Changes Of Chlorophyll Fluorescence Yield Research Articles

Flash-kinetics as a complementary analytical tool in PAM fluorimetry

A new measuring system based on the already existing Multi-Color-PAM Fluorimeter (Schreiber et al. in Photosynth Res 113:127–144, 2012) was developed that in addition to standard PAM measurements enables pump-and-probe flash measurements and allows simultaneous measurements of the changes in chlorophyll fluorescence yield (F) during application of saturating flashes (ST). A high-power Chip-on-Board LED array provides ST flashes with close to rectangular profiles at wide ranges of widths (0.5 µs to 5 ms), intensities (1.3 mmol to 1.3 mol 440 nm quanta m−2 s−1) and highly flexible repetition times. Using a dedicated rising-edge profile correction, sub-µs time resolution is obtained for assessment of initial fluorescence and rise kinetics. At maximal to moderate flash intensities the flash-kinetics (changes of F during course of ST, STK) are strongly affected by ‘High Intensity Quenching’ (HIQ), consisting of Car-triplet quenching, TQ, and donor-side-dependent quenching, DQ. The contribution of TQ is estimated by application of a second ST after 20 µs dark-time. Upon application of flash trains (ST sequences with defined repetition times) typical period-4 oscillations in dark fluorescence yield (F0) and ST-induced fluorescence yield, FmST, are obtained which can be measured in vivo both with suspensions and from the surface of leaves. Examples of application with dilute suspensions of Chlorella and an intact dandelion leaf are presented. It is shown that weak far-red light (730–740 nm) advances the S-state distribution of the water-splitting system by one step, resulting in substantial lowering of FmST and also of the I1-level in the polyphasic rise of fluorescence yield induced by a multiple-turnover flash (MT). Based on comparative measurements of STK and the polyphasic rise kinetics with the same Chlorella sample, it is concluded that the generally observed lower values of maximal fluorescence yields using ST-protocols compared to MT-protocols are due to a higher extent of HIQ (mainly DQ) and the contribution of variable PSI fluorescence to FmST.

Hydroxyectoine protects Mn-depleted photosystem II against photoinhibition acting as a source of electrons.

In the present study, we have investigated the effect of hydroxyectoine (Ect-OH), a heterocyclic amino acid, on oxygen evolution in photosystem II (PS II) membrane fragments and on photoinhibition of Mn-depleted PS II (apo-WOC-PS II) preparations. The degree of photoinhibition of apo-WOC-PS II preparations was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to restore the amplitude of light-induced changes of chlorophyll fluorescence yield (∆F). It was found that Ect-OH (i) stimulates the oxygen-evolving activity of PS II, (ii) accelerates the electron transfer from exogenous electron donors (K4[Fe(CN)6], DPC, TMPD, Fe2+, and Mn2+) to the reaction center of apo-WOC-PS II, (iii) enhances the protective effect of exogenous electron donors against donor-side photoinhibition of apo-WOC-PS II preparations. It is assumed that Ect-OH can serve as an artificial electron donor for apo-WOC-PS II, which does not directly interact with either the donor or acceptor side of the reaction center. We suggest that the protein conformation in the presence of Ect-OH, which affects the extent of hydration, becomes favorable for accepting electrons from exogenous donors. To our knowledge, this is the first study dealing with redox activity of Ect-OH towards photosynthetic pigment-protein complexes.

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO3−/CO2 as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α‑carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α‑carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO3−/CO2. Addition of exogenous HCO3− or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.

Trehalose protects Mn-depleted photosystem 2 preparations against the donor-side photoinhibition

Recently, it has been shown that the addition of 1M trehalose leads to the increase of the rate of oxygen photoconsumption associated with activation of electron transport in the reaction center of photosystem 2 (PS2) in Mn-depleted PS2 membranes (apo-WOC-PS2) [37]. In the present work the effect of trehalose on photoinhibition of apo-WOC-PS2 preparations (which are characterized by a high sensitivity to the donor side photoinhibition of PS2) was investigated. The degree of photoinhibition was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to reactivate the electron transport (measured by light-induced changes of chlorophyll fluorescence yield (∆F)) in apo-WOC-PS2. It was found that 1M trehalose enhanced the Mn2+-dependent suppression of photoinhibition of apo-WOC-PS2: in the presence of trehalose the addition of 0.2μM Mn2+ (corresponding to 2 Mn2+ per one reaction center) was sufficient for an almost complete suppression of the donor side photoinhibition of the complex. In the absence of trehalose it was necessary to add 100μM Mn2+ to achieve a similar result. The effect of trehalose was observed during photoinhibition of apo-WOC-PS2 at low (15μmolphotons−1m−2) and high (200μmolphotons−1m−2) light intensity. When Mn2+ was replaced by other PS2 electron donors (ferrocyanide, DPC) as well as by Ca2+ the protective effect of trehalose was not observed. It was also found that 1M trehalose decreased photoinhibition of apo-WOC-PS2 if the samples contained endogenous manganese (1–2 Mn ions per one RC was enough for the maximum protection effect). It is concluded that structural changes in PS2 caused by the addition of trehalose enhance the capability of photochemical reaction centers of apo-WOC-PS2 to accept electrons from manganese (both exogenous and endogenous), which in turn leads to a considerable suppression of the donor side photoinhibition of PS2.

Involvement of molecular oxygen in the donor-side photoinhibition of Mn-depleted photosystem II membranes.

It has been shownby Khorobrykh et al. (Biochemistry (Moscow)67:683-688, 2002); Yanykin et al. (Biochim Biophys Acta 1797:516-523, 2010); Khorobrykh et al. (Biochemistry 50:10658-10665, 2011) that Mn-depleted photosystem II (PSII) membrane fragments are characterized by an enhanced oxygen photoconsumption on the donor side of PSII which is accompanied with hydroperoxide formation and it was suggested that the events are related to the oxidative photoinhibition of PSII. Experimental confirmation of this suggestion is presented in this work. The degree of photoinhibition was determined by the loss of the capability of exogenous electron donors (Mn(2+) or sodium ascorbate) to the reactivation of electron transport [measured by the light-induced changes of chlorophyll fluorescence yield (∆F)] in Mn-depleted PSII membranes. The transition from anaerobic conditions to aerobic ones significantly activated photoinhibition of Mn-depleted PSII membranes both in the absence and in the presence of exogenous electron acceptor, ferricyanide. The photoinhibition of Mn-depleted PSII membranes was suppressed upon the addition of exogenous electron donors (Mn(2+), diphenylcarbazide, and ferrocyanide). The addition of superoxide dismutase did not affect the photoinhibition of Mn-depleted PSII membranes. It is concluded that the interaction of molecular oxygen (rather than superoxide anion radical formed on the acceptor side of PSII) with the oxidized components of the donor side of PSII reflects the involvement of O2 in the donor-side photoinhibition of Mn-depleted PSII membranes.

Chlorophyll fluorescence imaging of plant–pathogen interactions

Chlorophyll fluorescence imaging provides a noninvasive, non-destructive method with which to measure heterogenous changes in photosynthetic metabolism in plants infected by pathogens. The availability of commercial imaging fluorimeters has helped make this technique available to the wider scientific community, but considerable care is needed, both in experimental design and in the interpretation of results, to make the most of this powerful analytical tool. The origins of changes in chlorophyll fluorescence yield are discussed and the use of conventional and novel combinatorial imaging approaches explored, together with complementary techniques such as thermal imaging. This review examines the use of chlorophyll fluorescence imaging as a method for the early detection of viral, bacterial and fungal infection, before symptoms are visible by eye, and also as a means with which to probe underlying pathogen-induced changes in host physiology in both compatible and incompatible interactions. The use of chlorophyll fluorescence imaging to study host physiology is greatly enhanced when the atmosphere around the leaf is manipulated and simultaneous measurements of gas exchange made: The cost to the host plant of different resistance mechanisms can be calculated, the fate of the products of photosynthetic electron transport determined and localised alterations in the source-sink status of host tissue visualised.

Photo-electrochemical control of photosystem II chlorophyll fluorescence in vivo

The effect of electric field on chlorophyll fluorescence is considered on the basis of the reversible radical pair model. The hypothesis is presented that the electric fields generated by photosynthetic charge separation in reaction centers and propagated laterally through the thylakoid lumen are associated with changes in chlorophyll fluorescence yield.

Secondary stabilization reactions and proton-coupled electron transport in photosystem II investigated by electroluminescence and fluorescence spectroscopy.

The oxidized primary electron donor in photosystem II, P(680)(+), is reduced in several phases, extending over 4 orders of magnitude in time. Especially the slower phases may reflect the back-pressure exerted by water oxidation and provide information on the reactions involved. The kinetics of secondary electron-transfer reactions in the microseconds time range after charge separation were investigated in oxygen-evolving thylakoids suspended in H2O or D2O. Flash-induced changes of chlorophyll fluorescence yield and electric field-induced recombination luminescence were decomposed into contributions from oxidation states S(0), S(1), S(2), and S(3) of the oxygen-evolving complex and interpreted in terms of stabilization kinetics of the initial charge-separated state S(j)Y(Z)P(680)(+)Q(A)(-)Q(B). In approximately 10% of the centers, only charge recombination took place. Otherwise, no static heterogeneity was involved in the microsecond reduction of P(680)(+) by Y(Z) (stabilization) or Q(A)(-) (recombination). The recombination component in active centers occurs mainly upon charge separation in S(3), and, in the presence of D2O, in S(2) as well and is tentatively attributed to the presence of Y(Z)(ox)S(j-1) in equilibrium with Y(Z)S(j). A 20-30 micros stabilization occurs in all S-states, but to different extents. Possible mechanisms for this component are discussed. D2O was found to decrease: (i) the rate of the reaction Y(Z)(ox)S(1) to Y(Z)S(2), (ii) the equilibrium constant between P680(+)Y(Z)S(2) and P(680)Y(Z)(ox)S(2), (iii) the rate of the slow phase of P(680)(+) reduction for the S(3) --> S(0) transition, and (iv) the rate of electron transfer from Q(A)(-) to Q(B) /Q(B)(-). The increased 'miss probability' in D2O is due to (iii).

Study of the non-photochemical dark rise in chlorophyll fluorescence in pre-illuminated leaves of various C3 and C4 plants submitted to partial anaerobiosis

Changes in chlorophyll fluorescence yield were studied during a dark period in pre-illuminated leaves of various C3 and C4 plants. The oxygen content in the gaseous atmosphere was either normal (21 kPa) or low (1.5 or 0.36 kPa). C3 and C4 plants of the NAD malic enzyme subgroup showed an initial rise in fluorescence at the onset of the dark period with an amplitude depending on the O 2 level in the gas. In C4 plants belonging to the other two subgroups, the slow rise was absent or of very low size. At high [O 2], the fluorescence level decreased in some minutes to the initial F 0 level (determined in dark-adapted leaves). Conversely at low [O 2], the fluorescence yield remained higher than F 0 in all the C4 plants studied, whereas it decreased slowly to the F 0 level in the different C3 plants. At low [O 2], the fluorescence level decreased rapidly to F 0 when introducing for 30 s, a high O 2 level or when giving a 15-s far-red illumination. At the end of these treatments, the fluorescence level re-increased. These results demonstrate the presence at low [O 2] of highly fluorescent `closed' photosystem II centres containing Q - A in equilibrium with reduced plastoquinone molecules of the chloroplastic pool. Reoxidation of the plastoquinone pool would be dependent on the functioning of an oxidase probably dependent on a chlororespiration process fully active at O 2 levels higher than 2 kPa. The source of reducing equivalents for the plastoquinone pool is discussed.

Reconstruction of the Water‐Oxidizing Complex in Manganese‐Depleted Photosystem II Preparations Using Mononuclear Manganese Complexes

Abstract— The water‐oxidizing complex of chloroplast photosystem II is composed of a cluster of four manganese atoms that can accumulate four oxidizing redox equivalents. Depletion of manganese from the water‐oxidizing complex fully inhibits oxygen evolution. However, the complex can be reconstituted in the presence of exogenous manganese in a process called photoactivation. In the present study, mononuclear manganese complexes with ligands derived from either nitrosonaphthol and ethylenediamine (Niten) or from diaminohexane and salicylaldehyde (Salhxn) are used in photoactivation experiments. Measurements of photoinduced changes of chlorophyll fluorescence yield, thermal dissipation using photoacoustic spectroscopy, photoreduction of 2,6‐dichorophenolindophenol and oxygen evolution in manganese‐depleted and in reconstituted photosystem II preparations demonstrate that photoactivation is more efficient when Niten and Salhxn complexes are used instead of MnCl2. It is inferred that the aromatic ligands facilitate the interaction of the manganese atoms with photosystem II. The addition of CaCl2 and of the extrinsic polypeptide of 33 kDa known as the manganese‐stabilizing protein during photoactivation further enhances the recovery of electron transport and oxygen evolution activities. It is proposed that mononuclear manganese complexes are able to contribute to re‐constitution of the water‐oxidizing complex by sequential addition of single ions similarly to the current model for assembly of the tetranuclear manganese cluster and that these complexes constitute suitable model systems to study the assembly of the water‐oxidizing complex.

Light‐triggered electrical events in the thylakoid membrane of plant chloroplasts

An overview is given of electrical events that occur in plant chloroplasts in association with their energization and subsequent photosynthetic performance. Special emphasis is given to the measuring techniques, in particular application of patch‐clamp methods, which enable comparison of light‐induced photocurrent and ‐potential kinetics of the thylakoid with kinetics of changes in chlorophyll fluorescence yield.

Reconstruction of the Water-Oxidizing Complex in Manganese-Depleted Photosystem II Preparations Using Mononuclear Manganese Complexes

Abstract— The water-oxidizing complex of chloroplast photosystem II is composed of a cluster of four manganese atoms that can accumulate four oxidizing redox equivalents. Depletion of manganese from the water-oxidizing complex fully inhibits oxygen evolution. However, the complex can be reconstituted in the presence of exogenous manganese in a process called photoactivation. In the present study, mononuclear manganese complexes with ligands derived from either nitrosonaphthol and ethylenediamine (Niten) or from diaminohexane and salicylaldehyde (Salhxn) are used in photoactivation experiments. Measurements of photoinduced changes of chlorophyll fluorescence yield, thermal dissipation using photoacoustic spectroscopy, photoreduction of 2,6-dichorophenolindophenol and oxygen evolution in manganese-depleted and in reconstituted photosystem II preparations demonstrate that photoactivation is more efficient when Niten and Salhxn complexes are used instead of MnCl2. It is inferred that the aromatic ligands facilitate the interaction of the manganese atoms with photosystem II. The addition of CaCl2 and of the extrinsic polypeptide of 33 kDa known as the manganese-stabilizing protein during photoactivation further enhances the recovery of electron transport and oxygen evolution activities. It is proposed that mononuclear manganese complexes are able to contribute to re-constitution of the water-oxidizing complex by sequential addition of single ions similarly to the current model for assembly of the tetranuclear manganese cluster and that these complexes constitute suitable model systems to study the assembly of the water-oxidizing complex.

Comparison of the Functional Properties of the Monomeric and Dimeric Forms of the Isolated CP47-Reaction Center Complex

Chlorophyll fluorescence, thermoluminescence, and EPR spectroscopy have been used to investigate the functional properties of the monomeric and dimeric forms of the photosystem II CP47-reaction center (CP47-RC) subcore complex that was isolated (Zheleva, D., Sharma, J., Panico, M., Morris, H. R., and Barber, J. (1998) J. Biol. Chem. 273, 16122-16127). Chlorophyll fluorescence yield changes induced either by the initiation of continuous actinic light or by repetitive light flashes indicated that the dimeric, but not the monomeric, form of the CP47-RC complex showed secondary electron transport properties indicative of QA reduction. Thermoluminescence measurements also clearly distinguished the monomer from the dimer in that the latter showed a ZV band, which appeared at -55 degreesC, following illumination at -80 degreesC. This band has been determined to be an indicator of the photoaccumulation of QA-. The ability of the dimeric CP47-RC to show secondary electron transport properties was clearly demonstrated by EPR studies. The dimer was characterized by organic radical signals at about g = 2 induced either by illumination or by the addition of dithionite. The dithionite-induced signal was attributed to QA-, but there was no indication of any interaction with non-heme iron. The signal induced by light was more complex, being composed not only of the QA- radical but also of radicals generated on the donor side. Difference analyses indicated that one of these radicals is likely to be due to a D1 tyrosine 161 or D2 tyrosine 161. In contrast, the monomeric CP47-RC complex did not show similar EPR-detectable radicals and instead was dominated by a high yield of the spin-polarized triplet signal generated by recombination reactions between the oxidized primary reductant, pheophytin, and the primary donor, P680. It is also concluded from EPR analyses that both the monomeric and dimeric forms of the CP47-RC subcore complex contain one cytochrome b559 per reaction center. Overall the results suggest that photosystem II normally functions as a dimer complex and that monomerization at the level of the CP47-RC subcore complex leads to destabilization of the bound plastoquinone, which functions as QA.

Characteristic changes of function and structure of Photosystem II during strong-light photoinhibition under aerobic conditions

The effects of strong-light photoinhibition under aerobic condition on the function and structure of Photosystem (PS) II were studied by measuring flash-induced changes of chlorophyll fluorescence yield, thermoluminescence glow curves, Fourier transform infrared (FTIR) spectra and the amounts of the PS II proteins, including the D1 and D2 proteins, in PS II membranes. Aerobic photoinhibition resulted in decrease in light-induced FTIR difference spectra of PS II. The intensity of the positive band at 1479 cm −1 due to a semiquinone anion in the Q A pocket decreased with the same kinetics as the inhibition of O 2-evolving activity. In an early stage of photoinhibition, the very fast decaying component of flash-induced fluorescence relaxation due to rapid electron transfer from Q A − to Q B was eliminated, and the intensity of thermoluminescence band due to S 2Q B − charge recombination decreased concomitant with appearance of another band due to S 2Q A − charge recombination. These results suggested that the impairment of Q B function preceded the loss of Q A function and O 2 evolution. The aerobic photoinhibition also induced a decrease in the F m level of fluorescence, but its course was h much faster than the loss of O 2 evolution. Degradation of the D1 and D2 proteins followed the loss of O 2 evolution but it did not induce clear changes in whole structure of PS II as detected by FTIR even after prolonged photoinhibition. Based on these results, the functional and structural changes of PS II by the aerobic photoinhibition were discussed, in relation to the primary event of photoinhibition.

In situ evidence that chilling in the light does not cause uncoupling of photophosphorylation or detachment of coupling factor in chilling-sensitive plants

The potential involvement of impaired photophosphorylation in the chilling sensitivity of photosynthesis in warm climate plant species has been a topic of investigation for more than two decades. With recent advances in the analysis of photosynthetic energy transduction in intact leaves, experiments are now possible that either address or avoid important uncertainties in the significance and interpretation of earlier in vitro work. Nevertheless, different laboratories using different techniques to analyze the effects of chilling in the light on photophosphorylation in intact cucumber (Cucumis sativus) leaves have come to very different conclusions regarding the role of impaired ATP formation capacity in the inhibition of net photosynthesis. In order to evaluate these discrepancies and bring this issue to a final resolution, in this investigation, we have made a detailed analysis of the decay of the flash-induced electrochromic shift and changes in chlorophyll fluorescence yield in cucumber leaves before, during and after a 5 h light-chill at chill temperatures of between 4 and 10°C. We feel that our findings address the major discrepancies in both data and interpretation as well as provide convincing evidence that photophosphorylation is not disrupted in cucumber leaves during or after light and chilling exposure. It follows that impaired photophosphorylation is not a contributing element to the inhibition of net photosynthesis that is widely observed in warm climate plants as a result of chilling in the light.

Transient chlorophyll fluorescence yield changes from the isolated photosystem II reaction centre induced by electron transfer

It is shown that the photoaccumulation of reduced pheophytin can occur in the isolated D1/D2/cytochrome b559 photosystem II (PSII) reaction centre when Fe(II) is used as a donor to P680 + in a way similar to that observed in the presence of sodium dithionite. Using Fe(II) as an electron donor system it has been possible to induce chlorophyll fluorescence transients from the isolated reaction centre complex when it is reconstituted with decylplastoquinone. These transients seem to reflect classical changes in fluorescence yield of PSII which accompany the conversion of the quinone acceptor from its oxidised to its reduced state. In the case of the isolated complex these decyplastoquinone dependent changes in fluorescence yield are dominated by direct quenching by oxidised quinone and do not reflect changes in the extent of the recombination reaction between the primary radical pair, P680 + pheophytin _.

Effects of O2 and CO2 Concentrations on Quantum Yields of Photosystems I and II in Tobacco Leaf Tissue

The interactive effects of irradiance and O(2) and CO(2) levels on the quantum yields of photosystems I and II have been studied under steady-state conditions at 25 degrees C in leaf tissue of tobacco (Nicotiana tabacum). Assessment of radiant energy utilization in photosystem II was based on changes in chlorophyll fluorescence yield excited by a weak measuring beam of modulated red light. Independent estimates of photosystem I quantum yield were based on the light-dark in vivo absorbance change at 830 nanometers, the absorption band of P700(+). Normal (i.e. 20.5%, v/v) levels of O(2) generally enhanced photosystem II quantum yield relative to that measured under 1.6% O(2) as the irradiance approached saturation. Photorespiration is suspected to mediate such positive effects of O(2) through increases in the availability of CO(2) and recycling of orthophosphate. Conversely, at low intercellular CO(2) concentrations, 41.2% O(2) was associated with lower photosystem II quantum yield compared with that observed at 20.5% O(2). Inhibitory effects of 41.2% O(2) may occur in response to negative feedback on photosystem II arising from a build-up in the thylakoid proton gradient during electron transport to O(2). Covariation between quantum yields of photosystems I and II was not affected by concentrations of either O(2) or CO(2). The dependence of quantum yield of electron transport to CO(2) measured by gas exchange upon photosystem II quantum yield as determined by fluorescence was unaffected by CO(2) concentration.

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

Chlorophyll fluorescence yield changes as a tool in plant physiology I. The measuring system

The interaction of an amphipathic fluorescence probe, 2- p-toluidinonaphthalene-6-sulphonate, with isolated chloroplasts

The amphipathic fluorescence probe, 2- p-toluidinonaphthalene-6-sulphonate has been used to investigate the surface electrical properties of chloroplast thylakoid membranes. The fluorescence yield of 2- p-toluidinonaphthalene-6-sulphonate in aqueous solution increases on addition of hypotonically shocked chloroplast, and the emission maximum shifts towards the blue to 440 nm, although the emission spectrum is somewhat distorted by chloroplast pigment absorption. The intensity of 2- p-toluidinonaphthalene-6-sulphonate fluorescence is further increased on adding salts to the membrane suspension, and changes of >100% are routinely observed. Similar observations have also been made with soya bean phospholipid (azolectin) liposomes. The magnitude of the fluorescence increase is dependent on membrane concentration, being more pronounced at high surface area/suspending volume ratios. The effect of salt addition appears to be that of shielding the fixed negative charges on the membrane surface, thus increasing the fraction of 2- p-toluidinonaphthalene-6-sulphonate molecules at the surface, where the 2- p-toluidinonaphthalene-6-sulphonate has a higher fluorescence yield than in free aqueous solution. This concept is supported by the fact that the effectiveness of salts in increasing 2- p-toluidinonaphthalene-6-sulphonate fluorescence is as predicted by classical electrical double layer theory: governed mainly by the charge carried by the cation with an order of effectiveness C 3+ > C 2+ > C +, and not by the chemical nature of the cation or by the nature of its co-ion. It has been argued that the chlorophyll fluorescence yield, controlled by the cation composition of the suspending medium follows the total diffusible positive charge density at the thylakoid membrane surface (Barber, J., Mills, J. and Love, A. (1977) Febs. Lett. 74, 174–181). Although the cation induced 2- p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yield changes show similar characteristics, there are also distinct differences between the two phenomena particularly when cations are added to chloroplasts initially suspended in a virtually cation-free medium. Therefore it is concluded that although both 2- p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yields are governed by the electrical properties of the thylakoid membrane surface, the mechanism controlling their cation sensitivity is not the same.