Nitrate Reductase Gene Research Articles

Enhancing denitrification in constructed wetland with algae addition.

Constructed wetlands (CWs) can be used for tertiary treatment of wastewater; however, carbon source shortages limit denitrification. We studied the effect of algae addition as an external carbon source in CWs and found that the nitrogen removal efficiency of CWs is highly dependent on the algae dosage. Optimal nitrogen removal percentage (80.5%) can be achieved by adding 81.1 mg·L-1 dry weight algae to the influent when the chemical oxygen demand/nitrogen (COD/N) ratio reaches 5.3. Longitudinal changes in the nitrogen concentrations, organic matter concentrations, and nitrogen functional genes were also analyzed. The algae addition strengthened the anoxic environment, boosted the volatile fatty acid concentrations, and improved the ratio of nitrite reductase gene (nirS) and copper-containing nitrite reductase (nirK)/16S rRNA, as well as the ratio of nitrate reductase gene (narG)/16S rRNA, thereby expanding the active space for denitrification. The addition of algae could potentially provide enough carbon to enhance denitrification during treatment of wastewater with a low COD/N ratio.

Heterotrophic nitrification and aerobic denitrification by a novel Acinetobacter sp. TAC-1 at low temperature and high ammonia nitrogen

A novel strain was isolated from swinewastewater and identified as Acinetobacter sp. TAC-1 based on its phylogenetic and phenotypic characteristics. The strain TAC-1 was found to have a high ability to metabolize ammonium-N under low temperature condition. The strain TAC-1 could remove approximately 94.6% of ammonium-N (400 mg/L), 93.3% of nitrate-N (400 mg/L) and 42.4% of nitrite-N (400 mg/L) at 5 °C. The functional genes nitrate reductase gene (narG) and nitrite reductase gene (nirK, nirS) were successfully amplified by qPCR, further evidencing the heterotrophic nitrification and aerobic denitrification capability of Acinetobacter sp. TAC-1. The transcriptome data confirmed that the membrane transport protein and unsaturated fatty acid dehydrogenase-related genes of the strain TAC-1 were significantly up-regulated at 5 °C, enabling it to survive low temperatures. The high nitrogen removal ability at 5 °C makes this strain have a good application prospect.

Growth and physiological impairments in Fe-starved alfalfa are associated with the downregulation of Fe and S transporters along with redox imbalance

BackgroundIron (Fe) is an essential plant nutrient. Its deficiency is a major constraint in crop production systems, affecting crop yield and quality. It is therefore important to elucidate the responses and adaptive mechanisms underlying Fe-deficiency symptoms in alfalfa.Materials and methodsThe experiment was carried out on 12-day-old alfalfa plants grown in hydroponics under Fe-sufficient and Fe-deficient conditions.ResultsThe Fe-starved alfalfa showed decreased plant biomass, chlorophyll score, PSII efficiency, and photosynthesis performance index in young leaves under low Fe. Further, Fe shortage reduced the Fe, Zn, S and Ca concentration in root and shoot of alfalfa accompanied by the marked decrease of MsIRT1, MsZIP, MsSULTR1;1, MsSULTR1;2 and MsSULTR1;3 transcripts in root and shoot. It indicates that retardation caused by Fe-deficiency was also associated with the status of other elements, especially the reduced Fe and S may be coordinately attributed to the photosynthetic damages in Fe-deficient alfalfa. The ferric chelate reductase activity accompanied by the expression of MsFRO1 in roots showed no substantial changes, indicating the possible involvement of this Strategy I response in Fe-deficient alfalfa. However, the proton extrusion and expression of MsHAI1 were significantly induced following Fe-deficiency. In silico analysis further suggested their subcellular localization in the plasma membrane. Also, the interactome map suggested the partnership of MsFRO1 with plasma membrane H+-ATPase, transcription factor bHLH47, and nitrate reductase genes, while MsHAI1 partners include ferric reductase-like transmembrane component, plasma membrane ATPase, vacuolar-type H-pyrophosphatase, and general regulatory factor 2. In this study, SOD and APX enzymes showed a substantial increase in roots but unable to restore the oxidative damages in Fe-starved alfalfa.ConclusionThese findings promote further studies for the improvement of Fe-starved alfalfa or legumes through breeding or transgenic approaches.Graphic

What is the role of the nitrate reductase (euknr) gene in fungi that live in nitrate-free environments? A targeted gene knock-out study in Ampelomyces mycoparasites

Mycoparasitic fungi can be utilized as biocontrol agents (BCAs) of many plant pathogens. Deciphering the molecular mechanisms of mycoparasitism may improve biocontrol efficiency. This work reports the first functional genetic studies in Ampelomyces, widespread mycoparasites and BCAs of powdery mildew fungi, and a molecular genetic toolbox for future works. The nitrate reductase (euknr) gene was targeted to reveal the biological function of nitrate assimilation in Ampelomyces. These mycoparasites live in an apparently nitrate-free environment, i.e. inside the hyphae of powdery mildew fungi that lack any nitrate uptake and assimilation system. Homologous recombination-based gene knock-out (KO) was applied to eliminate the euknr gene using Agrobacterium tumefaciens-mediated transformation. Efficient KO of euknr was confirmed by PCR, and visible phenotype caused by loss of euknr was detected on media with different nitrogen sources. Mycoparasitic ability was not affected by knocking out euknr as a tested transformant readily parasitized Blumeria graminis and Podosphaera xanthii colonies on barley and cucumber, respectively, and the rate of mycoparasitism did not differ from the wild type. These results indicate that euknr is not involved in mycoparasitism. Dissimilatory processes, involvement in nitric oxide metabolism, or other, yet undiscovered processes may explain why a functional euknr is maintained in Ampelomyces.

Pseudomonas campi sp. nov., a nitrate-reducing bacterium isolated from grassland soil.

A novel strain was isolated from grassland soil that has the potential to assimilate ammonium by the reduction of nitrate in the presence of oxygen. Whole genome sequence analysis revealed the presence of an assimilatory cytoplasmic nitrate reductase gene nasA and the assimilatory nitrite reductase genes nirBD which are involved in the sequential reduction of nitrate to nitrite and further to ammonium, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate represents a member of the genus Pseudomonas. The closest phylogenetic neighbours based on 16S rRNA gene sequence analysis are the type strains of Pseudomonas peli (98.17%) and Pseudomonas guineae (98.03%). In contrast, phylogenomic analysis revealed a close relationship to Pseudomonas alcaligenes. Computation of the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) with the closest phylogenetic neighbours of S1-A32-2T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. On the basis of these results, it was concluded that the soil isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas campi sp. nov. (type strain S1-A32-2T=LMG 31521T=DSM 110222T) is proposed.

Preliminary functional analysis of the subgingival microbiota of cats with periodontitis and feline chronic gingivostomatitis

The subgingival microbial communities of domestic cats remain incompletely characterized and it is unknown whether their functional profiles are associated with disease. In this study, we used a shotgun metagenomic approach to explore the functional potential of subgingival microbial communities in client-owned cats, comparing findings between periodontally healthy cats and cats with naturally occurring chronic periodontitis, aggressive periodontitis, and feline chronic gingivostomatitis. Subgingival samples were subjected to shotgun sequencing and the metagenomic datasets were analyzed using the MG-RAST metagenomic analysis server and STAMP v2.1.3 (Statistical Analysis of Metagenomic Profiles) software. The microbial composition was also described to better understand the predicted features of the communities. The Respiration category in the level 1 Subsystems database varied significantly among groups. In this category, the abundance of V-Type ATP-synthase and Biogenesis of cytochrome c oxidases were significantly enriched in the diseased and in the healthy groups, respectively. Both features have been previously described in periodontal studies in people and are in consonance with the microbial composition of feline subgingival sites. In addition, the narH (nitrate reductase) gene frequency, identified using the KEGG Orthology database, was significantly increased in the healthy group. The results of this study provide preliminary functional insights of the microbial communities associated with periodontitis in domestic cats and suggest that the ATP-synthase and nitrate-nitrite-NO pathways may represent appropriate targets for the treatment of this common disease.

Shifts in bacterial community in response to conservation management practices within a soybean production system

A 3-year field study was conducted to assess effects of two winter cover crops, Elbon rye (Secale cereal L.) and Crimson clover (Trifolium incarnatum L.), under till and no till management, on bacterial community composition in soybean (Glycine max L.) field soils using high-throughput sequencing of the 16S rRNA gene. Effects of tillage and cover crop on bacterial composition at the phylum level were minor, with most significant differences between treatments occurring at finer taxonomic levels. Tilled plots displayed higher levels of Xanthomonadaceae, while cover cropped soils had greater Bradyrhizobium abundances. Functional gene prediction indicated that genes associated with decomposition of C and P compounds, as well as biocontrol agents, were elevated in tilled plots, genes associated with root growth promotion were elevated in cover crop treatments, and nitrate reductase and denitrification genes were elevated in both no till and cover crop plots. While valuable functional insights can be gained from sequence analyses, not all differences observed at the sequencing level will translate into functional differences due to variation in gene expression, and further study is needed to validate which functions can be predicted from sequencing data.

Genetic Characterization of Corynebacterium pseudotuberculosis Isolates in Egypt

Corynebacterium pseudotuberculosis is a small Gram-positive bacillus containing mycolic acid in the structure of the cell wall. The bacterium is responsible for Caseous Lymphadenitis (CLA) in small ruminants (sheep and goats). The bacteria are also responsible for Ulcerative Lymphangitis in equines. The disease causes great economic loss in the animal industry. This work aimed to check the ability of Quadruplex PCR (Q-PCR) for genotyping and identification of Egyptian isolates of C. pseudotuberculosis and sequence analysis of phospholipase D (PLD) gene of local isolates. Four of C. pseudotuberculosis local isolates previously biochemically identified were tested for narG gene (nitrate reductase gene). Both nitrate negative biovar (ovis) and nitrate positive biovar (equi) showed a positive result for 16S rRNA, rpoB and PLD genes of C. pseudotuberculosis species. The sequence analysis of our local isolates' PLD gene revealed minor changes in PLD proteins between ovine and equine strains compared with other published PLD sequences in GenBank. It was concluded that the Q-PCR method is able to differentiate between C. pseudotuberculosis equi and ovis biovars. Also, the sequence of PLD gene of local isolates representing the two biovars revealed some variation, which leads to an accurate diagnosis of C. pseudotuberculosis biovars and generates a mapping of immerged local isolates. Further, the PCR and sequence of these isolates provide rapid and accurate genotyping, especially with hyperimmune serum's unavailability.

Inhibition of nitric oxide production under alkaline conditions regulates iron homeostasis in rice.

Rice is one of the most susceptible plants to iron (Fe) deficiency under neutral and alkaline conditions. Alkaline stress induces H2 O2 production and increases the deposition of Fe on the root surface, which causes leaf chlorosis and Fe deficiency in rice. Gene chip and qRT-PCR analysis indicated that the expression of the nitrate reductase (NR) genes were downregulated by alkaline treatment, which resulted in significantly decreased nitrate activity and nitric oxide (NO) production in the epidermis and stele, where H2 O2 accumulated. In contrast, treatment with sodium nitroprusside (SNP), a NO donor, strongly alleviated alkaline-induced Fe deficiency by limiting Fe plaque formation. Increasing the NO signal significantly reduced the accumulation of H2 O2 and the lignin barrier but enhanced phenolic acid secretion in the root epidermis and stele under alkaline conditions. The secreted phenolic acid effectively mobilized the apoplast Fe and increased Fe uptake in roots, thereby alleviating the Fe-deficiency response and downregulating the expressions of Fe-uptake genes under alkaline conditions. In conclusion, alkaline stress inhibits NR activity and NO production in the roots of rice, which play vital roles in the mobilization of the apoplast Fe by regulation of H2 O2 and phenolic acid concentrations.

Nitrogen partitioning traits and expression patterns of N metabolism‐associated genes in maize hybrids with contrasting N utilization efficiencies

AbstractThe nitrogen (N) uptake and utilization in maize (Zea mays L.) plants are accomplished through a series of biochemical reactions and transformations involving the N assimilation enzymes. Nitrate reductase (NR) and glutamine synthetase (GS) exert important functions in the N assimilation and remobilization processes. The mechanisms underlying plant N partitioning and the associated expression patterns of NR and GS genes in maize hybrids with different N utilization efficiencies were still not clear. In this study, the expression of ZmNR and ZmGS genes that are related to N partitioning were analyzed in two maize hybrids (XY335 and HN138). Results showed that XY335 displayed higher grain yield, kernel numbers, shoot N uptake, and physiological N utilization efficiency (NUtE) than those shown in HN138 under low‐N conditions. The higher post‐silking N uptake and N remobilization in vegetative organs in XY335 were associated with the enhanced net photosynthetic rate (Pn), the NR and GS activities in ear‐leaf. Additionally, the expression levels of ZmGS1.3 and ZmGS1.4 were up‐regulated when exposed to low‐N stress, showing higher values in XY335 than those in HN138. Correlation analysis showed that the expression levels of ZmNR, ZmGS1.3, and ZmGS1.4 are correlated with leaf NR and GS activities, N remobilization, and grain yield. Our investigation suggested the critical roles of the N metabolism‐associated genes in the modulation of internal N translocation and plant productivity in maize. Future transgene analysis on ZmNR and ZmGS can provide insights into the molecular mechanisms underlying plant tolerance to low N stress in maize.

Rapid and marker-free gene replacement in citric acid-producing Aspergillus tubingensis (A. niger) WU-2223L by the CRISPR/Cas9 system-based genome editing technique using DNA fragments encoding sgRNAs

Strains belonging to Aspergillus section Nigri, including Aspergillus niger, are used for industrial production of citric acid from carbohydrates such as molasses and starch. The objective of this study was to construct the genome editing system that could enable rapid and efficient gene replacement in citric acid-producing fungi for genetic breeding. Using the citric acid-hyperproducer A.tubingensis (formerly A.niger) WU-2223L as a model strain, we developed a CRISPR/Cas9 system-based genome editing technique involving co-transformation of Cas9 and the DNA fragment encoding single guide RNA (sgRNA). Using this system, ATP-sulfurylase gene (sC) knock-out strain derived from WU-2223L was generated; the knock-out efficiency was 29 transformants when 5μg Cas9 was added to 5× 105 protoplasts. In the gene replacement method based on this system, a DNA fragment encoding sgRNAs that target both the gene of interest and marker gene was used, and replacement of nitrate reductase gene (niaD) using sC gene as a marker gene was attempted. More than 90% of the sC-knock-out transformants exhibited replaced niaD, indicating efficient gene replacement. Moreover, one-step marker rescue of the sC marker gene was accomplished by excising the knock-in donor via intramolecular homologous recombination, enabling marker-free genome editing and drastically shortening the gene replacement period by circumventing the transformation procedure to recover the sC gene. Thus, we succeeded in constructing a CRISPR/Cas9 system-based rapid and marker-free gene replacement system for the citric acid-hyperproducer strain WU-2223L.

Characterization of a novel marine aerobic denitrifier Vibrio spp. AD2 for efficient nitrate reduction without nitrite accumulation.

Aerobic denitrifiers have the potential to reduce nitrate in polluted water under aerobic conditions. A salt-tolerant aerobic denitrifier was newly isolated and identified as Vibrio spp. AD2 from a marine recirculating aquaculture system, in which denitrification performance was investigated via single-factor experiment, Box-Behnken experiment, and nitrogen balance analysis. Nitrate reductase genes were identified by polymerase chain reaction. Results showed that strain AD2 removed 98.9% of nitrate-nitrogen (NO3--N) with an initial concentration about 100 mg·L-1 in 48 h without nitrite-nitrogen (NO2--N) accumulation. Nitrogen balance indicated that approximately 17.5% of the initial NO3--N was utilized for bacteria synthesis themselves, 4.02% was converted to organic nitrogen, 39.8% was converted to nitrous oxide (N2O), and 31.1% was converted to nitrogen (N2). Response surface methodology experiment showed that the maximum removal of total nitrogen (TN) occurred under the condition of C/N ratio 11.5, shaking speed 127.9 rpm, and temperature 30.8 °C. Sequence amplification indicated that the denitrification genes, napA and nirS, were present in strain AD2. These results indicated that the strain AD2 has potential applications for removing NO3--N from high-salinity (3%) wastewater.

The driving effect of nitrogen-related functional microorganisms under water and nitrogen addition on N2O emission in a temperate desert

Nitrous oxide (N2O) is an important greenhouse gas and a precursor of ozone depletion in the upper atmosphere, thus contributing to climate change and biological safety. The mechanisms and response characteristics of N2O emission in desert soils to precipitation and nitrogen (N) deposition are still unclear. To further elucidate this, an in-situ experiment was conducted in the Gurbantunggut Desert, a temperate desert in China, between June and September 2015 and 2016. The response in N2O flux to water addition (equivalent to 5 mm precipitation) was very transient in summer, only lasting one to two days. This was attributed to the rapid decrease in soil moisture following the water addition, due to the high temperature and drought conditions, and there was no significant change in N2O emission or in the abundance of N-related key functional genes. In contrast, N2O emissions increased significantly in response to N addition. This was associated with an increase in functional gene abundances of amoA (ammonia oxidizing bacteria (AOB)) and ammonia-oxidizing archaea (AOA), which responded positively to increasing soil NH4+-N content, but were inhibited by increasing soil NO3−-N content. The abundance of the nirS (nitrate reductase) gene was significantly increased by increasing soil NO3−-N content. Interestingly, the indirect effect of increased soil moisture in enhancing N2O emission by increasing the abundance of AOA was offset by a direct effect of soil moisture in inhibiting soil N2O emission. Overall, N2O emissions were mainly controlled by AOA rather than AOB in summer, and were more sensitive to soil available N than to soil moisture in this temperate desert.

Genomic and Metabolic Insights into Two Novel Thiothrix Species from Enhanced Biological Phosphorus Removal Systems

Two metagenome-assembled genomes (MAGs), obtained from laboratory-scale enhanced biological phosphorus removal bioreactors, were analyzed. The values of 16S rRNA gene sequence identity, average nucleotide identity, and average amino acid identity indicated that these genomes, designated as RT and SSD2, represented two novel species within the genus Thiothrix, ‘Candidatus Thiothrix moscowensis’ and ‘Candidatus Thiothrix singaporensis’. A complete set of genes for the tricarboxylic acid cycle and electron transport chain indicates a respiratory type of metabolism. A notable feature of RT and SSD2, as well as other Thiothrix species, is the presence of a flavin adenine dinucleotide (FAD)-dependent malate:quinone oxidoreductase instead of nicotinamide adenine dinucleotide (NAD)-dependent malate dehydrogenase. Both MAGs contained genes for CO2 assimilation through the Calvin–Benson–Bassam cycle; sulfide oxidation (sqr, fccAB), sulfur oxidation (rDsr complex), direct (soeABC) and indirect (aprBA, sat) sulfite oxidation, and the branched Sox pathway (SoxAXBYZ) of thiosulfate oxidation to sulfur and sulfate. All these features indicate a chemoorganoheterotrophic, chemolithoautotrophic, and chemolithoheterotrophic lifestyle. Both MAGs comprise genes for nitrate reductase and NO-reductase, while SSD2 also contains genes for nitrite reductase. The presence of polyphosphate kinase and exopolyphosphatase suggests that RT and SSD2 could accumulate and degrade polyhosphates during the oxic-anoxic growth cycle in the bioreactors, such as typical phosphate-accumulating microorganisms.

Metagenomic insights into Chinese northeast suancai: Predominance and diversity of genes associated with nitrogen metabolism in traditional household suancai fermentation

Chinese northeast suancai represents a typical and valuable food product that has been handed down by traditional household procedures over centuries. Nitrite is formed and accumulated during the suancai fermentation process and commonly causes food safety problems. The biogeochemical cycle of nitrite may provide a reference and guidance for the enzymatic degradation of nitrite in fermented food. The potential nitrogen metabolic pathways in the microbially driven suancai fermentation were reasonably inferred through monitoring nitrogen conversions and detecting the genes of different functional enzymes. Complex microbial metabolism is responsible for the unique nitrogen conversions during suancai fermentation. The metagenomic results showed that Pseudomonas with nitrate reductase genes (narG, narH, narI) and nitrite reductase genes (nirB, nirD) contributed the most to both nitrite reduction and nitrate reduction. The majority of the sequences of nitrate reductase and nitrite reductase were derived from the families of Pseudomonadaceae, Erwiniaceae and Yersiniaceae. According to the physicochemical analysis, the nitrite concentration of the fermentation broth reached the peak value (0.48 mM) and gradually decreased to the minimum (0.02 mM). The downward trend of the pH and nitrite concentration were closely associated with the nitrite enzymatic degradation period before the acid degradation period. Our results indicated that nitrite removal in suancai fermentation involved the reduction of nitrite to ammonia and denitrification, which were mainly contributed by the reduction of nitrite to ammonia mediated by the nirB/nirD enzyme (Indentified ECs: 1.7.1.15). This research offers new insights into the metagenome-based bioinformatic roles of the previously unstudied microorganisms in spontaneous suancai fermentation for the enzymatic degradation of nitrite. It provides helpful basis for the detection and even elimination of nitrite in suancai and for improving the safety level of suancai.

A newly discovered function of nitrate reductase in chemoautotrophic vanadate transformation by natural mackinawite in aquifer

Mackinawite (FeS), a widely-distributed natural reducing mineral, can donate electron for various (bio)processes. However, little is known about mackinawite-driven chemoautotrophic bioreduction of toxic vanadate [V(V)] in aquifer. This study demonstrates that V(V) is successfully bioreduced by mackinawite under anaerobic condition via 150-d operation of constructed aquifer. Complete V(V) removal was achieved at the initial concentration of 10 mg/L and flow rate of 0.125 mL/min. Fluctuant hydrochemistry and hydrodynamics affected V(V) removal performance. Biotic activity was identified as the major contribution to V(V) transformation (76.4 ± 1.01%). Chemoautotrophic genera (e.g., Thiobacillus) could oxidize FeS coupled to direct V(V) reduction independently. Heterotrophic V(V) reducers (e.g., Pseudomonas and Spirochaeta) could also achieve V(V) detoxification by utilizing metabolic intermediates synthesized by autotrophic Fe(II) oxidizers (e.g., Thiobacillus) and S(-II) oxidizing genera (e.g., Sulfuricurvum). Gene abundance and enzymatic activity tests confirmed that nitrate reductase gene napA functioned crucially in chemoautotrophic V(V) reduction by Fe(II) and S(-II) donating electron. V(V) was reduced to insoluble V(IV) while elements in mackinawite were oxidized to Fe(III) and SO42−. This study reveals the coupling of iron, sulfur and vanadium in biogeochemical cycling, and offers a promising strategy for remediation of V(V)-polluted aquifer.

DNA-Stable Isotope Probing Shotgun Metagenomics Reveals the Resilience of Active Microbial Communities to Biochar Amendment in Oxisol Soil.

The functions and interactions of individual microbial populations and their genes in agricultural soils amended with biochar remain elusive but are crucial for a deeper understanding of nutrient cycling and carbon (C) sequestration. In this study, we coupled DNA stable isotope probing (SIP) with shotgun metagenomics in order to target the active community in microcosms which contained soil collected from biochar-amended and control plots under napiergrass cultivation. Our analyses revealed that the active community was composed of high-abundant and low-abundant populations, including Actinobacteria, Proteobacteria, Gemmatimonadetes, and Acidobacteria. Although biochar did not significantly shift the active taxonomic and functional communities, we found that the narG (nitrate reductase) gene was significantly more abundant in the control metagenomes. Interestingly, putative denitrifier genomes generally encoded one gene or a partial denitrification pathway, suggesting denitrification is typically carried out by an assembly of different populations within this Oxisol soil. Altogether, these findings indicate that the impact of biochar on the active soil microbial community are transient in nature. As such, the addition of biochar to soils appears to be a promising strategy for the long-term C sequestration in agricultural soils, does not impart lasting effects on the microbial functional community, and thus mitigates un-intended microbial community shifts that may lead to fertilizer loss through increased N cycling.

Efficient and multiplexable genome editing using Platinum TALENs in oleaginous microalga, Nannochloropsis oceanica NIES-2145.

Algae accumulate large amounts of lipids produced by photosynthesis, and these lipids are expected to be utilized as feedstocks for sustainable new energies, known as biodiesels. Nannochloropsis species are eukaryotic microalgae that produce high levels of lipids. However, since the production costs of algal biodiesels are higher than those of fossil fuels, the improved productivity of algal lipids by molecular breeding of algae is required for practical use. In the present study, we developed a highly efficient genome-editing system involving Platinum transcription activator-like effector nucleases (TALENs) in Nannochloropsis oceanica. Platinum TALENs codon-optimized for N.oceanica were synthesized, and their DNA-binding activity was confirmed by single-strand annealing assays in human HEK293T cells. All-in-one expression vectors for Platinum TALEN targeting the nitrate reductase gene, NoNR, and acyltransferase gene, LPAT1, were transfected into Nannochloropsis species. The introduction of each Platinum TALEN revealed high genome-editing efficiency with no detectable off-target mutations at the candidate sites in N.oceanica. By simultaneously introducing TALENs targeting two genes, we obtained double mutant strains. The loss-of-function phenotype of NoNR was also confirmed. These findings will provide an essential technology for molecular breeding in Nannochloropsis species.

Plant Nitrate Reductases Regulate Nitric Oxide Production and Nitrogen-Fixing Metabolism During the Medicago truncatula–Sinorhizobium meliloti Symbiosis

Nitrate reductase (NR) is the first enzyme of the nitrogen reduction pathway in plants, leading to the production of ammonia. However, in the nitrogen-fixing symbiosis between legumes and rhizobia, atmospheric nitrogen (N2) is directly reduced to ammonia by the bacterial nitrogenase, which questions the role of NR in symbiosis. Next to that, NR is the best-characterized source of nitric oxide (NO) in plants, and NO is known to be produced during the symbiosis. In the present study, we first surveyed the three NR genes (MtNR1, MtNR2, and MtNR3) present in the Medicago truncatula genome and addressed their expression, activity, and potential involvement in NO production during the symbiosis between M. truncatula and Sinorhizobium meliloti. Our results show that MtNR1 and MtNR2 gene expression and activity are correlated with NO production throughout the symbiotic process and that MtNR1 is particularly involved in NO production in mature nodules. Moreover, NRs are involved together with the mitochondrial electron transfer chain in NO production throughout the symbiotic process and energy regeneration in N2-fixing nodules. Using an in vivo NMR spectrometric approach, we show that, in mature nodules, NRs participate also in the regulation of energy state, cytosolic pH, carbon and nitrogen metabolism under both normoxia and hypoxia. These data point to the importance of NR activity for the N2-fixing symbiosis and provide a first explanation of its role in this process.

Photosynthetic efficiency and nutrient physiology of the diatom Thalassiosira pseudonana at three growth temperatures

Diatom cells utilize a variety of metabolic pathways to cope with internal energy imbalances caused by stressful environmental conditions. In this study, the model diatom species, Thalassiosira pseudonana, was grown in nutrient replete and nitrate (NO3−)- and dissolved silicate (Si)-depleted media at three growth temperatures (4, 17, 28 °C) to determine how nutrient enrichment and temperature affects diatom growth, photosynthetic efficiency, nitrate reductase (NR) enzyme activity, biogenic silica (bSiO2) deposition, and NR gene expression. Growth rates for nutrient-replete cultures were highest at 17 °C. Across all nutrient treatments, the cells grown at 17 °C had an average Fv/Fm of 0.44 ± 0.006, while the cells were grown at 4 °C and 28 °C had an average Fv/Fm of 0.37 ± 0.004 and 0.38 ± 0.01, respectively. Activity of NR was variable across treatments with no significant effect of temperature. The relative expression of the targeted NR gene was, on average, ~ 10 times higher in the 4 °C cultures and ~ 4 times higher in the 28 °C than in the 17 °C cultures, while the activity of the NR enzyme was generally highest in the cultures grown at 17 °C that were enriched with NO3−. Cells grown under nutrient-replete conditions had significantly higher bSiO2 deposition rates at 4 °C than cells grown at 17 and 28 °C. These data support the notion that cold, nutrient-replete conditions lead to increases in diatom silicification and that NR activity may be regulated downstream of mRNA transcription under specific environmental conditions.