LASER PULSE EXCITATION STUDIES OF THE FLUORESCENCE OF CHLOROPLASTS

Abstract. The fluorescence yield, φ, as a function of single picosecond laser pulse intensity was experimentally studied in spinach chloroplasts and for chlorophyll a in ethyl ether solution. The progressive decrease in φ with increasing incident intensity for in vivo chlorophyll was found to be adequately explained within the context of continuum bimolecular kinetics with a singlet-singlet fusion rate constant of γ=5×^-9cm^-3s^-1 at room temperature. We discuss qualitatively how the fluorescence quantum yield depends on the duration and intensity of the incident pulse. The identity of φ vs l (the number of absorbed quanta) curves at the emission maxima of 685 nm and 735 nm for single picosecond pulse mode of excitation is explained within the context of Butler's tripartite model of the fluorescence of chloroplasts at 77 K. Various models relating γ to the singlet exciton diffusion coefficient and the Förster energy transfer rate are used to infer lower bounds to these physical parameters. Predictions and supporting experimental evidence for the tripartite model are discussed.     

PHOTOCHEMICAL AND SPECTRAL PROPERTIES OF A PARTICULATE MODEL SYSTEM OF CHLOROPHYLL WITH AMPHIPHILES PREPARED FROM HISTAMINE

Abstract— In the photosynthesis model system described, chlorophyll a at an interface photosensitizes the transfer of hydrogen equivalents from a hydrocarbon phase to an aqueous phase. The hydrocarbon phase, to which chlorophyll is adsorbed, consists of polyethylene particles swollen with tetradecane. The particles are also charged positive by co-adsorption of dodecylpyridinium iodide. Furthermore, chlorophyll is ligated with the imidazole function of one of several amphiphiles derived from histamine, which may or may not contain a reducible nitroaromatic group that can serve as primary electron acceptor from photoexcited chlorophyll. The fluorescence quantum yield of chlorophyll on these particles is diminished by self-association of the pigment and by reaction with an oxidizing amphiphile; in the latter case, the quantum yield is correlated with the one-electron redox potential of the amphiphile. Fluorescence-lifetime analysis reveals that most excited singlet states of chlorophyll are quenched rather quickly, and that most of the fluorescence comes from a small fraction of chlorophyll with long lifetime. All preparations sensitize the photoreduction of 5,5′-dithiobis(2-nitrobenzoate) (DTNB) to the water-soluble thiolate by hydrazobenzene. When the amphiphile that ligates chlorophyll is not oxidizing, the quantum yield of photoreduction is unrelated to the fluorescence yield of the particles, but may be related to the degree of self-association of chlorophyll. When the amphiphile that ligates chlorophyll is oxidizing, the kinetics of photoreduction of DTNB require that the electron passes through the primary oxidant to DTNB. Quantum yields for photosensitized reducton of oxidizing amphiphiles in the absence of DTNB have a reversed correlation with redox potential, which can be rationalized in terms of the Marcus theory of electron transfer. All data are most consistently accounted for if the primary photoproduct is an ion pair of chlorophyll and primary oxidant when the latter is available, and a chlorophyll radical ion pair when it is not, both formed by electron transfer from the singlet excited state of chlorophyll.     

THE EFFECT OF COORDINATING LIGANDS ON THE TRIPLET STATE OF CHLOROPHYLL

Under laser excitation at 457.9 and 514.5 nm, a frozen solution of chlorophyll a in n -octane displays fluorescence peak maxima at 2K that may be assigned to two distinct monomeric chlorophyll species. Using zero-field fluorescence-detected magnetic resonance the triplet state properties of the two chlorophyll species have been assigned to the monoligated and biligated chlorophyll monomer in which water serves as the ligand coordinated to the magnesium metal center. These triplet state properties for chlorophyll in solution are then utilized in interpreting triplet state results for in vivo chlorophylls associated with the light harvesting chlorophyll protein complex. It is shown that the triplet state data are consistent with attachment of the chlorophyll molecule to the protein site with a single ligand coordinated to the chlorophyll metal center.     

Density Functional Study of the Structure and Infrared Spectrum of Ethyl Chlorophyllide (A)

A DFT/B3LYP calculation using the 6-31G(d) basis set was performed to obtain the structure, normal vibration frequencies, and absolute IR intensities of ethyl chlorophyllide (A). The calculated structural parameters agree well with the experimental X-ray diffraction data. The role of saturated substituents in stabilization of radical ion states of chlorophyll is discussed. The effective force field of ethyl chlorophyllide (A) was obtained in independent and dependent internal coordinates. The IR vibrational spectrum is modeled. The experimental IR spectrum of chlorophyll (A) is interpreted on the basis of the calculation.     

STUDIES ABOUT SYNTHESIS,CHARACTERIZATION, AND THE EFFECT OF 2-n-PROPYL-3-ETHYL-3-METHYL-1,4,2-BENZOXAZA-PHOSPHORINE-2-OXIDE ON GROWTH PARAMETERS AND CHLOROPHYLL CONTENT OF WHEAT

This article presents the synthesis and characterization of a new phosphorus heterocycle, namely 2-n-propyl-3-ethyl-3-methyl-1,4,2-benzoxaza-phosphorine-2-oxide, by a Mannich-type reaction between n-propyl-dichlorophosphine, methyl ethyl ketone, and o-aminophenol.

Taking into consideration that chlorophyll contents in plants were revealed to have connection with nitrogen and phosphorus concentrations at early growth stage, and because the above-mentioned heterocyclic compound can be considered as a phosphonic analogous of naturally occuring α-aminoacids, it was expected to develop biological activity from it. That is the reason why 2-n-propyl-3-ethyl-3-methyl-1,4,2-benzoxaza-phosphorine-2-oxide was subjected for biological tests on wheat.

The present study was also undertaken to investigate the interrelationship between chlorophyll content, dry matter, and other growth parameters of wheat, after treatment with different concentrations of 2-n-propyl-3-ethyl-3-methyl-1,4,2-benzoxaza-phosphorine-2-oxide.     

REACTIVITY OF CHLOROPHYLL a/b-PROTEINS AND MICELLAR TRITON X-100 COMPLEXES OF CHLOROPHYLLS a OR b WITH BOROHYDRIDE

The reaction of several plant chlorophyll-protein complexes with NaBH₄ has been studied by absorption spectroscopy. In all the complexes studied, chlorophyll b is more reactive than Chi a, due to preferential reaction of its formyl substituent at C-7. The complexes also show large variations in reactivity towards NaBH₄ and the order of reactivity is: LHCI > PSII complex > LHCII > PSI > P700 (investigated as a component of PSI). Differential pools of the same type of chlorophyll have been observed in several complexes.
Parallel work was undertaken on the reactivity of micellar complexes of chlorophyll a and of chlorophyll b with NaBH₄ to study the effect of aggregation state on this reactivity. In these complexes, both chlorophyll a and b show large variations in reactivity in the order monomer > oligomer > polymer with chlorophyll b generally being more reactive than chlorophyll a. It is concluded that aggregation decreases the reactivity of chlorophylls towards NaBH₄ in vitro, and may similarly decrease reactivity in naturally-occurring chlorophyll-protein complexes.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.     

CHLOROPHYLL METABOLISM IN ETIOLATED GHERKIN SEEDLINGS I. PHOTOINHIBITION OF CHLOROPHYLL ACCUMULATION

Abstract. Cotyledons of etiolated gherkin seedlings do not turn green upon transfer to high intensity red light (about 25 W/m²). A pre-irradiation with high intensity red light has an after-effect as chlorophyll accumulation during a subsequent exposure to white light (20 W/m²) is inhibited.
The capacity of protochlorophyll regeneration during a dark period depends on the length of a previous light period but is hardly affected by the light intensity. At high intensity light the rate of protochlorophyll regeneration, which also depends on the length of the foregoing irradiation, is lower than that at low intensity light only during the first 1.5h of the light period. It is concluded that high intensity red light inhibits chlorophyll accumulation mainly by photo-bleaching of chlorophyll. The after-effect is the result of a photooxidation which may lead to photo-bleaching of newly formed chlorophyll in relatively low intensity light.
Photoinhibition of chlorophyll accumulation is accompanied by a disturbed development of etioplasts into chloroplasts.     

Development of an efficient method for the preparative isolation and purification of chlorophyll a from a marine dinoflagellate Amphidinium carterae by high-speed counter-current chromatography coupled with reversed-phase high-performance liquid chromatography

In our research into chlorophylls of marine dinoflagellates, chlorophyll a was separated rapidly from the hexane extract of Amphidinium carterae in three steps. The first step was silica gel column chromatography, where elution was performed with 0–50% ethyl acetate in n-hexane. The second was high-speed counter-current chromatography using a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (5:5:5:1, v/v), and the third step was preparative reversed-phase high-performance liquid chromatography using a solvent system of acetone–water (89:11, v/v). HPLC analysis showed that the purity of chlorophyll a from the second step was over 83%, and after the third it was over 99%. Thirty milligrams of chlorophyll a was isolated from a crude sample of 250 mg of chlorophylls, and its structure was identified by analyzing its MS, ¹H NMR and ¹³C NMR spectra.     

LIQUID-CRYSTALLINE PHASE TRANSITION OF MONOMOLECULAR LAYERS OF CHLOROPHYLL a*

Monomolecular layers of chlorophyll a at the air-water interface were investigated by absorption spectroscopy and simultaneous thermodynamic measurements. Whereas at temperatures near 20°C at all pressures, only a liquid phase exists, at a temperature of 4°C, a liquid-crystalline phase transition is observed at a surface pressure of 5 dynes/cm. Pressure-induced changes in the chlorophyll arrangement become evident from a drastic change in the absorption spectra, accompanying the phase transition. The crystalline phase exhibits an extremely narrow absorption band (halfwidth below 9 nm) centered at 698 nm, indicative of a coplanar chlorophyll arrangement in a well-ordered environment. It is highly probable that in these model membranes a chlorophyll arrrangement could be established that is equivalent to the one proposed for the reaction center P700.     

ELECTROCHEMISTRY OF EXCITED MOLECULES: PHOTO-ELECTROCHEMICAL REACTIONS OF CHLOROPHYLLS*

Abstract— Semiconductors with a sufficiently large energy gap, in contact with an electrolyte, can be used as electrodes for the study of electrochemical reactions of excited molecules. The behavior of excited chlorophyll molecules at single crystal ZnO-electrodes has been investigated. These molecules inject electrons from excited levels into the conduction band of the electrode, thus giving rise to an anodic photocurrent. The influence of various agents on this electron transfer has been studied. In the presence of suitable electron donors (e.g., hydroquinone, phenylhydrazine) in the electrolyte chlorophyll molecules, absorbing quanta, mediate the pumping of electrons from levels of the reducing agents into the conduction band of the semiconductor-electron acceptor. The electron capture by the semiconductor electrode is irreversible, when an adequate electrochemical gradient is provided in the electrode surface. Some properties of excited chlorophyll at semiconductor electrodes (unidirectional electron transfer, highly efficient charge separation, chlorophyll as electron pump and able to convert electronic excitation into electric energy) show similarity to the behavior of chlorophyll in photosynthetic reaction centers.     

Dot immunobinding assay method with chlorophyll removal for the detection of southern rice black-streaked dwarf virus

Southern rice black-streaked dwarf virus (SRBSDV), a new virus from Fiji, has seriously damaged rice crops in southern China and northern Vietnam in recent years. This virus is difficult to diagnose in the early stages of infection, and is very destructive at the late stage. In the present study, a dot immunobinding assay (DIBA) that has a high sensitivity for diagnosing SRBSDV was developed. Two kinds of treatment for the DIBA were evaluated to determine the most effective one for removing chlorophyll interferences via rice extraction. The first included several reagents to remove chlorophyll, namely, the alkaline reagents like magnesium oxide and alumina oxide, the adsorbent reagents like activated carbon and bentonite, as well as the extraction agent acetone. The second and third treatments, which were used to remove chlorophyll in blot membrane-nitrocellulose and polyvinylidene fluoride (PVDF), included several organic solvents containing methanol, ethanol, acetone, ethyl acetate, and diethyl ether. The results showed that activated carbon and methanol yielded the best contrasting purple color for the infected samples by decreasing the chlorophyll content.     

PHOTOELECTRON TRANSFER BETWEEN A CHARGED DERIVATIVE OF CHLOROPHYLL AND FERRICYANIDE AT THE LIPID BILAYER-WATER INTERFACE

Abstract— Flash illumination of a lipid bilayer containing a positively charge pigment: chlorophyll b cholyl hydrazone and separating two salt solutions, one of which contained ferricyanide, resulted in a photovoltage of ∼20mV, acceptor side negative. The positive charge on the pigment resulted in several novel effects. (1) The photo-emf is twice that of chlorophyll a and five times that of chlorophyll b at a given concentration. A higher surface concentration of the charged derivative is the likely cause of this effect. (2) The pheophytin of chlorophyll b cholyl hydrazone produces about one-half the photo emf of the magnesium derivative whereas pheophytin a or b produced only one-tenth the signal. This may be a reflection of the changed redox potential of the cation chlorophyll b cholyl hydrazone. (3) A voltage drop of 100 mV across the membrane, the acceptor side negatively biased, causes a 3–4-fold increase in the charge recombination rate. Biasing the acceptor side 100 mV positive has no effect. Chlorophyll a or b do not show this field effect. This asymmetric effect is explained as a movement of the more polar chlorophyll dication towards the water interface, leading to more rapid reaction with donor. Thus the kinetics of the charge reversal are a sensitive and specific probe of the polar interfacial region of the lipid bilayer-water interface.     

OXYGEN QUENCHING OF CHLOROPHYLL FLUORESCENCE IN CHLOROPLASTS

Abstract— Three phases of chlorophyll a fluorescence quenching by O₂ are observed in green plants. The effects of various inhibitors on photosynthetic partial processes in chloroplasts were investigated in attempts to (1) localize the O₂-quenching sites and (2) assess possible physiological significance of O₂-quenching. Our results localize the most sensitive (and presumably functionally important) phase to a site between plastoquinone and the photosystem I acceptor, chlorophyll (P₇₀₀), possibly plastocyanin. It is suggested that PC may transfer electrons to oxygen in addition to P₇₀₀.     

ACTION SPECTRA FOR THE APPEARANCE OF DIFFERENCE ABSORPTION BANDS AT 480 AND 520 mμ IN ILLUMINATED CHLORELLA CELLS AND THEIR POSSIBLE SIGNIFICANCE TO A TWO-STEP MECHANISM OF PHOTOSYNTHESIS

Abstract— In the difference absorption spectrum of thin, actively growing* aerobic suspensions of Chlorella pyrenoidosa , both the 480 mμ (negative) and the 520 mμ (positive) bands are produced by light absorbed in chlorophyll b and chlorophyll a; the ratio of absorption changes caused by equal number of incident quanta of 650 mμ light and those of 680 mμ light, is about 1.2. Both effects are partially inhibited by DCMU. Upon replacing air with argon, the effects are increased several fold and become relatively insensitive to DCMU. The increase is stronger in the absorption region of chlorophyll a , than in that of chlorophyll b ; the ratio of the absorption changes, caused by equal numbers of 650 mμ and 680 mμ quanta decreases to about 0.8, for both effects. Variable (as regards the exact ratios of absorption changes), but parallel results for 480 and 520 mμ bands were obtained with cultures having low quantum yield of photosynthesis. This parallelism in the behavior of the 480 mμ and the 520 mμ band suggests that at least part of these two bands have a common origin. However, many observations suggest that both difference bands may have a multiple origin; as a working hypothesis, this origin is discussed in terms of three reactions: Reaction A—Photoreduction of chlorophyll a in system II; Reaction B—Photooxidation of chlorophyll b in system II; and Reaction C—Photooxidation (perhaps of a carotenoid) in system I.     

EFFECT OF CHLORIDE ON CHLOROPHYLL PHOTOCHEMISTRY IN SOLUTION: ENHANCEMENT OF CATION RADICAL AND SEMIQUINONE YIELDS

Abstract—Illumination with red light at low temperatures of a degassed ethanol solution of chlorophyll and ferric chloride reversibly produces an EPR signal due to the chlorophyll cation radical (Chl+). The magnitude of this signal is about ten times larger than is obtained with chlorophyll alone. When benzoquinone is also present, an EPR signal due to Chl+ and the neutral semiquinone radical (QH.) is photoproduced. The semiquinone signal is about five times larger than in the absence of ferric chloride. Both of these systems show first order radical decay when the light is turned off, suggesting that radical complexes are being formed. These results indicate that iron is capable of serving as an electron transfer bridge between chlorophyll and quinone.     

A FLASH PHOTOLYSIS STUDY OF INTERMEDIATES IN PHOTOCHEMICAL REACTIONS OF CHLOROPHYLL

Abstract— The photochemical reactions of chlorophyll intermediates in vitro have been studied by the flash photolysis method. The flash excitation of pigment solutions has been shown to involve the population of a chlorophyll triplet state where the oxidation-reduction processes occur. The mechanism and kinetics of pigment triplet decay have been investigated from 20°to — 50°C and the ability of chlorophyll molecules to carry out triplet-triplet energy transfer has been established. The latter phenomenon has been used to show up the role of chlorophyll triplets in the reversible photooxidation reaction with P -quinone. There have been studied initial products of pigment photoreduction with ascorbic acid and phenylhydrazine. Experimental data of the mechanism of the initial oxidation and reduction in chlorophyll photosensitized reactions have been analysed. There have been also obtained the differential spectra of chlorophyll triplets and radicals. A calculation has been made of rate constants for a few elementary reactions.     

Ab initio calculations on large molecules using molecular fragments. Preliminary investigation of ethyl chlorophyllide a and related molecules

Ab initio SCF calculations using the molecular fragment method are reported for four molecules related to chlorophyll a, i.e., free-base porphine, magnesium porphine, magnesium chlorin, and ethyl chlorophyllide a. Molecular orbital structure, the “four-orbital model”, reactivity sites, and Mg···N interactions are discussed.     

沙枣花净油化学成份的研究

用GC-MS-DS技术对沙枣花净油的化学成份进行了研究。其主要成份为反式肉桂酸乙酯。其它成份有:3-甲基己醛、苯甲醛、壬醛、苯甲酸乙酯、癸醛、苯乙酸乙酯、7-甲基十三烯-6、顺式肉桂酸乙酯、肉桂酸甲酯、肉桂酸异丙酯、正十七烷、苯乙烯基异丙基酮、6,10,14-三甲基十五酮-2、棕榈酸乙酯、棕榈酸、正二十一烷、油酸乙酯、硬酯酸乙酯、正二十八烷和花生油酸乙酯。     

THE EFFECT OF LEVULINIC ACID ON THE LIGHT INDUCED DEVELOPMENT OF PHOTOSYSTEM I AND II ACTIVITIES IN GREENING MAIZE LEAVES*

Photosystem I and Photosystem II activities were measured in chloroplasts isolated after 0–20 h illumination from etiolated maize leaves in which chlorophyll synthesis was specifically inhibited by levulinic acid. In control leaves not treated with levulinic acid, Photosystem I activity/chlorophyll developed rapidly during the first 2h in light, then fell off, and reached a constant level after 6h of illumination. In levulinic acid treated leaves, in which chlorophyll accumulation was inhibited up to 60%, a similar initial rise in Photosystem I activity was observed. However, the decrease in activity was much slower and continued for at least 20 h. The development of Photosystem I activity calculated on a leaf fresh weight basis was similar for control leaves or leaves treated with levulinic acid. This indicates that development of Photosystem I activity may not be related to chlorophyll accumulation during greening. Photosystem II activity/chlorophyll in leaves treated with or without levulinic acid increased similarly during the first 6h and then remained constant. Activity of Photosystem II per leaf fresh weight increased linearly, after the first h, for 20 h in the control leaves; in levulinic acid treated leaves this development was reduced by about 60%. Thus, development of Photosystem II activity can be related to chlorophyll accumulation. SDS gel electrophoresis of plastid membranes from control leaves illuminated for 12 h showed the presence of chlorophyll-protein complex I as well as Chl-protein 11; in the case of levulinic acid treated leaves only Chl-protein complex I was detectable, while Chl-protein complex II was markedly reduced.