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PubMed1994

ENDOR and special triple resonance studies of chlorophyll cation radicals in photosystem 2.

Electron nuclear double resonance (ENDOR) and special triple (ST) resonance spectroscopies have been used to study the cation radicals of the primary donor, P680, and two secondary donor chlorophylls (Chl) in photosystem 2 (PS2). Two different preparations were employed, Tris-washed PS2 membranes and PS2 reaction centers (D1-D2-I-Cytb559 complex). One secondary donor Chl a cation radical, Chl1.+, was generated in the Tris-washed preparation, while the P680.+ radical cation and a further Chl a cation radical, Chl2.+, were produced in the reaction center preparation. The ENDOR spectrum of the primary donor radical cation of photosystem 1 (P700.+) is also presented for comparison. Hyperfine coupling constants for methyl groups have been measured for all three PS2 radical species and assigned by comparison with previously published spectra of Chl a radicals in vitro. Electron spin densities were calculated from these hyperfine couplings. Comparison of ENDOR spectral features with those of Chla.+ in vitro indicates similar values for Chl1.+ and Chl2.+ radicals but an apparent reduction in unpaired electron spin density for P680.+. It has been proposed from the more detailed studies of purple bacterial reaction centers that such a reduction in spin density can be interpreted as a delocalization over two Chl a molecules. Our calculations therefore suggest that P680.+ is a weakly coupled chlorophyll pair with 82% of the unpaired electron spin located on one chlorophyll of the pair at 15 K. Environmental or geometrical changes to the chlorin ring structure to give a novel monomeric primary donor are also possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations↗
PubMed2001

Soret-excited Raman spectroscopy of the spinach cytochrome b6f complex. Structures of the b- and c-type hemes, chlorophyll a, and beta-carotene.

Soret-excited resonance Raman (RR) spectra of the spinach cytochrome b6f complex (cyt b6f) are reported for the oxidized, native, ascorbate-reduced, and dithionite-reduced forms. Using excitations at 441.6, 413.1, and 406.7 nm, RR contributions of chlorophyll a, beta-carotene, the c-type heme of cytochrome f, and the b-type hemes of cytochrome b6 of the b6f complex were identified and the data compared to those previously obtained for the Rhodospirillum rubrum bc1 complex [Le Moigne, C., Schoepp, B., Othman, S., Verméglio, A., and Desbois, A. (1999) Biochemistry 38, 1066-1076]. RR bands arising from the b(6)f-associated chlorophyll a and beta-carotene pigments were found to be particularly intense in the spectra excited at 441.6 nm. The frequencies of the phorbin skeleton of chlorophyll a at 1606, 1552, and 1525 cm(-1) are typical of a Mg atom with a single axial ligand. Strong RR bands corresponding to stretching or deformation modes of beta-carotene were detected at 1137, 1157, 1191, 1216, and 1531 cm(-1) in the different forms of cyt b6f. This set of frequencies is assigned to an all-trans configuration of the polyene chain. The redox titrations of the b(6)f complex allow the characterization of RR bands of the three hemes. The nu10, nu2, nu3, and nu8 modes of reduced cyt f are detected at 1619, 1591, 1492, and 356 cm(-1), respectively. From this set of frequencies, one can conclude that the particular histidine/amine heme coordination found in the truncated soluble domain of cyt f is a specific feature of the entire cyt f included in the b6f complex. The frequencies of the nu2, nu8, and nu10 marker modes are consistent with different conformations for the two b-type hemes of cyt b6f. One of these hemes is strongly distorted (nu2, nu8, and nu10 at 1581, 351, and 1610 cm(-1), respectively), while the other one is planar (1586, 345, and 1618 cm(-1), respectively). Largely different structures for the b-type hemes appear to be a common property for the bc1/b6f complexes.

Ascorbic Acid↗
PubMed2005

Aggregates of the chlorophyll-binding protein IsiA (CP43') dissipate energy in cyanobacteria.

In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of approximately 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light.

Bacterial Proteins↗
PubMed2004

Chlorophyll fluorescence imaging of individual algal cells: effects of herbicide on Spirogyra distenta at different growth stages.

Serious environmental degradation of aquatic ecosystems has been caused by eutrophication and by pollutants such as herbicides. Therefore, measurement of in situ algal photosynthetic activity is important for environmental monitoring. With ordinary nonimaging fluorometers, algal chlorophyll fluorescence can be measured easily, but heterogeneity within samples cannot be detected. Effects of a herbicide preparation containing 3-(3,4-dichlorophenyl)-1,1 -dimethylurea (DCMU) on photosynthetic activity at different growth stages of Spirogyra distenta were investigated by using a computer-aided microscopic imaging system for chlorophyll afluorescence. Photosystem II photochemical yield (phiPSII) images were used to diagnose photosynthetic activity of spiral filate chloroplasts in algal cells. The herbicide treatment caused a stronger decline in phiPSII values in younger than in mature algae cells. This result indicated that heterogeneity within algal samples should be considered when algae are used for environmental monitoring. Thus, measurement of chlorophyll fluorescence from young and mature chloroplasts with a microscopic imaging system makes it possible to improve the sensitivity for monitoring the environmental degradation of aquatic ecosystems.

Chlorophyll↗
PubMed2002

Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing.

The ChlH gene coding the H subunit of magnesium chelatase, an enzyme involved in chlorophyll biosynthesis, was silenced in Nicotiana benthamiana plants by infection with tobacco mosaic virus vectors (pTMV-30b) containing 67,214 or 549 nt long ChlH inserts. Silencing of the nuclear ChlH gene induced a chimeric phenotype with green and yellow/white leaves associated with alterations of chloroplast shape and ultrastructure. The symptoms became first evident around veins of young leaves, and only later in the mesophyll tissues. The efficiency of gene silencing was not dependent on the insert orientation, but was strongly correlated with the size of the ChlH insert, providing a flexible method to modulate the level of gene suppression. Silencing efficiency seemed to be strongly dependent on endogenous ChlH mRNA level of the target tissue. Silencing of the ChlH gene with the longest fragment of 549 nt also lowered the accumulation of ChlD and chlorophyll synthetase mRNAs, i.e. other genes involved in chlorophyll biosynthesis.

Carbon-Oxygen Ligases↗
PubMed2002

Visible light induced hydrogen production with Mg chlorophyll-a from spirulina and colloidal platinum.

Photoinduced hydrogen production with Mg chlorophyll-a from spirulina as a visible light photosensitizer by use of three component system consisting of nicotineamide adenine dinucleotide phosphate, reduced form (NADPH) as an electron donor, methylviologen as electron relay reagent and colloidal platinum as hydrogen evolution catalyst was investigated. By the addition of NADPH, the photostability of Mg chlorophyll-a was increased. The effective visible-light induced hydrogen production system with colloidal platinum was established using Mg chlorophyll-a.

Bacterial Proteins↗
PubMed2002

A chlorophyll a fluorescence-based Lemna minor bioassay to monitor microbial degradation of nanomolar to micromolar concentrations of linuron.

A plant-microbial bioassay, based on the aquatic macrophyte Lemna minor L. (duckweed), was used to monitor biodegradation of nano- and micromolar concentrations of the phenylurea herbicide linuron. After 7 days of exposure to linuron, log-logistic-based dose-response analysis revealed significant growth inhibition on the total frond area of L. minor when linuron concentrations > or = 80 nM were added to the bioassay. A plant-protective effect was obtained for all concentrations > 80 nM by inoculation with either a bacterial consortium or Variovorax paradoxus WDL1, which is probably the main actor in this consortium. The outcome of the plant-microbe-toxicant interaction was also assessed using pulse amplitude-modulated chlorophyll a fluorescence and chlorophyll a fluorescence imaging. Linuron toxicity to L. minor became apparent as a significant decrease in the effective quantum yield (Delta F/Fm') within 90 min after exposure of the plants to linuron concentrations > or = 160 nM. Inoculation of the bioassay with the linuron-degrading bacteria neutralized the effect on the effective quantum yield at concentrations > or = 160 nM, indicating microbial degradation of these concentrations. The chlorophyll a fluorescence-based Lemna bioassay described here offers a sensitive, fast and cost-effective approach to study the potential of biodegrading microorganisms to break down minute concentrations of photosynthesis-inhibiting xenobiotics.

Araceae↗
PubMed1997

Characterization of chlorophyll a and bacteriochlorophyll a synthases by heterologous expression in Escherichia coli.

Genes coding for putative chlorophyll a synthase (chlG) from Synechocystis sp. PCC 6803 and bacteriochlorophyll a synthase (bchG) from Rhodobacter capsulatus were amplified by the polymerase chain reaction and cloned into T7 RNA polymerase-based expression plasmids. In vitro enzymatic assays indicated that heterologous expression of the chlG and bchG gene products in Escherichia coli conferred chlorophyll a and bacteriochlorophyll a synthase activity, respectively. Chlorophyll a synthase utilized chlorophyllide a, but not bacteriochlorophyllide a, as a substrate, whereas bacteriochlorophyll a synthase utilized bacteriochlorophyllide a, but not chlorophyllide a. Both enzymes were also observed to exhibit a marked preference for phytyl diphosphate over geranylgeranyl diphosphate.

Bacteriochlorophylls↗
PubMed2003

The nature of a chlorophyll ligand in Lhca proteins determines the far red fluorescence emission typical of photosystem I.

Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at approximately 700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.

Amino Acid Sequence↗
PubMed2006

A chlorophyll a/b-binding protein homolog that is induced by iron deficiency is associated with enlarged photosystem I units in the eucaryotic alga Dunaliella salina.

Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.

Amino Acid Sequence↗
PubMed2001

Determination of double decker sandwich structured La-substituted chlorophyll a by EXAFS.

As a representative natural porphyrin derivative, it is interesting to know the chemical form of lanthanide in chlorophyll a. We select the natural fern dicranopteris dichotoma, the most lanthanide-concentrated plant known, to isolate the chlorophyll a for our study. It is found by EXAFS that lanthanum substitutes in the magnesium position in chlorophyll and coordinates with the porphyrin ring. The lanthanum is seen to have a similar coordination structure to a sandwich-type lanthanide complex, with the La surrounded by eight nitrogen atoms from two porphyrin rings with average La-N bond length of 2.65 A.

Chlorophyll↗
PubMed1991

Isolation, purification and partial characterization of a 30-kDa chlorophyll-a/b-binding protein from spinach.

A 30-kDa chlorophyll-a/b-binding protein was purified from photosystem II membrane fragments using Ca(2+)-chelating Sepharose 6B chromatography. The protein binds approximately four chlorophyll a molecules, one chlorophyll b molecule and carotenoids. Its 77-K fluorescence-emission spectrum exhibits a maximum at 680 +/- 1 nm. The protein has a high tendency to form a dimer in the presence of Ca2+.Ca2+ binding affects the low-temperature fluorescence-emission maximum, leading to a decrease in its intensity and a blue shift of 1 nm. Similar spectral changes were obtained in the presence of Mg2+, possibly indicating a common binding domain for both cations. We interpret these observations as cation-induced conformational changes of the protein, which were reversible upon subsequent incubation in EDTA. Evidence is presented for the involvement of carboxyl groups in the coordination sphere of the bivalent cations. The possible structural and functional role of the protein is discussed.

Blotting, Western↗
PubMed1994

Evidence from in vivo manipulations of lipid composition in mutants that the delta 3-trans-hexadecenoic acid-containing phosphatidylglycerol is involved in the biogenesis of the light-harvesting chlorophyll a/b-protein complex of Chlamydomonas reinhardtii.

The phosphatidylglycerol containing the unusual delta 3-trans hexadecenoic fatty acid is specifically found in photosynthetic membranes of eukaryotic organisms. Its involvement in the biogenesis and the structure of the light-harvesting chlorophyll a/b-protein complex has been evidenced by in vivo targeting this lipid to photosynthetic membranes of Chlamydomonas reinhardtii mutants lacking this lipid. In the mf1 and mf2 mutants, this deficiency results in (a) the absence of the oligomeric light-harvesting complex of photosystem 2; (b) an extensive destacking of thylakoid membranes; (c) a very low 77-K fluorescence emission in the photosystem-2 region. We show in this paper that these deficiencies result from modifications in the pigment and polypeptide compositions of the photosystem-2 light-harvesting complex; it contains less chlorophyll b and some of its constitutive polypeptides are absent or reduced in amount, while immunologically related polypeptides of lower molecular mass accumulate. The direct involvement of the lack of trans-C16: 1-phosphatidylglycerol in these deficiencies is evidenced by the partial restoration of normal characteristics of the light-harvesting complex (pigment and polypeptide composition, oligomerization) after liposome-mediated, in vivo incorporation of this lipid into the photosynthetic membranes of the mf2 mutant. Trans-C16:1-phosphatidylglycerol, therefore, is involved in the biogenesis of the photosystem-2 light-harvesting chlorophyll a/b-protein complex through a mechanism that may prevent degradation processes. Its contribution to the structural conformation of neosynthesized monomers and to their organization into stable oligomeric form is discussed.

Animals↗
PubMed2004

Reducing variability that is due to secondary pigments in the retrieval of chlorophyll a concentration from marine reflectance: a case study in the western equatorial Pacific Ocean.

A neural network is developed to retrieve chlorophyll a concentration from marine reflectance by use of the five visible spectral bands of the Sea-viewing Wide Field-of-view Sensor (SeaWiFS). The network, dedicated to the western equatorial Pacific Ocean, is calibrated with synthetic data that vary in terms of atmospheric content, solar zenith angle, and secondary pigments. Pigment variability is based on in situ data collected in the study region and is introduced through nonlinear modeling of phytoplankton absorption as a function of chlorophyll a, b, and c and photosynthetic and photoprotectant carotenoids. Tests performed on simulated yet realistic data show that chlorophyll a retrievals are substantially improved by use of the neural network instead of classical algorithms, which are sensitive to spectrally uncorrelated effects. The methodology is general, i.e., is applicable to regions other than the western equatorial Pacific Ocean.

Algorithms↗
PubMed2001

Photoinduced electron transfer between chlorophylls (a/b) and fullerenes (C60/C70) studied by laser flash photolysis.

Photoinduced electron-transfer processes in the systems of chlorophylls (Chl) (chlorophyll-a [Chl-a] and chlorophyll-b) and fullerenes (C60/C70) in both polar and non-polar solvents have been investigated with nanosecond laser photolysis technique, observing the transient spectra in the visible/near-IR regions. By the excitation of Chl in benzonitrile (BN) it has been proved that electron transfer takes place from the triplet excited states of Chl to the ground states of C60/C70. By the excitation of C70 in BN electron transfer takes place from the ground states of Chl to the triplet excited state of C70. In both Chl the rate constants and quantum yields for the electron-transfer processes are as high as those of zinc porphyrins and zinc phthalocyanines, indicating that the long alkyl chains of Chl play no role in retarding the electron transfer. The rate constant for the electron-mediating process from the radical anion of C70 to octylviologen dication yielding the octylviologen radical cation was evaluated. The back electron-transfer process from the viologen radical cation to the radical cation of Chl-a takes place in a longer time-scale, indicating that a photosensitized electron-transfer/electron-mediating cycle is achieved.

Carbon↗
PubMedDate not supplied

Vegetation stress detection through chlorophyll a + b estimation and fluorescence effects on hyperspectral imagery.

Physical principles applied to remote sensing data are key to successfully quantifying vegetation physiological condition from the study of the light interaction with the canopy under observation. We used the fluorescence-reflectance-transmittance (FRT) and PROSPECT leaf models to simulate reflectance as a function of leaf biochemical and fluorescence variables. A series of laboratory measurements of spectral reflectance at leaf and canopy levels and a modeling study were conducted, demonstrating that effects of chlorophyll fluorescence (CF) can be detected by remote sensing. The coupled FRT and PROSPECT model enabled CF and chlorophyll a + b (Ca + b) content to be estimated by inversion. Laboratory measurements of leaf reflectance (r) and transmittance (t) from leaves with constant Ca + b allowed the study of CF effects on specific fluorescence-sensitive indices calculated in the Photosystem I (PS-I) and Photosystem II (PS-II) optical region, such as the curvature index [CUR; (R675.R690)/R2(683)]. Dark-adapted and steady-state fluorescence measurements, such as the ratio of variable to maximal fluorescence (Fv/Fm), steady state maximal fluorescence (F'm), steady state fluorescence (Ft), and the effective quantum yield (delta F/F'm) are accurately estimated by inverting the FRT-PROSPECT model. A double peak in the derivative reflectance (DR) was related to increased CF and Ca + b concentration. These results were consistent with imagery collected with a compact airborne spectrographic imager (CASI) sensor from sites of sugar maple (Acer saccharum Marshall) of high and low stress conditions, showing a double peak on canopy derivative reflectance in the red-edge spectral region. We developed a derivative chlorophyll index (DCI; calculated as D705/D722), a function of the combined effects of CF and Ca + b content, and used it to detect vegetation stress.

Acer↗
PubMed1991

Superoxide mediated chlorophyll allomerization in a dimethyl sulphoxide-water mixture.

The interaction of chlorophyll a with superoxide anion was studied in an alkaline DMSO-water system. It was found that O2(-.), directly or via HO2., produces the chlorophyll enolate-ion (Molish's intermediate) that is oxidized to Mg-chlorine(s). The allomerization reaction was found to be inhibited by superoxide dismutase. A possible participation of oxygen radicals in chlorophyll degradation in plants is discussed.

Chlorophyll↗
PubMed1992

Characterization of photosystem 1 chlorophyll a/b-binding apoprotein accumulation in developing soybean using type-specific antibodies.

The structure and supramolecular assembly of the soybean photosystem 1 (PS 1) chlorophyll a/b-binding antenna (LHC 1) was examined. We identified the subunit composition of LHC 1 in soybean and followed the accumulation of individual subunits during light-induced assembly. We observed four LHC 1 subunits, at 23, 22, 21 and 20.5 kDa, obtained partial sequence information by amino-terminal sequence analysis, and classified the 20.5, 22, and 21 kDa subunits as being encoded by type I, II, and IV chlorophyll a/b binding protein genes, respectively. Antisera against LHC 1 subunits were used to follow the accumulation of individual subunits during the light-initiated transition from etioplast to chloroplast. Several points are noteworthy. First, monospecific antibody against the 22 kDa subunit decorated a 25 kDa peptide in etiolated tissue, which declined during maturation. This decline correlated with the light-induced appearance of mature 22 kDa peptide, suggesting a precursor/product relationship. Second, the same antibody identified a 22 kDa protein in mature corn, but not a larger band in etiolated corn, suggesting that LHC 1 accumulation is regulated differently between species before the onset of chlorophyll biosynthesis. Third, the mature 22 kDa subunit appeared somewhat later than the other LHC 1 peptides during greening, implying that this subunit is less intimately associated with the PS1 core than are the subunits appearing earlier in development.

Antibodies↗