A study of factors which regulate the membrane appression of lettuce thylakoids in relation to irradiance

Factors that may influence the extent of thylakoid membrane appression have been examined using lettuce (Lactuca sativa cv. Celtuce) grown under different irradiances. Electron microscopy and salt-induced chlorophyll fluorescence suggest that the percentage of membrane appression is increased in plants grown in low light (20 Wm^–2) compared with those grown in high light (150 Wm^–2). In high light plants surface charge, as measured by 9-aminoacridine, was found to be twice that measured in low light plants. There was a similar difference in ATPase activity of CF₁ and in light saturated photophosphorylation. The chlorophyll content of LHC-2 as a proportion of the total chlorophyll was greatest in thylakoids of low light plants. Measurement of non-cyclic photophosphorylation rates suggested that membrane appression has a stimulatory role in the photophosphorylation process. The importance of these inter-related factors for the mechanism of thylakoid appression is discussed.Abbreviations PS photosystem - chl chlorophyll - LHC-2 light harvesting chlorophyll-protein complex serving PS 2 - CF₁ coupling factor 1 - NADP nicotinamide-adenine dinucleotide phosphate     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack.These observations, in addition to a slight increase of charge density of the surface-as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies-and partial unstacking of the membranes-as monitored by digitonin method and 540 nm light scattering changes-after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.Abbreviations ATP adenosine triphosphate - 9-AA 9-aminoacridine - Chl chlorophyll - EDTA ethylenediaminetetraacetate - GDA glutaraldehyde - Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - LHC light-harvesting chlorophyll a/b complex PS photosystem     

Characterisation of the effects of Antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts

High energy state quenching of chlorophyll fluorescence (qE) is inhibited by low concentrations of the inhibitor antimycin A in intact and osmotically shocked chloroplasts isolated from spinach and pea plants. This inhibition is independent of any effect upon pH (as measured by 9-aminoacridine fluorescence quenching). A dual control of qE formation, by pH and the redox state of an unidentified chloroplast component, is implied. Results are discussed in terms of a role for qE in the dissipation of excess excitation energy within photosystem II.Abbreviations 9-AA_max = Maximum yield of 9-aminoacridine fluorescence - DCMU = 3(3,4-dichlorophenyl)-1,1-dimethylurea; F_max ± Maximum yield of chlorophyll fluorescence - hr = hour - PAR = Photosynthetically Active Radiation - Q_A = Primary stable electron acceptor within photosystem II - qE = High energy state quenching of chlorophyll fluorescence - qI = quenching of chlorophyll fluorescence related to photoinhibition - qP = Quenching of chlorophyll fluorescence by oxidised plastoquinone - qQ = photochemical quenching of chlorophyll fluorescence - qR = (F_max—maximum level of chlorophyll fluorescence induced by the addition of saturating DCMU) - qT = Quenching of chlorophyll fluorescence attributable to state transitions     

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12–58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E⁶⁵VIHARWAM⁷³ led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159–166] is proposed.Abbreviations BSA bovine serum albumin faction V - ELIPs early light-inducible proteins - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LHCP light-harvesting chlorophyll a/b-protein - LHC IIb light-harvesting complex associated with Photosystem II - pLHCP precursor to LHCP - Rubisco ribulose 1,5-biphosphate carboxylase-oxygenase - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis     

Photosynthetic control, “energy-dependent” quenching of chlorophyll fluorescence and photophosphorylation under influence of tertiary amines

The effects of the tertiary amines tetracaine, brucine and dibucaine on photophosphorylation and control of photosynthetic electron transport in isolated chloroplasts of Spinacia oleracea were investigated. Tertiary amines inhibited photophosphorylation while the related electron transport decreased to the rates, observed under non-phosphorylating conditions. Light induced quenching of 9-aminoacridine fluorescence and uptake of ¹⁴C-labelled methylamine in the thylakoid lumen declined in parallel with photophosphorylation, indicating a decline of the transthylakoid proton gradient. In the presence of ionophoric uncouplers such as nigericin, no effect of tertiary amines on electron transport was seen in a range of concentration where photophosphorylation was inhibited. Under the influence of the tertiary amines tested, pH-dependent feed-back control of photosystem II, as indicated by energy-dependent quenching of chlorophyll fluorescence, was unaffected or even increased in a range of concentration where 9-aminoacridine fluorescence quenching and photophosphorylation were inhibited. The data are discussed with respect to a possible involvement of localized proton flow pathways in energy coupling and feed-back control of electron transport.Abbreviations 9-AA 9-aminoacridine - J _e flux of photosynthetic electron transport - PC photosynthetic control - pH₁ H⁺ concentration in the thylakoid lumen - pmf proton motive force - _P potential quantum yield of photochemistry of photosystem II (with open reaction centers) - Q _A primary quinone-type electron acceptor of photosystem II - q _Q photochemical quenching of chlorophyll fluorescence - q _E energy-dependent quenching of chlorophyll fluorescence - q _AA light-induced quenching of 9-amino-acridine fluorescence     

Effects of nitrogen starvation on the function and organization of the photosynthetic membranes in Cryptomonas maculata (Cryptophyceae)

Nitrogen deficiency affects both photosystems and the antennae pigment systems in the photosynthetic apparatus of the marine alga, Cryptomonas maculata. Under increasing energy fluence rates, O₂ evolution in nitrogen-deficient (-N) cell suspensions never reached a positive value; in control cultures (+N), O₂ evolution increased and was saturated at about 6.4 W·m^-2 with about 100 mol O₂·mg chlorophyll^-1·h^-1. During fluorescence-induction experiments at room temperature, F_o and F_max were significantly increased in-N cells whereas the F_var/F_max ratio decreased from 0.6 to 0.1. These observations can be correlated with a significantly decreased population of 12.5-nm-size particles in the exoplasmic-fracture (EF) faces of freeze-cleaved thylakoid membranes in-N cells (Rhiel et al., 1985, Protoplasma 129, 62–73). The EF particles are suggested to represent photosystem II associated with chlorophyll a/c-protein complexes (LHCP). The banding pattern of isolated and Triton X-100-solubilized thylakoid membranes of both +N and-N cells in sucrose gradients showed that the LHCP is still present in-N cells. The same applies to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane fractions. The reduced number of the 12.5-nm particles in the EF faces of-N cells may be a result of decoupling of the LHCP constituents of the photosystem-II complex rather than their degradation. This is supported by high values for the initial fluorescence F_o in fluorescence-induction experiments and, in part, is indicated by the shift of the maximal fluorescence emission from 693 nm in +N to 684 nm in-N cells. The lack of the CP1 band in the gels of sodium dodecyl sulfate-solubilized thylakoid membranes from-N cells after electrophoresis demonstrates that photosystem I is also severely affected.Abbreviations Chl chlorophyll - CP1 chlorophyll-protein complex of PSI - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - LHCP light-harvesting chlorophyll a/c protein complex - +N/-N control/nitrogen-deficient cell suspension cultures - PSI (II) photosystem I (II) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol Dedicated to Prof. Wilhelm Nultsch on the occasion of his 60^th birthday     

Light-induced fluorescence decay during the greening of normal and lincomycin-treated maize leaves

Light-induced fluorescence decay was examined during the greening of control and lincomycintreated maize (Zea mays L.) leaves. Assuming that this decay to a first approximation is the result of two parallel first-order reactions, the fluorescence induction curves were linearized on the logarithm plot and the parameters were determined. The variable fluorescence increased, and the parameters of the two linear sections of the fluorescence decay—that is, the kinetics of the induction curves—changed during the greening of the control leaves. Lincomycin treatment caused some chlorophyll deficiency and the lowering of the chlorophyll a/b ratio, changed the fluorescence emission spectra and the effect of Mg²⁺ on the regulation of the excitation energy distribution. The structure of the thylakoids and the kinetics of the fluorescence decay were also changed in the treated leaves. The possible relationship between the change of the kinetics of the fluorescence decay and the change of spillover during greening and after lincomycin treatment is discussed.Abbreviations LHC light-harvesting complex - Chl chlorophyll - LM lincomycin - PS photosystem - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

The effect of high temperatures on leaf cells of Valerianella: relative heat stability of the tonoplast membrane of mesophyll vacuoles

The effect of short-term heat stress on the tonoplast membrane of lamb's lettuce (Valerianella locusta (L.) Betcke) mesophyll vacuoles has been investigated. The maintainance of a proton concentration difference (δpH) across the tonoplast membrane served as a criterion for the integrity of the vacuoles. After heat treatment, δpH was measured at room temperature using the fluorescent amine, 9-aminoacridine. It was found with this method that thermal damage to isolated vacuoles mainly occurred in the temperature range above 50°C. Compared with this results, the photosynthetic functions of isolated lettuce protoplasts proved to be markedly more thermolabile, e.g. photosynthetic CO₂ fixation and light-induced chlorophyll fluorescence were drastically reduced at temperatures between 40° and 50°C. Heating of whole leaves and protoplasts and subsequent isolation of vacuoles showed that tonoplast-membrane integrity is not affected by heat stress in situ up to 45°C. Measurement of 9-aminoacridine fluorescence in protoplasts, which allowed conclusions to be drawn regarding the integrity of the tonoplast membrane in its natural cytoplasmic environment, revealed that heat treatment up to 55°C did not significantly affect vacuolar compartmentation. The data provide evidence that the tonoplast membrane is relatively heat stable compared with photosynthetic membranes.     

Sodium Dependency of the Photosynthetic Electron Transport in the Alkaliphilic Cyanobacterium Arthrospira platensis

Arthrospira (Spirulina) platensis (A. platensis) is a model organism for investigation of adaptation of photosynthetic organisms to extreme environmental conditions: the cell functions in this cyanobacterium are optimized to high pH and high concentration (150–250 mM) of Na⁺. However, the mechanism of the possible fine-tuning of the photosynthetic functions to these extreme conditions and/or the regulation of the cellular environment to optimize the photosynthetic functions is poorly understood. In this work we investigated the effect of Na-ions on different photosynthetic activities: linear electron transport reactions (measured by means of polarography and spectrophotometry), the activity of photosystem II (PS II) (thermoluminescence and chlorophyll a fluorescence induction), and redox turnover of the cytochrome b ₆ f complex (flash photolysis); and measured the changes of the intracellular pH (9-aminoacridine fluorescence). It was found that sodium deprivation of cells in the dark at pH 10 inhibited, within 40 min, all measured photosynthetic reactions, and led to an alkalinization of the intracellular pH, which rose from the physiological value of about 8.3–9.6. These were partially and totally restored by readdition of Na-ions at 2.5–25 mM and about 200 mM, respectively. The intracellular pH and the photosynthetic functions were also sensitive to monensin, an exogenous Na⁺/H⁺ exchanger, which collapses both proton and sodium gradients across the cytoplasmic membrane. These observations explain the strict Na⁺-dependency of the photosynthetic electron transport at high extracellular pH, provide experimental evidence on the alkalization of the intracellular environment, and support the hypothesized role of an Na⁺/H⁺ antiport through the plasma membrane in pH homeostasis (Schlesinger et al. (1996). J. Phycol. 32, 608–613). Further, we show that (i) the specific site of inactivation of the photosynthetic electron transport at alkaline pH is to be found at the water splitting enzyme; (ii) in contrast to earlier reports, the inactivation occurs in the dark and, for short periods, without detectable damage in the photosynthetic apparatus; and (iii) in contrast to high pH, Na⁺ dependency in the neutral pH range is shown not to originate from PSII, but from the acceptor side of PSI. These data permit us to conclude that the intracellular environment rather than the machinery of the photosynthetic electron transport is adjusted to the extreme conditions of high pH and high Na⁺ concentration.     

The influence of metal cations and pH on the heat sensitivity of photosynthetic oxygen evolution and chlorophyll fluorescence in spinach chloroplasts

The heat-sensitivity of photosynthetic oxygen evolution of thylakoids isolated from spinach increases by increasing the pH above neutral value. The temperature for inactivation (transition temperature) is lowered from about 45° C (pH 6.0–7.4) to 33°C (pH 8.5). Similar results are obtained with intact chloroplasts. At pH 7.0 the transition temperature of washed thylakoids decreases by lowering the salt concentration below 20 mM with monovalent cations (Li⁺, Na⁺, K⁺) and below 3–4 mM with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺). Illumination decreases the heat-sensitivity of oxygen evolution in intact chloroplasts, but even increases the heat-sensitivity in uncoupled chloroplasts. In intact chloroplasts the transition temperature of the heat-induced rise in chlorophyll fluorescence yield (F_o; see Schreiber and Armond 1978) decreases from 44° C to 38° C when the pH of the suspending medium is increased from 6.5 to 8.5. At 20° C, F_o is almost insensitive to pH (6.0–8.5). At 40° C, however, F_o is constant between 6.0 and 7.0, but strongly increases by increasing the pH above neutral value. The results are discussed in terms of a close relation between electrostatic forces at the thylakoid membrane and thermal sensitivity of photosynthetic apparatus. It is suggested that the heat-sensitivity of the photosystem II complex partially depends on the ionization state of fixed groups having alkaline pK. The packed volume of thylakoids suspended in a low salt medium increases when the temperature is increased above 30° C (pH 7.0) and above 20° C (pH 8.0), respectively. This result suggests a heat-induced increase in surface charge density of the thylakoid membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES morpholinoethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - TRICIN N-[tris(hydroxymethyl)-methyl] glycine     

Correlation between thylakoid protein phosphorylation and molecular organization of the photosynthetic apparatus in a dynamic system

In-vitro thylakoid protein phosphorylation has been studied in synchronized cells of Scenedesmus obliquus at the 8- and 16-h of the life cycle, stages which are characterized by the maximum and minimum photosynthetic activities, respectively. The stage of maximum photosynthetic activity (8-h) is characterized by the highest protein phosphorylation in vitro and in vivo, by the largest proportion of the heavy subfraction of thylakoids, and by maximum oligomerization of the light-harvesting chlorophyll a/b-protein complex, altogether creating the highest energy charge of the thylakoid membranes. Protein phosphorylation in vitro decreases the amount of the heavy subfraction and increases the amount of oligomerization of the antenna of photosystem I (PSI) (increase of chlorophyll b in the light fraction). Concomittantly, PSII units become smaller (longer time for the rise in fluorescence induction) and photosynthetic efficiency increases (decrease of fluorescence yield). In-vivo protein phosphorylation is controlled mainly endogenously during the 8-h of the life cycle but is exogenously modulated by light to optimize the photosynthetic activity by redistribution of pigment-protein complexes. In-vitro protein phosphorylation seems to restore partially the conditions prevalent in vivo and lost during the preparation of membranes. The effect is greater in 16-h cells which have less-stable membranes. The regulatory mechanism between membrane stabilization and oligomerization on the one hand and redistribution of the light-harvesting chlorophyll a/b-protein complex from PSII to PSI on the other hand remains unexplained. We have confirmed that the mechanism of protein phosphorylation is regulated via plastohydroquinone, but experiments with the plastohydroquinone analogue 2,3,5,6-tetramethyl-p-benzoquinone demonstrated that plastohydroquinone is not solely responsible for the differences in protein phosphorylation of 8- and 16-h thylakoids. The inhibitory effect of ADP and the distinct rates of kinase reaction indicate that the adenylate energy charge and changes in the organization of the photosynthetic apparatus also contribute to the observed differences in protein phosphorylation. Phosphorylation in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicated that the 32-kDa phosphoprotein and the herbicide-binding Q_B protein may be the same. These experiments also indicated that 3-(3,4-dichlorophenyl)-1,1-dimethylurea-binding reduces kinase activity directly and not only by inhibiting electron transport.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSI, II photosystem I, II - TMQ 2,3,5,6-tetramethyl-p-benzoquinone Dedicated to Professor Dr. W. Nultsch on the occasion of his 60th brithday     

Some effects of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785) on the development of chloroplast thylakoid membranes in Hordeum vulgare L.

Chloroplast ultrastructural and photochemical features were examined in 6-d-old barley (Hordeum vulgare L. cv. Sundance) plants which had developed in the presence of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785). In spite of a substantial modification of the fatty-acid composition of thylakoid lipids there were no gross abnormalities in chloroplast morphology, and normal amounts of membrane and chlorophyll were present. Fluorescence kinetics at 77K demonstrated considerable energetic interaction of photosystem (PS)I and PSII chlorophylls within the altered lipid environment. An interference with electron transport was indicated from altered room-temperature fluorescence kinetics at 20°C. Subtle changes in the arrangements of chloroplast membranes were consistently evident and the overall effects of these changes was to increase the proportion of appressed to nonappressed membranes. This correlated with a lower chlorophyll a/b ratio, an increase in the amount of light-harvesting chlorophylls as determined by gel electrophoresis and fluorescence emission spectra, and an increase in excitation-energy transfer from PSII to PSI, as predicted from current ideas on the organisation of photosystems in appressed and non-appressed thylakoid membranes.Abbreviations CP1 P700-chlorophyll a protein - F_o, F_m, F_v minimal, maximal and variable fluorescence yield - LHCP light-harvesting chlorophyll-protein complex - PSI, PSII photosystem I, II - San 9785 4-chloro-5(dimethylamino)-2-phenyl-3(2H)-pyridazinone     

Acclimation of Arabidopsis thaliana to the light environment: Changes in composition of the photosynthetic apparatus

Acclimation to changes in the light environment was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Plants grown under four light regimes showed differences in their development, morphology, photosynthetic performance and in the composition of the photosynthetic apparatus. Plants grown under high light showed higher maximum rates of oxygen evolution and lower levels of light-harvesting complexes than their low light-grown counterparts; plants transferred to low light showed rapid changes in maximum photosynthetic rate and chlorophyll-a/b ratio as they became acclimated to the new environment. In contrast, plants grown under lights of differing spectral quality showed significant differences in the ratio of photosystem II to photosystem I. These changes are consistent with a model in which photosynthetic metabolism provides signals which regulate the composition of the thylakoid membrane.Abbreviations Aac1 gene encoding actin - Chl chlorophyll - F far-red-enriched light (R:FR = 0.72) - FR far-red light - H high light (400 mol · m^–2 · s^–1) - L low light (100 ml · m^–2 · s^–1) - LHCII light-harvesting complex of PSII - Lhcb genes encoding the proteins of LHCII - R red light - Rbcs genes encoding the small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - W white light (R:FR = 1.40) This work was supported by Natural Environment Research Council Grant No. GR3/7571A. We would like to thank H. Smith (Botany Department, University of Leicester) and E. Murchie (University of Sheffield) for helpful discussions.     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Pigment Interactions in Light-harvesting Complex II in Different Molecular Environments

Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions, at (−)-437 and (+)-484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids, at (+)-447 and (−)-494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at (−)-494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggregation-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spectral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, whereas a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

A comparative analysis of the effects of in-vivo and in-vitro abscisic-acid treatment on the surface electrical properties of barley chloroplast membranes

The effects of in-vivo and in-vitro abscisic acid (ABA) treatments on the surface charge density () of barley (Hordeum vulgare L.) thylakoids were compared using 9-aminoacridine fluorescence. The estimated surface charge density of isolated thylakoid membranes from control (non-treated) barley leaves was-0.065 C · m^-2. The net negative surface charge density decreased after application of various concentrations of ABA (10^-6, 10^-5 M) for 7 d in-vivo, the more pronounced effect being observed at 10^-5 M ABA. When ABA was added to the suspension of isolated thylakoids the opposite effect was observed. The average charge density increased in in-vitro-treated thylakoids at 10^-5 M ABA to -0.081 C · m^-2. The results are discussed in terms of a specific ABA-induced influence of the composition and-or stoicheometry of charged protein complexes within the thylakoid membranes.Abbreviations and Symbols ABA abscisic acid - 9AA 9-aminoacridine - C, C K⁺ and Mg²⁺ concentrations giving equal relative fluorescence - F 9AA-fluorescence intensity - F_max maximum 9AA fluorescence - surface charge density The authors are grateful to Professor L.P. Popova (Institute of Plant Physiology, Sofia, Bulgaria) for continuous support. This work was supported in part by the Bulgarian Ministry of Science and High Education under research contract No. 519.     

Photoinactivation of photosystem II during photoinhibition in the cyanobacterium Microcystis aeruginosa

Sites of photoinhibition and photo-oxidative damage to the photosynthetic electrontransport system of the unicellular cyanobacterium Microcystis aeruginosa were identified by studies of the kinetics of chlorophyll fluorescence induction by whole cells at room temperature and from partial photosynthetic electron-transport reactions in vitro in thylakoid preparations. Chlorophyll fluorescence intensity decreased following photoinhibitory light treatment. This was attributed to decreases both in the activity of photosystem II and in electron flow through the primary electron acceptor, Q. This inhibition was only partially reversed over a 50-min dark recovery period. Partial photosynthetic electron-transport experiments in vitro demonstrated that photosystem I was not affected by the photoinhibitory treatment. Light damage was associated exclusively with the light reactions, of photosystem II, at a site close to the reaction centre, between the site where diphenylcarbazide can donate electrons and the site where silicomolybdate can accept electrons. This damage presumably reduced production of ATP by noncyclic photophosphorylation and production of NADPH by photosystem I, decreasing the availability of these co-factors for reducing CO₂ in the dark reactions of photosynthesis. The importance of these findings is discussed.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenylcarbazide - PSI photosystem I - PSH photosystem II     

Acclimation of Arabidopsis thaliana to the light environment: changes in photosynthetic function

Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was grown under light regimes of differing spectral qualities, which results in differences in the stoichiometries of the two photosynthetic reaction centres. The acclimative value of these changes was investigated by assessing photosynthetic function in these plants when exposed to two spectrally distinct actinic lights. Plants grown in an environment enriched in far-red light were better able to make efficient use of non-saturating levels of actinic light enriched in long-wavelength red light. Simultaneous measurements of chlorophyll fluorescence and absorption changes at 820 nm indicated that differences between plants grown under alternative light regimes can be ascribed to imbalances in excitation of photosystems I and II (PSI, PSII). Measurements of chlorophyll fluorescence emission and excitation spectra at 77 K provided strong evidence that there was little or no difference in the composition or function of PSI or PSII between the two sets of plants, implying that changes in photosynthetic stoichiometry are primarily responsible for the observed differences in photosynthetic function.Abbreviations Chl chlorophyll - FR far-red light - HF highirradiance FR-enriched light (400 mol·m^–2·s^–1, RFR = 0.72) - HW high-irradiance white light (400 mol·m^–2 1·1 s^–1RFR = 1.40) - LHCI, LHCII light-harvesting complex of PSI, PSII - qO quenching of dark-level chlorophyll fluorescence - qN non-photochemical quenching of variable chlorophyll fluorescence - qP photochemical quenching of variable chlorophyll fluorescence - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Dr. Sasha Ruban for assistance with the 77 K fluorescence measurements and for helpful discussions. This work was supported by Natural Environment Research Council Grant GR3/7571A.