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Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme – the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions. Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. Here the authors determine the reduction potentials of the iron-sulphur clusters of PS-CI and thus the bioenergetics of the electron transfer relay.

Remote sensing methods enable detection of solar-induced chlorophyll a fluorescence. However, to unleash the full potential of this signal, intensive cross-disciplinary work is required to harmonize biophysical and ecophysiological studies. For decades, the dynamic nature of chlorophyll a fluorescence (ChlaF) has provided insight into the biophysics and ecophysiology of the light reactions of photosynthesis from the subcellular to leaf scales. Recent advances in remote sensing methods enable detection of ChlaF induced by sunlight across a range of larger scales, from using instruments mounted on towers above plant canopies to Earth-orbiting satellites. This signal is referred to as solar-induced fluorescence (SIF) and its application promises to overcome spatial constraints on studies of photosynthesis, opening new research directions and opportunities in ecology, ecophysiology, biogeochemistry, agriculture and forestry. However, to unleash the full potential of SIF, intensive cross-disciplinary work is required to harmonize these new advances with the rich history of biophysical and ecophysiological studies of ChlaF, fostering the development of next-generation plant physiological and Earth-system models. Here, we introduce the scale-dependent link between SIF and photosynthesis, with an emphasis on seven remaining scientific challenges, and present a roadmap to facilitate future collaborative research towards new applications of SIF.

Photosynthetic reaction centers (RC) catalyze the conversion of light to chemical energy that supports life on Earth, but they exhibit substantial diversity among different phyla. This is exemplified in a recent structure of the RC from an anoxygenic green sulfur bacterium (GsbRC) which has characteristics that may challenge the canonical view of RC classification. The GsbRC structure is analyzed and compared with other RCs, and the observations reveal important but unstudied research directions that are vital for disentangling RC evolution and diversity. Namely, (1) common themes of electron donation implicate a Ca2+ site whose role is unknown; (2) a previously unidentified lipid molecule with unclear functional significance is involved in the axial ligation of a cofactor in the electron transfer chain; (3) the GsbRC features surprising structural similarities with the distantly-related photosystem II; and (4) a structural basis for energy quenching in the GsbRC can be gleaned that exemplifies the importance of how exposure to oxygen has shaped the evolution of RCs. The analysis highlights these novel avenues of research that are critical for revealing evolutionary relationships that underpin the great diversity observed in extant RCs. Supplementary Information The online version contains supplementary material available at 10.1007/s11120-021-00857-9.

Construction of green nanodevices characterised by excellent long-term performance remains high priority in biotechnology and medicine. Tight electronic coupling of proteins to electrodes is essential for efficient direct electron transfer (DET) across the bio-organic interface. Rational modulation of this coupling depends on in-depth understanding of the intricate properties of interfacial DET. Here, we dissect the molecular mechanism of DET in a hybrid nanodevice in which a model electroactive protein, cytochrome c(553) (cyt c(553)), naturally interacting with photosystem I, was interfaced with single layer graphene (SLG) via the conductive self-assembled monolayer (SAM) formed by pyrene-nitrilotriacetic acid (pyr-NTA) molecules chelated to transition metal redox centers. We demonstrate that efficient DET occurs between graphene and cyt c(553) whose kinetics and directionality depends on the metal incorporated into the bio-organic interface: Co enhances the cathodic current from SLG to haem, whereas Ni exerts the opposite effect. QM/MM simulations yield the mechanistic model of interfacial DET based on either tunnelling or hopping of electrons between graphene, pyr-NTA-M2+ SAM and cyt c(553) depending on the metal in SAM. Considerably different electronic configurations were identified for the interfacial metal redox centers: a closed-shell system for Ni and a radical system for the Co with altered occupancy of HOMO/LUMO levels. The feasibility of fine-tuning the electronic properties of the bio-molecular SAM upon incorporation of various metal centers paves the way for the rational design of the optimal molecular interface between abiotic and biotic components of the viable green hybrid devices, e.g. solar cells, optoelectronic nanosystems and solar-to-fuel assemblies.

Achieving food security and sustainable food production is a major challenge for plant scientists. To accomplish this, the global food production needs not only to be remarkably boosted, but it has to be achieved under harsh environmental conditions. Moreover, the climate change scenarios estimate an enhanced pressure on crop yields in the upcoming decades. C-4 photosynthesis is highly promising to meet these challenges to global food production. Under current CO2 levels, C-4 photosynthesis is more efficient than C-3 photosynthesis, but more data is needed to map out its response under elevated CO2 (eCO(2)) conditions. Growing evidence also suggests that C-4 photosynthesis could be more efficient in water use under eCO(2). Production of reactive oxygen species (ROS) is an inevitable consequence of oxygenic photosynthesis and is also one of the first responses to environmental stresses. C-3 and C-4 plants have different ROS profiles, mainly because of reduced photorespiration in the latter. Moreover, the effects of eCO(2) on C-3 and especially C-4 plants remain poorly understood. Since C-3 and C-4 plants have different ROS production patterns, it is likely that ROS signalling and downstream effects on growth and development differ between C-3 and C-4 plants, which may result in different response to eCO(2). This would also be reflected in reproductive success and crop yields. Here we evaluate the recent literature on C-3 and C-4 plant responses to climate change conditions from the abiotic stress tolerance and food security perspectives, with redox connections. Current body of knowledge suggests engineering C-4 photosynthesis into major crops to be a viable way to increase yield, but such attempts have failed because of a lack of basic knowledge in this area. Therefore, this article also aims to fill this gap from the redox perspective., Scientific and Technological Research Council of Turkey (TUBITAK) [110 T289, 114Z991]; Ege University Scientific Research Grant [2011/BIL/009]; H2020 Marie Sklodowska-Curie Actions [101018915], The research on C4 photosynthesis of the authors (to I.T., R.O. and B.U.) were supported by The Scientific and Technological Research Council of Turkey (TUBITAK) (110 T289, 114Z991) and Ege University Scientific Research Grant (2011/BIL/009). S.A.G acknowledges the funding from H2020 Marie Sklodowska-Curie Actions, H2020-MSCA-IF-2021 #101018915.

Abstract An open hardware design and implementation for a transient absorption spectrometer are presented that has microsecond time resolution and measures full difference spectra in the visible spectral region from 380 to 750 nm. The instrument has been designed to allow transient absorption spectroscopy measurements of either low or high quantum yield processes by combining intense sub-microsecond excitation flashes using a xenon lamp together with stroboscopic non-actinic white light probing using LED sources driven under high pulsed current from a capacitor bank. The instrument is sensitive to resolve 0.15 mOD flash-induced differences within 1000 measurements at 20 Hz repetition rate using an inexpensive CCD sensor with 200 μm pixel dimension, 40 K electrons full well capacity and a dynamic range of 1800. The excitation flash has 230 ns pulse duration and the 2 mJ flash energy allows spectral filtering while retaining high power density with focussing to generate mOD signals in the 10–4–10–1 ΔOD range. We present the full electronics design and construction of the flash and probe sources, the optics as well as the timing electronics and CCD spectrometer operation and modification for internal signal referencing. The performance characterisation and example measurements are demonstrated using microsecond TAS of Congo red dye, as an example of a low quantum yield photoreaction at 2% with up to 78% of molecules excited. The instrument is fully open hardware and combines inexpensive selection of commercial components, optics and electronics and allows linear response measurements of photoinduced reactions for the purpose of accurate global analysis of chemical dynamics. Graphical abstract

Novel satellite measurements of solar-induced chlorophyll fluorescence (SIF) can improve our understanding of global photosynthesis; however, little is known about how to interpret the controls on its spectral variability. To address this, we disentangle simultaneous drivers of fluorescence spectra by coupling active and passive fluorescence measurements with photosynthesis. We show empirical and mechanistic evidence for where, why, and to what extent leaf fluorescence spectra change. Three distinct components explain more than 95% of the variance in leaf fluorescence spectra under both steady-state and changing illumination conditions. A single spectral shape of fluorescence explains 84% of the variance across a wide range of species. The magnitude of this shape responds to absorbed light and photosynthetic up/down regulation; meanwhile, chlorophyll concentration and nonphotochemical quenching control 9% and 3% of the remaining spectral variance, respectively. The spectral shape of fluorescence is remarkably stable where most current satellite retrievals occur (far-red, >740nm), and dynamic downregulation of photosynthesis reduces fluorescence magnitude similarly across the 670- to 850-nm range. We conduct an exploratory analysis of hourly red and far-red canopy SIF in soybean, which shows a subtle change in red:far-red fluorescence coincident with photosynthetic downregulation but is overshadowed by longer-term changes in canopy chlorophyll and structure. Based on our leaf and canopy analysis, caution should be taken when attributing large changes in the spectral shape of remotely sensed SIF to plant stress, particularly if data acquisition is temporally sparse. Ultimately, changes in SIF magnitude at wavelengths greater than 740 nm alone may prove sufficient for tracking photosynthetic dynamics. Plain Language Summary Satellite remote sensing provides a global picture of photosynthetic activity-allowing us to see when, where, and how much CO2 plants are assimilating. To do this, satellites measure a small emission of energy from the plants called chlorophyll fluorescence. However, this measurement is typically made across a narrow wavelength range, while the emission spectrum (650-850 nm) is quite dynamic. We show where, why, and to what extent leaf fluorescence spectra change across a diverse range of species and conditions, ultimately informing canopy remote sensing measurements. Results suggest that wavelengths currently used by satellites are stable enough to track the downregulation of photosynthesis resulting from stress, while spectral shape changes respond more strongly to dynamics in canopy structure and chlorophyll concentration.

The highly efficient bioelectrodes based on single layer graphene (SLG) functionalized with pyrene self-assembled monolayer and novel cytochrome c(553) (cyt c(553)) peptide linker variants were rationally designed to optimize the direct electron transfer (DET) between SLG and the heme group of cyt. Through a combination of photoelectrochemical and quantum mechanical (QM/MM) approaches we show that the specific amino acid sequence of a short peptide genetically inserted between the cyt c(553) - holoprotein and the surface anchoring C-terminal His s -tag plays a crucial role in ensuring the optimal orientation and distance of the heme group with respect to the SLG surface. Consequently, efficient DET occurring between graphene and cyt c(553) leads to a 20-fold enhancement of the cathodic photocurrent output compared to the previously reported devices of a similar type. The QM/MM modeling implies that a perpendicular or parallel orientation of the heme group with respect to the SLG surface is detrimental to DET, whereas the tilted orientation favors the cathodic photocurrent generation. Our work confirms the possibility of fine-tuning the electronic communication within complex bio-organic nanoarchitectures and interfaces due to optimization of the tilt angle of the heme group, its distance from the SLG surface and optimal HOMO/LUMO levels of the interacting redox centers. (C) 2021 The Authors. Published by Elsevier B.V.

We compare theoretical methods for calculating excitation energy transfer rates in multichromophoric systems. The employed methods are the multichromophoric Forster resonance energy transfer (MC-FRET), the numerical integration of the Schrodinger equation (NISE), and the Haken-Strobl-Reineker (HSR) model. As a reference, we use the numerically exact Hierarchy of Equations of Motion (HEOM). We examine these methods in various system and bath parameter regimes including the regime relevant to biological light-harvesting systems. We apply them to a model system of a monomeric donor coupled to a multichromophoric acceptor ring of varying size in two limiting configurations, namely symmetric and asymmetric. We find that the symmetric case is more sensitive to the approximations of the methods studied. The NISE method gives the most reasonable results throughout the parameter regimes tested, while still being computationally tractable.

The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O 2 , generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1 , we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1 . NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1 , but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1 , but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1 , in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MDS gene products may affect the Mehler reaction by altering the availability of O 2 . In green tissues, this putative effect is masked by photosynthetic O 2 evolution. However, O 2 evolution was rapidly suppressed in MV-treated plants. Transcriptomic meta-analysis indicated that MDS gene expression is linked to hypoxic response not only under MV, but also in standard growth conditions. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

A considerable literature has developed around the concept that the complexity of biological organisms, and the development of ever increasing complexity during biological evolution, is driven, in some way, by the maximization of entropy production (MEP). Most of these studies deal in very general terms with the living state and do not examine specific biological models. In the present study, we discuss some of the basic postulates of MEP as they are applied to living organisms. It is concluded that the MEP ideas seem to have little relevance either for the development of biological complexity or biological evolution.

ISSN: 0556-3860 WOS:000550058500006; International audience; Time-resolved infrared (IR) spectroscopy is a well established technique for the study of photobiological systems. In this review attention is focused on technical improvements (instrumental, data analysis, spectral simulation, methodology, sample preparation, isotope-labelling strategies, vibrational probes) and on the scientific results obtained since 2015 for time-resolved IR spectroscopy in the (sub-) ns - second time range. A peculiar emphasis will be given to studies in the field of photosynthesis. Finally, after the general conclusions, the perspectives for the application of time-resolved IR spectroscopy in photobiology are described.

We present ground and excited state frequency calculations of the recently discovered extremely red-shifted chlorophyll f. We discuss the experimentally available vibrational mode assignments of chlorophyll f and chlorophyll a which are characterised by particularly large downshifts of 131-keto mode in the excited state. The accuracy of excited state frequencies and their displacements are evaluated by the construction of Franck&ndash, Condon (FC) and Herzberg&ndash, Teller (HT) progressions at the CAM-B3LYP/6-31G(d) level. Results show that while CAM-B3LYP results are improved relative to B3LYP calculations, the displacements and downshifts of high-frequency modes are underestimated still, and that the progressions calculated for low temperature are dominated by low-frequency modes rather than fingerprint modes that are Resonant Raman active.

Successful measurements of chlorophyll fluorescence (ChlF) spectral properties (typically in the wavelength range of 650–850 nm) across plant species, environmental conditions, and stress levels are a first step towards establishing a quantitative link between solar-induced chlorophyll fluorescence (SIF), which can only be measured at discrete ChlF spectral bands, and photosynthetic functionality. Despite its importance and significance, the various methodologies for the estimation of leaf-level ChlF spectral properties have not yet been compared, especially when applied to leaves with complex morphology, such as needles. Here we present, to the best of our knowledge, a first comparison of protocols for measuring leaf-level ChlF spectra: a custom-made system designed to measure ChlF spectra at ambient and 77 K temperatures (optical chamber, OC), the widely used FluoWat leaf clip (FW), and an integrating sphere setup (IS). We test the three methods under low-light conditions, across two broadleaf species and one needle-like species. For the conifer, we characterize the effect of needle arrangements: one needle, three needles, and needle mats with as little gap fraction as technically possible. We also introduce a simple baseline correction method to account for non-fluorescence-related contributions to spectral measurements. Baseline correction was found especially useful in recovering the spectra nearby the filter cut-off. Results show that the shape of the leaf-level ChlF spectra remained largely unaffected by the measurement methodology and geometry in OC and FW methods. Substantially smaller red/far-red ratios were observed in the IS method. The comparison of needle arrangements indicated that needle mats could be a practical solution to investigate temporal changes in ChlF spectra of needle-like leaves as they produced more reproducible results and higher signals.

Epigenetics offers important opportunities in breeding to improve the potential yield in a wide variety of crops. Starting from a pure breeder seed lot of a rice (Oryza sativa ssp. indica) inbred population, repeated testing for improved cellular respiration rates and energy use efficiency (EUE) over three generations identified performant epilines with distinct epigenetic signatures and with improved seed yield in field trials. Epiline DNA methylomes were characterized by genome-wide bisulfite sequencing to discern cytosine methylation changes in relation to transcriptome and phenotype. Regional methylation changes were dispersed over the epiline genomes. A number of upstream-associated differentially methylated regions (DMRs) correlated with differentially expressed genes (DEGs) with a role in particular molecular functions like transmembrane transport and protein kinase activity. Targeted bisulfite sequencing confirmed epiline DMRs that anti-correlated with DEGs, identifying putative epialleles that were susceptible for cytosine methylation changes that might affect gene expression and contribute to the phenotype. Chromatin immunoprecipitation sequencing revealed the extensive enrichment of gene-associated histone H3 lysine-4 trimethylation (H3K4me3), which correlated with gene activation and reduced cytosine methylation. Our data indicate that seed formation is prone to epigenetic changes that might be used as a resource in crop improvement.

Oxygenic photosynthesis crucially depends on proteins that possess Fe (2+) or Fe/S complexes as co-factors or prosthetic groups. Here, we show that the small regulatory RNA (sRNA) IsaR1 (Iron-Stress-Activated RNA 1) plays a pivotal role in acclimation to low-iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe (2+)-containing proteins involved in photosynthetic electron transfer, detoxification of anion radicals, citrate cycle, and tetrapyrrole biogenesis. IsaR1 is essential for maintaining physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (1) directly, via posttranscriptional repression of gene expression; (2) indirectly, via suppression of pigment; and (3) Fe/S cluster biosynthesis. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a critically important riboregulator. These findings provide a new perspective for understanding the regulation of iron homeostasis in photosynthetic organisms. German Federal Ministry of Education and Research [0316165] DFG [HE 2544/9-1] Academy of Finland [253269, 271832, 273870] Portuguese Fundacao para a Ciencia e a Tecnologia [IF/00881/2013, UID/Multi/04326/2013-CCMAR] European Commission FP7 Marie Curie Initial Training Network "Photo.COMM'' [317184] info:eu-repo/semantics/publishedVersion

International audience; Pea (Pisum sativum L.) productivity is linked to its ability to cope with abiotic stresses such as low temperatures during fall and Winter. In this study, we investigate the chloroplast-related changes occurring during pea cold acclimation, in order to further lead to genetic improvement of its field performance. Champagne and Terese, two pea lines with different acclimation capabilities, were studied by physiological measurements, sub-cellular fractionation followed by relative protein quantification and two-dimensional DICE. The chilling tolerance might be related to an increase in protein related to soluble sugar synthesis, antioxidant potential, regulation of mRNA transcription and translation through the chloroplast. Freezing tolerance, only observed in Champagne, seems to rely on a higher inherent photosynthetic potential at the beginning of the cold exposure, combined with an early ability to start metabolic processes aimed at maintaining the photosynthetic capacity, optimizing the stoichiomety of the photosystems and inducing dynamic changes in carbohydrate and protein synthesis and/or turnover. (C) 2013 Elsevier B.V. All rights reserved.

Thomas et al. show that a novel protein, Eros, controls the abundance of components of the phagocyte NADPH oxidase, making it essential for the phagocyte respiratory burst and defense against common infections., The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox. Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.

The term 'photosynthetic control' describes the short- and long-term mechanisms that regulate reactions in the photosynthetic electron transport (PET) chain so that the rate of production of ATP and NADPH is coordinated with the rate of their utilization in metabolism. At low irradiances these mechanisms serve to optimize light use efficiency, while at high irradiances they operate to dissipate excess excitation energy as heat. Similarly, the production of ATP and NADPH in ratios tailored to meet demand is finely tuned by a sophisticated series of controls that prevents the accumulation of high NAD(P)H/NAD(P) ratios and ATP/ADP ratios that would lead to potentially harmful over-reduction and inactivation of PET chain components. In recent years, photosynthetic control has also been extrapolated to the regulation of gene expression because mechanisms that are identical or similar to those that serve to regulate electron flow through the PET chain also coordinate the regulated expression of genes encoding photosynthetic proteins. This requires coordinated gene expression in the chloroplasts, mitochondria, and nuclei, involving complex networks of forward and retrograde signalling pathways. Photosynthetic control operates to control photosynthetic gene expression in response to environmental and metabolic changes. Mining literature data on transcriptome profiles of C(3) and C(4) leaves from plants grown under high atmospheric carbon dioxide (CO(2)) levels compared with those grown with ambient CO(2) reveals that the transition to higher photorespiratory conditions in C(3) plants enhances the expression of genes associated with cyclic electron flow pathways in Arabidopsis thaliana, consistent with the higher ATP requirement (relative to NADPH) of photorespiration.

The light-harvesting complexes of photosystem I and II (Lhcas and Lhcbs) of plants display a high structural homology and similar pigment content and organization. Yet, the spectroscopic properties of these complexes, and accordingly their functionality, differ substantially. This difference is primarily due to the charge-transfer (CT) character of a chlorophyll dimer in all Lhcas, which mixes with the excitonic states of these complexes, whereas this CT character is generally absent in Lhcbs. By means of single-molecule spectroscopy near room temperature, we demonstrate that the presence or absence of such a CT state in Lhcas and Lhcbs can occasionally be reversed; i.e., these complexes are able to interconvert conformationally to quasi-stable spectral states that resemble the Lhcs of the other photosystem. The high structural similarity of all the Lhca and Lhcb proteins suggests that the stable conformational states that give rise to the mixed CT-excitonic state are similar for all these proteins, and similarly for the conformations that involve no CT state. This indicates that the specific functions related to Lhca and Lhcb complexes are realized by different stable conformations of a single generic protein structure. We propose that this functionality is modulated and controlled by the protein environment.

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structures at low, medium, and high resolution. Since electron micrographs of biological objects are very noisy, improvement of the signal-to-noise ratio by image processing is an integral part of EM, and this is performed by averaging large numbers of individual projections. Averaging procedures can be divided into crystallographic and non-crystallographic methods. The crystallographic averaging method, based on two-dimensional (2D) crystals of (membrane) proteins, yielded in solving atomic protein structures in the last century. More recently, single particle analysis could be extended to solve atomic structures as well. It is a suitable method for large proteins, viruses, and proteins that are difficult to crystallize. Because it is also a fast method to reveal the low-to-medium resolution structures, the impact of its application is growing rapidly. Technical aspects, results, and possibilities are presented.

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structures at low, medium, and high resolution. Since electron micrographs of biological objects are very noisy, improvement of the signal-to-noise ratio by image processing is an integral part of EM, and this is performed by averaging large numbers of individual projections. Averaging procedures can be divided into crystallographic and non-crystallographic methods. The crystallographic averaging method, based on two-dimensional (2D) crystals of (membrane) proteins, yielded in solving atomic protein structures in the last century. More recently, single particle analysis could be extended to solve atomic structures as well. It is a suitable method for large proteins, viruses, and proteins that are difficult to crystallize. Because it is also a fast method to reveal the low-to-medium resolution structures, the impact of its application is growing rapidly. Technical aspects, results, and possibilities are presented.

Summary There is a growing interest in the use of microalgae as low‐cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein‐coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome‐binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG→UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co‐introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae.

In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43′. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of ∼2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light. © 2005 American Chemical Society.

A significant part of global primary productivity is provided by cyanobacteria, which areabundant in most marine and freshwater habitats. In many oceanographic regions, however, theconcentration of iron can be so low that it limits growth. Cyanobacteria respond to this condition byexpressing a number of iron stress inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43?. It was recently shown that 18 IsiA proteins encircle trimeric photosystem I (PSI) under iron-deficient growth conditions. We report here that after prolonged growth of Synechocystis PCC 6803 in an iron-deficient medium, the number of bound IsiA proteins can be much higher than previously known. The largest complexes bind 12-14 units in an inner ring and 19-21 units in an outer ring around a PSI monomer. Fluorescence excitation spectra indicate an efficient light harvesting function for all PSI-bound chlorophylls. We also find that IsiA accumulates in cyanobacteria in excess of what is needed for functional light harvesting by PSI, and that a significant part of IsiA builds supercomplexes without PSI. Because the further decline of PSI makes photosystem II (PSII) increasingly vulnerable to photooxidation, we postulate that the surplus synthesis of IsiA shields PSII from excess light. We suggest that IsiA plays a surprisingly versatile role in cyanobacteria, by significantly enhancing the light harvestingability of PSI and providing photoprotection for PSII.

Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes., Author Summary Plastids originated from cyanobacteria that were incorporated into the eukaryotic cell through an endosymbiotic relationship. During the gradual evolution from endosymbiont to organelle, most genes of the cyanobacterial genome were transferred to the nuclear genome. Therefore, plastid biogenesis and function relies on nuclear gene expression and the import of these gene products into plastids, with the molecular dialogue between these two plant cell compartments therefore needing a precise coordination. Nuclei-to-chloroplast communication, and vice versa, are thus regulated through anterograde and retrograde signaling pathways, respectively. Post-transcriptional RNA editing of plastid RNAs by nuclear encoded regulatory proteins, such as pentatricopetide repeat (PPRs) proteins, represents one of such mechanisms of control. Through the characterization of the nuclear-encoded OCP3 protein, previously found to function as a disease resistance regulator in Arabidopsis, we have discovered a pathogen-sensitive editing-mediated control of the plastidial NDH complex involved in cyclic electron flow (CEF) around photosystem I. This led us to find that different PPRs controlling editing extent of transcripts for plastidial NDH complex are modulated by pathogenic cues. Our results thus represent the first series of evidence indicating engagement of chloroplast RNA editing and chloroplast NDH activity in plant immunity.

Chlorophyll fluorescence induction curves induced by an actinic pulse of red light follow different kinetics in dark-adapted plant leaves and leaves preilluminated with far-red light. This influence of far-red light was abolished in leaves infiltrated with valinomycin known to eliminate the electrical (Δφ) component of the proton-motive force and was strongly enhanced in leaves infiltrated with nigericin that abolishes the ΔpH component. The supposed influence of ionophores on different components of the proton motive force was supported by differential effects of these ionophores on the induction curves of the millisecond component of chlorophyll delayed fluorescence. Comparison of fluorescence induction curves with the kinetics of P700 oxidation in the absence and presence of ionophores suggests that valinomycin facilitates a build-up of a rate-limiting step for electron transport at the site of plastoquinone oxidation, whereas nigericin effectively removes limitations at this site. Far-red light was found to be a particularly effective modulator of electron flows in chloroplasts in the absence of ΔpH backpressure on operation of the electron-transport chain.

In addition to the linear electron flow, a cyclic electron flow (CEF) around photosystem I occurs in chloroplasts. In CEF, electrons flow back from the donor site of photosystem I to the plastoquinone pool via two main routes: one that involves the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (PGR) and one that is dependent of the NADH dehydrogenase-like complex. While the importance of CEF in photosynthesis and photoprotection has been clearly established, little is known about its regulation. We worked on the assumption of a redox regulation and surveyed the putative role of chloroplastic thioredoxins (TRX). Using Arabidopsis (Arabidopsis thaliana) mutants lacking different TRX isoforms, we demonstrated in vivo that TRXm4 specifically plays a role in the down-regulation of the NADH dehydrogenase-like complex-dependent plastoquinone reduction pathway. This result was confirmed in tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. In vitro assays performed with isolated chloroplasts and purified TRXm4 indicated that TRXm4 negatively controls the PGR pathway as well. The physiological significance of this regulation was investigated under steady-state photosynthesis and in the pgr5 mutant background. Lack of TRXm4 reversed the growth phenotype of the pgr5 mutant, but it did not compensate for the impaired photosynthesis and photoinhibition sensitivity. This suggests that the physiological role of TRXm4 occurs in vivo via a mechanism distinct from direct up-regulation of CEF. © 2012 American Society of Plant Biologists. All Rights Reserved.

Reverse engineering the whole-genome networks of complex multicellular organisms continues to remain a challenge. While simpler models easily scale to large number of genes and gene expression datasets, more accurate models are compute intensive limiting their scale of applicability. To enable fast and accurate reconstruction of large networks, we developed Tool for Inferring Network of Genes (TINGe), a parallel mutual information (MI)-based program. The novel features of our approach include: (i) B-spline-based formulation for linear-time computation of MI, (ii) a novel algorithm for direct permutation testing and (iii) development of parallel algorithms to reduce run-time and facilitate construction of large networks. We assess the quality of our method by comparison with ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) and GeneNet and demonstrate its unique capability by reverse engineering the whole-genome network of Arabidopsis thaliana from 3137 Affymetrix ATH1 GeneChips in just 9 min on a 1024-core cluster. We further report on the development of a new software Gene Network Analyzer (GeNA) for extracting context-specific sub-networks from a given set of seed genes. Using TINGe and GeNA, we performed analysis of 241 Arabidopsis AraCyc 8.0 pathways, and the results are made available through the web.

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochrome b6/f complex are discussed.

Five nitrogen fixing cyanobacterial strains have been found to contain PsaB2, an additional and divergent gene copy for the Photosystem I reaction center protein PsaB. In all five species the divergent gene, psaB2, is located separately from the normal psaAB operon in the genome. The protein, PsaB2, was recently identified in heterocysts of Nostoc punctiforme sp. strain PCC 73102. 12 conserved amino acid replacements and one insertion, were identified by a multiple sequence alignment of several PsaB2 and PsaB1 sequences. Several, including an inserted glutamine, are located close to the iron-sulfur cluster FX in the electron transfer chain. By homology modeling, using the Photosystem I crystal structure as template, we have found that the amino acid composition in PsaB2 will introduce changes in critical parts of the Photosystem I protein structure. The changes are close to FX and the phylloquinone (PhQ) in the B-branch, indicating that the electron transfer properties most likely will be affected. We suggest that the divergent PsaB2 protein produces an alternative Photosystem I reaction center with different structural and electron transfer properties. Some interesting physiologcial consequences that this can have for the function of Photosystem I in heterocysts, are discussed.

We have isolated cDNA and genomic clones for the potato (Solanum tuberosum) apoprotein 2 of the light harvesting complex of Photosystem 1, designated Lhca3.St.l. The protein shows all characteristics of the family of chlorophyll a/b-binding proteins. Potato Lhca3.1 gene expression occurs predominantly in leaves,and is transcriptionally regulated by light. One gene copy is present per haploid genome. The sequence of the 5' upstream region was determined. Most boxes identified in the promoter sequences of genes whose expression is light-regulated recur in the Lhca3.St.1 sequence. Functional analyses of the Lhca3. St.1 promoter and two deletion derivatives in transgenic potato transformed with a promoter-GUS fusion show high promoter activity in leaves and other green parts of the plant, which depends on light. Activity is absent in roots and potato tubers. The 500 bp promoter fragment is as active as the full 2.0 kb sequence, showing that all regulatory elements are present on the smallest deletion derivative. In transgenic tobacco (Nicotiana tabacum) plants carrying the largest promoter derivative a similar distribution of activity is found. Promoter activity is not restricted to the phloem, but also prominent in the xylem of the young stem, Which contrasts with promoters of other photosynthesis-associated genes.

The vesicle-inducing protein in plastids 1 (Vipp1) was found to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. In contrast to chloroplasts, it has been suggested that in cyanobacteria the protein is only tightly associated with the cytoplasmic membrane. In the present study we analyze and describe the subcellular localization and the oligomeric organization of Vipp1 from the cyanobacterium Synechocystis PCC 6803. Vipp1 forms stable dimers and higher-ordered oligomers in the cytoplasm as well as at both the cytoplasmic and thylakoid membrane. Vipp1 oligomers are organized in ring structures with a variable diameter of 25–33 nm and corresponding calculated molecular masses of ∼1.6–2.2 MDa. Six different types of rings were found with an unusual 12–17-fold symmetrical conformation. The simultaneous existence of multiple types of rings is very unusual and suggests a special function of Vipp1. Involvement of diverse ring structures in vesicle formation is suggested.

Two novel biomimetic materials have been designed and synthesized by using the sol-gel method combined with the imprinting technique. One material, [SiO2-histidine], bears histidines covalently attached in the pores of silica matrix, whereas the second material, [SiO2-tryptophan], bears tryptophans grafted in the pores of silica matrix. The pores have been formed by imprinted para-benzoquinone (pBQ) molecules which were used as templates and subsequently removed. After formation of the porous matrix, the quinones were reinserted in the pores forming the [SiO2-pBQ-histidine] biomimetic material M1 and [SiO2-pBQ-tryptophan] material M2, respectively. In M1 and M2, the quinones were stabilized via spatial confinement and weak interactions with the anchored aminoacids. Serniquinone (pBQ(center dot-)) radicals were then easily formed in the pores of MI and M2 materials. The pBQ(center dot) radicals showed remarkable stability remaining practically unaltered after several days. ESEEM spectroscopy revealed that in M1, spin delocalization from the pBQ(center dot-) radical onto the imidazoles of the anchored histidine occur. Density functional theory calculations show that the spin transfer from pBQ(center dot-) onto imidazole occurs via H bonds, in a way similar to that originally observed between semiquinones and protein imidazoles Q(A)-His219-D1 and Q(A)-His219 M in photosystem II and the bacterial reaction centers, respectively. In the M2 material, ESEEM spectroscopy and DFT calculations show that pBQ(center dot-) is pi-stacked with the indole of the anchored tryptophan. This interaction is similar to the pi-stacking in [indole from tryptophan W698-A(1) phyllosemiquinone] pair in photosystent I. Thus, M1 and M2 are the first example of synthetic material successfully mimicking an intriguing phenomenon, previously observed exclusively in the photosynthetic reaction centers. Journal of Physical Chemistry C

International audience; Chlorophyll a fluorescence can be used as an early stress indicator. Fluorescence is also connected to photosynthesis so it can be proposed for global monitoring of vegetation status from a satellite platform. Nevertheless, the correct interpretation of fluorescence requires accurate physical models. The spectral shape of the leaf fluorescence free of any re-absorption effect plays a key role in the models and is difficult to measure. We present a vegetation fluorescence emission spectrum free of re-absorption based on a combination of measurements and modelling. The suggested spectrum takes into account the photosystem I and II spectra and their relative contribution to fluorescence, This emission spectrum is applicable to describe vegetation fluorescence in biospectroscopy and remote sensing

The light-dependent production of ATP and reductants by the photosynthetic apparatus in vivo involves a series of electron and proton transfers. Consideration is given as to how electron fluxes through photosystem I (PSI), using absorption spectroscopy, and through photosystem II (PSII), using chlorophyll fluorescence analyses, can be estimated in vivo. Measurements of light-induced electrochromic shifts using absorption spectroscopy provide a means of analyzing the proton fluxes across the thylakoid membranes in vivo. Regulation of these electron and proton fluxes is required for the thylakoids to meet the fluctuating metabolic demands of the cell. Chloroplasts exhibit a wide and flexible range of mechanisms to regulate electron and proton fluxes that enable chloroplasts to match light use for ATP and reductant production with the prevailing metabolic requirements. Non-invasive probing of electron fluxes through PSI and PSII, and proton fluxes across the thylakoid membranes can provide insights into the operation of such regulatory processes in vivo.

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII fluorescence. A substantial decrease of the LHCII/PSII fluorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll fluorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but significantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P fluorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor- and donor side of PS II and an increase in antenna size. In CT pea leaves these effects were absent, except for a small but irreversible effect on PSII activity and antenna size. Thus, the differences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of different thylakoid supercomplexes rearrangements.

We investigate the exciton dynamics in a molecular crystal. The dynamics is based on the exciton Hamiltonian, the phonon Hamiltonian, and the exciton–phonon interaction linear in phonon coordinates. Using the interaction picture, expressions were previously obtained for the rate of coherent migration of the exciton clothed by phonons, and the rate of incoherent or hopping motion. In this work we derive an expression for the clothed exciton propagator, which permits an explicit calculation of the hopping rate. Thus, the rates of exciton generation, coherent transfer, and hopping are determined completely from theory. These velocity constants, along with the experimental fluorescence decay constants and reaction rate constants for the excited molecules, are used to write a generalized master equation that describes the rate of change of exciton population at each site. The master equation can be numerically solved by using a time step of the order of a few femtoseconds, while the excited-state reactions and exciton transfer occur in the picosecond scale. Exciton dynamics is numerically simulated for a simple model of thylakoid membrane in green plants. The model is based on the known characteristics of thylakoid architecture. The membrane is divided into 97 zones, each zone in the bulk containing 1,442 chlorophylls, one P700, and one P680. The latter two pigments are randomly placed in each zone, while keeping their distance between 55 and 60 A. This accounts for the randomness in orientation. The disorder of the chlorophyll molecules within the domains of photosystems is neglected. Our findings are as follows. On average, about 6 million photons within the range of 655–681 nm pass through a membrane in 1 s. About 2.3% of incident photons are absorbed by the membrane chlorophyll molecules. For an excitation bandwidth of 70–175 cm−1, the coherent transfer rate between two adjacent molecules is 1.138 ± 0.488 ps−1. Because the Debye frequency is expected to be much smaller, the slow phonon limit applies. The exciton–phonon coupling constant that can be determined from the transition dipole–transition dipole interaction model leads to a hopping rate of 0.088 ps−1 at 300 K. A steady state is effectively achieved for each zone in the bulk in ∼5 ns. After a period of 50 ns, P700 and P680 in average trap at the rate of 650–702 and 629–679 excitons per second, respectively. The average rate of charge separation and subsequent reactions of each excited photosystem varies as 592–639 per second for PSI and 630–680 per second for PSII. These numbers lead to a reasonable estimate for the amount of glucose produced in each square meter of leaf area in a day. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005