Encoding Nitrate Reductase Research Articles

The Interplay of Nitric Oxide and Nitrosative Modifications in Maize: Implications for Aphid Herbivory and Drought Stress.

Nitric oxide (NO) and other reactive nitrogen species (RNS) are considered to be signaling molecules in higher plants involved in the regulation of growth and development processes. However, the molecular mechanisms of their formation, removal, and participation in plant responses to adverse environmental stimuli remain largely unclear. Therefore, the aim of this study was to assess the influence of selected single stresses and combined stresses (i.e., Rhopalosiphum padi L. aphid infestation, drought, aphid infestation, and drought) and post-stress recovery on the contents of NO and peroxynitrite anion (ONOO-), as well as the levels of mRNA and protein nitration (i.e., the 8-nitroguanine and protein 3-nitrotyrosine amounts, respectively), in maize seedlings (Zea mays L.). Moreover, the expression patterns of the two tested genes (nos-ip, encoding nitric oxide synthase-interacting protein, and nr1, encoding nitrate reductase 1) involved in NO metabolism in maize plants were quantified. We identified significant intervarietal, time-course, and stress-dependent differences in the levels of the quantified parameters. Under the investigated stress conditions, the aphid-resistant Waza cv. seedlings were characterized by a higher and earlier NO accumulation and mRNA nitration level and an increased expression of the two target genes (nos-ip and nr1), compared to the aphid-susceptible Złota Karłowa cv. seedlings. Conversely, the Złota Karłowa plants responded with a greater elevation in the content of ONOO- and protein 3-nitrotyrosine than the Waza cv. plants The multifaceted role of NO and its derivatives in maize plants challenged by single and combined stresses, as well as during post-stress recovery, is discussed.

Plant growth and nitrate absorption and assimilation of two sweet potato cultivars with different N tolerances in response to nitrate supply

In sweet potato, rational nitrogen (N) assimilation and distribution are conducive to inhibiting vine overgrowth. Nitrate (NO3-) is the main N form absorbed by roots, and cultivar is an important factor affecting N utilization. Herein, a hydroponic experiment was conducted that included four NO3- concentrations of 0 (N0), 4 (N1), 8 (N2) and 16 (N3) mmol L-1 with two cultivars of Jishu26 (J26, N-sensitive) and Xushu32 (X32, N-tolerant). For J26, with increasing NO3- concentrations, the root length and root surface area significantly decreased. However, no significant differences were observed in these parameters for X32. Higher NO3- concentrations upregulated the expression levels of the genes that encode nitrate reductase (NR2), nitrite reductase (NiR2) and nitrate transporter (NRT1.1) in roots for both cultivars. The trends in the activities of NR and NiR were subject to regulation of NR2 and NiR2 transcription, respectively. For both cultivars, N2 increased the N accumulated in leaves, growth points and roots. For J26, N3 further increased the N accumulation in these organs. Under higher NO3- nutrition, compared with X32, J26 exhibited higher expression levels of the NiR2, NR2 and NRT1.1 genes, a higher influx NO3- rate in roots, and higher activities of NR and NiR in leaves and roots. Conclusively, the regulated effects of NO3- supplies on root growth and NO3- utilization were more significant for J26. Under high NO3- conditions, J26 exhibited higher capacities of NO3- absorption and distributed more N in leaves and in growth points, which may contribute to higher growth potential in shoots and more easily cause vine overgrowth.

Enhancing collaboration of anammox with heterotrophic microbes mediated selectively by iron of different valences: Activities balance, metabolic mechanism, and functional genes regulation

The partial denitrification/anammox (PD/A) process is receiving increasing attention due to its cost-effectiveness advantages. However, effective strategies to alleviate organic matter inhibition and promote anammox activity have been proven to be a big challenge. This study investigated the effects of three types of iron (nano zero-valent iron (nZVI), Fe(II), and Fe(III)) on the PD/A process. It is worth noting that nZVI of 5–50 mg/L and Fe(III) of 5–120 mg/L promoted both PD and anammox activity. Long-term intermittent addition of nZVI (50 mg/L) resulted in a nitrogen removal efficiency of 98.2% in the mixotrophic PD/A system driven by iron and organic matter. The contribution of anammox for nitrogen removal reached as high as 93.8%. The organic carbon demand decreased due to the external electron donor provided by nZVI for PD. Multiple Fe–N metabolic pathways, primarily involving ammonia oxidation by Fe(III) and nitrate reduction by nZVI, play a crucial role in facilitating nitrogen transformation. Conversely, the direct addition of 30–120 mg/L Fe (II) resulted in a significant decrease in pH to below 5.0 and severe inhibition of PD and anammox activity. Following prolonged operation in the presence of nZVI, it was demonstrated that there is an enhancing effect on robust nitrite production for anammox. This was accompanied by a remarkable up-regulation of genes encoding nitrate reductase and iron-transporting proteins dominated by Thauera. Overall, this study has provided an efficient approach for advanced nitrogen removal through organic- and iron-driven anammox processes.

Metagenomic insights into microbial metabolic mechanisms of a combined solid-phase denitrification and anammox process for nitrogen removal in mainstream wastewater treatment

To overcome the significant challenges associated with nitrite supply and nitrate residues in mainstream anaerobic ammonium oxidation (anammox)-based processes, this study developed a combined solid-phase denitrification (SPD) and anammox process for low-strength nitrogen removal without the addition of nitrite. The SPD step was performed in a packed-bed reactor containing poly-3-hydroxybutyrate-co-3-hyroxyvelate (PHBV) prior to employing the anammox granular sludge reactor in the continuous-flow mode. The removal efficiency of total inorganic nitrogen reached 95.7 ± 1.2% under a nitrogen loading rate of 0.18 ± 0.01 kg N·m3·d−1, and it required 1.02 mol of nitrate to remove 1 mol of ammonium nitrogen. The PHBV particles not only served as biofilm carriers for the symbiosis of hydrolytic bacteria (HB) and denitrifying bacteria (DB), but also carbon sources that facilitated the coupling of partial denitrification and anammox in the granules. Metagenomic sequencing analysis indicated that Burkholderiales was the most abundant HB genus in SPD. The metabolic correlations between DB (Betaproteobacteria, Rhodocyclaceae, and Anaerolineae) and anammox bacteria (Candidatus Brocadiac and Kuenenia) in the granules were confirmed through microbial co-occurrence networks analysis and functional gene annotations. Additionally, the genes encoding nitrate reductase (Nap) and nitrite reductase (Nir) in DB primarily facilitated nitrate reduction, thereby supplying nitric oxide to anammox bacteria for subsequent nitrogen removal with hydrazine synthase (Hzs) and hydrazine dehydrogenase (Hdh). The findings provide insights into microbial metabolism within combined SPD and anammox processes, thus advancing the development of mainstream anammox-based processes in engineering applications.

Transcriptome and metabolome reveal the effects of ABA promotion and inhibition on flavonoid and amino acid metabolism in tea plant.

Flavonoids (especially anthocyanins and catechins) and amino acids represent a high abundance of health-promoting metabolites. Although we observed abscisic acid accumulation in purple leaves and low levels in albino tea leaves, the specific mechanism behind its impact on flavor compounds remains unclear. In this study, we treated tea leaves with exogenous abscisic acid and abscisic acid biosynthesis inhibitors (Flu), measured physiological indicators and conducted comprehensive transcriptomic and metabolomic analyses to elucidate the potential mechanisms underlying color change. Our results demonstrate that abscisic acid treatment induces purple coloration, while Flu treatment causes discoloration in tea leaves. Metabolomic analysis revealed higher levels of four anthocyanins and six catechins in the group treated with abscisic acid in comparison with the control group. Additionally, there was a notable increase in 15 amino acids in the Flu-treated group. Notably, the levels of flavonoids and amino acids showed an inverse relationship between the two treatments. Transcriptomic comparison between the treatments and the control group revealed upregulation of differentially expressed genes encoding dihydroflavonol reductase and uridine diphosphate-glycose flavonoid glycosyltransferase in the abscisic acid-treated group, leading to the accumulation of identified anthocyanins and catechins. In contrast, differentially expressed genes encoding nitrate reductase and nitrate transporter exhibited elevated expression in the group treated with Flu, consequently facilitating the accumulation of amino acids, specifically L-theanine and L-glutamine. Furthermore, our co-expression network analysis suggests that MYB and bHLH transcription factors may play crucial roles in regulating the expression of differentially expressed genes involved in the biosynthesis of flavonoids and amino acids. This study provides insights for targeted genetic engineering to enhance the nutritional and market value of tea, together with the potential application of purple and albino tea leaves as functional beverages. It also offers guidance for future breeding programs and production.

Characterization of the prevalence of Salmonella in different retail chicken supply modes using genome-wide and machine-learning analyses

Salmonella is a foodborne pathogen that causes salmonellosis, of which retail chicken meat is a major source. However, the prevalence of Salmonella in different retail chicken supply modes and the threat posed to consumers remains unclear. The prevalence, serotype distribution, antibiotic resistance, and genomic characteristics of Salmonella in three supply modes of retail chicken (live poultry, frozen, and chilled) were investigated using whole-genome sequencing (WGS) and machine learning (ML). In this study, 480 retail chicken samples from live poultry, frozen, and chilled supply modes in Guangzhou from 2020 to 2021, as well as 253 Salmonella isolates (total isolation rate = 53.1 %), were collected. The prevalence of isolates in the live poultry mode (67.5 %, 81/120) was statistically higher than in the frozen (50.0 %, 120/240) and chilled (43.3 %, 52/120) (P < 0.05) modes. Serotype identification showed significant differences in the serotype distribution of Salmonella in different supply modes. S. Enteritis (46.7 %) and S. Indiana (14.2 %) were predominant in the frozen mode. S. Agona (23.5 %) and S. Saintpaul (13.6 %) were predominant in live poultry, while S. Enteritis (40.4 %) and S. Kentucky (17.3 %) were predominant in chilled mode. Antibiotic testing showed that frozen mode isolates were more resistant; the multidrug-resistant (MDR) rate of isolates in the frozen mode reached 91.8 %, significantly higher than in the chilled (86.5 %) and live (74.1 %) (P < 0.05) modes. WGS was performed on 155 top serotypes (S. Enteritidis, S. Kentucky, S. Indiana, and S. Agona). The antibiotic resistance gene analysis showed that the abundance and carrying rate of antibiotic resistance genes of Salmonella in the frozen mode (54 types, 16.1 %) were significantly higher than in other modes (live poultry: 36 types, 9.4 %, P < 0.05; chilled: 31 types, 11.6 %). The blaNDM-1 and blaNDM-9 genes encoding carbapenem resistance were found in frozen mode isolates on a complex transposon consisting of TnAS3-IS26. Virulence factors and plasmid replicons were abundant in the studied frozen mode isolates. In addition, single nucleotide polymorphism (SNP) phylogenetic tree results showed that in the frozen supply mode, the S. Enteritidis clonal clade continued to contaminate retail chicken meat and was homologous to S. Enteritidis strains found in farm chicken embryos, slaughterhouse chicken carcasses, and patients from hospitals in China (SNP 0 = 10). Notably, the pan-genome-based ML model showed that characteristic genes in frozen and live poultry isolates differed. The narZ gene was a key characteristic gene in frozen isolates, encoding nitrate reductase, relating to anaerobic bacterial growth. The ydgJ gene is a key characteristic gene in the live mode and encodes an oxidoreductase related to oxidative function in bacteria. The high prevalence of live poultry mode Salmonella and the transmission of frozen mode MDR Salmonella in this study pose serious risks to food safety and public health, emphasizing the importance of improving disinfection and cold storage measures to reduce Salmonella contamination and transmission. In conclusion, the continued surveillance of Salmonella across different supply models and the development of an epidemiological surveillance system based on WGS is necessary.

Do biodegradable microplastics cause soil inorganic carbon loss in calcareous soils?

The presence of biodegradable microplastics (MPs) has the potential to affect soil pH, and possibly accelerate or inhibit the loss of soil inorganic carbon (SIC) in calcareous soils. However, most researchers have focused on the release of biotic carbon dioxide (CO2) from soils following MP amendments, and few studies have investigated SIC-derived CO2. In this experiment, three typical biodegradable MPs were applied to three calcareous soils amended with 1 % 13C-labeled (99 % atom) carbonate, and the release of CO2 originating from SIC was quantified. The total CO2 emissions, soil pH, and microbial functional genes involved in soil nitrification and denitrification were also detected. Throughout the experiment, the contribution of 13C-labeled carbonate to total CO2 emissions ranged from 0.42 % to 3.31 %. The impact of biodegradable MPs on SIC-derived CO2 varied with incubation period. At the early stage (≤20 days), the amendment of three biodegradable MPs increased the abiotic CO2 in some cases, and the CO2 emissions from 13C-labeled SIC were positively correlated with the total CO2 originating from the decomposition of SOC and MPs. At the late stage (20–70 days), the presence of biodegradable MPs inhibited the release of CO2 from 13C-labeled carbonate in most treatments. Moreover, there were negative relationships of SIC-derived CO2 with soil pH and the amoA gene of ammonia-oxidizing archaea (AOA), but positive correlations of SIC-derived CO2 with amoA of ammonia oxidizing bacteria (AOB) and nirK and nirS genes encoding nitrate reductase in denitrification. Our results indicate that long-term exposure to biodegradable MPs probably regulates the release of H+ in the nitrification process by controlling AOB, and then controlling the dynamics of SIC in calcareous soils.

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.

Exploring recycled agricultural wastes for high-rate removal of nitrogen in wastewater: Emphasizing on the investigation of the inner driving force and comparison with conventional liquid carbon sources

In this study, four typical recycled agricultural wastes (AWs), corn cob, wheat straw, sawdust and walnut shells (named AW1, AW2, AW3 and AW4, respectively), were selected as external solid carbon sources to enhance the removal of nitrogen in wastewater, and specifically, the driving mechanism was thoroughly investigated. The leaching experiments showed that the dissolved organic carbon (DOC) release capacity followed the order of AW1>AW2>AW3>AW4, ranging from 6.21 to 31.92 mg/g. DOC released from AWs mainly consisted of protein-like substances, fulvic acid-like substances and humic-like substances. AW1 and AW2 achieved comparable NOx−-N removal performance with a liquid carbon source of sodium acetate (SA) during the long-term denitrification experiments (>94.2%) but not for the other two AWs (only 16.8%-38.1%). Denitrification performance relied on DOC released from AWs at the beginning, while the enrichment of the functional CAZymes (including glycoside hydrolase and carbohydrate esterase) involved in cellulose and hemicellulose decomposition of AWs and functional genes (GAPDH, gap 2, PK, etc.) related to glycolysis were the inner driving force, which guaranteed the continuous supply of electron donors for denitrification. The relatively high abundances of napAB, narGHI, nirKS, norBC and nosZ, which encode nitrate reductase, nitrite reductase, NO reductase and N2O reductase, assured the better denitrification performance in the SA, AW1 and AW2 groups. In addition to denitrification-related functional genes, the relative abundances of nirBD and nrfAH associated with dissimilatory nitrate reduction were much higher in AW1 and AW2 groups than in SA group, implying that the nitrogen removal mechanism should be different in liquid carbon source and AW-based solid carbon source systems. In addition, GLU, gltBD and glnA, which participate in ammonia assimilation were the highest in the AW2 group, resulting in a large amount of organic nitrogen accumulation (peak concentration of approximately 24.5 mg/L), and this finally ruled it out as an alternative external carbon source. The abovementioned microbial mechanism was verified based on the correlation analysis of nutrient removal and functional genes combined with host bacterial analysis. Our study can provide valuable information for understanding the mechanism of using AWs as alternative external carbon sources to promote the removal of nitrogen in wastewater.

Effect of Biochar Application to Fertile Soil on Tomato Crop Production under Saline Irrigation Regime

Biochar application is a promising sustainable strategy for enhancing soil properties thus crop production. However, biochar application to soil certainly alters its biological and physical properties, and could require extra costs. Therefore, biochar suitability to agroecosystems must be proactively estimated. The advantage of biochar addition to poor fertile or weathered soils has been well studies, however, its feasibility to fertile soil under low quality (saline) irrigation water was not frequently studied. Consequently, this work investigates the hypothesis of whether the application of biochar at a rate of 4.8 tons/ha to fertile soil (Nile Valley, Giza, Egypt) would ameliorate the negative effects of saline irrigation regime (3000 ppm) on tomato crop and soil. The results of two seasons experiments showed that saline irrigation significantly reduced tomato crop yield by an average reduction ratio of 51%, and biochar addition could not compensate such reduction. Furthermore, biochar did not reduce accumulated Na+ in fruits or roots. Tomato fruits produced from biochar-added soil were lower in TSS levels (41.7% reduction ratio) yet larger in diameter by approximately 1.5-fold increase. Interestingly, biochar addition into soil greatly promotes the length of stem-borne lateral roots and elevates the expression of LeNR (encodes nitrate reductase enzyme) in leaves yet under fresh irrigation regime. For soil properties, biochar application enhanced the soil properties under either saline or fresh water irrigation conditions. Collectively, it is assumed that biochar application to fertile soil in Nile Valley of Egypt could not alleviate tomato fruits yield reduction affected by applied saline irrigation regime.

The Dynamics of NO3- and NH4+ Uptake in Duckweed Are Coordinated with the Expression of Major Nitrogen Assimilation Genes.

Duckweed plants play important roles in aquatic ecosystems worldwide. They rapidly accumulate biomass and have potential uses in bioremediation of water polluted by fertilizer runoff or other chemicals. Here we studied the assimilation of two major sources of inorganic nitrogen, nitrate () and ammonium (), in six duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna turionifera, Lemna minor, and Wolffia globosa. All six duckweed species preferred over and started using only when was depleted. Using the available genome sequence, we analyzed the molecular structure and expression of eight key nitrogen assimilation genes in S. polyrhiza. The expression of genes encoding nitrate reductase and nitrite reductase increased about 10-fold when was supplied and decreased when was supplied. and induced the glutamine synthetase (GS) genes GS1;2 and the GS2 by 2- to 5-fold, respectively, but repressed GS1;1 and GS1;3. and upregulated the genes encoding ferredoxin- and NADH-dependent glutamate synthases (Fd-GOGAT and NADH-GOGAT). A survey of nitrogen assimilation gene promoters suggested complex regulation, with major roles for NRE-like and GAATC/GATTC cis-elements, TATA-based enhancers, repeats, and G-quadruplex structures. These results will inform efforts to improve bioremediation and nitrogen use efficiency.

Arms race in a cell: genomic, transcriptomic, and proteomic insights into intracellular phage\u2013bacteria interplay in deep-sea snail holobionts

BackgroundDeep-sea animals in hydrothermal vents often form endosymbioses with chemosynthetic bacteria. Endosymbionts serve essential biochemical and ecological functions, but the prokaryotic viruses (phages) that determine their fate are unknown.ResultsWe conducted metagenomic analysis of a deep-sea vent snail. We assembled four genome bins for Caudovirales phages that had developed dual endosymbiosis with sulphur-oxidising bacteria (SOB) and methane-oxidising bacteria (MOB). Clustered regularly interspaced short palindromic repeat (CRISPR) spacer mapping, genome comparison, and transcriptomic profiling revealed that phages Bin1, Bin2, and Bin4 infected SOB and MOB. The observation of prophages in the snail endosymbionts and expression of the phage integrase gene suggested the presence of lysogenic infection, and the expression of phage structural protein and lysozyme genes indicated active lytic infection. Furthermore, SOB and MOB appear to employ adaptive CRISPR–Cas systems to target phage DNA. Additional expressed defence systems, such as innate restriction–modification systems and dormancy-inducing toxin–antitoxin systems, may co-function and form multiple lines for anti-viral defence. To counter host defence, phages Bin1, Bin2, and Bin3 appear to have evolved anti-restriction mechanisms and expressed methyltransferase genes that potentially counterbalance host restriction activity. In addition, the high-level expression of the auxiliary metabolic genes narGH, which encode nitrate reductase subunits, may promote ATP production, thereby benefiting phage DNA packaging for replication.ConclusionsThis study provides new insights into phage–bacteria interplay in intracellular environments of a deep-sea vent snail.3bKHUh9dEq7Vm5ZK3_XS13Video

Soybean resistance to Phakopsora pachyrhizi infection is barely potentiated by boron

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most important diseases affecting soybean production. Nutrients play a crucial role in defending plants against pathogens. Soybean resistance against ASR as affected by boron (B) was investigated through physiological, biochemical, and molecular analyses (chlorophyll a fluorescence, concentrations of photosynthetic pigments, total soluble phenolic compounds (TSP), and lignin-thioglycolic acid (LTGA) derivatives as well as expression of defense- and B-related genes). There was no significant difference for ASR severity and TEF1 (translation elongation factor 1α) expression of P. pachyrhizi for plants grown hydroponically with nutrient solutions containing 0.25 (ideal) or 1.0 (high) mM B. Higher B foliar concentration occurred in inoculated plants than in non-inoculated plants regardless of B supply. Photosynthetic process was impaired during P. pachyrhizi infection regardless of B rates. Concentration of TSP was high at 15 days after inoculation for non-inoculated plants supplied with 1.0 mM B. Concentration of LTGA derivatives was higher for inoculated plants regardless of B supply. Defense-related genes PAL1.1, PAL3.1, CHIA1, CHI1B1, LOX7, and MMP2 were up-regulated in inoculated plants regardless of B supply. Regardless of B supply, fungal infection up-regulated expression of genes encoding nitrate reductase and nitrite reductase and down-regulated genes encoding for rhamnogalacturonan II. Taken together, this study provides molecular and physiological evidences that B was barely involved in the potentiation of soybean resistance against ASR.

Crenobacter intestini sp. nov., Isolated from the Intestinal Tract of Konosirus punctatus.

A novel Gram-stain-negative bacterium, designated strain GY 70310T, was isolated from the intestinal tract of Konosirus punctatus collected from Minjiang River, China. Cells of the strain were rod-shaped and motile with a single polar flagellum. The result of 16S rRNA gene sequence analyses showed that strain GY 70310T was moderately related to Crenobacter luteus YIM 78141T (94.7%), Paludibacterium paludis KBP-21T (93.8%) and Crenobacter cavernae K1W11S-77T (93.0%). The draft genome of strain GY 70310T consisted of 3.4 Mbp with DNA G+C content of 66.3mol%, which possessed genes putatively encoding nitrate reductase, nitrite oxidoreductase and urease. The novel strain showed a whole genome average nucleotide identity (OrthoANI) value of 77.1% and a digital DNA-DNA hybridization (dDDH) value of 22.4% with Crenobacter luteus DSM 27258T, followed by Crenobacter cavernae K1W11S-77T with OrthoANI and dDDH values of 76.4% and 20.6%, respectively. The major fatty acids (>10%) were identified as summed feature 3 (C16:1ω6c and/or iso-C15:0 2-OH,C16:1ω7c), C16:0 and C18:1ω7c. The major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified lipid and one unidentified phospholipid. On the basis of phylogenetic analyses, genotypic and chemotaxonomic characteristics, strain GY 70310T represents a novel species of the genus Crenobacter, for which the name Crenobacter intestini sp. nov., is proposed. The type strain is GY 70310T (= CGMCC 1.16821T = KCTC 62945T = NBRC 113900T).

Activation of MAPK signaling in response to nitrogen deficiency in Ulva prolifera (Chlorophyta)

Ulva prolifera (Ulvophyceae, Chlorophyta), one of the main seaweeds species that cause “green tides”, suffers changes in nitrogen levels during drifting. However, the mechanisms of how this alga resist to nitrogen deficiency remains unclear. Transcripts encoding nitrate reductase, nitrite reductase, and glutamine synthase involved in nitrogen assimilation were amplified by RT-PCR, suggesting that their levels were increased in response to nitrogen deficiency. Additionally, Fluorescent Brightener 28 and Evans Blue staining demonstrated that nitrogen deficiency had no influence on the cell-wall regeneration ratio but increased the apoptosis rates of protoplasts. Furthermore, western blot assays indicate that nitrogen limitation triggered the activation of mitogen-activated protein kinase (MAPK) signaling. An inhibitor of MAPK signaling enhanced the effects of nitrogen deficiency on the mortality and decrease the capacity for cell-wall regeneration of protoplasts. All these results provided evidence that MAPK signaling had functional roles in helping U. prolifera acclimate fluctuations in nitrogen availability within a short time.

Heterotrophic sulfide-oxidizing nitrate-reducing bacteria enables the high performance of integrated autotrophic-heterotrophic denitrification (IAHD) process under high sulfide loading

Micro-aerobic enhancement technology has been developed as an effective tool to enhance simultaneous removal of sulfide, nitrate and organic carbon during the integrated autotrophic-heterotrophic denitrification (IAHD) process under high loading; however, its mechanism of enhancement for functional bacteria remains ambiguous. In this study, we discovered that heterotrophic sulfide-oxidizing nitrate-reducing bacteria (h-soNRB) are responsible for enhancing IAHD performance under micro-aerobic conditions with high sulfide loading. In a continuous IAHD bioreactor, aeration rate of 2.6 mL min−1·L−1 promoted 2 to 4 times higher removal efficiencies of sulfide, nitrate and acetate with an influent sulfide concentration of 18.75 mmol/L. Metagenomic analysis revealed that trace oxygen stimulated the abundance of genes responsible for sulfide oxidation (sqr, glpE, pdo, sox and cysK), which were upregulated by 15.2%–129.9%, and the genes encoding nitrate reductase were up-regulated by 67.4%. The increased acetate removal efficiency was attributed to upregulation of ack, pta and TCA cycle related genes. The h-NRB Pseudomonas, Azoarcus, Thauera and Halomonas were detected and regarded as h-soNRB in our bioreactor. According to Illumina MiSeq sequencing, these genera were absolutely dominant in the micro-aerobic microbial community at relative abundances ranging from 82.72% to 90.84%. The sulfide, nitrate and acetate removal rates of Pseudomonas C27, a typical h-soNRB, were at least 10 times higher under micro-aerobic conditions than under anaerobic conditions. Besides, the sulfur, nitrogen and carbon metabolic network was constructed based on the Pseudomonas C27 genome. The pdo and cysK genes found in this strain may be the most advantageous for autotrophic sulfide oxidizing nitrate reducing bacteria (a-soNRB), which are closely related to the high-efficiency sulfide, nitrate and acetate removal performance under high sulfide concentrations and a limited oxygen supply. In addition, after micro-aerobic cultivation, the anaerobic sulfide loading tolerance of the IAHD bioreactor increased from 18.75 to 37.5 mmol/L with sulfide, nitrate and acetate removal efficiencies increasing 1.5 to 3 times, which suggests that intermittent micro-aeration might be a more economical and efficient regime for high-sulfide IAHD regulation.

Protein accumulation and expression of the nitrate reductase gene in Spirulina platensiscells depending on the spectral composition of led radiation

The effect of LED lighting of different spectral composition on the productivity of Spirulina platensis, an accumulation of protein in alga cells and an expression of the nitrate reductase gene has been studied. It was shown that LED lighting with a predominance of the red component in the emission spectrum allows achieving 9–29 % higher alga productivity compared to using fluorescent lamp illumination. Illumination with single blue light resulted in significant (83 %) decrease in the productivity of Spirulina platensiswhich apparently was due to the absence of the yellow and red components in the illuminator spectral composition, which are most effectively absorbed by phycocyanin. A positive correlation between an increase in the productivity of alga and the accumulation of protein in its cells was found. So, by using an illuminator with red LEDs, the protein content increased by 21 % calculated per gram of dry weight and 47 % calculated per liter of suspension relative to the control. Analysis of the expression of the Nar gene encoding nitrate reductase in Spirulina platensiscells did not reveal a direct dependence between an increasing protein accumulation and an expression level of the Nar gene in the most promising in terms of biomass and protein yield sample of alga, growing under red LEDs. This indicates the crucial role of the photosynthetic activity of Spirulina platensiscells in increasing productivity and protein synthesis.

Effect of nitrogen supply on nitrogen metabolism in the citrus cultivar 'Huangguogan'.

Nitrogen metabolism in citrus has received increased attention due to its effects on plant growth and productivity. However, little is known about the effects of nitrogen fertilization on nitrogen metabolism in young trees of citrus cultivar ‘Huangguogan’ (Citrus reticulata × Citrus sinensis). Here, genes encoding nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate dehydrogenase (GDH), and asparagine synthetase (AS), represented as HgNR, HgNiR, HgGS, HgGDH, and HgAS, respectively, were cloned from Huangguogan. Deduced protein sequences were analyzed and proteins were confirmed to be localized in their respective cellular organelles. Moreover, pot-cultured ‘Huangguogan’ seedlings were fertilized with 0 (N1), 1.36 (N2), 1.81 (N3), 2.26 (N4), or 2.72 (N5) kg N/year, for 12 months. Enzyme activity and enzyme-gene expression were studied in roots, leaves, and fruits at different stages. Finally, the effects of N application rate on root activity, leaf N, soluble protein, yield, and fruit quality at the ripening stage were measured. The results showed that: 1) HgNR, HgNiR, HgGDH, and HgAS gene products were found mainly in the cytoplasm and plasma membrane, while HgGS gene product was found mainly in cytoplasm and mitochondria. 2) Gene expression and enzyme activity differed among plant organs. As the root is in permanent direct contact with the soil we suggest that root gene expression and enzyme activity can be used as reference to determine N application rate. 3) Yield, fruit quality, enzyme activity, and enzyme-related gene expression were considerably lower at low than at high-N supply. However, they were all inhibited by excess nitrogen (i.e., 2.72 kg/year). Therefore, we recommend 1.81 kg N/year as the optimal N application rate for young ‘Huangguogan’ trees.

Overexpression of TaLEA3 induces rapid stomatal closure under drought stress in Phellodendron amurense Rupr

Late embryogenesis abundant (LEA) proteins participate in drought stress responses in plants. In the present study, the gene TaLEA3 from the drought-resistant plant Tamarix androssowii was transformed into Amur cork tree (Phellodendron amurense) via Agrobacterium tumefaciens to investigate the mechanism of stomatal closure in response to osmotic stress. Our results showed that P. amurense overexpressing TaLEA3 were resistant to drought stress by rapid stomatal closure. To study the stomatal movement regulated at the molecular level, a model system for stoma closure was established in in vitro P. amurense. In this work, we found that the increased Ca2+ accumulation in guard cells of transgenic plants caused stomatal closure and activated K+ efflux under polyethylene glycol (PEG) stress. Moreover, H+ changes might provide a needed pH condition for stomatal closure. Further, nitric oxide (NO) fluorescence was measured using an NO-specific fluorescent probe, diaminofluorescein-FM diacetate, which showed that guard cell NO fluorescence was stronger in transgenic plants compared with wild type plants. Additionally, five genes encoding nitrate reductase were up-regulated, indicating that TaLEA3 overexpression positively regulated NO biosynthesis and accumulation in the guard cells. This discovery will further our understanding of the LEA gene function and will help in engineering drought-resistant tree cultivars.

Copper excess reduces nitrate uptake by Arabidopsis roots with specific effects on gene expression

Nitrate uptake by plants is mediated by specific transport proteins in roots (NRTs), which are also dependent on the activity of proton pumps that energize the reaction. Nitrogen (N) metabolism in plants is sensitive to copper (Cu) toxicity conditions. To understand how Cu affects the uptake and assimilation processes, this study assesses the inhibitory effects of elevated Cu levels on the expression of genes related to N absorption, transport and assimilation in roots of Arabidopsis. Plants were grown hydroponically for 45 days, being exposed to a range of Cu concentrations in the last 72 h or alternatively exposed to 5.0 μM Cu for the last 15 days. High Cu levels decreased the uptake and accumulation of N in plants. It down-regulated the expression of genes encoding nitrate reductase (NR1), low-affinity nitrate transporters (NRT1 family) and bZIP transcription factors (TGA1 and TGA4) that regulate the expression of nitrate transporters. Cu toxicity also specifically down-regulated the plasma membrane proton pump, AHA2, whilst having little effect on AHA1 and AHA5. In contrast, there was an up-regulation of high-affinity nitrate transporters from the NRT2 family when exposed to medium level of Cu excess, but this was insufficient for restoring N absorption by roots to control levels. These results demonstrate that plants display specific responses to Cu toxicity, modulating the expression of particular genes related to nitrate uptake, such as low-affinity nitrate transporters and proton pumps.