Early reactions of light-induced protochlorophyllide and chlorophyllide transformations analyzed in vivo at room temperature with a diode array spectrofluorometer

The steps of protochlorophyllide (Pchlide) photoreduction and subsequent chlorophyllide (Chlide) transformations which occur in the seconds to minutes time-scale were studied using a diode array spectrofluorometer in dark-grown barley leaves. The intensity of the excitation light was varied between 3 and 2,500 micromol m(-2) s(-1) and a series of fluorescence spectra were recorded at room temperature in the seconds and minutes time scales. In certain experiments, 77-K emission spectra were measured with the same equipment. The high quality of the spectra allowed us to run spectral resolution studies which proved the occurrence, at room temperature, of multiple Pchlide and Chlide forms found previously in 77-K spectra. The comparison of the 77-K and room-temperature spectra showed that the fluorescence yields of the nonphotoactive 633-nm Pchlide form and of the Chlide product emitting at 678 nm were temperature independent. The fluorescence intensity of aggregated NADPH-pigment-POR complexes (photoactive 656-nm Pchlide and 693-nm Chlide forms) were strongly increased at 77 K, while that of the NADP(+)-Chlide-POR (684-686-nm Chlide form) was much less affected by temperature. Information was obtained also about the dynamics of the transformation of pigment forms in the light at different photon densities. At low light intensities, the phototransformation of the 642-644-nm Pchlide form was faster than that of the 654-656-nm form. The relative amplitudes of Gaussian components related to different Chlide forms found after exposure to a constant amount of photons strongly depended on the light intensity used. Strong quenching of all Chlide components occurred upon prolonged exposure to high intensity light. These effects are discussed by considering the interconversion processes between different forms of the pigment-protein complexes, their relative fluorescence yields and energy migration processes.     

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.     

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.     

The role of carotenoids in the photoadaptation of the brown-colored sulfur bacterium Chlorobium phaeobacteroides

The brown-colored sulfur bacterium Chlorobium (Cb.) phaeobacteroides 1549 (new name, Chlorobaculum limnaeum 1549) contains many kinds of carotenoids as well as bacteriochlorophyll (BChl) e. These carotenoids were identified with C18-high-performance liquid chromatography, absorption, mass and proton nuclear magnetic resonance spectroscopies and were divided into two groups: the first is carotenoid with one or two phi-end groups such as isorenieratene and beta-isorenieratene and the second is carotenoid with one or two beta-end groups such as p-zeacarotene, beta-carotene and 7,8-dihydro-beta-carotene. The latter 7,8-dihydro-beta-carotene was found to be a novel carotenoid in nature. OH-gamma-Carotene glucoside laurate and OH-chlorobactene glucoside laurate were also found as minor components. The distribution of BChl e homologs in Cb. phaeobacteroides cultivated under various light intensities did not change, but the carotenoid to BChl e ratio changed markedly: carotenoid with the phi-end group maintained the same ratio to BChl e, whereas that with the beta-end group increased with increasing light intensity. The cells cultured under low-light intensity contained more phi-end carotenoids than beta-end. In Cb. phaeobacteroides the wavelength of the Qy band of BChl e aggregates did not change. We suggested that Cb. phaeobacteroides photoadapts to light intensity by changing the carotenoid composition.     

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.     

THE FUNCTION OF CAROTENOIDS IN A PHOTOCHROMOGENIC BACTERIUM*

Abstract— The role of carotenoids in protecting a photochromogenic Mycobacterium has been investigated. Bacteria previously induced to synthesize carotenoids by low intensity white light are not killed by high intensity white light while uninduced (carotenoid-free bacteria) are. If high intensity irradiation with white light is carried out at O°, carotenoids still protect these organisms although at a reduced level. This finding is taken to mean that carotenoids can protect these organisnis from photodynamic death by shading photosensitizing pigments.     

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.     

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%.     

Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution

A study of the effect of light intensity and wavelengths on photodegradation reactions of riboflavin (RF) solutions in the presence of phosphate buffer using three UV and visible radiation sources has been made. The rates and magnitude of the two major photodegradation reactions of riboflavin in phosphate buffer (i.e., photoaddition and photoreduction) depend on light intensity as well as the wavelengths of irradiation. Photoaddition is facilitated by UV radiation and yields cyclodehydroriboflavin (CDRF) whereas photoreduction results from normal photolysis yielding lumichrome (LC) and lumiflavin (LF). The ratios of the photoproducts of the two reactions at 2.0 M phosphate concentration, CDRF/RF (0.09-0.22) and CDRF/LC (0.54-1.75), vary with the radiation source and are higher with UV radiation than those of the visible radiation. On the contrary, the ratios of LF/LC (0.15-0.25) increase on changing the radiation source from UV to visible. The rate is much faster with UV radiation causing 25% degradation of a 10(-5) M riboflavin solution in 7.5 min compared to that of visible radiations in 150-330 min.     

Selective photobleaching of chlorophylls and carotenoids in photosystem I particles under high-light treatment

Photosystem I particles (PSI-200) isolated from spinach leaves were studied by means of absorbance, 77K fluorescence and resonance Raman (RR) spectroscopy. The aim was to obtain better insight into the changes of the pigment spectral properties in those particles during prolonged exposure to high-light intensities and to reveal the involvement of these pigments in the photoprotection of the PSI. During prolonged exposure to high-light intensities of spinach PSI particles, a loss of a significant amount of photosynthetic pigments was observed. It was shown that various pigments exhibited different susceptibility to photodamage. In addition to bleaching of chlorophyll a (Chl a), bleaching of carotenoids was also clearly observed. RR technique allowed us to recognize the type and conformation of photobleached carotenoid molecules. Raman data revealed a nearly full photobleaching of the long-wavelength lutein molecules. The observed similar bleaching rate of the lutein molecules and the most-red shifted long-wavelength Chl a, located in the antenna membrane protein Lhca4, suggested that these molecules are located closely. Our results showed that the photobleached antenna pigments and especially luteins and the most long-wavelength absorbing chlorophylls are involved in photoprotection of PSI core complex.     

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.     

THE EFFECT OF NORFLURAZON ON PHOTOTROPIC REACTION IN Phaseolus aureus Roxb. SEEDLINGS

Abstract. The phototropic response of norflurazon-treated mung bean seedlings has been studied to evaluate the possible role of carotenoids, carotenoid-derived growth substances, or other factors in the perception/reaction system. Phototropism was slowed significantly in plants grown in white and blue light in the presence of norflurazon. This effect was evident in norflurazon-bleached seedlings, as well as in those whose pigment system was not affected, due to a shorter period of herbicide action. The possible modes of norflurazon action are discussed.     

STUDIES ON SOME INTERMEDIATES AND PRODUCTS OF THE PHOTOREDUCTION OF 9,10-ANTHRAQUINONE *,†

Abstract— The mechanism of the photoreduction of 9,10-anthraquinone (AQ) in alcohol and hexane has been studied by flash photolysis. The fluorescence spectrum of the photoproduct, 9,10-dihydroxy anthracene shows a large shift between hexane and ethanol. The quantum yields of photoreduction for AQ are solvent-dependent, the reaction between the solvent radical and AQ determining the quantum yield.
The absorption spectrum of the 9,10-anthrasemiquinone (AQH.) has a long-wavelength absorption band with peaks at 631 and 678 nm. The second-order decay constants for AQH. were estimated to be 1.3 × 10⁹, 6.7 × 10⁸ and 2.0 × 10⁸ M ^-1 sec^-1 in ethanol, 2-propanol and ethylene glycol, respectively.
A long-wavelength absorption band was observed for 9,10-anthrasemiquinone radical anion, having peaks at 776 and 860 nm; epsi;_max= 1900 at 776 nm. This spectrum is compared with the spectra of 9,10-dihydroxy anthracene mono- and di-anions. The 9,10-anthrasemiquinone radical anion was found to photoreduce quantitatively to 9,10-dihydroxy anthracene mono-anion with a quantum yield of 0.1.     

EFFECT OF PHYTOCHROME ON THE BIOSYNTHESIS OF ACYCLIC AND CYCLIC CAROTENOIDS IN HIGHER PLANTS

Radish plants were grown in the presence of three different herbicides that interfere with the formation of the normal range of cyclic carotenoids, leading to an accumulation of acyclic biosynthetic intermediates, mainly phytoene (SAN 6706 and amitrole) and zeta-carotene (3852). Plants were then irradiated by four different light programs in order to gain more insight into the first steps of carotenoid biosynthesis and their control by light and phytochrome. In all cases, herbicide-treated and control, carotenoid biosynthesis was greatly enhanced by red light consistent with an effect of phytochrome on the early steps of the pathway. However, similar enhancement was also obtained after treatment with far-red light. Indeed with SAN 6706-treated plants synthesis of phytoene was stimulated to a much greater extent by far-red light given alone, than by red light. The involvement of phytochrome in the regulation of carotenoid biosynthesis appears not to be as simple as previously supposed.     

Excited-State Energy Level Does Not Determine the Differential Effect of Violaxanthin and Zeaxanthin on Chlorophyll Fluorescence Quenching in the Isolated Light-Harvesting Complex of Photosystem II

Abstract— The mechanism of action of xanthophyll cycle carotenoids in controlling the quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II (LHCIIb) has been investigated. Auroxanthin, a diepoxy carotenoid with 7 conjugated carbon double bonds, violaxanthin (9 conjugated double bonds) and zeaxanthin (11 conjugated double bonds) have been compared with regard to their effects in vitro on fluorescence quenching and LHCIIb oligomerization. It was found that auroxanthin stimulated fluorescence quenching, similar to the effect of zeaxanthin and in contrast to the inhibition caused by violaxanthin. Auroxanthin caused an increase in the oligomerization of LHCIIb and an increase in relative emission of long-wavelength fluorescence at 77 K. It is concluded that auroxanthin can mimic the effect of zeaxanthin on LHCII, strongly suggesting that the xanthophyll cycle carotenoids control quenching in vitro by an indirect structural effect and not by direct quenching of chlorophyll excited states.     

REDOX DYES AS ARTIFICIAL PHOTORECEPTORS IN LIGHT-DEPENDENT CAROTENOID SYNTHESIS*

Abstract— The photodynamically active dyes methylene blue, toluidine blue, and neutral red act as artificial photoreceptors in light-dependent carotenoid synthesis in Fusarium aquaeductuum. Normally, carotenoid production is only induced by light of wavelengths shorter than 520 nm, but when mycelia are incubated with neutral red, methylene blue, and toluidine blue, red light is also effective in inducing carotenogenesis. Experiments with methylene blue and red light showed that pigments induced under these conditions are qualitatively the same as those induced with white light, and also that, in accord with the results found for photoinduction with white light, the amount of pigment synthesized was proportional to the logarithm of illumination time. In addition to their role in photoinduction, the dyes were also shown to interfere with the biosynthesis of carotenoids, whereas addition after irradiation caused an increase in pigment production that involves a quantitative change in the more unsaturated components.     

PROTOCHLOROPHYLL(IDE)630 PHOTOSENSITIZES ACTIVE Ca2+ ACCUMULATION IN MICROSOMAL AND MITOCHONDRIAL FRACTIONS ISOLATED FROM PLANTS

Abstract— White light irradiation of a microsomal fraction from etiolated plants affects their ATP-dependent Ca²⁺ accumulation by inhibiting active uptake and enhancing passive efflux. The succinate-dependent mitochondrial Ca²⁺ accumulation as well is decreased by light. The wavelength dependence of these light effects as well as their low quantum yield suggest non-phototransformable protochloro-phyll(ide) [PChl(ide)] to be the photoreceptor. PChlide has been isolated from corn leaves pretreated with 5-amino levulinic acid. Addition of Pchlide causes photosensitization of an otherwise light insensitive microsomal Ca²⁺ accumulation. The observed light effect may be due to contamination of the mitochondrial as well as the microsomal fractions with PChl(ide)‡ containing particles. Irradiation of the intact tissue leads to the almost complete disappearance of the light effect on the in vitro Ca²⁺ accumulation.     

A SHORT-LIVED INTERMEDIATE IN THE PHOTO-ENZYMATIC REDUCTION OF PROTOCHLOROPHYLL(IDE) INTO CHLOROPHYLL(IDE) AT A PHYSIOLOGICAL TEMPERATURE

The reduction of protochlorophyll(ide) into chlorophyll(ide) has been studied by flash absorption spectroscopy at 21°C, with a time resolution of 0.5 µs. The absorption changes have been recorded in the range 670–720 nm after the first and subsequent flashes given to an extract of etiolated bean leaves. At 695 nm the flash-induced absorption increase has its maximum value immediately after the flash and then partly decays with a half-time of about 7–10 µs. A complementary behaviour is observed at 675 nm where the absorption change is very small at time zero and then increases to a stationary value with a half-time of about 6–9 µs. From measurements at several wavelengths it is concluded that a species with an absorption peak around 695 nm is formed immediately after the flash and is then transformed into a stable species with an absorption peak around 675 nm. Measurements at lower temperatures, down to—50°C, show that the transformation is much slowed down at decreased temperatures. The species absorbing at 695 nm (P₆₉₅) is attributed to an intermediate in the photoreduction of protochlorophyll(ide) P_639,650 into chlorophyll(ide) P₆₇₆. When the protochlorophyll(ide) is photoreduced before the flash illumination, the newly formed chlorophyll(ide) gets to a triplet state, which decays with a half-time of 15 µs at 21°C. This result shows that carotenoid molecules do not exert their protective role at this stage of chlorophyll (Chi) formation.     

High-efficiency energy transfer from carotenoids to a phthalocyanine in an artificial photosynthetic antenna

Two carotenoid pigments have been linked as axial ligands to the central silicon atom of a phthalocyanine derivative, forming molecular triad 1. Laser flash studies on the femtosecond and picosecond time scales show that both the carotenoid S1 and S2 excited states act as donor states in 1, resulting in highly efficient singlet energy transfer from the carotenoids to the phthalocyanine. Triplet energy transfer in the opposite direction was also observed. In polar solvents efficient electron transfer from a carotenoid to the phthalocyanine excited singlet state yields a charge-separated state that recombines to the ground state of 1.     

Electron- and energy-transfer properties of hydrophilic carotenoids

The antioxidant activities-expressed as the electron-donating properties-of five hydrophilic carotenoids (carotenoid surfactants) and three related hydrophobic carotenoids were investigated by flash photolysis. The electron-transfer rates of the carotenoids to the triplet state of the sensitizer 2-nitronaphthalene and the energy transfer rates of triplet 2-nitronaphthalene to the carotenoids were determined. The results demonstrate that the electron-donating effects of the hydrophilic and hydrophobic carotenoids were comparable when evaluated in acetonitrile. In the presence of water, however, electron transfer (i.e., antioxidant efficiency) was enhanced by a factor of four for the hydrophilic carotenoids. The increased hydrophilicity of carotenoids, therefore, could expand their antioxidant properties, thus facilitating their use as aqueous-phase radical scavengers. At the same time, it was shown that supramolecular assembly ("aggregation") of the amphiphilic carotenoids prevented electron transfer, thus deactivating the antioxidant function. Modulation of the biophysical properties of carotenoids through synthetic modification is capable of increasing the biological and medical utility of this natural class of predominantly hydrophobic antioxidant compound.