Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K₃ Fe (CN)₆, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-Q_A interactions.Abbreviations -Car -Carotene - Chl Chlorophyll - DPC Diphenylcarbazide - EPR Electron Paramagnetic Resonance - FeCN potassium ferricyanide - HEPES N-2-hydroxyethylenepiperazine-N-2-ethanesulfonate - P680 reaction center of PSII - PA Photoacoustic - Pheo pheophytin - PSI photosystem I - PSII photosystem II - Q_A primary electron acceptor of PSII     

Changes in D1-protein turnover and recovery of photosystem II activity precede accumulation of chlorophyll in plants after release from mineral stress

After seven weeks of a combined magnesium and sulphur deficiency, spinach (Spinacea oleracea L.) plants showed a substantial accumulation of inactivated photosystem II (PSII) centres as indicated by a 40% decrease of the chlorophyll (Chl) fluorescence parameter F_v/F_m (F_v being the yield of variable fluorescence and F_m the yield of maximal fluorescence when all reaction centres are closed) together with a severe loss of leaf Chl content of 75%. The responses of the photosynthetic apparatus were examined when the deficient plants were transferred back to a rich nutrient medium. During the first 24 h of the recovery phase, thylakoid protein synthesis measured as incorporation of [¹⁴C]leucine per unit of Chl increased substantially. The synthesis rate of the D1 reaction-centre polypeptide of PSII, which in the deficient plants was reduced to 50% of the non-deficient control, was stimulated eight- to ninefold. D1-protein content, which in the deficient plants was reduced to 40% of the non-deficient control, started to increase 2 d later. Thus, D1-protein degradation was also enhanced. The increased D1-protein turnover led to a rapid repair of the existing PSII centres as indicated by the rise of F_v/F_m. It was completed at day 7 of the recovery phase. At day 2 of the recovery phase, the synthesis of other thylakoid proteins such as the D2 protein, cytochrome b ₅₅₉, CP 47 and the 33-kDa polypeptide of the water-splitting system, became stimulated. This process resulted in an accumulation of new PSII centres. During the first week, formation of new PSII centres was not associated with an increase in leaf Chl content. The Chl content of the recovering leaves only started to increase when the ratio of PSII polypeptides versus LHCII (light-harvesting complex of PSII), which was substantially diminished in the deficient plants, became comparable to that of the control. The recovery process was accompanied by substantial changes in thylakoid protein phosphorylation. Their relevance to thylakoid protein turnover and stability is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - F_o yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed (after a saturating flash) under steady-state conditions - F_v yield of variable fluorescence, (difference between F_oand F_m) - F yield of variable fluorescence under steady state conditions - LHC light-harvesting complex - PQ plastoquinone - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_P photochemical quenching - q_n non-photochemical quenching The authors like to thank Dipl. Biol. Britta Untereiser for determining the chlorophyll fluorescence quenching factors. This work was supported by grants from the Bundesminister für Forschung und Technologie, the Project Europäisches Forschungszentrum and the German Israeli Foundation in cooperation with Prof. I. Ohad, Hebrew University, Jerusalem, Israel.     

Effect of light quality on chloroplast-membrane organization and function in pea

The effect of light quality during plant growth of chloroplast membrane organization and function in peas (Pisum sativum L. cv. Alaska) was investigated. In plants grown under photosystem (PS) I-enriched (far-red enriched) illumination both the PSII/PSI stoichiometry and the electrontransport capacity ratios were high, about 1.9. In plants grown under PSII-enriched (far-red depleted) illumination both the PSII/PSI stoichiometry and the electron-transport capacity ratios were significantly lower, about 1.3. In agreement, steady-state electron-transport measurements under synchronous illumination of PSII and PSI demonstrated an excess of PSII in plants grown under far-red-enriched light. Sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of chlorophyll-containing complexes showed greater relative amounts of the PSII reaction center chlorophyll-protein complex in plants grown under farred-enriched light. Additional changes were observed in the ratio of light-harvesting chlorophyll a/b protein to PSII reaction center chlorophyll-protein under the two different light-quality regimes. The results demonstrate the dynamic nature of chloroplast structure and support the notion that light quality is an important factor in the regulation of chloroplast membrane organization and-function.Abbreviations and symbols Chl chlorophyll - CPa PSII reaction center chlorophyll protein complex - CPI PSI chlorophyll protein complex - FR-D light depleted in far-red sensitizing primarily PSII - FR-E light enriched in far-red sensitizing primarily PSI - LHCP PSII light-harvesting chlorophyll a/b protein complex - P ₇₀₀ primary electron donor of PSI - PSI, PSII photosystems I and II, respectively - Q primary electron acceptor of PSII     

Quantitation of plastoquinone photoreduction in spinach chloroplasts

The question of plastoquinone (PQ) concentration and its stoichiometry to photosystem I (PSI) and PSII in spinach chloroplasts is addressed here. The results from three different experimental approaches were compared. (a) Quantitation from the light-induced absorbance change at 263 nm (A₂₆₃) yielded the following ratios (mol:mol); Chl:PQ=70:1, PQ:PSI=9:1 and PQ:PSII=7:1. The kinetics of PQ photoreduction were a monophasic but non-exponential function of time. The deviation of the semilogarithmic plots from linearity reflects the cooperativity of several electron transport chains at the PQ pool level. (b) Estimates from the area over the fluorescence induction curve (A_fl) tend to exaggerate the PQ pool size because of electron transfer via PSI to molecular oxygen (Mehler reaction) resulting in the apparent increase of the pool of electron acceptors. The reliability of the A_fl method is increased substantially upon plastocyanin inhibition by KCN. (c) Quantitation of the number of electrons removed from PQH₂ by PSI, either under far-red excitation or after the addition of DCMU to preilluminated chloroplasts, is complicated due to the competitive loss of electrons from PQH₂ to molecular oxygen. The latter is biphasic reaction occurring with half-times of about 2 s (30–40% of PQH₂) and of about 60 s (60–70% of PQH₂).Abbreviations A_fl area over the fluorescence induction curve - Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PQ plastoquinone - PS photosystem - P700 reaction center of PSI - Q primary quinone acceptor of PSII - Tricine N-tris (hydroxymethyl) methyl glycine - Triton X-100 octyl phenoxy polyethoxyethanol     

Fluorescence Induction Kinetics in Membrane Preparations of Photosystem II with Heterogeneous Metal Clusters (Mn/Fe) in the Oxygen-Evolving Complex

Transport of electrons in spinach photosystem II (PSII) whose oxygen-evolving complex (OEC) contains heterogeneous metal clusters 2Mn2Fe and 3Mn1Fe was studied by measuring the fluorescence induction kinetics (FIK). PSII(2Mn,2Fe) and PSII(3Mn,1Fe) preparations were produced using Cadepleted PSII membranes (PSII(–Ca)). It was found that FIK in PSII(2Mn,2Fe) membranes is similar in form to FIK in PSII(–Ca) samples, but the fluorescence yield is lower in PSII(2Mn,2Fe). The results demonstrate that, just as in PSII(–Ca) preparations, there is electron transfer from the metal cluster in the OEC to the primary plastoquinone electron acceptor Q_A. They also show that partial substitution of Mn cations with Fe has no effect on the electron transport on the acceptor side of PSII. Thus, these data demonstrate the possibility of water oxidation either by the heterogeneous metal cluster or just by the manganese dimer. We established that FIK in PSII(3Mn,1Fe) preparations are similar in form to FIK in PSII(2Mn,2Fe) membranes but PSII(3Mn,1Fe) is characterized by a slightly higher maximal fluorescence yield, F_max. The electron transfer rate in PSII(3Mn,1Fe) preparations significantly (by a factor of two) increases in the presence of Ca²⁺, whereas Ca²⁺ has hardly any effect on the electron transport in PSII(2Mn,2Fe) membranes. In Mndepleted PSII membranes, FIK reaches its maximum (the so-called peak K), after which the fluorescence yield starts to decrease as the result of two factors: the oxidation of reduced primary plastoquinone Q _A ^? and the absence of electron influx from the donor side of PSII. The replacement of Mn cations by Fe in PSII(?Mn) preparations leads to fluorescence saturation and disappearance of the K peak. This is possibly due to the deceleration of the charge recombination process that takes place between reduced primary electron acceptor Q _A ^? and oxidized tyrosine Y _Z ^+. which is an electron carrier between the OEC and the primary electron donor P680.     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

Photoinactivation of the reactivation capacity of photosystem II in pea subchloroplast particles after a complete removal of manganese

After a complete removal of Mn from pea subchloroplast photosystem-II (PS II) preparations the electron phototransfer and oxygen evolution are restored upon addition of Mn²⁺ and Ca²⁺. Pre-illumination of the sample in the absence of Mn²⁺ leads to photoinhibition (PI) — irreversible loss of the capability of PS II to be reactivated by Mn²⁺. The effect of PI is considerably decreased in the presence of Mn²⁺ (4 Mn atoms per reaction center of PS II) and it is increased in the presence of ferricyanide or p-benzoquinone revealing the oxidative nature of the photoeffect. PI results in suppression of oxygen evolution, variable fluorescence, photoreduction of 2,6-dichlorophenol indophenol from either water or diphenylcarbazide. However, photooxidation of chlorophyll P₆₈₀, the primary electron donor of PS II as well as dark and photoinduced EPR signal II (ascribed to secondary electron donors D ₁ and Z) are preserved. PI is accompanied by photooxidation of 2–3 carotenoid molecules per PS II reaction center (RC) that is accelerated in the presence of ferricyanide and is inhibited upon addition of Mn²⁺ or diuron. The conclusion is made that PI in the absence of Mn leads to irreversible oxidative inactivation of electron transfer from water to RC of PS II which remains photochemically active. A loss of functional interaction of RC with the electron transport chain as a common feature for different types of PS II photoinhibition is discussed.Abbreviations A photoinduced absorbance changes - DPC diphenylcarbazide - DPIP 2,6-dichlorophenol indophenol - F _o constant fluorescence of chlorophyll - F photoinduced changes of Chl fluorescence yield - Mn manganese - P₆₈₀ the primary electron donor in PS II - PI photoinhibition - PS II photosystem II - Q the primary (quinone) electron acceptor in PS II - RC reaction center     

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1_mod and D2_mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1_mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2_mod contained D2/cytochrome b₅₅₉ with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1_mod but not D2_mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Analysis of isolated assembly complexes provides new insights into the early stages of photosystem II biogenesis.     

Characterization of O2 evolution by a wheat photosystem II reaction center complex isolated by a simplified method: disjunction of secondary acceptor quinone and enhanced Ca2+ demand

An O2-evolving photosystem II (PSII) reaction center complex was prepared from wheat by a simple method consisting of octylglucoside solubilization of Triton PSII particles followed by one-step sucrose density gradient centrifugation. The complex contained six species of proteins including the 33-kDa extrinsic protein with the same relative abundance as in the original PSII particles, one cytochrome b559, 4 Mn, and about 40 chlorophyll (Chl) per O2-evolving unit, and evolved O2 at a high rate of 1400-1700 mumol O2/mg Chl/h. O2 evolution by the complex was dependent on acceptor species, showing a hierarchy, ferricyanide greater than dichlorobenzoquinone greater than phenylbenzoquinone greater than dimethylbenzoquinone greater than duroquinone, and insensitive to DCMU, indicative of disjunction of the secondary quinone acceptor of PSII from the electron transport pathway. O2 evolution also showed a marked dependence on Cl- and Ca2+: about 10-fold acceleration by Cl- and an additional 2- to 3-fold by Ca2+. Comparison of the dissociation constants for Cl- and Ca2+ between the complex and NaCl-washed PSII particles revealed that octylglucoside treatment gives rise to a new Ca2+-sensitive site by removal of some unknown factor(s) other than the extrinsic 22- and 16-kDa proteins, while it preserves the Cl(-)-sensitive site as native as in NaCl-washed PSII particles. Analysis of the relationship between Cl- demand and Ca2+ demand revealed that Ca2+ absence noncompetitively inhibits the Cl(-)-supported O2 evolution, indicative of the independence of the binding site of these two factors.     

Photoinhibitory damage is modulated by the rate of photosynthesis and by the photosystem II light-harvesting chlorophyll antenna size

We investigated the effect of photosynthetic electron transport and of the photosystem II (PSII) chlorophyll (Chl) antenna size on the rate of PSII photoinhibitory damage. To modulate the rate of photosynthesis and the light-harvesting capacity in the unicellular chlorophyte Dunaliella salina Teod., we varied the amount of inorganic carbon in the culture medium. Cells were grown under high irradiance either with a limiting supply of inorganic carbon, provided by an initial concentration of 25 mM NaHCO₃, or with supplemental CO₂ bubbled in the form of 3% CO₂ in air. The NaHCO₃-grown cells displayed slow rates of photosynthesis and had a small PSII light-harvesting Chl antenna size (60 Chl molecules). The half-time of PSII photodamage was 40 min. When switched to supplemental CO₂ conditions, the rate of photodamage was retarded to a t_1/2 = 70 min. Conversely, CO₂-supplemented cells displayed faster rates of photosynthesis and a larger PSII light-harvesting Chl antenna size (500 Chl molecules). They also showed a rate of photodamage with t_1/2 = 40 min. When depleted of CO₂, the rate of photodamage was accelerated (t_1/2  = 20 min). These results indicate that the in-vivo susceptibility to photodamage is modulated by the rate of forward electron transport through PSII. Moreover, a large Chl antenna size enhances the rate of light absorption and photodamage and, therefore, counters the mitigating effect of forward electron transport. We propose that under steady-state photosynthesis, the rate of light absorption (determined by incident light intensity and PS Chl antenna size) and the rate of forward electron transport (determined by CO₂ availability) modulate the oxidation/reduction state of the primary PSII acceptor Q_A, which in turn defines the low/high probability for photodamage in the PSII reaction center. Received: 14 August 1997 / Accepted: 26 September 1997     

High correlation between thermotolerance and photosystem II activity in tall fescue

Heat stress affects a broad spectrum of cellular components and metabolism. The objectives of this study were to investigate the behavior of Photosystem II (PSII) in tall fescue (Festuca arundinacea Schreb) with various thermotolerance capacities and to broaden our comprehension about the relationship between thermotolerance and PSII function. Heat-tolerant and heat-sensitive accessions were incubated at 24 °C (control) and 46 °C (heat stress) for 5 h. The fluorescence transient curves (OJIP curves), slow Chl fluorescence kinetic, and light response curve were employed to study the behavior of PSII subjected to heat stress. After heat stress, performance index for energy conservation from photons absorbed by PSII antenna until the reduction of PSI acceptors (PI_Total), the value of electrons produced per photon (a), and the maximal rate of electron transport (ETR_max) of heat-tolerant accessions were lower than those of heat-sensitive accessions. Relatively lower reactive oxygen species (ROS) contents were detected in heat-tolerant accessions. Simultaneously, there was a significant decline in the quantum yield of photochemical energy conversion in PS II (Y(II)), probability that a PSII Chl molecule functions as reaction center (γ_RC), and the increase of quantum yield for non-regulated non-photochemical energy loss (Y(NO)) in heat-tolerant accessions. Moreover, a significant inverse correlation between heat tolerance indexes (HTI) and Y(II) was observed. Therefore, maintaining a lower photochemical activity in heat-tolerant accessions could be a crucial strategy to improve their thermotolerance. This finding could be attributed to the structural difference in the reaction center, and for heat-tolerant accessions, it could simultaneously limit energy input into linear electron transport, and dissipate more energy through non-regulated non-photochemical energy loss processes.     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

Kinetic study of PF and CarT states in the LM subunit purified from the wild-type Rhodobacter sphaeroides reaction centers

The kinetics of absorbance changes related to the charge-separated state, P^F, and to the formation and decay of the carotenoid triplet state (Car^T) were studied in the LM reaction center subunit isolated from a wild-type strain of the purple bacterium Rhodobacter sphaeroides (strain Y). The P^F lifetime is lengthened (20±1.5 ns) in the LM complex as compared to the intact reaction centers (11±1 ns). The yield of the carotenoid triplet formation is higher (0.28±0.01) in the LM complex than in native reaction centers. We interpret our results in terms of perturbations of a first-order reaction connecting the singlet and the triplet state of the radical-pair state. Our results, together with those of a recent work (Agalidis, I., Nuijs, A.M. and Reiss-Husson, F. (1987) Biochim. Biophys. Acta (in press)) are consistent with a high I to Q_A electron transfer rate in this LM subunit, which is metal-depleted.The LM complex is considerably more sensitive than the reaction centers to photooxidative damage in the presence of oxygen. This is not readily accounted for simply by the higher carotenoid triplet yield, and may suggest a greater accessibility of the internal structures in the absence of the H-subunit.The lifetime of the carotenoid triplet decay (6.4±0.3 s) in the LM subunit is unchanged compared to the native reaction centers.Abbreviations BChl bacteriochlorophyll - Bph bacteriopheophytin - Car carotenoid - Chl chlorophyll - cyt cytochrome - L, M and H subunits light, medium and heavy subunits of the reaction center complex - P^R triplet electronic state of the primary electron donor - P; Q_A the first stable electron acceptor, a bound quinone - RC reaction center - LDAO lauryldimethylamine N-oxide - SDS sodium dodecyl sulfate - UQ ubiquinone This paper is published in our new format. All future authors are requested to follow our new instructions (see Photosynthesis Research 10:519–526, 1986)—Editor.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: studies of the mechanism.

The effects of photosystem II (PSII) exogenous electron donors and acceptors on the kinetics of weak light photoinhibition of NH2OH/EDTA-extracted spinach PSII membranes were examined. Under aerobic conditions, Mn2+ (approximately 1 Mn/reaction center; Km approximately 400 nM) inhibited photoinactivation and approximately 1 Mn/reaction center plus 100 microM NH2NH2 gave almost complete protection. In the absence of electron donors, strict anaerobiosis greatly inhibited photoinactivation even in the presence of an electron acceptor. Under aerobic conditions, the addition of electron acceptors (FeCN, DCIP), oxyradical scavengers, or superoxide dismutase strongly suppressed rates of photodamages. Increase in the concentrations of superoxide above those produced by illuminated NH2OH/EDTA-photosystem II membranes increased the rates of damage in the light but gave no damage in the dark. Scavengers of hydroxyl radicals and singlet oxygen did not suppress the rates of aerobic photoinhibition. These findings, along with others, lead us to conclude that photodamage of the secondary donors of the PSII reaction center occurs by two mechanisms: (1) a rapid superoxide and tyrosine YZ+ dependent process and (2) a slower process in which P680+/Chl+ catalyze the damages.     

Latent manganese deficiency in barley can be diagnosed and remediated on the basis of chlorophyll a fluorescence measurements

Background and aims

Manganese (Mn) deficiency represents a major plant nutritional disorder in winter cereals. The deficiency frequently occurs latently and the lack of visual symptoms prevents timely remediation and cause significant yield reductions. These problems prompted us to investigate chlorophyll (Chl) a fluorescence as a tool for diagnosis of latent Mn deficiency.

Methods

Barley plants grown under controlled greenhouse conditions or in the field were exposed to different intensities of Mn deficiency. The responses were characterised by analysis of Chl a fluorescence, photosystem II (PSII) proteins and mineral elements.

Results

Analysis of the Chl a fluorescence induction kinetics (FIK) revealed distinct changes long before any visual symptoms of Mn deficiency were apparent. The changes were specific for Mn and did not occur in Mg, S, Fe or Cu deficient plants. The changes in Mn deficient plants were accompanied by a marked reduction of the D1 protein in PSII. Foliar Mn application fully restored PSII functionality, ensured winter survival, and increased grain yields under field conditions.

Conclusions

The efficiency and stability of PSII are markedly affected by latent Mn deficiency. Chlorophyll a fluorescence measurements constitute a powerful and valuable tool for diagnosis and remediation of latent Mn deficiency.     

Photon- and carbon-use efficiency in<Emphasis Type="Italic"> Ulva rigida</Emphasis> at different CO<Subscript>2</Subscript> and N levels

The seaweed Ulva rigida C. Agardh (Chlorophyta) was cultured under two CO₂ conditions supplied through the air bubbling system: non-manipulated air and 1% CO₂-enriched aeration. These were also combined with N sufficiency and N limitation, using nitrate as the only N source. High CO₂ in U. rigida led to higher growth rates without increasing the C fixed through photosynthesis under N sufficiency. Quantum yields for charge separation at photosystem II (PSII) reaction centres (_PSII) and for oxygen evolution (_O2) decreased at high CO₂ even in N-sufficient thalli. Cyclic electron flow around PSII as part of a photoprotection strategy accompanied by decreased antennae size was suspected. The new re-arrangement of the photosynthetic energy at high CO₂ included reduced investment in processes other than C fixation, as well as in carbon diverted to respiration. As a result, quantum yield for new biomass-C production (^growth) increased. The calculation of the individual quantum yields for the different processes involved allowed the completion of the energy flow scheme through the cell from incident light to biomass production for each of the CO₂ and N-supply conditions studied.Abbreviations A total thallus absorptance - A_pig absorptance due to pigments - A_str Absorptance due to non-pigmented structures - a* spectrally averaged in vivo absorption cross-section of chlorophyll a - CCM carbon-concentrating mechanism - Chl chlorophyll - DOC dissolved organic carbon - ETR electron transport rate - F_v/F_m optimum quantum yield for PSII charge separation - GP gross O₂ evolution rate - k_pig specific light absorption coefficient for pigments - k_str specific light absorption coefficient for non-pigmented structures - OP optimum O₂ evolution rate - PFR photon fluence rate - POC particulate organic carbon - PS photosystem - q_N non-photochemical quenching - q_P photochemical quenching - ^growth quantum yield for new biomass-C production - _O2 quantum yield for gross O₂ evolution - _PSII quantum yield for PSII charge separation     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein

A His-tagged PSII core complex was purified from recombinantChlamydomonas reinhardtii D2-H thylakoids by single-step Ni²⁺-affinitycolumn chromatography and its properties were partially characterizedin terms of their PSII functions and chemical compositions.The PSII core complex that has a His-tag extension at the C-terminusof the D2 protein evolved oxygen at a high rate of 2,400 µmol(mg Chl)^–1h^–1 at the optimum pH of 6.5 with ferricyanideand 2,6-dichlorobenzoquinone as electron acceptors in the presenceof Ca²⁺ as an essential cofactor, and approximately 90% of theactivity was blocked by 10 µM DCMU. The core complex exhibitedthe thermoluminescence Q-band but not the B-band regardlessof the presence or absence of DCMU, although both bands wereobserved in the His-tagged thylakoids. The core complex wasfree from PSI and contained one Y_D, Tyr 160 of the D2 protein,four Mn atoms, two cytochrome b-559, about 46 Chl a molecules,and probably one Q_A, the primary acceptor quinone of PSII. Itwas inferred from these results that His-tagging at the C-terminusof the D2 protein does not affect the functional and structuralintegrity of the PSII core complex, and that the ‘His-tagstrategy’ is highly useful for biochemical, physicochemical,and structural studies of Chlamydomonas PSII. (Received October 22, 1998; Accepted December 25, 1998)