Membrane-dependent heterogeneity of LHCII characterized using single-molecule spectroscopy

In green plants, light harvesting complex of Photosystem II (LHCII) absorbs and transports excitation energy toward the photosynthetic reaction centers and serves as a site for energy-dependent nonphotochemical quenching (qE), the photoprotective dissipation of energy as heat. LHCII is thought to activate dissipation through conformational changes that change the photophysical behaviors. Understanding this balance requires a characterization of how the conformations of LHCII, and thus its photophysics, are influenced by individual factors within the membrane environment. Here, we used ensemble and single-molecule fluorescence to characterize the excited-state lifetimes and switching kinetics of LHCII embedded in nanodisc- and liposome-based model membranes of various sizes and lipid compositions. As the membrane area decreased, the quenched population and the rate of conformational dynamics both increased because of interactions with other proteins, the aqueous solution, and/or disordered lipids. Although the conformational states and dynamics were similar in both thylakoid and asolectin lipids, photodegradation increased with thylakoid lipids, likely because of their charge and pressure properties. Collectively, these findings demonstrate the ability of membrane environments to tune the conformations and photophysics of LHCII.     

Thermodynamics of natural selection II: Chemical Carnot cycles

This is the second in a series of three papers devoted to energy flow and entropy changes in chemical and biological processes, and to their relations to the thermodynamics of computation. In the first paper of the series, it was shown that a general-form dimensional argument from the second law of thermodynamics captures a number of scaling relations governing growth and development across many domains of life. It was also argued that models of physiology based on reversible transformations provide sensible approximations within which the second-law scaling is realized. This paper provides a formal basis for decomposing general cyclic, fixed-temperature chemical reactions, in terms of the chemical equivalent of Carnot's cycle for heat engines. It is shown that the second law relates the minimal chemical work required to perform a cycle to the Kullback-Leibler divergence produced in its chemical output ensemble from that of a Gibbs equilibrium. Reversible models of physiology are used to create reversible models of natural selection, which relate metabolic energy requirements to information gain under optimal conditions. When dissipation is added to models of selection, the second-law constraint is generalized to a relation between metabolic work and the combined energies of growth and maintenance.     

On the relationship between dissipation and the rate of spontaneous entropy production from linear irreversible thermodynamics

When systems are far from equilibrium, the temperature, the entropy and the thermodynamic entropy production are not defined and the Gibbs entropy does not provide useful information about the physical properties of a system. Furthermore, far from equilibrium, or if the dissipative field changes in time, the spontaneous entropy production of linear irreversible thermodynamics becomes irrelevant. In 2000 we introduced a definition for the dissipation function and showed that for systems of arbitrary size, arbitrarily near or far from equilibrium, the time integral of the ensemble average of this quantity can never decrease. In the low-field limit, its ensemble average becomes equal to the spontaneous entropy production of linear irreversible thermodynamics. We discuss how these quantities are related and why one should use dissipation rather than entropy or entropy production for non-equilibrium systems.     

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.     

Mechanisms of non-photochemical chlorophyll fluorescence quenching in higher plants

The excitation energy of pigment molecules in photosynthetic antennae systems is utilised by photochemistry, partly it is thermally dissipated, and partly it is emitted as fluorescence. Changes in the quantum yield of chlorophyll (Chl) fluorescence reflect the changes in quantum yield of photochemical reaction and thermal dissipation of the excitation energy. Decrease of the Chl fluorescence quantum yield is called the Chl fluorescence quenching. The decrease of the quantum yield that is accompanied by photochemical reactions has been termed the photochemical quenching, and the decrease accompanied by thermal dissipation of the excitation energy is called the non-photochemical quenching. This review deals with mechanisms of the non-photochemical quenching.     

Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidata: How does it divert excess energy from photosynthetic reaction center?

Photosystems must efficiently dissipate absorbed light energy under freezing conditions. To clarify the energy dissipation mechanisms, we examined energy transfer and dissipation dynamics in needles of the evergreen plant Taxus cuspidata by time-resolved fluorescence spectroscopy. In summer and autumn, the energy transfer processes were similar to those reported in other higher plants. However, in winter needles, fluorescence lifetimes became shorter not only in PSII but also in PSI, indicating energy dissipation in winter needles. In addition, almost the same fluorescence spectra were obtained with different excitation wavelengths. In contrast, the fluorescence spectrum showed a large difference due to excitation wavelength in spring needles. The fluorescence spectrum of spring needles in 550-nm excitation showed similar spectra to that of winter needles, however, red-chlorophyll fluorescence was not observed in chlorophyll excitation. These observations suggest that some complexes with some kind of red-shifted carotenoid and red-chlorophyll unlink from the core complex in spring. Seasonal changes of excitation energy dynamics are also discussed in relation to changes in thylakoid stacking.     

Mechanisms of non-photochemical chlorophyll fluorescence quenching in higher plants

The excitation energy of pigment molecules in photosynthetic antennae systems is utilised by photochemistry, partly it is thermally dissipated, and partly it is emitted as fluorescence. Changes in the quantum yield of chlorophyll (Chl) fluorescence reflect the changes in quantum yield of photochemical reaction and thermal dissipation of the excitation energy. Decrease of the Chl fluorescence quantum yield is called the Chl fluorescence quenching. The decrease of the quantum yield that is accompanied by photochemical reactions has been termed the photochemical quenching, and the decrease accompanied by thermal dissipation of the excitation energy is called the non-photochemical quenching. This review deals with mechanisms of the non-photochemical quenching.     

Seasonal changes in xanthophyll cycle-dependent energy dissipation in Yucca glauca Nuttall

The evergreen species Yucca glauca was characterized at the end of September and following exposure to low temperatures at the end of November. In November the diurnal pattern of xanthophyll cycle-dependent energy dissipation was altered such that this thermal dissipation process was engaged at a high level throughout the day, whereas in September it only became engaged when leaves received direct sunlight. An analysis of the diurnal partitioning of the absorbed excitation energy into photochemistry versus thermal dissipation suggested that a smaller fraction of that energy was utilized in photochemistry and a greater fraction was dissipated thermally at the end of November compared to September. Lower ratios of Chl a / b and β -carotene/xanthophylls both suggested a decrease in the ratio of reaction centre plus core antenna proteins compared to light-harvesting proteins, and a lower leaf chlorophyll content suggested a decrease in light-harvesting capacity in November versus September. Thus adjustments to the photosynthetic apparatus occurred on several levels in response to the increase in excess excitation energy that Y. glauca experienced during the onset of winter.     

The dissipation of excess excitation energy in British plant species

The reversible dissipation of excitation energy in higher plants is believed to protect against light-induced damage to the photosynthetic apparatus. This dissipation is measured as the non-photochemical quenching of chlorophyll fluorescence. A method is described whereby the saturated capacity for rapidly reversible non-photochemical quenching can be compared between plant species. This method was applied to 22 common British plant species whose habitat was quantified using an index that describes shade tolerance. An association was found between occurrence in open habitats and a high capacity for non-photochemical quenching. It was found that, whilst this capacity was species dependent, it did not depend upon the conditions under which the plant was grown. The possible role of zeaxanthin as a determinant of quenching capacity was examined by measuring the contents of xanthophyll cycle carotenoids for each species. Comparing species, no correlation was seen between the saturated level of non-photochemical quenching and zeaxanthin content expressed relative to either total carotenoid or to chlorophyll. When zeaxanthin was expressed relative to the amount of xanthophyll cycle intermediates (zeaxanthin, antheraxanthin and violaxanthin), a weak correlation was seen.     

Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidata: how does it divert excess energy from photosynthetic reaction center?

Photosystems must efficiently dissipate absorbed light energy under freezing conditions. To clarify the energy dissipation mechanisms, we examined energy transfer and dissipation dynamics in needles of the evergreen plant Taxus cuspidata by time-resolved fluorescence spectroscopy. In summer and autumn, the energy transfer processes were similar to those reported in other higher plants. However, in winter needles, fluorescence lifetimes became shorter not only in PSII but also in PSI, indicating energy dissipation in winter needles. In addition, almost the same fluorescence spectra were obtained with different excitation wavelengths. In contrast, the fluorescence spectrum showed a large difference due to excitation wavelength in spring needles. The fluorescence spectrum of spring needles in 550-nm excitation showed similar spectra to that of winter needles, however, red-chlorophyll fluorescence was not observed in chlorophyll excitation. These observations suggest that some complexes with some kind of red-shifted carotenoid and red-chlorophyll unlink from the core complex in spring. Seasonal changes of excitation energy dynamics are also discussed in relation to changes in thylakoid stacking.     

The application of the minimal energy hypothesis to a casson fluid

Deakin (1967b) suggested that flow of blood might obey a law of minimal energy dissipation. The present paper presents a simpler derivation of Deakin’s equations pointing out several previously unrecognized features. It is shown that these equations are unlikely to be applicable. In particular, the solution obtained by Deakin and Jones (1968) does not yield a true minimum for energy dissipation. The solution for which energy dissipation is actually minimized is shown to possess features which render it unlikely to apply to a real flow.     

Thermal energy dissipation and xanthophyll cycles beyond the Arabidopsis model

Thermal dissipation of excitation energy is a fundamental photoprotection mechanism in plants. Thermal energy dissipation is frequently estimated using the quenching of the chlorophyll fluorescence signal, termed non-photochemical quenching. Over the last two decades, great progress has been made in the understanding of the mechanism of thermal energy dissipation through the use of a few model plants, mainly Arabidopsis. Nonetheless, an emerging number of studies suggest that this model represents only one strategy among several different solutions for the environmental adjustment of thermal energy dissipation that have evolved among photosynthetic organisms in the course of evolution. In this review, a detailed analysis of three examples highlights the need to use models other than Arabidopsis: first, overwintering evergreens that develop a sustained form of thermal energy dissipation; second, desiccation tolerant plants that induce rapid thermal energy dissipation; and third, understorey plants in which a complementary lutein epoxide cycle modulates thermal energy dissipation. The three examples have in common a shift from a photosynthetically efficient state to a dissipative conformation, a strategy widely distributed among stress-tolerant evergreen perennials. Likewise, they show a distinct operation of the xanthophyll cycle. Expanding the list of model species beyond Arabidopsis will enhance our knowledge of these mechanisms and increase the synergy of the current studies now dispersed over a wide number of species.     

Rapid regulation of excitation energy in two pennate diatoms from contrasting light climates

Non-photochemical quenching (NPQ) is a fast acting photoprotective response to high light stress triggered by over excitation of photosystem II. The mechanism for NPQ in the globally important diatom algae has been principally attributed to a xanthophyll cycle, analogous to the well-described qE quenching of higher plants. This study compared the short-term NPQ responses in two pennate, benthic diatom species cultured under identical conditions but which originate from unique light climates. Variable chlorophyll fluorescence was used to monitor photochemical and non-photochemical excitation energy dissipation during high light transitions; whereas whole cell steady state 77 K absorption and emission were used to measure high light elicited changes in the excited state landscapes of the thylakoid. The marine shoreline species Nitzschia curvilineata was found to have an antenna system capable of entering a deeply quenched, yet reversible state in response to high light, with NPQ being highly sensitive to dithiothreitol (a known inhibitor of the xanthophyll cycle). Conversely, the salt flat species Navicula sp. 110-1 exhibited a less robust NPQ that remained largely locked-in after the light stress was removed; however, a lower amplitude, but now highly reversible NPQ persisted in cells treated with dithiothreitol. Furthermore, dithiothreitol inhibition of NPQ had no functional effect on the ability of Navicula cells to balance PSII excitation/de-excitation. These different approaches for non-photochemical excitation energy dissipation are discussed in the context of native light climate.     

The molecular mechanism of the control of excitation energy dissipation in chloroplast membranes. Inhibition of delta pH-dependent quenching of chlorophyll fluorescence by dicyclohexylcarbodiimide.

Non-radiative dissipation of absorbed excitation energy in chloroplast membranes is induced in the presence of the trans-thylakoid proton motive force; this dissipation is measured as high energy state quenching of chlorophyll fluorescence, qE. It has been suggested that this results from a low pH-induced structural alteration in the light harvesting complex of photosystem II, LHCII [(1991) FEBS Letters 292, 1-4]. The effect of the carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), on energy dissipation in chloroplast membranes has been investigated. At concentrations below that required to inhibit electron transport, DCCD caused a decrease in the steady state delta pH, completely inhibited qE and also inhibited the low pH-dependent induction of qE. DCCD binding to polypeptides in the 22-28 kDa range correlated with inhibition of qE. It is suggested that DCCD reacts with amino acid residues in LHCII whose protonation is the primary event in the induction of energy dissipation. This LHCII domain may be identical to one forming a proton channel linking the site of PSII-dependent water oxidation to the thylakoid lumen [(1990) Eur. J. Biochem. 193, 731-736].     

Effects of aluminum on light energy utilization and photoprotective systems in citrus leaves

* BACKGROUND AND AIMS: Under high photon flux, excitation energy may be in excess in aluminum (Al)-treated leaves, which use a smaller fraction of the absorbed light in electron transport due to decreased CO2 assimilation compared with normal leaves. The objectives of this study were to test the hypothesis that the antioxidant systems are up-regulated in Al-treated citrus leaves and correlate with protection from photoxidative damage, and to test whether xanthophyll cycle-dependent thermal energy dissipation is involved in dissipating excess excitation energy. * METHODS: 'Cleopatra' tangerine seedlings were fertilized and irrigated daily for 8 weeks with quarter-strength Hoagland's nutrient solution containing Al at a concentration of 0 or 2 mM from Al2(SO4)3.18H2O. Thereafter, leaf absorptance, chlorophyll (Chl) fluorescence, Al, pigments, antioxidant enzymes and metabolites were measured on fully expanded leaves. * KEY RESULTS: Compared with control leaves, energy was in excess in Al-treated leaves, which had smaller thermal energy dissipation, indicated by non-photochemical quenching (NPQ). In contrast, conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) at midday increased in both treatments, but especially in Al-treated leaves, although A + Z accounted for less 40 % of the total xanthophyll cycle pool in them. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase (CAT), and concentrations of ascorbate (AsA), dehydroascorbate (DASA), reduced glutathione (GSH) and oxidized glutathione (GSSG) were higher in Al-treated than in control leaves. * CONCLUSIONS: These results corroborate the hypothesis that, compared with control leaves, antioxidant systems are up-regulated in Al-treated citrus leaves and protect from photoxidative damage, whereas thermal energy dissipation was decreased. Thus, antioxidant systems are more important than thermal energy dissipation in dissipating excess excitation energy in Al-treated citrus leaves.     

Analysis of LhcSR3, a protein essential for feedback de-excitation in the green alga Chlamydomonas reinhardtii

In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl) triplet states with O₂. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars), that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE) machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818) has been found to be essential for qE in Chlamydomonas reinhardtii. In this work, we report the production of two recombinant LhcSR isoforms from C. reinhardtii and their biochemical and spectroscopic characterization. We found the following: (i) LhcSR isoforms are Chl a/b– and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii) the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii) the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv) LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively active in energy dissipation in its purified form, opening the way to detailed molecular analysis. Owing to its protonatable residues and constitutive excitation energy dissipation, this protein appears to merge both pH-sensing and energy-quenching functions, accomplished respectively by PsbS and monomeric Lhcb proteins in plants.     

Mechanisms shaping the synergism of zeaxanthin and PsbS in photoprotective energy dissipation in the photosynthetic apparatus of plants

Safe operation of photosynthesis is vital to plants and is ensured by the activity of processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is considered to be the primary photoprotective mechanism and is most effective in the combined presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, using wild-type plants, mutants lacking PsbS (npq4), and mutants affected in the xanthophyll cycle (npq1), with the application of molecular spectroscopy and imaging techniques. The results lead to the conclusion that PsbS interferes with the formation of densely packed aggregates of thylakoid membrane proteins, thus allowing easy exchange and incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.     

Cyclic electron flow around photosystem 1 is required for adaptation to salt stress in wild soybean species Glycine cyrtoloba ACC547

A wild soybean species Glycine cyrtoloba ACC547 was found to possess a high salinity resistance trait. It maintained higher net photosynthetic rate (PN) and maximal photochemical efficiency (F_v/F_m) than the soybean Glycine max cultivar Melrose under salt stress. Saline treatment enlarged the post-illumination transient increase in chlorophyll fluorescence in ACC547 much more than that in Melrose, indicating that its cyclic electron flow around photosystem 1 (CEF1) was accelerated more by salt stress. Additionally, ACC547 maintained higher nonphotochemical dissipation of excitation energy than Melrose under salt stress. It is suggested that the salinity resistance of ACC547 might be due to the CEF1-coupled dissipation of excess excitation energy.     

The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves

It has been suggested previously that non-photochemical quenching of chlorophyll fluorescence is associated with a decrease in the rate of photosystem 2 (PS 2) photochemistry. In this study analyses of fluorescence yield changes, induced by flashes in leaves exhibiting different amounts of non-photochemical quenching of fluorescence, are made to determine the effect of non-photochemical excitation energy quenching processes on the rate of PS 2 photochemistry. It is demonstrated that both the high-energy state and the more slowly relaxing components of non-photochemical quenching reduce the rate of PS 2 photochemistry. Flash dosage response curves for fluorescence yield show that non-photochemical quenching processes effectively decrease the relative effective absorption cross-section for PS 2 photochemistry. It is suggested that non-photochemical quenching processes exert an effect on the rate of PS 2 photochemistry by increasing the dissipation of excitation energy by non-radiative processes in the pigment matrices of PS 2, which consequently results in a decrease in the efficiency of delivery of excitation energy for PS 2 photochemistry.     

Reaction centre quenching of excess light energy and photoprotection of photosystem II

In addition to the energy dissipation of excess light occurring in PSII antenna via the xanthophyll cycle, there is mounting evidence of a zeaxanthin-independent pathway for non-photochemical quenching based within the PSII reaction centre (reaction centre quenching) that may also play a significant role in photoprotection. It has been demonstrated that acclimation of higher plants, green algae and cyanobacteria to low temperature or high light conditions which potentially induce an imbalance between energy supply and energy utilization is accompanied by the development of higher reduction state of Q_A and higher resistance to photoinhibition (Huner et al., 1998). Although this is a fundamental feature of all photoautotrophs, and the acquisition of increased tolerance to photoinhibition has been ascribed to growth and development under high PSII excitation pressure, the precise mechanism controlling the redox state of Q_A and its physiological significance in developing higher resistance to photoinhibition has not been fully elucidated. In this review we summarize recent data indicating that the increased resistance to high light in a broad spectrum of photosynthetic organisms acclimated to high excitation pressure conditions is associated with an increase probability for alternative non-radiative P680⁺Q_A ^- radical pair recombination pathway for energy dissipation within the reaction centre of PSII. The various molecular mechanisms that could account for non-photochemical quenching through PSII reaction centre are also discussed.