Plant Nitrate Reductase Research Articles

Nitrate reductase enzymes in alga Chattonella subsalsa are regulated by environmental cues at the translational and post-translational levels

Nitrate reductase (NR) catalyzes the rate-limiting step in nitrate assimilation. Plant and algal NRs have a highly conserved domain architecture but differ in regulation. In plants, NR activity is regulated by reversible phosphorylation and subsequent binding of 14-3-3 proteins at a conserved serine residue. Algal NRs typically lack 14-3-3 binding motifs, which have only recently been identified in a few algal species. Previous research indicates that the alga, Chattonella subsalsa, possesses a novel NR, NR2-2/2HbN (NR2), which incorporates a 2/2 hemoglobin domain. A second NR (NR3) in C. subsalsa lacks the cytochrome b5 (heme-Fe) domain but includes a putative binding motif for 14-3-3 proteins. The expression of NR2 and NR3 genes indicates that NR2 transcript abundance was regulated by light, nitrogen source, and temperature, while NR3 transcript levels were only regulated by light. Here, we measured total NR activity in C. subsalsa and the potential for regulation of NR activity by putative 14-3-3 binding proteins. Results indicate that NR activity in C. subsalsa was regulated by light, nitrogen source, and temperature at the translational level. NR activity was also regulated by endogenous rhythm and temperature at the post-translational level, supporting the hypothesis that NR3 is regulated by 14-3-3 binding proteins. Together with a previous report describing the regulation of NR gene expression in C. subsalsa, results suggest that C. subsalsa responds to environmental conditions by differential regulation of NRs at transcriptional, translational, and post-translational levels. This flexibility may provide a competitive advantage for this species in the environment. To date, this is the first report which provides evidence for the potential post-translational regulation of NR by 14-3-3 proteins in algal species and suggests that regulatory mechanisms for NR activity may be shared between plants and some algal species.

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

BackgroundBrown 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.ResultsHere, 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.ConclusionThese 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.

Synergistic enhancement of the phytostabilization of a semiarid mine tailing by a combination of organic amendment and native microorganisms (Funneliformis mosseae and Bacillus cereus)

The goal of this work was to evaluate the effects of fermented sugar beet residue and inoculation with a native arbuscular mycorrhizal (AM) fungus, Funneliformis mosseae (Nicol. and Gerd.) Gerd. and Trappe, or a native bacterium, Bacillus cereus Frankland & Frankland, alone or in combination, on the establishment of Lygeum spartum L. seedlings grown in a mine tailing under semiarid conditions. We conducted a field study to analyse root and shoot dry biomass, shoot nutrient contents, mycorrhization, plant nitrate reductase (NR) and acid phosphomonoesterase activities, soil enzyme activities and aggregate stability. Ten months after field transplanting, it was found that the three experimental factors had interacted synergistically with regard to shoot and root biomass, with increases of about 410% and 370%, respectively relative to plants in the untreated soil. The treatment combining all three factors increased the root content of all heavy metals, and the levels of nitrogen (N), phosphorus, potassium and NR activity in shoot tissues, whereas it decreased root acid phosphomonoesterase activity. Soil dehydrogenase, protease and β-glucosidase activities, total N content and aggregate stability were increased by the combined treatment. In conclusion, the combination of the organic amendment, the native AM fungus and the native bacterium can be regarded as a suitable tool for phytostabilization with L. spartum due to its ability to enhance the tolerance of plants to heavy metals, improve the plant nutritional status and increase the soil microbial function related to the C cycling.

Effects of excessive nitrogen fertilizer and soil moisture deficiency on antioxidant enzyme system and osmotic adjustment in tomato seedlings

Long-term excessive application of nitrogen fertilizer induces secondary salinization of soil, which results in inhibiting plant growth. In addition, soil moisture deficiency also affects plant growth. To investigate the effects of excessive nitrogen fertilizer and soil moisture deficiency on the antioxidant enzyme system, plant water relations were analyzed through pressure-volume (P-V) curve, and photosynthetic light response parameters in tomato (Solanum lycopersicum L. Myoko) seedlings, an indoor experiment about 50 d was conducted using two irrigation water amounts based on field capacity (soil moisture deficiency: 50%-80%; adequate water: 70%-80%), two nitrogen fertilizer rates (moderate nitrogen; excessive nitrogen fertilizer: 0.585 g/pot) and two types of irrigation water (tap water and microbial diluent). The results showed that excessive nitrogen fertilizer (N) and soil moisture deficiency (W) reduced the biomass of tomato seedlings. In comparison to CK (combination of adequate water and tap water quality), microbial dilution (EM) increased plant biomass by 5.2%. Also, the nitrogen application increased chlorophyll relative contents (SPAD). The maximum net photosynthetic rate (Pc) decreased with nitrogen application and increased with EM application and irrigation amount. Excessive nitrogen application increased the plant nitrate reductase activity (NR). The plant NR in the N treatment showed a 13.0% increase compared to CK, and the plant NR in the treatment of nitrogen application with water deficiency (WN) increased 34.0% compared to water deficiency (W). After applying excessive nitrogen, N, EM-N, WN, EM-WN respectively increased the plant nitrate reductase activity by 13.0%, 22.9%, 34.0% and 28.6%, compared with the corresponding treatment with moderate nitrogen (i.e., CK, EM, W and EM-W). In addition, the activities of antioxidant enzymes [superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT)] in four treatments of nitrogen application (N, EM-N, WN, EM-WN) also increased significantly. Both soil moisture and nitrogen fertilizer significantly affect the parameters of osmotic adjustment, which is manifested in the reduction of osmotic potential (πFT), and the increase in the osmotic concentration (Cosm) and concentration difference (ΔCosm). But the decrease in the relative water content of apoplast (ζap), indicating that water deficiency and excessive nitrogen reduced the water absorption and water retention capacity of tomatoes to a certain extent. In conclusion, excessive nitrogen application and soil moisture deficiency inhibit plant growth significantly in this experiment. Meanwhile, microbial dilution can alleviate excessive nitrogen fertilizer and water stress to some extent, but the effect was not significant. Keywords: antioxidant enzyme, nitrogen, osmotic adjustment, pressure-volume curve, stress, tomato, water DOI: 10.25165/j.ijabe.20221502.5555 Citation: Zhang Y J, Yu S E, Li Z J, Chang T T, Xu Q C, Xu H L, et al. Effects of excessive nitrogen fertilizer and soil moisture deficiency on antioxidant enzyme system and osmotic adjustment in tomato seedlings. Int J Agric & Biol Eng, 2022; 15(2): 127–134.

Physiological Role of Aerobic Fermentation Constitutively Expressed in an Aluminum-Tolerant Cell Line of Tobacco (Nicotiana tabacum)

Aluminum (Al)-tolerant tobacco cell line ALT301 derived from SL (wild-type) hardly exhibits Al-triggered reactive oxygen species (ROS) compared with SL. Molecular mechanism leading to this phenotype was investigated comparatively with SL. Under normal growth condition, metabolome data suggested the activation of glycolysis and lactate fermentation but the repression of the tricarboxylic acid (TCA) cycle in ALT301, namely aerobic fermentation, which seemed to be transcriptionally controlled partly by higher expression of genes encoding lactate dehydrogenase and pyruvate dehydrogenase kinase. Microarray and gene ontology analyses revealed the upregulation of the gene encoding related to APETALA2.3 (RAP2.3)-like protein, one of the group VII ethylene response factors (ERFVIIs), in ALT301. ERFVII transcription factors are known to be key regulators for hypoxia response that promotes substrate-level ATP production by glycolysis and fermentation. ERFVIIs are degraded under normoxia by the N-end rule pathway of proteolysis depending on both oxygen and nitric oxide (NO), and NO is produced mainly by nitrate reductase (NR) in plants. In ALT301, levels of the NR gene expression (NIA2), NR activity and NO production were all lower compared with SL. Consistently, the known effects of NO on respiratory pathways were also repressed in ALT301. Under Al-treatment condition, NO level increased in both lines but was lower in ALT301. These results suggest that the upregulation of the RAP2.3-like gene and the downregulation of the NIA2 gene and resultant NO depletion in ALT301 coordinately enhance aerobic fermentation, which seems to be related to a higher capacity to prevent ROS production in mitochondria under Al stress.

Biochemical metabolism of young plants of Ucu\xfaba (Virola surinamensis) in the presence of cadmium

Virola surinamensis is a forest species widely distributed in the estuaries of the Amazon. These ecosystems are susceptible to contamination by Cadmium (Cd), indicating that the plant has strategies for tolerating this metal. The aim of this study was to assess the nitrogen and carbon metabolism of young plants of Ucuúba (Virola surinamensis) in the presence of cadmium with the perspective of the phytoremediation of contaminated environments. The used experimental design was a completely randomized design with five Cd concentrations (0, 15, 30, 45, and 60 mg L− 1), for 60 days. In general, Cd did not affect nitrate concentration in the root but had a positive effect on leaves. The reduction of nitrate reductase (NR) in plants exposed to Cd was followed by a decrease in ammonia, total soluble amino acids (TSA), and total soluble proteins (TSP). Cd promoted an increase in the concentration of total soluble carbohydrates (TSC), proline, sucrose, and reducing sugars in the plants. The increase in TSC, sucrose and proline, suggests a metabolic regulatory mechanism of V. surinamensis against Cd stress.

New Insights into the Transcriptional Regulation of Genes Involved in the Nitrogen Use Efficiency under Potassium Chlorate in Rice (Oryza sativa L.).

Potassium chlorate (KClO3) has been widely used to evaluate the divergence in nitrogen use efficiency (NUE) between indica and japonica rice subspecies. This study investigated the transcriptional regulation of major genes involved in the NUE in rice treated with KClO3, which acts as an inhibitor of the reducing activity of nitrate reductase (NR) in higher plants. A set of two KClO3 sensitive nitrate reductase (NR) and two nitrate transporter (NRT) introgression rice lines (BC2F7), carrying the indica alleles of NR or NRT, derived from a cross between Saeilmi (japonica, P1) and Milyang23 (indica, P2), were exposed to KClO3 at the seedling stage. The phenotypic responses were recorded 7 days after treatment, and samples for gene expression, physiological, and biochemical analyses were collected at 0 h (control) and 3 h after KClO3 application. The results revealed that Saeilmi (P1, japonica) and Milyang23 (P2, indica) showed distinctive phenotypic responses. In addition, the expression of OsNR2 was differentially regulated between the roots, stem, and leaf tissues, and between introgression lines. When expressed in the roots, OsNR2 was downregulated in all introgression lines. However, in the stem and leaves, OsNR2 was upregulated in the NR introgression lines, but downregulation in the NRT introgression lines. In the same way, the expression patterns of OsNIA1 and OsNIA2 in the roots, stem, and leaves indicated a differential transcriptional regulation by KClO3, with OsNIA2 prevailing over OsNIA1 in the roots. Under the same conditions, the activity of NR was inhibited in the roots and differentially regulated in the stem and leaf tissues. Furthermore, the transcriptional divergence of OsAMT1.3 and OsAMT2.3, OsGLU1 and OsGLU2, between NR and NRT, coupled with the NR activity pattern in the roots, would indicate the prevalence of nitrate (NO3¯) transport over ammonium (NH4+) transport. Moreover, the induction of catalase (CAT) and polyphenol oxidase (PPO) enzyme activities in Saeilmi (P1, KClO3 resistant), and the decrease in Milyang23 (P2, KClO3 sensitive), coupled with the malondialdehyde (MDA) content, indicated the extent of the oxidative stress, and the induction of the adaptive response mechanism, tending to maintain a balanced reduction–oxidation state in response to KClO3. The changes in the chloroplast pigments and proline content propose these compounds as emerging biomarkers for assessing the overall plant health status. These results suggest that the inhibitory potential of KClO3 on the reduction activity of the nitrate reductase (NR), as well as that of the genes encoding the nitrate and ammonium transporters, and glutamate synthase are tissue-specific, which may differentially affect the transport and assimilation of nitrate or ammonium in rice.

Post-Translational Modifications of Nitrate Reductases Autoregulates Nitric Oxide Biosynthesis in Arabidopsis.

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.

Bio-extraction of Potassium from Glauconite Nano-particle in an Alfisols of Southern India

ABSTRACT Glauconite is a potassium (K) bearing mineral, which generally contains 6–10% K2O and mostly available in various natural deposits in India. Glauconite mineral was converted to nano-particle form by grinding in a high-energy ball mill. The release of K from glauconite nano-particle in soil was studied in a pot culture experiment. Five different treatments viz., 0 mg K2O kg−1, 100 mg K2O kg−1, 150 mg K2O kg−1, 200 mg K2O kg−1 used glauconite nano-particle (GNP) as a K source and 100 mg K2O kg−1 from muriate of potash (MOP), were applied in soil to grow maize (Zea mays L.) plant. The highest K+ concentration was observed in shoot at the application of 200 mg K2O kg−1 through GNP and recorded 2 times more K+ concentration in shoots as compared to 0 mg K2O kg−1 treatment whereas 1.1 times more than the MOP treated soil at 5th harvesting stage. Selected plant physiological parameters namely electrical conductivity of plant cell, height, leaf area and nitrate reductase activity were also recorded maximum at 200 mg K2O kg−1 GNP treated soil. Overall, glauconite nano-particle could supply K throughout the growth period and enhanced biomass yield of maize plant without showing any K deficiency symptom.

Adaptation of nitrate reductase activity assay for high throughput screening of crops

The possibility of freeze drying of plant material and its grinding in a shaking bead mill to determine the activity of nitrate reductase (NR) was studied. The effectiveness of applying this approach to high throughput mass screening of crops was shown. The assay was carried out on seedlings of common wheat (Triticum aestivum) of the following cultivars: Altigo, Vassa, Grom, Doka, Soberbash, Starshina, Fisht and spring wheat Novosibirskaya 67. The crops were grown during 4-5 weeks on substrate without nitrogen and on substrate supplemented with 50 millimol / l (mM) of potassium nitrate. Nitrate reductase in plants retained its activity after lyophilization and grinding of dried leaves in a mill. The proposed protocol for NR activity assay is suitable for plant lysates with an NR activity suffi cient to form nitrite in the range of 5–120 micromoles / l (μM) in 800 μl of reaction mix (for instance, freeze-dried sample originated from 100 mg of wheat seedling leaves). Centrifugation of a plant lysate at 20,000 g almost did not change NR activity compared to 12,000 g that is achievable for most lab centrifuges. Lysates from fresh leaves contained signifi cantly more total protein than lysates from lyophilized leaves (with an equal amount of starting wet material). The difference in the nitrate-reducing activity in lysates from fresh and lyophilized leaves was not as high as the difference in protein concentration. Thus, the activity of NR calculated per g of total protein was higher in lyophilized leaves than in fresh leaves. The activity of NR was signifi cantly induced by nitrate for all cultivars. The basal and nitrate-induced NR activity varied widely between the cultivars, and the induction ranged from 2.5 fold for Novosibirskaya 67 variety and 2.7 fold for Vassa to 5.4 for Altigo and 5.7 fold for Grom.

Diazotrophic Paenibacillus beijingensis BJ-18 Provides Nitrogen for Plant and Promotes Plant Growth, Nitrogen Uptake and Metabolism.

Diazotrophic bacteria can reduce N2 into plant-available ammonium (NH4+), promoting plant growth and reducing nitrogen (N) fertilizer requirements. However, there are few systematic studies on the effects of diazotrophic bacteria on biological N2 fixation (BNF) contribution rate and host plant N uptake and metabolism. In this study, the interactions of the diazotrophic Paenibacillus beijingensis BJ-18 with wheat, maize, and cucumber were investigated when it was inoculated to these plant seedlings grown in both low N and high N soils, with un-inoculated plants as controls. This study showed that GFP-tagged P. beijingensis BJ-18 colonized inside and outside seedlings, forming rhizospheric and endophytic colonies in roots, stems, and leaves. The numbers of this bacterium in the inoculated plants depended on soil N levels. Under low N, inoculation significantly increased shoot dry weight (wheat 86.1%, maize 46.6%, and cucumber 103.6%) and root dry weight (wheat 46.0%, maize 47.5%, and cucumber 20.3%). The 15N-isotope-enrichment experiment indicated that plant seedlings derived 12.9–36.4% N from BNF. The transcript levels of nifH in the inoculated plants were 0.75–1.61 folds higher in low N soil than those in high N soil. Inoculation enhanced NH4+ and nitrate (NO3-) uptake from soil especially under low N. The total N in the inoculated plants were increased by 49.1–92.3% under low N and by 13–15.5% under high N. Inoculation enhanced activities of glutamine synthetase (GS) and nitrate reductase (NR) in plants, especially under low N. The expression levels of N uptake and N metabolism genes: AMT (ammonium transporter), NRT (nitrate transporter), NiR (nitrite reductase), NR, GS and GOGAT (glutamate synthase) in the inoculated plants grown under low N were up-regulated 1.5–91.9 folds, but they were not obviously changed under high N. Taken together, P. beijingensis BJ-18 was an effective, endophytic and diazotrophic bacterium. This bacterium contributed to plants with fixed N2, promoted plant growth and N uptake, and enhanced gene expression and enzyme activities involved in N uptake and assimilation in plants. However, these positive effects on plants were regulated by soil N status. This study might provide insight into the interactions of plants with beneficial associative and endophytic diazotrophic bacteria.

Identification of transcription factors of nitrate reductase gene promoters and NRE2 cis-element through yeast one-hybrid screening in Nicotiana tabacum

BackgroundThis study aimed to identify the transcription factors of nitrate reductase genes (NIA1 and NIA2) promoters and hypothetical cis-element of NRE2. Based on the constructed cDNA library of Nicotiana tabacum K326, a yeast one-hybrid system was established using the Matchmaker® Gold Yeast One-Hybrid Library Screening System from Clontech. The transcription factors of NIA1 andNIA2 promoters and NRE2 cis-elements were screened.ResultsAfter sequencing and bioinformatics analysis, 15 cDNA sequences were identified: 9 for NIA1 (including XP_016503563.1 and NP_001312236.1), 3 for NIA2 (including XP_016510250.1), and 3 for NRE2 (including XM_016576899.1). XP_016503563.1 was annotated in PREDICTED: CRM-domain containing factor CFM3, and NP_001312236.1chloroplastic/mitochondrial-like in Nicotiana tabacum. NP_001312236.1 was annotated in Sulfite oxidase-like of Nicotiana tabacum. XP_016510250.1 was annotated as PREDICTED: uncharacterized protein LOC107827596 in Nicotiana tabacum. XM_016576899.1 was annotated in PREDICTED: Nicotiana tabacum RING-H2 finger protein ATL16-like (LOC107759033).ConclusionA yeast one-hybrid library was successfully constructed. The identified transcription factors may provide a theoretical basis for the study of plant nitrate reductase.

Exploitation of new endophytic bacteria and their ability to promote sugarcane growth and nitrogen nutrition.

Few studies have evaluated endophytic bacteria in relation to plant growth promotion, nitrogen uptake and biological control. The aim of this study was to molecularly and physiologically characterize thirteen endophytic bacteria strains, evaluate their biological control properties and their ability to promote plant growth and plant N nutrition. All the strains produced indole acetic acid and promoted increase of plant biomass, N accumulative amount and N-use efficiency index. None of the strains carries the nifH gene. Four strains stimulated plant nitrate reductase activity, four solubilized phosphate, nine produced siderophores and none produced HCN. Seven strains inhibited Bipolaris sacchari growth and one was antagonistic to Ceratocystis paradoxa. The pathogens were inhibited by the production of diffusible and volatile metabolites by the bacterial strains. Moreover, this is the first study to demonstrate the effect of Delftia acidovorans on sugarcane plant growth, nitrogen metabolism improvement and antagonism to B. sacchari. The most efficient strains in promoting plant growth and exhibiting antagonistic activities towards fungal pathogens were Herbaspirillum frinsingense (IAC-BECa-152) and three Pantoea dispersa strains (IAC-BECa-128, IAC-BECa-129, and IAC-BECa-132). These bacteria show potential to be used as inoculants for sustainable agricultural management, mainly at the seedling production phase.

Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes.

Nitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants.

Cloning and characterization of nitrate reductase gene in Ulva prolifera (Ulvophyceae, Chlorophyta).

Ulva spp. dominates green tides around the world, which are occurring at an accelerated rate. The competitive nitrogen assimilation efficiency in Ulva is suggested to result in ecological success against other seaweeds. However, molecular characterization of genes involved in nitrogen assimilation has not been conducted. Here, we describe the identification of the nitrate reductase (NR) gene from a green seaweed Ulva prolifera, an alga which is responsible for the world's largest green tide in the Yellow Sea. Using rapid amplification of cDNA ends and genome walking, the NR gene from U.prolifera (UpNR) was cloned, which consisted of six introns and seven exons encoding 863 amino acids. According to sequence alignment, the NR in U.prolifera was shown to possess all five essential domains and 21 key invariant residues in plant NRs. The GC content of third codon position of UpNR (82.75%) was as high as those of green microalgae, and the intron number supported a potential loss issue from green microalga to land plant. Real-time quantitative PCR results showed that UpNR transcript level was induced by nitrate and repressed by ammonium, which could not be removed by addition of extra nitrate, indicating that U.prolifera preferred ammonium to nitrate. Urea would not repress NR transcription by itself, while it weakened the induction effect of nitrate, implying it possibly inhibited nitrate uptake rather than nitrate reduction. These results suggest the use of UpNR as a gene-sensor to probe the N assimilation process in green tides caused by Ulva.

In situ assay of nitrate reductase activity using portable water bath.

In environmental research (i.e., plant ecophysiology, environmental microbiology, and environmental chemistry), some assays require incubation of samples at controlled temperature and darkness. Until now, due to a lack of equipment providing such possibility in situ, researchers had to move collected samples to the laboratory for incubation. Obviously, a delayed incubation and the ex situ conditions could seriously affect the assays' results. A good example of analysis where water bath use is needed is the nitrate reductase activity (NRA) in vivo assay where plant tissue samples are incubated in buffer solution at a predetermined temperature. We designed a transportable water bath with a temperature control which enables in situ measurements in many types of environmental studies. The presented device is small in size featuring a thermally insulated chamber and an electronically controlled thermostat system powered by a 12-V battery. Due to its modular design, it can be transported comfortably in difficult terrain. The incubation process can be carried out continuously in stable temperature and darkness. In order to examine the field usability of the presented device, we conducted measurements of plant nitrate reductase activity in difficult field conditions. The in situ assays were carried out at high altitudes in the Karkonosze mountains, SW Poland. The NRA was studied in two alpine species (Deschampsia caespitosa and Homogyne alpina). Our results showed low NR activity in H. alpina (mean 0.31μM NO2 g-1 DW h-1) and higher NRA in D. caespitosa (mean 2.7μM NO2 g-1 DW h-1). The obtained results were highly reproducible and had small variability (low standard error values).

Effects of 5-aminolevulinic acid on nitrogen metabolism and ion distribution of watermelon seedlings under salt stress

The effects of foliar spray application of 5-aminolevulinic acid (ALA) on the growth, nitrogen metabolism, and ion distribution of salt-stressed watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) seedlings were investigated. Supplementation of the nutrient solution with 100 mM NaCl significantly reduced leaf and root biomass of watermelon plants. Foliar application of 1.25 mM ALA significantly alleviated the inhibition of plant growth under salt stress. Salinity induced significant accumulation of nitrate, ammonium, and soluble protein and a significant decrease in the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in watermelon plants. However, ALA significantly increased the activities of NR, GS, GOGAT, and GDH, but decreased the ammonium content and NiR activity. In addition, salt stress resulted in significant accumulation of Na+ and Cl− in plants, but decreased the contents of K+ and Mg2+. Application of ALA alleviated the salt stress-induced ion toxicity, and increased the contents of K+ and Mg2+. ALA also increased soluble protein and proline contents in salt-stressed watermelon plants. These results indicated that application of ALA alleviated the accumulation of Na+ and Cl− in salt-stressed watermelon plants, especially through regulating nitrogen metabolism and ion distribution, which were associated with an improvement in plant growth.

The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

Properties of nitrogen fertilization are decisive in determining the effects of elevated atmospheric CO2 on the activity of nitrate reductase in plants

ABSTRACTThe concentration of atmospheric CO2 is predicted to double by the end of this century. The response of higher plants to an increase in atmospheric CO2 often includes a change in nitrate reductase (NR) activity. In a recent study, we showed that, under elevated CO2 levels, NR induction in low-nitrate plants and NR inhibition in high-nitrate plants are regulated by nitric oxide (NO) generated via nitric oxide synthases. This finding provides an explanation for the diverse responses of plants to elevated CO2 levels, and suggests that the use of nitrogen fertilizers on soil will have a major influence on the nitrogen assimilation capacity of plants in response to CO2 elevation.

Nitrogen uptake efficiency of finger millet grown in hydroponic culture

ABSTRACTThe optimum nitrogen requirement along with nitrogen uptake efficiency of finger millet (Eleusine coracana) for obtaining plants of a high phenotypic quality was studied in this research using hydroponic culture. 20 days old seedlings were transferred in nutrient solution containing all the essential macro and micro nutrients. Plants were subjected to three treatments of nitrate viz. 0.05 mM, 0.1 mM and 0.5 mM by dissolving it in the nutrient solution. Plants grown in nutrient solution devoid of nitrate were treated as control. Plants grown in 0.5 mM nitrate conc. attained more height compared to other low nitrogen treatments (0.1 mM and 0.05 mM). Similarly plants of 0.5 mM nitrate treatments possessed more number of lateral roots, surface area of leaves, dry weight of plants and chlorophyll content compared to other low nitrogen treatments. Specific activity of nitrate reductase in plants treated with 0.5 mM concentration was found nearly 3 times higher than that of control plants; similarly, specific activity of nitrite reductase in 0.5 mM treated plants was also high compared to other low nitrogen treatments followed by control. The results of the present investigation therefore indicate that nitrogen uptake efficiency as revealed by the activities of nitrate reductase and nitrite reductase was high in 0.5 mM nitrate treatment. It also shows that 0.5 mM nitrate is optimum nitrogen concentration for the growth of finger millet in hydroponic condition.