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M3 strain cannot grow in NO3 − or NO2 − but metabolizes them. (a) Growth test of the nii1 mutants (G1, M3, and M4) and the WT (6145c) strain on the indicated N sources. Plate wells were inoculated with 0.1 × 10 6 cells ·ml −1 and cultured for 7 days. (b-d) Extracellular NO2 − quantification in cultures incubated in the presence of either NO3 − (red line) or NO2 − (black line). NH4 + -grown cells were washed and transferred to NO3 − -or NO2 − -containing media at 0.1 mM in non-sealed bottles (b), 0.1 mM in sealed bottles (c), or 1 mM in sealed bottles (d). Error bars represent ±SD, n ≥ 3.
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Nitrous oxide (N2O) is a powerful greenhouse gas and an ozone-depleting compound whose synthesis and release have traditionally been ascribed to bacteria and fungi. Although plants and microalgae have been proposed as N2O producers in recent decades, the proteins involved in this process have been only recently unveiled. In the green microalga Chla...
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... nii1 mutants (G1, M3, and M4) cannot reduce NO2 − to ammonium (NH4 + ) and, therefore, do not grow in media containing either NO3 − or NO2 − as the sole nitrogen (N) source (Figure 1a). The G1 strain is a deletion mutant that lacks the entire cluster of the NO3 − assimilation genes. ...
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... we studied NO2 − evolution in the M3 strain. NH4 + -grown cells were washed and transferred to fresh media containing 0.1 and 1 mM NO3 − or NO2 − , and NO2 − concentration in the medium was determined at different time points (Figure 1b-d). Cells exposed to 0.1 mM NO3 − showed a stoichiometric excretion of NO2 − after 4 h (Figure 1b), as previously reported [41]. ...
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... + -grown cells were washed and transferred to fresh media containing 0.1 and 1 mM NO3 − or NO2 − , and NO2 − concentration in the medium was determined at different time points (Figure 1b-d). Cells exposed to 0.1 mM NO3 − showed a stoichiometric excretion of NO2 − after 4 h (Figure 1b), as previously reported [41]. Subsequently, extracellular NO2 − concentration slowly decreased, being completely exhausted from the medium after 24 h. ...
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... same experiment was performed in sealed bottles, in which N2O emission would be retained and could be quantified. Under these conditions, similar rates of accumulation and depletion of NO2 − were observed (Figure 1c). However, NO2 − depletion was induced faster than in non-sealed cultures (2 h vs. 6-8 h); therefore, NO2 − excretion after NO3 − reduction was not stoichiometric and reached only a concentration of 86 µM. ...
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... as observed in non-sealed bottles, NO2 − was exhausted before 24 h. A similar pattern was observed when cells were exposed to 1 mM NO3 − or NO2 − , although total depletion required longer incubations (Figure 1d). ...
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... NO addition led to a three-fold increase in the CO2 emission rate in the light but not in the dark, where only a slight reduction was observed (Figure 4b). To further confirm whether NO reduces CO2 emission in the dark, the M3 strain was treated with NO donor in N-free medium in the dark, and after a short incubation time (75 min) (Supplementary Figure S1). Before NO donor addition, the CO2 emission rate was 242 ppm/h, but after NO donor addition, the CO2 emission rate decreased to 88 ppm/h. ...
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... NO donor addition, the CO2 emission rate was 242 ppm/h, but after NO donor addition, the CO2 emission rate decreased to 88 ppm/h. Accordingly, the N2O emission rate increased from 0 to 8 ppm/h (Supplementary Figure S1). CO2 emission was also studied in M3 cells under N deprivation and different NO2 − concentrations in the light (Supplementary Figure S2). ...
Citations
... Conversely, the ability of algae to assimilate nitrogen lowers the extent of N 2 O production during denitrification, which represents an advantage of HRAPs. However, nitrogen assimilation by microalgae does not necessarily prevent the potential total emissions of N 2 O due to the inherent ability of microalgae to synthesize N 2 O (Bellido-Pedraza et al., 2022;Burlacot et al., 2019;Plouviez and Guieysse, 2020). The mass of N 2 O emitted from an algal pond can vary depending on factors such as temperature, pH, nutrient concentrations and algal biomass concentration (Alcántara et al., 2015a). ...
... MB reduction to leucomethylene blue (MBH) by ascorbic acid in acetonitrile is studied by Hallock et al. [9] using cavity ring-down spectroscopy (CRDS) [10]. This technique allows for precise monitoring of concentration changes on a microsecond timescale, which was previously unattainable. ...
This paper offers a thorough investigation of hyperparameter tuning for neural network architectures using datasets encompassing various combinations of Methylene Blue (MB) Reduction by Ascorbic Acid (AA) reactions with different solvents and concentrations. The aim is to predict coefficients of decay plots for MB absorbance, shedding light on the complex dynamics of chemical reactions. Our findings reveal that the optimal model, determined through our investigation, consists of five hidden layers, each with sixteen neurons and employing the Swish activation function. This model yields an NMSE of 0.05, 0.03, and 0.04 for predicting the coefficients A, B, and C, respectively, in the exponential decay equation A + B · e−x/C. These findings contribute to the realm of drug design based on machine learning, providing valuable insights into optimizing chemical reaction predictions.
... Fungi have developed effective mechanisms for NO detoxification and removal toward reducing the cytotoxicity caused by an excessive accumulation of endogenous NO. Regarding the fate of NO produced in fungal cells, there are three alternatives. First, NO can be further reduced to N2O via catalysis of nitric oxide reductase (Nor), a type of cytochrome P450, during the denitrification process [2,69,86]. This mechanism has been demonstrated in the genera Fusarium, Trichoderma, and Guehomyces [31]. ...
Nitric oxide (NO) is synthesized in all kingdoms of life, where it plays a role in the regulation of various physiological and developmental processes. In terms of endogenous NO biology, fungi have been less well researched than mammals, plants, and bacteria. In this review, we summarize and discuss the studies to date on intracellular NO biosynthesis and function in fungi. Two mechanisms for NO biosynthesis, NO synthase (NOS)-mediated arginine oxidation and nitrate- and nitrite-reductase-mediated nitrite reduction, are the most frequently reported. Furthermore, we summarize the multifaceted functions of NO in fungi as well as its role as a signaling molecule in fungal growth regulation, development, abiotic stress, virulence regulation, and metabolism. Finally, we present potential directions for future research on fungal NO biology.
... Thus, the NR-mARC complex may play a critical role in modulating cellular NO levels. Furthermore, the NR-mARC complex of Chlamidomonas is able to regulate N 2 O production through NO synthesis [160]. ...
The optimization of all constituent conditions to obtain high and even maximum yields is a recent trend in agriculture. Legumes play a special role in this process, as they have unique characteristics with respect to storing protein and many other important components in their seeds that are useful for human and animal nutrition as well as industry and agriculture. A great advantage of legumes is the nitrogen fixation activity of their symbiotic nodule bacteria. This nitrogen self-sufficiency contributes directly to the challenging issue of feeding the world’s growing population. Molybdenum is one of the most sought-after nutrients because it provides optimal conditions for the maximum efficiency of the enzymes involved in nitrogen assimilation as well as other molybdenum-containing enzymes in the host plant and symbiotic nodule bacteria. In this review, we consider the most optimal way of providing legume plants with molybdenum, its distribution in ontogeny throughout the plant, and its accumulation at the end of the growing season in the seeds. Overall, molybdenum supply improves seed quality and allows for the efficient use of the micronutrient by molybdenum-containing enzymes in the plant and subsequently the nodules at the initial stages of growth after germination. A sufficient supply of molybdenum avoids competition for this trace element between nitrogenase and nodule nitrate reductase, which enhances the supply of nitrogen to the plant. Finally, we also consider the possibility of regulating molybdenum homeostasis using modern genetic approaches.
... We chose the wild type 6145c for experiments because it is an arginine prototroph and can utilize ammonium, nitrate, or nitrite as nitrogen sources [28]. In the first type of analysis, cell growth was compared on two nitrogen sources, ammonium and nitrate (Figure 1a). ...
N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.
... We chose the wild type 6145c for experiments because it is an arginine prototroph and can utilize ammonium, nitrate or nitrite as nitrogen sources [28]. In the first type of analysis, the cell growth was compared on two nitrogen sources, ammonium and nitrate ( Figure 1a). ...
N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, yet there is still much to be learnt on how NAGK is regulated in vivo. The use of unicellular green Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provided primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.
... These data suggest that the NR-mARC pair is a very efficient machinery for synthesizing NO under physiological conditions, in the presence of both nitrate and nitrite, and may have a very important role in modulating cellular NO levels. Apart from NO, the Chlamydomonas complex NR-mARC has been shown to regulate N 2 O production, a greenhouse gas, through NO synthesis [121]. The mARC proteins have been proposed as moonlighting proteins [122]. ...
Molybdenum (Mo) is vital for the activity of a small but essential group of enzymes called molybdoenzymes. So far, specifically five molybdoenzymes have been discovered in eukaryotes: nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and mARC. In order to become biologically active, Mo must be chelated to a pterin, forming the so-called Mo cofactor (Moco). Deficiency or mutation in any of the genes involved in Moco biosynthesis results in the simultaneous loss of activity of all molybdoenzymes, fully or partially preventing the normal development of the affected organism. To prevent this, the different mechanisms involved in Mo homeostasis must be finely regulated. Chlamydomonas reinhardtii is a unicellular, photosynthetic, eukaryotic microalga that has produced fundamental advances in key steps of Mo homeostasis over the last 30 years, which have been extrapolated to higher organisms, both plants and animals. These advances include the identification of the first two molybdate transporters in eukaryotes (MOT1 and MOT2), the characterization of key genes in Moco biosynthesis, the identification of the first enzyme that protects and transfers Moco (MCP1), the first characterization of mARC in plants, and the discovery of the crucial role of the nitrate reductase-mARC complex in plant nitric oxide production. This review aims to provide a comprehensive summary of the progress achieved in using C. reinhardtii as a model organism in Mo homeostasis and to propose how this microalga can continue improving with the advancements in this field in the future.
... The use of tungsten as an NR inhibitor was reported to inhibit N 2 O formation in plants [181], while NR inhibition challenged the plants' survival, as described in the above section, which further supports the concept that N 2 O formation also could play a role in plants' survival strategies. Moreover, recent results suggest that NR plays critical role in NO-mediated N 2 O formation in microalgae Chlamydomonas reinhardtii [182]. Both NO [183] and N 2 O [184] can increase the activities of phenylalanine ammonialyase, cinnamate-4-hydroxylase, and 4-coumaroyl-CoA ligase during pathogen attack in plants while increasing total phenolic, flavonoid, and lignin content. ...
Oxygen (O2) is the most crucial substrate for numerous biochemical processes in plants. Its deprivation is a critical factor that affects plant growth and may lead to death if it lasts for a long time. However, various biotic and abiotic factors cause O2 deprivation, leading to hypoxia and anoxia in plant tissues. To survive under hypoxia and/or anoxia, plants deploy various mechanisms such as fermentation paths, reactive oxygen species (ROS), reactive nitrogen species (RNS), antioxidant enzymes, aerenchyma, and adventitious root formation, while nitrate (NO3−), nitrite (NO2−), and nitric oxide (NO) have shown numerous beneficial roles through modulating these mechanisms. Therefore, in this review, we highlight the role of reductive pathways of NO formation which lessen the deleterious effects of oxidative damages and increase the adaptation capacity of plants during hypoxia and anoxia. Meanwhile, the overproduction of NO through reductive pathways during hypoxia and anoxia leads to cellular dysfunction and cell death. Thus, its scavenging or inhibition is equally important for plant survival. As plants are also reported to produce a potent greenhouse gas nitrous oxide (N2O) when supplied with NO3− and NO2−, resembling bacterial denitrification, its role during hypoxia and anoxia tolerance is discussed here. We point out that NO reduction to N2O along with the phytoglobin-NO cycle could be the most important NO-scavenging mechanism that would reduce nitro-oxidative stress, thus enhancing plants’ survival during O2-limited conditions. Hence, understanding the molecular mechanisms involved in reducing NO toxicity would not only provide insight into its role in plant physiology, but also address the uncertainties seen in the global N2O budget
Nitrate reductase (NR) catalyzes the rate-limiting step in nitrate assimilation. Plant and algal NRs have a highly conserved domain architecture but differ in regulation. In plants, NR activity is regulated by reversible phosphorylation and subsequent binding of 14-3-3 proteins at a conserved serine residue. Algal NRs typically lack 14-3-3 binding motifs, which have only recently been identified in a few algal species. Previous research indicates that the alga, Chattonella subsalsa, possesses a novel NR, NR2-2/2HbN (NR2), which incorporates a 2/2 hemoglobin domain. A second NR (NR3) in C. subsalsa lacks the cytochrome b5 (heme-Fe) domain but includes a putative binding motif for 14-3-3 proteins. The expression of NR2 and NR3 genes indicates that NR2 transcript abundance was regulated by light, nitrogen source, and temperature, while NR3 transcript levels were only regulated by light. Here, we measured total NR activity in C. subsalsa and the potential for regulation of NR activity by putative 14-3-3 binding proteins. Results indicate that NR activity in C. subsalsa was regulated by light, nitrogen source, and temperature at the translational level. NR activity was also regulated by endogenous rhythm and temperature at the post-translational level, supporting the hypothesis that NR3 is regulated by 14-3-3 binding proteins. Together with a previous report describing the regulation of NR gene expression in C. subsalsa, results suggest that C. subsalsa responds to environmental conditions by differential regulation of NRs at transcriptional, translational, and post-translational levels. This flexibility may provide a competitive advantage for this species in the environment. To date, this is the first report which provides evidence for the potential post-translational regulation of NR by 14-3-3 proteins in algal species and suggests that regulatory mechanisms for NR activity may be shared between plants and some algal species.
