Conservation and dissipation of light energy as complementary processes: homoiohydric and poikilohydric autotrophs

The relationship between photosynthetic energy conservation and thermal dissipation of light energy is considered, with emphasis on organisms which tolerate full desiccation without suffering photo-oxidative damage in strong light. As soon as water becomes available to dry poikilohydric organisms, they resume photosynthetic water oxidation. Only excess light is then thermally dissipated in mosses and chlorolichens by a mechanism depending on the protonation of a thylakoid protein and availability of zeaxanthin. Upon desiccation, another mechanism is activated which requires neither protonation nor zeaxanthin although the zeaxanthin-dependent mechanism of energy dissipation remains active, provided desiccation occurs in the light. Increased thermal energy dissipation under desiccation finds expression in the loss of variable, and in the quenching of, basal chlorophyll fluorescence. Spectroscopical analysis revealed the activity of photosystem II reaction centres in the absence of water. Oxidized beta-carotene (Car+) and reduced chlorophyll (Chl-), perhaps ChlD1 next to P680 within the D1 subunit, accumulates reversibly under very strong illumination. Although recombination between Car+ and Chl- is too slow to contribute significantly to thermal energy dissipation, a much faster reaction such as the recombination between P680+ and the neighbouring Chl- is suggested to form the molecular basis of desiccation-induced energy dissipation in photosystem II reaction centres. Thermal dissipation of absorbed light energy within a picosecond time domain deactivates excited singlet chlorophyll, thereby preventing triplet accumulation and the consequent photo-oxidative damage by singlet oxygen.     

Thermal energy dissipation in reaction centres and in the antenna of photosystem II protects desiccated poikilohydric mosses against photo-oxidation

Seasonal differences have been observed in the ability of desiccated mosses to dissipate absorbed light energy harmlessly into heat. During the dry summer season desiccation-tolerant mosses were more protected against photo-oxidative damage in the dry state than during the more humid winter season. Investigation of the differences revealed that phototolerance could be acquired or lost even under laboratory conditions. When a desiccated poikilohydric moss such as Rhytidiadelphus squarrosus is in the photosensitive state, the primary quinone, Q(A), in the reaction centre of photosystem II is readily reduced even by low intensity illumination as indicated by reversibly increased chlorophyll fluorescence. No such reduction is observed even under strong illumination in desiccated mosses after phototolerance has been acquired. In this state, reductive charge stabilization is replaced by energy dissipation. As a consequence, chlorophyll fluorescence is quenched. Different mechanisms are responsible for quenching. One is based on the presence of zeaxanthin provided drying occurs in the light. This mechanism is known to be controlled by a protonation reaction which is based on proton-coupled electron transport while the moss is still hydrated. Another mechanism which also requires light for activation, but no protonation, is activated during desiccation. While water is slowly lost, fluorescence is quenched. In this situation, an absorption band formed at 800 nm in the light is stabilized. It loses reversibility on darkening. Comparable kinetics of fluorescence quenching and 800 nm signals as well as the linear relationship between non-photochemical fluorescence quenching (NPQ) and loss of stable charge separation in photosystem II reaction centres suggested that desiccation-induced quenching is a property of photosystem II reaction centres. During desiccation, quenchers accumulate which are stable in the absence of water but revert to non-quenching molecular species on hydration. Together with zeaxanthin-dependent energy dissipation, desiccation-induced thermal energy dissipation protects desiccated poikilohydric mosses against photo-oxidation, ensuring survival during drought periods.     

Conservation and dissipation of light energy in desiccation-tolerant photoautotrophs, two sides of the same coin

Conservation of light energy in photosynthesis is possible only in hydrated photoautotrophs. It requires complex biochemistry and is limited in capacity. Charge separation in reaction centres of photosystem II initiates energy conservation but opens also the path to photooxidative damage. A main mechanism of photoprotection active in hydrated photoautotrophs is controlled by light. This is achieved by coupling light flux to the protonation of a special thylakoid protein which activates thermal energy dissipation. This mechanism facilitates the simultaneous occurrence of energy conservation and energy dissipation but cannot completely prevent damage by light. Continuous metabolic repair is required to compensate damage. More efficient photoprotection is needed by desiccation-tolerant photoautotrophs. Loss of water during desiccation activates ultra-fast energy dissipation in mosses and lichens. Desiccation-induced energy dissipation neither requires a protonation reaction nor light but photoprotection often increases when light is present during desiccation. Two different mechanisms contribute to photoprotection of desiccated photoautotrophs. One facilitates energy dissipation in the antenna of photosystem II which is faster than energy capture by functional reaction centres. When this is insufficient for full photoprotection, the other one permits energy dissipation in the reaction centres themselves.     

Thermal dissipation of light energy is regulated differently and by different mechanisms in lichens and higher plants

Modulated chlorophyll fluorescence was used to compare dissipation of light energy as heat in photosystem II of homoiohydric and poikilohydric photosynthetic organisms which were either hydrated or dehydrated. In hydrated chlorolichens with an alga as the photobiont, fluorescence quenching revealed a dominant mechanism of energy dissipation which was based on a protonation reaction when zeaxanthin was present. CO2 was effective as a weak protonating agent and actinic light was not necessary. In a hydrated cyanobacterial lichen, protonation by CO2 was ineffective to initiate energy dissipation. This was also true for leaves of higher plants. Thus, regulation of zeaxanthin-dependent energy dissipation by protonation was different in leaves and in chlorolichens. A mechanism of energy dissipation different from that based on zeaxanthin became apparent on dehydration of both lichens and leaves. Quenching of maximum or Fm fluorescence increased strongly during dehydration. In lichens, this was also true for so-called basal or Fo fluorescence. In contrast to zeaxanthin-dependent quenching, dehydration-induced quenching could not be inhibited by dithiothreitol. Both zeaxanthin-dependent and dehydration-induced quenching cooperated in chlorolichens to increase thermal dissipation of light energy if desiccation occurred in the light. In cyanolichens, which do not possess a zeaxanthin cycle, only desiccation-induced thermal energy dissipation was active in the dry state. Fluorescence emission spectra of chlorolichens revealed stronger desiccation-induced suppression of 685-nm fluorescence than of 720-nm fluorescence. In agreement with earlier reports of , fluorescence excitation data showed that desiccation reduced flow of excitation energy from chlorophyll b of the light harvesting complex II to emitting centres more than flow from chlorophyll a of core pigments. The data are discussed in relation to regulation and localization of thermal energy dissipation mechanisms. It is concluded that desiccation-induced fluorescence quenching of lichens results from the reversible conversion of energy-conserving to energy-dissipating photosystem II core complexes.     

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 F ₀ 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 F ₀ and F _m 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. Received: 8 April 2000 / Accepted: 31 August 2000     

Phototolerance of lichens, mosses and higher plants in an alpine environment: analysis of photoreactions

 Adaptation to excessive light is one of the requirements of survival in an alpine environment particularly for poikilohydric organisms which in contrast to the leaves of higher plants tolerate full dehydration. Changes in modulated chlorophyll fluorescence and 820-nm absorption were investigated in the lichens Xanthoria elegans (Link) Th. Fr. and Rhizocarpon geographicum (L.) DC, in the moss Grimmia alpestris Limpr. and the higher plants Geum montanum L., Gentiana lutea L. and Pisum sativum L., all collected at altitudes higher than 2000 m above sea level. In the dehydrated state, chlorophyll fluorescence was very low in the lichens and the moss, but high in the higher plants. It increased on rehydration in the lichens and the moss, but decreased in the higher plants. Light-induced charge separation in photosystem II was indicated by pulse-induced fluorescence increases only in dried leaves, not in the dry moss and dry lichens. Strong illumination caused photodamage in the dried leaves, but not in the dry moss and dry lichens. Light-dependent increases in 820-nm absorption revealed formation of potential quenchers of chlorophyll fluorescence in all dehydrated plants, but energy transfer to quenchers decreased chlorophyll fluorescence only in the moss and the lichens, not in the higher plants. In hydrated systems, coupled cyclic electron transport is suggested to occur concurrently with linear electron transport under strong actinic illumination particularly in the lichens because far more electrons became available after actinic illumination for the reduction of photo-oxidized P700 than were available in the pool of electron carriers between photosystems II and I. In the moss Grimmia, but not in the lichens or in leaves, light-dependent quenching of chlorophyll fluorescence was extensive even under nitrogen, indicating anaerobic thylakoid acidification by persistent cyclic electron transport. In the absence of actinic illumination, acidification by ca. 8% CO₂ in air quenched the initial chlorophyll fluorescence yield F_o only in the hydrated moss and the lichens, not in leaves of the higher plants. Under the same conditions, 8% CO₂ reduced the maximal fluorescence yield F_m strongly in the poikilohydric organisms, but only weakly or not at all in leaves. The data indicate the existence of deactivation pathways which enable poikilohydric organisms to avoid photodamage not only in the hydrated but also in the dehydrated state. In the hydrated state, strong nonphotochemical quenching of chlorophyll fluorescence indicated highly sensitive responses to excess light which facilitated the harmless dissipation of absorbed excitation energy into heat. Protonation-dependent fluorescence quenching by cyclic electron transport, P700 oxidation and, possibly, excitation transfer between the photosystems were effectively combined to produce phototolerance. Received: 10 December 1999 / Accepted: 13 April 2000     

Characterization of a nonphotochemical quenching-deficient Arabidopsis mutant possessing an intact PsbS protein, xanthophyll cycle and lumen acidification

Arabidopsis thaliana plants grown from ethyl methane sulfonate-treated seeds were screened for so-called que mutants, which are affected in non-photochemical energy quenching. Based on video imaging of chlorophyll fluorescence an energy dissipation mutant, que1, was identified, isolated and characterized. Similar to the npq mutants, the que1 mutant showed a drastically reduced capacity for pH-dependent energy dissipation, qE, but without affecting the Δ pH-dependent conformational changes at 535 nm (ΔA ₅₃₅), which have been supposed to be obligatorily correlated with qE and to reflect pH-regulated binding of zeaxanthin to the PsbS protein. Western blot and DNA sequence analysis revealed that neither a reduced expression of the PsbS protein nor a mutation in the PsbS gene was responsible for the missing qE in que1. Measurements of 9-aminoacridine fluorescence quenching showed that the acidification of the thylakoid lumen was also not affected in the mutant. Furthermore, que1 was able to convert violaxanthin to zeaxanthin. However, unusual characteristics of zeaxanthin formation in the mutant pointed at an altered availability of violaxanthin for de-epoxidation. This was further accompanied by a decrease of the photochemical quenching of chlorophyll fluorescence (qP), an increase of the portion of oxidized P700 and a reduction of the electron transport rate. These characteristics indicate changes in the organization of the thylakoid membrane that affect linear electron transport (but not lumen acidification) and the formation of energy dissipation in photosystem II. Preliminary genetic analysis revealed that the phenotype of que1 is related to two different mutations, mapped to the lower arms of chromosomes 1 and 4.     

The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

A pronounced light-induced zeaxanthin formation accompanied by an unusually slight increase in non-photochemical quenching: a study with barley leaves treated with methyl viologen at moderate light

Light-induced deepoxidation of violaxanthin to antheraxanthin and zeaxanthin in plants is associated with the induction of pronounced xanthophyll-dependent non-photochemical quenching (NPQ). To date, a misbalance between a high amount of zeaxanthin in thylakoid membranes and low NPQ has been explained by an absence of lumen acidification (e.g. when NPQ is measured in the dark after high light stress). In this study, we report that this misbalance can also be observed under moderate light. We found this result (deepoxidation state, DEPS, above 55% and NPQ0.9) in barley leaves treated with 10 μM methyl viologen (MV) under white light (100 μmol photons m⁻² s⁻¹, photosynthetically active radiation (PAR), growth irradiance). The addition of MV at this moderate light did not accelerate electron transport in thylakoid membranes, and induced only slight oxidative stress (no lipid peroxidation, almost unchanged maximum yield of photosystem II photochemistry, a decrease in activity of ascorbate peroxidase, and an increase in that of glutathion reductase). We suggest that, in leaves treated under the conditions used here, the lumen acidification induced by light-limited electron transport in thylakoid membranes was high enough to activate violaxanthin deepoxidase, but not sufficiently high to form the expected number of zeaxanthin-dependent quenching centers in photosystem II antennae.     

Inhibition of violaxanthin deepoxidation by ultraviolet-B radiation in isolated chloroplasts and intact leaves

The effect of pretreatment with ultraviolet-B (UV-B) light (280-320 nanometers) on the enzymatic conversion of the diepoxyxanthophyll violaxanthin to the epoxy-free zeaxanthin occurring in thylakoid membranes was investigated. When isolated chloroplasts of pea (Pisum sativum) were exposed to UV-B, a biologically effective fluence of 7000 joules per square meter caused about 50% inhibition of the activity of the violaxanthin deepoxidase, measured as the first order rate constant of the absorbance change at 505 nanometers. The dose requirement for the inhibition of the deepoxidase in intact leaves, however, was about 2 orders of magnitude higher. The inhibition of the rate constant was observed for both the dark deepoxidation at pH 5, and for the light-driven deepoxidation induced by the lumen acidification due to electron transport from H₂O to methylviologen or due to a photosystem I partial reaction with duroquinol as the electron donor. The availability of violaxanthin was not directly affected by UV-B radiation, as shown for UV-B-treated chloroplasts by the final extent of the 505 nanometer change measured in the dark at pH 5 or by the partial photosystem I reaction. A significant decrease in the violaxanthin availability was observed when lumen acidification was caused by electron transport from H₂O to methylviologen. That effect was probably caused by the wellknown UV-B inhibition of photosystem II with a subsequent decreased ability to reduce the plastoquinone pool, the redox state of which is believed to regulate the final amount of converted violaxanthin.     

Activation of mechanisms of photoprotection by desiccation and by light: poikilohydric photoautotrophs

Mechanisms of protection against photo-oxidation in selected desiccation-tolerant lichens and mosses have been investigated by measuring loss of light absorption during desiccation and chlorophyll fluorescence as indicators of photoprotection. Apparent absorption (1-T) spectra measured in the reflectance mode revealed stronger absorption of photosynthetic pigments in hydrated than in desiccated organisms, but differences were pronounced only in a cyanolichen, less so in some chlorolichens, and even less in mosses. Since the amplitude of chlorophyll fluorescence is a product of (1-T) light absorption by chlorophyll and quantum yield of fluorescence, and since fluorescence is inversely related to thermal energy dissipation, when chemical fluorescence quenching is negligible, fluorescence measurements were used to measure changes in energy dissipation. Preincubation of the hydrated organisms and desiccation in darkness excluded the contribution of mechanisms of energy dissipation to photoprotection which are dependent on the presence of zeaxanthin or on the light-dependent formation of a quencher of fluorescence within the reaction centre of photosystem II. Fast drying in darkness or in very low light was less effective in decreasing chlorophyll fluorescence than slow drying. Heating the desiccated organisms increased fluorescence by inactivating the mechanism responsible for fluorescence quenching. Glutaraldehyde inhibited fluorescence quenching during desiccation. Prolonged exposure of a desiccated moss or a desiccated lichen to very strong light caused more photo-induced damage after fast drying than after slow drying. The photo-oxidative nature of damage was emphasized by the observation that irreversible loss of fluorescence was larger in air than in a nitrogen atmosphere. It is concluded from these observations that desiccation-induced conformational changes of a chlorophyll protein complex result in the fast radiationless dissipation of absorbed light energy. This mechanism of photoprotection is more effective in preventing photo-oxidative damage than other mechanisms of energy dissipation which require light for activation such as zeaxanthin-dependent energy dissipation or quencher formation within the reaction centre of photosystem II.     

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.     

Measurements of cytochrome f and P-700 in intact leaves of Sinapis alba grown under high-light and low-light conditions

The oxidation and reduction of cytochrome f and P-700 is measured spectrophotometrically in leaves of low-light and high-light plants. After illumination with red light, an induction phenomenon for cytochrome f oxidation is observed which indicates a regulation of photosystem I activity through energy distribution between the pigment systems by the energy state of the membrane. After far-red excitation the reduction of cytochrome f in the dark is much slower in low-light leaves. This shows that cyclic electron transport is not improved in low-light plants under these conditions. P-700 is oxidized on excitation with far-red light. However, with high intensities of far-red light, P-700 is partially reduced again which is due to a low extent of photosystem II excitation with the far-red used in the experiments. The low-light leaves show greater sensitivity of photosystem II to this excitation. The initial rate of the cytochrome f oxidation-rate is the same in low-light and high-light leaves. This shows that several P-700 are connected with only one electron transport chain. The consequences of these results concerning the tripartite concept and the photosynthetic unit are discussed. In the high-light plants the experimental data can be well explained by the tripartite organization of the photosynthetic unit. In low-light plants, however, a multipartite organization has to be postulated. In the partition regions of the grana, several antennae systems I, antennae systems II, and light-harvesting complexes can communicate with one electron transport chain.Abbreviations CP I P-700-chlorophyll a-protein - Cyt f cytochrome f - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - LA leaf-area - PhAR photosynthetically active radiation - PS photosystem     

Activation of zeaxanthin is an obligatory event in the regulation of photosynthetic light harvesting.

By dynamic changes in protein structure and function, the photosynthetic membranes of plants are able to regulate the partitioning of absorbed light energy between utilization in photosynthesis and photoprotective non-radiative dissipation of the excess energy. This process is controlled by features of the intact membrane, the transmembrane pH gradient, the organization of the photosystem II antenna proteins and the reversible binding of a specific carotenoid, zeaxanthin. Resonance Raman spectroscopy has been applied for the first time to wild type and mutant Arabidopsis leaves and to intact thylakoid membranes to investigate the nature of the absorption changes obligatorily associated with the energy dissipation process. The observed changes in the carotenoid Resonance Raman spectrum proved that zeaxanthin was involved and indicated a dramatic change in zeaxanthin environment that specifically alters the pigment configuration and red-shifts the absorption spectrum. This activation of zeaxanthin is a key event in the regulation of light harvesting.     

Irrungen,Wirrungen? The Mehler reaction in relation to cyclic electron transport in C3 plants

Plants not only evolve but also reduce oxygen in photosynthesis. Considerable oxygen uptake occurs during photorespiration of C3 plants. Controversies exist on whether direct oxygen reduction in the Mehler reaction together with associated electron transport is also a major sink of electrons when leaves are exposed to sunlight. Here, preference is given to the view that it is not. Whereas photorespiration consumes ATP, the Mehler reaction does not. In isolated chloroplasts photosynthesizing in the presence of saturating bicarbonate, the Mehler reaction is suppressed. In the water – water cycle of leaves, which includes the Mehler reaction, water is oxidized and electrons flow through Photosystems II and I to oxygen producing water. The known properties of coupled electron transport suggest that the water – water cycle cannot act as an efficient electron sink. Rather, by contributing to thylakoid acidification it plays a role in the control of Photosystem II activity. Cyclic electron transport competes with the Mehler reaction for electrons. Both pathways can help to defray possible ATP deficiencies in the chloroplast stroma, but play a more important role by making intrathylakoid protein protonation possible. This is a necessary step for the dissipation of excess excitation energy as heat. Linear electron flow to oxygen relieves the inhibition of cyclic electron transport, which is observed under excessive reduction of intersystem electron carriers. In turn, cyclic electron transport replaces functions of the linear pathway in the control of Photosystem II when oxygen reduction is decreased at low temperatures or, experimentally, when the oxygen concentration of the gas phase is low. Thus, cyclic electron flow acts in flexible relationship with the water–water cycle to control Photosystem II activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Three different mechanisms of energy dissipation of a desiccation-tolerant moss serve one common purpose: to protect reaction centres against photo-oxidation

Three different types of non-photochemical de-excitation of absorbed light energy protect photosystem II of the sun- and desiccation-tolerant moss Rhytidium rugosum against photo-oxidation. The first mechanism, which is light-induced in hydrated thalli, is sensitive to inhibition by dithiothreitol. It is controlled by the protonation of a thylakoid protein. Other mechanisms are activated by desiccation. One of them permits exciton migration towards a far-red band in the antenna pigments where fast thermal deactivation takes place. This mechanism appears to be similar to a mechanism detected before in desiccated lichens. A third mechanism is based on the reversible photo-accumulation of a radical that acts as a quencher of excitation energy in reaction centres of photosystem II. On the basis of absorption changes around 800 nm, the quencher is suggested to be an oxidized chlorophyll. The data show that desiccated moss is better protected against photo-oxidative damage than hydrated moss. Slow drying of moss thalli in the light increases photo-protection more than slow drying in darkness.     

Rapid changes in xanthophyll cycle-dependent energy dissipation and photosystem II efficiency in two vines, Stephania japonica and Smilax australis, growing in the understory of an open Eucalyptus forest

Leaves of Stephania japonica and Smilax australis were characterized in situ on the coast of north-eastern New South Wales, Australia, where they were growing naturally in three different light environments: deep shade, in the understory of an open Eucalyptus forest where they received frequent sunflecks of high intensity, and in an exposed site receiving full sunlight. In deep shade the xanthophyll cycle remained epoxidized during the day and the vast majority of absorbed light was utilized for photosynthesis. In the exposed site both deepoxidation and epoxidation of the xanthophyll cycle and changes in the level of xanthophyll-dependent thermal energy dissipation largely tracked the diurnal changes in photon flux density (PFD). In the understory the xanthophyll cycle became largely deepoxidized to zeaxanthin and antheraxanthin upon exposure of the leaves to the first high intensity sunfleck and this high level of deepoxidation was maintained throughout the day both during and between subsequent sunflecks. In contrast, thermal energy dissipation activity, and the efficiency of photosystem II, fluctuated rapidly in response to the changes in incident PFD. These findings suggest a fine level of control over the engagement of zeaxanthin and antheraxanthin in energy dissipation activity, presumably through rapid changes in thylakoid acidification, such that they became rapidly engaged for photoprotection during the sunflecks and rapidly disengaged upon return to low light when continued engagement might limit carbon gain.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of external electric field on the electron transport system of spinach chloroplasts

Since the thylakoid membranes of an active chloroplast are constantly exposed to the electric fields generated by the electron transport system inside the membranes, we have studied the effects of pretreating chloroplasts of spinach ( Spinacia oleracea L.) leaves with an external AC (alternating current) electric field on their electron transport system. It was found that a few minutes electric field pretreatment (333 V cm^-1 across chloroplast samples), especially at low frequency, irreversibly inhibited the activity of photosystem II (PSII), but under certain conditions, stimulated that of photosystem I (PSI). From the measurements of fluorescence from PSII, we ascribe the inhibition to a lesion close to its reaction center P680, leading to increased dissipation of excitation energy to heat. The effect on PSI was investigated by the reduction of its reaction center, P700 by various artificial donors. We suggest that the stimulative effect can be attributed to a positive shift of the surface charge density of thylakoid membranes that brings about an increase in the accessibility of exogenous electronegative donors.     

Photophosphorylation Associated with Photosystem II: III. Characterization of Uncoupling, Energy Transfer Inhibition, and Proton Uptake Reactions Associated with Photosystem II Cyclic Photophosphorylation

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N′,N′-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.