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PubMed2005

Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants.

Chlorophyllide a oxygenase (CAO) that converts chlorophyllide a to chlorophyllide b was overexpressed in tobacco to increase chlorophyll (Chl) b biosynthesis and alter the Chl a/b ratio. Transgenic plants along with their wild-type cultivars were grown in low and high light intensities. In low light there was 20% increase in chlorophyll b contents in transgenic plants, which resulted in 16% reduction in the Chl a/b ratio. In high light, total Chl contents were 31% higher in transgenic plants than those of wild type. The increase in Chl a was 19% and that of Chl b was 72% leading to 31% decline of Chl a/b ratio. The increase in Chl b contents was accompanied by enhanced CAO expression that was highly pronounced in low light. As compared to low light, in high light Lhcb1 and Chl a/b transcripts abundance was significantly increased in transgenic plants suggesting a close relationship between Chl b synthesis and cab gene expression. However, there was a small increase in expression of LHCII proteins, which did not correspond to 72% increase in Chl b content in transgenic line, implying that LHCPII has the ability to bind more Chl b molecules.

Arabidopsis↗
PubMed2005

On the sub-maximal yield and photo-electric stimulation of chlorophyll a fluorescence in single turnover excitations in plant cells.

A set of expressions is derived which quantifies the chlorophyll fluorescence yield in terms of rate constants of primary light reactions of PSII, the fraction of open and semi-open RCs and of the electric field sensed by the RC in the thylakoid membrane. The decay kinetics of the chlorophyll fluorescence yield after a single turnover excitation in the presence of DCMU show at least two components, one reversible within approx. 1 s and one with a dark reversion lasting more than 30 s. The latter is attributed to photochemical quenching; the fast component is interpreted to be associated at least partially with photo-electrochemical control. It will be illustrated that (i) the sub-maximal fluorescence yield in single turnover excitation is associated with semi-closure of RCs, (ii) the trapping efficiency of semi-closed centers is less than 50% of that of open centers and (iii) the fluorescence yield of antennas with semi-closed RCs has the highest sensitivity to changes in strength of photo-electric fields.

Chenopodium album↗
PubMed2005

The occurrence of rapidly reversible non-photochemical quenching of chlorophyll a fluorescence in cyanobacteria.

Cyanobacteria have previously been considered to differ fundamentally from plants and algae in their regulation of light harvesting. We show here that in fact the ecologically important marine prochlorophyte, Prochlorococcus, is capable of forming rapidly reversible non-photochemical quenching of chlorophyll a fluorescence (NPQf or qE) as are freshwater cyanobacteria when they employ the iron stress induced chlorophyll-based antenna, IsiA. For Prochlorococcus, the capacity for NPQf is greater in high light-adapted strains, except during iron starvation which allows for increased quenching in low light-adapted strains. NPQf formation in freshwater cyanobacteria is accompanied by deep Fo quenching which increases with prolonged iron starvation.

Bacterial Proteins↗
PubMed2003

Effect of macromolecules and Triton X-100 on the triplet of aggregated chlorophyll in aqueous solution.

Chlorophyll a (Chla) in aqueous solution (2-6% acetone) is present as mono- and dihydrated aggregated forms which are characterized by specific ground state absorption spectra. The amount of dihydrated form is larger in the presence of macromolecules, such as bovine serum albumin (BSA), lysozime and polyvinyl alcohol (PVA), increasing from BSA to lysozime and PVA. Chla in aqueous acetone with and without macromolecules is characterized by low fluorescence and the absence of triplet-triplet (T-T) absorption. The ratio of dihydrated to monohydrated forms is significantly influenced by triton X-100. For lower triton X-100 concentrations, i.e. smaller than the critical micelle concentration of 0.26 mM (cmc), dihydrated forms are converted into monohydrated in both aqueous acetone and the presence of BSA or lysozime. In the presence of PVA dihydrated forms appeared to be resistant to triton X-100 action. Moreover, for triton X-100 concentrations of 2-3 times higher than cmc the amount of these forms is increased with time. T-T absorption of both mono- and dihydrated Chla aggregates was not detected in the presence of [triton X-100] < cmc. The lack of T-T absorption in aqueous acetone solution as well as in the presence of macromolecules implies that the triplet lifetime of the chlorophyll aggregates is short (tauT<10 ns) and/or the quantum yield of intersystem crossing is small (<5 x 10(-3)). The Chla monomers start to be formed as solubilized in the micelle for [triton X-100] larger than cmc, showing substantial fluorescence and T-T absorption.

Animals↗
PubMed2003

Chlorophylls of the c family: absolute configuration and inhibition of NADPH:protochlorophyllide oxidoreductase.

Using circular dichroism (CD) spectroscopy, the stereochemistry at C-13(2) of members of the chlorophyll (Chl) c family, namely Chls c(1), c(2), c(3) and [8-vinyl]-protochlorophyllide a (Pchlide a) was determined. By comparison with spectra of known enantiomers, all Chl c members turned out to have the (R) configuration, which is in agreement with considerations drawn from chlorophyll biosynthesis. Except for a double bond in the side chain at C-17, the chemical structure of Chl c(1) is identical with Pchlide a, the natural substrate of the light-dependent NADPH:protochlorophyllide oxidoreductase (POR). Thus, lack of binding to the active site due to the wrong configuration at C-13(2), which had been proposed previously, cannot be an explanation for inactivity of Chl c in this enzymic reaction. Our results show rather that Chl c(1) is a competitive inhibitor for this enzyme, tested with Pchlide a and Zn-protopheophorbide a (Zn-Ppheide a) as substrates.

Chlorophyll↗
PubMed1995

Aggregation of chlorophyll a probed by resonance light scattering spectroscopy.

We report the resonance light scattering (RLS) spectra of chlorophyll a aggregated in a 9:1 solution of formamide and pH 6.8 phosphate buffer. The aggregate formed after 2 h of mixing, referred to as Chl469, shows a strong scattering feature at 469 nm (Soret band) and a much weaker feature at 699 nm (Qy band). A kinetic investigation confirmed that the aggregation process is cooperative, and also detected one intermediate (Chl458) with a strong RLS spectrum but only a weak CD spectrum. We propose that aggregation proceeds via at least three steps: 1) formation of a nucleating species, probably a dimer of chlorophylls; 2) formation of large aggregates with little or no secondary structure (e.g., Chl458); and 3) conformational change to form helical aggregate (Chl469).

Biophysical Phenomena↗
PubMed2001

Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light-harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b-antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl-binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77-K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long-wavelength absorption and 730-nm fluorescence emission. In Lhcb1 with only Chl b long-wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long-wavelength properties.

Absorption↗
PubMed2003

Rapid separation of chlorophylls a and b and their demetallated and dephytylated derivatives using a monolithic silica C18 column and a pyridine-containing mobile phase.

A monolithic C18-bonded silica rod column (Merck Chromolith) was applied to the separation of mixtures of chlorophylls a and b and their derivatives originated by hydrolysis of the phytyl ester linkage (chlorophyllides), loss of the central Mg atom (pheophytins), or both processes (pheophorbides). Mobile phases containing two different ion-pair reagents, ammonium acetate and buffered pyridine, were tested. Both eluents achieved the resolution of the eight pigments in less than 5 min. The method based on the pyridine-containing mobile phase was applied to the separation of chlorophylls and their green coloured degradation products in senescing leaves of deciduous trees, green fruits and a marine microalga.

Chlorophyll↗
PubMed2003

Simultaneous analysis of prompt and delayed chlorophyll a fluorescence in leaves during the induction period of dark to light adaptation.

An attempt is made to reveal the relation between the induction curves of delayed fluorescence (DF) registered at 0.35-5.5 ms and the prompt chlorophyll fluorescence (PF). A simple formulation was proposed to link the ratio of the transient values of delayed and variable fluorescence with the redox state of the primary electron acceptor of Photosystem II--QA, and the thylakoid membrane energization. The term luminescence potential (UL) was introduced, defined as the sum of the redox potential of QA and the transmembrane proton gradient. It was shown that UL is proportional to the ratio of DF to the variable part of PF. The theoretical model was verified and demonstrated by analysing induction courses of PF and millisecond DF, simultaneously registered from leaves of barley--wild-type and the chlorophyll b-less mutant chlorina f2. A definitive correlation between PF and DF was established. If the luminescence changes are strictly due to UL, the courses of DF and PF are reciprocal and the millisecond DF curve resembles the first derivative of the PFt function.

Adaptation, Physiological↗
PubMed2003

A chemotaxonomic method to quantify phytoplankton groups in freshwater lentic mesocosms: an approach including chlorophyll a breakdown products.

The use of HPLC methods to determine and quantify phytoplankton population composition, is sometimes less time-consuming than microscopic identification. However, its general application poses problems since high discrepancies between chlorophyll a calculated using chemotaxonomic methods and direct measurements were noticed. For instance, chemotaxonomic protocols generally employed can lead to a poor estimation of total and relative abundance when high amounts of chlorophyll a breakdown products are present. Therefore, we propose a new approach to calculate relative abundance of algal groups in a phytoplankton population, based on integration of these degradation products in the chemotaxonomic assessment in lentic and shallow freshwater ecosystems.

Chlorophyll↗
PubMed2004

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae.

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the Förster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.

Ammonium Sulfate↗
PubMed2005

Quenching of chlorophyll triplet states by carotenoids in reconstituted Lhca4 subunit of peripheral light-harvesting complex of photosystem I.

In this study, triplet quenching, the major photoprotection mechanism in antenna proteins, has been studied in the light-harvesting complex of photosystem I (LHC-I). The ability of carotenoids bound to LHC-I subunit Lhca4, which is characterized by the presence of the red-most absorption components at wavelength >700 nm, to protect the system through quenching of the chlorophyll triplet states, has been probed, by analyzing the induction of carotenoid triplet formation. We have investigated this process at low temperature, when the funneling of the excitation toward the low-lying excited states of the Chls is stronger, by means of optically detected magnetic resonance (ODMR), which is well-suited for investigation of triplet states in photosynthetic systems. The high selectivity and sensitivity of the technique has made it possible to point out the presence of specific interactions between carotenoids forming the triplet states and specific chlorophylls characterized by red-shifted absorption, by detection of the microwave-induced Triplet minus Singlet (T-S) spectra. The effect of the red forms on the efficiency of triplet quenching was specifically probed by using the Asn47His mutant, in which the red forms have been selectively abolished (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Lack of the red forms yields into a reduced efficiency of the triplet quenching in LHC-I thus suggesting that the "red Chls" play a role in enhancing triplet quenching in LHC-I and, possibly, in the whole photosystem I.

Amino Acid Substitution↗
PubMed2006

Femtosecond time-resolved absorption spectroscopy of main-form and high-salt peridinin-chlorophyll a-proteins at low temperatures.

Steady-state and femtosecond time-resolved optical methods have been used to compare the spectroscopic features and energy transfer dynamics of two systematically different light-harvesting complexes from the dinoflagellate Amphidinium carterae: main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a-proteins. Pigment analysis and X-ray diffraction structure determinations [Hofmann, E., Wrench, P. M., Sharples, F. P., Hiller, R. G., Welte, W., Diederichs, K. (1996) Science 272, 1788-1791; T. Schulte, F. P. Sharples, R. G. Hiller, and E. Hofmann, unpublished results] have revealed the composition and geometric arrangements of the protein-bound chromophores. The MFPCP contains eight peridinins and two chlorophyll (Chl) a, whereas the HSPCP has six peridinins and two Chl a, but both have very similar pigment orientations. Analysis of the absorption spectra has shown that the peridinins and Chls absorb at different wavelengths in the two complexes. Also, in the HSPCP complex, the Qy transitions of the Chls are split into two well-resolved bands. Quantum computations by modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) methods have revealed that charged amino acid residues within 8 A of the pigment molecules are responsible for the observed spectral shifts. Femtosecond time-resolved optical spectroscopic kinetic data from both complexes show ultrafast (<130 fs) and slower (approximately 2 ps) pathways for energy transfer from the peridinin excited singlet states to Chl. The Chl-to-Chl energy transfer rate constant for both complexes was measured and is discussed in terms of the Förster mechanism. It was found that, upon direct Chl excitation, the Chl-to-Chl energy transfer rate constant for MFPCP was a factor of 4.2 larger than for HSPCP. It is suggested that this difference arises from a combination of factors including distance between Chls, spectral overlap, and the presence of two additional peridinins in MFPCP that act as polarizable units enhancing the rate of Chl-to-Chl energy transfer. The study has revealed specific pigment-protein interactions that control the spectroscopic features and energy transfer dynamics of these light-harvesting complexes.

Carotenoids↗
PubMed1997

Quenching of chlorophyll a fluorescence in the aggregates of LHCII: steady state fluorescence and picosecond relaxation kinetics.

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).

Calcium↗
PubMed2003

Simultaneous fluorometric determination of chlorophylls a and B and pheophytins a and B in olive oil by partial least-squares calibration.

The resolution of quaternary mixtures of chlorophylls a and b and pheophytins a and b has been accomplished by partial least-squares (PLS) multivariate calibration, applied to the fluorescence signals of these pigments. The total luminescence information of the compounds has been used to optimize the spectral data set to perform the calibration. After preliminary studies, a method is described in acetone media, to avoid emulsions with the olive oil samples. Different scanning paths have been selected for each method. For the simultaneous determination of the pigments in olive oil samples, a comparative study of the results found by using excitation, emission, and synchronous spectral data, as analytical signal, was performed. The excitation spectra were selected as the better analytical signals for the determination of the pigments in olive oil samples. The optimum wavelength range to record the excitation spectra (lambda(em) = 662 nm) was selected to minimize the contribution of pheophytin a and to maximize the contribution of the other pigments, which are the minor constituents in olive oil. Determination of these pigments in olive oil samples was effected from the excitation spectra of dissolutions o suitable aliquots in acetone. Recovery values from olive oil, spiked with chlorophylls a and b and pheophytins a and b, were in the ranges of 70-112, 71-111, 76-105, and 82-109%, respectively.

Chlorophyll↗
PubMed2005

Effect of cyclodextrins on the physicochemical properties of chlorophyll a in aqueous solution.

The interactions between chlorophyll a and two beta-cyclodextrins, that have the same cavity size but different substituents, were studied in aqueous solutions. These supramolecular host-guest complexes were examined by a combination of UV/vis absorption, circular dichroism, NMR, and steady-state and time-resolved fluorescence measurements. The results indicate that all cyclodextrins solubilize the pigment mainly in monomeric form in water. The pigment forms 1:1 complexes with the heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin and 1:2 complexes with the hydroxypropyl-beta-cyclodextrin. In such complexes the methyl groups of the cyclodextrin inner cavity are involved in the interaction with the pigment as evidenced by NMR measurements. We also measured the luminescence of singlet oxygen photosensitized by chlorophyll a in the inclusion complexes.

Chemical Phenomena↗
PubMed1998

The 38 kDa chlorophyll a/b protein of the prokaryote Prochlorothrix hollandica is encoded by a divergent pcb gene.

The chlorophyll (Chl) a/b proteins of the photosynthetic prokaryotes appear to have evolved by gene duplication and divergence of the core Chl a antenna family, which also includes CP43 and CP47 and the iron-stress induced Chl a-binding IsiA proteins. We show here that Prochlorothrix hollandica has a cluster of three pcb (prochlorophyte chlorophyll b) genes which are co-transcribed. The major antenna polypeptides of 32 and 38 kDa are encoded by pcbA and pcbC respectively. The pcbC gene is significantly divergent from the other two and may have originated by a gene duplication independent of the one that led to isiA and the other prochlorophyte pcb genes. The distant relatedness of the three prochlorophyte genera implies that not only the ability to make Chl b and use it for light-harvesting arose independently in the three lineages, but also that the pcb genes may have arisen as the result of independent gene duplications in each lineage.

Amino Acid Sequence↗
PubMed2006

A model considering light reabsorption processes to correct in vivo chlorophyll fluorescence spectra in apples.

Chlorophyll-a contained in the peel of Granny Smith apples emits fluorescence upon excitation with blue light. The observed emission, collected by an external detector and corrected by its spectral response, is still distorted by light reabsorption processes taking place in the fruit skin and differs appreciably from the true spectral distribution of fluorescence emerging from chlorophyll molecules in the biological tissue. Reabsorption processes particularly affect the ratio of fluorescence intensities at 680 nm and at 730 nm. A model to obtain the correct spectral distribution of the emission, from the experimental fluorescence recorded at a fluorometer detector and corrected for the detector spectral sensitivity, is developed in the present work. Measurements of the whole fruit reflectance, the peel transmittance and the flesh reflectance allow the calculation of the reabsorption-corrected spectra. The model is validated by comparing the corrected emission spectra with that obtained for a thin layer of apple-peel-chloroplasts, where no reabsorption takes place. It is recommended to correct distortions in emission spectra of intact fruits due to light reabsorption effects whenever a correlation between the physiological state of the fruit and its fluorescence spectra is investigated.

Absorption↗