unknown energy storing process. 2. If the competitive process would be a futile cycle the life-times of the involved intermediates had to be faster than 50 s.The results of the photoacoustic measurements are in line with the idea that q_E quenching originates from an increased probability of thermal deactivation of excited chlorophylls.Abbreviations F actual fluorescence - F_m fluorescence yield with all PS II reaction centers closed in a light adapted state - F₀ fluorescence yield with all PS II reaction centers open in a light adapted state - PS Photosystem - _p intrinsic photochemical yield - q_E energy-dependent quenching - q_I photoinhibition quenching - q_N non-photochemical quenching - q_P photochemical quenching - q_T state transition quenching     

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.     

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.     

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.     

Rapid light-response curves of chlorophyll fluorescence in microalgae: relationship to steady-state light curves and non-photochemical quenching in benthic diatom-dominated assemblages

Rapid light-response curves (RLC) of variable chlorophyll fluorescence were measured on estuarine benthic microalgae with the purpose of characterising its response to changes in ambient light, and of investigating the relationship to steady-state light-response curves (LC). The response of RLCs to changes in ambient light (E, defined as the irradiance level to which a sample is acclimated to prior to the start of the RLC) was characterised by constructing light-response curves for the RLC parameters α _RLC, the initial slope, ETR_m,RLC, the maximum relative electron transport rate, and E _k,RLC, the light-saturation parameter. Measurements were carried out on diatom-dominated suspensions of benthic microalgae and RLC and LC parameters were compared for a wide range of ambient light conditions, time of day, season and sample taxonomic composition. The photoresponse of RLC parameters was typically bi-phasic, consisting of an initial increase of all parameters under low ambient light (E < 21–181 μmol m⁻² s⁻¹), and of a phase during which α _RLC decreased significantly with E, and the increase of ETR_m,RLC and E _k,RLC was attenuated. The relationship between RLC and LC parameters was dependent on ambient irradiance, with significant correlations being found between α _RLC and α, and between ETR_m,RLC and ETR_m, for samples acclimated to low and to high ambient irradiances, respectively. The decline of α _RLC under high light (Δα _RLC) was strongly correlated (P < 0.001 in all cases) with the level of non-photochemical quenching (NPQ) measured before each RLC. These results indicate the possibility of using RLCs to characterise the steady-state photoacclimation status of a sample, by estimating the LC parameter E _k, and to trace short-term changes in NPQ levels without dark incubation.     

On the relationship between the non-photochemical quenching of the chlorophyll fluorescence and the Photosystem II light harvesting efficiency. A repetitive flash fluorescence induction study

Plants respond to excess light by a photoprotective reduction of the light harvesting efficiency. The notion that the non-photochemical quenching of chlorophyll fluorescence can be reliably used as an indicator of the photoprotection is put to a test here. The technique of the repetitive flash fluorescence induction is employed to measure in parallel the non-photochemical quenching of the maximum fluorescence and the functional cross-section (sigma(PS II)) which is a product of the photosystem II optical cross-section a(PS II) and of its photochemical yield Phi(PS II) (sigma (PS II) = a(PS II) Phi(PS II)). The quenching is measured for both, the maximum fluorescence found in a single-turnover flash (F(M) (ST)) and in a multiple turnover light pulse (F(M) (MT)). The experiment with the diatom Phaeodactylum tricornutum confirmed that, in line with the prevalent model, the PS II functional cross-section sigma (PS II) is reduced in high light and restored in the dark with kinetics and amplitude that are closely matching the changes of the F(M) (ST) and F(M) (MT) quenching. In contrast, a poor correlation between the light-induced changes in the PS II functional cross-section sigma (PS II) and the quenching of the multiple-turnover F(M) (MT) fluorescence was found in the green alga Scenedesmus quadricauda. The non-photochemical quenching in Scenedesmus quadricauda was further investigated using series of single-turnover flashes given with different frequencies. Several mechanisms that modulate the fluorescence emission in parallel to the Q(A) redox state and to the membrane energization were resolved and classified in relation to the light harvesting capacity of Photosystem II.     

Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer

A newly developed fluorescence measuring system is employed for the recording of chlorophyll fluorescence induction kinetics (Kautsky-effect) and for the continuous determination of the photochemical and non-photochemical components of fluorescence quenching. The measuring system, which is based on a pulse modulation principle, selectively monitors the fluorescence yield of a weak measuring beam and is not affected even by extremely high intensities of actinic light. By repetitive application of short light pulses of saturating intensity, the fluorescence yield at complete suppression of photochemical quenching is repetitively recorded, allowing the determination of continuous plots of photochemical quenching and non-photochemical quenching. Such plots are compared with the time courses of variable fluorescence at different intensities of actinic illumination. The differences between the observed kinetics are discussed. It is shown that the modulation fluorometer, in combination with the application of saturating light pulses, provides essential information beyond that obtained with conventional chlorophyll fluorometers.     

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.     

Photoprotection in bryophytes: rate and extent of dark relaxation of non-photochemical quenching of chlorophyll fluorescence

Abstract

Vascular plants are typically endohydric and are killed by drying beyond 30% relative water content. Bryophytes are ectohydric and are typically desiccation tolerant (DT). Mosses in open sun-exposed habitats show major electron flow to oxygen and high levels of non-photochemical quenching (NPQ) in chlorophyll fluorescence measurements. This has been regarded as a main source of photoprotection for these plants. The aim of the work described in this paper was to explore the rate and extent of relaxation of this quenching, and to seek evidence of its nature and consequences. Sequences of measurements were made during illumination at various intensities and a subsequent dark period. Light-response curves were constructed using dithiothreitol (DTT) as an inhibitor of violaxanthin de-epoxidase to provide additional evidence of the proportion of NPQ mediated by the xanthophyll cycle. The relaxation curves were fitted by exponential decay curves. A double-exponential fit to curves for the sun-adapted species gave a fast phase with a halflife of ca 6–16 seconds, and a slow phase with a halflife of ca 100–300 seconds. Shade species were best fitted by single-exponential curves. A persistent offset remained of ca 5–23% of the pre-darkening NPQ. Light-response curves for several species showed NPQ reduced in the presence of DTT to similar proportions of the control. Around 70–95% of NPQ in the bryophytes investigated relaxed with a halflife of ca 2–5 minutes. The fast phase of the double-exponential fit is consistent with likely rates of decay of the trans-thylakoid pH gradient and re-epoxidation of zeaxanthin. This leads to the same conclusion as the effect of DTT in depressing NPQ. The contrast in physiology between bryophytes and vascular plants reflects the different selection pressures facing leaf cells of poikilohydric plants and the mesophyll cells of vascular plants, and their divergent evolutionary histories since the mid-Palaeozoic.     

Determination of the quantum efficiency of photosystem II and of non-photochemical quenching of chlorophyll fluorescence in the field

A newly developed portable chlorophyll fluorometer in combination with a special leaf clip holder was used for assessing photosynthetic activity of attached sun leaves of Fagus sylvatica and Cucurbita pepo under field conditions. During diurnal time courses, fluorescence yield, photosynthetic photon flux density (PPFD) incident on the leaf plane, and leaf temperature were measured and quantum efficiency of photosystem II (PS II), apparent relative electron transport rates, and non-photochemical fluorescence quenching (NPQ) calculated. In both species, quantum efficiency followed closely the incident PPFD and no hysteresis could be observed during the day. Apparent electron transport rate showed light saturation above a PPFD of 700 mol m^–2 s^–1 in F. sylvatica, while in C. pepo no saturation was visible up to 1400 mol m^–2 s^–1. NPQ was closely correlated to excessive PPFD calculated from the PS II quantum yield. Maximal NPQ observed was 3.3 Although the beech leaf was exposed for a considerable time to PPFD values of 1400–1500 mol m^–2 s^–1 and leaf temperatures between 30 and 35°C, no obvious signs for sustained photodamage could be observed. The data demonstrate the potential of chlorophyll fluorescence measurements to analyse photosynthetic performance under field conditions with minimal disturbance of the plant. Potential error sources due to the geometry of the leaf clip holder used are discussed.Dedicated to Prof. Dr. F.-C. Czygan on the occasion of his 60th birthday     

Influence of ascorbate and the Mehler peroxidase reaction on non-photochemical quenching of chlorophyll fluorescence in maize mesophyll chloroplasts

 Non-photochemical quenching of chlorophyll fluorescence (NPQ) and quantum yield of photosystem II (PSII) were studied with intact mesophyll chloroplasts of maize (Zea mays L.) during the initial minutes of illumination using the pulse-modulated chlorophyll fluorescence technique. Non-photochemical quenching was rapidly reversible in the dark at any point during illumination, which is indicative of energy-dependent dissipation of energy (mediated via thylakoid ΔpH changes and ascorbate-dependent synthesis of zeaxanthin). In chloroplasts suspensions including 15 mM ascorbate in the medium, with addition of oxaloacetate and pyruvate, the PSII yield, rate of reduction of oxaloacetate and phosphorylation of pyruvate reached a maximum after approximately 2 min of illumination. Under these conditions, which promote phosphorylation and a decreased ΔpH across the thylakoid membrane, NPQ rose to a maximum after 2–3 min of illumination, dropped to a minimum after about 6 min, and then increased to a steady-state level. A rather similar pattern was observed when leaves were illuminated following a 30-min dark period. Providing chloroplasts with higher levels of ascorbate (60 mM), prevented the transient drop in NPQ. Anaerobic conditions or addition of potassium cyanide caused a decrease in PSII yield, providing evidence for operation of the ascorbate-dependent Mehler-peroxidase reaction. These conditions also strongly suppressed the transient drop in NPQ. Dithiothreitol, an inhibitor of violaxanthin de-epoxidase, caused a large drop in NPQ even in the presence of high levels of ascorbate. The results suggest that the decline of NPQ occurs in response to an increase in lumen pH after initiation of phosphorylation, that this decline can be suppressed by conditions where ascorbate is not limiting for violaxanthin de-epoxidase, and that the increase of NPQ after such a decline is the result of development of energy dissipation in PSII reaction centers. Received: 13 August 1999 / Accepted: 17 September 1999     

O2-dependent electron flow,membrane energization and the mechanism of non-photochemical quenching of chlorophyll fluorescence

Recent progress in chlorophyll fluorescence research is reviewed, with emphasis on separation of photochemical and non-photochemical quenching coefficients (q_P and q_N) by the saturation pulse method. This is part of an introductory talk at the Wageningen Meeting on The use of chlorophyll fluorescence and other non-invasive techniques in plant stress physiology. The sequence of events is investigated which leads to down-regulation of PS II quantum yield in vivo, expressed in formation of q_N. The role of O₂-dependent electron flow for pH- and q_N-formation is emphasized. Previous conclusions on the rate of pseudocyclic transport are re-evaluated in view of high ascorbate peroxidase activity observed in intact chloroplasts. It is proposed that the combined Mehler-Peroxidase reaction is responsible for most of the q_N developed when CO₂-assimilation is limited. Dithiothreitol is shown to inhibit part of q_N-formation as well as peroxidase-induced electron flow. As to the actual mechanism of non-photochemical quenching, it is demonstrated that quenching is favored by treatments which slow down reactions at the PS II donor side. The same treatments are shown to stimulate charge recombination, as measured via 50 s luminescence. It is suggested that also in vivo internal thylakoid acidification leads to stimulation of charge recombination, although on a more rapid time scale. A unifying model is proposed, incorporating reaction center and antenna quenching, with primary control of pH at the PS II reaction center, involving radical pair spin transition and charge recombination to the triplet state in a first quenching step. In a second step, triplet excitation is trapped by zeaxanthin (if present) which in its triplet excited state causes additional quenching of singlet excited chlorophyll.Abbreviations q_P coefficient of photochemical quenching - q_N coefficient of non-photochemical quenching - q_E coefficient of energy-dependent quenching - LED light emitting diode     

Spectroscopy of non-photochemical and photochemical quenching of chlorophyll fluorescence in leaves; evidence for a role of the light harvesting complex of Photosystem II in the regulation of energy dissipation

Dissipation of absorbed excitation energy as heat, measured by its effect on the quenching of chlorophyll fluorescence, is induced under conditions of excess light in order to protect the photosynthetic apparatus of plants from light-dependent damage. The spectral characteristics of this quenching have been compared to that due to photochemistry in the Photosystem II reaction centre using leaves of Guzmania monostachia. This was achieved by making measurements at 77K when fluorescence emission bands from each type of chlorophyll protein complex can be distinguished. It was demonstrated that photochemistry and non-photochemical dissipation preferentially quench different emission bands and therefore occur by dissimilar mechanisms at separate sites. It was found that photochemistry was associated with a preferential quenching of emission at 688 nm whereas the spectrum for rapidly reversible non-photochemical quenching had maxima at 683 nm and 698 nm, suggesting selective quenching of the bands originating from the light harvesting complexes of Photosystem II. Further evidence that this was occurring in the light harvesting system was obtained from the fluorescence excitation spectra recorded in the quenched and relaxed states.Abbreviations pH transthylakoid pH gradient - F_o minimum level of chlorophyll fluorescence when Photosystem II reaction centres are open - F_m maximum level of fluorescence when Photosystem II reaction centres are closed - F_v variable fluorescence F_m minus F_o - F'_o F_o in any quenched state - F_m F_m in any quenched state - LHCII light harvesting complexes of Photosystem II - PSI Photosystem I - PS II Photosystem II - qN non-photochemical quenching of chlorophyll fluorescence - qE non-photochemical quenching of chlorophyll fluorescence that occurs in the presence of a pH