Current status of the role of Cl− ion in the oxygen-evolving complex

This minireview summarizes the current state of knowledge concerning the role of Cl⁻ in the oxygen-evolving complex (OEC) of photosystem II (PSII). The model that proposes that Cl⁻ is a Mn ligand is discussed in light of more recent work. Studies of Cl⁻ specificity, stoichiometry, kinetics, and retention by extrinsic polypeptides are discussed, as are the results that fail to detect Cl⁻ ligation to Mn and results that show a lack of a requirement for Cl⁻ in PSII-catalyzed H₂O oxidation. Mutagenesis experiments in cyanobacteria and higher plants that produce evidence for a correlation between Cl⁻ retention and stable interactions among intrinsic and extrinsic polypeptides are summarized, and spectroscopic data on the interaction between PSII and Cl⁻ are discussed. Lastly, the question of the site of Cl⁻ action in PSII is discussed in connection with the current crystal structures of the enzyme.     

Differential proteomic analysis using iTRAQ reveals changes in thylakoids associated with Photosystem II‐acquired thermotolerance in Synechocystis sp. PCC 6803

Growth temperature has a marked influence on the thermotolerance of photosystem II (PSII), which is the most heat‐sensitive component of photosynthesis. Using Synechocystis sp. PCC 6803 we have established that thylakoids isolated from cells grown at 38°C have a greater degree of thermotolerance than those isolated from cells grown at 25°C. Reconstitution experiments using Triton X‐100 protein extracts of these thylakoids added to Triton‐treated thylakoid membranes further indicated that the 38°C Triton extract contains proteins that are directly capable of enhancing PSII thermotolerance. We have used 4‐plex iTRAQ, extensive off‐line fractionation and sample re‐injection to comprehensively identify the differences between these two preparations that may be responsible for the observed effects on PSII thermotolerance. This has resulted in the reproducible identification of 168 proteins out of a total of 385 distinct proteins. Our results have identified 15 proteins whose levels are increased in extracts that result in increased thermotolerance of PSII and 33 proteins whose levels decrease. Notably, components of the cytochrome b₆/f and NADH dehydrogenase complexes, crucial components in electron transport, are approximately twofold more abundant in 38°C thylakoid extracts. The possible biological importance of these changes is discussed.     

Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25–50°C) for 4 h in the dark and under growth light intensity (300 μmol m⁻² s⁻¹). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H₂O₂, O₂⁻ and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.     

Fluoride inhibition of photosystem II and the effect of removal of the PsbQ subunit

Photosystem II (PSII), the light-absorbing complex of photosynthesis that evolves oxygen, requires chloride for activation of the oxygen evolving complex (OEC). In this study, fluoride was characterized as an inhibitor of Cl⁻-activated oxygen evolution in higher plant PSII. It was confirmed to be primarily a competitive inhibitor in intact PSII, with Cl⁻-competitive inhibition constant K_i = 2 mM and uncompetitive inhibition constant \textK_\texti^￠ {\text{K}}_{\text{i}}^{\prime }  = 79 mM. A pH dependence study showed that fluoride inhibition was more pronounced at lower pH values. In order to determine the location of the fluoride effect, PSII preparations lacking various amounts of the PsbQ subunit were prepared. The competitive F⁻ inhibition constant and the Michaelis constant for Cl⁻ activation increased with loss of the PsbQ subunit, while the uncompetitive F⁻ inhibition constant was relatively insensitive to loss of PsbQ. The S₂ state EPR signals from PSII lacking PsbQ responded to Ca²⁺ and Cl⁻ removal and to F⁻ treatment similar to intact PSII, with enhancement of the g = 4.1 signal and suppression of the multiline signal, but the effects were more pronounced in PSII lacking PsbQ. Together, these results support the interpretation that the PsbQ subunit has a role in retaining anions within the OEC.     

Phosphorylation and disassembly of the photosystem II core as an early stage of photoinhibition

The presence of heterogeneity in phosphorylated PSII core populations in grana membranes of spinach (Spinacia oleracea L.) was previously demonstrated (Giardi et al., 1991, Biochem. Biophys. Res. Commun. 176, 1298–1304). The effect of photoinhibitory conditions on the distribution of these phosphorylated PSII core populations in thylakoids and PSII particles has been investigated. The sensitivity of the PSII core to strong illumination depended on the phosphorylation state of D₁ and D₂ proteins as well as on the content of the 9-kDa PsbH phosphoprotein. When D₁ and D₂ proteins are under-phosphorylated, the 9-kDa phosphoprotein is tightly bound to the PSII core; thus, a partial protection from photoinhibition is observed. Of the different PSII core populations isolated from membranes photoinhibited for 10 min, the highly phosphorylated populations lack internal antennae CP43 and CP47; perhaps these migrate out to the non-appressed regions of thylakoids. The degradation of the D₁ protein seems to follow the disassembly of the PSII core.     

Desiccation of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> at high temperature

Haberlea rhodopensis plants, growing under low irradiance in their natural habitat, were desiccated to air-dry state at a similar light intensity (about 30 μmol m⁻² s⁻¹) under optimal (23/20°C, day/night) or high (38/30°C) temperature. Dehydration of plants at high temperature increased the rate of water loss threefold and had a more detrimental effect than either drought or high temperature alone. Water deficit decreased the photochemical activity of PSII and PSI and the rate of photosynthetic oxygen evolution, and these effects were stronger when desiccation was carried out at 38°C. Some reduction in the amount of the main PSI and PSII proteins was observed especially in severely desiccated Haberlea leaves. The results clearly showed that desiccation of the homoiochlorophyllous poikilohydric plant Haberlea rhodopensis at high temperature had more damaging effects than desiccation at optimal temperature and in addition recovery was slower. Increased thermal energy dissipation together with higher proline and carotenoid content in the course of desiccation at 38°C compared to desiccation at 23°C probably helped in overcoming the stress.     

Molecular organization of oxygen-evolution system in chloroplast

The main function of Photosystem II in chloroplast is to oxidize water molecules to produce oxygen. Strong oxidant produced by photoreaction at Photosystem II reaction center derives electrons from water and the electrons are transferred via Photosystem I to NADP⁺. The components required for water oxidation in Photosystem II were identified and their molecular properties as well as their roles in the oxygen evolution process were elucidated. The entity of the oxygen evolution system is a supramolecular complex of Photosystem II in the thylakoid membrane where reaction center binding polypeptides, three extrinsic polypeptides, managenese atoms, Ca²⁺ and Cl⁻ ions are the essential components, and they constitute a specific catalytic domain for water oxidation. Recipient of the Botanical Society Award for Young Scientists, 1988.     

Acclimation of photosystem II to high temperature in a suspension culture of soybean (Glycine max) cells requires proteins that are associated with the thylakoid membrane

In a study of the responses of photosystem II (PSII) to high temperature in suspension-cultured cells of soybean (Glycine max L. Merr.), we found that high temperatures inactivated PSII via two distinct pathways. Inactivation of PSII by moderately high temperatures, such as 41°C, was reversed upon transfer of cells to 25°C. The recovery of PSII required light, but not the synthesis of proteins de novo. By contrast, temperatures higher than 45°C inactivated PSII irreversibly. An increase in the growth temperature from 25 to 35°C resulted in an upward shift of 3°C in the profile of the heat-induced inactivation of PSII, which indicated that the thermal stability of PSII had been enhanced. This acclimative response was reflected by the properties of isolated thylakoid membranes: PSII in thylakoid membranes from cells that had been grown at 35°C exhibited greater thermal stability than that from cells grown at 25°C. Disruption of the vesicular structure of thylakoid membranes with 0.05% Triton X-100 decreased the thermal stability of PSII to a similar level in both types of thylakoid membrane. Proteins released by Triton X-100 from thylakoid membranes from cells grown at 35°C were able to increase the thermal stability of Triton-treated thylakoid membranes. These observations suggest that proteins that are associated with thylakoid membranes might be involved in the enhancement of the thermal stability of PSII.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chloride relations of photosystem II membrane preparations depleted of,and resupplied with,their 17 and 23 kDa extrinsic polypeptides

Photosystem II membranes prepared from thylakoids of Phytolacca americana chloroplasts were depleted of their intrinsic 17 and 23 kDa polypeptides, and the effects of a reconstitution of these polypeptides on the Cl^– requirements of O₂ evolution activity were analyzed. It was found that the activating effectiveness of limiting amounts of added Cl^– was increased several fold by an addition of the 23 kDa polypeptide. When it was supplemented by the 17 kDa species, only a small additional increase occurred, but Cl^– retention in Cl^– free media was enhanced greatly. Addition of the 17 kDa polypeptide alone was without effect because it is known that it cannot bind to its native site unless the 23 kDa polypeptide is in place.Optimal enhancements of the effectiveness of activating added Cl^– were observed when the assays were done in the presence of the reconstituting polypeptides. When the reconstituting treatment with the polypeptides, and the assay of the Cl^– relations, were separated, it was advantageous to have Cl^– present in the reconstituting medium, and not to add Ca²⁺, another cofactor of photosynthetic water oxidation. Those requirements are attributed to the labilizing effects Cl^– free conditions and divalent cations have on the association of especially the 23 kDa polypeptides with the water oxidizing complex, and to a possible aggregation of the membranes under the influence of Ca²⁺ which might have impeded proper polypeptide binding.Abbreviations Chl Chlorophyll a+b - Mes 4-morpholineethanesulfonic acid - PSII photosystem II - SDS and LDS sodium or lithium dodecylsulfate     

Highly efficient photoactivation of Mn-depleted photosystem II by imidazole-liganded manganese complexes

The oxygen-evolving complex (OEC) of Mn-depleted photosystem II (PSII) can be reconstituted in the presence of exogenous Mn or a Mn complex under weak illumination, a process called photoactivation. Synthetic Mn complexes could provide a powerful system to analyze the assembly of the OEC. In this work, four mononuclear Mn complexes, [(terpy)₂Mn^II(OOCH₃)]·2H₂O (where terpy is 2,2′:6′,2″-terpyridine), Mn^II(bzimpy)₂, Mn^II(bp)₂(CH₃CH₂OH)₂ [where bzimpy is 2,6-bis(2-benzimidazol-2-yl)pyridine] and [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH (where py is pyridine) were used in photoactivation experiments. Measurements of the photoreduction of 2,6-dichorophenolindophenol and oxygen evolution demonstrate that photoactivation is more efficient when Mn complexes are used instead of MnCl₂ in reconstructed PSII preparations. The most efficient recoveries of oxygen evolution and electron transport activities are obtained from a complex, [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH, that contains both imidazole and phenol groups. Its recovery of the rate of oxygen evolution is as high as 79% even in the absence of the 33-kDa peptide. The imidazole ligands of the Mn complex probably accelerate P ₆₈₀ ^•+ reduction and consequently facilitate the process of photoactivation. Also, the strong intermolecular hydrogen bond probably facilitates interaction with the Mn-depleted PSII via reorganization of the hydrogen-bonding network, and therefore promotes the recovery of oxygen evolution and electron transport activities.     

A two-dimensional many-body system with competing interactions as a model for segregation of photosystems in thylakoids of green plants

 We address the segregation of photosystems I (PSI) and II (PSII) in thylakoid membranes by means of a molecular dynamics method. We assume a two-dimensional (in-plane) problem with PSI and PSII being represented by particles with different values of negative charge. The pair interactions between particles include a screened Coulomb repulsive part and am exponentially decaying attractive part. Our modeling results suggest that the system may have a complicated phase behavior, including a quasi-crystalline phase at low ionic screening, a disordered phase and, in addition, a possible “clotting” agglomerate phase at high screening where the photosystems tend to clot together. The relevance of the observed phenomena to the stacking of thylakoid membranes is discussed. Received: 14 October 1999 / Revised version: 30 March 2000 / Accepted: 30 March 2000     

Microgravity effects on thylakoid,single leaf,and whole canopy photosynthesis of dwarf wheat

The concept of using higher plants to maintain a sustainable life support system for humans during long-duration space missions is dependent upon photosynthesis. The effects of extended exposure to microgravity on the development and functioning of photosynthesis at the leaf and stand levels were examined onboard the International Space Station (ISS). The PESTO (Photosynthesis Experiment Systems Testing and Operations) experiment was the first long-term replicated test to obtain direct measurements of canopy photosynthesis from space under well-controlled conditions. The PESTO experiment consisted of a series of 21–24 day growth cycles of Triticum aestivum L. cv. USU Apogee onboard ISS. Single leaf measurements showed no differences in photosynthetic activity at the moderate (up to 600 μmol m⁻² s⁻¹) light levels, but reductions in whole chain electron transport, PSII, and PSI activities were measured under saturating light (>2,000 μmol m⁻² s⁻¹) and CO₂ (4000 μmol mol⁻¹) conditions in the microgravity-grown plants. Canopy level photosynthetic rates of plants developing in microgravity at ∼280 μmol m⁻² s⁻¹ were not different from ground controls. The wheat canopy had apparently adapted to the microgravity environment since the CO₂ compensation (121 vs. 118 μmol mol⁻¹) and PPF compensation (85 vs. 81 μmol m⁻² s⁻¹) of the flight and ground treatments were similar. The reduction in whole chain electron transport (13%), PSII (13%), and PSI (16%) activities observed under saturating light conditions suggests that microgravity-induced responses at the canopy level may occur at higher PPF intensity.     

Photosynthetic response and recovery of Antarctic marine benthic microalgae exposed to elevated irradiances and temperatures

Exposure to high temperatures affects the photosynthetic processes in marine benthic microalgae by limiting the transport of electrons, thus reducing the ability of the cell to use light. This causes damage to the Photosystem II (PSII) and may lead to photoinhibition. However, the PSII of benthic microalgal communities from Brown Bay, eastern Antarctica, were relatively unaffected by significant changes in temperature. Benthic microalgae exposed to temperatures up to 8°C and an irradiance of 450 μmol photons m⁻² s⁻¹ did not experience any photosynthetic damage or irreversible photoinhibition. The effective quantum yield (∆F/F _m′) at 8°C (0.433 ± 0.042) was higher by comparison to cell incubated at −0.1°C (0.373 ± 0.015) with similar irradiances. Temperatures down to −5°C at a similar irradiance showed a decrease in photosynthesis with decreasing temperature, but no severe photoinhibition as the cells were able to dissipate excess energy via non-photochemical quenching and recover from damage. These responses are consistent with those recorded in past studies on Antarctic benthic microalgae and suggest that short-term temperature change (from −5 to 8°C) will not do irreversible damage to the PSII and will not affect the photosynthesis of the benthic microalgae.     

Effects of Hyperthermia on Intracellular Chloride

Hyperthermia induces transient changes in [Na⁺] _i and [K⁺] _i in mammalian cells. Since Cl⁻ flux is coupled with Na⁺ and K⁺ in several processes, including cell volume control, we have measured the effects of heat on [Cl⁻] _i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl⁻] _i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl⁻] _i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na⁺] _i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl⁻] _i depended upon time and temperature. The [Cl⁻] _i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl⁻] _i increase was completely blocked. Since furosemide inhibits the Na⁺/K⁺/2Cl⁻ cotransporter, Cl⁻ channels, and even Cl⁻HCO₃ exchange, these ion transporters may be involved in the heat-induced increase in [Cl⁻] _i . Received: 15 June 1995/Revised: 9 April 1996     

Oxygen evolution from single- and multiple-turnover light pulses: temporal kinetics of electron transport through PSII in sunflower leaves

Oxygen evolution per single-turnover flash (STF) or multiple-turnover pulse (MTP) was measured with a zirconium O₂ analyzer from sunflower leaves at 22°C. STF were generated by Xe arc lamp, MTP by red LED light of up to 18000 μmol quanta m⁻² s⁻¹. Ambient O₂ concentration was 10–30 ppm, STF and MTP were superimposed on far-red background light in order to oxidize plastoquinone (PQ) and randomize S-states. Electron (e⁻) flow was calculated as 4 times O₂ evolution. Q _A → Q _B electron transport was investigated firing double STF with a delay of 0 to 2 ms between the two. Total O₂ evolution per two flashes equaled to that from a single flash when the delay was zero and doubled when the delay exceeded 2 ms. This trend was fitted with two exponentials with time constants of 0.25 and 0.95 ms, equal amplitudes. Illumination with MTP of increasing length resulted in increasing O₂ evolution per pulse, which was differentiated with an aim to find the time course of O₂ evolution with sub-millisecond resolution. At the highest pulse intensity of 2.9 photons ms⁻¹ per PSII, 3 e⁻ initially accumulated inside PSII and the catalytic rate of PQ reduction was determined from the throughput rate of the fourth and fifth e⁻. A light response curve for the reduction of completely oxidized PQ was a rectangular hyperbola with the initial slope of 1.2 PSII quanta per e⁻ and V _m of 0.6 e⁻ ms⁻¹ per PSII. When PQ was gradually reduced during longer MTP, V _m decreased proportionally with the fraction of oxidized PQ. It is suggested that the linear kinetics with respect to PQ are apparent, caused by strong product inhibition due to about equal binding constants of PQ and PQH₂ to the Q _B site. The strong product inhibition is an appropriate mechanism for down-regulation of PSII electron transport in accordance with rate of PQH₂ oxidation by cytochrome b₆f.     

Electron spin-lattice relaxation studies of different forms of the S2 state multiline EPR signal of the Photosystem II oxygen-evolving complex

The pulsed EPR inversion recovery sequence has been utilized to monitor the temperature dependence of the electron spin-lattice relaxation rate of the Mn cluster of the Photosystem II oxygen evolving complex poised in a variety of S ₂ state forms giving rise to g = 2 multiline EPR signals. A previous study (Lorigan and Britt (1994) Biochemistry 33: 12072–12076) showed that for PS II membranes treated with 5% ethanol, the S ₂ state Mn cluster relaxes via the Orbach spin-lattice relaxation mechanism, where the relaxation is enhanced via phonon scattering off an excited state spin manifold, in this case at an energy of Δ = 36.5 cm⁻¹ above the S = 1/2 ground state giving rise to the multiline EPR signal. Parallel experiments are reported for PS II membranes with 5% methanol, treated with ammonia, and following short and long term dark adaptation. In each case, the temperature dependence of the electron spin-lattice relaxation rate is consistent with Orbach relaxation, and the range of excited state energies is relatively narrow (33.8 cm⁻¹ ≤ Δ ≤ 39.7 cm⁻¹). In addition, short term dark adapted (6 min, ‘active state’) PS II membranes show biphasic recovery traces which indicate that a minority fraction of the oxygen evolving complexes are trapped in a form with greatly slowed spin-lattice relaxation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Frost hardening and photosynthetic performance of Scots pine (Pinus sylvestris L.). II. Seasonal changes in the fluidity of thylakoid membranes

The fluidity of chloroplast thylakoid membranes of frost-tolerant and frost-sensitive needles of␣three- to four-year-old Scots pine (Pinus sylvestris L.) trees, of liposomes produced from the lipids of the thylakoids of these needles, and of liposomes containing varying amounts of light-harvesting complex (LHC) II protein was investigated by means of electron paramagnetic resonance (EPR) measurements using spin-labelled fatty acids as probes. Broadening of the EPR-resonance signals of 16-doxyl stearic acid in chloroplast membranes of frost-sensitive needles and changes in the amplitudes of the peaks were observed upon a decrease in temperature from +30 °C to −10 °C, indicating a drastic loss in rotational mobility. The lipid molecules of the thylakoid membranes of frost-tolerant needles exhibited greater mobility. Moderate frost resistance could be induced in Scots pine needles by short-day treatment (Vogg et al., 1997, Planta, this issue), and growth of the trees under short-day illumination (9 h) resulted in a higher mobility of the chloroplast membrane lipids than did growth under long-day conditions (16 h). The EPR spectrum of thylakoids from frost-tolerant needles at −10 °C was typical of a spin label in highly fluid surroundings. However, an additional peak in the low-field range appeared in the subzero temperature range for the chloroplast membranes of frost-sensitive needles, which represents spin-label molecules in a motionally restricted surrounding. The EPR spectra of thylakoids and of liposomes of thylakoid lipids from frost-hardy needles were identical at +30 °C and −10 °C. The corresponding spectra from frost-sensitive plants revealed an additional peak for the thylakoids, but not for the pure liposomes. Hence, the domains with restricted mobility could be attributed to protein-lipid interactions in the membranes. Broadening of the spectrum and the appearance of an additional peak was observed with liposomes of pure distearoyl phosphatidyl glycerol modified to contain increasing amounts of LHC II. These results are discussed with respect to a loss of chlorophyll and chlorophyll-binding proteins in thylakoids of Scots pine needles under winter conditions. Received: 3 March 1997 / Accepted: 16 July 1997     

Comparison of thermostability of PSII between the chromatic and green leaf cultivars of <Emphasis Type="Italic">Amaranthus tricolor</Emphasis> L.

In the present study, we investigated the antioxidative potential in leaves of the chromatic (CC) versus green (GC) Amaranthus tricolor L. under moderate high-temperature stress at 45°C. Before heat stress, CC had significantly higher levels of betacyanins [about 3.2 mg g⁻¹(FM)] than the green [1.8 mg g⁻¹(FM) (p<0.01), while similar chlorophyll (Chl) content [about 2 mg g⁻¹(FM)] was observed between both cultivars. After exposure to high temperature (45°C) for 6 days, betacyanins in leaves of CC were remarkably increased (about 2 times of that in control samples grown at 30°C). In contrast, betacyanins in GC significantly decreased by 56% in comparison with that of the control. Chl level in CC was higher than that in GC after heat stress for 6 days. Flavonoids and total phenolics in both cultivars were increased, but much more in CC. Significantly less H₂O₂ accumulation was observed in the leaves and stems of CC than in those of GC under heat stress. Interestingly, much stronger circadian oscillation in fluorescence was observed in both cultivars after treatment at 45°C, which suggested that heat stress stimulates endogenous rhythms of photosystem II (PSII). Under moderate high-temperature stress, Chl fluorescence parameters F_v/F_m (maximum quantum yield of PSII), q_P (coefficient of photochemical quenching), Φ_PSII (effective PSII quantum yield), and ETR (electron transport rate) exhibited a gradual decrease, NPQ (nonphotochemical quenching) showed a slight increase followed by a gradual decline, whereas F_o (minimum fluorescence of a dark-adapted leaf) increased continuously. In contrast to GC, after 120 h of high-temperature treatment, CC exhibited significantly lower Fo level, and higher levels of F_v/F_m and NPQ. It is clear that PSII in CC was more stable than that in GC. The results indicate that betacyanins are an effective antioxidant, and probably contribute greatly to the higher thermal stability of PSII and higher tolerance to heat stress.     

Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids

The non-bilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant type of lipid in the thylakoid membrane and plays an important role in regulating the structure and function of photosynthetic membrane proteins. In this study, we have reconstituted the isolated major light-harvesting complexes of photosystem II (PSII) (LHCIIb) and a preparation consisting of PSII core complexes and minor LHCII of PSII (PSIICC) into liposomes that consisted of digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), with or without MGDG. Transmission electron microscopy and freeze-fracture studies showed unilamellar proteoliposomes, and demonstrated that most of the MGDG is incorporated into bilayer structures. The impact of MGDG on the functional interaction between LHCIIb and PSIICC was investigated by low temperature (77 K) fluorescence emission spectra and the photochemical activity of PSII. The additional incorporation of LHCIIb into liposomes containing PSIICC markedly increased oxygen evolution of PSIICC. Excitation at 480 nm of chlorophyll (Chl) b in LHCIIb stimulated a characteristic fluorescence emission of the Chl a in PSII (684.2 nm), rather than that of the Chl a in LHCIIb (680 nm) in the LHCIIb–PSIICC proteoliposomes, which indicated that the energy was transferred from LHCIIb to PSIICC in liposome membranes. Increasing the percentage of MGDG in the PSIICC–LHCIIb proteoliposomes enhanced the photochemical activity of PSII, due to a more efficient energy transfer from LHCIIb to PSIICC and, thus, an enlarged antenna cross section of PSII.