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The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH2) into ATP, the cell’s energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress. Complex I, the NADH-dehydrogenase, is part of the largest supercomplex (CI + CIII2 + CIVn). Here, we demonstrate the role of NDUFB10, a subunit of the membrane arm of complex I, in complex I and supercomplex assembly on the one hand and bioenergetics function on the other. NDUFB10 knockout was correlated with a decrease of SCAF1, a supercomplex assembly factor, and a reduction of respiration and mitochondrial membrane potential. This likely is due to loss of proton pumping since the CI P P -module is downregulated and the P D -module is completely abolished in NDUFB10 knock outs.

Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI–light-harvesting complex I monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH and thus partially overlaps with the surface area that would bind one of the light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI–light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways.

Vibrational spectroscopy in supersonic jet expansions is a powerful tool to assess molecular aggregates in close to ideal conditions for the benchmarking of quantum chemical approaches. The low temperatures achieved as well as the absence of environment effects allow for a direct comparison between computed and experimental spectra. This provides potential benchmarking data which can be revisited to hone different computational techniques, and it allows for the critical analysis of procedures under the setting of a blind challenge. In the latter case, the final result is unknown to modellers, providing an unbiased testing opportunity for quantum chemical models. In this work, we present the spectroscopic and computational results for the first HyDRA blind challenge. The latter deals with the prediction of water donor stretching vibrations in monohydrates of organic molecules. This edition features a test set of 10 systems. Experimental water donor OH vibrational wavenumbers for the vacuum-isolated monohydrates of formaldehyde, tetrahydrofuran, pyridine, tetrahydrothiophene, trifluoroethanol, methyl lactate, dimethylimidazolidinone, cyclooctanone, trifluoroacetophenone and 1-phenylcyclohexane-cis-1,2-diol are provided. The results of the challenge show promising predictive properties in both purely quantum mechanical approaches as well as regression and other machine learning strategies.

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H2) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H2 production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H2 photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H2 photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H2 photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast ofChlamydomonas reinhardtiiwith largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild type cells in comparison withcrxinsertional mutant (IMcrx), two CRISPR/Cas9 KO mutants and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnects redox control with active photosynthetic electron transport and metabolism as well as Ca2+signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding, that the absence of CRX under HL conditions severely inhibited light-driven CO2fixation underpins the importance of CRX for redox tuning as well as for efficient photosynthesis.One-sentence summaryCalredoxin dependent redox regulation ensures efficient photosynthesis.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.

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.

In Chlamydomonas reinhardtii, iron levels are strictly controlled by iron-assimilating protein 1 (FEA1). Recombinant FEA1 protein was successfully purified and crystallized by hanging-drop vapor diffusion. The phase problem was solved by the native sulfur SAD method using long-wavelength X-rays (2.7 Å). Laser-cutting technology was used to increase the signal-to-noise ratio and derive an initial structure., As an essential component of protein cofactors, iron is important for all photosynthetic organisms. In Chlamydomonas reinhardtii, iron levels are strictly controlled by proteins such as iron-assimilating protein 1 (FEA1). This periplasmic protein is expressed under conditions of iron deficiency, but its mechanisms of function remain unknown. Because FEA1 has no amino-acid similarity to protein structures in the Protein Data Bank, its crystal structure cannot be solved by molecular replacement. Here, recombinant FEA1 protein lacking the N-terminal signal sequence was successfully purified and crystals of apo FEA1 were obtained by hanging-drop vapor diffusion. Neither selenomethionine substitution nor heavy-atom derivatization was successful; therefore, the phase problem of FEA1 crystals was solved by the native sulfur SAD method using long-wavelength X-rays (2.7 Å). Laser-cutting technology was used to increase the signal-to-noise ratio and derive an initial structure. This study will lead to further structural studies of FEA1 to understand its function and its links to the iron-assimilation pathway.

Most of life’s energy comes from sunlight, and thus, photosynthesis underpins the survival of virtually all life forms. The light-driven electron transfer at photosystem I (PSI) is certainly the most important generator of reducing power at the cellular level and thereby largely determines the global amount of enthalpy in living systems (Nelson 2011). The PSI is a light-driven plastocyanin:ferredoxin oxidoreductase, which is embedded into thylakoid membranes of cyanobacteria and chloroplasts of eukaryotic photosynthetic organism. Structural determination of complexes of the photosynthetic machinery is vital for the understanding of its mode of action. Here, we describe new structural and functional insights into PSI and associated light-harvesting proteins, with a focus on the plasticity of PSI.

Nitrate and nitrite reduction to ammonia and nitrous oxide by anaerobic E. coli batch cultures is investigated by advanced spectroscopic analytical techniques with 15N-isotopic labelling. Non-invasive, in situ analysis of the headspace is achieved using White cell FTIR and cavity-enhanced Raman (CERS) spectroscopies alongside liquid-phase Raman spectroscopy. For gas-phase analysis, White cell FTIR measures CO2, ethanol and N2O while CERS allows H2, N2 and O2 monitoring. The 6 m pathlength White cell affords trace gas detection of N2O with a noise equivalent detection limit of 60 nbar or 60 ppbv in 1 atm. Quantitative analysis is discussed for all four 14N/15N-isotopomers of N2O. Monobasic and dibasic phosphates, acetate, formate, glucose and NO3− concentrations are obtained by liquid-phase Raman spectroscopy, with a noise equivalent detection limit of 0.6 mM for NO3− at 300 s integration time. Concentrations of the phosphate anions are used to calculate the pH in situ using a modified Henderson–Hasselbalch equation. NO2− concentrations are determined by sampling for colorimetric analysis and NH4+ by basifying samples to release 14N/15N-isotopomers of NH3 for measurement in a second FTIR White cell. The reductions of 15NO3−, 15NO2−, and mixed 15NO3− and 14NO2− by anaerobic E. coli batch cultures are discussed. In a major pathway, NO3− is reduced to NH4+via NO2−, with the bulk of NO2− reduction occurring after NO3− depletion. Using isotopically labelled 15NO3−, 15NH4+ production is distinguished from background 14NH4+ in the growth medium. In a minor pathway, NO2− is reduced to N2O via the toxic radical NO. With excellent detection sensitivities, N2O serves as a monitor for trace NO2− reduction, even when cells are predominantly reducing NO3−. The analysis of N2O isotopomers reveals that for cultures supplemented with mixed 15NO3− and 14NO2− enzymatic activity to reduce 14NO2− occurs immediately, even before 15NO3− reduction begins. Optical density and pH measurements are discussed in the context of acetate, formate and CO2 production. H2 production is repressed by NO3−; but in experiments with NO2− supplementation only, CERS detects H2 produced by formate disproportionation after NO2− depletion.

Polymerization-induced self-assembly (PISA) is exploited to design hydrogen-bonded poly(stearyl methacrylate)-poly(benzyl methacrylate) [PSMA-PBzMA] worm gels in n-dodecane. Using a carboxylic acid-based RAFT agent facilitates hydrogen bonding between neighboring worms to produce much stronger physical gels than those prepared using the analogous methyl ester-based RAFT agent. Moreover, tuning the proportion of these two types of end-groups on the PSMA chains enables the storage modulus (G′) of a 20% w/w worm gel to be tuned from ∼4.5 kPa up to ∼114 kPa. This is achieved via two complementary routes: (i) an in situ approach using binary mixtures of acid- and ester-capped PSMA stabilizer chains during PISA or (ii) a post-polymerization processing strategy using a thermally-induced worm-to-sphere transition to mix acid- and ester-functionalized spheres at 110 °C that fuse to form worms on cooling to 20 °C. SAXS and rheology studies of these hydrogen-bonded worm gels provide detailed insights into their inter-worm interactions and physical behavior, respectively. In the case of the carboxylic acid-functionalized worms, SAXS provides direct evidence for additional inter-worm interactions, while rheological studies confirm both a significant reduction in critical gelation concentration (from approximately 10% w/w to 2–3% w/w) and a substantial increase in critical gelation temperature (from 41 °C to 92 °C). It is remarkable that a rather subtle change in the chemical structure results in such improvements in gel strength, gelation efficiency and gel cohesion.

MotivationProtein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes.ResultsTherefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae.AvailabilityThe source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems.Contactmhippler@uni-muenster.de and pohlschr@uni-muenster.deSupplementary informationSupplementary data are available online.

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6–8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 μbar for acetaldehyde and 0.26 μbar for ethanol in the gas phase, corresponding to 3.2 μM acetaldehyde and 22 μM ethanol in solution, using Henry’s law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation of Escherichia coli is studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO2 and H2 is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor with E. coli is expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production.

International audience; Nowadays, little information is available regarding the N‐glycosylation pathway in the green microalga Chlamydomonas reinhardtii. Recent investigation demonstrated that C. reinhardtii synthesizes linear oligomannosides. Maturation of these oligomannosides results in N‐glycans that are partially methylated and carry one or two xylose residues. One xylose residue was demonstrated to be a core β(1,2)‐xylose. Recently, N‐glycoproteomic analysis performed on glycoproteins secreted by C. reinhardtii demonstrated that the xylosyltransferase A (XTA) was responsible for the addition of the core β(1,2)‐xylose. Furthermore, another xylosyltransferase candidate named XTB was suggested to be involved in the xylosylation in C. reinhardtii. In the present study, we focus especially on the characterization of the structures of the xylosylated N‐glycans from C. reinhardtii taking advantage of insertional mutants of XTA and XTB, and of the XTA/XTB double‐mutant. The combination of mass spectrometry approaches allowed us to identify the major N‐glycan structures bearing one or two xylose residues. They confirm that XTA is responsible for the addition of the core β(1,2)‐xylose, whereas XTB is involved in the addition of the xylose residue onto the linear branch of the N‐glycan as well as in the partial addition of the core β(1,2)‐xylose suggesting that this transferase exhibits a low substrate specificity. Analysis of the double‐mutant suggests that an additional xylosyltransferase is involved in the xylosylation process in C. reinhardtii. Additional putative candidates have been identified in the C. reinhardtii genome. Altogether, these results pave the way for a better understanding of the C. reinhardtii N‐glycosylation pathway.

Calcium (Ca2+) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca2+-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca2+-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 A resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca2+ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane used to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH are variable, cyclic electron flow (CEF) between PSI and cytochrome b6f complex (b6f) produces extra ATP. The b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 mutant of the vascular plant Arabidopsis thaliana, carrying Pro194Leu in the b6f Rieske Iron-sulfur protein. In this work, we introduced pgr1 mutation in the green alga Chlamydomonas reinhardtii (PETC-P171L). Consistent with pgr1 phenotype, PETC-P171L displayed an impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provides evidence that the ΔpH component in PETC-P171L is dependent on oxygen availability. Only under low oxygen conditions the ΔpH component was sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the PQ pool and showed a diminished electron flow through the b6f complex.One sentence summaryChange of PETC to P171L via site directed mutagenesis alters the pH dependency of the photosynthetic control mechanism

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2conditions. When light energy exceeds CO2fixation capacity,Chlamydomonas reinhardtiiactivates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2Concentrating Mechanism (CCM). How light and CO2signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2concentrations and that depletion of CO2drives these responses, even in total darkness. High CO2levels, derived from respiration or impaired photosynthetic fixation, repressLHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controllingLHCSR3andPSBStranscript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2levels.

Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here, we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Our cryo-EM structure identifies that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-LHCI monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH, and thus partially overlaps with the surface area that would bind one of the LHCII complexes in state transitions. We also define the most accurate available PSI-LHCI model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations of all the pigments, as well as 621 water molecules that affect energy transfer pathways.

The expression of antigens in their immunologically-active form remains a challenge, both in the analysis of regulatory pathways exploited by parasitic nematodes or in the development of vaccines. Despite the success of native proteins to induce protective immunity, recombinant proteins expressed in bacteria, yeast or insect cells offer only limited protective capacities, presumably due to incorrect folding or missing complex posttranslational modifications. The present study investigates the feasibility of using the free-living nematode Caenorhabditis elegans as an alternative expression system for proteins found in the secretome of parasitic nematodes. Exemplified by the expression of the extracellular superoxide dismutase from Haemonchus contortus (HcSODe) and the extracellular and glycosylated glutathione S-transferase from the filarial parasite Onchocerca volvulus (OvGST1), we continue our efforts to improve production and purification of recombinant proteins expressed in C. elegans. We demonstrate that sufficient quantities of functional proteins can be expressed in C. elegans for subsequent immunological and biochemical studies.

Polymerization-induced self-assembly (PISA) is exploited to design hydrogen-bonded poly(stearyl methacrylate)-poly(benzyl methacrylate) [PSMA-PBzMA] worm gels in n-dodecane. Using a carboxylic acid-based RAFT agent facilitates hydrogen bonding between neighboring worms to produce much stronger physical gels than those prepared using the analogous methyl ester-based RAFT agent. Moreover, tuning the proportion of these two types of end-groups on the PSMA chains enables the storage modulus (G′) of a 20% w/w worm gel to be tuned from ∼4.5 kPa up to ∼114 kPa. This is achieved via two complementary routes: (i) an in situ approach using binary mixtures of acid- and ester-capped PSMA stabilizer chains during PISA or (ii) a post-polymerization processing strategy using a thermally-induced worm-to-sphere transition to mix acid- and ester-functionalized spheres at 110 °C that fuse to form worms on cooling to 20 °C. SAXS and rheology studies of these hydrogen-bonded worm gels provide detailed insights into their inter-worm interactions and physical behavior, respectively. In the case of the carboxylic acid-functionalized worms, SAXS provides direct evidence for additional inter-worm interactions, while rheological studies confirm both a significant reduction in critical gelation concentration (from approximately 10% w/w to 2–3% w/w) and a substantial increase in critical gelation temperature (from 41 °C to 92 °C). It is remarkable that a rather subtle change in the chemical structure results in such improvements in gel strength, gelation efficiency and gel cohesion., Carboxylic acid-capped diblock copolymer worms are prepared in n-dodecane via polymerization-induced self-assembly. Varying the proportion of terminal carboxylic acid groups modulates the inter-worm H-bonding interactions and hence the gel modulus.

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 Å resolution reveals strong hydrophobic interactions in the plant PSI–Pc ternary complex, leading to exclusion of water molecules from PsaA–PsaB/Pc interface once the PSI–Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared with reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover.

Microalgal H2 photoproduction has the potential of being an affordable method for producing this alternative fuel; however extreme sensitivity of hydrogenase enzyme to photosynthetic O2 naturally prevents large-scale H2 production upon illumination. Although a two-phase sulfur deficiency method has been established to deal with this incompatibility, its time and cost demanding, so that this model is not commercially scalable. Despite much research has been conducted, no proper economic alternative for the sulfur deprivation model, with higher or even same productivity and sustainability, has been presented till now. Herein we propose a simple and viable alternative, through introducing a chemical O2 scavenger system, called oxysorb, to algal cultures. Oxysorb, in non-cytotoxic concentrations (including 50 or 100 mM sodium ascorbate and 5 ppm cupric sulfate) for CC124 as well as pgr5 cultures (containing 30 μg/ml chlorophyll) showed a fast, safe and persisted O2 removal capacity, initiating H2 production in the sulfur-containing cultures, either in photoheterotrophic or in autotrophic conditions. Total H2 production obtained with CC124 and pgr5 cultures, containing 100 mM oxysorb, was 2–5.5 times higher than sulfur-deprived ones (measured in a small closed system). This higher H2 productivity in the oxysorb approach was achieved due to anoxia establishment with no ROS production and without impacting PSII activity.

Summary Phosphorylation dynamics of LHCSR3 were investigated in Chlamydomonas reinhardtii by quantitative proteomics and genetic engineering. LHCSR3 protein expression and phosphorylation were induced in high light. Our data revealed synergistic and dynamic N‐terminal LHCSR3 phosphorylation. Phosphorylated and nonphosphorylated LHCSR3 associated with PSII‐LHCII supercomplexes. The phosphorylation status of LHCB4 was closely linked to the phosphorylation of multiple sites at the N‐terminus of LHCSR3, indicating that LHCSR3 phosphorylation may operate as a molecular switch modulating LHCB4 phosphorylation, which in turn is important for PSII‐LHCII disassembly. Notably, LHCSR3 phosphorylation diminished under prolonged high light, which coincided with onset of CEF. Hierarchical clustering of significantly altered proteins revealed similar expression profiles of LHCSR3, CRX, and FNR. This finding indicated the existence of a functional link between LHCSR3 protein abundance and phosphorylation, photosynthetic electron flow, and the oxidative stress response., Significance Statement We analyzed light‐dependent phosphorylation of LHCSR3 and LHCB4 by quantitative proteomics, genetic engineering, and functional measurements. N‐terminal LHCSR3 phosphorylation is light dependent and modulates phosphorylation of LHCB4 and the association of LHCSR3 with photosynthetic multi‐protein complexes. Our data revealed the functional importance of LHCSR3 phosphorylation, pointing to unforeseen functions in algal photosynthesis.

Abscisic acid (ABA) regulates growth and developmental processes in response to limiting water conditions. ABA functions through a core signaling pathway consisting of PYR1/PYL/RCAR ABA receptors, type 2C protein phosphatases (PP2Cs), and SnRK2-type protein kinases. Other signaling modules might converge with ABA signals through the modulation of core ABA signaling components. We have investigated the role of the protein kinase WNK8 in ABA signaling. WNK8 interacted with PP2CA and PYR1, phosphorylated PYR1 in vitro, and was dephosphorylated by PP2CA. A hypermorphic wnk8-ct Arabidopsis mutant allele suppressed ABA and glucose hypersensitivities of pp2ca-1 mutants during young seedling development, and WNK8 expression in protoplasts suppressed ABA-induced reporter gene expression. We conclude that WNK8 functions as a negative modulator of ABA signaling.

RBOHF from Arabidopsis thaliana represents a multifunctional NADPH oxidase regulating biotic and abiotic stress tolerance, developmental processes and guard cell aperture. The molecular components and mechanisms determining RBOHF activity remain to be elucidated. Here we combined protein interaction studies, biochemical and genetic approaches, and pathway reconstitution analyses to identify and characterize proteins that confer RBOHF regulation and elucidated mechanisms that adjust RBOHF activity. While the Ca2+ sensor-activated kinases CIPK11 and CIPK26 constitute alternative paths for RBOHF activation, the combined activity of CIPKs and the kinase open stomata 1 (OST1) triggers complementary activation of this NADPH oxidase, which is efficiently counteracted through dephosphorylation by the phosphatase ABI1. Within RBOHF, several distinct phosphorylation sites (p-sites) in the N-terminus of RBOHF appear to contribute individually to activity regulation. These findings identify RBOHF as a convergence point targeted by a complex regulatory network of kinases and phosphatases. We propose that this allows for fine-tuning of plant reactive oxygen species (ROS) production by RBOHF in response to different stimuli and in diverse physiological processes.