Far-red light enrichment affects gene expression and architecture as well as growth and photosynthesis in rice.

Plants use light as a resource and signal. Photons within the 400-700 nm waveband are considered photosynthetically active. Far-red photons (FR, 700-800 nm) are used by plants to detect nearby vegetation and elicit the shade avoidance syndrome. In addition, FR photons have also been shown to contribute to photosynthesis, but knowledge about these dual effects remains scarce. Here, we study shoot-architectural and photosynthetic responses to supplemental FR light during the photoperiod in several rice varieties. We observed that FR enrichment only mildly affected the rice transcriptome and shoot architecture as compared to established model species, whereas leaf formation, tillering and biomass accumulation were clearly promoted. Consistent with this growth promotion, we found that CO2-fixation in supplemental FR was strongly enhanced, especially in plants acclimated to FR-enriched conditions as compared to control conditions. This growth promotion dominates the effects of FR photons on shoot development and architecture. When substituting FR enrichment with an end-of-day FR pulse, this prevented photosynthesis-promoting effects and elicited shade avoidance responses. We conclude that FR photons can have a dual role, where effects depend on the environmental context: in addition to being an environmental signal, they are also a potent source of harvestable energy.

An artificial metalloenzyme that can oxidize water photocatalytically: design, synthesis, and characterization.

In nature, light-driven water oxidation (WO) catalysis is performed by photosystem II via the delicate interplay of different cofactors positioned in its protein scaffold. Artificial systems for homogeneous photocatalytic WO are based on small molecules that often have limited solubility in aqueous solutions. In this work, we alleviated this issue and present a cobalt-based WO-catalyst containing artificial metalloenzyme (ArM) that is active in light-driven, homogeneous WO catalysis in neutral-pH aqueous solutions. A haem-containing electron transfer protein, cytochrome B5 (CB5), served to host a first-row transition-metal-based WO catalyst, CoSalen (CoIISalen, where H2Salen = N,N'-bis(salicylidene)ethylenediamine), thus producing an ArM capable of driving photocatalytic WO. The CoSalen ArM formed a water-soluble pre-catalyst in the presence of [Ru(bpy)3](ClO4)2 as photosensitizer and Na2S2O8 as the sacrificial electron acceptor, with photocatalytic activity similar to that of free CoSalen. During photocatalysis, the CoSalen-protein interactions were destabilized, and the protein partially unfolded. Rather than forming tens of nanometer sized CoOx nanoparticles as free CoSalen does under photocatalytic WO conditions, the CB5 : CoSalen ArM showed limited protein cross-linking and remained soluble. We conclude that a weak, dynamic interaction between a soluble cobalt species and apoCB5 was formed, which generated a catalytically active adduct during photocatalysis. A detailed analysis was performed on protein stability and decomposition processes during the harsh oxidizing reaction conditions of WO, which will serve for the future design of WO ArMs with improved activity and stability.

Pd12MnL24 (for n = 6, 8, 12) nanospheres by post-assembly modification of Pd12L24 spheres.

In this contribution, we describe a post-assembly modification approach to selectively coordinate transition metals in Pd12L24 cuboctahedra. The herein reported approach involves the preparation of Pd12L24 nanospheres with protonated nitrogen donor ligands that are covalently linked at the interior. The so obtained Pd12(LH+)24 nanospheres are shown to be suitable for coordinative post-modification after deprotection by deprotonation. Selective formation of tetra-coordinated MB in Pd12MB6L24, tri-coordinated MB in Pd12MB8L24 nanospheres and two-coordinated MB in Pd12MB12L24 nanospheres is achieved as a result of different nitrogen donor ligands. A combination of pulsed EPR spectroscopy (DEER) to measure Cu-Cu distances in the different spheres, NMR studies and computational investigations, support the presence of the complexes at precise locations of the Pd12MB6L24 nanosphere. The general post-assembly modification methodology can be extended using other transition metal precursors or supramolecular systems and can guide precise formation and investigation of novel transition metal-complex containing nanospheres with well-defined composition.

In-depth magnetometry and EPR analysis of the spin structure of human-liver ferritin: from DC to 9 GHz.

Ferritin, the major iron storage protein in organisms, stores iron in the form of iron oxyhydroxide most likely involving phosphorous as a constituent, the mineral form of which is not well understood. Therefore, the question of how the ca. 2000 iron atoms in the ferritin core are magnetically coupled is still largely open. The ferritin core, with a diameter of 5-8 nm, is encapsulated in a protein shell that also catalyzes the uptake of iron and protects the core from outside interactions. Neurodegenerative disease is associated with iron imbalance, generating specific interest in the magnetic properties of ferritin. Here we present 9 GHz continuous wave EPR and a comprehensive set of magnetometry techniques including isothermal remanent magnetization (IRM) and AC susceptibility to elucidate the magnetic properties of the core of human liver ferritin. For the analysis of the magnetometry data, a new microscopic model of the ferritin-core spin structure is derived, showing that magnetic moment is generated by surface-spin canting, rather than defects. The analysis explicitly includes the distribution of magnetic parameters, such as the distribution of the magnetic moment. This microscopic model explains some of the inconsistencies resulting from previous analysis approaches. The main findings are a mean magnetic moment of 337µB with a standard deviation of 0.947µB. In contrast to previous reports, only a relatively small contribution of paramagnetic and ferrimagnetic phases is found, in the order of maximally 3%. For EPR, the over 30 mT wide signal of the ferritin core is analyzed using the model of the giant spin system [Fittipaldi et al., Phys. Chem. Chem. Phys., 2016, 18, 3591-3597]. Two components are needed minimally, and the broadening of these components suggests a broad distribution of the magnetic resonance parameters, the zero-field splitting, D, and the spin quantum number, S. We compare parameters from EPR and magnetometry and find that EPR is particularly sensitive to the surface spins of the core, revealing the potential to use EPR as a diagnostic for surface-spin disorder.

Effects of sublethal single, simultaneous and sequential abiotic stresses on phenotypic traits of Arabidopsis thaliana.

Plant responses to abiotic stresses are complex and dynamic, and involve changes in different traits, either as the direct consequence of the stress, or as an active acclimatory response. Abiotic stresses frequently occur simultaneously or in succession, rather than in isolation. Despite this, most studies have focused on a single stress and single or few plant traits. To address this gap, our study comprehensively and categorically quantified the individual and combined effects of three major abiotic stresses associated with climate change (flooding, progressive drought and high temperature) on 12 phenotypic traits related to morphology, development, growth and fitness, at different developmental stages in four Arabidopsis thaliana accessions. Combined sublethal stresses were applied either simultaneously (high temperature and drought) or sequentially (flooding followed by drought). In total, we analysed the phenotypic responses of 1782 individuals across these stresses and different developmental stages. Overall, abiotic stresses and their combinations resulted in distinct patterns of effects across the traits analysed, with both quantitative and qualitative differences across accessions. Stress combinations had additive effects on some traits, whereas clear positive and negative interactions were observed for other traits: 9 out of 12 traits for high temperature and drought, 6 out of 12 traits for post-submergence and drought showed significant interactions. In many cases where the stresses interacted, the strength of interactions varied across accessions. Hence, our results indicated a general pattern of response in most phenotypic traits to the different stresses and stress combinations, but it also indicated a natural genetic variation in the strength of these responses. This includes novel results regarding the lack of a response to drought after submergence and a decoupling between leaf number and flowering time after submergence. Overall, our study provides a rich characterization of trait responses of Arabidopsis plants to sublethal abiotic stresses at the phenotypic level and can serve as starting point for further in-depth physiological research and plant modelling efforts.

Ferritin-Based Single-Electron Devices.

We report on the fabrication of single-electron devices based on horse-spleen ferritin particles. At low temperatures the current vs. voltage characteristics are stable, enabling the acquisition of reproducible data that establishes the Coulomb blockade as the main transport mechanism through them. Excellent agreement between the experimental data and the Coulomb blockade theory is demonstrated. Single-electron charge transport in ferritin, thus, establishes a route for further characterization of their, e.g., magnetic, properties down to the single-particle level, with prospects for electronic and medical applications.

Quantification of different iron forms in the aceruloplasminemia brain to explore iron-related neurodegeneration.

AIMS: Aceruloplasminemia is an ultra-rare neurodegenerative disorder associated with massive brain iron deposits, of which the molecular composition is unknown. We aimed to quantitatively determine the molecular iron forms in the aceruloplasminemia brain, and to illustrate their influence on iron-sensitive MRI metrics. METHODS: The inhomogeneous transverse relaxation rate (R2*) and magnetic susceptibility obtained from 7 T MRI were combined with Electron Paramagnetic Resonance (EPR) and Superconducting Quantum Interference Device (SQUID) magnetometry. The basal ganglia, thalamus, red nucleus, dentate nucleus, superior- and middle temporal gyrus and white matter of a post-mortem aceruloplasminemia brain were studied. MRI, EPR and SQUID results that had been previously obtained from the temporal cortex of healthy controls were included for comparison. RESULTS: The brain iron pool in aceruloplasminemia detected in this study consisted of EPR-detectable Fe3+ ions, magnetic Fe3+ embedded in the core of ferritin and hemosiderin (ferrihydrite-iron), and magnetic Fe3+ embedded in oxidized magnetite/maghemite minerals (maghemite-iron). Ferrihydrite-iron represented above 90% of all iron and was the main driver of iron-sensitive MRI contrast. Although deep gray matter structures were three times richer in ferrihydrite-iron than the temporal cortex, ferrihydrite-iron was already six times more abundant in the temporal cortex of the patient with aceruloplasminemia compared to the healthy situation (162 µg/g vs. 27 µg/g), on average. The concentrations of Fe3+ ions and maghemite-iron in the temporal cortex in aceruloplasminemia were within the range of those in the control subjects. CONCLUSIONS: Iron-related neurodegeneration in aceruloplasminemia is primarily associated with an increase in ferrihydrite-iron, with ferrihydrite-iron being the major determinant of iron-sensitive MRI contrast.

In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers.

Knowledge of the mechanisms of assembly of amyloid proteins into aggregates is of central importance in building an understanding of neurodegenerative disease. Given that oligomeric intermediates formed during the aggregation reaction are believed to be the major toxic species, methods to track such intermediates are clearly needed. Here we present a method, electron paramagnetic resonance (EPR), by which the amount of intermediates can be measured over the course of the aggregation, directly in the reacting solution, without the need for separation. We use this approach to investigate the aggregation of α-synuclein (αS), a synaptic protein implicated in Parkinson's disease and find a large population of oligomeric species. Our results show that these are primary oligomers, formed directly from monomeric species, rather than oligomers formed by secondary nucleation processes, and that they are short-lived, the majority of them dissociates rather than converts to fibrils. As demonstrated here, EPR offers the means to detect such short-lived intermediate species directly in situ. As it relies only on the change in size of the detected species, it will be applicable to a wide range of self-assembling systems, making accessible the kinetics of intermediates and thus allowing the determination of their rates of formation and conversion, key processes in the self-assembly reaction.

Light signalling shapes plant-plant interactions in dense canopies.

Plants growing at high densities interact via a multitude of pathways. Here, we provide an overview of mechanisms and functional consequences of plant architectural responses initiated by light cues that occur in dense vegetation. We will review the current state of knowledge about shade avoidance, as well as its possible applications. On an individual level, plants perceive neighbour-associated changes in light quality and quantity mainly with phytochromes for red and far-red light and cryptochromes and phototropins for blue light. Downstream of these photoreceptors, elaborate signalling and integration takes place with the PHYTOCHROME INTERACTING FACTORS, several hormones and other regulators. This signalling leads to the shade avoidance responses, consisting of hyponasty, stem and petiole elongation, apical dominance and life cycle adjustments. Architectural changes of the individual plant have consequences for the plant community, affecting canopy structure, species composition and population fitness. In this context, we highlight the ecological, evolutionary and agricultural importance of shade avoidance.

Lipid and protein dynamics of stacked and cation-depletion induced unstacked thylakoid membranes.

Chloroplast thylakoid membranes in plants and green algae form 3D architectures of stacked granal membranes interconnected by unstacked stroma lamellae. They undergo dynamic structural changes as a response to changing light conditions that involve grana unstacking and lateral supramolecular reorganization of the integral membrane protein complexes. We assessed the dynamics of thylakoid membrane components and addressed how they are affected by thylakoid unstacking, which has consequences for protein mobility and the diffusion of small electron carriers. By a combined nuclear and electron paramagnetic-resonance approach the dynamics of thylakoid lipids was assessed in stacked and cation-depletion induced unstacked thylakoids of Chlamydomonas (C.) reinhardtii. We could distinguish between structural, bulk and annular lipids and determine membrane fluidity at two membrane depths: close to the lipid headgroups and in the lipid bilayer center. Thylakoid unstacking significantly increased the dynamics of bulk and annular lipids in both areas and increased the dynamics of protein helices. The unstacking process was associated with membrane reorganization and loss of long-range ordered Photosystem II- Light-Harvesting Complex II (PSII-LHCII) complexes. The fluorescence lifetime characteristics associated with membrane unstacking are similar to those associated with state transitions in intact C. reinhardtii cells. Our findings could be relevant for understanding the structural and functional implications of thylakoid unstacking that is suggested to take place during several light-induced processes, such as state transitions, photoacclimation, photoinhibition and PSII repair.

A Two-Armed Probe for In-Cell DEER Measurements on Proteins*.

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed GdIII complexes, GdIII -CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly GdIII labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy.

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant KD of 20±4×10-6  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.

Quantitative comparison of different iron forms in the temporal cortex of Alzheimer patients and control subjects.

We present a quantitative study of different molecular iron forms found in the temporal cortex of Alzheimer (AD) patients. Applying the methodology we developed in our previous work, we quantify the concentrations of non-heme Fe(III) by Electron Paramagnetic Resonance (EPR), magnetite/maghemite and ferrihydrite by SQUID magnetometry, together with the MRI transverse relaxation rate [Formula: see text], to obtain a systematic view of molecular iron in the temporal cortex. Significantly higher values of [Formula: see text], a larger concentration of ferrihydrite, and a larger magnetic moment of magnetite/maghemite particles are found in the brain of AD patients. Moreover, we found correlations between the concentration of the iron detected by EPR, the concentration of the ferrihydrite mineral and the average iron loading of ferritin. We discuss these findings in the framework of iron dis-homeostasis, which has been proposed to occur in the brain of AD patients.

Coiled-coil formation of the membrane-fusion K/E peptides viewed by electron paramagnetic resonance.

The interaction of the complementary K (Ac-(KIAALKE)3-GW-NH2) and E (Ac-(EIAALEK)3-GY-NH2) peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009) is investigated by electron paramagnetic resonance (EPR). By frozen solution continuous-wave EPR and double electron-electron resonance (DEER), the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm) and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.

["StigMa" - Evaluation of a Psychological Therapy Program for Stigma-Management]. / "StigMa" ­ Evaluation eines psychologischen Therapieprogramms zu Stigma-Management.

OBJECTIVE: The project "Stigma Management - StigMa" aims on the evaluation of an adaptive therapy program for patients with psychiatric illness to help them in managing internalized stigma and self-stigmatization. METHODS: The patients for this pilot-study were recruited in day-hospitals of pro mente tirol. 26 patients participated in 11 group sessions, following 6 modules: "Education", "Activation of Resources", "Social Network", "Self-Esteem", "Social competence in public places" and "My personal stigma management". The control group consisted of 20 patients who did not participate in StigMa. Pre-post-evaluation was done by the Internalized Stigma of Mental Illness-Scale 1. RESULTS: No significant interaction effects could be observed, although in the treatment group, the burden of perceived discrimination was significantly less pronounced after training than before it. The program, however, was evaluated as being extremely positive by the participants. CONCLUSIONS: The program StigMa will be adapted in accordance with the suggestions of the participants and reevaluated taking into consideration methodological optimization.

Human-brain ferritin studied by muon spin rotation: a pilot study.

Muon spin rotation is employed to investigate the spin dynamics of ferritin proteins isolated from the brain of an Alzheimer's disease (AD) patient and of a healthy control, using a sample of horse-spleen ferritin as a reference. A model based on the Néel theory of superparamagnetism is developed in order to interpret the spin relaxation rate of the muons stopped by the core of the protein. Using this model, our preliminary observations show that ferritins from the healthy control are filled with a mineral compatible with ferrihydrite, while ferritins from the AD patient contain a crystalline phase with a larger magnetocrystalline anisotropy, possibly compatible with magnetite or maghemite.

Membrane Binding of Parkinson's Protein α-Synuclein: Effect of Phosphorylation at Positions 87 and 129 by the S to D Mutation Approach.

Human α-synuclein, a protein relevant in the brain with so-far unknown function, plays an important role in Parkinson's disease. The phosphorylation state of αS was related to the disease, prompting interest in this process. The presumed physiological function and the disease action of αS involves membrane interaction. Here, we study the effect of phosphorylation at positions 87 and 129, mimicked by the mutations S87A, S129A (nonphosphorylated) and S87D, S129D (phosphorylated) on membrane binding. Local binding is detected by spin-label continuous-wave electron paramagnetic resonance. For S87A/D, six positions (27, 56, 63, 69, 76, and 90) are probed; and for S129A/D, three (27, 56, and 69). Binding to large unilamellar vesicles of 100 nm diameter of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in a 1 : 1 composition is not affected by the phosphorylation state of S129. For phosphorylation at S87, local unbinding of αS from the membrane is observed. We speculate that modulating the local membrane affinity by phosphorylation could tune the way αS interacts with different membranes; for example, tuning its membrane fusion activity.

Integrated Computational Approach to the Electron Paramagnetic Resonance Characterization of Rigid 310-Helical Peptides with TOAC Nitroxide Spin Labels.

We address the interpretation, via an integrated computational approach, of the experimental continuous-wave electron paramagnetic resonance (cw-EPR) spectra of a complete set of conformationally highly restricted, stable 310-helical peptides from hexa- to nonamers, each bis-labeled with nitroxide radical-containing TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) residues. The usefulness of TOAC for this type of analysis has been shown already to be due to its cyclic piperidine side chain, which is rigidly connected to the peptide backbone α-carbon. The TOAC α-amino acids are separated by two, three, four, and five intervening residues. This set of compounds has allowed us to modulate both the radical···radical distance and the relative orientation parameters. To further validate our conclusion, a comparative analysis has been carried out on three singly TOAC-labeled peptides of similar main-chain length.

A novel approach to quantify different iron forms in ex-vivo human brain tissue.

We propose a novel combination of methods to study the physical properties of ferric ions and iron-oxide nanoparticles in post-mortem human brain, based on the combination of Electron Paramagnetic Resonance (EPR) and SQUID magnetometry. By means of EPR, we derive the concentration of the low molecular weight iron pool, as well as the product of its electron spin relaxation times. Additionally, by SQUID magnetometry we identify iron mineralization products ascribable to a magnetite/maghemite phase and a ferrihydrite (ferritin) phase. We further derive the concentration of magnetite/maghemite and of ferritin nanoparticles. To test out the new combined methodology, we studied brain tissue of an Alzheimer's patient and a healthy control. Finally, we estimate that the size of the magnetite/maghemite nanoparticles, whose magnetic moments are blocked at room temperature, exceeds 40-50 nm, which is not compatible with the ferritin protein, the core of which is typically 6-8 nm. We believe that this methodology could be beneficial in the study of neurodegenerative diseases such as Alzheimer's Disease which are characterized by abnormal iron accumulation in the brain.