PSI) And II Research Articles

PGRL1 Is the Elusive Ferredoxin-Plastoquinone Reductase in Photosynthetic Cyclic Electron Flow

During plant photosynthesis, photosystems I (PSI) and II (PSII), located in the thylakoid membranes of the chloroplast, use light energy to mobilize electron transport. Different modes of electron flow exist. Linear electron flow is driven by both photosystems and generates ATP and NADPH, whereas cyclic electron flow (CEF) is driven by PSI alone and generates ATP only. Two variants of CEF exist in flowering plants, of which one is sensitive to antimycin A (AA) and involves the two thylakoid proteins, PGR5 and PGRL1. However, neither the mechanism nor the site of reinjection of electrons from ferredoxin into the thylakoid electron transport chain during AA-sensitive CEF is known. Here, we show that PGRL1 accepts electrons from ferredoxin in a PGR5-dependent manner and reduces quinones in an AA-sensitive fashion. PGRL1 activity itself requires several redox-active cysteine residues and a Fe-containing cofactor. We therefore propose that PGRL1 is the elusive ferredoxin-plastoquinone reductase (FQR).

Probing the picosecond kinetics of the photosystem II core complex in vivo

Photosystems I (PSI) and II (PSII) are two major pigment-protein complexes of photosynthetic organisms that function in series to convert sunlight energy into chemical energy. We have studied the picosecond fluorescence behaviour of the core of both photosystems in vivo by using two Synechocystis PCC 6803 mutants: BE cells contain PSI but are lacking both PSII and the light-harvesting complexes called phycobilisomes (PBs) whereas PAL cells contain both PSI and PSII but lack the PBs. Measurements were performed at room temperature and at 77 K. The fluorescence kinetics of PSI and PSII can nicely be separated, en passant providing the PSI/PSII ratio. At room temperature, the PSI kinetics are identical to those of isolated PSI whereas the PSII kinetics can equally well be described by the in vitro trap-limited model of Y. Miloslavina, M. Szczepaniak, M. G. Muller, J. Sander, M. Nowaczyk, M. Rogner and A. R. Holzwarth, Biophys J., 2009, 96(2), 621-631, and by the transfer-to-the-trap-limited model of C. D. van der Weij-de Wit, J. P. Dekker, R. van Grondelle and I. H. M. van Stokkum, J. Phys. Chem. A, 2011, 115(16), 3947-3956, albeit that the in vivo kinetics turn out to be somewhat slower. At 77 K several low-energy pigments are observed in both photosystems which complicate the overall dynamics but the PSII kinetics can still be described by both a trap-limited and a transfer-to-the-trap-limited model.

Simultaneous analysis of photosystem responses of Microcystis aeruginoga under chromium stress

Chromium (Cr) is a toxic metal that poses a great threat to aquatic ecosystems. Information is limited on coinstantaneous responses of photosystems I (PSI) and II (PSII) to Cr(VI) stress due to lack of instruments that can simultaneously measure PSI and PSII activities. In the present study, responses of quantum yields of energy conversion and electron transport rates of PSI and PSII in Microcystis aeruginosa cells to Cr(VI) stress were simultaneously analyzed by a DUAL-PAM-100 system. Quantum yield of cyclic electron flow (CEF) under Cr(VI) stress and its physiological role in alleviating toxicity of Cr(VI) were also analyzed. At 5mgL−1 Cr(VI), quantum yield and electron transport rate of PSII decreased significantly, and light-induced non-photochemical fluorescence quenching lost. Cr(VI) also inhibited efficiency of PSII to use energy under high light more than of PSI. PSII showed lower maximal electron transport rate and light adaptability than PSI. Electron transport rate of PSI was higher and decreased less than that of PSII, implying less sensitivity of PSI to high light and Cr(VI). Energy dissipation through non-light-induced non-photochemical fluorescence quenching increased with increasing Cr(VI) concentration. CEF was stimulated under Cr(VI) treatment and made a significant contribution to quantum yield and electron transport of PSI, which was essential for protection of PSI from stresses of Cr(VI) and high light.

Functional analyses of the plant photosystem I-light-harvesting complex II supercomplex reveal that light-harvesting complex II loosely bound to photosystem II is a very efficient antenna for photosystem I in state II.

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.

Disturbed structure and function of chloroplasts during blocked biosynthesis of 5-aminolevulinic acid in the light

Xantha-702 mutant of cotton (Gossypium hirsutum L.) proved to have blocked synthesis of 5'-aminolevulinic acid in the light. Accordingly, mutant leaves accumulated 2-5% chlorophyll of baseline. Mutant plants demonstrated disturbed production of pigment-protein complexes of photosystems I (PSI) and II (PSII) and generation of the chloroplast membrane system blocked at the early stages, largely, at the stages of vesicles and single short thylakoid. The functional activity of the PSI and PSII reaction centers was close to zero. Only the chlorophyll a/b light-harvesting complexes of PSI and PSII with the chlorophyll fluorescence peaks at 728 and 681 nm, respectively, were produced in the xantha-702 mutant. We propose that the genetic block of 5-aminolevunilic acid biosynthesis in the light in the xantha-702 mutant disturbs the formation and activity of the complexes of the reaction centers of PS-I and PS-II and inhibits the development of the whole membrane system of chloroplasts.

Extinction coefficients and critical solubilisation concentrations of photosystems I and II from Thermosynechococcus elongatus

The absorption properties of chlorophyll a (Chl a) in active core complexes of photosystems I (PSI) and II (PSII) isolated in high purity from the thermophilic cyanobacterium Thermosynechococcus elongatus were correlated with those of extracts in 80% acetone to determine effective extinction coefficients of protein-bound Chl a and molar extinction coefficients of core complexes and reaction centers (RC). These coefficients allow a quick determination of Chl a and protein concentrations from steady-state absorption spectra of intact samples without the need for pigment extraction and protein destruction. In the visible range, ε 680 p = 57 mM −1 cm −1 for trimeric PSI (PSIt) and ε 674 p = 70 mM −1 cm −1 for dimeric (PSIId) and monomeric (PSIIm) PSII (error ±6%; superscript “ p” refers to Chl a bound to intact protein, subscripts are the peak maxima in nm). The integral extinction coefficient ϕ p = 2.8 nm μM −1 cm −1 for the wavelength interval between 550 and 800 nm and the extinction coefficient ε B p = 14 mM −1 cm −1 for the smaller absorption maximum (B = 632 nm for PSI and 627 nm for PSII) were found to be essentially the same for both types of PS. The coefficients of PSIt are shown to remain unaltered when 65% (v/v) of the buffer is replaced with glycerol. Molar extinction coefficients of core complexes were determined using Chl a/RC ratios of 96±1 for PSI and 35±2 for PSII based on X-ray data. In addition, the critical solubilisation concentration of n-dodecyl-β- d-maltoside (βDM), necessary to keep the core complexes in solution, was determined by turbidimetric titrations. It was found that at least ∼500 βDM molecules per PSIt (∼2 βDM per Chl a) and 190 βDM molecules per PSIIm (∼5 βDM per Chl a, also for PSIId) in excess of the critical micelle concentration of 0.16 ± 0.03 mM are necessary for a complete solubilisation of the core complexes.

Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry.

The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80,000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning alpha-helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.

Thylakoid Membrane Proteome: Separation by HPLC and Identification by Accurate Molecular Mass Determinations

This review covers the vast array of methods that appeared in the last few years for separation and identification of thylakoid membrane proteins present in chloroplast, a good model for setting up analytical methods suitable for membrane proteins. Although the major method for protein separation is gel electrophoresis (GE), we will summarise in this review the results of several studies performed to develop a rapid and straightforward method based on high-performance liquid chromatography (HPLC) to resolve most of the antenna proteins of the two photosystems, photosystem I (PSI) and II (PSII) and identify them by intact molecular mass determination using electrospray ionisation mass spectrometry (ESI-MS). The complex mixtures of these strongly hydrophobic membrane proteins are prefractionated by sucrose gradient ultracentrifugation in the first dimension followed by reversed-phase HPLC in the second dimension. The separation achieved is by far superior to that possible with GE. It is based on small differences in hydrophobicity of these very similar proteins. The correspondence between the intact molecular masses measured with those deduced from the DNA sequence enabled the identification of the different protein components of antenna complex. Isomeric forms of the proteins were readily revealed. On the contrary, peptide mass fingerprinting, commonly used for soluble proteins, does not seem to be very helpful for identification of these highly hydrophobic membrane proteins due to the limited number of peptides which can be obtained by proteolysis. Thus, separations and identifications of antenna proteins presented in this study may serve as reference for confident identification in future studies dealing with any membrane proteins. Keywords: proteomics, thylakoid membrane, photosystems, antenna proteins, mass spectrometry, 2d-gel electrophoresis, liquid chromatography, intact molecular mass

The lower cell density of leaf parenchyma in the Arabidopsis thaliana mutant lcd1-1 is associated with increased sensitivity to ozone and virulent Pseudomonas syringae.

Under optimal growth conditions (120 micro mol photons m-2 sec-1 photosynthetically active radiation (PAR), 16-h photoperiod), the recessive ozone-sensitive Arabidopsis thaliana L. Heynh. mutant lcd1-1 exhibits a pale phenotype compared to the wild type. Confocal and multiphoton microscopy revealed that the paleness of lcd1-1 is because of a lower cell density in the leaf palisade parenchyma, resulting in decreased chlorophyll content. When exposed to ozone, lcd1-1 leaves become paler and contain an increased amount of the lipid peroxidation product malondialdehyde compared to the wild type, suggesting that lcd1-1 suffers from elevated levels of reactive oxygen species (ROS) generated in the apoplast. Infection of leaves with virulent Pseudomonas syringae reveals higher bacterial growth as well as lower pathogenesis-related protein 1 (PR-1) and PR-5 expression in lcd1-1 than in the wild type. When the wild type and lcd1-1 are exposed to short-term high-light stress, leaves do not bleach in lcd1-1 and potential activities of photosystems I (PSI) and II (PSII) decrease to a similar extent in both the genotypes, indicating that the photosynthetic apparatus is not affected by lcd1-1 mutation. The LCD1 gene, found to contain a nonsense mutation in the mutant, has been identified. It is located at the bottom of chromosome 2 of the Arabidopsis genome. However, the function of the protein encoded by LCD1 is not yet known. We hypothesize that LCD1 plays a role in normal leaf development, and that the increased sensitivity to ozone and virulent P. syringae is a secondary effect that presumably results from the lower-cell-density phenotype in lcd1-1.

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.

Action Spectra of Photosystems I and II in State 1 and State 2 in Intact Sugar Maple Leaves.

Photochemical activity, measured as energy storage of photosystems I (PSI) and II (PSII) together and individually, is studied in sugar maple (Acer saccharum Marsh.) leaves in the spectral range between 400 and 700 nm in state 1 and state 2. Total photochemical activity remains the same in both state 1 and state 2 between 580 and 700 nm, but it is lower in state 2 between 400 and 580 nm. Both PSI and PSII activities change significantly during the state transition due to the migration of light-harvesting chlorophyll a/b protein complex of PSII (LHCII). In the action spectra of PSI and PSII, peak positions vary depending on the association or dissociation of LHCII, except for the peak at 470 nm in the PSII spectrum. PSII activity is about 3 times higher than or equal to PSI in state 1 or state 2, respectively, over most of the spectrum except in the blue and far-red regions. At 470 nm, PSII activity is 8 or 1.6 times higher than PSI in state 1 or state 2, respectively. The amplitude of LHCII coupling-induced change is the same in both PSI and PSII between 580 and 700 nm, but it is less in PSI than in PSII between 400 and 580 nm, which explains the lower photochemical activity of the leaf in state 2 than in state 1. This may be due to a decrease in energy transfer efficiency of carotenoids to chlorophylls in LHCII when it is associated with PSI.

Fractionation of thylakoid membranes from Porphyridium purpureum using the detergent N-lauryl-?-iminodipropionate

Two green fractions, thought to represent the chlorophyll-antennae of photosystems I (PSI) and II (PSII), were isolated from the red alga Porphyridium purpureum by solubilisation of the thylakoid membranes using the detergent N-lauryl-ß-iminodipropionate and subsequent sucrose-density-gradient centrifugation. No release of pigments from the pigment-protein complexes was detected during isolation. The fractions were analyzed with respect to their chlorophyll-protein pattern, spectral properties and pigment composition. The supposed PSII antenna fraction contained both the major carotenoids of P. purpureum, β-carotene and zeaxanthin, and showed a long-wavelength absorption maximum at 672 nm and a low-temperature fluorescence maximum at 692-694 nm. Polypeptides of this fraction cross-reacted with antibodies raised against the PSII polypeptides D1, CP43 and CP47 from higher plants. The PSI fraction could perform P-700 photooxidation and showed a long-wavelength absorbance maximum at 679 nm and a low-temperature fluorescence maximum at 718 nm. It contained β-carotene as the only carotenoid. The fluorescence excitation spectrum of the fraction and measurements of the photochemical activity of a thylakoid preparation excited with light that is preferentially absorbed either by chlorophyll (433 nm) or by carotenoids (495 nm) indicate that β-carotene serves as a very efficient antenna-pigment in PSI. In contrast, only a small amount of energy transfer from the carotenoids to chlorophyll could be observed with the supposed PSII fraction.