Studies on chlorophyll fluorescence in chloroplasts III. Effect of artificial electron donors for photosystexn 2 on reoxidation of the fluorescence quencher, Q, in spinach chloroplasts

1. Effects of various reducing reagents including dithionite,whichserve as artificial electron donors for photosystem 2,on therecovery of fluorescence induction in the presence of3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU) during the darkincubation of preilluminated chloroplastswere investigated.
2. The dark recovery of fluorescence induction was not affectedby the addition of the p-phenylenediamine-ascorbate couple,the hydroquinone-ascorbate couple or manganese. Incubation ofchloroplasts with dithionite caused gradual suppression of thedark recovery.
3. Preillumination of chloroplasts caused partialinhibition ofthe recovery of fluorescence induction.
4. At lowintensities of excitation light, the fluorescence yieldincreasedvery slowly and continuously, and never reached asteady state.This continuous increase in fluorescence yieldunder weak lightwas due to photoinhibition of the dark recovery.A techniquewas devised to determine the steady state yieldof fluorescence,without the intervention of photoinhibition,at weak light intensities.The steady state yield of fluorescencein the presence of DCMUwas suppressed at lower excitation intensities.This drop inthe fluorescence yield was not altered by the presenceof addedreducing reagents but was suppressed after long preincubationof chloroplasts with dithionite.
5. The delayed fluorescencewith a decay time of seconds was affectedby dithionite butnot by other reductants.
6. Results are discussed in terms ofreoxidation of the reducedprimary electron acceptor, Q, bythe oxidized primary electrondonor for photosystem 2. A modelof the electron transport associatedwith photoreaction 2 isproposed to account for the experimentalresults obtained.

(Received February 27, 1973; )     

Photorespiration is Essential for the Protection of the Photosynthetic Apparatus of C3 Plants Against Photoinactivation Under Sunlight

CO₂ assimilation, transpiration and modulated chlorophyll fluorescence of leaves of Chenopodium bonus-henricus (L.) were measured in the laboratory and, at a high altitude location, in the field. Direct calibration of chlorophyll fluorescence parameters against carbon assimilation in the presence of 1 or 0.5% oxygen (plus CO₂) proved necessary to calculate electron transport under photorespiratory conditions in individual experiments. Even when stomata were open in the field, total electron transport was two to three times higher in sunlight than indicated by net carbon gain. It decreased when stomata were blocked by submerging leaves under water or by forcing them to close in air by cutting the petiole. Even under these conditions, electron transport behind closed stomata approached 10 nmol electrons m^?2 leaf area s^?1 at temperatures between 25 and 30 °C. No photoinactivation of photosystem II was indicated by fluorescence analysis after a day's exposure to full sunlight. Only when leaves were submerged in ice was appreciable photoinactivation noticeable after 4 h exposure to sunlight. Even then almost full recovery occurred overnight. Electron transport behind blocked stomata was much decreased when leaves were darkened for 70 min (in order to deactivate light-regulated enzymes of the Calvin cycle) before exposure to full sunlight. Brief exposure of leaves to HCN (to inhibit photoassimilation and photorespiration) also decreased electron transport drastically compared to electron transport in unpoisoned leaves with blocked stomata. Non-photochemical fluorescence quenching and reduction of Q_A, the primary electron acceptor of photosystem II was increased by HCN-poisoning. Very similar observations were made when glyceraldehyde was used instead of HCN to inhibit photosynthesis and photorespiration. In HCN-poisoned leaves, residual electron transport increased linearly with temperature and showed early light saturation revealing characteristics of the Mehler reaction. During short exposure of these leaves to photon flux densities equivalent to 25% of sunlight, no or only little photoinactivation of photosystem II was observed. However, prolonged exposure to sunlight caused inactivation even though non-photochemical quenching of chlorophyll fluorescence was extensive. Simultaneously, oxidation of cellular ascorbate and glutathione increased. Inactivation of photosystem II was reversible in dim light and in the dark only after short times of exposure to sunlight. Glyceraldehyde was very similar to HCN in increasing the sensitivity of photosystem II in leaves to sunlight. We conclude from the observations that the electron transport permitted by the interplay of photoassimilatory and photorespiratory electron transport is essential to prevent the photoinactivation of photosynthetic electron transport. The Mehler and Asada reactions, which give rise to strong nonphotochemical fluorescence quenching, are insufficient to protect the chloroplast electron transport chain against photoinactivation.     

Studies on chlorophyll fluorescence in chloroplasts II. Effect of ferricyanide on the induction of fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea

1. The effect of preincubating spinach chloroplasts with ferricyanideon the time courses of chlorophyll- fluorescence in the presenceof 3-(3,4-dichlorophyl)-1,1-dimethylurea (DCMU) was studied.When DCMU was absent from the preincubation mixture, but wasadded just before the onset of excitation light, preincubationof chloroplasts with ferricyanide markedly affected the fluorescencekinetics. The rise-rate was lowered and consequently the areaabove the induction curve (S/Fv), which is proportional to thepool size of the electron acceptor(s) for photosystem 2, increased.The maximum increase in the S/Fv was attained after 3 min and10 min, respectively, of preincubation with 5?10^–4M and3?10^–5M ferricyanide.
2. When DCMU was present during preincubationwith ferricyanide,the effect of ferricyanide in increasingthe S/Fv, was completelyeliminated.
3. The effect of ferricyanidewas also suppressed by addition offerrocyanide to the preincubationmixture. The redox potentialof the ferri-ferrocyanide mixturewhich produced 50% suppressionof the ferricyanide effect wasabout 360 mV.
4. A similar dependency of the ferricyanide effecton the redoxpotential was observed in Tris-treated chloroplasts.However,the redox potential of cytochrome b-559 was markedlyloweredby Tris-treatment.
5. These results were explained byassuming the occurrence of asecondary electron acceptor, R,between the reaction centerof photosystem 2 and the DCMU-sensitivesite.

(Received February 27, 1973; )     

Photorespiration is More Effective than the Mehler Reaction in Protecting the Photosynthetic Apparatus against Photoinhibition

I. Isolated intact chloroplasts: Photosystem II, but not photosystem I, of the electron transport chain is rapidly photoinactivated even by very low intensities of red light when no large proton gradient can be formed and the electron transport chain becomes over-reduced in the absence of oxygen and other reducable substrates. Electron acceptors including oxygen provide protection against photoinactivation. Nevertheless, photosystem II is rapidly, and photosystem I more slowly, photoinactivated by high intensities of red light when oxygen is the only electron acceptor available. Increased damage is observed at increased oxygen concentrations although catalase is added to destroy H₂O₂ formed during oxygen reduction in the Mehler reaction. Photoinactivation can be decreased, but not prevented by ascorbate which reduces hydrogen peroxide inside the chloroplasts and increases coupled electron flow. II. Leaves: Simple measurements of chlorophyll fluorescence permit assessment of damage to photosystem II after exposure of leaves to high intensity illumination. In contrast to isolated chloroplasts, chloroplasts suffer more damage in situ at reduced than at elevated oxygen concentrations. The difference in the responses is due to photorespiration which is active in leaves, but not in isolated chloroplasts. After photosynthesis and photorespiration are inhibited by feeding glyceraldehyde to leaves, photoinactivation is markedly increased, although oxygen reduction in the Mehler reaction is not affected by glyceraldehyde. In the presence of reduced CO₂ levels, photorespiratory reactions, but not the Mehler reaction, can prevent the overreduction of the electron transport chain. Over-reduction indicates ineffective control of photosystem II activity. Effective control is needed for protection of the electron transport chain against photoinactivation. It is suggested to be made possible by coupled cyclic electron flow around photosystem I which is facilitated by the redox poising resulting from the interplay between photorespiratory carbohydrate oxidation and the refixation of evolved CO₂.     

Short term acclimation of spinach to high temperatures: effect on chlorophyll fluorescence at 293 and 77 Kelvin in intact leaves

Using intact leaves of Spinacia oleracea (L.), reversible temperature-induced changes in chlorophyll fluorescence emitted at room temperature and at 77K were studied. Interpretation of fluorescence at 77K was largely facilitated by developing a new method to minimize reabsorption artifacts (`diluted leaf-powder'). Leaves of plants grown at 15 to 20°C were exposed for several hours to different temperatures. Upon incubation at 35°C in the dark or in the light, the following changes in 77K fluorescence occurred with a half-time of less than 1 hour: (a) the initial fluorescence (F₀) of photosystem I increased by 15%, while that one of photosystem II somewhat decreased; (b) although variable fluorescence declined in both photosystems, the decrease in photosystem II (40%) was more severe; (c) the changes were less significant after 480-nanometer excitation light was replaced by 430-nanometer light. The data were interpreted in terms of a reversible, temperature-induced change in thylakoid structure and related change in the distribution of the absorbed energy in favor of photosystem I, at the expense of photosystem II excitation, probably accompanied by an increase in the rate of thermal deactivation of excited states. The considerable decrease in the variable part of room temperature fluorescence gives rise to the suggestion that this transition has lowered the reduction level of plastoquinone, i.e. has increased electron flow through photosystem I, relative to photosystem II. Possible physiological and mechanistic analogies between this temperature-induced state transition and the light-dependent state 1-state 2 regulation has been discussed.     

A New Pathway of Photoinactivation of Photosystem II. Irreversible Photoreduction of Pheophytin Causes Loss of Photochemical Activity of Isolated D1–D2–Cytochrome b559 Complex

A new pathway of photoinactivation of photosystem II (PS II) connected with irreversible photoaccumulation of reduced pheophytin (Ph) in isolated D1–D2–cytochrome b ₅₅₉ complexes of reaction center (RC) of PS II was discovered. The inhibitory effects of white light illumination on photochemical activity of D1–D2–cytochrome b ₅₅₉ complexes of RCs of photosystem II, isolated from pea chloroplasts, have been compared under anaerobic conditions in the absence and in the presence of sodium dithionite, electron transfer from which to the oxidized primary electron donor P680⁺ results in the photoaccumulation of anion-radical of the primary electron acceptor, PH. In both cases, prolonged illumination (1-5 min, 120 W/m²) led to a pronounced loss of the photochemical activity as it was monitored by measuring the amplitude of the reversible photoinduced absorbance changes at 682 nm related to the photoreduction of Ph. The extent of the photoinactivation depended on the illumination time and pH of the medium. At pH 8.0, the presence of dithionite during photoinactivation brought about a protective effect compared to that in a control sample. In contrast, lowering pH to 6.0 increased the sensitivity to photoinactivation in the dithionite containing samples. For 5 min irradiation, the photochemical activity in the absence and in the presence of dithionite decreased by 35 and 72%, respectively (this was accompanied by an irreversible bleaching of the pheophytin Q_x absorption band at 542 nm). Degradation of the D1 and D2 proteins was not observed under these conditions. A subsequent addition of an electron acceptor, potassium ferricyanide, to the illuminated samples restored neither the amplitude of the signal at 682 nm nor absorption at 542 nm. It is suggested that at pH < 7.0 the photoaccumulated PH is irreversibly converted into a secondary, most probably protonated form, that does not lead to destruction of the RCs but prevents the photoformation of the primary radical pair [P680⁺PH]. A possible application of this effect to photoinactivation of PS II in vivo is discussed.     

Electron transport to oxygen mitigates against the photoinactivation of Photosystem II in vivo

The role of electron transport to O₂ in mitigating against photoinactivation of Photosystem (PS) II was investigated in leaves of pea (Pisum sativum L.) grown in moderate light (250 mol m^–2 s^–1). During short-term illumination, the electron flux at PS II and non-radiative dissipation of absorbed quanta, calculated from chlorophyll fluorescence quenching, increased with increasing O₂ concentration at each light regime tested. The photoinactivation of PS II in pea leaves was monitored by the oxygen yield per repetitive flash as a function of photon exposure (mol photons m^–2). The number of functional PS II complexes decreased nonlinearly with increasing photon exposure, with greater photoinactivation of PS II at a lower O₂ concentration. The results suggest that electron transport to O₂, via the twin processes of oxygenase photorespiration and the Mehler reaction, mitigates against the photoinactivation of PS II in vivo, through both utilization of photons in electron transport and increased nonradiative dissipation of excitation. Photoprotection via electron transport to O₂ in vivo is a useful addition to the large extent of photoprotection mediated by carbon-assimilatory electron transport in 1.1% CO₂ alone.Abbreviations Fm, Fo, Fv- maximal, initial (corresponding to open PS II traps) and variable chlorophyll fluorescence yield, respectively - NPQ- non-photochemical quenching - PS- photosystem - Q_A- primary quinone acceptor - qP- photochemical quenching coefficient     

Light-induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans II. The fast changes and the effect of photosynthetic inhibitors on both the fast and slow fluorescence induction

1. The intensity dependence and spectral variations during thefast transient of chlorophyll a (Chl a) fluorescence have beenanalyzed in the blue-green alga Anacystis nidulans. (Unlikethe case of eukaryotic unicellular green or red algae, the fastfluorescence induction characteristics of the prokaryotic blue-greenalgae had not been documented before.)
2. Dark adapted cellsof Anacystis exhibit two types of fluctuationsin the fluorescenceyield when excited with bright orange light(absorbed mainlyin phycocyanin). The first kinetic patterncalled the fast (sec)fluorescence transient exhibits a characteristicoriginal levelO, intermediary hump I, a pronounced dip D, peakP and a transitorysmall decline to a quasi steady state S.After attaining S,fluorescence yield slowly rises to a maximumlevel M. From M,the decline in fluorescence yield to a terminalT level is extremelyslow as shown earlier by Papageorgiou andGovindjee (8). Ascompared with green and red algae, blue-greenalgae seem tohave a small PS decline and a very characteristicslow SM rise,with a M level much higher than the peak P.
3. A prolonged darkadaptation and relatively high intensity ofexciting illuminationare required to evoke DPS type yield fluctuationsin Anacystis.At low to moderate intensities of exciting light,the time forthe development of P depends on light doses, butfor M, thisremains constant at these intensities.
4. Fluorescence emissionwas heterogeneous during the inductionperiod in Anacystis;the P and the M levels were relativelyenriched in short-wavelengthsystem II Chi a emission as comparedto D and S levels.
5. Thefast DPS transient was found to be affected by electrontransportcofactor (methyl viologen), and inhibitors (e.g.,DCMU, NH₂OH)in a manner suggesting that these changes are mostlyrelatedto the oxido-reduction level of intermediates betweenthe twophotosystems. On the other hand, the slow SM changesin fluorescenceyield, as reported earlier (5, 15), paralleloxygen evolution.These changes were found to be resistant toa variety of electrontransport inhibitors (O-phenanthroline,HOQNO, salicylaldoxime,DCMU, NH₂OH and Antimycin a). It issuggested that, in Anacystis,even in the presence of so-calledinhibitors of cyclic electronflow, a "high energy state" isstill produced.
6. Measurementsof Chlorophyll a fluorescence and delayed lightemission inthe presence of both DCMU and NH₂OH indicate thatthe slow SMchanges are not due to the recovery of the reactioncenter IIin darkness preceeding illumination.
7. Our results, thus, suggestthat in Anacystis a net electrontransport supported oxidation-reductionstate of the quencherQ regulates only partially the developmentof the DPS transient,but the development of the slow fluorescenceyield changes seemsnot to be regulated by these reactions.It appears, from datapresented elsewhere, that the slow risein the yield resultsdue to a structural modification of thethylakoid membrane.

¹We are grateful to the National Science Foundation for financialsupport. (Received November 21, 1972; )     

Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging

High resolution imaging of chlorophyll a fluorescence was used to identify the sites at which ozone initially induces perturbations of photosynthesis in leaves of Phaseolus vulgaris. Leaves were exposed to 250 and 500 nmol mol(-1) ozone at a photosynthetically active photon flux density of 300 micromol m(-2) s(-1) for 3 h. Images of fluorescence parameters indicated that large decreases in both the maximum and operating quantum efficiencies of photosystem II had occurred in cells adjacent to stomata in the upper, but not lower, leaf surfaces. However, this treatment did not produce any significant changes in the maximum or operating quantum efficiencies of photosystem II in the leaves when estimated from fluorescence parameters measured with a conventional, integrating fluorometer. The localized decreases in photosystem II photochemical efficiencies were accompanied by an increase in the minimal fluorescence level, which is indicative of photoinactivation of photosystem II complexes and a decrease in stomatal conductance. Perturbations of photochemical efficiencies were not observed in cells associated with all of the stomata on the upper leaf surface or within cells distant from the upper leaf surface. It is concluded that ozone penetrates the leaf through stomata and initially damages only cells close to stomatal pores.     

Salt-induced alterations of the fluorescence yield and of emission spectra in Chlorella pyrenoidosa

1. The addition of salts to the suspending medium induces a decreasein the yield of chlorophyll a fluorescence in normal and DCMU-poisonedintact algal cells of Chlorella pyrenoidosa. Potassium and sodiumacetate cause a pronounced lowering of the fluorescence at relativelylow concentrations (0.01–0.1 M). MgCl₂ and KCl cause asimilar lowering of fluorescence but at much higher concentrations(0.1–0.4 M). In contrast to sodium acetate, ammonium acetatedoes not cause any significant change in the fluorescence transient.
2. Unlike the case in isolated chloroplasts, MgCl₂ decreasestheratio of short wavelength (mainly system 2) to long wavelength(mainly system 1) emission bands in both DCMU poisoned and normalcells. Since these salt-induced changes do not appear to berelated to the redox reactions of photosynthesis, the saltsmight have caused a decrease in the mutual distance betweenthe two photosystems by changing the microstructure of the chloroplastsin vivo thereby facilitating the spillover of excitation energyfrom strongly fluorescent system 2 to weakly fluorescent system1.
3. The light induced turbidity changes in intact algal cells,asmeasured by the increase in optical density at 540 nm, isreducedin the presence of these salts. However, MgCl₂ producesthegreatest reduction while Na acetate the least, even thoughbothof these salts (at the concentrations used) cause largesuppressionof the fluorescence transient. Moreover, the lightinduced turbiditychanges were, essentially irreversible.
4. Ashigh concentrations of salts increase the osmotic potentialof the bathing medium, it seems that the osmotic changes aswell as the ionic changes in the intact algal cells are responsiblefor the fluorescence quenching and changes in the mode of excitationtransfer observed in this study. In the case of low concentrationsof salts (e.g., 0.1 M Na or K acetate) the effects are predominantlyionic, and in the case of very high concentrations of MgCl₂(0.4 M), the osmotic effects play a much larger role.

(Received July 30, 1973; )     

Analysis of chlorophyll a fluoresence changes in weak light in heat treated Amaranthus chloroplasts

After preheating of Amaranthus chloroplasts at elevated temperatures (up to 45°C), the chlorophyll a fluorescence level under low excitation light rises as compared to control (unheated) as observed earlier in other chloroplasts (Schreiber U and Armond PA (1978) Biochim Biophys Acta 502: 138–151). This elevation of heat induced fluorescence yield is quenched by addition of 0.1 mM potassium ferricyanide, suggesting that with mild heat stress the primary electron acceptor of photosystem II is more easily reduced than the unheated samples. Furthermore, the level of fluorescence attained after illumination of dithionite-treated samples is independent of preheating (up to 45°C). Thus, these experiments indicate that the heat induced rise of fluorescence level at low light can not be due to changes in the elevation in the true constant F₀ level, that must by definition, be independent of the concentration of Q_A. It is supposed that the increase in the fluorescence level by weak modulated light is either partly associated with dark reduction of Q_A due to exposure of chloroplasts to elevated temperature or due to temperature induced fluorescence rise in the so called inactive photosystem II centre where Q_A are not connected to plastoquinone pool. In the presence of dichlorophenyldimethylurea the fluorescence level triggered by weak modulated light increases at alkaline pH, both in control and heat stressed chloroplasts. This result suggests that the alkaline pH accelerates electron donation from secondary electron donor of photosystem II to Q_A both in control and heat stressed samples. Thus the increase in fluorescence level probed by weak modulated light due to preheating is not solely linked to increase in true F₀ level, but largely associated with the shift in the redox state of Q_A, the primary stable electron acceptor of photosystem II.Abbreviations ADRY Acceleration of Deactivation of Reaction of Enzyme Y - CCCP Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone - Chl Chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FeCN potassium ferricyanide - HEPES 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid - LHCP Light harvesting chlorophyll protein - MES (4-morpholine ethane sulfonic acid) - PS photosystem - Q_A and Q_B first and second consecutive electron acceptors of photosystem II - TES (2-[tris(hydroxymethyl)-methylamino]-1-ethanesulfonic acid) sulfonic acid - TRICINE N-[tris(hydroxymethyl)methyl] glycine     

Purification and properties of the photoactive particle corresponding to photosystem II

1. 1) Purification of the photoactive particle corresponding tophotosystem II (particle II) was achieved using a combinationof procedures including digitonin- and Triton X-100-treatments,sonication, and differential- and density-gradient centrifugations.
2. 2) The "purified" particle II preparation showed only oxygenevolution activity and showed no NADP⁺ photoreduction activityeven when an electron donor couple was added.
3. 3) The chemicalcompositions of this particle and of the particlecorrespondingto photosystem I (particle I) were compared withrespect tothe composition and contents of their chlorophylls,carotenoids,cytochromes, plastoquinones, protein and lipid.Marked differenceswere found.
4. 4) Using the two photoactive particles I and IIlacking therespective counterpart activity, a partial successwas attainedin reconstituting a system, in which photoinducedelectron flowfrom water to NADP⁺ was observed, provided thatsuitable intermediateelectron carriers, e. g. plastoquinoneand plastocyanin, wereadded.
5. 5) The nature of the two photoactiveparticles obtained andtheir relationship to particles so farreported are discussed.

¹This study was aided by grants from the Ministry of Education(407160-1965, 91402-1966, 1967). Financial support from SanyoBroadcasting Scientific Foundation is also acknowledged withcordial thanks. (Received October 23, 1968; )     

Antenna Size Dependency of Photoinactivation of Photosystem II in Light-Acclimated Pea Leaves

Utilization of absorbed light energy by photosystem (PS) II for O2 evolution depends on the light-harvesting antenna size, but the role of antenna size in the photoinactivation of PSII seems controversial. To address this controversy, pea (Pisum sativum L.) plants were grown in low (50 [mu]mol m-2 s-1) or high (650 [mu]mol m-2 s-1) light. The doubled functional antenna size of PSII in low light allows each PSII to utilize twice as many photons at given flash light energies for O2 evolution. The application of a target theory to depict the photon dose dependency of PSII photoinactivation measured by repetitive-flash O2 yield and the ratio of variable to maximal chlorophyll fluorescence indicates that photoinactivation of PSII is probably a single-hit process in which repair or photoprotective mechanisms are only slightly involved. Furthermore, the exacerbation of photoinactivation of PSII with greater antenna size under anaerobic conditions strongly indicates that photoinactivation of PSII depends on antenna size.     

CHANGES IN EMISSION SPECTRA OF PHOTOSYNTHETIC PIGMENTS IN VIVO

1. The effects of 3-(4'-chlorophenyl)-1, 1-dimethylurea (CMU)onthe fluorescence of photosynthetic pigments in vivo wereinvestigatedin blue-green, red and brown algae and in isolatedspinach chloroplasts.CMU caused an increase in steady statelevel of fluorescenceof chlorophyll a, but did not influencethe fluorescence ofphycobilins. The spectrum of the fluorescenceincrement hada peak at 685 m/µ and a shoulder at 730–740mµ.These two bands probably arise from chlorophyll a(C_f684) belongingto pigment system II.
2. On excitation of chlorophylla in a red alga, Porphyra yezoensis,a fluorescence band witha peak at 720 mµ was observedbesides a shoulder at 685mµ. The 720 m band is inferredto arise from chlorophylla (probably, C_f-1) in pigment systemI.
3. On addition of CMUto the algal cells, the induction of fluorescencewas modifiedto take a simple time course. The induction wasobserved onlywith respect to the fluorescence of chlorophylla, but not inthe fluorescence of phycobilins. The spectrumof the "transient"fluorescence showed two emission bands ofchlorophyll a at 685mµ and 740 mµ, and was quitesimilar in form tothe spectrum of the CMU-caused increase insteady state fluorescence.
4. These facts were interpreted in terms of the correlation offluorescence of chlorophyll a and the photochemical reactionsof photosynthesis

(Received July 20, 1967; )     

Kinetics Studies on the Fluorescence Quencher in Isolated Chloroplasts

The chlorophyll fluorescence yield in isolated chloroplasts without an added electron acceptor is increased by actinic illumination. The decline in the fluorescence yield when the actinic illumination is extinguished can be accurately represented by three, independent, exponential decays with half-times of approximately 0.8, 5, and 30 sec. These results have been interpreted using Duysens' theory of fluorescence quenching by a compound (Q) on the reducing side of photosystem II. This theory states that changes in fluorescence yield are indicative of electron flow through Q. The most rapid decay is eliminated by an EDTA washing of the chloroplasts and the half-time is increased by uncoupling with ammonia and by added electron acceptors in suboptimal concentrations. Thus, this decay may represent electron flow from Q to intermediates on the oxidizing side of photosystem I. The decay with a half-time of 5 sec is affected in the same manner as the decay with the shortest half-time by the same procedures. However, electron donors to photosystem II lengthen the half-time of the 5 sec decay while eliminating the most rapid decay. This 5 sec decay can be interpreted as electron flow from Q to intermediates either on the reducing side of photosystem II or on the oxidizing side of photosystem I. The decay with the longest half-time is affected only by pH and electron donors to photosystem II. Therefore, this decay may indicate electron flow from Q to intermediates on the oxidizing side of photosystem II which may be connected to the regeneration of the oxygen burst.     

Light Energy Distribution in the Brown Alga Macrocystis pyrifera (Giant Kelp)

The brown alga Macrocystis pyrifera (giant kelp) was studied by a combination of fluorescence spectroscopy at 77 kelvin, room temperature modulated fluorimetry, and photoacoustic techniques to determine how light energy is partitioned between photosystems I and II in states 1 and 2. Preillumination with farred light induced the high fluorescence state (state 1) as determined by fluorescence emission spectra measured at 77K and preillumination with green light produced a low fluorescence state (state 2). Upon transition from state 1 to state 2, there was an almost parallel decrease of all of the fluorescence bands at 693, 705, and 750 nanometers and not the expected decrease of fluorescence of photosystem II and increase of fluorescence in photosystem I. The momentary level of room temperature fluorescence (fluorescence in the steady state, F_s), as well as the fluorescence levels corresponding to all closed (F_m) or all open (F_o) reaction-center states were measured following the kinetics of the transition between states 1 and 2. Calculation of the distribution of light 2 (540 nanometers) between the two photosystems was done assuming both the `separate package' and `spill-over' models. Unlike green plants, red algae, and cyanobacteria, the changes here of the light distribution were rather small in Macrocystis so that there was approximately an even distribution of the photosystem II light at 540 nanometers to photosystem I and photosystem II in both states 1 and 2. Photoacoustic measurements confirmed the conclusions reached as a result of fluorescence measurements, i.e. an almost equal distribution of light-2 quanta to both photosystems in each state. This conclusion was reached by analyzing the enhancement phenomenon by light 2 of the energy storage measured in far red light. The effect of light 1 in decreasing the energy storage measured in light 2 is also consistent with this conclusion. The photoacoustic experiments showed that there was a significant energy storage in light 1 which could be explained by cyclic electron transport around photosystem I. From a quantitative analysis of the enhancement effect of background light 2 (maximum enhancement of 1.4-1.5) it was shown that around 70% of light 1 was distributed to this cyclic photosystem I transport.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

A New Mechanism for Adaptation to Changes in Light Intensity and Quality in the Red Alga Porphyra perforata: III. Fluorescence Transients in the Presence of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea

In the red alga Porphyra perforata, the level of chlorophyll fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased during illumination of the thallus. The results showed that: (a) this decay was related to the photooxidative activity of photosystem I; (b) Q, the primary electron acceptor of photosystem II, became oxidized during the decay of the fluorescence; (c) reagents which inhibit the back reaction of photosystem II inhibited the decay.

From these results, it is suggested that, when conditions in the chloroplasts of this red alga become too oxidative, excess light energy can be converted to heat as a result of an accelerated back reaction of photosystem II. This may be one of the mechanisms by which this alga can cope with the high salt and high light conditions that can occur in its natural habitat.

     

Kinetics of photosystem II electron transport: a mathematical analysis based on chlorophyll fluorescence induction

The OJDIP rise in chlorophyll fluorescence during induction at different light intensities was mathematically modeled using 24 master equations describing electron transport through photosystem II (PSII) plus ordinary differential equations for electron budgets in plastoquinone, cytochrome f, plastocyanin, photosystem I, and ferredoxin. A novel feature of the model is consideration of electron in- and outflow budgets resulting in changes in redox states of Tyrosine Z, P680, and Q_A as sole bases for changes in fluorescence yield during the transient. Ad hoc contributions by transmembrane electric fields, protein conformational changes, or other putative quenching species were unnecessary to account for primary features of the phenomenon, except a peculiar slowdown of intra-PSII electron transport during induction at low light intensities. The lower than F _m post-flash fluorescence yield F _f was related to oxidized tyrosine Z. The transient J peak was associated with equal rates of electron arrival to and departure from Q_A and requires that electron transfer from Q_A ^? to Q_B be slower than that from Q_A ^? to Q_B ^?. Strong quenching by oxidized P680 caused the dip D. Reduced plastoquinone, a competitive product inhibitor of PSII, blocked electron transport proportionally with its concentration. Electron transport rate indicated by fluorescence quenching was faster than the rate indicated by O₂ evolution, because oxidized donor side carriers quench fluorescence but do not transport electrons. The thermal phase of the fluorescence rise beyond the J phase was caused by a progressive increase in the fraction of PSII with reduced Q_A and reduced donor side.     

Enhanced NPQ affects long-term acclimation in the spring ephemeral Berteroa incana

The spring ephemeral Berteroa incana is a familial relative of Arabidopsis thaliana and thrives in a diverse range of terrestrial ecosystems. Within this study, the novel chlorophyll fluorescence parameter of photochemical quenching in the dark (qPd) was used to measure the redox state of the primary quinone electron acceptor (Q_A) in order to estimate the openness of photosystem II (PSII) reaction centres (RC). From this, the early onset of photoinactivation can be sensitively quantified alongside the light tolerance of PSII and the photoprotective efficiency of nonphotochemical quenching (NPQ). This study shows that, with regards to A. thaliana, NPQ is enhanced in B. incana in both low-light (LL) and high-light (HL) acclimation states. Moreover, light tolerance is increased by up to 500%, the rate of photoinactivation is heavily diminished, and the ability to recover from light stress is enhanced in B. incana, relative to A. thaliana. This is due to faster synthesis of zeaxanthin and a larger xanthophyll cycle (XC) pool available for deepoxidation. Moreover, preferential energy transfer via CP47 around the RC further enhances efficient photoprotection. As a result, a high functional cross-section of photosystem II is maintained and is not downregulated when B. incana is acclimated to HL. A greater capacity for protective NPQ allows B. incana to maintain an enhanced light-harvesting capability when acclimated to a range of light conditions. This enhancement of flexible short-term protection saves the metabolic cost of long-term acclimatory changes.