Spectra Of Chlorophyll Research Articles

Changes in the Carbon−Carbon and Carbon−Nitrogen Bond Orders in the Macrocycle of Chlorophyll a upon Singlet and Triplet Excitation As Probed by Resonance Raman Spectroscopy of Natural-Abundance and Singly and Doubly Labeled Species with 15N, 13C, and 2H Isotopes

The Raman spectra of chlorophyll a (1700−1000 cm-1) in the S0, S1, and T1 states were recorded for the species of natural-abundance (NA) isotopic composition and those species that were totally labeled with the 15N, 13C, 13C + 15N, 2H, and 2H + 15N isotopes. An empirical normal-coordinate analysis using the S0 Raman lines of all the isotope species was performed to establish their assignments and to determine a set of force constants including the stretching, bending, nonbonded repulsive force constants and their cross-terms. The Ca−Cm and Ca−N stretching force constants in the S1 and T1 states were also determined, on the basis of the assumption that other force constants were the same as those in the S0 state, to achieve the classification of the observed Raman lines of the NA-, 13C-, and 2H-Chl a into two groups: those which shift largely upon the 15N-substitution and are associated with the Ca−N stretching vibrations and those which do not and are associated with the carbon−oxygen and carbon−carbon s...

Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements

Summary The in vivo emission spectra of chlorophyll (Chi) fluorescence of green leaves, taken at room temperature, show two maxima near 685 nm and 735 nm. The shape of the spectra is modified by re-absorption processes and depends on the Chl content of leaves. By performing reflectance, transmission and fluorescence measurements, we investigated the reason for the repeatedly reported increase of the fluorescence ratio F685/F735 during stress or damage-induced breakdown of chlorophyll in plants. The in vivo spectra of Chi fluorescence (F), transmittance (T) and reflectance (R) were taken at room temperature at the same leaf spot. Leaves with a wide range of green colour (Chl content varied from 70 to 670 mg m 2 ) were chosen from a beech ( Fagus sylvatica L.) and an elm tree ( Ulmus minor Miller) as well as from a wild vine shrub ( Parthenocissus tricuspidata L.). Strict linear correlations (with a determination coefficient of r 2 >0.92) were found between the ratio F685/F735 on the one hand, and (i) the ratio of the non-absorbed radiation (R685+T685)/(R735+T735), (ii) the reflectance ratio R685/R735, and (iii) the reflectance at 685 nm, on the other hand. The results demonstrate that 92% or more of the ratio F685/F735 variation in leaves during development or at damage and stress events is determined by the variation in Chl content and corresponding changes of the optical properties of leaves. The Chi fluorescence emission spectrum has been corrected at each wavelength for re-absorption by means of non-absorbed radiation (R + T) and reflectance yielding the actually emitted retrieved Chl fluorescence. The retrieved Chl fluorescence at 685 and 735 nm is found to linearily increase with the Chl content. The shape of the retrieved Chl fluorescence spectrum was very similar to that of strongly diluted Chl a in solution, only the position of the emission maxima is different (673 nm of Chl a in ethanol solution, and 685 nm in leaves). Studies on the actually emitted «true» Chl fluorescence, as retrieved from room temperature and outdoor fluorescence measurements, may show new ways of plant stress detection.

Effects of Simulated Acid Rain on Pinus densiflora: Inhibition of Net Photosynthesis by the Pheophytization of Chlorophyll

This research addressed the effects of simulated acid rain on the chlorophyll contents of leaves of Pinus densiflora, on the net photosynthetic rate on a unit chlorophyll basis, and on chlorophyll degradation. When NO3- concentrations in rain water increased, the chlorophylla+b contents of the leaves increased in parallel, even though the pH of the simulated rain decreased. The nitrogen content of the rain was sufficient to stimulate the production of chlorophyll and was able to override any degradative effect expected due to the H+ input from acid rain. The absorption spectrum of chlorophyll extracted from the leaves exposed to acid rain showed peaks at 665, 606 and 535 nm, characteristic of pheophytin a, indicating degradation of chlorophyll to pheophytin. Net photosynthetic rate on the basis of chlorophyll content was reduced by acid rain, even though the total chlorophyll content of the leaves was increased. The acid rain does, therefore, simultaneously increase chlorophyll content and reduce the efficiency of the use of chlorophyll in photosynthesis. This reduced efficiency may be linked to the increase in the rate of degradation of chlorophyll to pheophytin.

Optical properties of solvated molecules calculated by a QMMM method Chlorophyll a and bacteriochlorophyll a

We have applied a hybrid quantum mechanical–molecular mechanical (QMMM) approach to the calculation of electronic–vibrational coupling. To test the validity of this approach, we compare results to the steady state absorption and emission spectra of chlorophyll a (Chl-a) and bacterio-chlorophyll a (BChl-a). We find that the Stokes shift and the widths of the spectra are reasonably well represented while the amplitudes of the vibrational side band in both molecules are greatly underestimated.

Spectrophotometric Analysis of Carotenoids in Plant Extracts Based on Elimination of Chlorophyll Absorption

Subtraction of the absorption spectra of chlorophyll(s) from the total spectra of plant cell extracts reveals the carotenoid absorption spectra in both the ultraviolet and visible regions, from which the total carotenoid content can be easily evaluated. The applications of this method to Spirulina platensis cells treated with the herbicide norflurazon and to Anabaena siamensis cells grown under high light irradiation are demonstrated. The possibility of subtraction of the absorption of both chlorophyll a and b is demonstrated in an extract of the leaves of a higher plant (grape).

Fourier transform multipixel spectroscopy for quantitative cytology

A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 104 pixels in a single cell. The net result was I(xy,λ), the fluorescence intensity (I) for each pixel of the image (xy), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food‐digestive cycle in the unicellular Paramecium vulgaris fed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a ‘similarity mapping’ algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were similarly imaged.

Optics of a Bifacial Leaf: 2. Light Regime as Affected by the Leaf Structure and the Light Source

Abstract— This paper presents a theoretical analysis of the light regime in a bifacial leaf performed on the basis of optical parameters estimated from the measurements of internal light fluxes with optical microprobes. Pathlength distributions, absorptances and gradients of absorption rates are calculated as affected by different aspects of leaf optics and morphology: scattering of tissue layers, overall scattering, surface reflection, thickness and absorption to attenuation ratio in single tissue layers. The in vivo measured transmission spectrum of a leaf is correlated with the in vitro chlorophyll spectrum by accounting for two main disturbing factors: multiple scattering (pathlength statistics) and distributional error (absorption statistics).

Multivariate calibration of chlorophyll a using partial least squares and electronic absorption spectroscopy

Partial least squares (PLS) calibration has been applied in the study of electronic absorption spectra of pure chlorophyll a. Inverse univariate calibration was applied separately at each wavelength. The effectiveness of the calibration models was quantitatively expressed as root mean square errors of both calibration and prediction. Wavelengths with the best linear behaviour were selected, and PLS performed on an increasing number of wavelengths, sorted by their prediction efficiency. PLS calibration results in a substantial reduction in the prediction error relative to the best single wavelength result and is an alternative to using molar absorbance (extinction) coefficients.

Time-resolved delayed luminescence of chlorophyll a in anhydrous polymer systems

Abstract Absorption, fluorescence and time-resolved delayed luminescence spectra of chlorophyll a in thin anhydrous polymer matrices of polyvinyl alcohol and nitrocellulose have been measured. Delayed luninescence spectra were measured in the microsecond time range in the 600–800 nm region at different temperatures (8–300 K). Delayed luminescence kinetics for the following chlorophyll a species: monomeric form, chlorophyll-chlorophyll dimer and chlorophyll-nitrocellulose complex, have been obtained and decay times are evaluated (9–17 μs). Decay times are found to be independent of temperature. The possible mechanisms responsible for delay luminescence are discussed. It is found that the delayed luminescence intensity slowly and linearly increases with decreasing temperature. Thermal repopulation of the singlet excited state of chlorophyll a from the triplet state together with annihilation processes have been excluded as mechanisms for generation delayed luminescence. Charge separation and ion recombination are discussed as more likely mechanisms of delayed luminescence. Delayed luminescence results are compared to those reported in the literature chlorophyll a under a variety of conditions.

Vibrational equilibration in absorption difference spectra of chlorophyll a

We describe Franck-Condon simulations of vibrational cooling effects on absorption difference spectra in chlorophyll a (Chl a). The relative contributions of vibrational equilibration in the electronic ground and excited states depend on the pump and probe wavelengths. For Franck-Condon-active vibrational modes exhibiting small Huang-Rhys factors (S < 0.1, characteristic in Chl a pigments), vibrational thermalization causes essentially no spectral changes when the origin band is excited. Significant spectral evolution does occur for S < 0.1 when the 0-1 and 1.0 (hot) vibronic bands are excited. However, vibrational equilibration in these cases causes no spectral shifting in the empirical photobleaching/stimulated emission band maximum. This result bears on the interpretation of time-resolved absorption difference spectra of Chl a-containing antennae such as the Chl a/b light-harvesting peripheral antenna of photosystem II.

UV-VIS absorption, CD and MCD spectra of chlorophylls and their derivatives

UV-VIS absorption, CD and MCD spectra of chlorophylls and their derivatives

Red‐band resonance raman spectroscopy of chlorophyll cofactors in photosynthetic proteins

AbstractOnly recently have various methodological approaches been successful in producing Raman spectra of chlorophylls resonantly with their lowest singlet electronic transitions (Qy bands). The information content of these new approaches and their impact on current research on photosynthetic proteins are discussed. It is shown that resonance methods can contribute very valuable data on the dynamics of the primary photophysical events, while preresonance methods are yielding many new data on the structural bases of these events at atomic level. © 1995 John Wiley &amp; Sons, Inc.

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).

Effects of vinyl substitutions on resonance Raman spectra of (bacterio)chlorophylls

AbstractSoret resonance and Qy preresonance Raman spectra are reported and compared for a series of (bacterio)chlorophylls. Chlorophyll a, 2‐acetylchlorophyll a, bacteriochlorophyll a and 2‐vinylbacteriochlorophyll a were studied in the non‐protic solvent tetrahydrofuran. These experiments were designed to identify Raman bands corresponding to the stretching mode(s) of the vinyl group at the C‐2 position of ring I of chlorophyll a and 2‐vinylbacteriochlorophyll a, and to ascertain whether additional bands corresponding to Ca Cm and/or Cb Cb vibrations could be observed in the 1615–1660 cm−1 region. Raman spectra of chlorophyll a and 2‐vinylbacteriochlorophyll a exhibit a 1625 cm−1 band, which is absent from the Raman spectra of 2‐acetylchlorophyll a and bacteriochlorophyll a. It is assigned to the vC2a C2b mode of the vinyl group. No other band can be definitively assigned to any mode predominantly arising from vinyl motions. The acetyl‐containing molecules 2‐acetylchlorophyll a and bacteriochlorophyll a give rise to a ca. 1070 cm−1 band, which appears to be related to the presence of the acetyl substituent. The 1615–1660 cm−1 region of the Raman spectra of all four derivatives did not contain any additional band which could be ascribed to modes involving the vCa Cm and/or νCb Cb coordinates.

327 LEAF WATER POTENTIAL, RELATIVE WATER CONTENT AND GAS EXCHANGE OF STRAWBERRY LEAVES IN RESPONSE TO WATE.R STRESS

Leaf water potential (LWP). relative water content (RWC), gas exchange rates and 4th-derivative spectra were measured in water-stressed and normally Irrigated plank of Totem' strawberry (Fragaria × ananassa) grown in a growth chamber. CO2 assimilation rate (A) dropped sharply when LWP decreased from -0.5 to -1.2 MPa and almost ceased as LWP fell below -1.5 MPa. There was a significant but more gradual decline of A as RWC decreased form 90% to 55%. An exponential relationship with A was observed across a wide range of LWP and RWC (Rz= 0.64, 0.86, respectively). LWP was more closely related with transpiration and leaf and stomatal conductances than with A and water use efficiency. RWC was highly correlated with all gas exchange parameters. Under moderate water stress, younger leaves maintain higher RWC and A than older leaves. There was no relationship between LWP and leaf age. RWC and A were both negatively correlated with peak amplitudes of Ca 684 and Ca 697 and positively correlated with Ca 693 in their 4th-derivative spectra of chlorophyll. LWP had a negative correlation with Cb 640.

Energy transfer, charge separation and pigment arrangement in the reaction center of Photosystem II

Energy transfer and charge separation in the isolated Photosystem II reaction center at room temperature were studied with transient absorption difference spectroscopy upon selective excitation of the reaction center pigments. The measurements were performed with two dye lasers, which had a spectral bandwidth of less than 1 nm, and with an instrument response function of 5 or 18 ps depending on the type of experiment. Small changes with time constants of 0.6 ns and 120 ps are attributed to damaged reaction centers. Selective excitation of the long-wavelength pigments, presumably P680 and the pheophytins, led to charge separation in 3 ps. Selective excitation of the short-wavelength pigments, presumably accessory chlorophylls, led to charge separation in 30 ps with the same quantum efficiency. This excludes equilibration of the excited state between accessory chlorophyll and P680 in less than 30 ps. The overlap of the fluorescence spectrum of accessory chlorophyll with the absorption of P680 is very good and the slow energy transfer is attributed to an about 30 Å center-to-center distance, which makes the histidines 118 in helix II of the D1 and D2 proteins likely binding sites of the chlorophylls nearest to the long-wavelength pigments, P680 and pheophytin. Reevaluation of the literature in the light of these data suggests that P680 is a dimer with nearly (anti) parallel Q Y-transition moments of the constituent monomers, making an angle with their connecting axis close to the magic angle, and that the geometry of P680 and the pheophytins is not C 2-symmetrical around an axis perpendicular to the membrane.

AUTHENTIC in vivo ABSORPTION SPECTRA FOR CHLOROPHYLL IN LEAVES AS DERIVED FROM in situ AND in vitro MEASUREMENTS*

AbstractOn the basis of a comprehensive treatment of light propagation in plant tissue the first authentic in vivo absorption spectrum of chlorophyll in a leaf (of Catalpa bignonioides) has been elucidated. An experimental‐theoretical procedure is described, which permits derivation of the authentic chlorophyll spectrum by purifying the in vivo absorption spectrum of a leaf from the distortions of optical origin: multiple scattering and distributional error. The procedure involves measurements of internal and external light fluxes, spatial characterization of tissue layers, cells, chloroplasts and grana stacks. Furthermore, one has to determine the local chlorophyll concentrations. The theories of radiation transfer and absorption statistics make up the theoretical basis of the procedure. The derived authentic chlorophyll spectrum differs both from the in vivo spectrum of the leaf and (although to a smaller degree) from the in vitro spectrum of chlorophyll‐protein complexes extracted from the same leaf.

The derivation of the formyl-group oxygen of chlorophyll b in higher plants from molecular oxygen. Achievement of high enrichment of the 7-formyl-group oxygen from 18O2 in greening maize leaves.

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark-grown plants, by illumination under white light in the presence of either H2(18)O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7-formyl substituent by reversible formation of chlorophyll b-7(1)-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process. Mass spectra of chlorophyll a and [7-hydroxymethyl]-chlorophyll b extracted from leaves greened in the presence of either H2(18)O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 13(1)-oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599-602]. The incorporation of isotope led to as much as 77% enrichment of the 13(1)-oxo group of chlorophyll a: assuming identical incorporation into the 13(1) oxygen of chlorophyll b, then enrichment of the 7-formyl oxygen was as much as 93%. Isotope dilution by re-incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The high enrichment using 18O2, and the absence of labelling by H2(18)O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase-type enzyme.

Stark spectra of chlorophylls and carotenoids in antenna pigment-proteins LHC-II and CP-II

The band structure of low-temperature absorption and Stark spectra of light-harvesting chlorophyll a/ b protein complexes LHC-II and CP-II is analysed. In the red spectral region, the Stark spectrum serves as an independent experimental reference allowing for the resolution of eight bands at 639, 642–3, 649, 656, 662, 665 671 and 678 nm, consistently with the gaussian deconvolution of the absorption spectrum. Different parts of the Stark spectrum are fitted with combinations of the derivatives of either the total absorption spectrum or of individual gaussian components. Two electronic transitions at 671 nm and 678 nm and two others at 639 nm and 642–3 nm are found to excite, respectively, localized chlorophyll a and chlorophyll b states. Increased changes in permanent dipole moment, Δμ ≅ 2.2 Debye units, corresponding to the transitions at 656 nm and at 665 nm, and an increase in polarizability ≅ 70 Å 3 corresponding to the first of them indicate that these transitions originate from strong exciton coupling. In the violet, the Stark spectra are dominated by the contribution from xanthophylls. A value of Δμ ≥ 10 D was estimated for xanthophyll bands at 484 nm and at 511–513 nm. The mechanism of the Stark effect in xanthophylls is briefly discussed.

IS ZEAXANTHIN CAPABLE OF ENERGY TRANSFER TO CHLOROPHYLL a IN PARTIALLY GREENED LEAVES? A STUDY OF FLUORESCENCE EXCITATION SPECTRA DURING VIOLAXANTHIN DEEPOXIDATION

AbstractAbsorbance spectra and excitation spectra of chlorophyll a fluoresence were recorded during the light‐induced deepoxidation of violaxauthin to zeaxanthin in bean leaves (Phaseolus coccineus) greened under intermittent light. Light minus dark fluorescence excitation difference spectra showed distinct minima at 440, 465, and 500 nm corresponding to maxima in the absorbance difference spectra. Both difference spectra were prevented by the deepoxidase inhibitor dithiothreitol and were inverted when zeaxanthin was epoxidized. The fluorescence excitation difference spectra were successfully modeled by considering the absorbance differences between violaxanthin and zeaxanthin, assuming no energy transfer from the two pigments to chlorophyll a, and accounting for light‐induced scattering changes. The pigment stoichiometry and the scattering changes of the simulation were in accordance with experimental data. The results indicate that, in the early stage of leaf development, light absorbed by the cycle pigments violaxanthin and zeaxanthin is not transferred to chlorophyll.