Photodynamic effect of iron excess on photosystem II function in pea plants

An earlier mechanistic phase of iron toxicity in photosynthetic cells was interpreted in terms of enhanced photodynamic action by the cytochrome b6/f complex (Cyt b6/f) via singlet oxygen (1O2) on the photosystem II complex (PS II). Iron excess was induced in hydroponically cultured pea (Pisum sativum L.) plants, and its effect on the function of PS II in vivo as well as in vitro was studied under high-irradiance conditions. Iron excess in plants gave rise to a significant increase in Cyt b6/f content of thylakoids. It appeared that the larger the content of Cyt b6/f, the more susceptible PS II was to photoinhibition, and the higher the rate of 1O2 photoproduction in thylakoids was. The action spectrum for degradation of the D1 protein in thylakoids revealed that photosensitization by nonporphyrin chromophore(s) was apparently associated with near UV to blue light-induced deterioration of PS II. The results are pertinent to the concept that photooxidative damage to PS 11, exacerbated by iron accumulation in thylakoid membranes in the form of Cyt b6/f, is involved in the mechanism of iron toxicity in leaf cells.     

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.     

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.     

Photodamage of a Mn(III/IV)-oxo mixed-valence compound and photosystem II: evidence that a high-valent manganese species is responsible for UV-induced photodamage of the oxygen-evolving complex in photosystem II

The Mn cluster in photosystem II (PS II) is believed to play an important role in the UV photoinhibition of green plants, but the mechanism is still not clear at a molecular level. In this work, the photochemical stability of [Mn(III)(O)(2)Mn(IV)(H(2)O)(2)(Terpy)(2)](NO(3))(3) (Terpy=2,2':6',2'-terpyridine), designated as Mn-oxo mixed-valence dimer, a well characterized functional model of the oxygen-evolving complex in PS II, was examined in aqueous solution by exposing the complex to excess light irradiation at six different wavelengths in the range of 250 to 700 nm. The photodamage of the Mn-oxo mixed-valence dimer was confirmed by the decrease of its oxygen-evolution activity measured in the presence of the chemical oxidant oxone. Ultraviolet light irradiation induced a new absorption peak at around 400-440 nm of the Mn-oxo mixed-valence dimer. Visible light did not have the same effect on the Mn-oxo mixed-valence dimer. We speculate that the spectral change may be caused by conversion of the Mn(III)O(2)Mn(IV) dimer into a new structure--Mn(IV)O(2)Mn(IV). In the processes, the appearance of a 514 nm fluorescence peak was observed in the solution and may be linked to the hydration or protonation of Terpy ligand in the Mn-oxo dimer. In comparing the response of the PS II functional model compound and the PS II complex to excess light radiation, our results support the idea that UV photoinhibition is triggered at the Mn(4)Ca center of the oxygen-evolution complex in PS II by forming a modified structure, possibly a Mn(IV) species, and that the reaction of Mn ions is likely the initial step.     

Isolation and characterization of a photosystem I-associated antenna (LHC I) and a photosystem I—core complex from the chlorophyll c-containing alga Pleurochloris meiringensis (Xanthophyceae)

A photosystem (PS) I holocomplex was isolated from Pleurochloris meiringensis Vischer (Xanthophyceae) using sucrose density centrifugation. This complex exhibited a fluorescence emission maximum at 715 nm, which is in accordance with the long wavelength emission of whole cells. The complex was further dissociated into a core complex and a light-harvesting protein (LHC I). The core protein contains mainly Chl a and β-carotene, is 8.25 times enriched in P700 and has its main emission maximum at 715 nm. Therefore, the longest wavelength emission of P. meiringensis is due to the PS I core, which is in contrast to higher plants. The LHC I differs from LHC II with regard to its polypeptide pattern as well as its spectral properties. The arrangement of antennae is discussed in relation to the regulation of energy transfer between the photosystems.     

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.     

CHLOROPHYLL b: AN INTEGRAL COMPONENT OF PHOTOSYSTEM I OF HIGHER PLANT CHLOROPLASTS

Abstract— An undissociated photosystem I complex may be isolated from spinach thylakoids by mild gel electrophoresis (CP1a) or Triton X-100. CP1a has a Chl a / b ratio of 11 and a Chl/P700 ratio of 120. while the Triton X-100 PS I complex (Chl a / b ratio of 5.9) has a larger antenna unit size (Chl/P700 ratio of 180). None of the Chl a / b -proteins of the main light-harvesting complex (apoproteins of 30–27 kD) are present in CP1a, and they account for less than 10% of the total chlorophyll in the Triton X-100 PS I complex. Instead, these PS I complexes have specific, but as yet little characterized, Chi a / b -proteins (apoproteins in the 26–21 kD range). With both PS I complexes, Chi b transfers light excitation to the 735 nm low temperature fluorescence band characteristic of photosystem I. We suggest that Chi b is an integral but minor component of photosystem I.     

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.     

UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities

The reaction center of photosystem II is susceptible to photodamage. In particular the D1 protein located in the photosystem II core has a rapid, light-dependent turnover termed the photosystem II repair cycle that, under illumination, degrades and resynthesizes D1 protein to limit accumulation of photodamaged photosystem II. Most studies concerning the effects of UVB (280-320 nm) on this cycle have been on cyanobacteria or specific phytoplankton species rather than on natural communities of phytoplankton. During a 5-year multidisciplinary project on the effects of UV radiation (200-400 nm) on natural systems, the effects of UVB on the D1 protein of natural phytoplankton communities were assessed. This review provides an overview of photoinhibitory effects of light on cultured and natural phytoplankton, with an emphasis on the interrelation of UVB exposure, D1 protein degradation and the repair of photosystem II through D1 resynthesis. Although the UVB component of the solar spectrum contributes to the primary photoinactivation of photosystem II, we conclude that, in natural communities, inhibition of the rate of the photosystem II repair cycle is a more important influence of UVB on primary productivity. Indeed, exposing tropical and temperate phytoplankton communities to supplemented UVB had more inhibitory effect on D1 synthesis than on the D1 degradation process itself. However, the rate of net D1 damage was faster for the tropical communities, likely because of the effects of high ambient light and water temperature on mechanisms of protein degradation and synthesis.     

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.     

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.     

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.     

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.     

Enzymatic function of cytochrome b559 in photosystem II

Cytochrome b(559) (cyt b(559)) is a heme-bridged protein heterodimer in photosystem II (PSII) of all oxygenic photosynthetic organisms. In spite of the fact that cyt b(559) is strictly required for proper function of PSII, it is not involved in the linear electron transport chain from water to plastoquinone. Instead of that the participation of cyt b(559) in the cyclic electron transport around PSII has been proposed mainly based on the ability of the heme iron to accept and donate an electron form the electron acceptor and to the electron donor side of PSII, respectively. In addition to the involvement of cyt b(559) in the cyclic electron transport around PSII, several lines of evidence have been provided on the enzymatic function of cyt b(559). The ability of oxygenic photosynthetic organisms to oxidize water and reduce plastoquinone is connected to the formation of reactive oxygen species (ROS) and thus required to develop an effective antioxidant defense system against ROS. The review attempts to summarize a recent progress on the role of cyt b(559) as oxygen reductase, superoxide reductase, superoxide oxidase and plastoquinol oxidase. The focus is mainly given on the characterization of redox, redox potential and acid-base properties of the heme iron in the putative enzymatic cycles. The possible oxidase and reductase enzymatic activity of cyt b(559) in protection from photoinhibition is discussed.     

光合放氧复合物结构及其放氧机理的研究

光合水氧化是地球上最重要的生化过程之一.光合放氧生物包括光系统Ⅰ(PSⅠ)和光系统Ⅱ(PSⅡ)两种类型反应中心,光系统Ⅱ反应中心能以水作为电子给体,利用光能氧化水产生质子和氧气.对于水如何被氧化这个难题前人已做了大量的工作,但到目前为止放氧复合物(OEC)的结构及水氧化的机理仍不清楚.本文结合当前研究结果,就光合放氧复合物的结构及光合放氧机理进行了综述,希望能有助于推进这方面的工作.     

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.     

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.     

Spectroscopic studies on electron transfer between plastocyanin and cytochrome b6f complex

This paper reports the results of the research on the interaction between the highly active cytochrome b(6)f complex and plastocyanin, both isolated from the same source - spinachia oleracea plants. An equilibrium constant K between the cytochrome f of the cytochrome b(6)f complex and plastocyanin has been estimated by two independent spectroscopic techniques: steady-state absorption spectroscopy and stopped-flow. The second-order rate constants k2 for forward and backward electron transfer between cytochrome f and plastocyanin have been found between 1.4-2 x 10(7) and 8-10 x 10(6) M(-1)s(-1), respectively, giving the value of an equilibrium constant of about 2+/-0.4 or a difference in redox potential between plastocyanin and cytochrome f of cytochrome b(6)f complex of ca. 17 mV. The value of K=1.7+/-0.3 has been estimated from steady-state experiments in which the initial and final concentrations of participating components after mixing have been estimated via differential spectra analysis or spectra deconvolution. We propose a method of evaluation of the final plastocyanin concentration after the electron transfer reaction between cytochrome bf complex and plastocyanin that overcomes the interference by the strong chlorophyll absorption in the spectral region where oxidised plastocyanin has its low extinction absorption band. The data from both experiments, in the system devoid of quinol being the electron donor to cytochrome b(6), suggest that in case of electron transfer from cytochrome f to plastocyanin electron transfer can either bypass cytochrome f or the Rieske iron-sulfur protein can be reduced prior to its movement to the quinol binding site of cytochrome b(6). The role of the Rieske protein in forward and backward electron transfer reactions is discussed.     

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.