Origin of chlorophyll fluorescence in plants at 55-75 degrees C

The origin of heat-induced chlorophyll fluorescence rise that appears at about 55-60 degrees C during linear heating of leaves, chloroplasts or thylakoids (especially with a reduced content of grana thylakoids) was studied. This fluorescence rise was earlier attributed to photosystem I (PSI) emission. Our data show that the fluorescence rise originates from chlorophyll a (Chl a) molecules released from chlorophyll-containing protein complexes denaturing at 55-60 degrees C. This conclusion results mainly from Chl a fluorescence lifetime measurements with barley leaves of different Chl a content and absorption and emission spectra measurements with barley leaves preheated to selected temperatures. These data, supported by measurements of liposomes with different Chl a/lipid ratios, suggest that the released Chl a is dissolved in lipids of thylakoid membranes and that with increasing Chl a content in the lipid phase, the released Chl a tends to form low-fluorescing aggregates. This is probably the reason for the suppressed fluorescence rise at 55-60 degrees C and the decreasing fluorescence course at 60-75 degrees C, which are observable during linear heating of plant material with a high Chl a/lipid ratio (e.g. green leaves, grana thylakoids, isolated PSII particles).     

ON THE FORMATION OF PHOTOSYNTHETIC MEMBRANES IN BEAN PLANTS*

Abstract— The formation of lamellar chlorophyll-protein complexes I and II, solubilized by sodium dodecyl sulfate, was studied by hydroxylapatite column chromatography during greening of etiolated Phaseohis vulgaris leaves.
The protein moiety of both complexes preexists in the prolamellar body of etiolated tissue. The complex II to complex I protein ratio is of the order of 0.5. During greening in intermittent illumination the 'proto'-chloroplast is agranal, and contains 'primary' thylakoids and chlorophyll a (Chl a ). At this stage the complex II to complex I protein ratio increases only slightly. Further greening of the plant tissue in continuous illumination results in grana, Chi b (chlorophyll b ) and more Chl a formation. The complex II to complex I protein ratio in unfractionated thylakoids is now of the order of 2.5, while in grana it is of the order of 4.0.
The binding of chlorophyll formed during greening to the protein moiety of the two complexes is found to be selective. The Chi a selectively formed under intermittent illumination is more strongly bound to the complex I protein. The Chi b and Chl a formed in continuous illunination are found bound to both complex I and complex II proteins.
Analysis by hydroxylapatite column chromatography of subchloroplast fractions obtained by different fractionation procedures have shown that these two chlorophyll-protein complexes are most probably derived from the PSI (photosystem I) and PSII (photosystem II) particles of the photosynthetic membrane. These findings suggest that PSI units are assembled ahead of PSII units. Moreover, they indicate that the complex I protein is the main protein component in the prolamellar body membranes, the 'primary' thylakoids. and the stroma lamellae, while in the grana membranes the major protein is the complex II protein. Finally our results show that formation of the photosynthetic membranes is a multi-step process.     

The action of oxygen on chlorophyll fluorescence quenching and absorption spectra in pea thylakoid membranes under the steady-state conditions

The effect of oxygen concentration on both absorption and chlorophyll fluorescence spectra was investigated in isolated pea thylakoids at weak actinic light under the steady-state conditions. Upon the rise of oxygen concentration from anaerobiosis up to 412 microM a gradual absorbance increase around both 437 and 670 nm was observed, suggesting the disaggregation of LHCII and destacking of thylakoids. Simultaneously, an increase in oxygen concentration resulted in a decline in the Chl fluorescence at 680 nm to about 60% of the initial value. The plot of normalized Chl fluorescence quenching, F(-O(2))/F(+O(2)), showed discontinuity above 275 microM O(2), revealing two phases of quenching, at both lower and higher oxygen concentrations. The inhibition of photosystem II by DCMU or atrazine as well as that of cyt b(6)f by myxothiazol attenuated the oxygen-induced quenching events observed above 275 microM O(2), but did not modify the first phase of oxygen action. These data imply that the oxygen mediated Chl fluorescence quenching is partially independent on non-cyclic electron flow. The second phase of oxygen-induced decline in Chl fluorescence is diminished in thylakoids with poisoned PSII and cyt b(6)f activities and treated with rotenone or N-ethylmaleimide to inhibit NAD(P)H-plastoquinone dehydrogenase. The data suggest that under weak light and high oxygen concentration the Chl fluorescence quenching results from interactions between oxygen and PSI, cyt b(6)f and Ndh. On the contrary, inhibition of non-cyclic electron flow by antimycin A or uncoupling of thylakoids by carbonyl cyanide m-chlorophenyl hydrazone did not modify the steady-state oxygen effect on Chl fluorescence quenching. The addition of NADH protected thylakoids against oxygen-induced Chl fluorescence quenching, whereas in the presence of exogenic duroquinone the decrease in Chl fluorescence to one half of the initial level did not result from the oxygen effect, probably due to oxygen action as a weak electron acceptor from PQ pool and an insufficient non-photochemical quencher. The data indicate that mechanism of oxygen-induced Chl fluorescence quenching depends significantly on oxygen concentration and is related to both structural rearrangement of thylakoids and the direct oxygen reduction by photosynthetic complexes.     

Thermally Induced Chemiluminescence of Barley Leaves

Abstract— An unconventional band in the thermoluminescence glow curve of barley leaves at about +50°C was examined. In contrast to bands usually observed around +50°C, this band (designated as CL) is not related to photosynthetic electron transport in photosystem II. The appearance of the CL band (1) requires previous freezing of the sample, (2) is not influenced by light excitation and (3) depends on the presence of oxygen. In pure oxygen the glow curves for both leaves and chloroplast suspension exhibit three maxima at about +40°C, +65°C and +90°C. Based on the emission spectra of the CL band and measurements with etiolated leaves, we suppose that the majority of emission corresponding to the CL band originates from chlorophyll. A lipoxygenase inhibitor, butylated hydroxytoluene, and sodium azide decrease the intensity of the CL band. We propose that the mechanism leading to emission of the CL band involves thermally stimulated production of an active oxygen species that results in lipid peroxidation.     

Changes in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F(O) (basal level of fluorescence yield at transient O) and F(M) (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F(O) to F(M), a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F(M) to F(T) (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII.     

Photoinhibition in vivo and in vitro involves weakly coupled chlorophyll-protein complexes

In the present study the analysis of the relation between the excited state population in the photosystem II (PSII) antenna and photoinactivation has been extended from an in vitro system, isolated thylakoids, to an in vivo system, Chlamydomonas reinhardtii cells. The results indicate that the excited state quenching by an added singlet quencher induces maximal protection against photoinhibition of about 30% of that expected on the basis of the observed light intensity-treatment time reciprocity rule. Similar results, obtained previously with thylakoids, have been interpreted in terms of damaged or incorrectly assembled complexes that play an important role in photoinhibition in the thylakoid membranes (Santabarbara, S., K. Neverov, F. M. Garlaschi, G. Zucchelli and R. C. Jennings [2001] Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids. FEBS Lett. 491, 109-113.). In an attempt to better define this aspect, the photoinhibition action spectra were determined for mutant barley thylakoids, lacking the chlorophyll (Chl) a-b complexes of the outer antenna, and for its wild type. The results indicate that in both systems the action spectra are significantly blueshifted (2-4 nm) and are broader than the PSII absorption in the membranes. These data are interpreted in terms of a heterogeneous population of outer and inner antenna pigment-protein complexes that contain significant levels of uncoupled Chl.     

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.     

ANOTHER ROLE OF CHLOROPHYLL a/b BINDING PROTEINS OF HIGHER PLANTS: THEY MODULATE PROTOLYTIC REACTIONS ASSOCIATED WITH PHOTOSYSTEM II

Chlorophyll alb binding proteins serve as light-harvesting antennae. Additionally they seem to modulate proton pumping by photosystem II, because their covalent modification by N, N-dicyclohexylcarbodiimide is paralleled by a short-circuit of this activity. This side action was further characterized in comparative study with control thylakoids and thylakoids lacking chlorophyll alb binding proteins. The latter were derived from peas grown under intermittent light. They differed from controls in the following: (i) after incubation with A^A^-dicyclohexylcarbodiimide there was no protonic short circuit, (ii) under flashing light the rate of proton consumption at the acceptor side of photosystem II was accelerated and (iii) the periodical pattern of proton release from water oxidation was flattened out. It was obvious that chlorophyll alb binding proteins modulated the kinetics and the stoichiometry of proton release from water oxidation and proton uptake at the quinone binding pocket.     

GENETIC VARIATIONS AND LINOLENIC ACID INDUCED CHANGES IN THE ORIENTATION PATTERN OF CHLOROPHYLL a IN THYLAKOID MEMBRANES

Abstract Orientation pattern of the Q_y absorption and emission dipoles of chlorophyll a were studied in wild type Scenedesmus obliquus and in mutants deficient in chlorophyll b and carotenoids. Fluorescence polarization ratio at –140°C and linear dichroism at 25°C were measured in whole cells and thylakoids aligned in polyacrylamide gel. Unlike normal thylakoids, mutants displayed fluorescence polarization ratios significantly lower than 1.0 and showed a negative LD signal around 672 nm, indicating the tendency of the Q_y dipoles to tilt out from the membrane plane. Such an orientation pattern can also be artificially induced by treating normal thylakoids with linolenic acid.     

Transformation of the Photoacoustic Signal after Treatment of Barley Leaves with Methylviologen or High Temperatures

Abstract— The photoacoustic (PA) signal at the modulation frequency of 35 Hz was studied in MV-treated barley leaves or leaves preheated at different temperatures. Saturating illumination enhanced the magnitude of the PA signal in MV-treated leaves in contrast with the opposite result usually found in untreated intact leaves where saturating illumination abolishes the photobaric component of the PA signal due to oxygen evolution and thus decreases the total PA signal. A linear relationship was found between the changes induced by continuous background light in the negative response of PA signal to saturating light in intact leaves and in the positive response in MV-treated leaves. A linear relationship was also observed in MV-treated leaves between the positive changes in the PA signal and the changes in the rate of electron transport through photosystem II (PSII) calculated from chlorophyll fluorescence data. The conclusion was drawn that only the thermal component contributes to the PA signal measured at low modulated frequency in MV-treated leaves because the enhanced O2 uptake provides a zero net oxygen exchange by superimposing with O2 evolution. The leaves preheated at temperatures above 43°C demonstrated the positive response of the PA signal to saturating light at 35 Hz. In leaves preheated at 41.5°C, the first and second saturating pulses induced the enhancement of PA signal, whereas other pulses decreased the PA signal due to onset of oxygen evolution. The energy storage activity measured in the absence of oxygen evolution in heat-treated leaves is proposed to be associated with cyclic electron transport activities around PSII and PSI.     

Photocontrol and processing of LHCP II apoprotein in Euglena: possible role of Golgi and other cytoplasmic sites.

Like other green photosynthetic eukaryotes, cells of Euglena gracilis var. bacillaris and strain Z contain a light-harvesting chlorophyll a/b complex associated with photosystem II. In Euglena, the formation of the 26.5 kDa principal light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP II) has a number of unusual features. The precursors to LHCP II are large polyproteins containing multiple copies of LHCP II, and photocontrol of their formation is largely translational. Under conditions favoring LHCP II accumulation in the thylakoids, a reaction with anti-LHCP II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHCP II passes through the Golgi on the way to the thylakoids. The compartmentalized osmiophilic structure (COS) also shows an immunoreaction. These observations, and other discussed in this paper, suggest that light permits translation of polyprotein LHCP II precursors on cytoplasmic ribosomes of the rough endoplasmic reticulum (ER) and that these pass through the ER to the Golgi where, presumably, further modifications take place. Since an LHCP II immunoreaction is found in Golgi vesicles, these may transport the nascent LHCP II to the plastid and facilitate its uptake.     

LONG-LIVED AFTERGLOW OF PHOTOSYNTHETIC PIGMENTS IN SOLUTION: PHOTOCHEMILUMINESCENCE

Abstract —Our recent research on photochemiluminescence (PCL) of pigments in solutions is reviewed. PCL was observed in the course of photooxidation by oxygen of chlorophyll a , bacteriochlorophyll, protochlorophyll, their analogs, synthetic dyes and aromatic hydrocarbons. The PCL of chlorophyll was studied in detail. It depends on oxygen concentration, intensity of exciting light, pH, nature of pigments, solvents etc. The thermochemiluminescence was observed after illumination of liquid and solid pigment solutions at low temperature (down to - 170C). The excitation spectra of PCL coincide with the pigment absorption spectra. The PCL emission spectra in most cases differ from those of pigment fluorescence. Electron acceptors, electron donors, radical inhibitors and β-carotene quench PCL. The quenching efficiency of electron acceptors is similar to their action on the chlorophyll triplet state. The quenching effect of radical inhibitors and β-carotene correlates with their activity in reaction with singlet oxygen. The effect of quenchers on the chlorophyll fluorescence, photobleaching and pigment sensitized oxygenation was studied. Analysis of experimental data allowed the assumption that chemiluminescence accompanies the decomposition of labile pigment peroxides. The accumulation of peroxides is probably due to the reaction in the complex of pigment and singlet oxygen, formed as a result of energy transfer from photoexcited (triplet) pigment molecules to oxygen. The terminal chemiluminescence emission proceeds from the singlet excited states of molecules of pigments and products of their oxidation.     

Time-resolved resonance Raman observation of the dimerization of didehydroazepines in solution

Time-resolved resonance Raman (TR3) studies of the photochemistry of phenyl azide, 3-hyroxyphenyl azide, 3-methoxyphenyl azide and 3-nitrophenyl azide in acetonitrile:water solutions is reported. After photolysis of these four aryl azides in room temperature solutions, only one species was observed in the TR3 spectra for each azide, respectively at the probe wavelengths employed in the TR3 experiments. The species observed after photolysis of 3-nitrophenyl azide was assigned to 3,3'-dinitroazobenzene, an azo compound formed from the dimerization reaction of triplet 3-nitrophenylnitrene. In contrast, the species observed after photolysis of phenyl azide, 3-hydroxyphenyl azide and 3-methoxyphenyl azide were tentatively assigned to intermediates formed from the dimerization of didehydroazepines that are produced from the ring expansion reaction of the respective singlet arylnitrene. To our knowledge, this is the first time-resolved vibrational spectroscopic observation of the dimerization reaction of didehydroazepines in solution. In addition, these are the first resonance Raman spectra reported for dimers formed from didehydroazepines. We briefly discuss the structures, properties and chemical reactivity of the dimer species observed in the TR3 spectra and possible implications for the photochemistry of aryl azides.     

PHOTOSENSITIZATION OF MELANIN: AN ELECTRON SPIN RESONANCE STUDY OF SENSITIZED RADICAL PRODUCTION AND OXYGEN CONSUMPTION

Abstract Rose Bengal is shown to photosensitize free-radical production and oxygen consumption in solutions of melanin from autooxidation of 3,4-dihydroxyphenylalanine (DOPA). In anaerobic solutions the sensitizer enhances rates of free-radical production by up to a factor of 20. In aerobic solutions, rates of oxygen consumption can be increased by a factor of several hundred. The reactions appear to involve the triplet state of the sensitizer. The effect of the sensitizer in increasing oxygen consumption is quenched by low concentrations of azide and enhanced by D₂O, suggesting that a singlet oxygen mechanism is involved.     

Involvement of type I and type II mechanisms in the linoleic acid peroxidation photosensitized by tiaprofenic acid

Analysis of the photomixtures resulting from irradiation of aqueous solutions of linoleic acid sensitized by tiaprofenic acid (TPA) or its major photoproduct (DTPA) by HPLC has shown the formation of all the four possible conjugated dienic hydroperoxides. According to laser flash photolysis experiments the rate constants for hydrogen abstraction from linoleic acid by the excited triplet states of TPA and DTPA are 2 x 10(5) and 3.2x 10(5) M(-1) s(-1), respectively. These data, together with the known rate constants for oxygen quenching of triplet (D)TPA and for the reaction of singlet oxygen with linoleic acid, show that the mechanism is mixed type I/type II. Finally, typical radical scavengers such as BHA and singlet oxygen quenchers such as DABCO and sodium azide are efficient quenchers of the triplet excited state of DTPA. This shows the risk of assigning mechanisms based on indirect 'evidences' using 'specific' additives.     

Effect of ultraviolet-B radiation on intact cells of the cyanobacterium Spirulina platensis: characterization of the alterations in the thylakoid membranes

Intact trichomes of Spirulina platensis are exposed to ultraviolet- B (UV-B) radiation (270-320 nm; 1.9 mW m(-2)) for 9 h. This UV-B exposure results in alterations in the pigment-protein complexes and in the fluorescence emission profile of the chlorophyll-protein complexes of the thylakoids as compared with thylakoids isolated from control dark-adapted Spirulina cells. The UV-B exposure causes a significant decrease in photosystem II activity, but no loss in photosystem I activity. Although there is no change in the photosystem I activity in thylakoids from UV-B-exposed cells, the chlorophyll a emission at room temperature and at 77 K indicates alterations associated with photosystem I. Additionally, the results clearly demonstrate that the photosystem II core antennae of chlorophyll proteins CP47 and CP43 are affected by UV-B exposure, as revealed by Western blot analysis. Furthermore, a prominent 94 kDa protein band appears in the sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of UV-B-exposed cell thylakoids, which is absent from the control thylakoids. This 94 kDa protein appears not to be newly induced by UV-B exposure, but could possibly have originated from the UV-B-induced cross-linking of the thylakoid proteins. The exposure of isolated Spirulina thylakoids to the same intensity of UV-B radiation for 1-3 h induces losses in the CP47 and CP43 levels, but does not induce the appearance of the 94 kDa protein band in SDS-PAGE. These results clearly demonstrate that prolonged exposure of Spirulina cells to moderate levels of UV-B affects the chlorophyll a-protein complexes and alters the fluorescence emission spectral profile of the pigment-protein complexes of the thylakoid membranes. Thus, it is clear that chlorophyll a antennae of Spirulina platensis are significantly altered by UV-B radiation.     

Azide Quenching of Singlet Oxygen in Suspensions of Microenvironments of Neutral and Surface Charged Liposomes and Micelles

The azide anion is often used as a physical quencher of singlet oxygen, the important active intermediate in photosensitized oxidation. An observed effect of azide on the rate of a reaction is considered an indication to the involvement of singlet oxygen. In most biological photosensitizations, the light‐absorbing sensitizer is located in a membrane or in an intracellular organelle, whereas azide is water soluble. The quenching it causes relies on a physical encounter with singlet oxygen during the latter's short lifetime. This can happen either if azide penetrates into the membrane's lipid phase or if singlet oxygen is intercepted when diffusing in the aqueous phase. We demonstrate in this article the difference, in liposomes’ suspension, between the effect of azide when using a water‐soluble and membrane‐bound chemical targets of singlet oxygen, whereas this difference does not exist when micelles are used. We explain the difference on the population of sensitizer and target in the liposome vs micelle. We also show the effect that exists on azide quenching of singlet oxygen by electrically charged lipids in liposomes. This is a result of the accumulation or dilution of azide in the debye layer near the membranes’ surface, due to the surface Gouy–Chapman potential.     

Contribution of LHC II complex to the electric properties of thylakoid membranes: an electric light scattering study of Chl b-less barley mutant

Electric light scattering measurements demonstrate a strong decline in the permanent electric dipole moment and electric polarizability of both thylakoid membranes and photosystem II-enriched particles of the Chlorina f2 mutant which has severely reduced levels of light-harvesting chlorophyll a/b-binding proteins compared to the wild type barley chloroplasts. The shift in the electric polarizability relaxation to higher frequencies in thylakoids and photosystem II particles from Chlorina f2 reflects higher mobility of the interfacial charges of the mutant than that of the wild type membranes. The experimental data strongly suggest that the major light-harvesting complex of photosystem II directly contribute to the electric properties of thylakoid membranes.     

Superoxidation of retinoic acid

Atmospheric pressure chemical ionization mass spectroscopy (APCI-MS) was used to examine the light-induced oxidation products of retinoic acid under conditions that favor and preclude its aggregation. We observed that in conditions that favor aggregation, i.e. in aqueous solutions, retinoic acid undergoes superoxidation to yield highly oxidized species. Oxidation is limited, however, in the absence of such communication, i.e. when the polyene is fully solvated. From a comparison of the measured MS with that obtained from chemical oxidation of retinoic acid under conditions that promote radical oxidation and singlet oxygen-mediated oxidation, we conclude that superoxidation is mediated by reactive oxygen species other than singlet oxygen.     

Spectroscopic observations on photofading and fluorescence changes of eosin Y solutions in the presence of sodium azide

Eosin Y solutions exposed to illumination with visible light showed a decreased absorption at 513 nm with increasing irradiation time. In the presence of the singlet oxygen quencher sodium azide, photofading at 513 nm was somewhat lower and a new and small absorption band at 420 nm was observed. Eosin Y fluorescence at 538 nm also decreased with illumination, while a new emission peak appeared at 478 nm. The presence of sodium azide largely increased the photoinduced emission at 478 nm.