In Vivo NADH/NAD+ Biosensing Reveals the Dynamics of Cytosolic Redox Metabolism in Plants.

NADH and NAD+ are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD+ in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis (Arabidopsis thaliana) and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism.

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics.

Mitochondria host vital cellular functions, including oxidative phosphorylation and co-factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity-based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage-Dependent Anion Channel 1 to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.

Structure of a PSI-LHCI-cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions.

Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.

Multiparametric real-time sensing of cytosolic physiology links hypoxia responses to mitochondrial electron transport.

Hypoxia regularly occurs during plant development and can be induced by the environment through, for example, flooding. To understand how plant tissue physiology responds to progressing oxygen restriction, we aimed to monitor subcellular physiology in real time and in vivo. We establish a fluorescent protein sensor-based system for multiparametric monitoring of dynamic changes in subcellular physiology of living Arabidopsis thaliana leaves and exemplify its applicability for hypoxia stress. By monitoring cytosolic dynamics of magnesium adenosine 5'-triphosphate, free calcium ion concentration, pH, NAD redox status, and glutathione redox status in parallel, linked to transcriptional and metabolic responses, we generate an integrated picture of the physiological response to progressing hypoxia. We show that the physiological changes are surprisingly robust, even when plant carbon status is modified, as achieved by sucrose feeding or extended night. Inhibition of the mitochondrial respiratory chain causes dynamics of cytosolic physiology that are remarkably similar to those under oxygen depletion, highlighting mitochondrial electron transport as a key determinant of the cellular consequences of hypoxia beyond the organelle. A broadly applicable system for parallel in vivo sensing of plant stress physiology is established to map out the physiological context under which both mitochondrial retrograde signalling and low oxygen signalling occur, indicating shared upstream stimuli.

The fluorescent protein sensor roGFP2-Orp1 monitors in vivo H2 O2 and thiol redox integration and elucidates intracellular H2 O2 dynamics during elicitor-induced oxidative burst in Arabidopsis.

Hydrogen peroxide (H2 O2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2 O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H2 O2 sensing in living plants. We established H2 O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H2 O2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2 O2 , pharmacologically-induced H2 O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2 O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants. We provided a well defined toolkit for H2 O2 monitoring in planta and showed that intracellular H2 O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2 O2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.

Mitochondria Affect Photosynthetic Electron Transport and Photosensitivity in a Green Alga.

Photosynthetic organisms use sunlight as the primary source of energy to support their metabolism. In eukaryotes, reactions responsible of the conversion of light into chemical energy occur in specific organelles, the chloroplasts. In this study, we showed that mitochondria also have a seminal influence on cells' energy metabolism and on photosynthetic reactions. This is illustrated by the observation that the strong photosensitivity of Chlamydomonas reinhardtii cells depleted of the chloroplast protein PGRL1 was rescued by the introduction of a mitochondrial mutation affecting respiratory complex I. Functional analysis showed that such a reduced respiratory activity influenced chloroplast electron transport with consequent overreduction of plastoquinone and donor-side limitation of photosystem I (PSI). As a consequence, damage due to excess light affected more photosystem II (PSII) rather than PSI. Double mutant cells are able to grow under excess illumination, while single pgrl1 are not, thanks to the presence of an efficient repair mechanism of PSII. These results also underline the seminal biological relevance of the regulation of electron transport reactions within the photosynthetic complexes. Photosynthetic organisms evolved a strategy to respond to excess light where damage is targeting preferentially to a specific complex, PSII. Cells are able to endure extensive damage targeting this complex thanks to an efficient repair mechanisms, while if PSI is affected, there are drastic consequences on growth.

Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii.

Ferredoxins (FDX) and the FDX:NADP+ oxidoreductase (FNR) represent a key junction of electron transport downstream of photosystem I (PSI). Dynamic recruitment of FNR to the thylakoid membrane has been considered as a potential mechanism to define the fate of photosynthetically derived electrons. In this study, we investigated the functional importance of the association of FNR with the photosynthetic apparatus in Chlamydomonas reinhardtii. In vitro assays based on NADP+ photoreduction measurements as well as NMR chemical shift perturbation analyses showed that FNR preferentially interacts with FDX1 compared to FDX2. Notably, binding of FNR to a PSI supercomplex further enhanced this preference for FDX1 over FDX2, suggesting that FNR is potentially capable of channelling electrons towards distinct routes. NADP+ photoreduction assays and immunoblotting revealed that the association of FNR with the thylakoid membrane including the PSI supercomplex is impaired in the absence of Proton Gradient Regulation 5 (PGR5) and/or Proton Gradient Regulation 5-Like photosynthetic phenotype 1 (PGRL1), implying that both proteins, directly or indirectly, contribute to the recruitment of FNR to the thylakoid membrane. As assessed via in vivo absorption spectroscopy and immunoblotting, PSI was the primary target of photodamage in response to high-light stress in the absence of PGR5 and/or PGRL1. Anoxia preserved the activity of PSI, pointing to enhanced electron donation to O2 as the source of the observed PSI inactivation and degradation. These findings establish another perspective on PGR5/PGRL1 knockout-related phenotypes and potentially interconnect FNR with the regulation of photosynthetic electron transport and PSI photoprotection in C. reinhardtii.

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes.

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of light-harvesting complex stress-related protein3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-light harvesting complex I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.

Proton Gradient Regulation5-Like1-Mediated Cyclic Electron Flow Is Crucial for Acclimation to Anoxia and Complementary to Nonphotochemical Quenching in Stress Adaptation.

To investigate the functional importance of Proton Gradient Regulation5-Like1 (PGRL1) for photosynthetic performances in the moss Physcomitrella patens, we generated a pgrl1 knockout mutant. Functional analysis revealed diminished nonphotochemical quenching (NPQ) as well as decreased capacity for cyclic electron flow (CEF) in pgrl1. Under anoxia, where CEF is induced, quantitative proteomics evidenced severe down-regulation of photosystems but up-regulation of the chloroplast NADH dehydrogenase complex, plastocyanin, and Ca2+ sensors in the mutant, indicating that the absence of PGRL1 triggered a mechanism compensatory for diminished CEF. On the other hand, proteins required for NPQ, such as light-harvesting complex stress-related protein1 (LHCSR1), violaxanthin de-epoxidase, and PSII subunit S, remained stable. To further investigate the interrelation between CEF and NPQ, we generated a pgrl1 npq4 double mutant in the green alga Chlamydomonas reinhardtii lacking both PGRL1 and LHCSR3 expression. Phenotypic comparative analyses of this double mutant, together with the single knockout strains and with the P. patens pgrl1, demonstrated that PGRL1 is crucial for acclimation to high light and anoxia in both organisms. Moreover, the data generated for the C. reinhardtii double mutant clearly showed a complementary role of PGRL1 and LHCSR3 in managing high light stress response. We conclude that both proteins are needed for photoprotection and for survival under low oxygen, underpinning a tight link between CEF and NPQ in oxygenic photosynthesis. Given the complementarity of the energy-dependent component of NPQ (qE) and PGRL1-mediated CEF, we suggest that PGRL1 is a capacitor linked to the evolution of the PSII subunit S-dependent qE in terrestrial plants.

Proton gradient regulation 5-mediated cyclic electron flow under ATP- or redox-limited conditions: a study of ΔATpase pgr5 and ΔrbcL pgr5 mutants in the green alga Chlamydomonas reinhardtii.

The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence.

Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

Deletion of Proton Gradient Regulation 5 (PGR5) and PGR5-Like 1 (PGRL1) proteins promote sustainable light-driven hydrogen production in Chlamydomonas reinhardtii due to increased PSII activity under sulfur deprivation.

Continuous hydrogen photo-production under sulfur deprivation was studied in the Chlamydomonas reinhardtii pgr5 pgrl1 double mutant and respective single mutants. Under medium light conditions, the pgr5 exhibited the highest performance and produced about eight times more hydrogen than the wild type, making pgr5 one of the most efficient hydrogen producer reported so far. The pgr5 pgrl1 double mutant showed an increased hydrogen burst at the beginning of sulfur deprivation under high light conditions, but in this case the overall amount of hydrogen produced by pgr5 pgrl1 as well as pgr5 was diminished due to photo-inhibition and increased degradation of PSI. In contrast, the pgrl1 was effective in hydrogen production in both high and low light. Blocking photosynthetic electron transfer by DCMU stopped hydrogen production almost completely in the mutant strains, indicating that the main pathway of electrons toward enhanced hydrogen production is via linear electron transport. Indeed, PSII remained more active and stable in the pgr mutant strains as compared to the wild type. Since transition to anaerobiosis was faster and could be maintained due to an increased oxygen consumption capacity, this likely preserves PSII from photo-oxidative damage in the pgr mutants. Hence, we conclude that increased hydrogen production under sulfur deprivation in the pgr5 and pgrl1 mutants is caused by an increased stability of PSII permitting sustainable light-driven hydrogen production in Chlamydomonas reinhardtii.

A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling ((14)N/(15)N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii.

Single cell green algae (microalgae) are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA) is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels). Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3) in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) compared to 100% control levels, and improved light to H2 (180%) and biomass (165%) conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m(-2) s(-1)) and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression and 5) reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems.