Blue light-modulation of chlorophyll a fluorescence transients in guard cell chloroplasts

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO₂ concentration on the fluorescence transients was investigated in the presence of HCO₃⁻ at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO₂ concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.     

Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components

The light-induced decline of chlorophyll a fluorescence from a peak (P) to a low stationary level (S) in intact, physiologically active isolated chloroplasts and in intact Chlorella cells is shown to be predominantly composed of two components: (1) fluorescence quenching by partial reoxidation of the quencher Q, the primary acceptor of Photosystem II and (2) energy-dependent fluorescence quenching related to the photoinduced acidification of the intrathylakoid space. These two mechanisms of fluorescence quenching can be distinguished by the different kinetics of the relaxation of quenching observed upon addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation of quenching by addition of DCMU is biphasic. The fast phase with a half-time of about 1 s is attributed to the reversal of Q-dependent quenching. The slow phase with a half-time of about 15 s in chloroplasts and 5 s in Chlorella cells is ascribed to relaxation of energy-dependent quenching. As shown by fluorescence spectroscopy at 77 K, the energy-dependent fluorescence quenching essentially is not caused by increased transfer of excitation energy to Photosystem I. By analyzing the energy- and Q-dependent components of quenching, information on the energy state of the thylakoid membranes and on the redox state of Q under various physiological conditions is obtained.     

Regulation of electron transport in maize mesophyll chloroplasts: The relationship between chlorophyll a fluorescence quenching and O2 evolution

The relationship between the redox state of the primary electron acceptor of photosystem II (Q_A) and the rate of O₂ evolution in isolated mesophyll chloroplasts from Zea mays L. is examined using pulse-modulated chlorophyll a fluorescence techniques. A linear relationship between photochemical quenching of chlorophyll fluorescence (q_Q) and the rate of O₂ evolution is evident under most conditions with either glycerate 3-phosphate or oxaloacetate as substrates. There appears to be no effect of the transthylakoid pH gradient on the rate of electron transfer from photosystem II into Q_A in these chloroplasts. However, the proportion of electron transport occurring through cyclic-pseudocyclic pathways relative to the non-cyclic pathway appears to be regulated by metabolic demand for ATP. The majority of non-photochemical quenching in these chloroplasts at moderate irradiances appeared to be energy-dependent quenching.Abbreviations and symbols PSII photosystem II - Fm maximum fluorescence obtained on application of a saturating light pulse - Fo basal fluorescence recorded in the absence of actinic light (i.e. all PSII traps are open) - Fv Fm-Fo - q_Q photochemical quenching - q_NP non-photochemical quenching - q_E energy-dependent quenching of chlorophyll fluorescence     

Non-photochemical quenching of chlorophyll a fluorescence in isolated chloroplasts under conditions of stressed photosynthesis

Non-photochemical quenching of chlorophyll a fluorescence after short-time light, heat and osmotic stress was investigated with intact chloroplasts from Spinacia oleracea L. The proportions of non-photochemical fluorescence quenching (q _N ) which are related (q _E ) and unrelated (q _I ) to the transthylakoid proton gradient (pH) were determined. Light stress resulted in an increasing contribution of q _Ito total q _N.The linear dependence of q. _Eand pH, as seen in controls, was maintained. The mechanisms underlying this type of quenching are obviously unaffected by photoin-hibition. In constrast, q _Ewas severely affected by heat and osmotic stress. In low light, the response of q _Eto changes in pH was enhanced, whereas it was reduced in high light. The data are discussed with reference to the hypothesis that q _Eis related to thermal dissipation of excitation energy from photosystem II. It is shown that q _Eis not only controlled by pH, but also by external factors.Abbreviations and symbols 9-AA 9-aminoacridine - F _o basic chlorophyll fluorescence - F _o variable chlorophyll fluorescence - L ₂ saturating light pulse - PS photosystem - q _E pH-dependent, non-photochemical quenching of fluorescence - q _I pH-independent, non-photochemical quenching - q _N entire non-photochemical quenching - q _Q photochemical quenching     

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (_p), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (q_I).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (_exe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q _N and the efficiency of excitation capture (_exe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.Abbreviations F₀ basic fluorescence - F₀ basic fluorescence, thylakoid in energized state - F_m maximal fluorescence - F_m maximum fluorescence in energized state - F_s steady state fluorescence - F_v maximal variable fluorescence - PFD photon flux density - PS II_rc Photosystem II reaction center - q_F0 quenching of basic fluorescence - q_E energy related quenching - q_N non-photochemical quenching:-q_f-total quenching - q_I inhibition-related quenching - q_p photochemical quenching - q_r quenching due to state transition - R_d dark respiration - _p PS II efficiency of excitation capture of open PS II_rc - _pe extrapolated minimal value of _p - _p0 extrapolated maximal value of _p - _si quantum efficiency of linear electron transport, calculated from gas exchange measurements based on incident light - _sf quantum efficiency of linear electron transport, calculated from fluorescence measurements, based on incident measuring light     

Chlorophyll fluorescence transients in a barley mutant lacking Photosystem I

We have compared the properties of a mutant of barley lacking Photosystem I (viridis-zb ⁶³ ) with the corresponding wild type using modulated fluorescence measurements. The mutant showed two unexpected characteristics. Firstly, there was a slow decline in the fluorescence signal in the light which was dependent on the presence of O₂ at concentrations similar to that in air; 2% O₂ in N₂ had no effect. The observed decline was mainly due to an increase in the non-photochemical quenching. Secondly, in the absence of O₂, saturating light pulses caused a pronounced transient decrease in the fluorescence signal; a similar effect could also be observed in wild type plants when neither CO₂ nor O₂ was present.Abbreviations PPFD- photosynthetic photon flux density - q_N- non-photochemical quenching of chlorophyll fluorescence - q_p- photochemical quenching of chlorophyll fluorescence     

Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander

The effect of exposing intact leaves and isolated chloroplast membranes of Nerium oleander L. to excessive light levels under otherwise favorable conditions was followed by measuring photosynthetic CO₂ uptake, electron transport and low-temperature (77K=-196°C) fluorescence kinetics. Photoinhibition, as manifested by a reduced rate and photon (quantum) yield of photosynthesis and a reduced electron transport rate, was accompanied by marked changes in fluorescence characteristics of the exposed upper leaf surface while there was little effect on the shaded lower surface. The most prominent effect of photoinhibitory treatment of leaves and chloroplasts was a strong quenching of the variable fluorescence emission at 692 nm (F_v,692) while the instantaneous fluorescence (F_o,692) was slightly increased. The maximum and the variable fluorescence at 734 nm were also reduced but not as much as F_M,692 and F_v,692. The results support the view that photoinhibition involves an inactivation of the primary photochemistry of photosystem II by damaging the reaction-center complex. In intact leaves photoinhibition increased with increased light level, increased exposure time, and with decreased temperature. Increased CO₂ pressure or decreased O₂ pressure provided no protection against photoinhibition. With isolated chloroplasts, inhibition of photosystem II occurred even under essentially anaerobic conditions. Measurements of fluorescence characteristics at 77K provides a simple, rapid, sensitive and reproducible method for assessing photoinhibitory injury to leaves. The method should prove especially useful in studies of the occurrence of photoinhibition in nature and of interactive effects between high light levels and major environmental stress factors.Abbreviations and symbols PFD photon flux area density - PSI, PSII photosystem I, II - F_M, F_O, F_V maximum, instantaneous, variable fluorescence emission C.I.W.-D.P.B. Publication No. 773     

Chlorophyll fluorescence at 680 and 730 nm and leaf photosynthesis

Chlorophyll fluorescence constitutes a simple, rapid, and non-invasive means to assess light utilization in Photosystem II (PS II). This study examines aspects relating to the accuracy and applicability of fluorescence for measurement of PS II photochemical quantum yield in intact leaves. A known source of error is fluorescence emission at 730 nm that arises from Photosystem I (PS I). We measured this PS I offset using a dual channel detection system that allows measurement of fluorescence yield in the red (660 nm < F < 710 nm) or far red (F > 710 nm) region of the fluorescence emission spectrum. The magnitude of the PS I offset was equivalent to 30% and 48% of the dark level fluorescence F₀ in the far red region for Helianthus annuus and Sorghum bicolor, respectively. The PS I offset was therefore subtracted from fluorescence yields measured in the far red spectral window prior to calculation of PS II quantum yield. Resulting values of PS II quantum yield were consistently higher than corresponding values based on emission in the red region. The basis for this discrepancy lies in the finite optical thickness of the leaf that leads to selective reabsorption by chlorophyll of red fluorescence emission originating in deeper cell layers. Consequently, red fluorescence measurements preferentially sense emission from chloroplasts in the uppermost layer of the leaf where levels of photoprotective nonphotochemical quenching are higher due to increased photon density. It is suggested that far red fluorescence, corrected for the PS I offset, provides the most reliable quantitative basis for calculation of PS II quantum yield because of reduced sensitivity of these measurements to gradients in leaf transmittance and quenching capacity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Salts and chloroplast fluorescence

Chloroplast fluorescence was excited by a weak measuring beam. A time-separated actinic light was used to modify the redox states of Q which in turn induced a change in the fluorescence yield. In salt-depleted chloroplasts, fluorescence saturated at a low actinic light intensity. CaCl₂ increased the “variable” fluorescence as well as the rate of ferricyanide-Hill reaction. With Tris-washed chloroplasts, Photosystem II donor couple, phenylenediamine and ascorbate, did not increase the fluorescence to a large extent without the presence of CaCl₂. It is suggested that salt-depletion inactivates the Photosystem II reaction center of chloroplasts.     

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, F_VF_M, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and F_VF_M ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations F_M maximum total fluorescence - F₀ initial fluorescence - F_V maximum variable fluorescence - PS Photosystem - Q_A, Q_B primary and secondary electron acceptors of Photosystem II     

On the relationship between the quantum yield of Photosystem II electron transport,as determined by chlorophyll fluorescence and the quantum yield of CO2-dependent O2 evolution

We tested the two empirical models of the relationship between chlorophyll fluorescence and photosynthesis, previously published by Weis E and Berry JA 1987 (Biochim Biophys Acta 894: 198–208) and Genty B et al. 1989 (Biochim Biophys Acta 990: 87–92). These were applied to data from different species representing different states of light acclimation, to species with C₃ or C₄ photosynthesis, and to wild-type and a chlorophyll b-less chlorina mutant of barley. Photosynthesis measured as CO₂-saturated O₂ evolution and modulated fluorescence were simultaneously monitored over a range of photon flux densities. The quantum yields of O₂ evolution (ØO₂) were based on absorbed photons, and the fluorescence parameters for photochemical (q_p) and non-photochemical (q_N) quenching, as well as the ratio of variable fluorescence to maximum fluorescence during steady-state illumination (F'_v/F'_m), were determined. In accordance with the Weis and Berry model, most plants studied exhibited an approximately linear relationship between ØO₂/q_p (i.e., the yield of O₂ evolution by open Photosystem II reaction centres) and q_N, except for wild-type barley that showed a non-linear relationship. In contrast to the linear relationship reported by Genty et al. for q_p×F'_v/F'_m (i.e., the quantum yield of Photosystem II electron transport) and ØCO₂, we found a non-linear relationship between q_p×F'_v/F'_m and ØO₂ for all plants, except for the chlorina mutant of barley, which showed a largely linear relationship. The curvilinearity of wild-type barley deviated somewhat from that of other species tested. The non-linear part of the relationship was confined to low, limiting photon flux densities, whereas at higher light levels the relationship was linear. Photoinhibition did not change the overall shape of the relationship between q_p×F'_v/F'_m and ØO₂ except that the maximum values of the quantum yields of Photosystem II electron transport and photosynthetic O₂ evolution decreased in proportion to the degree of photoinhibition. This implies that the quantum yield of Photosystem II electron transport under high light conditions may be similar for photoinhibited and non-inhibited plants. Based on our experimental results and theoretical analyses of photochemical and non-photochemical fluoresce quenching processes, we conclude that both models, although not universal for all plants, provide useful means for the prediction of photosynthesis from fluorescence parameters. However, we also discuss that conditions which alter one or more of the rate constants that determine the various fluorescence parameters, as well as differential light penetration in assays for oxygen evolution and fluorescence emission, may have direct effect on the relationships of the two models.Abbreviations F₀ and F'₀ fluorescence when all Photosystem II reaction centres are open in dark- and light-acclimated leaves, respectively - F_m and F'_m fluorescence when all Photosystem II reaction centres are closed in dark and light, respectively - F_v variable fluorescence equal to F_m-F₀ - F_s steady state level of fluorescence in light - F'_v and F'_m variable (F'_m-F'₀) and maximum fluorescence under steady state light conditions - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - Q_A the primary, stabile quinone acceptor of Photosystem II - q_N non-photochemical quenching of fluorescence - q_p photochemical quenching of fluorescence - ØO₂ quantum yield of CO₂-saturated O₂ evolution based on absorbed photons     

Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH₄Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN₃ inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.Abbreviations PS II Photosystem II - qQ photochemical quenching of chlorophyll fluorescence - qNP non-photochemical quenching of chlorophyll fluorescence - qE energy-dependent quenching of chlorophyll fluorescence - F _m maximum level of chlorophyll fluorescence for dark adapted cells - F _m level of fluorescence at any time when qQ is zero     

Effects of uncouplers on Mg2+-dependent fluorescence quenching in isolated chloroplasts

Uncoupling concentrations (about 1 mol l^-1) of desaspidin or carbonyl cyanide-4-trifluoromethoxyphenyl hydrazone reverse the slow light-induced, Mg²⁺-dependent quenching of fluorescence of chlorophyll a in isolated (intact and broken) spinach chloroplasts. Likewise, uncoupling inhibits the light-induced increase of the Mg²⁺ concentration in the stroma of intact chloroplasts, as determined with Eriochrome Blue SE. Addition of higher amounts of the uncouplers to the chloroplasts leads to a slow, light-dependent fluorescence lowering which appears to be promoted by high light intensities and is not reversed in the dark. The reversal of the fluorescence quenching by uncoupling is interpreted to reflect exchange of protons for Mg²⁺ ions at negative sites of the inner thylakoid face, caused by the collapse of the proton gradient across the membrane. The secondary fluorescence lowering caused by high levels of the uncouplers and high light intensities is suggested to be related to an inhibition of non-cyclic photosynthetic electron transport.Abbreviation FCCP carbonyl cyanide-4-trifluoromethoxyphenyl hydrazone     

Energy-dependent quenching of dark-level chlorophyll fluorescence in intact leaves

A new type of modulation fluorometer was used in the study of energy-dependent chlorophyll fluorescence quenching (q_E) in intact leaves. Under conditions of strong energization of the thylakoid membrane (high light intensity, absence of CO₂) not only variable fluorescence, F_V, but also dark-level fluorescence, F_O, was quenched, leading to definition of a quenching coefficient, q_O. Information on q_O was shown to be essential for correct determination of photochemical (q_Q) and energy dependent quenching (q_E) by the saturation pulse method. The relationship between q_E and q_O was analysed over a range of light intensities at steady state conditions. q_E was found to consist of two components, the second of which is linearly correlated with q_O. q_O and the second component of q_E are interpreted to reflect the state 1 — state 2 shift caused by LHC II phosphorylation.     

Effects of anaerobiosis as probed by the polyphasic chlorophyll a fluorescence rise kinetic in pea (Pisum sativum L.)

We analysed the changes of the chlorophyll (Chl)a fluorescence rise kinetic (from 50 s to 1 s) that occur when leaves or chloroplasts of pea ( Pisum sativum L.) are incubated under anaerobic conditions in the dark. In control leaves, Chl a fluorescence followed a typical O-J-I-P polyphasic rise [Strasser et al. (1995) Photochem Photobiol 61: 32–42]. Anaerobiosis modified the shape of the transient with the main effect being a time-dependent increase in the fluorescence yield at the J-step (2 ms). Upon prolongation of the anaerobic treatment (> 60 min), the O-J-I-P fluorescence rise was eventually transformed to an O-J (J = P) rise. A similar transformation was observed when pea leaves were treated with DCMU or sodium dithionite. Anaerobiosis resulted in a 10–20% reduction in the maximum quantum yield of the primary photochemistry of Photosystem II, as measured by the ratio of the maximal values of variable and total fluorescence (F_V/F_M). When the leaves were returned to the air in the dark, the shape of the fluorescence transient showed a time-dependent recovery from the anaerobiosis-induced change. The original O-J-I-P shape could also be restored by illuminating the anaerobically treated samples with far-red light but not with blue or white light. Osmotically broken chloroplasts displayed under anaerobic conditions fluorescence transients similar to those observed in anaerobically treated leaves, but only when they were incubated in a medium comprising reduced pyridine nucleotides (NADPH or NADH). As in intact leaves, illumination of the anaerobically treated chloroplasts by far-red light restored the original O-J-I-P transient, although only in the presence of methyl viologen. The results provide additional evidence for the existence of a chlororespiratory pathway in higher plant cells. Furthermore, they suggest that the J-level of the fluorescence transient is strongly determined by the redox state of the electron carriers at the PS II acceptor side.     

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R<Subscript>Fd</Subscript> of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters F_v, F_m, F₀, F_m′, F_v′, NF, and ΔF, the Chl fluorescence ratios F_v/F_m, F_v/F₀, ΔF/F_m′, as well as the photochemical (q_P) and non-photochemical quenching coefficients (q_N, q_CN, and NPQ). q_N consists of three components (q_N = q_E + q_T + q_I), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, q_N, q_CN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. q_N was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas q_CN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio R_Fd (F_d/F_s), which, when measured at saturation irradiance is directly correlated to the net CO₂ assimilation rate (_{P N}) of leaves. This R_Fd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the R_Fd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.     

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     

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II.

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E_m7.6 = + 25 mV and the second with E_m7.6 = -270 mV. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm (R. Malkin, 1978, FEBS Lett.87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.