Role of Nitrate Reductase in NO Production in Photosynthetic Eukaryotes

Nitric oxide is a gaseous secondary messenger that is critical for proper cell signaling and plant survival when exposed to stress. Nitric oxide (NO) synthesis in plants, under standard phototrophic oxygenic conditions, has long been a very controversial issue. A few algal strains contain NO synthase (NOS), which appears to be absent in all other algae and land plants. The experimental data have led to the hypothesis that molybdoenzyme nitrate reductase (NR) is the main enzyme responsible for NO production in most plants. Recently, NR was found to be a necessary partner in a dual system that also includes another molybdoenzyme, which was renamed NO-forming nitrite reductase (NOFNiR). This enzyme produces NO independently of the molybdenum center of NR and depends on the NR electron transport chain from NAD(P)H to heme. Under the circumstances in which NR is not present or active, the existence of another NO-forming system that is similar to the NOS system would account for NO production and NO effects. PII protein, which senses and integrates the signals of the C–N balance in the cell, likely has an important role in organizing cell responses. Here, we critically analyze these topics

Algae-Bacteria Consortia as a Strategy to Enhance H2 Production

Biological hydrogen production by microalgae is a potential sustainable, renewable and clean source of energy. However, many barriers limiting photohydrogen production in these microorganisms remain unsolved. In order to explore this potential and make biohydrogen industrially affordable, the unicellular microalga Chlamydomonas reinhardtii is used as a model system to solve barriers and identify new approaches that can improve hydrogen production. Recently, Chlamydomonas–bacteria consortia have opened a new window to improve biohydrogen production. In this study, we review the different consortia that have been successfully employed and analyze the factors that could be behind the improved H2 production

Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1

The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction of several N-hydroxylated compounds (NHC) and nitrite. Some NHC are prodrugs containing an amidoxime structure or mutagens such as 6-hydroxylaminopurine (HAP). We have studied this protein in the green alga Chlamydomonas reinhardtii (crARC). Interestingly, all the ARC proteins need the reducing power supplied by other proteins. It is known that crARC requires a cytochrome b5 (crCytb5-1) and a cytochrome b5 reductase (crCytb5-R) that form an electron transport chain from NADH to the substrates. Here, we have investigated NHC reduction by crARC, the interaction with its partners and the function of important conserved amino acids. Interactions among crARC, crCytb5-1 and crCytb5-R have been studied by size-exclusion chromatography. A protein complex between crARC, crCytb5-1 and crCytb5-R was identified. Twelve conserved crARC amino acids have been substituted by alanine by in vitro mutagenesis. We have determined that the amino acids D182, F210 and R276 are essential for NHC reduction activity, R276 is important and F210 is critical for the Mo Cofactor chelation. Finally, the crARC C-termini were shown to be involved in protein aggregation or oligomerizatio

Identification of the MAPK Cascade and its Relationship with Nitrogen Metabolism in the Green Alga Chlamydomonasreinhardtii

The mitogen activated protein kinases (MAPKs) form part of a signaling cascade through phosphorylation reactions conserved in all eukaryotic organisms. The MAPK cascades are mainly composed by threeproteins, MAPKKKs, MAPKKs and MAPKs. Some signals induce MAPKKK-mediated phosphorylation and activation of MAPKK that phosphorylate and activate MAPK. Afterward, MAPKs can act either in the cytoplasm or be imported into the nucleus to activate other proteins or transcription factors. In the green microalga Chlamydomonasreinhardtii the pathway for nitrogen (N) assimilation is well characterized, yet its regulation still has many unknown features. Nitric oxide (NO) is a fundamental signal molecule for N regulation, where nitrate reductase (NR) plays a central role in its synthesis. The MAPK cascades could be regulating N assimilation, since it has been described that the phosphorylation of NR by MAPK6 promotes NO production in Arabidopsis thaliana. We have identified the proteins involved in the MAPK cascades in Chlamydomonasreinhardtii, finding 17 MAPKs, 2 MAPKKs and 108 MAPKKKs (11 MEKK-, 94 RAF- and 3 ZIK-type) that have been structurally and phylogenetically characterized. The genetic expressions of MAPKs and the MAPKK were slightly regulated by N. However, the genetic expressions of MAPKKKs RAF14 and RAF79 showed a very strong repression by ammonium, which suggests that they may have a key role in the regulation of N assimilation, encouraging to further analyze in detail the role of MAPK cascades in the regulation of N metabolism

Arginine is a component of the ammonium- CYG56 signalling cascade that represses genes of the nitrogen assimilation pathway in Chlamydomonas reinhardtii

Nitrogen assimilation and metabolism are essential processes for all living organisms, yet there is still much to be learnt on how they are regulated. The use of Chlamydomonas reinhardtii as a model system has been instrumental not only in identifying conserved regulation mechanisms that control the nitrogen assimilation pathway, but also in understanding how the intracellular nitrogen status regulates metabolic processes of industrial interest such as the synthesis of biolipids. While the genetic regulators that control the nitrogen pathway are successfully being unravelled, other layers of regulation have received less attention. Amino acids, for example, regulate nitrogen assimilation in certain organisms, but their role in Chlamydomonas has not thoroughly been explored. Previous results had suggested that arginine might repress key genes of the nitrogen assimilation pathway by acting within the ammonium negative signalling cascade, upstream of the nitric oxide (NO) inducible guanylate cyclase CYG56. We tested this hypothesis with a combination of genetic and chemical approaches. Antagonising the effects of arginine with an arginine biosynthesis mutant or with two chemical analogues released gene expression from ammonium mediated repression. The cyg56 and related non1 mutants, which are partially insensitive to ammonium repression, were also partially insensitive to repression by arginine. Finally, we show that the addition of arginine to the medium leads to an increase in intracellular NO. Our data reveal that arginine acts as a negative signal for the assimilation of nitrogen within the ammonium-CYG56 negative signalling cascade, and provide a connection between amino acid metabolism and nitrogen assimilation in microalgae

Characterization of a Mutant Deficient for Ammonium and Nitric Oxide Signalling in the Model System Chlamydomonas reinhardtii

The ubiquitous signalling molecule Nitric Oxide (NO) is characterized not only by the variety of organisms in which it has been described, but also by the wealth of biological processes that it regulates. In contrast to the expanding repertoire of functions assigned to NO, however, the mechanisms of NO action usually remain unresolved, and genes that work within NO signalling cascades are seldom identified. A recent addition to the list of known NO functions is the regulation of the nitrogen assimilation pathway in the unicellular alga Chlamydomonas reinhardtii, a well-established model organism for genetic and molecular studies that offers new possibilities in the search for mediators of NO signalling. By further exploiting a collection of Chlamydomonas insertional mutant strains originally isolated for their insensitivity to the ammonium (NH4 +) nitrogen source, we found a mutant which, in addition to its ammonium insensitive (AI) phenotype, was not capable of correctly sensing the NO signal. Similarly to what had previously been described in the AI strain cyg56, the expression of nitrogen assimilation genes in the mutant did not properly respond to treatments with various NO donors. Complementation experiments showed that NON1 (NO Nitrate 1), a gene that encodes a protein containing no known functional domain, was the gene underlying the mutant phenotype. Beyond the identification of NON1, our findings broadly demonstrate the potential for Chlamydomonas reinhardtii to be used as a model system in the search for novel components of gene networks that mediate physiological responses to NO

From the Eukaryotic Molybdenum Cofactor Biosynthesis to the Moonlighting Enzyme mARC

All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue

Nitrous Oxide Emissions from Nitrite Are Highly Dependent on Nitrate Reductase in the Microalga Chlamydomonas reinhardtii

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 Chlamydomonas reinhardtii, flavodiiron proteins (FLVs) and cytochrome P450 (CYP55) are two nitric oxide (NO) reductases responsible for N2O synthesis in the chloroplast and mitochondria, respectively. However, the molecular mechanisms feeding these NO reductases are unknown. In this work, we use cavity ring-down spectroscopy to monitor N2O and CO2 in cultures of nitrite reductase mutants, which cannot grow on nitrate or nitrite and exhibit enhanced N2O emissions. We show that these mutants constitute a very useful tool to study the rates and kinetics of N2O release under different conditions and the metabolism of this greenhouse gas. Our results indicate that N2O production, which was higher in the light than in the dark, requires nitrate reductase as the major provider of NO as substrate. Finally, we show that the presence of nitrate reductase impacts CO2 emissions in both light and dark conditions, and we discuss the role of NO in the balance between CO2 fixation and release

Low oxygen levels contribute to improve photohydrogen production in mixotrophic non‑stressed Chlamydomonas cultures

Background: Currently, hydrogen fuel is derived mainly from fossil fuels, but there is an increasing interest in clean and sustainable technologies for hydrogen production. In this context, the ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen is a promising alternative for renewable, clean-energy production. Among a diverse array of photosynthetic microorganisms able to produce hydrogen, the green algae Chlamydomonas reinhardtii is the model organism widely used to study hydrogen production. Despite the well-known fact that acetate-containing medium enhances hydrogen production in this algae, little is known about the precise role of acetate during this process. Results: We have examined several physiological aspects related to acetate assimilation in the context of hydrogen production metabolism. Measurements of oxygen and CO2 levels, acetate uptake, and cell growth were performed under different light conditions, and oxygenic regimes. We show that oxygen and light intensity levels control acetate assimilation and modulate hydrogen production. We also demonstrate that the determination of the contribution of the PSII-dependent hydrogen production pathway in mixotrophic cultures, using the photosynthetic inhibitor DCMU, can lead to dissimilar results when used under various oxygenic regimes. The level of inhibition of DCMU in hydrogen production under low light seems to be linked to the acetate uptake rates. Moreover, we highlight the importance of releasing the hydrogen partial pressure to avoid an inherent inhibitory factor on the hydrogen production. Conclusion: Low levels of oxygen allow for low acetate uptake rates, and paradoxically, lead to efficient and sustained production of hydrogen. Our data suggest that acetate plays an important role in the hydrogen production process, during non-stressed conditions, other than establishing anaerobiosis, and independent of starch accumulation. Potential metabolic pathways involved in hydrogen production in mixotrophic cultures are discussed. Mixotrophic nutrient-replete cultures under low light are shown to be an alternative for the simultaneous production of hydrogen and biomass

Understanding nitrate assimilation and its regulation in microalgae

Nitrate assimilation is a key process for nitrogen (N) acquisition in green microalgae. Among Chlorophyte algae, Chlamydomonas reinhardtii has resulted to be a good model system to unravel important facts of this process, and has provided important insights for agriculturally relevant plants. In this work, the recent findings on nitrate transport, nitrate reduction and the regulation of nitrate assimilation are presented in this and several other algae. Latest data have shown nitric oxide (NO) as an important signal molecule in the transcriptional and posttranslational regulation of nitrate reductase and inorganic N transport. Participation of regulatory genes and proteins in positive and negative signaling of the pathway and the mechanisms involved in the regulation of nitrate assimilation, as well as those involved in Molybdenum cofactor synthesis required to nitrate assimilation, are critically reviewed