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The cyanobacterium Synechocystis sp. PCC 6803 has a bidirectional [NiFe]-hydrogenase (Hox hydrogenase) which reversibly reduces protons to H2. This enzyme is composed of a hydrogenase domain and a diaphorase moiety, which is distinctly homologous to the NADH input module of mitochondrial respiratory Complex I. Hox hydrogenase physiological function is still unclear, since it is not required for Synechocystis fitness under standard growth conditions. We analyzed the phenotype under prolonged darkness of three Synechocystis knock-out strains, lacking either Hox hydrogenase (ΔHoxE-H) or one of the proteins responsible for the assembly of its NiFe active site (ΔHypA1 and ΔHypB1). We found that Hox hydrogenase is required for Synechocystis growth under this condition, regardless of the functional status of its catalytic site, suggesting an additional role beside hydrogen metabolism. Moreover, quantitative proteomic analyses revealed that the expression levels of several subunits of the respiratory NADPH/plastoquinone oxidoreductase (NDH-1) are reduced when Synechocystis is grown in the dark. Our findings suggest that the Hox hydrogenase could contribute to electron transport regulation when both photosynthetic and respiratory pathways are down-regulated, and provide a possible explanation for the close evolutionary relationship between mitochondrial respiratory Complex I and cyanobacterial [NiFe]-hydrogenases.

Violaxanthin-chlorophyll a binding protein (VCP) is the major light harvesting complex (LHC) of the Heterokonta Nannochloropsis gaditana. It binds chlorophyll a, violaxanthin and vaucheriaxanthin, the last in the form of 19' deca/octanoate esters. Photosynthetic apparatus of algae belonging to this group have been poorly characterized in the past, but they are now receiving an increasing interest also because of their possible biotechnological application in biofuel production. In this work, isolated VCP proteins have been studied by means of advanced EPR techniques in order to prove the presence of the photoprotective mechanism based on the triplet-triplet energy transfer (TTET), occurring between chlorophyll and carotenoid molecules. This process has been observed before in several light harvesting complexes belonging to various photosynthetic organisms. We used Optically Detected Magnetic Resonance (ODMR) to identify the triplet states populated by photo-excitation, and describe the optical properties of the chromophores carrying the triplet states. In parallel, time-resolved EPR (TR-EPR) and pulse EPR have been employed to get insight into the TTET mechanism and reveal the structural features of the pigment sites involved in photoprotection. The analysis of the spectroscopic data shows a strong similarity among VCP, FCP from diatoms and LHC-II from higher plants. Although these antenna proteins have differentiated sequences and bind different pigments, results suggest that in all members of the LHC superfamily there is a protein core with a conserved structural organization, represented by two central carotenoids surrounded by five chlorophyll a molecules, which plays a fundamental photoprotective role in Chl triplet quenching through carotenoid triplet formation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

One of the main parameters influencing microalgae production is light, which provides energy to support metabolism but, if present in excess, can lead to oxidative stress and growth inhibition. In this work, the influence of illumination on Scenedesmus obliquus growth was assessed by cultivating cells at different light intensities in a flat plate photobioreactor. S. obliquus showed a maximum growth rate at 150 μmol photons m(-2) s(-1). Below this value, light was limiting for growth, while with more intense illumination photosaturation effects were observed, although cells still showed the ability to duplicate. Looking at the biochemical composition, light affected the pigment contents only while carbohydrate, lipid, and protein contents remained stable. By considering that in industrial photobioreactors microalgae cells are subjected to light-dark cycles due to mixing, algae were also grown under pulsed illumination (5, 10, and 15 Hz). Interestingly, the ability to exploit pulsed light with good efficiency required a pre-acclimation to the same conditions, suggesting the presence of a biological response to these conditions.

Nannochloropsis gaditana belongs to Eustigmatophyceae, a class of eukaryotic algae resulting from a secondary endosymbiotic event. Species of this class have been poorly characterized thus far but are now raising increasing interest in the scientific community because of their possible application in biofuel production. Nannochloropsis species have a peculiar photosynthetic apparatus characterized by the presence of only chlorophyll a, with violaxanthin and vaucheriaxanthin esters as the most abundant carotenoids. In this study, the photosynthetic apparatus of this species was analyzed by purifying the thylakoids and isolating the different pigment-binding complexes upon mild solubilization. The results from the biochemical and spectroscopic characterization showed that the photosystem II antenna is loosely bound to the reaction center, whereas the association is stronger in photosystem I, with the antenna-reaction center super-complexes surviving purification. Such a supramolecular organization was found to be conserved in photosystem I from several other photosynthetic eukaryotes, even though these taxa are evolutionarily distant. A hypothesis on the possible selective advantage of different associations of the antenna complexes of photosystems I and II is discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae. Here, we report a high-quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of the chromosomes. We identified 10646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing, and cell wall synthesis. We also analyzed the modification of the transcriptional profile in nitrogen deficiency-a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time, we observed a very significant down-regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway. Finally, we released an information resource of the genomic data of N. gaditana, available online at www.nannochloropsis.org.

A major challenge for next decades is development of competitive renewable energy sources, highly needed to compensate fossil fuels reserves and reduce greenhouse gas emissions. Among different possibilities, which are currently under investigation, there is the exploitation of unicellular algae for production of biofuels and biodiesel in particular. Some algae species have the ability of accumulating large amount of lipids within their cells which can be exploited as feedstock for the production of biodiesel. Strong research efforts are however still needed to fulfill this potential and optimize cultivation systems and biomass harvesting. Light provides the energy supporting algae growth and available radiation must be exploited with the highest possible efficiency to optimize productivity and make microalgae large scale cultivation energetically and economically sustainable. Investigation of the molecular bases influencing light use efficiency is thus seminal for the success of this biotechnology. In this work factors influencing light use efficiency in algal biomass production are reviewed, focusing on how algae genetic engineering and control of light environment within photobioreactors can improve the productivity of large scale cultivation systems., (© 2013.)

Photosynthesis converts light energy into chemical energy, and supplies ATP and NADPH for CO2 fixation into carbohydrates and for the synthesis of several compounds which are essential for autotrophic growth. Oxygenic photosynthesis takes place in thylakoid membranes of chloroplasts and photosynthetic prokaryote cyanobacteria. An ancestral photoautotrophic prokaryote related to cyanobacteria has been proposed to give rise to chloroplasts of plants and algae through an endosymbiotic event. Indeed, photosynthetic complexes involved in the electron transport coupled to H(+) translocation and ATP synthesis are similar in higher plants and cyanobacteria. Furthermore, some of the protein and solute/ion conducting machineries also share common structure and function. Electrophysiological and biochemical evidence support the existence of ion channels in the thylakoid membrane in both types of organisms. By allowing specific ion fluxes across thylakoid membranes, ion channels have been hypothesized to either directly or indirectly regulate photosynthesis, by modulating the proton motive force. Recent molecular identification of some of the thylakoid-located channels allowed to obtain genetic proof in favor of such hypothesis. Furthermore, some ion channels of the envelope membrane in chloroplasts have also been shown to impact on this light-driven process. Here we give an overview of thylakoid/chloroplast located ion channels of higher plants and of cyanobacterium Synechocystis sp. PCC 6803. We focus on channels shown to be implicated in the regulation of photosynthesis and discuss the possible mechanisms of action., (© 2013 Elsevier B.V. All rights reserved.)

The size of the light-induced proton motive force (pmf) across the thylakoid membrane of chloroplasts is regulated in response to environmental stimuli. Here, we describe a component of the thylakoid membrane, the two-pore potassium (K(+)) channel TPK3, which modulates the composition of the pmf through ion counterbalancing. Recombinant TPK3 exhibited potassium-selective channel activity sensitive to Ca(2+) and H(+). In Arabidopsis plants, the channel is found in the thylakoid stromal lamellae. Arabidopsis plants silenced for the TPK3 gene display reduced growth and altered thylakoid membrane organization. This phenotype reflects an impaired capacity to generate a normal pmf, which results in reduced CO2 assimilation and deficient nonphotochemical dissipation of excess absorbed light. Thus, the TPK3 channel manages the pmf necessary to convert photochemical energy into physiological functions.

Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel, SynCaK, in the genome of the freshwater cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a calcium-dependent potassium channel isolated from Methanobacterium thermoautotrophicum. Expression of SynCaK in fusion with enhanced GFP in mammalian Chinese hamster ovary cells' plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasma membrane. To understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803, ΔSynCaK, has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in the wild type, ΔSynCaK was characterized by a depolarized resting membrane potential, as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress and that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor that also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Although light is the source of energy for photosynthetic organisms, it causes oxidative stress when in excess. Plants and algae prevent reactive oxygen species (ROS) formation by activation of nonphotochemical quenching (NPQ), which dissipates excess excitation energy as heat. Although NPQ is found in both algae and plants, these organisms rely on two different proteins for its activation, Light harvesting complex stress-related (LHCSR) and Photosystem II subunit S (PSBS). In the moss Physcomitrella patens, both proteins are present and active. Several P. patens lines depleted in or over-expressing PSBS and/or LHCSR at various levels were generated by exploiting the ability of Physcomitrella to undergo homologous recombination. The analysis of the transgenic lines showed that either protein is sufficient, alone, for NPQ activation independently of the other, supporting the idea that they rely on different activation mechanisms. Modulation of PSBS and/or LHCSR contents was found to be correlated with NPQ amplitude, indicating that plants and algae can directly modulate their ability to dissipate energy simply by altering the accumulation level of one or both of these proteins. The availability of a large range of P. patens genotypes differing in PSBS and LHCSR content allowed comparison of their activation mechanisms and discussion of implications for the evolution of photoprotection during land colonization., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

In this work we present an optically detected magnetic resonance (ODMR) study on the triplet states populated under illumination in the isolated fucoxanthin-chlorophyll light-harvesting complex from the diatom Cyclotella meneghiniana. Evidence for the quenching of chlorophyll triplet states by fucoxanthin is provided, showing that this carotenoid is able to perform the photoprotective role. For the first time, the magnetic parameters characterizing the fucoxanthin triplet state have been determined. The results reveal analogies but also differences with respect to the triplet-triplet energy transfer process, which involves chlorophylls a and carotenoids in the LHC complex from dinoflagellates and LHCII from higher plants. The degree of efficiency of the photoprotection mechanism, in these light harvesting complexes, is discussed in terms of pigment-protein structure., (Copyright © 2012 Elsevier Inc. All rights reserved.)

[FeFe]-hydrogenases are iron-sulfur proteins characterized by a complex active site, the H-cluster, whose assembly requires three conserved maturases. HydE and HydG are radical S-adenosylmethionine enzymes that chemically modify a H-cluster precursor on HydF, a GTPase with a dual role of scaffold on which this precursor is synthesized, and carrier to transfer it to the hydrogenase. Coordinate structural and functional relationships between HydF and the two other maturases are crucial for the H-cluster assembly. However, to date only qualitative analysis of this protein network have been provided. In this work we showed that the interactions of HydE and HydG with HydF are distinct events, likely occurring in a precise functional order driven by different kinetic properties, independently of the HydF GTPase activity, which is instead involved in the dissociation of the maturases from the scaffold. We also found that HydF is able to interact with the hydrogenase only when co-expressed with the two other maturases, indicating that under these conditions it harbors per se all the structural elements needed to transfer the H-cluster precursor, thus completing the maturation process. These results open new working perspectives aimed at improving the knowledge of how these complex metalloenzymes are biosynthesized.

Plants live in variable environments in which light intensity can rapidly change, from limiting to excess conditions. Non-photochemical quenching (NPQ) is a regulatory mechanism which protects plants from oxidative stress by dissipating excess Chl singlet excitation. In this work, the physiological role of NPQ was assessed by monitoring its influence on the population of the direct source of light excess damage, i.e., Chl triplets ((3)Chl*). (3)Chl* formation was evaluated in vivo, with the moss Physcomitrella patens, by exploiting the high sensitivity of fluorescence-detected magnetic resonance (FDMR). A dark adapted sample was compared with a pre-illuminated sample in which NPQ was activated, the latter showing a strong reduction in (3)Chl* yield. In line with this result, mutants unable to activate NPQ showed only a minor effect in (3)Chl* yield upon pre-illumination.The decrease in (3)Chl* yield is equally experienced by all the Chl pools associated with PSII, suggesting that NPQ is effective in protecting both the core and the peripheral antenna complexes. Moreover, the FDMR results show that the structural reorganization in the photosynthetic apparatus, required by NPQ, does not lead to the formation of new (3)Chl* traps in the LHCs. This work demonstrates that NPQ activation leads to effective photoprotection, promoting a photosystem II state characterized by a reduced probability of (3)Chl* formation, due to a decreased singlet excited state population, while maintaining an efficient quenching of the (3)Chl* eventually formed by carotenoids., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.

Biofuels from algae are highly interesting as renewable energy sources to replace, at least partially, fossil fuels, but great research efforts are still needed to optimize growth parameters to develop competitive large-scale cultivation systems. One factor with a seminal influence on productivity is light availability. Light energy fully supports algal growth, but it leads to oxidative stress if illumination is in excess. In this work, the influence of light intensity on the growth and lipid productivity of Nannochloropsis salina was investigated in a flat-bed photobioreactor designed to minimize cells self-shading. The influence of various light intensities was studied with both continuous illumination and alternation of light and dark cycles at various frequencies, which mimic illumination variations in a photobioreactor due to mixing. Results show that Nannochloropsis can efficiently exploit even very intense light, provided that dark cycles occur to allow for re-oxidation of the electron transporters of the photosynthetic apparatus. If alternation of light and dark is not optimal, algae undergo radiation damage and photosynthetic productivity is greatly reduced. Our results demonstrate that, in a photobioreactor for the cultivation of algae, optimizing mixing is essential in order to ensure that the algae exploit light energy efficiently.

Mixotrophy can be exploited to support algal growth over night or in dark-zones of a photobioreactor. In order to achieve the maximal productivity, however, it is fundamental also to provide CO(2) in excess to maximize photosynthetic activity and phototropic biomass production. The aim of this paper is to verify the possibility of exploiting mixotrophy in combination with excess CO(2). Two species with high biomass productivity were selected, Nannochloropsis salina and Chlorella protothecoides. Different organic substrates available at industrial scale were tested, and glycerol chosen for its ability to support growth of both species. In mixotrophic conditions, excess CO(2) stimulated photosynthesis but blocked the metabolization of the organic substrate, thus canceling the advantages of mixotrophy. By cultivating microalgae under day-night cycle, organic substrate supported growth during the night, but only if CO(2) supply was not provided. This represents thus a possible method to reconcile CO(2) stimulation of photosynthesis with mixotrophy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

[FeFe]-hydrogenases catalyze the reversible production of H2 in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 Å resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous β-sheet comprising eight β-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.

Photosynthetic organisms respond to strong illumination by activating several photoprotection mechanisms. One of them, non-photochemical quenching (NPQ), consists in the thermal dissipation of energy absorbed in excess. In vascular plants NPQ relies on the activity of PSBS, whereas in the green algae Chlamydomonas reinhardtii it requires a different protein, LHCSR. The moss Physcomitrella patens is the only known organism in which both proteins are present and active in triggering NPQ, making this organism particularly interesting for the characterization of this protection mechanism. We analysed the acclimation of Physcomitrella to high light and low temperature, finding that these conditions induce an increase in NPQ correlated to overexpression of both PSBS and LHCSR. Mutants depleted of PSBS and/or LHCSR showed that modulation of their accumulation indeed determines NPQ amplitude. All mutants with impaired NPQ also showed enhanced photosensitivity when exposed to high light or low temperature, indicating that in this moss the fast-responding NPQ mechanism is also involved in long-term acclimation., (© 2011 Blackwell Publishing Ltd.)

Algae are interesting potential sources of biodiesel, although research is still needed to develop efficient large scale productions. One major factor affecting productivity is light use efficiency. The effect of different light regimes on the seawater alga Nannochloropsis gaditana was accessed monitoring growth rate and photosynthetic performances. N. gaditana showed the capacity of acclimating to different light intensities, optimizing its photosynthetic apparatus to illumination. Thanks to this response, N. gaditana maintained similar growth rates under a wide range of irradiances, suggesting that this organism is a valuable candidate for outdoor productions in variable conditions. In the conditions tested here, without external CO(2) supply, light intensity alone was not found to be a major signal affecting lipids accumulation showing the absence of a direct regulatory link between the light stress and lipids accumulation. Strong illumination can nevertheless indirectly influences lipid accumulation if combined with other stresses or in the presence of excess CO(2)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bioinformatic approaches have allowed the identification in Arabidopsis thaliana of twenty genes encoding for homologues of animal ionotropic glutamate receptors (iGLRs). Some of these putative receptor proteins, grouped into three subfamilies, have been located to the plasmamembrane, but their possible location in organelles has not been investigated so far. In the present work we provide multiple evidence for the plastid localization of a glutamate receptor, AtGLR3.4, in Arabidopsis and tobacco. Biochemical analysis was performed using an antibody shown to specifically recognize both the native protein in Arabidopsis and the recombinant AtGLR3.4 fused to YFP expressed in tobacco. Western blots indicate the presence of AtGLR3.4 in both the plasmamembrane and in chloroplasts. In agreement, in transformed Arabidopsis cultured cells as well as in agroinfiltrated tobacco leaves, AtGLR3.4::YFP is detected both at the plasmamembrane and at the plastid level by confocal microscopy. The photosynthetic phenotype of mutant plants lacking AtGLR3.4 was also investigated. These results identify for the first time a dual localization of a glutamate receptor, revealing its presence in plastids and chloroplasts and opening the way to functional studies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

[FeFe]-hydrogenases have been claimed as the most promising catalysts of hydrogen bioproduction and several efforts have been accomplished to express and purify them. However, previous attemps to obtain a functional recombinant [FeFe]-hydrogenase in heterologous systems such as Escherichia coli failed due to the lack of the specific maturation proteins driving the assembly of its complex active site. The unique exception is that of [FeFe]-hydrogenase from Clostridium pasteurianum that has been expressed in active form in the cyanobacterium Synechococcus PCC 7942, which holds a bidirectional [NiFe]-hydrogenase with a well characterized maturation system, suggesting that the latter is flexible enough to drive the synthesis of a [FeFe]-enzyme. However, the capability of cyanobacteria to correctly fold a [FeFe]-hydrogenase in the absence of its auxiliary maturation proteins is a debated question. In this work, we expressed the [FeFe]-hydrogenase from Chlamydomonas reinhardtii as an active enzyme in the cyanobacterium Synechocystis sp. PCC 6803. Our results, using a different experimental system, confirm that cyanobacteria are able to express a functional [FeFe]-hydrogenase even in the absence of additional chaperones., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We present an optically detected magnetic resonance (ODMR) and electron paramagnetic resonance (EPR) spectroscopic study on the quenching of photo-induced chlorophyll triplet states by carotenoids, in the intrinsic light-harvesting complex (LHC) from the dinoflagellate Amphidinium carterae. Two carotenoid triplet states, differing in terms of optical and magnetic spectroscopic properties, have been identified and assigned to peridinins located in different protein environment. The results reveal a parallelism with the triplet-triplet energy transfer (TTET) process involving chlorophyll a and luteins observed in the LHC-II complex of higher plants. Starting from the hypothesis of a conserved alignment of the amino acid sequences at the cores of the LHC and LHC-II proteins, the spin-polarized time-resolved EPR spectra of the carotenoid triplet states of LHC have been calculated by a method which exploits the conservation of the spin momentum during the TTET process. The analysis of the spectra shows that the data are compatible with a structural model of the core of LHC which assigns the photo-protective function to two central carotenoids surrounded by the majority of Chl a molecules present in the protein, as found in LHC-II. However, the lack of structural data, and the uncertainty in the pigment composition of LHC, leaves open the possibility that this complex posses a different arrangement of the pigments with specific centers of Chl triplet quenching., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.

Light is the source of energy for photosynthetic organisms; when in excess, however, it also drives the formation of reactive oxygen species and, consequently, photoinhibition. Plants and algae have evolved mechanisms to regulate light harvesting efficiency in response to variable light intensity so as to avoid oxidative damage. Nonphotochemical quenching (NPQ) consists of the rapid dissipation of excess excitation energy as heat. Although widespread among oxygenic photosynthetic organisms, NPQ shows important differences in its machinery. In land plants, such as Arabidopsis thaliana, NPQ depends on the presence of PSBS, whereas in the green alga Chlamydomonas reinhardtii it requires a different protein called LHCSR. In this work, we show that both proteins are present in the moss Physcomitrella patens. By generating KO mutants lacking PSBS and/or LHCSR, we also demonstrate that both gene products are active in NPQ. Plants lacking both proteins are more susceptible to high light stress than WT, implying that they are active in photoprotection. These results suggest that NPQ is a fundamental mechanism for survival in excess light and that upon land colonization, photosynthetic organisms evolved a unique mechanism for excess energy dissipation before losing the ancestral one found in algae.

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K(+)-uptake-system deficient E. coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese hamster ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K(+) channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

Thylakoid membranes in higher plant chloroplasts are composed by two distinct domains: stacked grana and stroma lamellae. We developed a procedure for biochemical isolation of grana membranes using mild detergent to maintain membrane structure. Pigment and polypeptide analyses of membrane preparation showed the preparations were indeed enriched in grana membranes. The method was shown to be effective in four different plant species, although with small changes in detergent concentration. Electron microscopy analyses also showed that the preparation consisted of large membrane patches with roughly round shape and diameter comparable with grana membranes in vivo. Furthermore, protein complexes distribution was shown to be maintained with respect to freeze fracture studies, demonstrating that the protocol was successful in isolating membranes close to their in vivo state.

Bioinformatic approaches have allowed the identification of twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iGLRs) in the Arabidopsis thaliana model plant. Indirect evidence suggests that plant iGLRs function as non-selective cation channels. In the present work we provide biochemical and electrophysiological evidences for the chloroplast localization of glutamate receptor(s) of family 3 (iGLR3) in spinach. A specific antibody, recognizing putative receptors of family 3 locates iGLR3 to the inner envelope membrane of chloroplasts. In planar lipid bilayer experiments, purified inner envelope vesicles from spinach display a cation-selective electrophysiological activity which is inhibited by DNQX (6,7-dinitroquinoxaline-2,3-dione), considered to act as an inhibitor on both animal and plant iGLRs. These results identify for the first time the intracellular localization of plant glutamate receptor(s) and a DNQX-sensitive, glutamate-gated activity at single channel level in native membrane with properties compatible with those predicted for plant glutamate receptors., (Copyright 2010 S. Karger AG, Basel.)

Indirect evidence points to the presence of K(+) channels in plant mitochondria. In the present study, we report the results of the first patch clamp experiments on plant mitochondria. Single-channel recordings in 150 mM potassium gluconate have allowed the biophysical characterization of a channel with a conductance of 150 pS in the inner mitochondrial membrane of mitoplasts obtained from wheat (Triticum durum Desf.). The channel displayed sharp voltage sensitivity, permeability to potassium and cation selectivity. ATP in the mM concentration range completely abolished the activity. We discuss the possible molecular identity of the channel and its possible role in the defence mechanisms against oxidative stress in plants., (Copyright © 2010 S. Karger AG, Basel.)

Background: Photosystems are composed of two moieties, a reaction center and a peripheral antenna system. In photosynthetic eukaryotes the latter system is composed of proteins belonging to Lhc family. An increasing set of evidences demonstrated how these polypeptides play a relevant physiological function in both light harvesting and photoprotection. Despite the sequence similarity between antenna proteins associated with the two Photosystems, present knowledge on their physiological role is mostly limited to complexes associated to Photosystem II., Results: In this work we analyzed the physiological role of Photosystem I antenna system in Arabidopsis thaliana both in vivo and in vitro. Plants depleted in individual antenna polypeptides showed a reduced capacity for photoprotection and an increased production of reactive oxygen species upon high light exposure. In vitro experiments on isolated complexes confirmed that depletion of antenna proteins reduced the resistance of isolated Photosystem I particles to high light and that the antenna is effective in photoprotection only upon the interaction with the core complex., Conclusion: We show that antenna proteins play a dual role in Arabidopsis thaliana Photosystem I photoprotection: first, a Photosystem I with an intact antenna system is more resistant to high light because of a reduced production of reactive oxygen species and, second, antenna chlorophyll-proteins are the first target of high light damages. When photoprotection mechanisms become insufficient, the antenna chlorophyll proteins act as fuses: LHCI chlorophylls are degraded while the reaction center photochemical activity is maintained. Differences with respect to photoprotection strategy in Photosystem II, where the reaction center is the first target of photoinhibition, are discussed.

The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at approximately 620 and approximately 640 nm in the Triplet-minus-Singlet (T-S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.

The photoexcited triplet state of the carotenoid peridinin in the high-salt peridinin-chlorophyll a-protein (HSPCP) of the dinoflagellate Amphidinium carterae was investigated by ODMR (optically detected magnetic resonance), pulse EPR and pulse ENDOR spectroscopies. The properties of peridinins associated to the triplet state formation in HSPCP were compared to those of peridinins involved in triplet state population in the main-form peridinin-chlorophyll protein (MFPCP), previously reported. In HSPCP no signals due to the presence of chlorophyll triplet state have been detected, during either steady-state illumination or laser-pulse excitation, meaning that peridinins play the photo-protective role with 100% efficiency as in MFPCP. The general spectroscopic features of the peridinin triplet state are very similar in the two complexes and allow drawing the conclusion that the triplet formation pathway and the triplet localization in one specific peridinin in each subcluster are the same in HSPCP and MFPCP. However some significant differences also emerged from the analysis of the spectra. Zero field splitting parameters of the peridinin triplet states are slightly smaller in HSPCP and small changes are also observed for the hyperfine splittings measured by pulse ENDOR and assigned to the beta-protons belonging to one of the two methyl groups present in the conjugated chain, (a(iso)=10.3 MHz in HSPCP vs a(iso)=10.6 MHz in MFPCP). The differences are explained in terms of local distortion of the tails of the conjugated chains of the peridinin molecules, in agreement with the conformational data resulting from the X-ray structures of the two complexes.

The photoexcited triplet state of the carotenoid peridinin in the Peridinin-chlorophyll a-protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the alpha- and beta-protons of the peridinin conjugated chain. Assignments of beta-protons belonging to two different methyl groups, with aiso=7.0 MHz and aiso=10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole pi-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186-195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll-peridinin pair, is functioning as a bridge in the triplet-triplet energy transfer between the two pigments.

O(2)carriers (extracellular and intracellular as well as monomeric and multimeric) have evolved over the last billion of years, displaying iron and copper reactive centers; very different O(2)carriers may co-exist in the same organism. Circulating O(2)carriers, faced to the external environment, are responsible for maintaining an adequate delivery of O(2)to tissues and organs almost independently of the environmental O(2)partial pressure. Then, intracellular globins facilitate O(2)transfer to mitochondria sustaining cellular respiration. Here, molecular aspects of multiple strategies evolved for O(2)transport and delivery are examined, from the simplest myoglobin to the most complex giant O(2)carriers and the red blood cell, mostly focusing on the aspects which have been mainly addressed by the so called 'Rome Group'.