Isolation and characterization of a main porin from the outer membrane of Salinibacter ruber.

Salinibacter ruber is an extremophilic bacterium able to grow in high-salts environments, such as saltern crystallizer ponds. This halophilic bacterium is red-pigmented due to the production of several carotenoids and their derivatives. Two of these pigment molecules, salinixanthin and retinal, are reported to be essential cofactors of the xanthorhodopsin, a light-driven proton pump unique to this bacterium. Here, we isolate and characterize an outer membrane porin-like protein that retains salinixanthin. The characterization by mass spectrometry identified an unknown protein whose structure, predicted by AlphaFold, consists of a 8 strands beta-barrel transmembrane organization typical of porins. The protein is found to be part of a functional network clearly involved in the outer membrane trafficking. Cryo-EM micrographs showed the shape and dimensions of a particle comparable with the ones of the predicted structure. Functional implications, with respect to the high representativity of this protein in the outer membrane fraction, are discussed considering its possible role in primary functions such as the nutrients uptake and the homeostatic balance. Finally, also a possible involvement in balancing the charge perturbation associated with the xanthorhodopsin and ATP synthase activities is considered.

The Low Molecular Mass Photosystem II Protein PsbTn Is Important for Light Acclimation.

Photosystem II (PSII) is a supramolecular complex containing over 30 protein subunits and a large set of cofactors, including various pigments and quinones as well as Mn, Ca, Cl, and Fe ions. Eukaryotic PSII complexes contain many subunits not found in their bacterial counterparts, including the proteins PsbP (PSII), PsbQ, PsbS, and PsbW, as well as the highly homologous, low-molecular-mass subunits PsbTn1 and PsbTn2 whose function is currently unknown. To determine the function of PsbTn1 and PsbTn2, we generated single and double psbTn1 and psbTn2 knockout mutants in Arabidopsis (Arabidopsis thaliana). Cross linking and reciprocal coimmunoprecipitation experiments revealed that PsbTn is a lumenal PSII protein situated next to the cytochrome b 559 subunit PsbE. The removal of the PsbTn proteins decreased the oxygen evolution rate and PSII core phosphorylation level but increased the susceptibility of PSII to photoinhibition and the production of reactive oxygen species. The assembly and stability of PSII were unaffected, indicating that the deficiencies of the psbTn1 psbTn2 double mutants are due to structural changes. Double mutants exhibited a higher rate of nonphotochemical quenching of excited states than the wild type and single mutants, as well as slower state transition kinetics and a lower quantum yield of PSII when grown in the field. Based on these results, we propose that the main function of the PsbTn proteins is to enable PSII to acclimate to light shifts or intense illumination.

Establishment of Photosynthesis through Chloroplast Development Is Controlled by Two Distinct Regulatory Phases.

Chloroplasts develop from undifferentiated proplastids present in meristematic tissue. Thus, chloroplast biogenesis is closely connected to leaf development, which restricts our ability to study the process of chloroplast biogenesis per se. As a consequence, we know relatively little about the regulatory mechanisms behind the establishment of the photosynthetic reactions and how the activities of the two genomes involved are coordinated during chloroplast development. We developed a single cell-based experimental system from Arabidopsis (Arabidopsis thaliana) with high temporal resolution allowing for investigations of the transition from proplastids to functional chloroplasts. Using this unique cell line, we could show that the establishment of photosynthesis is dependent on a regulatory mechanism involving two distinct phases. The first phase is triggered by rapid light-induced changes in gene expression and the metabolome. The second phase is dependent on the activation of the chloroplast and generates massive changes in the nuclear gene expression required for the transition to photosynthetically functional chloroplasts. The second phase also is associated with a spatial transition of the chloroplasts from clusters around the nucleus to the final position at the cell cortex. Thus, the establishment of photosynthesis is a two-phase process with a clear checkpoint associated with the second regulatory phase allowing coordination of the activities of the nuclear and plastid genomes.

The PsbY protein of Arabidopsis Photosystem II is important for the redox control of cytochrome b559.

Photosystem II is a protein complex embedded in the thylakoid membrane of photosynthetic organisms and performs the light driven water oxidation into electrons and molecular oxygen that initiate the photosynthetic process. This important complex is composed of more than two dozen of intrinsic and peripheral subunits, of those half are low molecular mass proteins. PsbY is one of those low molecular mass proteins; this 4.7-4.9kDa intrinsic protein seems not to bind any cofactors. Based on structural data from cyanobacterial and red algal Photosystem II PsbY is located closely or in direct contact with cytochrome b559. Cytb559 consists of two protein subunits (PsbE and PsbF) ligating a heme-group in-between them. While the exact function of this component in Photosystem II has not yet been clarified, a crucial role for assembly and photo-protection in prokaryotic complexes has been suggested. One unique feature of Cytb559 is its redox-heterogeneity, forming high, medium and low potential, however, neither origin nor mechanism are known. To reveal the function of PsbY within Photosystem II of Arabidopsis we have analysed PsbY knock-out plants and compared them to wild type and to complemented mutant lines. We show that in the absence of PsbY protein Cytb559 is only present in its oxidized, low potential form and plants depleted of PsbY were found to be more susceptible to photoinhibition.

Fingerprinting the macro-organisation of pigment-protein complexes in plant thylakoid membranes in vivo by circular-dichroism spectroscopy.

Macro-organisation of the protein complexes in plant thylakoid membranes plays important roles in the regulation and fine-tuning of photosynthetic activity. These delicate structures might, however, undergo substantial changes during isolating the thylakoid membranes or during sample preparations, e.g., for electron microscopy. Circular-dichroism (CD) spectroscopy is a non-invasive technique which can thus be used on intact samples. Via excitonic and psi-type CD bands, respectively, it carries information on short-range excitonic pigment-pigment interactions and the macro-organisation (chiral macrodomains) of pigment-protein complexes (psi, polymer or salt-induced). In order to obtain more specific information on the origin of the major psi-type CD bands, at around (+)506, (-)674 and (+)690nm, we fingerprinted detached leaves and isolated thylakoid membranes of wild-type and mutant plants and also tested the effects of different environmental conditions in vivo. We show that (i) the chiral macrodomains disassemble upon mild detergent treatments, but not after crosslinking the protein complexes; (ii) in different wild-type leaves of dicotyledonous and monocotyledonous angiosperms the CD features are quite robust, displaying very similar excitonic and psi-type bands, suggesting similar protein composition and (macro-) organisation of photosystem II (PSII) supercomplexes in the grana; (iii) the main positive psi-type bands depend on light-harvesting protein II contents of the membranes; (iv) the (+)506nm band appears only in the presence of PSII-LHCII supercomplexes and does not depend on the xanthophyll composition of the membranes. Hence, CD spectroscopy can be used to detect different macro-domains in the thylakoid membranes with different outer antenna compositions in vivo.

PsbN is required for assembly of the photosystem II reaction center in Nicotiana tabacum.

The chloroplast-encoded low molecular weight protein PsbN is annotated as a photosystem II (PSII) subunit. To elucidate the localization and function of PsbN, encoded on the opposite strand to the psbB gene cluster, we raised antibodies and inserted a resistance cassette into PsbN in both directions. Both homoplastomic tobacco (Nicotiana tabacum) mutants psbN-F and psbN-R show essentially the same PSII deficiencies. The mutants are extremely light sensitive and failed to recover from photoinhibition. Although synthesis of PSII proteins was not altered significantly, both mutants accumulated only ∼25% of PSII proteins compared with the wild type. Assembly of PSII precomplexes occurred at normal rates, but heterodimeric PSII reaction centers (RCs) and higher order PSII assemblies were not formed efficiently in the mutants. The psbN-R mutant was complemented by allotopic expression of the PsbN gene fused to the sequence of a chloroplast transit peptide in the nuclear genome. PsbN represents a bitopic trans-membrane peptide localized in stroma lamellae with its highly conserved C terminus exposed to the stroma. Significant amounts of PsbN were already present in dark-grown seedling. Our data prove that PsbN is not a constituent subunit of PSII but is required for repair from photoinhibition and efficient assembly of the PSII RC.

Comparison of methods for extracting thylakoid membranes of Arabidopsis plants.

Robust and reproducible methods for extracting thylakoid membranes are required for the analysis of photosynthetic processes in higher plants such as Arabidopsis. Here, we compare three methods for thylakoid extraction using two different buffers. Method I involves homogenizing the plant material with a metal/glass blender; method II involves manually grinding the plant material in ice-cold grinding buffer with a mortar and method III entails snap-freezing followed by manual grinding with a mortar, after which the frozen powder is thawed in isolation buffer. Thylakoid membrane samples extracted using each method were analyzed with respect to protein and chlorophyll content, yields relative to starting material, oxygen-evolving activity, protein complex content and phosphorylation. We also examined how the use of fresh and frozen thylakoid material affected the extracts' contents of protein complexes. The use of different extraction buffers did not significantly alter the protein content of the extracts in any case. Method I yielded thylakoid membranes with the highest purity and oxygen-evolving activity. Method III used low amounts of starting material and was capable of capturing rapid phosphorylation changes in the sample at the cost of higher levels of contamination. Method II yielded thylakoid membrane extracts with properties intermediate between those obtained with the other two methods. Finally, frozen and freshly isolated thylakoid membranes performed identically in blue native-polyacrylamide gel electrophoresis experiments conducted in order to separate multimeric protein supracomplexes.

Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle.

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Presence of state transitions in the cryptophyte alga Guillardia theta.

Plants and algae have developed various regulatory mechanisms for optimal delivery of excitation energy to the photosystems even during fluctuating light conditions; these include state transitions as well as non-photochemical quenching. The former process maintains the balance by redistributing antennae excitation between the photosystems, meanwhile the latter by dissipating excessive excitation inside the antennae. In the present study, these mechanisms have been analysed in the cryptophyte alga Guillardia theta. Photoprotective non-photochemical quenching was observed in cultures only after they had entered the stationary growth phase. These cells displayed a diminished overall photosynthetic efficiency, measured as CO2 assimilation rate and electron transport rate. However, in the logarithmic growth phase G. theta cells redistributed excitation energy via a mechanism similar to state transitions. These state transitions were triggered by blue light absorbed by the membrane integrated chlorophyll a/c antennae, and green light absorbed by the lumenal biliproteins was ineffective. It is proposed that state transitions in G. theta are induced by small re-arrangements of the intrinsic antennae proteins, resulting in their coupling/uncoupling to the photosystems in state 1 or state 2, respectively. G. theta therefore represents a chromalveolate algae able to perform state transitions.

An Arabidopsis soluble chloroplast proteomic analysis reveals the participation of the Executer pathway in response to increased light conditions.

The Executer1 and Executer2 proteins have a fundamental role in the signalling pathway mediated by singlet oxygen in chloroplast; nonetheless, not much is known yet about their specific activity and features. Herein, we have followed a differential-expression proteomics approach to analyse the impact of Executer on the soluble chloroplast protein abundance in Arabidopsis. Because singlet oxygen plays a significant role in signalling the oxidative response of plants to light, our analysis also included the soluble chloroplast proteome of plants exposed to a moderate light intensity in the time frame of hours. A number of light- and genotype-responsive proteins were detected, and mass-spectrometry identification showed changes in abundance of several photosynthesis- and carbon metabolism-related proteins as well as proteins involved in plastid mRNA processing. Our results support the participation of the Executer proteins in signalling and control of chloroplast metabolism, and in the regulation of plant response to environmental changes.

Family-wide characterization of matrix metalloproteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity.

MMPs (matrix metalloproteases) are a family of zinc-dependent endopeptidases widely distributed throughout all kingdoms of life. In mammals, MMPs play key roles in many physiological and pathological processes, including remodelling of the extracellular matrix. In the genome of the annual plant Arabidopsis thaliana, five MMP-like proteins (At-MMPs) are encoded, but their function is unknown. Previous work on these enzymes was limited to gene expression analysis, and so far proteolytic activity has been shown only for At1-MMP. We expressed and purified the catalytic domains of all five At-MMPs as His-tagged proteins in Escherichia coli cells to delineate the biochemical differences and similarities among the Arabidopsis MMP family members. We demonstrate that all five recombinant At-MMPs are active proteases with distinct preferences for different protease substrates. Furthermore, we performed a family-wide characterization of their biochemical properties and highlight similarities and differences in their cleavage site specificities as well as pH- and temperature-dependent activities. Detailed analysis of their sequence specificity using PICS (proteomic identification of protease cleavage sites) revealed profiles similar to human MMPs with the exception of At5-MMP; homology models of the At-MMP catalytic domains supported these results. Our results suggest that each At-MMP may be involved in different proteolytic processes during plant growth and development.

Metabolomic analysis of extreme freezing tolerance in Siberian spruce (Picea obovata).

Siberian spruce (Picea obovata) is one of several boreal conifer species that can survive at extremely low temperatures (ELTs). When fully acclimated, its tissues can survive immersion in liquid nitrogen. Relatively little is known about the biochemical and biophysical strategies of ELT survival. We profiled needle metabolites using gas chromatography coupled with mass spectrometry (GC-MS) to explore the metabolic changes that occur during cold acclimation caused by natural temperature fluctuations. In total, 223 metabolites accumulated and 52 were depleted in fully acclimated needles compared with pre-acclimation needles. The metabolite profiles were found to develop in four distinct phases, which are referred to as pre-acclimation, early acclimation, late acclimation and fully acclimated. Metabolite changes associated with carbohydrate and lipid metabolism were observed, including changes associated with increased raffinose family oligosaccharide synthesis and accumulation, accumulation of sugar acids and sugar alcohols, desaturation of fatty acids, and accumulation of digalactosylglycerol. We also observed the accumulation of protein and nonprotein amino acids and polyamines that may act as compatible solutes or cryoprotectants. These results provide new insight into the mechanisms of freezing tolerance development at the metabolite level and highlight their importance in rapid acclimation to ELT in P. obovata.

Measurements of Oxygen Evolution in Photosynthesis.

This chapter compares two different techniques for monitoring photosynthetic O2 production; the wide-spread Clark-type O2 electrode and the more sophisticated membrane inlet mass spectrometry (MIMS) technique. We describe how a simple membrane inlet for MIMS can be made out of a commercial Clark-type cell and outline the advantages and drawbacks of the two techniques to guide researchers in deciding which method to use. Protocols and examples are given for measuring O2 evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center.

Cryo-electron microscopy reveals hydrogen positions and water networks in photosystem II.

Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thus sustains life on Earth. It catalyzes two chemical reactions: water oxidation to molecular oxygen and plastoquinone reduction. Coupling of electron and proton transfer is crucial for efficiency; however, the molecular basis of these processes remains speculative owing to uncertain water binding sites and the lack of experimentally determined hydrogen positions. We thus collected high-resolution cryo-electron microscopy data of fully hydrated photosystem II from the thermophilic cyanobacterium Thermosynechococcus vestitus to a final resolution of 1.71 angstroms. The structure reveals several previously undetected partially occupied water binding sites and more than half of the hydrogen and proton positions. This clarifies the pathways of substrate water binding and plastoquinone B protonation.

Photosystem II, a growing complex: updates on newly discovered components and low molecular mass proteins.

Photosystem II is a unique complex capable of absorbing light and splitting water. The complex has been thoroughly studied and to date there are more than 40 proteins identified, which bind to the complex either stably or transiently. Another special feature of this complex is the unusually high content of low molecular mass proteins that represent more than half of the proteins. In this review we summarize the recent findings on the low molecular mass proteins (<15kDa) and present an overview of the newly identified components as well. We have also performed co-expression analysis of the genes encoding PSII proteins to see if the low molecular mass proteins form a specific sub-group within the Photosystem II complex. Interestingly we found that the chloroplast-localized genes encoding PSII proteins display a different response to environmental and stress conditions compared to the nuclear localized genes. This article is part of a Special Issue entitled: Photosystem II.

Isolation of monomeric photosystem II that retains the subunit PsbS.

Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221-226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content. A "milder" procedure than the protocol reported by Fey et al. (Biochim Biophys Acta 1777:1501-1509, 2008) led to almost exclusively monomeric PSII complexes which in part still bind the PsbS protein. This finding might support a role for PSII monomers in higher plants.

The PsbW protein stabilizes the supramolecular organization of photosystem II in higher plants.

PsbW, a 6.1-kDa low-molecular-weight protein, is exclusive to photosynthetic eukaryotes, and associates with the photosystem II (PSII) protein complex. In vivo and in vitro comparison of Arabidopsis thaliana wild-type plants with T-DNA insertion knock-out mutants completely lacking the PsbW protein, or with antisense inhibition plants exhibiting decreased levels of PsbW, demonstrated that the loss of PsbW destabilizes the supramolecular organization of PSII. No PSII-LHCII supercomplexes could be detected or isolated in the absence of the PsbW protein. These changes in macro-organization were accompanied by a minor decrease in the chlorophyll fluorescence parameter F(V) /F(M) , a strongly decreased PSII core protein phosphorylation and a modification of the redox state of the plastoquinone (PQ) pool in dark-adapted leaves. In addition, the absence of PsbW protein led to faster redox changes in the PQ pool, i.e. transitions from state 1 to state 2, as measured by changes in stationary fluorescence (F(S) ) kinetics, compared with the wild type. Despite these dramatic effects on macromolecular structure, the transgenic plants exhibited no significant phenotype under normal growth conditions. We suggest that the PsbW protein is located close to the minor antenna of the PSII complex, and is important for the contact and stability between several PSII-LHCII supercomplexes.

Extraordinary µs-ms backbone dynamics in Arabidopsis thaliana peroxiredoxin Q.

Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin-dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted the resolving cysteine, generating an intramolecular disulfide bond and release of a second water molecule. PrxQ is recycled to the active state by a thioredoxin-dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here, we have performed a detailed study of the activity, structure and dynamics of PrxQ in both the oxidized and reduced states. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Analysis of NMR spin relaxation rates shows that PrxQ is monomeric in both oxidized and reduced states. As evident from R(2) relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow µs-ms timescale. The ground state of this conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.

Arabidopsis plants grown in the field and climate chambers significantly differ in leaf morphology and photosystem components.

BACKGROUND: Plants exhibit phenotypic plasticity and respond to differences in environmental conditions by acclimation. We have systematically compared leaves of Arabidopsis thaliana plants grown in the field and under controlled low, normal and high light conditions in the laboratory to determine their most prominent phenotypic differences. RESULTS: Compared to plants grown under field conditions, the "indoor plants" had larger leaves, modified leaf shapes and longer petioles. Their pigment composition also significantly differed; indoor plants had reduced levels of xanthophyll pigments. In addition, Lhcb1 and Lhcb2 levels were up to three times higher in the indoor plants, but differences in the PSI antenna were much smaller, with only the low-abundance Lhca5 protein showing altered levels. Both isoforms of early-light-induced protein (ELIP) were absent in the indoor plants, and they had less non-photochemical quenching (NPQ). The field-grown plants had a high capacity to perform state transitions. Plants lacking ELIPs did not have reduced growth or seed set rates, but their mortality rates were sometimes higher. NPQ levels between natural accessions grown under different conditions were not correlated. CONCLUSION: Our results indicate that comparative analysis of field-grown plants with those grown under artificial conditions is important for a full understanding of plant plasticity and adaptation.