Carotenoid dependence of the protochlorophyllide to chlorophyllide phototransformation in dark-grown wheat seedlings.

The influence of carotenoids on partial protochlorophyllide (Pchlide) photoreduction and the successive formation of long-wavelength chlorophyllide (Chlide) forms was studied by low-temperature fluorescence spectroscopy (77 K). Wheat leaves with a decreased content of carotenoids obtained from norflurazon-treated seedlings (10 and 100 micromol l(-1)) were compared with leaves containing normal amounts of these pigments. Partial photoreduction of Pchlide was achieved by irradiation of the leaves with one light flash in combination with a number of neutral gray and/or red Perspex filters. There were significant differences between the fluorescence emission spectra (the position and height of the peaks) of dark-grown normal and carotenoid-deficient leaves irradiated with non-saturating white light of increasing intensity. The long-wavelength Chlide forms appeared first in the leaves nearly devoid of carotenoids (treated with 100 micromol l(-1) norflurazon), then in the leaves with carotenoid deficiency (treated with 10 micromol l(-1) norflurazon), and finally in normal leaves. After irradiation with non-saturating light of the same intensity, the ratio Chlide/Pchlide(657) was always the highest in the leaves nearly deficient of carotenoids, medium in the leaves with carotenoid deficiency and lowest in the normal leaves. Similarly to white light, red light of low intensity induced faster formation of long-wavelength Chlide species in the leaves with carotenoid deficiency in comparison to the normal leaves. We propose that, in leaves with reduced carotenoid content, a greater number of Pchlide molecules transform to Chlide per light flash than in normal leaves. The results are discussed in relation to the involvement of carotenoids in competitive absorption and light screening, as well as to their influence on Pchlide-Chlide interactions.     

Fluorescence lifetimes of protochlorophyllide in plants with different proportions of short-wavelength and long-wavelength protochlorophyllide spectral forms

Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide(650-656) form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3-0.8 ns and a slow component in the range 5.1-7.1 ns were present in all samples, but the values varied, depending on species. The long-wavelength Pchlide(650-656) form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short-wavelength Pchlide(628-633) form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein-bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.     

Analysis of Fluorescence Lifetime of Protochlorophyllide and Chlorophyllide in Isolated Etioplast Membranes Measured from Multifrequency Cross-correlation Phase Fluorometry

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple-exponential model at 14°C and a double-exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady-state fluorescence spectra. The fluorescence decay of the main Pchlide form, photoactive Pchlide-F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5–6.0 ns are ascribed to the second photoactive form, Pchlide-F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating light pulse, we found a single 5.0 ns component for Chlide-F688 (the Chlide-NADPH: protochlorophyllide oxidoreductase [PORJ-NADP⁺complex) and an additional 1.6 ns component when the formation of Chlide-F696 (the Chlide-POR-NADPH complex) was promoted by exogenous NADPH. From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide-F696 as being 70%.     

Photoactive protochlorophyllide regeneration in cotyledons and leaves from higher plants

Chlorophyll accumulation during greening implies the continuous transformation of photoactive protochlorophyllide (Pchlide) to chlorophyllide. Since this reaction is a light-dependent step, the study of regeneration of photoactive Pchlide under a continuous illumination is difficult. Therefore this process is best studied on etiolated plants during a period of darkness following the initial photoreduction of photoactive Pchlide. In this study, the regeneration process has been studied using spinach cotyledons, as well as barley and bean leaves, illuminated by a single saturating flash. The regeneration was characterized using 77 K fluorescence emission and excitation spectra and high-performance liquid chromatography. The fluorescence data indicated that the same spectral forms of photoactive Pchlide are regenerated by different pathways: (1) photoactive Pchlide regeneration starts immediately after the photoreduction through the formation of a nonphotoactive Pchlide form, emitting fluorescence at approximately 651 nm. This form is similar to the large aggregate of photoactive Pchlide present before the illumination, but it contains oxidized form of nicotinamide adenine dinucleotide phosphate, instead of the reduced form (NADPH), in the ternary complexes; and (2) after the dislocation of the large aggregates of chlorophyllide-light-dependent NADPH:Pchlide a photooxidoreductase-NADPH ternary complexes, the regeneration occurs at the expense of the several nonphotoactive Pchlide spectral forms present before the illumination.     

Single and multi-exciton dynamics in aqueous protochlorophyllide aggregates

In plants, the oxidoreductase enzyme POR reduces protochlorophyllide (Pchlide) into chlorophyllide (Chlide), using NADPH as a cofactor. The reduction involves the transfer of two electrons and two protons to the C17═C18 double bond of Pchlide, and the reaction is initiated by the absorption of light by Pchlide itself. In this work we have studied the excited state dynamics of Pchlide dissolved in water, where it forms excitonically coupled aggregates, by ultrafast time-resolved transient absorption and fluorescence experiments performed in the 480-720 nm visible region and in the 1780-1590 cm(-1) mid-IR region. The ground state visible absorption spectrum of aqueous Pchlide red shifts and broadens in comparison to the spectrum of monomeric Pchlide in organic solvents. The population of the one-exciton state occurs at low excitation densities, of <1 photon per aggregate. We characterized the multiexciton manifolds spectra by measuring the absorption difference spectra at increasingly higher photon densities. The multiexciton states are characterized by blue-shifted stimulated emission and red-shifted excited state absorption in comparison to those of the one-exciton manifold. The relaxation dynamics of the multiexciton manifolds into the one-exciton manifold is found to occur in ～10 ps. This surprisingly slow rate we suggest is due to the intrinsic charge transfer character of the PChlide excited state that leads to solvation, stabilizing the CT state, and subsequent charge recombination, which limits the exciton relaxation.     

Chloroplast Biogenesis 81: Transient Formation of Divinyl Chlorophyll a Following a 2.5 ms Light Flash Treatment of Etiolated Cucumber Cotyledons

Abstract— The possible conversion of nascent divinyl (DV) chloro-phyllide a (Chlide a ) to DV chlorophyll a (Chi a during the early stages of greening in a dark divinyl-light divinyl-light/dark divinyl (DDV-LDV-LDDV) plant species was investigated. Etiolated cucumber cotyledons ( Cucu-mis sativus L .) were subjected to a 2.5 ms light flash followed by darkness. The DV and monovinyl (MV) components of the protochlorophyllide a (Pchlide a ), Chlide a , Pchlide a ester and Chi a pools were monitored quantitatively by high-resolution spectrofluorometry, immediately following the light treatments and after various periods in darkness. The light treatment photoconverted DV and MV Pchlide a to DV and MV Chlide a . Some photoconversion of MV Pchlide a ester to MV Chi a also appeared to take place. A sharp rise in the level of DV Chi a following the light treatment could not be accounted for by photoconversion of DV Pchlide a ester. It must have arisen by rapid esterification of nascent DV Chlide a. After illumination, the level of DV Chi a rose for 5 s and then declined. The implications of the transient rise and fall of DV Chi a content following illumination to the Chi a biosynthetic heterogeneity is discussed.     

Spectroscopic properties of protochlorophyllide analyzed in situ in the course of etiolation and in illuminated leaves

The spectroscopic properties of photoactive (i.e. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two phases in the process of Pchl(ide) accumulation were apparent from quantitative measurements of pigment content: a lag phase (first week) during which photoactive Pchl(ide) accumulated faster than nonphotoactive Pchl(ide); and a fast phase (second week), showing parallel accumulation of both types of Pchl(ide). 'Flashed-minus-dark' absorbance difference spectra recorded in situ at 77 K showed that P650-655 was the predominant form of photoactive protochlorophyllide regardless of developmental stage. Quantitative analysis of energy migration processes between the Pchl(ide) forms showed the existence of energy transfer units containing a 1:8 ratio of nonphotoactive and photoactive Pchl(ide)s during development. Gaussian deconvolution of in situ 77 K fluorescence spectra indicated that the 633 nm band of nonphotoactive Pchl(ide) was made of four bands, at 625, 631, 637 and 643 nm, whose relative amplitudes only slightly changed during development. The emission band of photoactive Pchlide was also analyzed using the same method. Three components were found at 644, 652 and 657 nm. The emission band of P650-655 included the last two components, which become predominant only in fully etiolated plants. Photoactive Pchlide with an emission maximum at 653 nm was detected in the light during development of leaves of photoperiodically grown plants.     

Enzyme activation and catalysis: characterisation of the vibrational modes of substrate and product in protochlorophyllide oxidoreductase

The light-dependent reduction of protochlorophyllide, a key step in the synthesis of chlorophyll, is catalyzed by the enzyme protochlorophyllide oxidoreductase (POR) and requires two photons (O. A. Sytina et al., Nature, 2008, 456, 1001-1008). The first photon activates the enzyme-substrate complex, a subsequent second photon initiates the photochemistry by triggering the formation of a catalytic intermediate. These two events are characterized by different spectral changes in the infra-red spectral region. Here, we investigate the vibrational frequencies of the POR-bound and unbound substrate, and product, and thus provide a detailed assignment of the spectral changes in the 1800-1250 cm(-1) region associated with the catalytic conversion of PChlide:NADPH:TyrOH into Chlide:NADP(+):TyrO(-). Fluorescence line narrowed spectra of the POR-bound Pchlide reveal a C=O keto group downshifted by more than 20 cm(-1) to a relatively low vibrational frequency of 1653 cm(-1), as compared to the unbound Pchlide, indicating that binding of the chromophore to the protein occurs via strong hydrogen bond(s). The frequencies of the C=C vibrational modes are consistent with a six-coordinated state of the POR-bound Pchlide, suggesting that there are two coordination interactions between the central Mg atom of the chromophore and protein residues, and/or a water molecule. The frequencies of the C=C vibrational modes of Chlide are consistent with a five-coordinated state, indicating a single interaction between the central Mg atom of the chromophore and a water molecule. Rapid-scan FTIR measurements on the Pchlide:POR:NADPH complex at 4 cm(-1) spectral resolution reveal a new band in the 1670 cm(-1) region. The FTIR spectra of the enzyme activation phase indicate involvement of a nucleotide-binding structural motif, and an increased exposure of the protein to solvent after activation.     

Changes in the structure of chlorophyll-protein complexes and excitation energy transfer during photoinhibitory treatment of isolated photosystem I submembrane particles

The activity of light-induced oxygen consumption, absorption spectra, low temperature (77 K) chlorophyll fluorescence emission and excitation spectra were studied in suspensions of photosystem (PS) I submembrane particles illuminated by 2000 microE m(-2) s(-1) strong white light (WL) at 4 degrees C. A significant stimulation of oxygen uptake was observed during the first 1-4 h of photoinhibitory treatment, which rapidly decreased during further light exposure. Chlorophyll (Chl) content gradually declined during the exposure of isolated PSI particles to strong light. In addition to the Chl photobleaching, pronounced changes were found in Chl absorption and fluorescence spectra. The position of the major peak in the red part of the absorption spectrum shifted from 680 nm towards shorter wavelengths in the course of strong light exposure. A 6-nm blue shift of that peak was observed after 5-h illumination. Even more pronounced changes were found in the characteristics of Chl fluorescence. The magnitude of the dominating long-wavelength emission band at 736 nm located in untreated particles was five times reduced after 2-h exposure, whereas the loss in absolute Chl contents did not exceed 10% of its initial value. The major peak in low-temperature Chl fluorescence emission spectra shifted from 736 to 721 nm after 6-h WL treatment. Individual Chl-protein complexes differed in the response of their absorption spectra to strong WL. Unlike light-harvesting complexes (LHC), LHCI-680 and LHC-730, which did not exhibit changes in the major peak position, its maximum was shifted from 678 to 671 nm in CPIa complex after PSI submembrane particles were irradiated with strong light for 6 h. The results demonstrated that excitation energy transfer represents the stage of photosynthetic utilization of absorbed quanta which is most sensitive to strong light in isolated PSI particles.     

TRIPLET ESR IN l-TYROSINE

Abstract— Triplet state electron spin resonances have been observed from L-tyrosine in low temperature glasses at 77° K on irradiation by u.v. light in the band between 220 and 300 millimicrons. Two different resonances have been observed. and ESR spectra and optical absorption spectra as a function of degree of ionization show that the resonances are due to the ionized and un-ionized forms of tyrosine respectively. Solvent composition and temperature affect the ratio of the concentrations of the two forms but not their resonant fields. The resonances have half lives of from two to three seconds at 77° K.     

Fluorescence lifetimes and spectral properties of protochlorophyllide in organic solvents in relation to the respective parameters in vivo

In this work, absorption and fluorescence spectra of protochlorophyllide (Pchlide), as well as its fluorescence lifetime, were investigated in organic solvents having different physical properties. The obtained Pchlide spectral features are discussed in relation to the parameters describing solvent properties (refractive index and dielectric constant) and taking into account the specific solvent-Pchlide interaction. The correlation of Pchlide Qy and Soret absorption bands with solvent polarizability function ((n2 - 1)/(n2 + 2)) has been found; however, the dispersion of the observed points was rather high. A small Stokes shift of a magnitude between 50 and 300 cm(-1) was found, which indicates low sensitivity of Pchlide to nonspecific solvation. The fluorescence decay of Pchlide was single exponential in all the investigated solvents, with the lifetime value ranging from 5.2 ns for dioxane to 3.5 ns for methanol. Dependence of the obtained fluorescence lifetimes on the solvent orientation polarizability, a parameter being the function of both refractive index and dielectric constant, was discussed. In water-methanol mixtures, a further decrease of the fluorescence lifetime was observed, giving values of 2.9 ns for 25% methanol. Double-exponential decay of Pchlide fluorescence was found for Pchlide in a solution of 15% methanol with the lifetimes of 4.5 +/- 0.5 ns and 1.2 +/- 0.3 ns and in pure water with the lifetimes of 2.5 +/- 0.5 ns and 0.4 +/- 0.1 ns. The obtained results are discussed in relation to spectroscopic properties of Pchlide in vivo.     

Chloroplast Biogenesis 77: Two Novel Monovinyl and Divinyl Light-Dark Greening Groups of Plants and Their Relationship to the Chlorophyll a Biosynthetic Heterogeneity of Green Plants

Abstract— On the basis of the steady-state accumulation of divinyl (DV) or monovinyl (MV) protochlorophyllide (Pchlide) a in darkness (D) or in the light (L), green plants have been classified into three different greening groups namely dark divinyl-light divinyl (DDV-LDV), dark monovinyl-light divinyl (DMV-LDV) and dark monovinyl-light monovinyl (DMV-LMV) (Ionannides et al., Biochem. Syst. Ecol. 22, 211-220,1994). Interruption of the L phase of the photoperiod by a brief period of darkness (LD condition) revealed a predominance of different chlorophyll (Chl) a biosynthetic routes, depending upon the greening group affiliation of the plant species. For example, in DMV-LDV and DMV-LMV plants, the predominant Chl a biosynthetic routes under the LD condition appear to be the MV Chi a biosynthetic route and/or a mixed DV-MV Chi a biosynthetic route that bifurcates at the level of DV Pchlide a. On the basis of DV and MV Pchlide a accumulation rates after re-darkening, this greening group is designated as a light-dark MV (LDMV) subgroup. In DDV-LDV plants, the predominant LD Chi a biosynthetic routes appear to be the DV Chi a biosynthetic route and/or a mixed DV-MV Chi a biosynthetic route that bifurcates at the level of DV Chlide a. This greening group is designated as a light-dark DV (LDDV) subgroup. It is proposed that upon inhibiting the conversion of Pchlide a to Chi a by interruption of the L phase of the photoperiod by a brief period of D, the rates of DV and MV Pchlide a regeneration may reflect the carryover rates of DV and MV Pchlide a biosynthesis in L instead of reflecting a differential use of DV and MV carboxylic biosynthetic rates in D. It is also shown that in LDMV plants, MV Chlide a and MV Chi a are formed without the participation of [4-vinyl] Chlide a reductase. On the basis of recently published evidence, it is also argued that Pchlide oxidoreductase-A (POR-A) may be active in LDDV plants, while POR-B may predominate in LDMV plant species. The evolutionary significance of the LDDV and LDMV greening subgroups is discussed.     

Changes of Chlorophyll(ide) Fluorescence Yield Induced by a Short Light Pulse as a Probe to Monitor the Early Steps of Etioplast Phototransformation in Dark-Grown Leaves

The fluorescence yield of chlorophyll(ide) (Chl[ide]) excited by weak modulated light was recorded at room temperature during a 2 h period after a short actinic light pulse that transformed all photoactive protochlorophyllide in dark-grown barley leaves. A typical pattern of fluorescence yield variations was found whatever the age of the leaf but with age-dependent changes in rates. Its successive phases were related to the Chl(ide) spectral shifts observed in low-temperature emission spectra. The fluorescence yield started at a high level and strongly declined during the formation of Chlide₆₉₅ from Chlide₆₆₈ within a few seconds. It increased to a transient maximum during the Shibata shift (15–25 min) that resulted in Chl(ide)₆₈₂. A final, slow decrease to a steady state occurred during the final red shift to Chl₆₈₅. Pretreatments with δ-aminolevulinic acid, chloramphenicol or 1, 10-phenanthroline resulted in correlated modifications of Chl(ide) fluorescence yield transients and shifts of the low-temperature Chl(ide) emission band. The complex response of the final decrease phase of the fluorescence yield to these compounds suggests that it results both from the assembly of photosynthetic Chl proteins and from the reorganization of the etioplast membrane system. From these results it is concluded that continuous recordings of Chl(ide) fluorescence yield after a short light pulse represent a useful tool to monitor the kinetics of pigment–protein organization and primary thylakoid assembly triggered by Pchlide photoreduction.     

ENERGY TRANSFER FROM PHYCOBILINS TO CHLOROPHYLL a IN HEAT-STRESSED CELLS OF Anacystis nidulans: CHARACTERIZATION OF THE LOW TEMPERATURE 683 nm FLUORESCENCE EMISSION BAND*

Abstract— Heat-induced changes of the characteristics of fluorescence spectra of Anacystis nidulans cells were studied after 39°C-grown cells were heated at 55°C. Heat-treatment of the cells induced no changes in the absorption properties or photosystem I-catalyzed cytochrome oxidation, but induced a dramatic change in the fluorescence characteristics of the cells. The low temperature fluorescence emission spectra of heated cells showed a large increase of fluorescence emission at683–685 nm (F683) and at 695 nm, while the bands at 660 nm (allophycocyanin) and at 718 nm (chlorophyll a of photosystem I) were not affected when the cells were excited with light absorbed by phycobilins. When the cells were heated for various periods, a progressive increase of the intensity of F683 occurred with the loss in oxygen evolution capacity. The increase of the F683 band was observed prior to the increase of the F695 band. Quenching of emission spectra by the addition of quinones indicates that the F683 band emanated mainly from a long wavelength form of allophycocyanin. Excitation spectra of heated cells measured at 77 K showed that light absorbed by phycobilins was effective in exciting F685, F695, and F715 emission. A possible energy distribution pathway in Anacystis nidulans is discussed.     

Proton transfer reaction of a new orthohydroxy Schiff base in protic solvents at room temperature

Ground and excited state inter- and intramolecular proton transfer reactions of a new o-hydroxy Schiff base, 7-ethylsalicylidenebenzylamine (ESBA) have been investigated by means of absorption, emission and nanosecond spectroscopy in different protic solvents at room temperature and 77 K. The excited state intramolecular proton transfer (ESIPT) is evidenced by a large Stokes shifted emission (approximately 11000 cm(-1)) at a selected excited energy in alcoholic solvents. Spectral characteristics obtained reveal that ESBA exists in more than one structural form in most of the protic solvents, both in the ground and excited states. From the nanosecond measurements and quantum yield of fluorescence we have estimated the decay rate constants, which are mainly represented by nonradiative decay rates. At 77 K the fluorescence spectra are found to be contaminated with phosphorescence spectra in glycerol and ethylene glycol. It is shown that the fluorescence intensity and nature of the species present are dependent upon the excitation energy.     

LUMINESCENCE STUDIES ON FORMYCIN, ITS AGLYCONE, AND THEIR N-METHYL DERIVATIVES: TAUTOMERISM, SITES OF PROTONATION AND PHOTOTAUTOMERISM

Abstract— A detailed study has been made of the luminescence spectra of 3-β-d -ribofuranosyl-7-amino-pyrazolo(4,3-d)pyrimidine (formycin A), 3-propyl-7-aminopyrazolo(4,3-d)pyrimidine (7APP), and their various N-methyl derivatives, at room temperature and in methanol-water glasses at 77 K. Comparisons of the foregoing, together with the observed dependence of the emission spectra of formycin and 7APP on excitation wavelength, demonstrated that these consist of two tautomeric species, N(1)H and N(2)H, both of which emit at 300 and 77 K. The two tautomers may be distinguished by the location of the emission maxima, especially for phosphorescence, and quantum yields for emission. Comparisons of the emission spectra of the protonated forms of 7APP and its N-methyl derivatives showed that the fluorescence of the cations of 7APP and its N,- and N₂-methyl derivatives originates from the forms protonated on N(4). By contrast, the forms protonated on N(6) contribute appreciably to the phosphorescence at 77 K. On the basis of the emission spectra at 77 K, it is concluded that the major tautomeric form of the formycin cation is N(1)H,N(4)H₊, but there is also some contribution by the form N(2)H,N(4)H₊. In acid medium at room temperature, there is photodissociation of a proton from the pyrazole ring of the formycin cation. This leads to formation in the state S! of the tautomeric species N(4)H, which does not exist in the ground state. This conclusion, similar to that previously reported for the analogous isomeric 4-aminopyrazolo(3,4-d)pyrimidines, is derived from a comparison of the fluorescence spectra of the cations of formycin and N₄-methylformycin, which exhibit two bands at 375 and 440 nm, the latter corresponding to the emission of the neutral form of N,i-methylformycin. The proposed mechanism of phototautomerization is supported by a study of solvent and salt effects.     

High light-induced changes of 77 K fluorescence emission of pea thylakoid membranes with altered membrane fluidity

The effect of lipid phase order of isolated thylakoid membranes on fluorescent characteristics of both photosystems during illumination with high light intensity at 22 degrees C and 4 degrees C was investigated. For artificial modification of membrane fluidity two membrane perturbing agents were applied-cholesterol and benzyl alcohol. 77 K fluorescence emission and excitation spectra of control, cholesterol- and benzyl alcohol-treated thylakoid membranes were analysed in order to determine the high light-induced changes of emission bands attributed to different chlorophyll-protein complexes-F 735, emitted by photosystem I-light-harvesting complex I; and F 685 and F 695, emitted by photosystem II-light-harvesting complex II. Analysis of emission bands showed that high light treatment leads to a decrease of the area of band at 695 nm and a concomitant increase of intensity of the band at 735 nm. The involvement of different pigment pools (chlorophyll a and chlorophyll b) in the energy supply of both photosystems before and after photoinhibitory treatment was estimated on the basis of excitation fluorescence spectra. The dependence of the ratios F 735/F 685 and the band areas at 685 and 695 nm on the illumination time was studied at both temperatures. Data presented indicate that cholesterol incorporation stabilized the intersystem structure in respect to light-induced changes of fluorescence emission of PSI and PSII. It was shown that the effect of fluid properties of thylakoid membranes on the 77 K fluorescence characteristics of main pigment protein complexes of pea thyalkoid membranes depends on the temperature during high light treatment.     

Effects of alcohols on fluorescence intensity and color of a discharged-obelin-based biomarker

Photoproteins are responsible for bioluminescence of marine coelenterates; bioluminescent and fluorescent biomarkers based on photoproteins are useful for monitoring of calcium-dependent processes in medical investigations. Here, we present the analysis of intensity and color of light-induced fluorescence of Ca²⁺-discharged photoprotein obelin in the presence of alcohols (ethanol and glycerol). Complex obelin spectra obtained at different concentrations of the alcohols at 350- and 280-nm excitation (corresponding to polypeptide-bound coelenteramide and tryptophan absorption regions) were deconvoluted into Gaussian components; fluorescent intensity and contributions of the components to experimental spectra were analyzed. Five Gaussian components were found in different spectral regions—ultraviolet (tryptophan emission), blue-green (coelenteramide emission), and red (hypothetical indole-coelenteramide exciplex emission). Inhibition coefficients and contributions of the components to experimental fluorescent spectra showed that presence of alcohols increased contributions of ultraviolet, violet, and red components, but decreased contributions of components in the blue-green region. The effects were related to (1) changes of proton transfer efficiency in fluorescent S*₁ state of coelenteramide in the obelin active center and (2) formation of indole-coelenteramide exciplex at 280-nm photoexcitation. The data show that variation of fluorescence color and intensity in the presence of alcohols and dependence of emission spectra on excitation wavelength should be considered while applying the discharged obelin as a fluorescence biomarker.     

Chloroplast biogenesis 87: Evidence of resonance excitation energy transfer between tetrapyrrole intermediates of the chlorophyll biosynthetic pathway and chlorophyll a

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination of chlorophyll (Chl) and thylakoid apoprotein biosynthesis. As a working model for future investigations, we have proposed three Chl-thylakoid apoprotein biosynthesis models, namely, a single-branched Chl biosynthetic pathway (SBP) single-location model, an SBP multilocation model and a multibranched Chl biosynthetic pathway (MBP) sublocation model. Rejection or validation of these models can be probed by determination of resonance excitation energy transfer between various tetrapyrrole intermediates of the Chl biosynthetic pathway and various thylakoid Chl-protein complexes. In this study we describe the detection of resonance energy transfer between protoporphyrin IX (Proto), Mg-Proto and its monomethyl ester (Mp(e)) and divinyl and monovinyl protochlorophyllide a (Pchlide a) and several Chl-protein complexes. Induction of various amounts of tetrapyrrole accumulation in green photoperiodically grown cucumber cotyledons and barley leaves was achieved by dark incubation of excised tissues with delta-aminolevulinic acid (ALA) and various concentrations of 2,2'-dipyridyl for various periods of time. Controls were incubated in distilled water. After plastid isolation, treated and control plastids were diluted in buffered glycerol to the same Chl concentration. Excitation spectra were then recorded at 77 K at emission maxima of about 686, 694 and 738 nm. Resonance excitation energy transfer from Proto, Mp(e) and Pchlide a to Chl-protein complexes emitting at 686, 694 and 738 nm was observed by calculation of treated minus control difference excitation spectra. The occurrence of resonance excitation energy transfer between anabolic tetrapyrroles and Chl-protein complexes appeared as well-defined excitation bands with excitation maxima corresponding to those of Proto, Mp(e) and Pchlide a. Furthermore, it appeared that resonance excitation energy transfer from multiple short-wavelength, medium-wavelength and long-wavelength Proto, Mp(e) and Chlide a sites to various Chl-protein complexes took place. Because resonance excitation transfer from donors to acceptors cannot take place at distances larger than 100 A, it is proposed that the observed resonance excitation energy transfers are not compatible with the SBP single-location Chl biosynthesis thylakoid membrane biogenesis model. The latter assumes that a single-branched Chl biosynthetic pathway located in the center of a 450 x 130 A photosynthetic unit generates all of the Chl needed for the assembly of all Chl-protein complexes.     

Spectral characterization of chlorophyll fluorescence in barley leaves during linear heating. Analysis of high-temperature fluorescence rise around 60 degrees C

The spectral characteristics of chlorophyll fluorescence and absorption during linear heating of barley leaves within the range 25-75 degreesC (fluorescence temperature curve, FTC) were studied. Leaves with various content of light harvesting complexes (green, Chl b-less chlorina f2 and intermittent light grown) revealing different types of FTC were used. Differential absorption, emission and excitation spectra documented four characteristic phases of the FTC. The initial two FTC phases (a rise in the 46-49 degreesC region and a subsequent decrease to about 55 degreesC) mostly reflected changes in the fluorescence quantum yield peaking at about 685 nm. A steep second fluorescence rise at 55-61 degreesC was found to originate from a short-wavelength Chl a spectral form (emission maximum at 675 nm) causing a gradual blue shift of the emission spectra. In this temperature range, a clear correspondence of the blue shift in the emission and absorption spectra was found. We suggest that the second fluorescence rise in FTC reflects a weakening of the Chl a-protein interaction in the thylakoid membrane.