The effect of some antiseptic drugs on the energy transfer in chromatophore photosynthetic membranes of purple non-sulfur bacteria Rhodobacter sphaeroides.

Chromatophores of purple non-sulfur bacteria (PNSB) are invaginations of the cytoplasmic membrane that contain a relatively simple system of light-harvesting protein-pigment complexes, a photosynthetic reaction center (RC), a cytochrome complex, and ATP synthase, which transform light energy into the energy of synthesized ATP. The high content of negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) in PNSB chromatophore membranes makes these structures potential targets that bind cationic antiseptics. We used the methods of stationary and kinetic fluorescence spectroscopy to study the effect of some cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine at concentrations up to 100 µM) on the spectral and kinetic characteristics of the components of the photosynthetic apparatus of Rhodobacter sphaeroides chromatophores. Here we present the experimental data on the reduced efficiency of light energy conversion in the chromatophore membranes isolated from the photosynthetic bacterium Rb. sphaeroides in the presence of cationic antiseptics. The addition of antiseptics did not affect the energy transfer between the light-harvesting LH1 complex and reaction center (RC). However, it significantly reduced the efficiency of the interaction between the LH2 and LH1 complexes. The effect was maximal with 100 µM octenidine. It has been proved that molecules of cationic antiseptics, which apparently bind to the heads of negatively charged cardiolipin molecules located in the rings of light-harvesting pigments on the cytoplasmic surface of the chromatophores, can disturb the optimal conditions for efficient energy migration in chromatophore membranes.

Digitonin-sensitive LHCII enlarges the antenna of Photosystem I in stroma lamellae of Arabidopsis thaliana after far-red and blue-light treatment.

Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and PSII work in series, it is important that the excitation pressure on the two photosystems is balanced. When plants are exposed to illumination that overexcites PSII, a special pool of the major light-harvesting complex LHCII is phosphorylated and moves from PSII to PSI (state 2). If instead PSI is over-excited the LHCII complex is dephosphorylated and moves back to PSII (state 1). Recent findings have suggested that LHCII might also transfer energy to PSI in state 1. In this work we used a combination of biochemistry and (time-resolved) fluorescence spectroscopy to investigate the PSI antenna size in state 1 and state 2 for Arabidopsis thaliana. Our data shows that 0.7 ± 0.1 unphosphorylated LHCII trimers per PSI are present in the stroma lamellae of state-1 plants. Upon transition to state 2 the antenna size of PSI in the stroma membrane increases with phosphorylated LHCIIs to a total of 1.2 ± 0.1 LHCII trimers per PSI. Both phosphorylated and unphosphorylated LHCII function as highly efficient PSI antenna.

ß-Carotene influences the phycobilisome antenna of cyanobacterium Synechocystis sp. PCC 6803.

We investigated the relation between the carotenoid composition and the structure of phycobilisome (PBS) antenna of cyanobacterium Synechocystis sp. PCC 6803. PBS is a large soluble protein complex enhances the light harvesting efficiency of the cells. It is composed of a central allophycocyanin core and radial phycocyanin rods, but it does not contain carotenoids. However, the absence or low level of carotenoids were previously shown to lead the co-existence of unconnected rod units and assembled PBS with shorter peripheral rods. Here we show that the lack of ß-carotene, but not of xanthophylls or the distortion of photosystem structure, evoked unconnected rods. Thus, these essential ß-carotene molecules are not bound by Photosystem I or Photosystem II. Our results do not show correlation between the reactive oxygen species (ROS) and PBS distortion despite the higher singlet oxygen producing capacity and light sensitivity of the mutant cells. Reduced cellular level of those linker proteins attaching the rod units together was also observed, but the direct damage of the linkers by ROS are not supported by our data. Enzymatic PBS proteolysis induced by nitrogen starvation in carotenoid mutant cells revealed a retarded degradation of the unconnected rod units.

Peripheral Light-Harvesting LH2 Complex Can Be Assembled in Cells of Nonsulfur Purple Bacterium Rhodoblastus acidophilus without Carotenoids.

The effect of carotenoids on the assembly of LH2 complex in cells of the purple nonsulfur bacterium Rhodoblastus acidophilus was investigated. For this purpose, the bacterial culture was cultivated with an inhibitor of carotenoid biosynthesis - 71 µM diphenylamine (DPA). The inhibitor decreased the level of biosynthesis of the colored carotenoids in membranes by ~58%. It was found that a large amount of phytoene was accumulated in them. This carotenoid precursor was bound nonspecifically to LH2 complex and did not stabilize its structure. Thermostability testing of the isolated LH2 complex together with analysis of carotenoid composition revealed that the population of this complex was heterogeneous with respect to carotenoid composition. One fraction of the LH2 complex with carotenoid content around 90% remains stable and was not destroyed under heating for 15 min at 50°C. The other fraction of LH2 complex containing on average less than one molecule of carotenoid per complex was destroyed under heating, forming a zone of free pigments (and polypeptides). The data suggest that a certain part of the LH2 complexes is assembled without carotenoids in cells of the nonsulfur bacterium Rbl. acidophilus grown with DPA. These data contradict the fact that the LH2 complex from nonsulfur bacteria cannot be assembled without carotenoids, but on the other hand, they are in good agreement with the results demonstrated in our earlier studies of the sulfur bacteria Allochromatium minutissimum and Ectothiorhodospira haloalkaliphila. Carotenoidless LH2 complex was obtained from these bacteria with the use of DPA (Moskalenko, A. A., and Makhneva, Z. K. (2012) J. Photochem. Photobiol., 108, 1-7; Ashikhmin, A., et al. (2014) Photosynth. Res., 119, 291-303).

Alteration of the structure and function of photosystem I by Pb2+.

The toxic effects of Pb(2+) on photosynthetic electron transport were studied in photosystem I (PSI) submembrane fractions isolated from spinach. Structural and spectroscopic analysis using FTIR, fluorescence and X-ray photoelectron spectroscopy (XPS) showed that Pb(2+) binds with proteins via oxygen and nitrogen atoms with an overall binding constant of KPb-PSI=4.9×10(3) (±0.2) M(-1) and the number of bound Pb(2+) cation was 0.9 per PSI complex. Pb(2+) binding altered the protein conformation indicating a partial protein destabilization. Electron transport and P700 photooxidation/reduction measurements showed that the interaction of Pb(2+) cations with PSI produced a donor side limitation of electron transport presumably due to Pb(2+) binding to or in the vicinity of plastocyanin.

Effect of vulculic acid produced by Nimbya alternantherae on the photosynthetic apparatus of Alternanthera philoxeroides.

The effect of the toxin vulculic acid produced by Nimbya alternantherae, on the photosynthetic apparatus of Alternanthera philoxeroides, was investigated via the photochemical activity and SDS-PAGE of protein on thylakoid membranes, fast chlorophyll a fluorescence transient measurements and the JIP-test. The electron transport rate of photosystem II (PSII), non-cyclic photophosphorylation activity, as well as the activity of chloroplast ATPase and Rubisco reduced significantly after vulculic acid treatment. Vulculic acid affected the O-J-I-P fluorescence induction kinetics, showing an increase of the parameters FV/FO, VK and VJ and a decrease of FO, FM, PIABS, φPo, ψEo, φEo, φRo, δRo and PItotal. In addition, it significantly decreased the amounts of major photosystem I (PSI) and PSII proteins. It is concluded that vulculic acid is a photosynthetic inhibitor with multiple action sites. The main targets are the light harvesting complex (LHC) and the oxygen evolving complex (OEC) on the PSII donor side. Vulculic acid blocks electron transport beyond QA and on the PSI acceptor side by digesting major PSI and PSII proteins.

PMS: photosystem I electron donor or fluorescence quencher.

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.

Lutein can act as a switchable charge transfer quencher in the CP26 light-harvesting complex.

Energy-dependent quenching of excitons in photosystem II of plants, or qE, has been positively correlated with the transient production of carotenoid radical cation species. Zeaxanthin was shown to be the donor species in the CP29 antenna complex. We report transient absorbance analyses of CP24 and CP26 complexes that bind lutein and zeaxanthin in the L1 and L2 domains, respectively. For CP24 complexes, the transient absorbance difference profiles give a reconstructed transient absorbance spectrum with a single peak centered at approximately 980 nm, consistent with zeaxanthin radical cation formation. In contrast, CP26 gives constants for the decay components probed at 940 and 980 nm of 144 and 194 ps, a transient absorbance spectrum that has a main peak at 980 nm, and a substantial shoulder at 940 nm. This suggests the presence of two charge transfer quenching sites in CP26 involving zeaxanthin radical cation and lutein radical cation species. We also show that lutein radical cation formation in CP26 is dependent on binding of zeaxanthin to the L2 domain, implying that zeaxanthin acts as an allosteric effector of charge transfer quenching involving lutein in the L1 domain.

Treatments with acetic acid followed by curing reduce postharvest decay on Citrus fruit.

Citrus fruit are susceptible to many postharvest diseases and disorders, but Penicillium digitatum and Penicillium italicum are the most common and serious pathogens during storage and marketing. The continuous employ in packing houses of synthetic fungicides such as imazalil (IMZ) or thiabendazote for the control of these pathogens is promoting the selection of resistant biotypes. These considerations together with an increased attention for human health and the environment have multiplied the studies on new ecological technologies. In recent years researchers studies focused on alternatives to the chemical control of post-harvest decay, such as the utilization of GRAS compounds as well as physical methods. In the present study is reported the sequential use of acetic acid (AAC) followed by curing. The lemon variety "Verna" and the orange variety "Jaffa", naturally inoculated, were treated with vapours of AAC performed at three different concentration (15, 25 and 50 microL/L) for 15 minutes, after an incubation period of 24 hours at 27 degrees C and 90% relative humidity (RH). After treatments fruits were cured at 36 degrees C for 36 hours with 90% RH and subsequently stored at 8 degrees C and 90% of RH for eight weeks. Both citrus varieties were also treated with IMZ at a concentration of 200 mL/HL. At the end of the experiment decay and weight loss were evaluated. After 8 weeks of storage, in the lemon variety, the lowest percentage of infected wounds was 1.5% for both the fruit treated with IMZ or with AAC at 25 microL/L. Fruit treated with 15 mciroL/L or untreated (control) showed similar results with 13.6% and 16.6% of rotted fruit respectively. Different results were obtained with the orange variety, in this case the synthetic fungicide was the most effective at the end of the storage period, with 18.0% of decay. AAC treatments were not a successful as on lemons, the best result was achieved even in this case with AAC performed at 25 pL/L, but with 39.9% of decay. In both species the weight loss was not affected by the treatments. These results show that a good control of postharvest decay could be achieved, on lemon fruit, by combining the effect of a GRAS compound such as AAC with curing. Conversely the results obtained, by applying this control method to the orange variety were not so promising. Further researches are needed to shed light on the different behaviour between the two species.

Abscisic acid, high-light, and oxidative stress down-regulate a photosynthetic gene via a promoter motif not involved in phytochrome-mediated transcriptional regulation.

In etiolated seedlings, light perceived by phytochrome promotes the expression of light-harvesting chlorophyll a/b protein of photosystem II (Lhcb) genes. However, excess of photosynthetically active radiation can reduce Lhcb expression. Here, we investigate the convergence and divergence of phytochrome, high-light stress and abscisic acid (ABA) signaling, which could connect these processes. Etiolated Arabidopsis thaliana seedlings bearing an Lhcb promoter fused to a reporter were exposed to continuous far-red light to activate phytochrome and not photosynthesis, and treated with ABA. We identified a cis-acting region of the promoter required for down-regulation by ABA. This region contains a CCAC sequence recently found to be necessary for ABI4-binding to an Lhcb promoter. However, we did not find a G-box-binding core motif often associated with the ABI4-binding site in genes promoted by light and repressed by ABI4. Mutations involving this motif also impaired the responses to reduced water potential, the response to high photosynthetic light and the response to methyl viologen but not the response to low temperature or to Norflurazon. We propose a model based on current and previous findings, in which hydrogen peroxide produced in the chloroplasts under high light conditions interacts with the ABA signaling network to regulate Lhcb expression. Since the mutation that affects high-light and methyl viologen responses does not affect phytochrome-mediated responses, the regulation by retrograde and phytochrome signaling can finally be separated at the target promoter level.

Evolution of a bacteriophytochrome from light to redox sensor.

Bacteriophytochromes are red/far-red photoreceptors that bacteria use to mediate sensory responses to their light environment. Here, we show that the photosynthetic bacterium Rhodopseudomonas palustris has two distinct types of bacteriophytochrome-related protein (RpBphP4) depending upon the strain considered. The first type binds the chromophore biliverdin and acts as a light-sensitive kinase, thus behaving as a bona fide bacteriophytochrome. However, in most strains, RpBphP4 does not to bind this chromophore. This loss of light sensing is replaced by a redox-sensing ability coupled to kinase activity. Phylogenetic analysis is consistent with an evolutionary scenario, where a bacteriophytochrome ancestor has adapted from light to redox sensing. Both types of RpBphP4 regulate the synthesis of light harvesting (LH2) complexes according to the light or redox conditions, respectively. They modulate the affinity of a transcription factor binding to the promoter regions of LH2 complex genes by controlling its phosphorylation status. This is the first complete description of a bacteriophytochrome signal transduction pathway involving a two-component system.

A polyamine- and LHCII protease activity-based mechanism regulates the plasticity and adaptation status of the photosynthetic apparatus.

In the present study we aim to dissect the basis of the polyamine mode of action in the structure and function of the photosynthetic apparatus. Although the modulating effects of polyamines in photosynthesis have been reported since long [K. Kotzabasis, A role for chloroplast-associated polyamines? Bot. Acta 109 (1996) 5-7], the underlying mechanisms remained until today largely unknown. The diamine putrescine was employed in this study, by being externally added to Scenedesmus obliquus cultures acclimated to either low or high light conditions. The results revealed the high efficiency by which putrescine can alter the levels of the major photosynthetic complexes in a concerted manner inducing an overall structure and function of the photosynthetic apparatus similar to that under higher light conditions. The revealed mechanism for this phenomenon involves alterations in the level of the polyamines putrescine and spermine which are bound to the photosynthetic complexes, mainly to the LHCII oligomeric and monomeric forms. In vitro studies point out to a direct impact of the polyamines on the autoproteolytic degradation of LHCII. Concomitantly to the reduction of the LHCII size, exogenously supplied putrescine, induces the reaction centers' density and thus the photosynthetic apparatus is adjusted as if it was adapted to higher light conditions. Thus polyamines, through LHCII, play a crucial role in the regulation of the photosynthetic apparatus' photoadaptation. The protective role of polyamines on the photosynthetic apparatus under various environmental stresses is also discussed in correlation to this phenomenon.

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess.

Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10 mM MnSO4. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72 h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96 h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion-tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.

Effects of acid and alkali on the light absorption, energy transfer and protein secondary structures of core antenna subunits CP43 and CP47 of photosystem II.

The effects of acid and alkali treatment on the light absorption, energy transfer and protein secondary structure of the photosystem II core antenna CP43 and CP47 of spinach were investigated by the absorption spectra, fluorescence emission spectra and circular dichroism spectra. It has been found that acid treatment caused the appearance of absorption characteristic of pheophytin a (Pheo a), whereas alkali treatment induced a new absorption peak at 642 nm. The energy transfer between beta-carotene and chlorophyll a (Chl a) in CP43 was easily disturbed by alkali, whereas in CP47 was readily affected by acid. As to the effects on the secondary structure of proteins in CP43 and CP47, effects of acid were far less than those of alkali. Both acid and alkali disturbed the microenvironment of Chl a and interfered exciton interaction between Chl a molecules. It was suggested that acid and alkali affect the light absorption, energy transfer and protein secondary structure of CP43 and CP47 in a different way. H+ can permeate into the internal space of alpha-helix, change Chl a into Pheo a and disturb the microenvironment of pigments without damaging the secondary structure of protein, whereas OH- can induce the protein unfolding at first, then saponify Chl a to chlorophyllide and disturb the microenvironment of pigments.

A novel role of water-soluble chlorophyll proteins in the transitory storage of chorophyllide.

All chlorophyll (Chl)-binding proteins involved in photosynthesis of higher plants are hydrophobic membrane proteins integrated into the thylakoids. However, a different category of Chl-binding proteins, the so-called water-soluble Chl proteins (WSCPs), was found in members of the Brassicaceae, Polygonaceae, Chenopodiaceae, and Amaranthaceae families. WSCPs from different plant species bind Chl a and Chl b in different ratios. Some members of the WSCP family are induced after drought and heat stress as well as leaf detachment. It has been proposed that this group of proteins might have a physiological function in the Chl degradation pathway. We demonstrate here that a protein that shared sequence homology to WSCPs accumulated in etiolated barley (Hordeum vulgare) seedlings exposed to light for 2 h. The novel 22-kD protein was attached to the outer envelope of barley etiochloroplasts, and import of the 27-kD precursor was light dependent and induced after feeding the isolated plastids the tetrapyrrole precursor 5-aminolevulinic acid. HPLC analyses and spectroscopic pigment measurements of acetone-extracted pigments showed that the 22-kD protein is complexed with chlorophyllide. We propose a novel role of WSCPs as pigment carriers operating during light-induced chloroplast development.

Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes.

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b(6)f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.

Temperature-induced greening of Chlorella vulgaris. The role of the cellular energy balance and zeaxanthin-dependent nonphotochemical quenching.

When cells of the green alga Chlorella vulgaris Beij. are transferred from growth at 5 degrees C and an irradiance of 150 micromol photons m(-2) s(-1) to 27 degrees C and the same irradiance, they undergo what is normally considered a high-light to low-light phenotypic change. This involves a 3-fold increase in cellular chlorophyll content with a concomitant increase in light-harvesting complex polypeptide levels. This process appears to occur in response to the cellular capacity to utilize the products of photosynthesis, with the redox state of the plastoquinone pool sensing the cellular energy balance. The phenotypic adjustment can be enhanced or blocked using chemical inhibitors that modulate the redox state of the plastoquinone pool. The functional changes in the photosynthetic apparatus that occurred during the high-light to low-light acclimation were examined with special consideration paid to the paradox that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated cells, with non-functional photosystem II (PSII), accumulate light-harvesting polypeptides. At the structural and basic functional levels, the light-harvesting complex of the cells treated with DCMU was indistinguishable from that of the untreated, control cells. To examine how PSII was protected in the DCMU-treated cells, we measured the content of xanthophyll-cycle pigments. It appeared that a zeaxanthin-dependent nonphotochemical quenching process was involved in PSII protection during greening in the presence of DCMU. Metabolic inhibitors of mitochondrial respiration were used to examine how the change in cellular energy balance regulates the greening process. Apparently, the mitochondrion acts to supply energy to the chloroplast during greening, and inhibition of mitochondrial respiration diminishes chlorophyll accumulation apparently through an increase in the redox state of the plastoquinone pool.

Isolation and characterisation of phycobiliprotein rich mutant of cyanobacterium Synechocystis sp.

Chemical (N' methyl-N'-nitro-N-nitrosoguanidine) mutagenesis and penicillin selection were utilised to isolate a phenotypically altered mutant of cyanobacterium Synechocystis sp. This mutant (ntm60A) exhibited enhanced protein content and nitrogen fixing potential but lower amount of chlorophyll and nitrate reductase activity. A remarkable and significant increase was observed in the total phycobiliprotein content of the mutant, especially in relation to the amount of phycoerythrin. This strain can be exploited as a rich source of natural coloring agent such as phycobilins in the bioindustry.