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Mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) is a central enzyme in primary carbon metabolism, since its function is required to drive four multienzymes involved in photorespiration, the tricarboxylic acid (TCA) cycle, and the degradation of branched-chain amino acids. However, in illuminated, photosynthesizing tissue a vast amount of mtLPD1 is necessary for glycine decarboxylase (GDC), the key enzyme of photorespiration. In light of the shared role, the functional characterization of mtLPD1 is necessary to understand how the three pathways might interact under different environmental scenarios. This includes the determination of the biochemical properties and all potential regulatory mechanisms, respectively. With regards to the latter, regulation can occur through multiple levels including effector molecules, cofactor availability, or posttranslational modifications (PTM), which in turn decrease or increase the activity of each enzymatic reaction. Gaining a comprehensive overview on all these aspects would ultimately facilitate the interpretation of the metabolic interplay of the pathways within the whole subcellular network or even function as a proof of concept for genetic engineering approaches. Here, we describe the typical workflow how to clone, express, and purify plant mtLPD1 for biochemical characterization and how to analyze potential redox regulatory mechanisms in vitro and in planta.
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Dihidrolipoamida Deshidrogenasa , Oxidación-Reducción , Dihidrolipoamida Deshidrogenasa/metabolismo , Dihidrolipoamida Deshidrogenasa/genética , Mitocondrias/metabolismo , Mitocondrias/genética , Mitocondrias/enzimología , Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Clonación Molecular/métodosRESUMEN
Two recent studies reinvestigated the phenomenon of photorespiration as a photoprotective mechanism. Smith et al. suggest alleviated negative feedback regulation of chloroplast ATP synthase as an alternative hypothesis. Von Bismarck et al. discuss how photorespiration-impaired mutants cope somewhat better with fluctuating light (FL) environments because of downregulated photosynthesis and complex metabolic re-routing.
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Pollution from personal care products, such as UV-filters like avobenzone and nano-zinc oxide (nZnO), poses a growing threat to marine ecosystems. To better understand this hazard, especially for lesser-studied sediment-dwelling marine organisms, we investigated the physiological impacts of simultaneous exposure to nZnO and avobenzone on the lugworm Arenicola marina. Lugworms were exposed to nZnO, avobenzone, or their combination for three weeks. We assessed pollutant-induced metabolic changes by measuring key metabolic intermediates in the body wall and coelomic fluid, and oxidative stress by analyzing antioxidant levels and oxidative lesions in proteins and lipids of the body wall. Exposure to UV filters resulted in shifts in the concentrations of Krebs' cycle and urea cycle intermediates, as well as alterations in certain amino acids in the body wall and coelomic fluid of the lugworms. Pathway enrichment analyses revealed that nZnO induced more pronounced metabolic shifts compared to avobenzone or their combination. Exposure to avobenzone or nZnO alone prompted an increase in tissue antioxidant capacity, indicating a compensatory response to restore redox balance, which effectively prevented oxidative damage to proteins or lipids. However, co-exposure to nZnO and avobenzone suppressed superoxide dismutase and lead to accumulation of lipid peroxides and methionine sulfoxide, indicating oxidative stress and damage to lipids and proteins. Our findings highlight oxidative stress as a significant mechanism of toxicity for both nZnO and avobenzone, especially when combined, and underscores the importance of further investigating the fitness implications of oxidative stress induced by these common UV filters in benthic marine organisms.
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Estrés Oxidativo , Poliquetos , Protectores Solares , Animales , Poliquetos/efectos de los fármacos , Poliquetos/fisiología , Poliquetos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Protectores Solares/toxicidad , Óxido de Zinc/toxicidad , Minerales , Antioxidantes/metabolismo , Contaminantes Químicos del Agua/toxicidad , Rayos UltravioletaRESUMEN
When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. It has been hypothesized that the excreted nitrite represents excess nitrogen that cannot be further assimilated due to the missing carbon, but the underlying molecular mechanisms are unclear. Here, we identified a protein that interacts with nitrite reductase, regulates nitrogen metabolism and promotes nitrite excretion. The protein, which we named NirP1, is encoded by an unannotated gene that is upregulated under low carbon conditions and controlled by transcription factor NtcA, a central regulator of nitrogen homeostasis. Ectopic overexpression of nirP1 in Synechocystis sp. PCC 6803 resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments indicated that NirP1 interacts with nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting nitrite reductase.
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Nitritos , Synechocystis , Nitritos/metabolismo , Nitratos/metabolismo , Nitrito Reductasas/genética , Nitrito Reductasas/metabolismo , Amoníaco/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Synechocystis/genética , Synechocystis/metabolismo , Nitrógeno/metabolismo , Carbono/metabolismo , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismoRESUMEN
Thioredoxins (TRXs) are central to redox regulation, modulating enzyme activities to adapt metabolism to environmental changes. Previous research emphasized mitochondrial and microsomal TRX o1 and h2 influence on mitochondrial metabolism, including photorespiration and the tricarboxylic acid (TCA) cycle. Our study aimed to compare TRX-based regulation circuits towards environmental cues mainly affecting photorespiration. Metabolite snapshots, phenotypes and CO2 assimilation were compared among single and multiple TRX mutants in the wild-type and the glycine decarboxylase T-protein knockdown (gldt1) background. Our analyses provided evidence for additive negative effects of combined TRX o1 and h2 deficiency on growth and photosynthesis. Especially metabolite accumulation patterns suggest a shared regulation mechanism mainly on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1)-dependent pathways. Quantification of pyridine nucleotides, in conjunction with 13C-labelling approaches, and biochemical analysis of recombinant mtLPD1 supported this. It also revealed mtLPD1 inhibition by NADH, pointing at an additional measure to fine-tune it's activity. Collectively, we propose that lack of TRX o1 and h2 perturbs the mitochondrial redox state, which impacts on other pathways through shifts in the NADH/NAD+ ratio via mtLPD1. This regulation module might represent a node for simultaneous adjustments of photorespiration, the TCA cycle and branched chain amino acid degradation under fluctuating environmental conditions.
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Dihidrolipoamida Deshidrogenasa , Mitocondrias , Tiorredoxinas , Dihidrolipoamida Deshidrogenasa/metabolismo , Dihidrolipoamida Deshidrogenasa/genética , Mitocondrias/metabolismo , Tiorredoxinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Fotosíntesis , Oxidación-Reducción , NAD/metabolismo , Ambiente , Mutación , Dióxido de Carbono/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMEN
Life on tidal coasts presents physiological major challenges for sessile species. Fluctuations in oxygen and temperature can affect bioenergetics and modulate metabolism and redox balance, but their combined effects are not well understood. We investigated the effects of intermittent hypoxia (12h/12h) in combination with different temperature regimes (normal (15 °C), elevated (30 °C) and fluctuating (15 °C water/30 °C air)) on the Pacific oyster Crassostrea (Magallana) gigas. Fluctuating temperature led to energetic costly metabolic rearrangements and accumulation of proteins in oyster tissues. Elevated temperature led to high (60%) mortality and oxidative damage in survivors. Normal temperature had no major negative effects but caused metabolic shifts. Our study shows high plasticity of oyster metabolism in response to oxygen and temperature fluctuations and indicates that metabolic adjustments to oxygen deficiency are strongly modulated by the ambient temperature. Co-exposure to constant elevated temperature and intermittent hypoxia demonstrates the limits of this adaptive metabolic plasticity.
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Crassostrea , Animales , Temperatura , Crassostrea/fisiología , Inmersión , Metabolismo Energético , Oxígeno/metabolismo , Hipoxia/metabolismoRESUMEN
Photorespiration is inevitable for oxygenic photosynthesis. It has fascinated researchers over decades because of its multicompartmental organization. Recently, Lin and Tsay identified a vacuole glycerate transporter contributing to photorespiratory metabolism under short-term nitrogen depletion. This key finding adds a fifth interacting subcellular compartment and extends the photorespiratory metabolic repair module.
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Fotosíntesis , Vacuolas , Vacuolas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Oxígeno/metabolismo , Nitrógeno/metabolismoRESUMEN
Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , NADP/metabolismo , Dióxido de Carbono/metabolismo , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Arabidopsis/metabolismo , Fotosíntesis/fisiología , Cloroplastos/metabolismo , Oxidación-Reducción , Hojas de la Planta/metabolismo , Tiorredoxinas/metabolismo , AclimataciónRESUMEN
Benthic species are exposed to oxygen fluctuations that can affect their performance and survival. Physiological effects and ecological consequences of fluctuating oxygen are not well understood in marine bioturbators such as the soft-shell clam Mya arenaria. We explored the effects of different oxygen regimes (21 days of exposure to constant hypoxia (~4.1 kPa PO2), cyclic hypoxia (~2.1-~10.4 kPa PO2) or normoxia (~21 kPa PO2)) on energy metabolism, oxidative stress and ecological behaviors (bioirrigation and bioturbation) of M. arenaria. Constant hypoxia and post-hypoxic recovery in cyclic hypoxia led to oxidative injury of proteins and lipids, respectively. Clams acclimated to constant hypoxia maintained aerobic capacity similar to the normoxic clams. In contrast, clams acclimated to cyclic hypoxia suppressed aerobic metabolism and activated anaerobiosis during hypoxia, and strongly upregulated aerobic metabolism during recovery. Constant hypoxia led to decreased lipid content, whereas in cyclic hypoxia proteins and glycogen accumulated during recovery and were broken down during the hypoxic phase. Digging of clams was impaired by constant and cyclic hypoxia, and bioirrigation was also suppressed under constant hypoxia. Overall, cyclic hypoxia appears less stressful for M. arenaria due to the metabolic flexibility that ensures recovery during reoxygenation and mitigates the negative effects of hypoxia, whereas constant hypoxia leads to depletion of energy reserves and impairs ecological functions of M. arenaria potentially leading to negative ecological consequences in benthic ecosystems.
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Mya , Animales , Mya/metabolismo , Oxígeno/metabolismo , Ecosistema , Metabolismo Energético , Proteínas/metabolismo , HipoxiaRESUMEN
Nanopollutants such as nZnO gain importance as contaminants of emerging concern due to their high production volume and potential toxicity. Coastal sediments serve as sinks for nanoparticles but the impacts and the toxicity mechanisms of nZnO in sediment-dwelling organisms are not well understood. We used metabolomics to assess the effects of nZnO-contaminated sediments on a benthic ecosystem engineer, an infaunal polychaete Arenicola marina. The worms were exposed to unpolluted (control) sediment or to the sediment spiked with 100 or 1000 µg Zn kg-1 of nZnO. Oxidative lesions (lipid peroxidation and protein carbonyls) were measured in the body wall as traditional biomarkers of nanopollutant toxicity. Metabolite profiles (including amino acids, tricarboxylic acid (TCA) cycle and urea cycle intermediates) were determined in the body wall and the coelomic fluid. Exposure to nZnO altered metabolism of the lugworms via suppression of the metabolism of gluconeogenic and aromatic amino acids, and altered the TCA cycle likely via suppression of fumarase activity. These metabolic changes may negatively affect carbohydrate metabolism and energy storage, and impair hormonal signaling in the worms. The total pool of free amino acids was depleted in nZnO exposures with potentially negative consequences for osmoregulation and protein synthesis. Exposure to nZnO led to accumulation of the lipid peroxidation products demonstrating high susceptibility of the cellular membranes to nZnO-induced oxidative stress. The nZnO-induced shifts in the metabolite profiles were more pronounced in the coelomic fluid than the body wall. This finding emphasizes the important metabolic role of the coelomic fluid as well as its suitability for assessing the toxic impacts of nZnO and other metabolic disruptors. The metabolic disruptions caused by environmentally relevant concentrations of nZnO can have negative effects on the organisms' fitness impairing growth and reproduction of the populations of marine bioturbators like the lugworms in nanoparticle-polluted sediments.
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Nanopartículas , Poliquetos , Contaminantes Químicos del Agua , Óxido de Zinc , Animales , Ecosistema , Contaminantes Químicos del Agua/análisis , Nanopartículas/toxicidad , Aminoácidos/metabolismo , Óxido de Zinc/toxicidadRESUMEN
The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. It is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both enzyme systems. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic and highly reducing conditions while the PDH complex is inactivated. We found that cells rely on a general shift from utilizing NAD(H)- to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs-free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, the utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.
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Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Piruvato-Sintasa/metabolismo , Catálisis , Ferredoxinas/metabolismo , NAD/metabolismoRESUMEN
Fluctuating light (FL) is a typical natural light stress that can cause photodamage to photosystem I (PSI). However, the effect of growth light on FL-induced PSI photoinhibition remains controversial. Plants grown under high light enhance photorespiration to sustain photosynthesis, but the contribution of photorespiration to PSI photoprotection under FL is largely unknown. In this study, we examined the photosynthetic performance under FL in tomato (Lycopersicon esculentum) plants grown under high light (HL-plants) and moderate light (ML-plants). After an abrupt increase in illumination, the over-reduction of PSI was lowered in HL-plants, resulting in a lower FL-induced PSI photoinhibition. HL-plants displayed higher capacities for CO2 fixation and photorespiration than ML-plants. Within the first 60 s after transition from low to high light, PSII electron transport was much higher in HL-plants, but the gross CO2 assimilation rate showed no significant difference between them. Therefore, upon a sudden increase in illumination, the difference in PSII electron transport between HL- and ML-plants was not attributed to the Calvin-Benson cycle but was caused by the change in photorespiration. These results indicated that the higher photorespiration in HL-plants enhanced the PSI electron sink downstream under FL, which mitigated the over-reduction of PSI and thus alleviated PSI photoinhibition under FL. Taking together, we here for the first time propose that photorespiration acts as a safety valve for PSI photoprotection under FL.
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Desiccation-tolerant cyanobacteria can survive frequent hydration/dehydration cycles likely affecting inorganic carbon (Ci) levels. It was recently shown that red/far-red light serves as signal-preparing cells toward dehydration. Here, the effects of desiccation on Ci assimilation by Leptolyngbya ohadii isolated from Israel's Negev desert were investigated. Metabolomic investigations indicated a decline in ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation activity, and this was accelerated by far-red light. Far-red light negatively affected the Ci affinity of L. ohadii during desiccation and in liquid cultures. Similar effects were evident in the non-desiccation-tolerant cyanobacterium Synechocystis The Synechocystis Δcph1 mutant lacking the major phytochrome exhibited reduced photosynthetic Ci affinity when exposed to far-red light, whereas the mutant ΔsbtB lacking a Ci uptake inhibitory protein lost the far-red light inhibition. Collectively, these results suggest that red/far-red light perception likely via phytochromes regulates Ci uptake by cyanobacteria and that this mechanism contributes to desiccation tolerance in strains such as L. ohadii.
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Small proteins are critically involved in the acclimation response of photosynthetic cyanobacteria to nitrogen starvation. NblD is the 66-amino-acid effector of nitrogen-limitation-induced phycobilisome breakdown, which is believed to replenish the cellular amino acid pools. To address the physiological functions of NblD, the concentrations of amino acids, intermediates of the arginine catabolism pathway and several organic acids were measured during the response to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 wild type and in an nblD deletion strain. A characteristic signature of metabolite pool composition was identified, which shows that NblD-mediated phycobilisome degradation is required to maintain the cellular amino acid and organic acid pools during nitrogen starvation. Specific deviations from the wild type suggest wider-reaching effects that also affect such processes as redox homeostasis via glutathione and tetrapyrrole biosynthesis, both of which are linked to the strongly decreased glutamate pool, and transcriptional reprogramming via an enhanced concentration of 2-oxoglutarate, the metabolite co-regulator of the NtcA transcription factor. The essential role played by NblD in metabolic homeostasis is consistent with the widespread occurrence of NblD throughout the cyanobacterial radiation and the previously observed strong positive selection for the nblD gene under fluctuating nitrogen supply. Importance Cyanobacteria play important roles in the global carbon and nitrogen cycles. In their natural environment, these organisms are exposed to fluctuating nutrient conditions. Nitrogen starvation induces a coordinated nitrogen-saving program that includes the breakdown of nitrogen-rich photosynthetic pigments, particularly phycobiliproteins. The small protein NblD was recently identified as an effector of phycobilisome breakdown in cyanobacteria. In this study, we demonstrate that the NblD-mediated degradation of phycobiliproteins is needed to sustain cellular pools of soluble amino acids and other crucial metabolites. The essential role played by NblD in metabolic homeostasis explains why genes encoding this small protein are conserved in almost all members of cyanobacterial radiation.
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Photorespiration is an integral component of plant primary metabolism. Accordingly, it has been often observed that impairing the photorespiratory flux negatively impacts other cellular processes. In this study, the metabolic acclimation of the Arabidopsisthaliana wild type was compared with the hydroxypyruvate reductase 1 (HPR1; hpr1) mutant, displaying only a moderately reduced photorespiratory flux. Plants were analyzed during development and under varying photoperiods with a combination of non-targeted and targeted metabolome analysis, as well as 13C- and 14C-labeling approaches. The results showed that HPR1 deficiency is more critical for photorespiration during the vegetative compared to the regenerative growth phase. A shorter photoperiod seems to slowdown the photorespiratory metabolite conversion mostly at the glycerate kinase and glycine decarboxylase steps compared to long days. It is demonstrated that even a moderate impairment of photorespiration severely reduces the leaf-carbohydrate status and impacts on sulfur metabolism. Isotope labeling approaches revealed an increased CO2 release from hpr1 leaves, most likely occurring from enhanced non-enzymatic 3-hydroxypyruvate decarboxylation and a higher flux from serine towards ethanolamine through serine decarboxylase. Collectively, the study provides evidence that the moderate hpr1 mutant is an excellent tool to unravel the underlying mechanisms governing the regulation of metabolic linkages of photorespiration with plant primary metabolism.
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The ubiquitous freshwater cyanobacterium Microcystis is remarkably successful, showing a high tolerance against fluctuations in environmental conditions. It frequently forms dense blooms which can accumulate significant amounts of the hepatotoxin microcystin, which plays an extracellular role as an infochemical but also acts intracellularly by interacting with proteins of the carbon metabolism, notably with the CO2 fixing enzyme RubisCO. Here we demonstrate a direct link between external microcystin and its intracellular targets. Monitoring liquid cultures of Microcystis in a diel experiment revealed fluctuations in the extracellular microcystin content that correlate with an increase in the binding of microcystin to intracellular proteins. Concomitantly, reversible relocation of RubisCO from the cytoplasm to the cell's periphery was observed. These variations in RubisCO localization were especially pronounced with cultures grown at higher cell densities. We replicated these effects by adding microcystin externally to cultures grown under continuous light. Thus, we propose that microcystin may be part of a fast response to conditions of high light and low carbon that contribute to the metabolic flexibility and the success of Microcystis in the field.
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The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and non-targeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaA and, to a lesser extent, in the ΔkaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.
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Photorespiration, an essential component of plant metabolism, is concerted across four subcellular compartments, namely, chloroplast, peroxisome, mitochondrion, and the cytoplasm. It is unclear how the pathway located in different subcellular compartments respond to stress occurring exclusively in one of those. We attempted to assess the inter-organelle interaction during the photorespiratory pathway. For that purpose, we induced oxidative stress by menadione (MD) in mitochondria and photo-oxidative stress (high light) in chloroplasts. Subsequently, we examined the changes in selected photorespiratory enzymes, known to be located in other subcellular compartments. The presence of MD upregulated the transcript and protein levels of five chosen photorespiratory enzymes in both normal and high light. Peroxisomal glycolate oxidase and catalase activities increased by 50% and 25%, respectively, while chloroplastic glycerate kinase and phosphoglycolate phosphatase increased by ~30%. The effect of MD was maximum in high light, indicating photo-oxidative stress was an influential factor to regulate photorespiration. Oxidative stress created in mitochondria caused a coordinative upregulation of photorespiration in other organelles. We provided evidence that reactive oxygen species are important signals for inter-organelle communication during photorespiration. Thus, MD can be a valuable tool to modulate the redox state in plant cells to study the metabolic consequences across membranes.
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Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-l-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it PII-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-PII interaction requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell.IMPORTANCE Cyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g., as major primary producers. Due to their photosynthetic lifestyle, cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis. Besides its role as a proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. Therefore, the obtained results will not only enhance our understanding of flux control in these organisms but also help to provide a scientific basis for targeted metabolic engineering and, hence, the design of photosynthesis-driven biotechnological applications.