APPEARANCE AND DEVELOPMENT OF PHOTOSYNTHETIC ACTIVITIES IN WHEAT ETIOPLASTS GREENED UNDER CONTINUOUS OR INTERMITTENT LIGHT—EVIDENCE FOR WATER-SIDE PHOTOSYSTEM II DEFICIENCY AFTER GREENING UNDER INTERMITTENT LIGHT*

Abstract— Etiolated wheat seedlings are greened under continuous or intermittent light. Under continuous light the onset of photosystem II (PS II) activity appears after 4 h of illumination. Under intermittent light (1 ms flashes alternating with 15 min dark periods), PS II activity cannot be detected after 300–400 flashes, although the pigment composition and structural development of these plastids are similar to those observed after 4 h of continuous light. However, the appearance of PS II activity in isolated plastids can be observed in two different ways: (1) in vivo by exposing the seedlings to a short period of continuous light after the intermittent light; or (2) in vitro by addition to the isolated plastids of an electron donor for PS II, such as diphenylcarbazide. It is concluded that the intermittent light induces development of the electron transport chain from PS II to PS I, but that a deficiency occurs on the water-side of PS II.     

DCMU-INDUCED STIMULATION OF PHOTOSYSTEM I ELECTRON TRANSPORT. INVOLVEMENT OF MEMBRANE STRUCTURAL CHANGES

DCMU-induced stimulation of the rate of photosystem I (PS I) electron transport in DCIPH₂→ MV photoreaction occurs through the action of DCMU on the rate-limiting step which contains the site of electron donation of DCIPH₂ (Ramanujam et al. , 1981). The magnitude of stimulation of the rate by 50 μ M DCMU decreased with increasing concentration of chlorophyll (Chl), implying that DCMU is stoichiometrically related to Chl with respect to the stimulation of the PS I rate.
DCMU-induced stimulation was sensitive to the ionic condition of the thylakoids, the effect being reduced at low cation concentration. Cation-induced scattering changes in thylakoid suspension were partially reversed by DCMU, and the percent Chl in the 10 K fraction of the thylakoid decreased upon addition of DCMU, indicating that grana structure is disrupted by DCMU. Hydroquinone-mediated reduction of cytochrome f in thylakoids in the dark was accelerated in the presence of DCMU. The DCMU effect was not observed in isolated PS I particles.
It is concluded that DCMU binds to the thylakoid membranes and brings about structural changes leading to unstacking of the thylakoids accompanied by an altered interaction of the electron transfer chain components with the added electron donor. This binding of DCMU must have an affinity lower than the well-known binding of DCMU to photosystem II (PS II), because the concentration required is markedly higher.     

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.     

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.     

OXYGEN MEDIATED PHOTOSYSTEM I ACTIVITY IN THYLAKOID MEMBRANES MONITORED BY A PHOTOELECTROCHEMICAL CELL

Abstract— A photoelectrochemical cell has been used to monitor the effects of three enzymes on the photocurrent produced by isolated spinach thylakoids. The enzymes were glucose oxidase, superoxide dismutase and catalase. It is shown that all three inhibit the photocurrent to varying degrees. The results demonstrate that electron transport to the working electrode is mediated by oxygen. Further, the activity monitored originated from photosystem I with oxygen as the acceptor and photosystem II/plastoquinone as the donor. Thus, the photoelectrochemical cell constitutes a potential new approach for the monitoring of pseudocyclic electron transport.     

SINGLET OXYGEN IS NOT PRODUCED IN PHOTOSYSTEM I UNDER PHOTOINHIBITORY CONDITIONS

Abstract— Excess illumination of photosynthetic systems brings about the complex functional and structural damage known as photoinhibition. According to the generally accepted and experimentally confirmed model, photoinhibition involves singlet oxygen production and subsequent oxidative damage in the photosystem II reaction center. However, it was recently suggested that singlet oxygen is not necessarily produced in photosystem II itself but rather in the non-heme iron-containing Fe-S centers of photosystem I (Chung, S.K. & J. Jung, Photochem. Photobiol. 61, 383–389, 1995). Contrary to this suggestion, our electron paramagnetic resonance spectroscopy experiments with the singlet oxygen trap 2,2,6,6-tetramethylpiperidine demonstrate that under photoinhibitory conditions, singlet oxygen is present in thylakoids and photosystem II core complex preparations but is not produced in photosystem I particles.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

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.     

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.     

Effects of light on the photosynthetic apparatus and a novel type of degradation of the photosystem I peripheral antenna complexes under darkness

A novel type of degradation of photosystem I peripheral antenna complexes has been observed in rice leaves under darkness in the present study. Photosynthesis, chlorophyll content, the chlorophyll a/b ratio, and relative amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase decrease during dark treatment. The levels of photosystem II reaction-center complex and cytochrome f on the basis of units of chlorophyll also decline rapidly under darkness. In contrast, the levels of photosystem I reaction-center complex remain stable under darkness for six days. Low-temperature fluorescence emission spectra ascribed to photosystem I antennae clearly show a blue shift. A similar shift is also observed in the photosystem I complexes resolved with dodecyl maltoside-polyacrylamide gel electrophoresis. Moreover, polypeptide analysis of the thylakoids and photosystem I complexes isolated from the green gels shows that some polypeptides originating from photosystem I peripheral antenna complexes disappear during the dark treatment. A curve-fitting method also displays remarkable changes in the chlorophyll components between the light and dark treatments. It is likely that these results indicate the disconnection/disassembly of the photosystem I antenna as well as the photosystem II complexes induced by dark treatment. Moreover, these findings also imply the existence of different degradation mechanisms for the photosystem I and II complexes.     

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.     

2-[(R-phenyl)amine]-1,4-naphthalendiones as photosystem I electron acceptors: structure-activity relationship of m- and p-PAN compounds with QSAR analysis

Nineteen 2-[(R-phenyl)amine]-1,4-naphthalendione derivatives (PAN) were tested on spinach thylakoids for their activity as electron acceptors. These molecules act as photosystem I electron acceptors in the micromolar range. AC(50) values varied from 5 nM to 24 microM. QSAR analysis revealed a linear correlation of the m-PAN derivative log [1/AC(50)] with the energy difference of the LUMO and HOMO orbitals. The biological activity of p-PAN derivatives correlates linearly with structural parameters. Electron affinity is being the most important. The half wave I potential values (E(1/2)) of PAN compounds (from -213 to -569 mV vs. NHE) match with the mid-point potentials of the A(0) to F(X) niche of PSI electron transport carriers. The logP values of PAN derivatives were 3.35 and 3.88, indicating that they are hydrophobic compounds. Therefore PAN compounds accept electrons at the hydrophobic A(0) to F(X) niche of PSI.     

THE PHOTOSYNTHETIC APPARATUS OF Acetabularia mediterranea GROWN UNDER RED OR BLUE LIGHT. BIOPHYSICAL QUANTIFICATION and CHARACTERIZATION OF PHOTOSYSTEM II and ITS CORE COMPONENTS

Abstract— The photosynthetic activity of white light-grown Acetabularia mediterranea Lamouroux (= A. acetabulum (L.) Silva) decreases under continuous red light to less than 20% within 3 weeks. Subsequent blue light reactivates photosynthesis within a relatively short period of 3 days. In a former publication (Wennicke and Schmid, Plant Physiol. 84 ,1252–1256, 1987) we have shown that the regulated rate limiting step, which is an immediate light driven reaction, is part of photosystem II (PS II). The following biophysical properties of PS II were analyzed in thylakoids isolated from algae grown 3 weeks under either blue or red light with or without subsequent 3 days of blue light illumination: (a) fluorescence induction in the short time domain dominated by Q_A reduction, (b) the slow fluorescence decline reflecting pheophytin photoaccumulation, (c) absorption changes at 320 and 830 nm under repetitive flash excitation as indicator for the turnover of Q_A and P₆₈₀, respectively, (d) oscillation pattern of the oxygen yield by a flash train in dark adapted samples and (e) the binding capacity for atrazine. None of these PS II functions were severely affected, but a minor impairment of20–30% was observed in the thylakoids from algae grown for 3 weeks in red irradiation. The changes do not fully account for the drastic reduction of the electron transport through PS II which was 80% after red light treatment. Therefore, the regulated rate-limiting step appears to not be mainly located in the PS II core complex itself. It seems likely that the regulation process predominantly comprises the antenna system.     

PHOTOSYNTHETIC ACTIVITY AND CHLOROPHYLL ABSORPTION SPECTRA OF FRACTIONS FROM THE ALGA,DUNALIELLA*

Abstract— Dunaliella chloroplasts were fractionated according to C. Arntzen et al, Biochim. Biophys. Acta 256 , 85–107, 1972. The initial French-press treatment and differential centrifugation produced Fraction 1 (Fr 1) enriched in photosystem I activity and a heavier Fraction 2 (Fr 2). When Fr 2 was treated with digitonin followed by either gradient or differential centrifugation, two more fractions were recovered: Fr 1 g with a photosystem 1 activity similar to that of Fr 1, and Fr 2 g with very low photosystem II activity. Photosystem II activity was considerably lower in these Dunaliella chloroplasts and fractions than in spinach particles measured under the same conditions, but the relative activities between the fractions were similar to those for spinach. Fr 2 always had greater photosystem II activity than Fr 1, but the digitonin fractions were low and similar in photosystem II activity. Photosystem II activity was measured as the reduction of 2, 6–dichlorophenol indophenol (DCIP) with H₂O, diphenylcarbazide (DPC) or Mn²⁺ as electron donor. The results indicated that exogenous manganous ion competed with H₂O as an electron donor to photosystem II in broken chloroplasts initially, but after 10–15 s of illumination, the Mn³⁺ formed began to reoxidize DCIP and a cyclic reaction ensued. DPC and Mn²⁺ appeared to react at different sites. Computer-assisted curve analysis of the absorption spectrum of each fraction revealed four major component curves representing the absorbing forms of chlorophyll a at 663, 670, 679 and 684 nm seen in numerous other in vivo chlorophyll spectra (C. S. French et al., Plant Physiol. 49 , 421–429, 1972). However, Fr 2g had approx. 20 percent more of Ca663 and Ca670 and 10% more absorption by chl b than Fr 1 which correlated with the difference in photosystem II activity. On the long wavelength side, Fr 2 g had no Ca694 and almost no photosystem I activity. The results are not sufficient to answer the question of whether the photosystem I particle obtained from the original homogenate is significantly similar to or different from the corresponding fraction obtained from Fr 2 with digitonin.     

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.     

Outward photocurrent component in chloroplasts of Peperomia metallica and its assignment to the 'closed thylakoid' recording configuration

The photoinduced electrical events at energy-conserving chloroplast membranes can be studied in whole plastids using suction electrodes. In chloroplasts of Peperomia metallica the kinetic profile of photocurrent contains a minor outward component that occurs prior to and differs in polarity from the main component. The origin of this outward current was analyzed using single-turnover flashes in combination with prolonged light exposures and differential physicochemical treatments of tip-located (internal) and the exposed parts of a chloroplast. The outward current signal was higher after 10- to 20-s preillumination and gradually reduced in darkness. The relative amplitude of the outward peak current was enhanced when photosystem II (PS II) was excited by flashes given in the presence of far-red background light (lambda = 712 nm). The outward current was small or absent under conditions promoting activity of photosystem I (cyclic electron transport supported by artificial redox mediators in the presence of diuron) and was particularly high in the presence of PS II electron acceptors (e.g., p-phenylenediamine). This indicates the predominant association of the outward current with activity of PS II. The external application of diuron strongly inhibited the inward current, giving rise to a temporal increase in the outward current. On the contrary, when diuron was added into the suction pipette, the outward current was inhibited soon after sealing. The data suggest that the outward current originated in the tip-located portions of the thylakoid membrane that have orientation opposite to the exposed part of 'whole thylakoid'. These tip-located membrane portions are least accessible for inhibitors added into the outer medium and are highly sensitive to inhibitors (diuron), ionophores (gramicidin D), and detergents (Triton X-100) added into the pipette. Differential involvement of two photosystems in generation of the outward current may be caused by uneven structural distribution of photosystems I and II between appressed (granal) and nonappressed (stromal) thylakoids and by different recording configurations for these thylakoids.     

Redox state of the photosynthetic electron transport chain in wild-type and mutant leaves of Arabidopsis thaliana: Impact on photosystem II fluorescence

In addition to the photosynthetic linear electron transport, several alternative electron transport routes exist in thylakoids of higher plants. The plastoquinone (PQ) pool acts as a common electron carrier in these pathways. In the cyclic electron flow around photosystem I (PSI), reduced ferredoxin is used by the ferredoxin-quinone reductase (FQR) to reduce the PQ pool. Chlororespiratory pathway consists in the reduction of the PQ pool by the NAD(P)H dehydrogenase (NDH). These alternative pathways and their role in photosynthesis are still not fully understood. In the present study, the accumulation kinetics of quinone acceptors was measured by fluorescence induction in leaves of Arabidopsis thaliana wild-type and mutants altered in alternative electron pathways after various light- and dark-adaptation conditions. Results show that NDH activity can be probed by fluorescence induction during light-to-dark transition of plants. Also, the activity of FQR pathway did not affect directly the FI kinetics. However, the accumulation kinetics of reduced PQ under actinic light was dependant on the redox state of PSI acceptors prior to illumination.     

Polymer-Modified Single-Walled Carbon Nanotubes Affect Photosystem II Photochemistry,Intersystem Electron Transport Carriers and Photosystem I End Acceptors in Pea Plants

Single-walled carbon nanotubes (SWCNT) have recently been attracting the attention of plant biologists as a prospective tool for modulation of photosynthesis in higher plants. However, the exact mode of action of SWCNT on the photosynthetic electron transport chain remains unknown. In this work, we examined the effect of foliar application of polymer-grafted SWCNT on the donor side of photosystem II, the intersystem electron transfer chain and the acceptor side of photosystem I. Analysis of the induction curves of chlorophyll fluorescence via JIP test and construction of differential curves revealed that SWCNT concentrations up to 100 mg/L did not affect the photosynthetic electron transport chain. SWCNT concentration of 300 mg/L had no effect on the photosystem II donor side but provoked inactivation of photosystem II reaction centres and slowed down the reduction of the plastoquinone pool and the photosystem I end acceptors. Changes in the modulated reflection at 820 nm, too, indicated slower re-reduction of photosystem I reaction centres in SWCNT-treated leaves. We conclude that SWCNT are likely to be able to divert electrons from the photosynthetic electron transport chain at the level of photosystem I end acceptors and plastoquinone pool in vivo. Further research is needed to unequivocally prove if the observed effects are due to specific interaction between SWCNT and the photosynthetic apparatus.     

STRUCTURAL AND FUNCTIONAL INTEGRITY OF THE PHOTOSYSTEM II REACTION CENTER ON SILVER ELECTRODES: FLUORESCENCE AND REDOX PROBES

Two simple and sensitive methods have been developed to assess the structural and functional integrity of isolated photosystem II reaction centers deposited on a roughened Ag electrode. Surface-enhanced resonance Raman scattering (SERRS) spectra useful for ascertaining structural information can be obtained from biological materials with this technique. The first method presented is based on observing differences in the fluorescence emission properties of reaction centers; these depend on the activity of the material. The second is based on the observation of changes in Raman bands that are sensitive to the redox state of cytochrome b₅₅₉ present in the reaction center complex. It is concluded that the conditions used here to obtain SERRS spectra do not affect the structural or functional integrity of the isolated photosystem II reaction center complex. In principle these approaches also could be used with other chromoproteins.     

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.