Uptake of diuron and concomitant loss of photosynthetic activity in leaves as visualized by imaging the red chlorophyll fluorescence

The principles of the chlorophyll (Chl) fluorescence induction kinetics (known as Kautsky effect) and their change by the photosystem II herbicide diuron are presented together with the Chl fluorescence emission spectra of a normal and diuron-inhibited leaf. By imaging the Chl fluorescence emission of green leaves the successive uptake of diuron and the concomitant loss of photosynthetic quantum conversion from the leaf base to the leaf tip are documented.     

Optical properties of mixtures of beta-carotene and chlorophyll a adsorbed on bovine serum albumin

The fluorescence of the natural photosynthetic pigments beta-carotene (beta-K) and chlorophyll a (Chl) and their mixtures with bovine serum albumin (BSA) in different molar ratios has been studied. An increase in the fluorescence intensity in a pigment mixture-BSA complex was found. The highest possible (four- to sixfold) increase in the fluorescence intensity compared with fluorescence intensity of one-pigment BSA complexes BSA (beta-K) and BSA (Chl) was achieved at the ratio 11-27% beta-K/89-73% Chl in the BSA complex. A considerable overlap of fluorescence spectra of BSA (Chl) complex (lambda(max) at 690 nm) and BSA (beta-K) complex (lambda(max) at 684 nm) was observed.     

Differential changes in degradation of chlorophyll-protein complexes of photosystem I and photosystem II during flag leaf senescence of rice.

The stability of chlorophyll-protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO(2) assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll-protein complexes of PSI was greater than in chlorophyll-protein complexes of PSII. The fluorescence yields for all chlorophyll-protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll-protein complex was different and the order for the stability of chlorophyll-protein complexes during leaf senescence was: LHCII>CP43>CP47>LHCI>PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll-protein complexes.     

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 F?rster 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.     

Pigment compositions,spectral properties,and energy transfer efficiencies between the xanthophylls and chlorophylls in the major and minor pigment-protein complexes of photosystem II

Absorption, fluorescence, and fluorescence excitation spectra have been measured from CP26, CP29, and monomeric and trimeric LHCIIb light-harvesting complexes isolated from Photosystem II subchloroplast particles from spinach. The complexes were purified using a combination of isoelectric focusing and sucrose gradient ultracentrifugation. The chlorophyll (Chl) and xanthophyll pigment compositions were measured using high-performance liquid chromatography (HPLC). Using the pigment compositions from the HPLC analysis as a starting point, the absorption spectral profiles of the complexes have been reconstructed from the individual absorption spectra obtained for each of the pigments. Also, the fluorescence excitation spectra of the complexes have been deconvoluted. The data reveal the energy transfer efficiencies between Chl b and Chl a and between specific xanthophylls and Chl a in the complexes. The spectral analyses reveal the underlying features of the highly congested spectral profiles associated with the complexes and are expected to be beneficial to researchers employing spectroscopic methods to investigate the mechanisms of energy transfer between the pigments bound in these complexes.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

Photosynthetic acclimation to light in woody and herbaceous species: a comparison of leaf structure, pigment content and chlorophyll fluorescence characteristics measured in the field

Acclimation of foliage photosynthetic properties occurs with varying time kinetics, but structural, chemical and physiological factors controlling the kinetics of acclimation are poorly understood, especially in field environments. We measured chlorophyll fluorescence characteristics, leaf total carotenoid (Car), chlorophyll (Chl) and nitrogen (N) content and leaf dry mass per area (LMA) along vertical light gradients in natural canopies of the herb species, Inula salicina and Centaurea jacea, and tree species, Populus tremula and Tilia cordata, in the middle of the growing season. Presence of stress was assessed on the basis of night measurements of chlorophyll fluorescence. Our aim was to compare the light acclimation of leaf traits, which respond to light availability at long (LMA and N), medium (Chl a/b ratio, Car/Chl ratio) and short time scales (fluorescence characteristics). We found that light acclimation of nitrogen content per unit leaf area (N(area)), chlorophyll content per unit dry mass (Chl(mass)) and Chl/N ratio were related to modifications in LMA. The maximum PSII quantum yield (F(v) /F(m)) increased with increasing growth irradiance in I. salicina and P. tremula but decreased in T. cordata. Leaf growth irradiance, N content and plant species explained the majority of variability in chlorophyll fluorescence characteristics, up to 90% for steady-state fluorescence yield, while the contribution of leaf total carotenoid content was generally not significant. Chlorophyll fluorescence characteristics did not differ strongly between growth forms, but differed among species within a given growth form. These data highlight that foliage acclimation to light is driven by interactions between traits with varying time kinetics.     

Characteristics of the tertiary structure of histone H1 from the calf thymus

By optical methods it has been previously shown that the globular "head" of histone H1 forms a hydrophobic cavity containing Tyr72. The latter is screened from the polar water surrounding and its intramolecular mobility is drastically hindered. As a consequence of the alteration in the micromilieu are a long wave shift (lambda max = 279,5 nm) and a more pronounced longwave absorption spectra, higher anisotropy (A = 0,11), augmented quantum yield of fluorescence (approximately 0,2) and a decrease of the Stern-Volmer constant for Hl at fluorescence quenching by acrylamide. It was found that changes in fluorescence intensity of histones are connected with alterations in the quantum yield of fluorescence at lambda exc = = 265 nm, but not at lambda exc = 280 nm. The changes in fluorescence intensity at light excitation 280 nm (F280) and 265 nm (F265) are in good accordance with shift delta E286 in differential absorption spectra. Introduction of parameter Cf = F280/F265 allows to study shifts of excitation spectra instead of shifts in absorption spectra of histones. This method has certain advantages, since it permits investigations with lower protein concentrations and in turbid solutions. The data obtained allow to draw out Tyr72 of histone Hl into a special class of fluorescent-tyrosyls, that differ in properties from those of other tryptophandevoided proteins: RNAse, insulin and core-histones H2A, H2B, H3 and H4.     

Structure and Function of Chloroplast Membrane XVI. Comparative Studies on Some Photosynthetic Characteristics between Normal and Mutant Barleys

Some photosynthetic characteristics of mutant barley Chlorina f, were studied in comparison with that of normal variety. They were quite different in chlo- roplast membrane structures, pigment protein complexes, the content of electron transport components and photosynthetic functions. The absence of Chlb in mutant barley, as demonstrated by absorption and fluorescence excitation spectra, caused some defects of membrane structure and lose of the ability to regulate the distribution of excitation energy between PSII and PSⅠ. In comparison with the normal variety, the mutant barley contained much less chlorophyll per leaf area, but more P700, Cyt f and PQ on the chlorophyll basis. These differences surely affect their photochemical activities. As envisaged by fluorescence spectra, peripheral antenna of PSⅠ is absent in mutant barley membrane besides the lacking of Chl a/b-protein of PSⅡ. Fluorescence induction transient of mutant barley leaf did not show the typical time course of O→P→S→M→T. The coexistence of light harvesting Chl a/b-protein eomplex of PSⅡ and peripheral antenna of PSI and their cooperation with each other seem to be necessary for the occurence of typical fluorescence induction transient.     

Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.     

黄化油菜突变体Cr3529子叶类囊体膜光谱性质研究

以发育10d的黄化油菜突变体为材料,分析了突变体油菜子叶类囊体膜的色素含量、室温吸收光谱、叶绿素荧光发射和激发光谱以及蛋白内源荧光光谱的变化。数据显示：与野生型相比,突变体油菜子叶类囊体膜的光合色素Chl α和Chl b含量均减少．但Chl α／b比值升高;突变体油菜子叶类囊体膜叶绿素捕光能力和受激发能力均下降,且较依赖于Chl α捕光并将光能激发传递给PSⅡ反应中心;突变体油菜子叶类囊体膜的蛋白内源荧光也明显异于野生型。进一步表明突变体油菜子叶类囊体膜蛋白组成发生了改变。     

Replacement of chlorophyll with di-vinyl chlorophyll in the antenna and reaction center complexes of the cyanobacterium Synechocystis sp. PCC 6803: Characterization of spectral and photochemical properties

Chlorophyll (Chl) a in a cyanobacterium Synechocystis sp. PCC 6803 was replaced with di-vinyl (DV)-Chl a by knock-out of the specific gene (slr1923), responsible for the reduction of a 8-vinyl group, and optical and photochemical properties of purified photosystem (PS) II complexes (DV-PS II) were investigated. We observed differences in the peak wavelengths of absorption and fluorescence spectra; however, replacement of Chl a with DV-Chl a had limited effects. On the contrary, photochemical reactions were highly sensitive to high-light treatments in the mutant. Specifically, DV-Chl a was rapidly bleached under high-light conditions, and we detected significant dissociation of complexes and degradation of D1 proteins (PsbA). By comparing the SDS-PAGE patterns observed in this study to those observed in spinach chloroplasts, this degradation is assigned to the acceptor-side photoinhibition. The delayed fluorescence in the nanosecond time region at 77 K was suppressed in DV-PS II, possibly increasing triplet formation of Chl molecules. Our findings provide insight into the evolutionary processes of cyanobacteria. The effects of pigment replacement on the optimization of reactions are discussed.     

Some physiological and biochemical changes in marine eukaryotic red tide alga Heterosigma akashiwo during the alleviation from iron limitation

Some physiological and biochemical changes in the marine eukaryotic red tide alga Heterosigma akashiwo (Hada) were investigated during the alleviation from iron limitation. Chlorophyll a/carotenoid ratio increases as a result of iron alleviation. In vivo absorption spectra of iron-limited cells showed a chlorophyll (Chl) absorption peak at 630 nm, 2 nm blue-shifted from the normal position. Low-temperature fluorescence emission spectra of the cells have one prominent Chl emission peak at 685 nm. The cells showed a decrease in fluorescence yield from 685 nm band during alleviation from iron limitation. Low-temperature fluorescence excitation spectra and room-temperature fluorescence spectra indicated an efficient excitation energy transfer in the cells alleviated from iron limitation. Photosynthetic efficiency and carbohydrate content per cell increased after alleviation from iron limitation. Total protein decreased in iron-limited cells, while iron deficiency induced the appearance of specific soluble proteins (17 and 55 kDa).     

Spectral analysis on origination of the bands at 437 nm and 475.5 nm of chlorophyll fluorescence excitation spectrum in Arabidopsis chloroplasts

Chlorophyll fluorescence has been often used as an intrinsic optical molecular probe to study photosynthesis. In this study, the origin of bands at 437 and 475.5 nm in the chlorophyll fluorescence excitation spectrum for emission at 685 nm in Arabidopsis chloroplasts was investigated using various optical analysis methods. The results revealed that this fluorescence excitation spectrum was related to the absorption characteristics of pigment molecules in PSII complexes. Moreover, the excitation band centred at 475.5 nm had a blue shift, but the excitation band at 437 nm changed relatively less due to induction of non‐photochemical quenching (NPQ). Furthermore, fluorescence emission spectra showed that this blue shift occurred when excitation energy transfer from both chlorophyll b (Chl b) and carotenoids (Cars) to chlorophyll a (Chl a) was blocked. These results demonstrate that the excitation band at 437 nm was mainly contributed by Chl a, while the excitation band at 475.5 nm was mainly contributed by Chl b and Cars. The chlorophyll fluorescence excitation spectrum, therefore, could serve as a useful tool to describe specific characteristics of light absorption and energy transfer between light‐harvesting pigments. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of Chromatic Adaptation on the Photochemical Apparatus of Photosynthesis in Porphyridium cruentum

Cells of Porphyridium cruentum were grown in different colors of light which would be absorbed primarily by chlorophyll (Chl) (red and blue light) or by the phycobilisomes (green or two intensities of cool-white fluorescent light), and samples of these cells were frozen to −196 C for measurements of absorption and fluorescence emission spectra. Cells grown in the high intensity white light had least of all of the photosynthetic pigments, a higher ratio of carotenoid/Chl, but essentially the same ratio of phycobilin to Chl as cells grown in the low intensity white light. The ratio of photosystem II (PSII) to photosystem I (PSI) pigments was affected by light quality; the ratios of phycobilin to Chl and of short wavelength (PSII) Chl to long wavelength (PSI) Chl were both greater in the cells grown in red or blue light.     

Peridinin chlorophyll a protein: relating structure and steady-state spectroscopy

Peridinin chlorophyll a protein (PCP) from Amphidinium carterae has been studied using absorbance (OD), linear dichroism (LD), circular dichroism (CD), fluorescence emission, fluorescence anisotropy, fluorescence line narrowing (FLN), and triplet-minus-singlet spectroscopy (T-S) at different temperatures (4-293 K). Monomeric PCP binds eight peridinins and two Chls a. The trimeric structure of PCP, resolved at 2 A [Hofmann et al. (1996) Science 27, 1788-1791], allows modeling of the Chl a-protein and Chl a-Chl a interactions. The FLN spectrum shows that Chl a is not or is very weakly hydrogen-bonded and that the central magnesium of the emitting Chl a is monoligated. Simulation of the temperature dependence of the absorption spectra indicates that the Huang-Rhys factor, characterizing the electron-phonon coupling strength, has a value of approximately 1. The width of the inhomogeneous distribution function is estimated to be 160 cm(-)(1). LD experiments show that the two Chls a in PCP are essentially isoenergetic at room temperature and that a substantial amount of PCP is in a trimeric form. From a comparison of the measured and simulated CD, it is concluded that the interaction energy between the two Chls a within one monomer is very weak, <10 cm(-)(1). In contrast, the Chls a appear to be strongly coupled to the peridinins. The 65 cm(-)(1) band that is visible in the low-frequency region of the FLN spectrum might indicate a Chl a-peridinin vibrational mode. The efficiency of Chl a to peridinin triplet excitation energy transfer is approximately 100%. On the basis of T-S, CD, LD, and OD spectra, a tentative assignment of the peridinin absorption bands has been made.     

Deriving room temperature excitation spectra for photosystem I and photosystem II fluorescence in intact leaves from the dependence of <Emphasis Type="Italic">F</Emphasis><Subscript>V</Subscript>/<Emphasis Type="Italic">F</Emphasis><Subscript>M</Subscript> on excitation wavelength

The F ₀ and F _M level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated (PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of F _V/F _M (=(F _M − F ₀)/F _M) have been derived: within each of the three spectra of F _V/F _M, statistically meaningful variations were detected. Also, at distinct wavelength regions, the F _V/F _M differed significantly between leaf types. From spectra of F _V/F _M, excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of F _V/F _M between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the F _V/F _M at these wavelengths. All three leaves exhibited low values of F _V/F _M around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550–650 nm region, the F _V/F _M in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in maize, which is a NADP-ME C₄ species, than in barley, a C₃ species. Finally, low values of F _V/F _M at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra of the F _V/F _M ratio to derive information on spectral absorption properties of PS I and PS II is discussed.     

Differences in pigment composition, photosynthetic rates and chlorophyll fluorescence images of sun and shade leaves of four tree species.

The differential pigment composition and photosynthetic activity of sun and shade leaves of deciduous (Acer pseudoplatanus, Fagus sylvatica, Tilia cordata) and coniferous (Abies alba) trees was comparatively determined by studying the photosynthetic rates via CO(2) measurements and also by imaging the Chl fluorescence decrease ratio (R(Fd)), which is an in vivo indicator of the net CO(2) assimilation rates. The thicker sun leaves and needles in all tree species were characterized by a lower specific leaf area, lower water content, higher total chlorophyll (Chl) a+b and total carotenoid (Cars) content per leaf area unit, as well as higher values for the ratio Chl a/b compared to the much thinner shade leaves and needles that possess a higher Chl a+b and Cars content on a dry matter basis and higher values for the weight ratio Chls/Cars. Sun leaves and needles exhibited higher rates of maximum net photosynthetic CO(2) assimilation (P(Nmax)) measured at saturating irradiance associated with higher maximum stomatal conductance for water vapor efflux. The differences in photosynthetic activity between sun and shade leaves and needles could also be sensed via imaging the Chl fluorescence decrease ratio R(Fd), since it linearly correlated to the P(Nmax) rates at saturating irradiance. Chl fluorescence imaging not only provided the possibility to screen the differences in P(N) rates between sun and shade leaves, but in addition permitted detection and quantification of the large gradients in photosynthetic rates across the leaf area existing in sun and shade leaves.     

Long-wavelength chlorophyll forms in Photosystem I from pea thylakoids

Absorption maximum positions of three LW Chl forms in pea chloroplasts were estimated using 77 K excitation spectra of fluorescence detected in their maxima (720, 732 and 746 nm). The 705, 714 and 723 nm components were revealed in the second derivative plots of the excitation spectra. The same maxima were found in normalized excitation spectra obtained with dividing excitation spectra by absorption spectrum. It was confirmed that the observed maxima belong to absorption of LW fluorescing Chl forms. The same maxima were displayed in an action spectrum of P700 oxidation measured at room temperature. It confirms the energy transfer from LW Chl forms to P700. Close to 50% efficiency of bulk Chl forms in both excitation of LW fluorescence and P700 oxidation was found. Analysis of the shape of normalized excitation spectra suggests that there is no energy exchange among LW Chl forms. Their location and physiological role are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.