Nitrate Assimilation Research Articles

Physical and Biogeochemical Influences on Nutrients Through the Canadian Arctic Archipelago: Insights From Nitrate Isotope Ratios

AbstractThe Canadian Arctic Archipelago (CAA) provides a gateway for the flow of nutrients from the North Pacific to the North Atlantic. The transport and biogeochemical cycling of nutrients throughout the CAA, however, are sparsely documented. Here, we report water column nitrogen and oxygen isotope ratios of nitrate (δ15NNO3 and δ18ONO3) collected in a throughflow of the CAA into Baffin Bay, providing insights on inherent nutrient dynamics. The nitracline shoaled eastward into the CAA, wherein large subsurface chlorophyll maxima and coincident increases in δ15NNO3 and δ18ONO3 indicated enhanced nitrate assimilation relative to the oligotrophic Canada Basin. High δ15NNO3 values characteristic of Pacific Winter Water (PWW) pervaded the Archipelago and Baffin Bay, decreasing eastward due to mixing with underlying Atlantic water (AW)—from the Canada Basin west of Barrow Strait, and from Baffin Bay east thereof. Nearly 25% of nutrients in the central CAA were of Atlantic origin, a substantially larger fraction than surmised previously. Nutrient properties in the CAA were notably not influenced by benthic denitrification. Those in underlying AW in the western CAA, however, were modified by remineralization of Pacific‐sourced nutrients, manifested from increases in δ15NNO3 relative to the Canada Basin. Properties in the eastern CAA exposed the complex hydrography of Baffin Bay, characterized by lateral intrusions of nutrient‐poor winter waters into PWW and of cold mode waters in underlying AW. High δ15NNO3 values in AW revealed the presence of a substantial fraction of N from remineralization, stemming from the high productivity in Baffin Bay fueled by Pacific nutrients.

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

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

Multi-Omic Investigation of Low-Nitrogen Conditional Resistance to Clubroot Reveals Brassica napus Genes Involved in Nitrate Assimilation.

Nitrogen fertilization has been reported to influence the development of clubroot, a root disease of Brassicaceae species, caused by the obligate protist Plasmodiophora brassicae. Our previous works highlighted that low-nitrogen fertilization induced a strong reduction of clubroot symptoms in some oilseed rape genotypes. To further understand the underlying mechanisms, the response to P. brassicae infection was investigated in two genotypes “Yudal” and HD018 harboring sharply contrasted nitrogen-driven modulation of resistance toward P. brassicae. Targeted hormone and metabolic profiling, as well as RNA-seq analysis, were performed in inoculated and non-inoculated roots at 14 and 27 days post-inoculation, under high and low-nitrogen conditions. Clubroot infection triggered a large increase of SA concentration and an induction of the SA gene markers expression whatever the genotype and nitrogen conditions. Overall, metabolic profiles suggested that N-driven induction of resistance was independent of SA signaling, soluble carbohydrate and amino acid concentrations. Low-nitrogen-driven resistance in “Yudal” was associated with the transcriptional regulation of a small set of genes, among which the induction of NRT2- and NR-encoding genes. Altogether, our results indicate a possible role of nitrate transporters and auxin signaling in the crosstalk between plant nutrition and partial resistance to pathogens.

Root or shoot nitrate assimilation in terrestrial vascular plants – does it matter?

Root or shoot nitrate assimilation in terrestrial vascular plants – does it matter?

Metagenomic Insights into the Structure of Microbial Communities Involved in Nitrogen Cycling in Two Integrated Multitrophic Aquaculture (IMTA) Ponds

The microbial structure and metabolic potential, particularly with regard to nitrogen (N) cycling, in integrated multitrophic aquaculture (IMTA) ponds with shrimp remain unclear. In this study, an analysis of microbial community taxonomic diversity and a metagenomic analysis of N-related genes were performed in a shrimp-crab pond (Penaeus japonicus-Portunus trituberculatus, SC) and a shrimp-crab-clam pond (P. japonicus-P. trituberculatus-Sinonovacula constricta, SCC) to evaluate microbial structure and N transformation capacities in these two shrimp IMTA ponds. The composition of the microbial communities was similar between SC and SCC, but the water and sediments shared few common members in either pond. The relative abundances of N cycling genes were significantly higher in sediment than in water in both SC and SCC, except for assimilatory nitrate reduction genes. The main drivers of the differences in the relative abundances of N cycling genes in SC and SCC were salinity and pH in water and the NO2− and NH4+ contents of pore water in sediment. These results indicate that the coculture of S. constricta in a shrimp-crab pond may result in decreased N cycling in sediment. The reduced N flux in the shrimp IMTA ponds primarily originates within the sediment, except for assimilatory nitrate reduction.

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

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

New insights into the role of chrysanthemum calcineurin B\u2013like interacting protein kinase CmCIPK23 in nitrate signaling in Arabidopsis roots

Nitrate is an important source of nitrogen and also acts as a signaling molecule to trigger numerous physiological, growth, and developmental processes throughout the life of the plant. Many nitrate transporters, transcription factors, and protein kinases participate in the regulation of nitrate signaling. Here, we identified a gene encoding the chrysanthemum calcineurin B-like interacting protein kinase CmCIPK23, which participates in nitrate signaling pathways. In Arabidopsis, overexpression of CmCIPK23 significantly decreased lateral root number and length and primary root length compared to the WT when grown on modified Murashige and Skoog medium with KNO3 as the sole nitrogen source (modified MS). The expression of nitrate-responsive genes differed significantly between CmCIPK23-overexpressing Arabidopsis (CmCIPK23-OE) and the WT after nitrate treatment. Nitrate content was significantly lower in CmCIPK23-OE roots, which may have resulted from reduced nitrate uptake at high external nitrate concentrations (≥ 1 mM). Nitrate reductase activity and the expression of nitrate reductase and glutamine synthase genes were lower in CmCIPK23-OE roots. We also found that CmCIPK23 interacted with the transcription factor CmTGA1, whose Arabidopsis homolog regulates the nitrate response. We inferred that CmCIPK23 overexpression influences root development on modified MS medium, as well as root nitrate uptake and assimilation at high external nitrate supply. These findings offer new perspectives on the mechanisms by which the chrysanthemum CBL interacting protein kinase CmCIPK23 influences nitrate signaling.

Triplication is the main evolutionary driving force of NLP transcription factor family in Chinese cabbage and related species

The NODULE-INCEPTION-like protein (NLP) is a plant-specific transcription factor (TF) family that plays an important role in both signal transduction and nitrate assimilation. However, the NLP gene family in Chinese cabbage (Brassica rapa) has yet to be studied. Here we identified 17, 16, and 32 NLP genes in Chinese cabbage, Brassica oleracea, and Brassica napus, respectively. We found that duplication of those NLP genes almost always originated from genome-wide duplication events. Further analysis (using Arabidopsis as a reference) revealed that the NLP family in Chinese cabbage and B. oleracea was characterized by direct expansion caused by whole-genome duplication. By contrast, indirect expansion characterized B. napus, which arose from hybridization and fusion of the two species. In addition, phylogenetic and homology analyses showed that the Brassica NLP gene family has been highly conserved in evolution. Finally, we also identified optimal codons for four studied species. Altogether, through comparative genome analysis methods, we presented compelling evidence that triplication is the main driving force for the NLP TF family's evolution in Chinese cabbage and related Brassica plants, a process evidently highly conserved. This work will help in better understanding the impact of genome-wide duplication on gene families of plants.

Transcriptomics and proteomics revealed the psychrotolerant and antibiotic-resistant mechanisms of strain Pseudomonas psychrophila RNC-1 capable of assimilatory nitrate reduction and aerobic denitrification

Aerobic denitrification has been proved to be profoundly affected by temperature and antibiotics, but little is known about how aerobic denitrifiers respond to temperature and antibiotic stress. In this study, the nitrate reduction performance and the intracellular metabolism by a psychrotolerant aerobic denitrifying bacteria, named Pseudomonas psychrophila RNC-1, were systematically investigated at different temperatures (10 °C, 20 °C, 30 °C) and different sulfamethoxazole (SMX) concentrations (0 mg/L, 0.1 mg/L, 0.5 mg/L, 1.0 mg/L, and 5.0 mg/L). The results showed that strain RNC-1 performed satisfactory nitrate removal at 10 °C and 20 °C, but its growth was significantly inhibited at 30 °C. Nitrate removal by strain RNC-1 was slightly promoted in the presence of 0.5 mg/L SMX, whereas it was significantly suppressed with 5.0 mg/L SMX. Nitrogen balance analysis indicated that assimilatory nitrate reduction and dissimilatory aerobic denitrification jointly dominated in the nitrate removal process of strain RNC-1, in which the inhibition effected on assimilation process was much higher than that on the aerobic denitrification process under SMX exposure. Further transcriptomics and proteomics analysis revealed that the psychrotolerant mechanism of strain RNC-1 could be attributed to the up-regulation of RNA translation, energy metabolism, ABC transporters and the over-expression of cold shock proteins, while the down-regulation of oxidative phosphorylation pathway was the primary reason for the deteriorative cell growth at 30 °C. The promotion of nitrate reduction with 0.5 mg/L SMX was related to the up-regulation of amino acid metabolism pathways, while the down-regulation of folate cycle, glycolysis/gluconeogenesis and bacterial chemotaxis pathways were responsible for the inhibition effect at 5.0 mg/L SMX. This work provides a mechanistic understanding of the metabolic adaption of strain RNC-1 under different stress, which is of significance for its application in nitrogen contaminated wastewater treatment processes.

Seasonal changes in nitrate assimilation of boreal woody species: importance of the leaf-expansion period

Key messageNitrate served as an important nitrogen source for dominant deciduous tree species, especially during their leaf expansion period, even in boreal forests, where nitrate availability was assumed to be low.Temporal changes in leaf nitrate assimilation with leaf growth were intensively investigated in boreal tree species to demonstrate the contribution of nitrate as a N source and to determine temporal changes in the contribution of nitrate during leaf ontogeny. Leaf area, mass, nitrate reductase activity (NRA), N concentration, and δ15N were repeatedly measured in developing leaves of naturally grown Alnus crispa, Betula neoalaskana, and Populus tremuloides during their leaf expansion period. Alnus crispa and B. neoalaskana showed distinct peaks in NRA during leaf expansion, whereas P. tremuloides did not. The highest peak in NRA occurred for A. crispa, whereas it had low NRA during the summer. Peak NRA in B. neoalaskana was lower than that of A. crispa (p < 0.01, ANOVA), although it showed higher NRA during summer (p < 0.01, ANOVA). All species showed clear decrease in N concentration through the leaf expansion period, but total N content per leaf increased. Only the N-fixing species A. crispa showed a rapid change in δ15N during the leaf expansion, and the decline indicated the changes in N source during the leaf development. The results indicate leaves of target species assimilated nitrate during the leaf expansion period, consuming immense energy, although leaves were considered a carbon sink during the early leaf expansion period. We suggest the early onset of leaf growth due to climate warming could influence plant nutrition via asynchrony between supply and demand for energy during spring.

MdMYB10 affects nitrogen uptake and reallocation by regulating the nitrate transporter MdNRT2.4-1 in the red flesh apple.

ABSTRACTNitrate is the major nitrogen source for higher plants. In addition to serving as a nutrient, it is also a signaling molecule that regulates plant growth and development. Although membrane-bound nitrate transporter/peptide transporters (NRT/PTRs) have been extensively studied and shown to regulate nitrate uptake and movement, little is known about how these factors are regulated by the external nitrogen environment. Red-fleshed apple, whose coloration is determined by the transcription factor MdMYB10, had higher nitrate uptake efficiency than non-red-fleshed apple. Nitrate assimilation and utilization were higher in red-fleshed apple cultivars, and comparative transcriptome analysis showed that the expression of genes encoding the NRT2s was increased in red-fleshed apple. In vitro and in vivo experiments showed that MdMYB10 directly bound to the MdNRT2.4-1 promoter to transcriptionally activate its expression, resulting in enhanced nitrate uptake. MdMYB10 also controlled nitrate reallocation from old leaves to new leaves through MdNRT2.4-1. Overall, our findings provide novel insights into the mechanism by which MdMYB10 controls nitrate uptake and reallocation in apple, which facilitates adaptation to a low-nitrogen environment.

Arabidopsis Growth-Promotion and Root Architecture Responses to the Beneficial Rhizobacterium Phyllobacterium brassicacearum Strain STM196 Are Independent of the Nitrate Assimilatory Pathway.

Phyllobacterium brassicacearum STM196, a plant growth-promoting rhizobacterium isolated from roots of oilseed rape, stimulates Arabidopsis growth. We have previously shown that the NRT2.5 and NRT2.6 genes are required for this growth promotion response. Since these genes are members of the NRT2 family of nitrate transporters, the nitrogen assimilatory pathway could be involved in growth promotion by STM196. We address this hypothesis using two nitrate reductase mutants, G5 deleted in the major nitrate reductase gene NIA2 and G′4-3 altered in both NIA1 and NIA2 genes. Both mutants had a reduced growth rate and STM196 failed to increase their biomass production on a medium containing NO3− as the sole nitrogen source. However, they both displayed similar growth promotion by STM196 when grown on an NH4+ medium. STM196 was able to stimulate lateral roots development of the mutants under both nutrition conditions. Altogether, our results indicate that the nitrate assimilatory metabolism is not a primary target of STM196 interaction and is not involved in the root developmental response. The NIA1 transcript level was reduced in the shoots of nrt2.5 and nrt2.6 mutants suggesting a role for this nitrate reductase isoform independently from its role in nitrate assimilation.

Reducing phosphorylation of nitrate reductase improves nitrate assimilation in rice

Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and post-translational phosphorylation regulates NR activity. To evaluate the impact of the dephosphorylation of nitrate reductase 1 (NIA1) protein on NR activity, nitrogen metabolism and plant growth, NIA1 phosphorylation site directed mutant lines (S532D and S532A) and an OsNia1 over-expression line (OE) were constructed, and the phenotype, NIA1 protein and its phosphorylation level, NR activity, nitrate metabolism and reactive oxygen metabolism of the transgenic lines were analysed. Exogenous NIA1 protein was not phosphorylated in S532D and S532A mutant lines, and their NR activities, activity states of NR and assimilation efficiencies of NO3−-N were higher than those in Kitaake (WT) and OE. The changes in these physiological and biochemical indexes in the OE line were less than in S532D and S532A compared to WT. These results suggest that the removal of transcriptional level control had little effect on nitrogen metabolism, but the removal of post-translational modification had a profound effect on it. With the removal of NIA1 phosphorylation and the improvement in the nitrate assimilation efficiency, the plant height and chlorophyll content of S532D and S532A decreased and the hydrogen peroxide and malondialdehyde contents of rice seedlings increased, which may be related to the excessive accumulation of nitrite as an intermediate metabolite. These results indicated that the phosphorylation of NR may be a self-protection mechanism of rice. The reduced phosphorylation level of nitrate reductase improved the assimilation of nitrate, and the increased phosphorylation level reduced the accumulation of nitrite and prevented the toxic effects of reactive oxygen species in rice.

Boosting nitrogen fertilization by a slow releasing nitrate-intercalated biocompatible layered double hydroxide-hydrogel composite loaded with Azospirillum brasilense.

Indiscriminate use of chemical fertilizers leads to soil environmental disbalance and therefore, preparation and application of environment-friendly slow-release multifunctional fertilizers are of paramount importance for sustainable crop production in the present scenario. In this study, we propose a slow-release multifunctional composite nitrogen (N) fertilizer, which possesses the ability to supply plant accessible N in the form of ammonium (NH4+) and nitrate (NO3−) to improve nitrate assimilation coupled with zinc (Zn, a major micronutrient for plants in the soil) after its degradation. For this purpose, NO3−-intercalated zinc–aluminum (Zn–Al) layered double hydroxide (LDH) was synthesized using a co-precipitation protocol. The prepared LDH was added as 25.45% of total polymer weight to a sodium carboxymethyl cellulose/hydroxyethyl cellulose citric acid (NaCMC/HEC-CA) biodegradable hydrogel. A. brasilense, commonly used nitrogen-fixing bacteria in soils, was added to the LDH–hydrogel composite along with LDH alone to augment the availability of NH4+ and NO3−. Adjusting the pH under acidic (pH 5.25) and neutral (pH 7) conditions, the release pattern of NO3− from LDH and the composite was monitored for 30 days at normal temperature. The pH was selected based on the soil analysis data of North East India. The LDH-composite released 90% (w/w) and 85.45% (w/w) of intercalated NO3− at pH 5.25 and 7.00 respectively in 30 days. However, 100% (w/w) and 87% (w/w) of intercalated NO3− at pH 5.25 and 7.00 respectively were released in 30 days when only LDH was applied, which indicated the lower performance of LDH alone in comparison to the LDH-composite for the nitrate holding pattern. The pH of the bacteria-loaded system was observed to be acidic (pH = 5–6) during the study of nitrate assimilation and Zn2+ release. A. brasilense improved nitrate assimilation and increased the NH4+ ion concentration in the studied system. A significant increase in Zn2+ release was observed from day 5 in the presence of A. brasilense in the LDH-composite compared with that in the absence of A. brasilense. In conclusion, the prepared LDH–hydrogel–A. brasilense composite fertilizer system increases the availability of plant accessible N form (both NO3− and NH4+) and can potentially improve soil fertility with the addition of Zn and bacteria to the soil in the extended course.

Root Growth and Nitrate Absorption and Assimilation of Two Sweet Potato Cultivars with Different N Tolerances in Response to Nitrate Supply

Root Growth and Nitrate Absorption and Assimilation of Two Sweet Potato Cultivars with Different N Tolerances in Response to Nitrate Supply

Acclimation to elevated CO2 affects the C/N balance by reducing de novo N-assimilation.

Plants exposed to elevated atmospheric CO2 concentrations show an increased photosynthetic activity. However, after prolonged exposure, the activity declines. This acclimation to elevated CO2 is accompanied by a rise in the carbon-to-nitrogen ratio of the biomass. Hence, increased sugar accumulation and sequential downregulation of photosynthetic genes, as well as nitrogen depletion and reduced protein content, have been hypothesized as the cause of low photosynthetic performance. However, the reason for reduced nitrogen content in plants at high CO2 is unclear. Here, we show that reduced photorespiration at increased CO2 -to-O2 ratio leads to reduced de novo assimilation of nitrate, thus shifting the C/N balance. Metabolic modeling of acclimated and non-acclimated plants revealed the photorespiratory pathway to function as a sink for already assimilated nitrogen during the light period, providing carbon skeletons for de novo assimilation. At high CO2 , low photorespiratory activity resulted in diminished nitrogen assimilation and eventually resulted in reduced carbon assimilation. For the hpr1-1 mutant, defective in reduction of hydroxy-pyruvate, metabolic simulations show that turnover of photorespiratory metabolites is expanded into the night. Comparison of simulations for hpr1-1 with those for the wild type allowed investigating the effect of a perturbed photorespiration on N-assimilation.

Physiological and Transcriptomic Characterization of Sea-Wheatgrass-Derived Waterlogging Tolerance in Wheat.

Waterlogging, causing hypoxia stress and nitrogen depletion in the rhizosphere, has been an increasing threat to wheat production. We developed a wheat–sea wheatgrass (SWG) amphiploid showing superior tolerance to waterlogging and low nitrogen. Validated in deoxygenated agar medium for three weeks, hypoxia stress reduced the dry matter of the wheat parent by 40% but had little effect on the growth of the amphiploid. To understand the underlying mechanisms, we comparatively analyzed the wheat–SWG amphiploid and its wheat parent grown in aerated and hypoxic solutions for physiological traits and root transcriptomes. Compared with its wheat parent, the amphiploid showed less magnitude in forming root porosity and barrier to radial oxygen loss, two important mechanisms for internal O2 movement to the apex, and downregulation of genes for ethylene, lignin, and reactive oxygen species. In another aspect, however, hypoxia stress upregulated the nitrate assimilation/reduction pathway in amphiploid and induced accumulation of nitric oxide, a byproduct of nitrate reduction, in its root tips, and the amphiploid maintained much higher metabolic activity in its root system compared with its wheat parent. Taken together, our research suggested that enhanced nitrate assimilation and reduction and accumulation of nitric oxide play important roles in the SWG-derived waterlogging tolerance.

Certain Environmental Conditions Maximize Ammonium Accumulation and Minimize Nitrogen Loss During Nitrate Reduction Process by Pseudomonas putida Y-9.

Realizing the smallest nitrogen loss is a challenge in the nitrate reduction process. Dissimilatory nitrate reduction to ammonium (DNRA) and nitrate assimilation play crucial roles in nitrogen retention. In this study, the effects of the carbon source, C/N ratio, pH, and dissolved oxygen on the multiple nitrate reduction pathways conducted by Pseudomonas putida Y-9 are explored. Strain Y-9 efficiently removed nitrate (up to 89.79%) with glucose as the sole carbon source, and the nitrogen loss in this system was 15.43%. The total nitrogen decrease and ammonium accumulation at a C/N ratio of 9 were lower than that at 12 and higher than that at 15, respectively (P < 0.05). Besides, neutral and alkaline conditions (pH 7–9) favored nitrate reduction. Largest nitrate removal (81.78%) and minimum nitrogen loss (10.63%) were observed at pH 7. The nitrate removal and ammonium production efficiencies of strain Y-9 increased due to an increased shaking speed. The expression patterns of nirBD (the gene that controls nitrate assimilation and DNRA) in strain Y-9 were similar to ammonium patterns of the tested incubation conditions. In summary, the following conditions facilitated nitrate assimilation and DNRA by strain Y-9, while reducing the denitrification: glucose as the carbon source, a C/N ratio of 9, a pH of 7, and a shaking speed of 150 rpm. Under these conditions, nitrate removal was substantial, and nitrogen loss from the system was minimal.

Role of protein phosphatases in the regulation of nitrogen nutrition in plants

The reversible protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases regulate different biological processes and their response to environmental cues, including nitrogen (N) availability. Nitrate assimilation is under the strict control of phosphorylation-dephosphorylation mediated post-translational regulation. The protein phosphatase family with approximately 150 members in Arabidopsis and around 130 members in rice is a promising player in N uptake and assimilation pathways. Protein phosphatase 2A (PP2A) enhances the activation of nitrate reductase (NR) by deactivating SnRK1 and reduces the binding of inhibitory 14-3-3proteins on NR. The functioning of nitrate transporter NPF6.3 is regulatedby phosphorylation of CBL9 (Calcineurin B like protein 9) and CIPK23 (CBL interacting protein kinase 23) module. Phosphorylation by CIPK23 inhibits theactivity of NPF6.3, whereas protein phosphatases (PP2C) enhance the NPF6.3-dependentnitrate sensing. PP2Cs and CIPK23 also regulate ammonium transporters (AMTs). Under either moderate ammonium supply or high N demand, CIPK23 is bound and inactivated by PP2Cs. Ammoniumuptake is mediated by nonphosphorylated and active AMT1s. Whereas, under high ammonium availability, CIPK23 gets activated and phosphorylate AMT1;1 and AMT1;2 rendering them inactive. Recent reports suggest the critical role of protein phosphatases in regulating N use efficiency (NUE). In rice, PP2C9 regulates NUE by improving N uptake and assimilation. Comparative leaf proteome of wild type and PP2C9 over-expressing transgenic rice lines showed 30 differentially expressed proteins under low N level. These proteins are involved in photosynthesis, N metabolism, signalling, and defence.

Genetic Inactivation of D-Amino Acid Oxidase Genes in Methylotrophic Yeast Hansenula Polymorpha

Cellular D-amino acid oxidases (ODA-FAD containing flavoproteins) are widely used in biotechnology. In this regard, it is of particular interest to investigate the functional role of the composition and regulation of individual ODA genes. The aim of this work was to investigate physiological and biochemical characteristics of ODA genes of strain DL1x Hansenula (O.) polymorpha in vivo using gene knockout methodology as well as to determine the nature of these genes expression and regulation of ODA activity as a function of nitrogen and carbon source composition in the cultivation medium. H. polymorpha is a thermotolerant methylotrophic yeast. They are used to study the mechanisms of peroxisomal biogenesis and degradation, regulation of methanol metabolism, nitrate assimilation and stress response. A serial dilution method has been used for rapid assessment of strain growth and stress tolerance. Vector design for genetic inactivation of ODA genes in H. polymorpha was performed using yeast vector pAM773. Selection of "knockout" cell clones was performed using PCR analysis. To complete the transformation process, H. polymorpha was deleted from the pAM773 vector and the obtained DNA was used in the experiment. In vitro cultivation of knockout strains of H. polymorpha was found to exhibit substratespecificity of the ODA. According to the authors, the HP2914 gene is important for D-alanine oxidation, while the gene complex 2400 and 2914 is important for ODA activation in the presence of D-Phe in the medium. It is likely that the gene complex 2082 and 2165 regulates ODA activation when cells are cultured in medium with D-Ala and D-Asp. It was found that in the absence of 2165, 2400, 2914 genes, increased ODA activity to D-Ala only was observed in cell culture medium. The presence of D-alanine combined with 1 % glycerol and 1 % methanol in the culture medium stimulated the activity of the three major ODAs of H. polymorpha through the expression of the HP2914 gene, while the presence of glucose and L-alanine in the culture medium suppressed their activity