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Chlorophyll a fluorescence imaging of ozone-stressed Brassica napus L. plants differing in glucosinolate concentrations.

Brassicaceae are characterised by glucosinolates (GS), which appear to be involved not only in biotic but also in abiotic stress responses of plants. We investigated the effect of O (3) stress on leaf GS concentrations in two lines of BRASSICA NAPUS L., differing in GS content. Ozone fumigation decreased GS concentrations in leaves of B. NAPUS of one line. In control conditions, chlorophyll content, rates of saturating photosynthesis, and quantum yield of photosystem 2 differed between the two BRASSICA lines, but differences were smaller in O (3)-stress conditions, suggesting that the relationship between leaf GS concentration and sensitivity to abiotic stress merits further research. In agreement with other ecophysiological measurements, chlorophyll fluorescence imaging clearly distinguished both lines and in some cases also treatments. A method for analysis of fluorescence images accounting for the two-dimensional leaf heterogeneity is presented.

Brassica napus↗

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680(.+) primary donor chlorophyll.

We report (13)C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo-CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance (13)C provide information on the electronic structure of the primary electron donor P(680) (chlorophyll a molecules absorbing around 680 nm) and on the p(z) spin density pattern in its oxidized form, P(680)(.+). Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P(680)(.+). PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P(680) by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Carbon Isotopes↗

Global spectral-kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley.

This study presents a novel measurement, and simulation, of the time-resolved room temperature chlorophyll a fluorescence emission spectra from leaves of the barley wild-type and chlorophyll-b-deficient chlorina (clo) f2 and f104 mutants. The primary data were collected with a streak-camera-based picosecond-pulsed fluorometer that simultaneously records the spectral distribution and time dependence of the fluorescence decay. A new global spectral-kinetic analysis programme method, termed the double convolution integral (DCI) method, was developed to convolve the exciting laser pulse shape with a multimodal-distributed decay profile function that is again convolved with the spectral emission band amplitude functions. We report several key results obtained by the simultaneous spectral-kinetic acquisition and DCI methods. First, under conditions of dark-level fluorescence, when photosystem II (PS II) photochemistry is at a maximum at room temperature, both the clo f2 and clo f104 mutants exhibit very similar PS II spectral-decay contours as the wild-type (wt), with the main band centred around 685 nm. Second, dark-level fluorescence is strongly influenced beyond 700 nm by broad emission bands from PS I, and its associated antennae proteins, which exhibit much more rapid decay kinetics and strong integrated amplitudes. In particular a 705-720 nm band is present in all three samples, with a 710 nm band predominating in the clo f2 leaves. When the PS II photochemistry becomes inhibited, maximizing the fluorescence yield, both the clo f104 mutant and the wt exhibit lifetime increases for their major distribution modes from the minimal 205-500 ps range to the maximal 1500-2500 ps range for both the 685 nm and 740 nm bands. The clo f2 mutant, however, exhibits several unique spectral-kinetic properties, attributed to its unique PS I antennae and thylakoid structure, indicating changes in both PS II fluorescence reabsorption and PS II to PS I energy transfer pathways compared to the wt and clo f104. Photoprotective energy dissipation mediated by the xanthophyll cycle pigments and the PsbS protein was uninhibited in the clo f104 mutant but, as commonly reported in the literature, significantly inhibited in the clo f2; the inhibited energy dissipation is partly attributed to its thylakoid structure and PS II to PS I energy transfer properties. It is concluded that it is imperative with steady-state fluorometers, especially for in vivo studies of PS II efficiency or photoprotective energy dissipation, to quantify the influence of the PS I spectral emission.

Chlorophyll↗

Improved chlorophyll extraction method.

Filtered plankton samples subjected to sonic oscillations yielded 11 to 30 percent more chlorophyll a with cvacetone extraction than similar, nonsonificated samples. The improved chlorophyll extraction was attributed to algal cell-wall rupture.

Chlorophyll↗

LOCATION OF CHLOROPHYLL IN RHODOSPIRILLUM RUBRUM.

Holt, Stanley C. (University of California, Davis), and Allen G. Marr. Location of chlorophyll in Rhodospirillum rubrum. J. Bacteriol. 89:1402-1412. 1965.-If cells of Rhodospirillum rubrum are broken by sonic and ballistic disruption, the chlorophyll is not found in discrete cytoplasmic structures, but is located in a more extensive structure of the cell, the intracytoplasmic membrane. Direct electron microscopy of sonically disrupted cells of R. rubrum and stereo-electron microscopy of osmotically shocked cells reveal the presence of a tubular network of internal membranes originating from the periphery of the cell.

Cell Biology↗

Effect of bio-optical parameter variability and uncertainties in reflectance measurements on the remote estimation of chlorophyll-a concentration in turbid productive waters: modeling results.

Most algorithms for retrieving chlorophyll-a concentration (Chla) from reflectance spectra assume that bio-optical parameters such as the phytoplankton specific absorption coefficient (aPhi*) or the chlorophyll-a fluorescence quantum yield (eta) are constant. Yet there exist experimental data showing large ranges of variability for these quantities. The main objective of this study was to analyze the sensitivity of two Chla algorithms to variations in bio-optical parameters and to uncertainties in reflectance measurements. These algorithms are specifically designed for turbid productive waters and are based on red and near-infrared reflectances. By means of simulated data, it is shown that the spectral regions where the algorithms are maximally sensitive to Chla overlap those of maximal sensitivity to variations in the above bio-optical parameters. Thus, to increase the accuracy of Chla retrieval, we suggest using spectral regions where the algorithms are less sensitive to Chla, but also less sensitive to these interferences. aPhi* appeared to be one of the most important sources of error for retrieving Chla. However, when the phytoplankton backscattering coefficient (bb,Phi) dominates the total backscattering, as is likely during algal blooms, variations in the specific bb,Phi may introduce large systematic uncertainties in Chla estimation. Also, uncertainties in reflectance measurements, which are due to incomplete atmospheric correction or reflected skylight removal, seem to affect considerably the accuracy of Chla estimation. Instead, variations in other bio-optical parameters, such as eta or the specific backscattering coefficient of total suspended particles, appear to have minor importance. Suggestions regarding the optimal band locations to be used in the above algorithms are finally provided.

Algorithms↗

Extended X-ray absorption fine-structure study of the bound form of lanthane in chlorophyll-a from Dicranopteris dichotoma.

The in vivo coordination structure of lanthanum ions interacting with chlorophyll-a of the fern Dicranopteris dichotoma grown in a rare earth minefield in southern China was determined by the extended x-ray absorption fine structure (EXAFS). The results show that lanthanum includes two porphyrin rings in its coordination sphere. It is postulated that the La-chlorophyll-a complex may have a bilayer structure. The analytical method may serve as a new tool to gain insight in the in vivo interactions of rare earth elements.

China↗

[Effect of Suining Tributary ecoremediation to all size-fractionated chlorophyll-a contents].

Through analysis of the size-fractionated chlorophyll-a contents in the remedied part, control part and non-remedied part in Suining Tributary, this paper discussed the effect of bioremediation to all size-fractionated chlorophyll-a contents. The averaged Chlorophy-a content of nano-plus pico-phytoplankton made up 85.232%, 92.402%, 95.205% of the total, respectively for the remedied, control and non-remedied part. Among these, the nano fraction alone made up 78.460%, 87.943%, 87.211%, respectively. Nano-phytoplankton contributed most to the total biomass of Chl a. Its average contribution was 84.538% to the whole tributary, whereas net-phytoplankton contribued only 9.054%. Nano- and pico-phytoplankton was most sensitive to the test eco-remedy. The remedy reduced the relative biomass of net-phytoplankton, and increased the relative biomass of Pico-enhance. The relative biomass of nano- and ultra-phytoplankton fraction was little effected.

Biodegradation, Environmental↗

Subunit stoichiometry of the chloroplast photosystem II antenna system and aggregation state of the component chlorophyll a/b binding proteins.

Photosystem (PS) II membranes, obtained by the method of Berthold et al. (Berthold, D. A., Babcock, G. T., and Yocum, C. F. (1981) FEBS Lett. 134, 231-234), have been fractionated by a sucrose gradient ultracentrifugation method which allows the quantitative separation of the three major chlorophyll binding complexes in these membranes: the chlorophyll (chl) a binding PSII reaction center core, the major light-harvesting complex II, and the minor chl a/b proteins called CP26, CP29, and CP24. Each fraction has been analyzed for its subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results show that 12 mol of light-harvesting complex II and 1.5 mol of each of the minor chl a/b proteins are present per mol of the PSII reaction center complex in PSII membranes. These data suggest a dimeric organization of PSII, in agreement with a recent crystallographic study (Bassi, R., Ghiretti Magaldi, A., Tognon, G., Giacometti, G. M., and Miller, K. (1989) Eur. J. Cell Biol. 50, 84-93) and imply that such a dimeric complex is served by antenna chl a/b proteins whose minimal aggregation state includes three polypeptides. This was confirmed by covalent cross-linking of purified antenna complexes.

Centrifugation, Density Gradient↗

The 110-kDa reaction center protein of photosystem I, P700-chlorophyll a-protein 1, is an iron-sulfur protein.

Germination and growth of barley (Hordeum vulgare L.) in the presence of 59Fe2+ or 35SO4(2-) allows heavy incorporation of both isotopes into the thylakoid membranes and into isolated photosystem I particles. Analysis of 59Fe-labeled preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under mild conditions demonstrates that a minimum of four iron atoms/P700 is carried on P700-chlorophyll a-protein 1. When isolated from 35S-labeled preparations, P700-chlorophyll a-protein 1 binds zero valence 35S, which is converted into acid-labile [35S]sulfide by dithiothreitol reduction. Isolated photosystem I particles contain 14 acid-labile sulfide atoms and 10 iron atoms for each molecule of P700 and are composed of polypeptides of 110, 18, 15, 10, and 8 kDa of which the 10-kDa component is loosely bound. Under the electrophoretic conditions used, none of the low molecular weight polypeptides could be shown to be specifically associated with iron or acid-labile sulfide. Carboxymethylation of cysteine residues shows a high cysteine content in the 8-kDa polypeptide and an intermediate content in the 110- and 18-kDa polypeptides, whereas the 15-kDa polypeptide is devoid of sulfur amino acids. The experiments with the 59Fe-labeled thylakoids reveal other labeled polypeptides not associated with photosystem I, namely cytochrome f and possibly cytochromes b6 and b559.

Chlorophyll↗

Chlorophyll and pheophytin derivatives in geochemical transformation pathways: a surface-enhanced resonance Raman spectroscopic study.

Protected surface-enhanced resonance Raman spectroscopy (PSERRS) has been used to study a number of chlorophyll transformation products that have been suggested as intermediates in the so-called Treibs hypothesis which describes the transformation of ancient chlorophyll a (chl a) in the biosphere into desoxophylloerythroetio-porphyrin (DPEP) found in sedimentary environments. Both Soret- and Qy-resonant PSERR spectra have been recorded, providing two-dimensional structural fingerprints containing a number of bands which enable the presence of specific peripheral substituents to be identified. Some of these marker bands can be assigned directly to vibrational modes of the particular substituents. This has allowed further characterization of the vibrational spectrum of chl a; in particular, a vinyl mode has been identified which previously was thought to be Raman silent.

Chlorophyll↗

The field of possible structures for the chlorophyll a dimer in photosystem I of green plants delineated by polarized photochemistry.

Photoselection experiments with immobilized photosystem I particles have been done to determine the mutual orientation of pigments in the reaction centre. When these particles are excited and interrogated with linearly polarized light, the flash-induced transient absorption changes (mainly from the chlorophyll a dimer) reveal linear dichroism, which yields information on the mutual orientation between the excited and the interrogated transition moments. The interpretation of the data, however, is ambiguous, (1) for reasons of principles inherent in the photoselection technique when applied to complex systems and (2) because of incomplete knowledge about the relative contribution of x- and of y-polarized transitions of chlorophyll a to absorption or to absorption changes at a given wavelength. We find it impossible to attribute any particular structure to the photooxidizable dimer based on photoselection data alone. Instead we present a field of possible structures, imposing constraints on proposed models for the dimer structure.

Chlorophyll↗

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves.

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.

Chlorophyll↗

Separation of mono- and divinyl chlorophyll species by high-performance liquid chromatography using an octadecyl polyvinyl alcohol polymer column.

Most of the chlorophylls and their related compounds from nonesterified chlorophyllide to esterified pheophytin were separated by high- performance liquid chromatography using a wide pore, C(18) polyvinyl alcohol polymer column with an elution using a binary gradient starting from a buffered mobile phase. The high selectivity of this system enabled not only the separation of common chlorophyll species but also the resolution of structurally similar compounds such as mono- and divinyl chlorophyllide and pheophorbide species which usually coelute on monomeric bonded phases. The method is successfully applied to analyses of the pigments extracted from green and etiolated tissues of higher plants and photosynthetic bacterial cells.

Chlorophyll↗

Changes in Arabidopsis leaf ultrastructure, chlorophyll and carbohydrate content during spaceflight depend on ventilation.

Leaf structure and function under spaceflight conditions have received little study despite their important implications for biological life support systems using plants. Previous reports described disruption of the membrane apparatus for photosynthesis and a general decrease in carbohydrate content in foliage. During a series of three short-duration experiments (Chromex-03, -04, -05) on the US space shuttle (STS-54, STS-51, STS-68), we examined Arabidopsis thaliana leaves. The plants were at the rosette stage at the time of loading onto the space shuttle, and received the same light, temperature, carbon dioxide and humidity regimes in the orbiter as in ground controls. The experiments differed according to the regime provided in the headspace around the plants: this was either sealed (on mission STS-54); sealed with high levels of carbon dioxide (on mission STS-51) or vented to the cabin air through a filtration system (on mission STS-68). Immediately post-flight, leaf materials were fixed for microscopy or frozen in liquid nitrogen for subsequent analyses of chlorophyll and foliar carbohydrates. At the ultrastructural level, no aberrations in membrane structure were observed in any of the experiments. When air-flow was provided, plastids developed large starch grains in both spaceflight and ground controls. In the experiments with sealed chambers, spaceflight plants differed from ground controls with regard to measured concentrations of carbohydrate and chlorophyll, but the addition of airflow eliminated these differences. The results point to the crucial importance of consideration of the foliage microenvironment when spaceflight effects on leaf structure and metabolism are studied.

Arabidopsis↗

Chlorophyll a fluorescence: a tool for the investigation of toxic effects in the photosynthetic apparatus.

The effects of environmental pollutants, such as triazines, urea herbicides, phenols, nitro aromatic compounds, aldehydes, hydrogen sulfide, volatile halogenated aliphatic and aromatic compounds, and alkylbenzenes, the photosynthesis of green algae were investigated by using detailed analysis of chlorophyll a fluorescence. Application of quenching analysis by means of a pulse amplitude-modulated fluorometer allows the discrimination of different modes of action. Blocks of the electron-transport chain, the action of uncouplers, and fluorescence quenching by nitroaormatic compounds could be distinguished. Hydrogen sulfide and short-chain aldehydes seem to react in the same way, probably by reaction with components of the electron-transport chain. Nonreactive volatile compounds affect chlorophyll fluorescence in a strictly hydrophobicity-dependent manner. The quenching analysis suggests an inhibition of utilization of proton-motive force for ATP synthesis. Detailed analysis of fluorescence patterns may be of great help for identification of toxic pollutants and their sources.

Adenosine Triphosphate↗

Arsenate toxicity to Pisum sativum: mineral nutrients, chlorophyll content, and phytase activity.

A significant mechanism of arsenate toxicity to Pisum sativum is interference with its mineral nutrient balance. This conclusion is supported by assessments made after exposing P. sativum L. cv. "Phenomen" for 12 days to 12.5, 20.8, and 33.3 mg, and for 32 days to 7.5, 22.1, 36.7, and 73.3 mg of sodium arsenate/kg dry wt soil in the greenhouse. At 20.8 mg of arsenate, mobilization of manganese from the cotyledons was significantly increased and that of zinc decreased. Nitrogen accumulated in the roots. On Day 32, at 22.1 mg of arsenate, magnesium, zinc, and manganese contents of the roots increased, but that of phosphorus of the shoot decreased. The distribution pattern and the ratios between individual elements were severely altered. Relatively more arsenic accumulated from the low than the high soil concentrations. Growth of the shoot was more affected than that of the roots. After a 32-day exposure, chlorophyll content of the leaves increased, but the chlorophyll a/b ratio decreased. On Day 12, at 12.5 mg and 20.8 mg of arsenate, in vivo phytase activity was 64 and 66% that of the controls, respectively.

6-Phytase↗

A 3-D structural model of solid self-assembled chlorophyll a/H(2)O from multispin labeling and MAS NMR 2-D dipolar correlation spectroscopy in high magnetic field.

Magic angle spinning (MAS) NMR with Lee-Goldburg cross-polarization (LG-CP) is used to promote long-range heteronuclear transfer of magnetization and to constrain a structural model for uniformly labeled chlorophyll a/H(2)O. An effective maximum transfer range d(max) can be determined experimentally from the detection of a gradually decreasing series of intramolecular correlations with the (13)C along the molecular skeleton. To probe intermolecular contacts, d(max) can be set to approximately 4.2 A by choosing an LG-CP contact time of 2 ms. Long-range (1)H-(13)C correlations are used in conjunction with carbon and proton aggregation shifts to establish the stacking of the chlorophyll a (Chl a) molecules. First, high-field (14.1 T) 2-D MAS NMR homonuclear ((13)C-(13)C) dipolar correlation spectra provide a complete assignment of the carbon chemical shifts. Second, proton chemical shifts are obtained from (1)H-(13)C heteronuclear dipolar correlation spectroscopy in high magnetic field. The shift constraints and long-range (1)H-(13)C intermolecular correlations reveal a 2-D stacking homologous to the molecular arrangement in crystalline solid ethyl-chlorophyllide a. A doubling of a small subset of the carbon resonances, in the 7-methyl region of the molecule, provides evidence for two marginally different well-defined molecular environments. Evidence is found for the presence of neutral structural water molecules forming a hydrogen-bonded network to stabilize Chl a sheets. In line with the microcrystalline order observed for the rings, the long T(1)'s, and absence of conformational shifts for the (13)C in the phytyl tails, it is proposed that the Chl a form a rigid 3-D space-filling structure. Probably the only way this can be realized with the sheets is by forming bilayers with interpenetration of elongated tails. Such a 3-D space-filling organization of the aggregated Chl a from MAS NMR would match existing models inferred from electron microscopy and low-resolution X-ray powder diffraction, while a micellar model based on neutron diffraction and antiparallel stacking observed in solution can be discarded.

Carbon Isotopes↗