Effect of modification of light-harvesting complex II on fluorescence properties of thylakoid membranes of Arabidopsis thaliana

The 77 K chlorophyll fluorescence spectra of Arabidopsis thaliana mutants deficient in lipid fatty acid desaturation have been used in order to further explore the influence of the modification of LHC II after mutation and proteolitic treatment on the energy transfer between the chlorophyll-protein complexes, as well as on the structure-function relationship in the supramolecular complex of Photosystem II. The gaussian decomposition and analysis of the fluorescence bands associated with PS II complex show the controversial action of the trypsin in the investigated thylakoid membranes. This reveals that the organization of PS II complexes is different in the wild type and both mutants indicating altered connection between the LHC II and the RC core complexes of PS II in both mutants. The results obtained demonstrate that different amounts of oligomer and monomer forms of LHC II in the mutants (LK3 and JB67), arising from lipid modification, are responsible for different proteolytic action in their thylakoid membranes.     

Changes in the energy distribution between chlorophyll-protein complexes of thylakoid membranes from pea mutants with modified pigment content. I. Changes due to the modified pigment content

The low-temperature (77 K) emission and excitation chlorophyll fluorescence spectra in thylakoid membranes isolated from pea mutants were investigated. The mutants have modified pigment content, structural organization, different surface electric properties and functions [Dobrikova et al., Photosynth. Res. 65 (2000) 165]. The emission spectra of thylakoid membranes were decomposed into bands belonging to the main pigment protein complexes. By an integration of the areas under them, the changes in the energy distribution between the two photosystems as well as within each one of them were estimated. It was shown that the excitation energy flow to the light harvesting, core antenna and RC complexes of photosystem II increases with the total amount of pigments in the mutants, relative to the that to photosystem I complexes. A reduction of the fluorescence ratio between aggregated trimers of LHC II and its trimeric and monomeric forms with the increase of the pigment content (chlorophyll a, chlorophyll b, and lutein) was observed. This implies that the closer packing in the complexes with a higher extent of aggregation regulates the energy distribution to the PS II core antenna and reaction centers complexes. Based on the reduced energy flow to PS II, i.e., the relative increased energy flow to PS I, we hypothesize that aggregation of LHC II switches the energy flow toward LHC I. These results suggest an additive regulatory mechanism, which redistributes the excitation energy between the two photosystems and operates at non-excess light intensities but at reduced pigment content.     

Modulation of photosystem II chlorophyll fluorescence by electrogenic events generated by photosystem I.

In an attempt to uncover electric field interactions between PS I and PS II during their functioning, fluorescence induction curves were measured on hydroxylamine-treated thylakoids of Chenopodium album under conditions ensuring low and high levels of photogenerated membrane potentials. In parallel experiments with Peperomia metallica chloroplasts, the photocurrents were measured with patch-clamp electrodes and served as indicator of electrogenic activity of thylakoid membranes in continuous light. Inhibition of linear electron flow at PS II donor side by hydroxylamine (0.1 mM) eliminated a slow rise of chlorophyll fluorescence to a peak level and suppressed photoelectrogenesis. Activation of PS I-dependent electron transport using cofactors of either cyclic (phenazine methosulfate) or noncyclic electron transport (reduced TMPD or DCPIP in combination with methyl viologen) restored photoelectrogenesis in hydroxylamine-treated chloroplasts and led to reappearance of slow components in the fluorescence induction curve. Exposure of thylakoids to valinomycin reduced the peak fluorescence in the presence of KCl but not in the absence of KCl. Combined application of valinomycin and nigericin in the presence of KCl exerted stronger suppression of fluorescence than valinomycin alone but was ineffective in the absence of KCl. In samples treated with hydroxylamine and PS I cofactors (DCPIP/ascorbate and methyl viologen), preillumination with a single-turnover flash or a multiturnover pulse shifted the induction curves of both membrane potential and chlorophyll fluorescence to shorter times, which confirms the supposed influence of PS I-generated electrical field on PS II fluorescence. A model is presented that describes modulating effect of the membrane potential on chlorophyll fluorescence and roughly simulates the fluorescence induction curves measured at low and high membrane potentials.     

In situ SUSCEPTIBILITIES OF PHOTOSYSTEMS I AND II TO PHOTOSENSITIZED DEACTIVATION via SINGLET OXYGEN MECHANISM

Abstract— A comparative study was carried out on the in situ susceptibilities to photoinactivation of the photosystem I (PS I) and II (PS II) complexes of spinach thylakoids treated with efficient type II sensitizers. While the presence of the exogenous sensitizers caused a substantial increase in the extent of photoinactivation of whole chain electron transport, it did not affect PS I activity of thylakoids in light but exerted an enhanced photoinactivating effect only on PS II. The measurements of the action spectrum for the inhibition of PS II activity of the sensitizer-incorporated thylakoids and that for the generation of singlet oxygen (¹O₂) from them revealed that photosensitized inactivation of PS II is directly related to the photoproduction of ¹O₂ in thylakoid membranes. The results obtained in the present work clearly demonstrate an exceptional sensitivity of PS II to ¹O₂, providing circumstantial evidence that high light-induced damage to PS II may result from photosensitization reactions mediated by ¹O₂, which is not necessarily produced within the PS II complex.     

Relationship between the organization of the PS II super complex and the functions of the photosynthetic apparatus

The chlorophyll fluorescence and the photosynthetic oxygen evolution (flash-induced oxygen yield patterns and oxygen bursts under continuous irradiation) were investigated in the thylakoid membranes with different stoichiometry and organization of the chlorophyll-protein complexes. Data show that the alteration in the organization of the photosystem II (PS II) super complex, i.e. the amount and the organization of the light-harvesting chlorophyll a/b protein complex (LHCII), which strongly modifies the electric properties of the membranes, influences both the energy redistribution between the two photosystems and the oxygen production reaction. The decrease of surface electric parameters (charge density and dipole moments), associated with increased degree of LHCII oligomerization, correlates with the strong reduction of the energy transfer from PS II to PSI. In the studied pea thylakoid membranes (wild types Borec, Auralia and their mutants Coeruleovireus 2/16, Costata2/133, Chlorotica XV/1422) with enhanced degree of oligomerization of LHCII was observed: (i) an increase of the S(0) populations of PS II in darkness; (ii) an increase of the misses; (iii) an alteration of the decay kinetics of the oxygen bursts under continuous irradiation. There is a strict correlation between the degree of LHCII oligomerization in the investigated pea mutants and the ratio of functionally active PS II alpha to PS II beta centers, while in thylakoid membranes without oligomeric structure of LHCII (Chlorina f2 barley mutant) the PS II alpha centers are not registered.     

Changes in the energy distribution in mutant thylakoid membranes of pea with modified pigment content. II. Changes due to magnesium ions concentration

Low-temperature (77K) steady-state chlorophyll fluorescence emission spectra, room temperature fluorescence and light scattering of thylakoid membranes isolated from pea mutants were studied as a function of Mg2+ concentration. The mutants have modified pigment content and altered structural organization of the pigment-protein complexes, distinct surface electric properties and functions. The analysis of the 77K emission spectra revealed that Mg2+-depletion of the medium caused not only an increased energy flow toward photosystem I in all investigated membranes but also changes in the quenching of the fluorescence, most probably by internal conversion. The results indicated that the macroorganization of the photosynthetic apparatus of mutants at supramolecular level (distribution and segregation of two photosystems in thylakoid membranes) and at supermolecular level (stacking of photosystem II supercomplexes) required different Mg ion concentrations. The data confirmed that the segregation of photosystems and the stacking of thylakoid membranes are two distinct phenomena and elucidated some features of their mechanisms. The segregation is initiated by changes in the lateral microorganization of light harvesting complexes II, their migration (repulsion from photosystem I) and subsequent separation of the two photosystems. Most likely 3D aggregation and formation of macrodomains, containing only photosystem II antenna complexes, play a certain precursory role for the increasing degree of the membrane stacking and the energy coupling between the light harvesting complexes II and the core complexes of photosystem II in the frame of photosystem II supercomplexes.     

INFLUENCE OF CHANGES IN THE PHOTON PROTECTIVE ENERGY DISSIPATION ON RED LIGHT-INDUCED DETRAPPING OF THE THERMOLUMINESCENCE Z-BAND

-Thermoluminescence emission at 110 K (Z-band) was markedly diminished when thylakoid membranes were exposed to red light during or after Z-band charging with blue light. Analysis of this phenomenon showed that deactivation of Z-band-emitting chlorophyll species occurred preferentially on the low temperature side of the glow curve, and red light of670–680 nm was most efficient in the deactivation. In order to test our hypothesis that this detrapping is related to local heating effects caused by dissipation of absorbed energy, we measured thermoluminescence glow curves and Z-band emission spectra from spinach leaf discs and thylakoid membranes during induction of nonphotochemical chlorophyll fluorescence quenching. Pretreatment of the plant material was designed to achieve different levels of (1) de-epoxidized xanthophylls in the photosynthetic apparatus and (2) the proton concentration in the thylakoid lumen. In comparison, measurements were performed in aggregated and trimeric light-harvesting pigment-protein complexes of photosystem II. We observed on all three levels of organization that a higher capacity of excitation energy dissipation was accompanied by a stronger red light-induced detrapping of Z-band thermoluminescence.     

Photoinhibition of photosystem I is accelerated by dimethyldithiocarbamate, an inhibitor of superoxide dismutase, during light-chilling of spinach leaves

In vivo photoinhibition of photosystem I (PS I) was investigated at chilling temperature using the leaves of the chilling-resistant spinach plant treated with an inhibitor of superoxide dismutase, diethyldithiocarbamate (DDC). When spinach leaves were treated with DDC during chilling at 4 degrees C for 12 h with a light intensity of 120 micromol m(-2) s(-1), the activity of PS I and the content of iron-sulfur centers declined to about 50% and 25% of the non-DDC-treated controls, respectively. A native green gel analysis of thylakoid membranes isolated from the DDC-treated leaves resolved a novel chlorophyll-protein complex, which was identified as the light-harvesting complex I (LHC I)-deficient PS I complex when examined by 77 K fluorescence spectroscopy and two-dimensional sodium dodecyl sulfate gel electrophoresis. The possible dissociation of LHC I as an early structural change in the PS I complex after DDC-induced photoinhibition of PS I is discussed.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

Cytochrome b6/f complex as an indigenous photodynamic generator of singlet oxygen in thylakoid membranes

Possible association of photodynamic sensitization by cytochrome b6/f complex (cyt b6/f) via singlet oxygen (1O2) mechanism with photoinhibition damage to photosystem II (PS II) was studied using such subthylakoid preparations as photosystem I (PS I) particles, PS II core complex and cyt b6/f from spinach leaves. Upon exposure to bright light, PS II core complex lost photosynthetic electron transport activity to a certain extent, whose-spectral dependence implied that pheophytin a is likely involved in photoinactivation of PS II core complex in itself. The presence of PS I particles exerted virtually no effect on PS II core photoinactivation. However, the inclusion of cyt b6/f in samples resulted in a marked exacerbation of the photoinactivation, particularly in UV-A and blue light. Such effect of cyt b6/f was suppressed by azide and enhanced by the medium deuteration. Photogeneration of 1O2 from cyt b6/f was confirmed by ESR and spectrophotometry, chemically trapping 1O2. Action spectra for both 1O2 photoproduction and PS II core photoinactivation by cyt b6/f bore a close resemblance to each other, seemingly carrying the absorption characteristics of the Rieske Fe-S protein. A complex deficient in the Rieske protein prepared from intact cyt b6/f showed virtually no generation of 1O2 in light, whereas an efficient photoformation of 1O2 was seen in the Rieske protein preparation. The results suggest that cyt b6/f, rather specifically the Rieske center, may play a prominent role in photoinhibition processes through type II photosensitization in thylakoids.     

PARTICIPATION OF THE TWO PHOTOSYSTEMS IN LIGHT DEPENDENT HYDROGEN EVOLUTION IN Scenedesmus obliquus

Abstract— The H₂-photoproduction in the presence of dithionite measured in wild type and mutant cells of Scenedesmus obliquus demonstrates two sequential phases. In mutants showing only PS I activity phase 1 of H₂-photoproduction is visible with its core activity. When PS II is developed during greening, considerable activity is added to the core of phase I and phase II activity appears. Addition of DCMU reduces H₂-photoproduction by about 90%. The residual activity is completely attributed to the core of phase I. It was concluded that the core of phase I is dependent upon PS I only and can use sources different from water as electron donors. Phase II is dependent upon the capacity of PS II, a functioning photosynthetic apparatus and water as electron donor. The results are supported by studies of wavelength dependent activity of the two separate phases of H₂-photoproduction.     

INACTIVATION OF THE ACCEPTOR SIDE AND DEGRADATION OF THE D1 PROTEIN OF PHOTOSYSTEM II BY SINGLET OXYGEN PHOTOGENERATED FROM THE OUTSIDE

Abstract— The possible association of photodynamic sensitization with photoinhibition damage to the photosystem II complex (PS II) has been investigated using isolated intact thylakoids from pea leaves. For this study singlet oxygen (¹O₂), photoproduced by endogenous chromophores that are independent of the function of PS II, was assumed to be the major reactive intermediate involved in the photoinhibition process. When thylakoid samples preincubated with rose bengal were subjected to exposure to relatively weak green light (500–600 nm) under aerobic conditions, PS II was severely damaged. The pattern of the rose bengal-sensitized inhibition of PS II was similar to that of high light-induced damage to PS II: (1) the secondary quinone (Q_B)-dependent electron transfer through PS II is inactivated much faster than the Q_B-independent electron flow, (2) PS II activity is lost prior to degradation of the D1 protein, (3) diuron, an herbicide that binds to the Q_B domain on the D1 protein, prevents D1 degradation, and (4) PS II is damaged to a greater extent by the deuteration of thylakoid suspensions but to a lesser extent by the presence of histidine. Furthermore, it was observed that destroying thylakoid Fe-S centers resulted in a marked reduction of high light-induced PS II damage. These results may suggest that the primary processes of photoinhibition are mediated by ¹O₂ and that Fe-S centers, which are located in some membrane components, but not in PS II, play an important role in photogenerating the activated oxygen immediately responsible for the initiation of photodamage to PS II.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

Ultrafast spectroscopy studies on the mechanism of electron transfer and energy conversion in the isolated pseudo ginseng, water hyacinth and spinach chloroplasts

The spectroscopy characteristics and the fluorescence lifetime for the chloroplasts isolated from the pseudo ginseng, water hyacinth and spinach plant leaves have been studied by absorption spectra, low temperature steady-state fluorescence spectroscopy and single photon counting measurement under the same conditions and by the same methods. The similarity of the absorption spectra for the chloroplasts at room temperature suggests that different plants can efficiently absorb light of the same wavelength. The fluorescence decays in PS II measured at the natural QA state for the chloroplasts have been fitted by a three-exponential kinetic model. The three fluorescence lifetimes are 30, 274 and 805 ps for the pseudo ginseng chloroplast; 138, 521 and 1494 ps for the water hyacinth chloroplast; 197, 465 and 1459 ps for the spinach chloroplast, respectively. The slow lifetime fluorescence component is assigned to a collection of associated light harvesting Chl a/b proteins, the fast lifetime component to the react     

Preparation protocols for high-activity photosystem II membrane particles of green algae and higher plants, pH dependence of oxygen evolution and comparison of the S2-state multiline signal by X-band EPR spectroscopy

Photosystem II (PS II) membrane particles are particularly well suited for various types of spectroscopic investigations on the PS II manganese complex. Here we present: (1) a preparation protocol for PS II membrane particles of higher plants, which yields exceptionally high oxygen-evolution activity due to the use of glycinebetaine as a PS II-stabilizing agent; (2) preparation protocols for highly active PS II membrane particles for the green algae Scenedesmus obliquus and Chlamydomonas reinhardtii; (3) a determination of pH dependence of oxygen evolution for spinach and Scenedesmus; (4) a comparison of the EPR multiline signal observed in the S2-state of green algae and higher plants of PS II membrane particles. A clearly broader type of multiline EPR signal is observed in green algae.     

Determination of cadmium and zinc in fertilizer samples by FAAS after solid-phase extraction with freshly precipitated manganese-diethyldithiocarbamate.

Using freshly precipitated manganese-diethyldithiocarbamate (Mn(DDTC)(2)) as a new reagent, a solid phase extraction method (SPE) has been developed for the extraction of Cd(II) and Zn(II) in aqueous fertilizer samples. A sample solution of 300 mL was taken and 0.10 g of freshly precipitated Mn(DDTC)(2) was added. After adding a phosphate buffer solution, the mixture was stirred at 10 min, filtered with a glass filter and washed with deionized water. The solid product containing Mn(DDTC)(2)-Cd(DDTC)(2)-Zn(DDTC)(2) complexes was dissolved in concentrated nitric acid and its volume was made complete up to 10 mL with deionized water. The metal contents of the solution were measured by an atomic-absorption spectrometer.     

Enrichment of cardiolipin content throughout the purification procedure of photosystem II

Photosystem II is a multisubunit membrane complex which performs the water oxidation process in the higher plants. Core dimers and monomers of photosystem II have been isolated from thylakoid membranes by sucrose density gradient centrifugation. Lipids extracted from different photosystem II-enriched fractions obtained from spinach thylakoids have been analysed by thin layer chromatography. Cardiolipin is enriched throughout the purification of photosystem II complexes; in particular dimers contained two times more cardiolipin than their monomeric counterparts.     

EVIDENCE FOR A HETEROGENOUS ORGANIZATION OF VIOLAXANTHIN IN THYLAKOID MEMBRANES

Abstract Two functionally different species of violaxanthin have been observed in thylakoid membranes, one that can be de-epoxidised to zeaxanthin under light and one not available for light-induced zeaxanthin formation (Siefermann, D. and H. Y. Yamamoto, 1974, Biochim. Biophys. Acta 357 , 144–150). Here the distribution of available and unavailable violaxanthin is examined between membrane subfractions obtained from Triton X-100 solubilized spinach thylakoids by isoelectric focusing: (1) Only 40% of the available violaxanthin is detected in isolated Chl-proteins, while the residual 60% occur in a fraction of'free'pigments; (2) Almost 80% of the unavailable violaxanthin is recovered from the light-harvesting Chl a/b -protein complex (36%) and from photochemically active complexes containing photosystem I (20%) or photosystem II (20%). The results suggest a heterogenous organization of available and unavailable violaxanthin in thylakoid membranes.     

Comparative study of the photochemistry of chloroplast membranes and photosystem II particles

A comparative study of the photoreducing potentials of spinach thylakoid membranes and spinach photosystem II particles has been made. Hexachloroplatinate ions have been used as electron acceptors in a Hill-like assay for oxygen evolution measurements with both thylakoid membranes and photosystem II particles. However, unlike other Hill acceptors, such as ferricyanide, hexachloroplatinate can be fully reduced to metallic platinum that is catalytically active for hydrogen evolution. This is experimentally confirmed in the ability of chloroplast membranes to photoprecipitate platinum and photoproduce molecular hydrogen. Although similar experiments with photosystem II particles resulted in hexachloroplatinate-supported oxygen evolution, hydrogen evolution was not observed. Moreover, photosystem II particles coupled to ferredoxin and hydrogenase resulted in neither hydrogen nor oxygen evolution—a distinct contrast to the results obtained with chloroplast membranes.

     

SURFACE-ENHANCED RESONANCE RAMAN SCATTERING SPECTROSCOPY AS A SURFACE TOPOGRAPHY PROBE IN PLANT PHOTOSYNTHETIC MEMBRANES

Strong resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) signals from carotenoids were detected from thylakoid (stromal-side out) vesicles and inside-out (lumenal-side out) vesicles isolated from spinach chloroplasts. The intensity of the signals from both types of membranes was comparable, indicating that plant carotenoids are exposed on or close to both surfaces or sides of the thylakoid membrane. This is in contrast to previous studies with bacterial photosynthetic membranes (Picorel et al., 1988, J. Biol. Chem. 263 , 4374–4380; and 1990, Biochemistry 29 , 707–712) that show carotenoids selectively located on the cytoplasmic side. In addition_; strong RR and SERRS signals were detected from stacked and unstacked photosystem-II-enriched membrane fragments, demonstrating that carotenoids are also exposed on both surfaces of the appressed region of the thylakoid membrane. Antibodies against the photosystem (PS) II extrinsic proteins blocked SERRS signals from stacked PS II membrane fragments, but only partially affected the SERRS signals from unstacked membranes. The results indicate that these antibodies, which preferentially cover the surface of the original lumenalside of the appressed region, act as spacers between the membrane and SERRS electrode surfaces. The original stromal-side of the appressed region is unaffected. These findings verify the distance sensitivity of the SERRS technique and underscore the above conclusion about the location of carotenoids in the appressed regions. Finally, SERRS signals are sensitive to membrane aging and storage temperature; caution is suggested to those applying SERRS spectroscopy to intact membrane systems.