Nonphotosynthetic reduction of the intersystem electron transport chain of chloroplasts following heat stress. The pool size of stromal reductants.

The properties of a negative transient signal (negative peak) observed during the first seconds of the induction of the photoacoustic (PA) signal in dark-adapted barley leaves treated with methyl viologen (MV) and diuron and then exposed to high temperatures have been examined. Under those conditions no electron donation from photosystem II (PSII) occurred, and electron flow through PSI could be supported only by soluble reductants located in the chloroplast stroma. The negative peak was observed only if the PA signal had been monitored at low, and not high, frequencies. The peak obviously originated from the oxygen consumption by PSI. The size of the peak increased as the temperature of preheating was raised from 39 to 45 degrees C. The size of the peak decreased exponentially with a half-time of 3.7 s during illumination under low light. This decrease was found to be much faster under strong light. The recovery of the peak during dark acclimation required several minutes. It is concluded that the negative peak reflects the oxygen consumption supported by stromal reductants, their pool being rapidly exhausted under light in the presence of MV. The maximal size of the pool was calculated as 140 eq: P700 in dark-adapated leaves.

Protection of the photosynthetic apparatus against damage by excessive illumination in homoiohydric leaves and poikilohydric mosses and lichens.

Experimental work on the control of photosystem II in the photosynthetic apparatus of higher plants, mosses and lichens is reviewed on a background of current literature. Transmembrane proton transport during photoassimilatory and photorespiratory electron flows is considered insufficient for producing the intrathylakoid acidification necessary for control of photosystem II activity under excessive illumination. Oxygen reduction during the Mehler reaction is slow. Together with associated reactions (the water-water cycle), it poises the electron transport chain for coupled cyclic electron transport rather than acting as an efficient electron sink. Coupled electron transport not accompanied by ATP consumption in associated reactions provides the additional thylakoid acidification needed for the binding of zeaxanthin to a chlorophyll-containing thylakoid protein. This results in the formation of energy-dissipating traps in the antennae of photosystem II. Competition for energy capture decreases the activity of photosystem II. In hydrated mosses and lichens, but not in leaves of higher plants, protein protonation and zeaxanthin availability are fully sufficient for effective energy dissipation even when photosystem II reaction centres are open. In leaves, an additional light reaction is required, and energy dissipation occurs not only in the antennae but also in reaction centres. Loss of chlorophyll fluorescence during the drying of predarkened poikilohydric mosses and lichens indicates energy dissipation in the dry state which is unrelated to protonation and zeaxanthin availability. Excitation of photosystem II by sunlight is not destructive in these dry organisms, whereas photosystem II activity of dried leaves is rapidly lost under strong illumination.

A few molecules of zeaxanthin per reaction centre of photosystem II permit effective thermal dissipation of light energy in photosystem II of a poikilohydric moss.

The relationship between thermal dissipation of light energy (as indicated by the quenching of chlorophyll fluorescence), zeaxanthin availability and protonation reactions was investigated in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. In the absence of zeaxanthin and actinic illumination, acidification by 20% CO2 in air was incapable of quenching basal, so-called F0 fluorescence either in the moss or in spinach (Spinacia oleracea L.) leaves. However, 1-s light pulses given either every 40, 60 or 200 s increased thermal dissipation as indicated by F0 and Fm quenching in the presence of 20% CO2 in air in the moss, but not in spinach while reaction centres of photosystem II (PSII) were photochemically open. In the moss, a few short light pulses, which were separated by prolonged dark times, were sufficient to raise zeaxanthin levels in the presence of 20% CO2 in air. Simultaneously, quantum efficiency of charge separation in PSII was decreased. Increasing the CO2 concentration beyond 20% further decreased quantum efficiency even in the absence of short light pulses. Under conditions optimal for fluorescence quenching, one molecule of zeaxanthin per reaction centre of PSII was sufficient to decrease quantum efficiency of charge separation in PSII by 50%. Thus, in combination with a protonation reaction, one molecule of zeaxanthin was as efficient at capturing excitation energy as a photochemically open reaction centre. The data are discussed in relation to the interaction between zeaxanthin and thylakoid protonation, which enables effective thermal dissipation of light energy in the antennae of PSII in the moss but not in higher plants when actinic illumination is absent.

Energy dissipation in photosynthesis: does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?

Dissipation of light energy was studied in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst., and in leaves of Spinacia oleracea L. and Arabidopsis thaliana (L.) Heynh., using chlorophyll fluorescence as an indicator reaction. Maximum chlorophyll fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated spinach leaves, as produced by saturating light and studied between and -20 degrees C, revealed an activation energy delta E of 0.11 eV. As this suggested recombination fluorescence produced by charge recombination between the oxidized primary donor of photosystem II and reduced pheophytin, a mathematical model explaining fluorescence, and based in part on known characteristics of primary electron-transport reactions, was developed. The model permitted analysis of different modes of fluorescence quenching, two localized in the reaction center of photosystem II and one in the light-harvesting system of the antenna complexes. It predicted differences in the relationship between quenching of variable fluorescence Fv and quenching of basal, so-called F0 fluorescence depending on whether quenching originated from antenna complexes or from reaction centers. Such differences were found experimentally, suggesting antenna quenching as the predominant mechanism of dissipation of light energy in the moss Rhytidiadelphus, whereas reaction-center quenching appeared to be important in spinach and Arabidopsis. Both reaction-center and antenna quenching required activation by thylakoid protonation but only antenna quenching depended on or was strongly enhanced by zeaxanthin. De-protonation permitted relaxation of this quenching with half-times below 1 min. More slowly reversible quenching, tentatively identified as so-called qI or photoinhibitory quenching, required protonation but persisted for prolonged times after de-protonation. It appeared to originate in reaction centers.

Nonphotosynthetic reduction of the intersystem electron transport chain of chloroplasts following heat stress. Steady-state rate.

The consequence of elevated temperatures in the range of 39-51 degrees C on the steady-state rate of light-induced electron transport through photosystem I (PSI) supported by stromal reductants was studied in intact barley leaves using photoacoustic and chlorophyll fluorescence techniques. Measurable electron flow through PSI in diuron-treated leaves occurred only after exposure to temperatures above 37 degrees C. The steady-state rate of the above diuron-insensitive electron flow with methyl viologen as electron acceptor was estimated to be 3.7 mu eq m-2 s-1 or 0.018 mu eq mumol chlorophyll-1 s-1 in leaves exposed for 5 min to 45 degrees C.

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping.

The role of cyclic electron transport has been re-examined in leaves of C3 plants because the bioenergetics of chloroplasts (H +/e = 3 in the presence of a Q-cycle; H+/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C3 photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by farred light and a large increase in the number of electrons available to reduce P700+ in the dark. At low CO2 and O2 molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, Fv/Fm) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a trans-thylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-redoxidised P700 and the decline in Fv/Fm brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO2-dependent O2 evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease Fv/Fm. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H+/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory.

Control of Photosystem II in spinach leaves by continuous light and by light pulses given in the dark.

The light-induced induction of components of non-photochemical quenching of chlorophyll fluorescence which are distinguished by different rates of dark relaxation (qNf, rapidly relaxing and qNs, slowly relaxing or not relaxing at all in the presence brief saturating light pulses which interrupt darkness at low frequencies) was studied in leaves of spinach.After dark adaptation of the leaves, a fast relaxing component developed in low light only after a lag phase. Quenching increased towards a maximum with increasing photon flux density. This 'fast' component of quenching was identified as energy-dependent quenching qE. It required formation of an appreciable transthylakoid ΔpH and was insignificant when darkened spinach leaves received 1 s pulses of light every 30 s even though zeaxanthin was formed from violaxanthin under these conditions.Another quenching component termed qNs developed in low light without a lag phase. It was not dependent on a transthylakoid pH gradient, decayed exponentially with a long half time of relaxation and was about 20% of total quenching irrespective of light intensity. When darkened leaves were flashed at frequencies higher than 0.004 Hz with 1 s light pulses, this quenching also appeared. Its extent was very considerable, and it did not require formation of zeaxanthin. Relaxation was accelerated by far-red light, and this acceleration was abolished by NaF.We suggest that qNs is the result of a so-called state transition, in which LHC II moves after its phosphorylation from fluorescent PS II to nonfluorescent PS I. This state transition was capable of decreasing in darkened leaves the potential maximum quantum efficiency of electron flow through Photosystem II by about 20%.

The efficiency of electron transfer from QA (-) to the donor side of Photosystem II decreases during induction of photosynthesis: Evidences from chlorophyll fluorescence and photoacoustic techniques.

The amplitudes ratio of the fast and slow phases (Afast/Aslow) in the kinetics of the dark relaxation of variable chlorophyll fluorescence (FV) was studied after various periods of illumination of dark-adapted primary barley leaves. Simultaneously, photosynthetic activity was monitored using the photoacoustic technique and the photochemical and non-photochemical fluorescence quenching parameters. The ratio Afast/Aslow changed with the preceding illumination time in a two-step manner. During the first stage of photosynthetic induction (0-20 s of illumination), characterized by a drop in O2-dependent photoacoustic signal following an initial spike and by a relatively stable small value of photochemical FV quenching, the ratio Afast/Aslow remained practically unaltered. During the second stage (20-60 s of illumination), when both the rate of O2 evolution and the photochemical FV quenching were found to be sharply developed, a marked increase in the above ratio was also observed. A linear correlation was found between the value of the photochemical quenching and the ratio Afast/Aslow during the second phase of photosynthetic induction. It is concluded that the slow phase appearing in the kinetics of FV dark relaxation is not due to the existence of Photosystem II reaction centres lacking the ability to reduce P700(+) with high rates, but is instead related to the limitation of electron release from Photosystem I during the initial stage of the induction period of photosynthesis. This limitation keeps the intersystem electron carriers in the reduced state and thus increases the probability of back electron transfer from QA (-) to the donor side of Photosystem II.

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves.

The dark-relaxation kinetics of variable fluorescence, Fv, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t1/2 = 1 s) and slow (t1/2 = 7-8 s) phases in fv dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of Fv dark decay decreased from 250 ms in dark-adapted leaves to 12-15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on Fv dark decay indicates that the slow phase develops when a fraction of QA (-) (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700(+) (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. Fv rise appears which is prevented by far-red (PSI-absorbed) light. This transient fv rise reflects the accumulation of QA (-) in the dark. The observation of this transient Fv rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in Fv dark-decay kinetics represents the reoxidation of QA (-) by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of QA (-) with the donor side of PSII.

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 QA. It is supposed that the increase in the fluorescence level by weak modulated light is either partly associated with dark reduction of QA due to exposure of chloroplasts to elevated temperature or due to temperature induced fluorescence rise in the so called inactive photosystem II centre where QA 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 QA 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 QA, the primary stable electron acceptor of photosystem II.

[Effect of secondary structure on the rate dark reduction of P700+ with ascorbate of the photosystem I complex]. / Vliianie vtorichnoi struktury na skorost' temnovogo vosstanovleniia P700+ askorbatom kompleksa fotosistemy I.

Relationship was studied between the spectra of KD, absorption and fluorescence and kinetics of P700+ dark reduction with exogenous donor and sodium dodecyl concentration inducing a conformation transition of the protein globule. It has been shown that with a decrease of the detergent concentration from 0.01 to 0.06% an increase of the portion of the protein globule-helical regions (from 10 to 30%) and an essential decrease of the time of P700+ dark reduction with ascorbate take place. The changes of KD, absorption and fluorescence spectra of chlorophyll a observed in the same region of detergent concentration do not correlate with the changes of the secondary structure of the protein part of the pigment-protein complex. It is suggested that accelerated reduction of P700+ with ascorbate is conditioned by an increase of accessibility of exogenous donor to the reaction centre resulting from conformation changes of the complex protein part.

[Dark and photo-induced changes in absorption and fluorescence spectra of phycobilisomes in the presence of dithionite]. / Temnovye i fotoindutsirovannye izmeneniia pogloshcheniia i fluorestsentsii fikobilisom v prisutstvii dintonita.

In order to test the possibility of photochemical participation of phycobilin pigments in photosynthesis, the ability of phycobilisomes for reversible photo-induced redox reactions was studied. The photo-induced fast reversible changes in the absorption and fluorescence spectra of phycobilisomes in the presence of dithionite were found. Simultaneously dithionite induced dark changes revealed by the decrease of the absorption and fluorescence yields. However, the dark and photo-induced changes differ in spectral parameters depending on dithionite concentration. The ability of phycobilisomes to photosensitive redox reactions was demonstrated. A possible nature of dark and photo-induced changes in the absorption and fluorescence spectra of phycobilisomes is discussed.

[Temperature independence of the rate of the rapid phase of P700+ dark reduction]. / Nezavisimost' ot temperatury skorosti bystroi fazy temnovogo vosstanovleniia P700+.

To elucidate the mechanism of interaction between P700+ and reduced primary electron acceptor in the reaction centres of photosystem I dark relaxation time course of absorption changes at 700 nm was studied at different temperatures. It is shown that in photosystem I subchloroplast fragments with partially inactivated endogenous secondary electron acceptors (treatment by ether or preliminary heating) the recombination of reduced primary acceptor and P700+ is found at -15 degrees C. In fragments with undamaged system of secondary acceptors the recovery of primary photoact in darkness is observed only at temperatures lower than -95 degrees C. At temperatures from -60 degrees to -170 degrees C the electron transfer from reduced primary acceptor to P700+ is described by the first order reaction with half time 250 ms; the rate of this process does not depend on the presence of secondary electron acceptor. The temperature independence of the rate of rapid phase P700+ dark reduction is interpreted as an indication of tunneling mechanisms from reduced primary acceptor to P700+.

[Effect of dehydration on functioning of photosystems of higher plants]. / Vliianie degidratatsii na funktsionirovanie fotosistem vysshikh rastenii.

The functional activity of both photosystems of higher plants and their thermoresistance in conditions of dehydratation of chloroplasts or subchloroplast fragments were studied. It is shown that dehydratation of the sample does not change the P700 amount capable to photooxidation. At 20 degrees in the time course of dark reduction of photooxidized P700 P(700+) in films two phases differing in rate were found. The relative contribution of each phase depends on the illumination duration. Since dehydratation blocks electron transfer between photosystems, the double phase dark reduction of P700+ in films reflects the electron flow from various components of potosystem 1 acceptor part. Dehydratation has little effect on properties of photosystem 1 acceptor part, because at low temperature the time courses of P700+ dark reduction in films and chloroplast or subchloroplast suspensions are similar. In contrast with potosystem 1, the functioning of photosystem 2, studied by light induced increase of fluorescence yield of chloroplasts, is blocked abruptly by water removal, but it could be partly restored by rehydratation of dried chloroplasts. The water removal increases the thermostability of both photosystems, however in suspension of the studied samples and also in their films photosystem 1 is more thermostable in comparison with photosystem 2.

[Photoinduced changes in adsorption at 800 nm related to photosystem I functioning]. / Fotoindutsirovannye izmeneniia pogloshcheniia pri 800 nm, sviazannye s funktsionirovaniem fotosistemy I.

The spectra and kinetics of light-induced absorbance changes in the near-infrared region of subchloroplast fragments enriched by P700 were studied. An increase in absorbancy within the region of 725--900 nm upon illumination was characterized by a maximum around 810 nm and by "shoulders" around 760 and 870 nm. Similar effects of thermal inactivation and low temperatures on the duration of dark recovery of light-induced absorbance changes at 700 nm and within the region of 725--900 nm suggest that the absorbance changes in the near-infrared region are due to photooxidation of P700. The values of P700 differential extinction coefficients at 810 nm are 8,2.10(3) M-1.cm-1 for digitonin fragments and 7,7.10(3) M-1.cm-1 for fragments prepared with the use of diethyl ester. It was shown that the value of midpoint oxidation-reduction potential measured for the absorbance changes at 810 nm (+492 mv) is higher than that measured at 700 nm (+475 mv).

[Nature of changes in the fluorescence process of P700-enriched chloroplast fragments]. / Pripoda izmenenii bykhoda fluorestsentsii fragmentov khloroplastov, obogashchennykh P700.

Spectral and photochemical properties of P700-enriched chloroplast fragments, obtained by ether treatment of liophylized digitonin fragments, were studied. It was shown that time course of fluorescence changes of isolated fragments (in contrast to digitonin fragments) at 20 degrees does not correspond to time course of absorption changes at 700 nm. Differences in low temperature fluorescence spectra of fragments, initially distinguished by redox states of photosystem 1 reaction centers were found. However, the fragments under study were incapable of light-induced changes of fluorescence yield at--196 degrees, independently of spectral region of measured fluorescence (lambda greater than 660 nm or lambda greater than 710 nm), though these fragments reveal phototransformation of P700. Thus changes in the low temperature fluorescence spectra cannot be accounted for by redox changes of P700. The fragments, isolated by ether treatment at 20 degrees as well at--196 degrees do not reveal light-induced fluorescence changes caused by redox changes of P700. The fluorescence changes observed may be due to accessory photoprocesses of chlorophyllprotein complex.

[Study of the acceptor portion of photosystem I according to the temperature relationships of P700 photoconversions]. / Issledovanie aktseptornoi chasti fotosistemy I po temperaturnoi zavisimosti fotoprevrashchenii P700.

The functioning of the acceptor part of photosystem I was studied by temperature dependence of time course of light induced absorbtion changes at 700 nm of digitonin chloroplast fragments, enriched by photosystem I. Partial irreversibility of P700 photooxidation at low temperatures and appearance of two components (rapid and slow) in the time course of P700+ dark reduction reflect the contribution of different acceptors in electron transport. Thermoinactivation of fragments incubation at acid pH or treatment by glutaraldehyde cause complete inhibition of irreversible P700 photooxidation and slow dark reduction of P700+ at -170 degrees. The slow component of P700+ reduction and irreversible photooxidation of P700 are ascribed to contribution of secondary ferredoxin acceptors. The accurence of rapid component of P700+ dark reduction in light induced signal of treated fragments indicate that this component is due to recombination of reduced primary acceptor and P700+. Because only one electron transport takes at -170 degrees, the occurence of rapid and slow components in dark decay kinetics of P700+ suggests, that secondary acceptors of some reaction centers are incapable to reduction at -170 degrees. The shape of temperature dependence curve of the slow P700+ reduction component is interpreted as an indication of the tunneling electron transport.

[Characteristics of photosystem I from grana thylakoids and from stroma lamellae]. / Kharakteristika fotosistemy i Iz tilakoidov grany i mezhgrannykh lamell khloroplastov.

Photochemical and spectral properties of photosystem I particles isolated from grana thylakoids and stroma lamellae of chloroplasts of higher plants are studied. The similarity of P700 content in these particles and the same dependence of P700+ reduction on temperature and the time course of variable fluorescence from artificial electron donors and acceptors indicate on the same type of reaction center for both photosystems I. Stroma particles have a higher rate of dark relaxation of variable fluorescence and a higher rate of dark reduction of P700+ at room temperature, than grana particles. Moreover, on the basis of data on low temperature fluorescence spectra, the stroma particles contain less shortwave chlorophyll a forms than grana particles. Thus, photosystem I from grana thylakoids and from stroma lamellae have the same type of reaction centers and can differ only by their nearest environment.