Protein NirP1 regulates nitrite reductase and nitrite excretion in cyanobacteria.

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.

A low red/far-red ratio restricts nitrogen assimilation by inhibiting nitrate reductase associated with downregulated TaNR1.2 and upregulated TaPIL5 in wheat (Triticum aestivum L.).

Understanding the physiological mechanism underlying nitrogen levels response to a low red/far-red ratio (R/FR) can provide new insights for optimizing wheat yield potential but has been not well documented. This study focused on the changes in nitrogen levels, nitrogen assimilation and nitrate uptake in wheat plants grown with and without additional far-red light. A low R/FR reduced wheat nitrogen accumulation and grain yield compared with the control. The levels of total nitrogen, free amino acid and ammonium were decreased in leaves but nitrate content was temporarily increased under a low R/FR. The nitrate reductase (NR) activity in leaves was more sensitive to a low R/FR than glutamine synthetase, glutamate synthase, glutamic oxalacetic transaminase and glutamic-pyruvic transaminase. Further analysis showed that a low R/FR had little effect on the NR activation state but reduced the level of NR protein and the expression of encoding gene TaNR1.2. Interestingly, a low R/FR rapidly induced TaPIL5 expression rather than TaHY5 and other members of TaPILs in wheat, suggesting that TaPIL5 was the key transcription factor response to a low R/FR in wheat and might be involved in the downregulation of TaNR1.2 expression. Besides, a low R/FR downregulated the expression of TaNR1.2 in leaves earlier than that of TaNRT1.1/1.2/1.5/1.8 in roots, which highlights the importance of NR and nitrogen assimilation in response to a low R/FR. Our results provide revelatory evidence that restricted nitrate reductase associated with downregulated TaNR1.2 and upregulated TaPIL5 mediate the suppression of nitrogen assimilation under a low R/FR in wheat.

Pseudomonas aeruginosa LasI-dependent plant growth promotion requires the host nitrate transceptor AtNRT1.1/CHL1 and the nitrate reductases NIA1 and NIA2.

MAIN CONCLUSION: In P. aeruginosa, mutation of the gene encoding N-acyl-L-homoserine lactone synthase LasI drives defense and plant growth promotion, and this latter trait requires adequate nitrate nutrition. Cross-kingdom communication with bacteria is crucial for plant growth and productivity. Here, we show a strong induction of genes for nitrate uptake and assimilation in Arabidopsis seedlings co-cultivated with P. aeruginosa WT (PAO1) or ΔlasI mutants defective on the synthesis of the quorum-sensing signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone. Along with differential induction of defense-related genes, the change from plant growth repression to growth promotion upon bacterial QS disruption, correlated with upregulation of the dual-affinity nitrate transceptor CHL1/AtNRT1/NPF6.3 and the nitrate reductases NIA1 and NIA2. CHL1-GUS was induced in Arabidopsis primary root tips after transfer onto P. aeruginosa ΔlasI streaks at low and high N availability, whereas this bacterium required high concentrations of nitrogen to potentiate root and shoot biomass production and to improve root branching. Arabidopsis chl1-5 and chl1-12 mutants and double mutants in NIA1 and NIA2 nitrate reductases showed compromised growth under low nitrogen availability and failed to mount an effective growth promotion and root branching response even at high NH4NO3. WT P. aeruginosa PAO1 and P. aeruginosa ΔlasI mutant promoted the accumulation of nitric oxide (NO) in roots of both the WT and nia1nia2 double mutants, whereas NO donors SNP or SNAP did not improve growth or root branching in nia1nia2 double mutants with or without bacterial cocultivation. Thus, inoculation of Arabidopsis roots with P. aeruginosa drives gene expression for improved nitrogen acquisition and this macronutrient is critical for the plant growth-promoting effects upon disruption of the LasI quorum-sensing system.

Molecular hydrogen positively regulates nitrate uptake and seed size by targeting nitrate reductase.

Although the sources of molecular hydrogen (H2) synthesis in plants remain to be fully elucidated, ample evidence shows that plant-based H2 can regulate development and stress responses. Here, we present genetic and molecular evidence indicating that nitrate reductase (NR) might be a target of H2 sensing that positively regulates nitrogen use efficiency (NUE) and seed size in Arabidopsis (Arabidopsis thaliana). The expression level of NR and changes of NUE under control and, in particular, low nitrogen supply were positively associated with H2 addition supplied exogenously or through genetic manipulation. The improvement in nitrate assimilation achieved by H2 was also mediated via NR dephosphorylation. H2 control of seed size was impaired by NR mutation. Further genetic evidence revealed that H2, NR, and nitric oxide can synergistically regulate nitrate assimilation in response to N starvation conditions. Collectively, our data indicate that NR might be a target for H2 sensing, ultimately positively regulating nitrate uptake and seed size. These results provide insights into H2 signaling and its functions in plant metabolism.

Enhanced growth performance of abi5 plants under high salt and nitrate is associated with reduced nitric oxide levels.

Numerous environmental stresses have a significant impact on plant growth and development. By 2050, it is anticipated that high salinity will destroy more than fifty percent of the world's agricultural land. Understanding how plants react to the excessive use of nitrogen fertilizers and salt stress is crucial for enhancing crop yield. However, the effect of excessive nitrate treatment on plant development is disputed and poorly understood; so, we evaluated the effect of excessive nitrate supply and high salinity on abi5 plant growth performance. We demonstrated that abi5 plants are tolerant to the harmful environmental conditions of excessive nitrate and salt. abi5 plants have lower amounts of endogenous nitric oxide than Arabidopsis thaliana Columbia-0 plants due to their decreased nitrate reductase activity, caused by a decrease in the transcript level of NIA2, a gene encoding nitrate reductase. Nitric oxide appeared to have a critical role in reducing the salt stress tolerance of plants, which was diminished by an excess of nitrate. Discovering regulators such as ABI5 that can modulate nitrate reductase activity and comprehending the molecular activities of these regulators are crucial for the application of gene-editing techniques. This would result in the appropriate buildup of nitric oxide to increase the production of crops subjected to a variety of environmental stresses.

Nitrate Reductase NarGHJI Modulates Virulence via Regulation of agr Expression in Methicillin-Resistant Staphylococcus aureus Strain USA300 LAC.

Staphylococcus aureus is a pathogenic bacterium with a widespread distribution that can cause diverse severe diseases. The membrane-bound nitrate reductase NarGHJI serves respiratory function. However, little is known about its contribution to virulence. In this study, we demonstrated that narGHJI disruption results in the downregulation of virulence genes (e.g., RNAIII, agrBDCA, hla, psmα, and psmß) and reduces the hemolytic activity of the methicillin-resistant S. aureus (MRSA) strain USA300 LAC. Moreover, we provided evidence that NarGHJI participates in regulating host inflammatory response. A mouse model of subcutaneous abscess and Galleria mellonella survival assay demonstrated that the ΔnarG mutant was significantly less virulent than the wild type. Interestingly, NarGHJI contributes to virulence in an agr-dependent manner, and the role of NarGHJI differs between different S. aureus strains. Our study highlights the novel role of NarGHJI in regulating virulence, thereby providing a new theoretical reference for the prevention and control of S. aureus infection. IMPORTANCE Staphylococcus aureus is a notorious pathogen that poses a great threat to human health. The emergence of drug-resistant strains has significantly increased the difficulty of preventing and treating S. aureus infection and enhanced the pathogenic ability of the bacterium. This indicates the importance of identifying novel pathogenic factors and revealing the regulatory mechanisms through which they regulate virulence. The nitrate reductase NarGHJI is mainly involved in bacterial respiration and denitrification, which can enhance bacterial survival. We demonstrated that narGHJI disruption results in the downregulation of the agr system and agr-dependent virulence genes, suggesting that NarGHJI participates in the regulation of S. aureus virulence in an agr-dependent manner. Moreover, the regulatory approach is strain specific. This study provides a new theoretical reference for the prevention and control of S. aureus infection and reveals new targets for the development of therapeutic drugs.

Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy of Rapaza viridis.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here, we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified gene RvNaRL, which had sequence similarity to genes encoding nitrate reductases in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify the function of the protein product RvNaRL, we established RNAi-mediated knock-down and CRISPR-Cas9-mediated knock-out experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knock-down and knock-out cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in the absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains, as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to the acquisition of nitrate assimilation via horizontal gene transfer.

DksA, ppGpp, and RegAB Regulate Nitrate Respiration in Paracoccus denitrificans.

The periplasmic (NAP) and membrane-associated (Nar) nitrate reductases of Paracoccus denitrificans are responsible for nitrate reduction under aerobic and anaerobic conditions, respectively. Expression of NAP is elevated in cells grown on a relatively reduced carbon and energy source (such as butyrate); it is believed that NAP contributes to redox homeostasis by coupling nitrate reduction to the disposal of excess reducing equivalents. Here, we show that deletion of either dksA1 (one of two dksA homologs in the P. denitrificans genome) or relA/spoT (encoding a bifunctional ppGpp synthetase and hydrolase) eliminates the butyrate-dependent increase in nap promoter and NAP enzyme activity. We conclude that ppGpp likely signals growth on a reduced substrate and, together with DksA1, mediates increased expression of the genes encoding NAP. Support for this model comes from the observation that nap promoter activity is increased in cultures exposed to a protein synthesis inhibitor that is known to trigger ppGpp synthesis in other organisms. We also show that, under anaerobic growth conditions, the redox-sensing RegAB two-component pair acts as a negative regulator of NAP expression and as a positive regulator of expression of the membrane-associated nitrate reductase Nar. The dksA1 and relA/spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. We also show that the second dksA homolog (dksA2) and relA/spoT have roles in regulation of expression of the flavohemoglobin Hmp and in biofilm formation. IMPORTANCE Paracoccus denitrificans is a metabolically versatile Gram-negative bacterium that is used as a model for studies of respiratory metabolism. The organism can utilize nitrate as an electron acceptor for anaerobic respiration, reducing it to dinitrogen via nitrite, nitric oxide, and nitrous oxide. This pathway (known as denitrification) is important as a route for loss of fixed nitrogen from soil and as a source of the greenhouse gas nitrous oxide. Thus, it is important to understand those environmental and genetic factors that govern flux through the denitrification pathway. Here, we identify four proteins and a small molecule (ppGpp) which function as previously unknown regulators of expression of enzymes that reduce nitrate and oxidize nitric oxide.

Identification and examination of nitrogen metabolic genes in Lelliottia amnigena PTJIIT1005 for their ability to perform nitrate remediation.

Lelliottia amnigena PTJIIT1005 is a bacterium that utilizes nitrate as the sole nitrogen source and can remediate nitrate from media. The annotation was done related to nitrogen metabolic genes using the PATRIC, RAST tools, and PGAP from the genome sequence of this bacterium. Multiple sequence alignments and phylogenetic analysis of respiratory nitrate reductase, assimilatory nitrate reductase, nitrite reductase, glutamine synthetase, hydroxylamine reductase, nitric oxide reductase genes from PTJIIT1005 were done to find out sequence identities with the most similar species. The identification of operon arrangement in bacteria was also identified. The PATRIC KEGG feature mapped the N-metabolic pathway to identify the chemical process, and the 3D structure of representative enzymes was also elucidated. The putative protein 3D structure was analyzed using I-TASSER software. It gave good quality protein models of all nitrogen metabolism genes and showed good sequence identity with reference templates, approximately 81-99%, except for two genes; assimilatory nitrate reductase and nitrite reductase. This study suggested that PTJIIT1005 can remove N-nitrate from water because of having N-assimilation and denitrification genes.

Cloning and characterization of nitrate reductase gene in kelp Saccharina japonica (Laminariales, Phaeophyta).

BACKGROUND: Brown macroalgae dominate temperate coastal ecosystems, and their productivity is typically limited by nitrate availability. As an economically important kelp, Saccharina japonica is the most productive farmed seaweed and needs to be supplemented with sufficient nitrate throughout the cultivation process. However, molecular characterization of genes involved in nitrogen assimilation has not been conducted in brown macroalgae. RESULTS: Here, we described the identification of the nitrate reductase (NR) gene from S. japonica (SjNR). Using two different cloning methods for SjNR, i.e. rapid amplification of cDNA ends (RACE) and cDNA cloning alone, a single fragment was obtained respectively. According to results of sequence analysis between these two fragments, the tentative coding sequence in two clones, SjNR-L and SjNR-S, were suggested to represent two transcripts of the single copy SjNR, and the ATG of SjNR-S was located inside the third exon of SjNR-L. In the 5' upstream sequence of each transcript, promoter core elements, response elements, especially multiple N response elements which occurred in microalgal NR, were all predicted. Further sequence analysis revealed that both transcripts encoded all five domains conserved in eukaryotic plant NRs. RT-qPCR results showed that the transcription level of SjNR in juvenile sporophytes could be significantly induced by nitrate and inhibited by ammonium, which was in line with plant NRs. The recombinant SjNR-L and SjNR-S were all proved to have NR activity, suggesting that the single-copy gene SjNR might be regulated on transcription level based on alternative promoters and multiple transcriptional start sites. Moreover, both NADH and NADPH were found to be able to act as electron donors for SjNR alone, which is the first confirmation that brown algal NR has a NAD(P)H-bispecific form. CONCLUSION: These results will provide a scientific basis for understanding the N demand of kelp in various stages of cultivation and evaluating the environmental remediation potential of kelp in eutrophic sea areas.

An apple NITRATE REDUCTASE 2 gene positively regulates nitrogen utilization and abiotic stress tolerance in Arabidopsis and apple callus.

Nitrogen (N) is an essential element that plays an important role in crop biomass accumulation and quality formation. Increased crop yield is relied on excessive application of fertilizers, which usually leads to environmental pollution and unsustainable development. Thus, identification and characterization of genes involved in promoting nitrogen use efficiency is of high priority in crop breeding. The activity of nitrate reductase (NR) plays a critical role in nitrogen metabolism. In model plant Arabidopsis, NITRATE REDUCTASE 2 (NIA2), one of the two NRs, is responsible for about 90% of the NR activity. In this study, MdNIA2 gene in apple (Malus domestica) genome was screened out and identified by using AtNIA2 as bait. Phylogenetic analysis revealed that MdNIA2 had the closest evolutionary relationship with MbNIA from Malus baccata. Ectopic expression of MdNIA2 in Arabidopsis elevated the nitrogen use efficiency and increased root hair elongation and formation, resulting in promoted plant growth. Furthermore, the overexpression of MdNIA2 improved salt and drought tolerance in transgenic Arabidopsis and improved the salt tolerance of transgenic apple callus, and MdNIA2-reagualted NO metabolism might contribute to the abiotic stress tolerance. Overall, our data indicate the critical role of MdNIA2 in regulating nitrogen utilization efficiency and abiotic stress responses.

Physio-biochemical, agronomical, and gene expression analysis reveals different responsive approach to low nitrogen in contrasting rice cultivars for nitrogen use efficiency.

BACKGROUND: Nitrogen (N) is an essential macronutrient for plant growth and development as it is an essential constituent of biomolecules. Its availability directly impacts crop yield. Increased N application in crop fields has caused environmental and health problems, and decreasing nitrogen inputs are in demand to maintain crop production sustainability. Understanding the molecular mechanism of N utilization could play a crucial role in improving the nitrogen use efficiency (NUE) of crop plants. METHODS AND RESULTS: In the present study, the effect of low N supply on plant growth, physio-biochemical, chlorophyll fluorescence attributes, yield components, and gene expression analysis were measured at six developmental stages in rice cultivars. Two rice cultivars were grown with a supply of optimium (120 kg ha-1) and low N (60 kg ha-1). Cultivar Vikramarya excelled Aditya at low N supply, and exhibits enhanced plant growth, physiological efficiency, agronomic efficiency, and improved NUE due to higher N uptake and utilization at low N treatment. Moreover, plant biomass, leaf area, and photosynthetic rate were significantly higher in cv. Vikramarya than cv. Aditya at different growth stages, under low N treatment. In addition, enzymatic activities in cultivar Vikramarya were higher than cultivar Aditya under low nitrogen, indicating its greater potential for N metabolism. Gene expression analysis was carried out for the most important nitrogen assimilatory enzymes, such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT). Expression levels of these genes at different growth stages were significantly higher in cv. Vikramarya compared to cv. Aditya at low N supply. Our findings suggest that improving NUE needs specific revision in N metabolism and physiological assimilation. CONCLUSION: Overall differences in plant growth, physiological efficiency, biochemical activities, and expression levels of N metabolism genes in N-efficient and N-inefficient rice cultivars need a specific adaptation to N metabolism. Regulatory genes may separately or in conjunction, enhance the NUE. These results provide a platform for selecting crop cultivars for nitrogen utilization efficiency at low N treatment.

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.

Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism.

Gaining insight into the behavior of bacteria at the single-cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize the expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels across heterogeneous populations such as biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression remained at peak levels deeper into anoxic environments than other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and they demonstrate the utility of HCR in unveiling cellular activities at the microscale level in heterogeneous populations. IMPORTANCE To understand bacteria in diverse contexts, we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations. By focusing on energy conservation, we demonstrate that HCR v3.0 can visualize nuances in gene expression, allowing us to understand how metabolism in Pseudomonas aeruginosa biofilms responds to microenvironmental variation at high spatial resolution. We validated probes for four catabolic genes, including a constitutively expressed oxidase, acetate kinase, nitrite reductase, and nitrous oxide reductase. We showed that the genes for different modes of metabolism are expressed in overlapping but distinct subpopulations according to oxygen concentrations in a predictable fashion. The spatial transcriptomic technique described here has the potential to be used to map microbial activities across diverse environments.

A mechanistic study of the influence of nitrogen and energy availability on the NH4+ sensitivity of nitrogen assimilation in Synechococcus.

In most algae, NO3- assimilation is tightly controlled and is often inhibited by the presence of NH4+. In the marine, non-colonial, non-diazotrophic cyanobacterium Synechococcus UTEX 2380, NO3- assimilation is sensitive to NH4+ only when N does not limit growth. We sequenced the genome of Synechococcus UTEX 2380, studied the genetic organization of the nitrate assimilation related (NAR) genes, and investigated expression and kinetics of the main NAR enzymes, under N or light limitation. We found that Synechococcus UTEX 2380 is a ß-cyanobacterium with a full complement of N uptake and assimilation genes and NAR regulatory elements. The nitrate reductase of our strain showed biphasic kinetics, previously observed only in freshwater or soil diazotrophic Synechococcus strains. Nitrite reductase and glutamine synthetase showed little response to our growth treatments, and their activity was usually much higher than that of nitrate reductase. NH4+ insensitivity of NAR genes may be associated with the stimulation of the binding of the regulator NtcA to NAR gene promoters by the high 2-oxoglutarate concentrations produced under N limitation. NH4+ sensitivity in energy-limited cells fits with the fact that, under these conditions, the use of NH4+ rather than NO3- decreases N-assimilation cost, whereas it would exacerbate N shortage under N limitation.

Characterization of the amidoxime reducing components ARC1 and ARC2 from Arabidopsis thaliana.

Five molybdenum-dependent enzymes are known in eukaryotes. While four of them are under investigation since decades, the most recently discovered, (mitochondrial) amidoxime reducing component ((m)ARC), has only been characterized in mammals and the green algae Chlamydomonas reinhardtii. While mammalian mARCs have been shown to be involved in various signalling pathways, Chlamydomonas ARC was shown to be a nitric oxide (NO)-forming nitrite reductase. Similar to mammals, higher plants possess two ARC proteins. To test whether plant ARCs have a similar function in NO production to the function they have in C. reinhardtii, we analysed the enzymes from the model plant Arabidopsis thaliana. Both ARC1 and ARC2 from Arabidopsis could reduce N-hydroxylated compounds, while nitrite reduction to form NO could only be demonstrated for ARC2. Searching for physiological electron donors, we found that both ARC enzymes accept electrons from NADH via cytochrome b5 reductase and cytochrome b5 , but only ARC2 is able to accept electrons from nitrate reductase at all. Furthermore, arc-deficient mutant plants were similar to wildtype plants regarding growth and also nitrite-dependent NO-formation. Altogether, our results did not confirm the hypothesis that either ARC1 or ARC2 from Arabidopsis are involved in physiologically relevant nitrite-dependent NO-formation. In contrast, our data suggest that ARC1 and ARC2 have distinct, yet unknown physiological roles in higher plants.

The mechanism by which Enteromorpha Linza polysaccharide promotes Bacillus subtilis growth and nitrate removal.

In this study, we discussed the relationship between Entermorpha linza polysaccharide (EP) and Bacillus subtilis, which can transform nitrate. A sole carbon source experiment showed that Bacillus subtilis could utilize EP, and the bacterial density was maximally increased by 54.43% in the EP groups. The results of reducing sugar determination proved the secretion of polysaccharide-degrading enzymes. Scanning electron microscopy (SEM) showed that the EP groups had fewer spores and shrunken bacteria, indicating that EP could improve the growth environment and maintain bacterial integrity. Additionally, the ratios of periplasmic nitrate reductase (NAP), nitrite reductase (NIR), and dissimilatory nitrate reductase (D-NRase) in the EP groups were maximally increased by 107.22%, 84.70% and 36.10%, respectively. Transcriptome analysis further confirmed the above mentioned results. For example, the high expression of quorum sensing genes indicated that EP groups had higher bacterial density. Moreover, the high expression of antioxidant genes in the EP groups may be related to morphological integrity. Our study provides a basis for further discussion of the mechanism.

The Regulation of Nitrate Reductases in Response to Abiotic Stress in Arabidopsis.

The two homologous genes, NIA1 and NIA2, encode nitrate reductases in Arabidopsis, which govern the reduction of nitrate to nitrite. This step is the rate-limiting step of the nitrate assimilation and utilization. Therefore, the regulation of NIA1 and NIA2 is important for plant development and growth. Although they are similar in sequence and structure, their regulations are different. Genetic analysis uncovers that NIA1, rather than NIA2, plays a predominant role in adopting to ABA stress. Although both long-term stress conditions can cause an improvement in NIA1 levels, a decrease in NIA1 levels under short-term treatments seems to be necessary for plants to switch from the growth status into the adopting status. Interestingly, the downregulation of the NR is distinct under different stress conditions. Under ABA treatment, the NR proteins are degraded via a 26S-proteasome dependent manner, while the transcriptional regulation is the main manner to rapidly reduce the NIA1 levels under nitrogen deficiency and NaCl stress conditions. These results indicate that under stress conditions, the regulation of NIA1 is complex, and it plays a key role in regulating the balance between growth and adaptation.

Understanding the effect of GO on nitrogen assimilation in wheat through transcriptomics and metabolic process analysis.

The extensive use of graphene oxide (GO) has resulted in its inevitable entry into the environment. It has been established that GO is detrimental to nitrogen accumulation in plants, as nitrogen is one of the most important nutrient for plant growth. However, its influence on nitrogen assimilation has not yet been investigated comprehensively. Based on the analysis of transcriptomics and nitrogen metabolites, this study showed that 400 mg L-1 GO exposure downregulated most of the genes encoding nitrogen-assimilating enzymes, including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH). The activities of the above enzymes in wheat roots were also reduced with GO addition, and the activities of NR and GS, the rate-limiting enzymes of nitrate and ammonium assimilation, were approximately 75% and 76% lower with 400 mg L-1 GO supply, respectively, compared to those upon control treatment. Correspondingly, GO appears to exert a negative effect on multiple nitrogen assimilation products, including nitrous nitrogen, ammonium nitrogen, glutamine, glutamate, and soluble protein. In summary, this study showed that GO has adverse effects on the nitrogen assimilation of plants, and NR and GS are the most affected sites. Our findings would provide deeper insights into the physiological and molecular mechanisms underlying GO phytotoxicity.

Analysis of mRNA-derived siRNAs in mutants of mRNA maturation and surveillance pathways in Arabidopsis thaliana.

Defects in RNA maturation and RNA decay factors may generate substrates for the RNA interference machinery. This phenomenon was observed in plants where mutations in some RNA-related factors lead to the production of RNA-quality control small interfering RNAs and several mutants show enhanced silencing of reporter transgenes. To assess the potential of RNAi activation on endogenous transcripts, we sequenced small RNAs from a set of Arabidopsis thaliana mutants with defects in various RNA metabolism pathways. We observed a global production of siRNAs caused by inefficient pre-mRNA cleavage and polyadenylation leading to read-through transcription into downstream antisense genes. In addition, in the lsm1a lsm1b double mutant, we identified NIA1, SMXL5, and several miRNA-targeted mRNAs as producing siRNAs, a group of transcripts suggested being especially sensitive to deficiencies in RNA metabolism. However, in most cases, RNA metabolism perturbations do not lead to the widespread production of siRNA derived from mRNA molecules. This observation is contrary to multiple studies based on reporter transgenes and suggests that only a very high accumulation of defective mRNA species caused by specific mutations or substantial RNA processing defects trigger RNAi pathways.