NR Expression Research Articles

Nitric Oxide Modulates Glycine Max L. Growth and Physio-Molecular Responses during Flooding Stress

Long-term exposure to flooding creates hypoxic conditions, affecting plant growth and development. Nitric oxide is a stress signaling and evading molecule involved in many physiological and biochemical response in plants stress tolerance, however, its potential role in response to flooding stress is not fully understood. We illuminate the role of NO in regulation of stress-related biochemical and genetic influence in soybean plants after 3, and 7days of flooding. Exogenous nitric oxide donor (SNP) application improve plants growth and development, and chlorophyll content, which may correlate to increase in the antioxidant enzyme activity such as peroxidase (POD), catalase (CAT), superoxide dismutase [1] and reduced glutathione (GSH), and protect plants from oxidative damage through scavenge the H2O2 accumulation, reduced the MDA level and increased the proline accumulation and further improved the photosynthesis and stomata conductance. Furthermore SNP treatments reduced the ABA content and down regulate the relative expression of ABAreceptors ABAR1, ABAR2 and ABA biosynthesis gene NCED3 after 3, and 7days of flooding stress. In the case of endogenous NO signaling, GSNOR and NR expression was enhanced by SNP treatments and improve the cellular SNO level, showing a positive effects on flooding stress tolerance. These positive effects of NO on stress tolerance were completely reversed by NO scavenger cPTIO, and NO inhibitor L-NAME treatments by enhance the ABA accumulation, relative expression of ABAR1, ABAR2 and NCED3, and cause reduction in antioxidant activity and increase the H2O2 content and MDA level. NO treatments improved plant tolerance to flooding stress and improved various biochemical and transcriptional programs that are ameliorative to plant growth during long-term flooding stress. The potential benefit of SNP or related NO sources during flooding can be attributed to its antagonistic effects on ABA biosynthesis, which in turn regulated flooding tolerance.

Effect of LED Spectrum on the Quality and Nitrogen Metabolism of Lettuce Under Recycled Hydroponics.

Light quality optimization is an efficient method for improving the growth and quality of lettuce in plant factories. In this study, lettuce seedlings were illuminated under different light-emitting diode (LED) lights, namely, red-blue (RB), red-blue-green (RBG), red-blue-purple (RBP), and red-blue-far-red (RBF) LED lights, to investigate the effect of light quality on growth, quality, and nitrogen metabolism. The combination of 75% red and 25% blue light was set as the basic light source, and 20% of green, purple and far-red light were added to basic light source, respectively. All the treatments were set to 200 μmol m–2 s–1. Results showed that the fresh weight and dry weight of aboveground lettuce under RBG, RBP, and RBF treatments were significantly lower than those under the RB treatment because of the decrease in the effective photon flux density for chlorophyll absorption. The vitamin C content of the lettuce leaves was increased by about 23% with the addition of purple light. For nitrate reduction, the addition of green light significantly increased the nitrite content of the lettuce leaves. It also promoted the reduction from nitrite to ammonium through the activation of the nitrite reductase (NiR) expression and enzyme activity. The nitrate and ammonium content decreased with the addition of purple light because of the inhibited NR and NiR expression and enzyme activity. For nitrogen assimilation, individual (e.g., Asp, Glu, and Leu) and total amino acids were induced to increase by adding green, purple, and far-red light. The addition of light was hypothesized to have inhibited protein biosynthesis, thereby causing the accumulation of amino acids. Correlation analysis showed that the relative expression levels between HY5 and NR/NiR presented a significantly negative correlation. Transcription factor HY5 might mediate the regulation of light quality on nitrogen metabolism by inhibiting NR and NiR expressions. It might also exert a negative effect on nitrate reduction. Further studies via genome editing techniques on the identification of HY5 functions for nitrate assimilation will be valuable. Nevertheless, the results of this work enrich the understanding of the effect of light quality on nitrate metabolism at the level of gene expression and enzyme activity.

High-Dimensional Data Approaches to Understanding Nuclear Hormone Receptor Signaling.

Bioinformatics applies unbiased approaches to develop statistically robust insight into health and disease. At the global, or "20,000ft" view bioinformatic analyses of NR signaling can measure how the NRs are implicated in human health and disease through the impact of genome-wide significant genetic variation, family-wide NR expression patterns or considering where NRs are significantly identified in other high-dimensional data analyses. With a more NR-centric, or "2000ft" view, bioinformatic approaches can interrogate events downstream of a given NR. Integrative approaches aim to combine multiple NR-centric high-dimensional data both derived in cell models and primary human tissue to reveal how NR-transcriptional networks relate to human health and disease. Bioinformatic approaches to such high-dimensional data are central and require specialist statistical insight and computational skills, coupled with a dexterous understanding of the biological question. A current challenge is determining the optimal mechanism to share such bioinformatic approaches through the biological research community.

High expression of orphan nuclear receptor NR4A1 in a subset of ovarian tumors with worse outcome

ObjectiveNuclear receptors (NRs) play a vital role in the development and progression of several cancers including breast and prostate. Using TCGA data, we sought to identify critical nuclear receptors in high grade serous ovarian cancers (HGSOC) and to confirm these findings using in vitro approaches. MethodsIn silico analysis of TCGA data was performed to identify relevant NRs in HGSOC. Ovarian cancer cell lines were screened for NR expression and functional studies were performed to determine the significance of these NRs in ovarian cancers. NR expression was analyzed in ovarian cancer tissue samples using immunohistochemistry to identify correlations with histology and stage of disease. ResultsThe NR4A family of NRs was identified as a potential driver of ovarian cancer pathogenesis. Overexpression of NR4A1 in particular correlated with worse progression free survival. Endogenous expression of NR4A1 in normal ovarian samples was relatively high compared to that of other tissue types, suggesting a unique role for this orphan receptor in the ovary. Expression of NR4A1 in HGSOC cell lines as well as in patient samples was variable. NR4A1 primarily localized to the nucleus in normal ovarian tissue while co-localization within the cytoplasm and nucleus was noted in ovarian cancer cell lines and patient tissues. ConclusionsNR4A1 is highly expressed in a subset of HGSOC samples from patients that have a worse progression free survival. Studies to target NR4A1 for therapeutic intervention should include HGSOC.

Adaptation involved in nitrogen metabolism in sea ice alga Chlamydomonas sp. ICE-L to Antarctic extreme environments

There are several well-described acclimation mechanisms in sea ice-based algae, but little is known about their primary nitrogen metabolism. This study examined nitrogen uptake, nitrate reductase gene (nr) expression, and nitrate reductase activity (NRA) in an Antarctic microalga Chlamydomonas sp. ICE-L relative to altered photoperiod, nitrogen source, and salinity. Both nr expression and NRA varied diurnally over two 24-h light/dark cycles. nr expression reached maximum levels at the end of the light cycle and decreased during the dark period, and the peak in NRA occurred 12 h later than the peak in nr expression. After transfer to continuous light (LL) or continuous dark (DD), this periodicity was abolished, and both parameters were regulated by light availability. Chlamydomonas sp. ICE-L could assimilate both NO3–N and NH4–N as nitrogen sources, but the alga preferred NH4–N when both were present in culture media at the same concentration. The presence of NH4 + suppressed nr expression and NRA. Consistent with the regulation of nr expression, the highest NRA was also observed in cells grown in media supplemented with KNO3. Salinity significantly (P <0.05) affected nr expression and NRA. Both of these parameters were down-regulated with an increase in salinity from 32 to 160 ‰.

Effects of Dizocilpine (MK-801) via Up-modulation of N-methyl-D-aspartate (NMDA) Receptors on Hypoxic-Ischemic Brain Injury in Neonatal Rats

Objective : Several studies have demonstrated the neuroprotective effects of (+)-MK-801 hydrogen maleate (dizocilpine), in various animal models of hypoxic-ischemic (HI) brain injury. However limited data are available on the neonatal model of HI brain injury. The aim of the present study was to investigate the effects of dizocilpine and its mechanisms associated with NMDARs expression in neonatal rat model of HI brain injury. Methods : In in vivo model, 7d-old rat pups underwent permanent unilateral carotid ligation. The animals were divided into six groups: N, normoxia; H, hypoxia without operation; HS, hypoxia with Sham operation; HO, hypoxia with operation; HV, HO treated with vehicle; HD, HO treated with dizocilpine. Dizocilpine (10 mg/kg) was administered intracerebrally to the rats 30 min before HI brain injury. Rat pups were exposed to hypoxia by placing them for 2 hours in hypoxic incubator (92% N2, 8% O2). In in vitro model, embryonic cortical neuronal cell cultures (from SD rats of embryonic days of 18) were done. The normoxia (N) group was prepared in 5% CO2 incubators. The hypoxia (H), and hypoxia treated with dizocilpine (HD) groups were placed in 1% O2 incubators (94% N2, 5% CO2) for 16 hours. In order to estimation of cell viability and growth, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was done. The degree of neuronal death was evaluated by morphometric method and the protein expression of each NMDARs was quantified by Real Time-PCR and Western blot. Results : Both in the in vitro and in vivo models, the expressions of NMDAR subunits were lower in the hypoxia group than in the normoxia group, whereas they increased in the hypoxia treated with dizocilpine group compared to the hypoxia group. In vitro model, however, the expressions of NR1, NR2A mRNAs decreased in the H group when compared to the N group, whereas they increased a little in the HD group when compared to the H group. Conclusion : Dizocilpine was modulated the degeneration of neuronal cell death in neonatal rat model of HI by preservation of NR expression.

Abstract 3558: Single cell-based image analysis of nuclear receptor levels and activity in breast cancer cells reveals heterogeneous subpopulations: Development of biological response fingerprints for ligands and endocrine disruptors.

Abstract Modern microscopy- and flow cytometry-based analyses indicate tumor cell populations can be markedly heterogeneous. Yet, the vast majority of approaches used to investigate tumorigenic mechanisms and therapeutic responses are based upon average single-point analysis of bulk populations of cells, where subpopulation responses are undetectable and therefore unexplored. For example, breast cancers are generally classified by IHC as either estrogen receptor-alpha positive (ER+) or negative (ER-); however, in ER+ tumors, there is a broad range of ER levels in individual cells. Despite a growing awareness of the importance of tumor cell heterogeneity and outlier biology in cancer therapeutics, there is a paucity of data on the potential interplay between multiple nuclear receptors and coregulators, their heterogeneity of expression and functions, and their combinatorial role in determining therapeutic response, or in sensing environmental stressors (e.g., endocrine disruptors). To specifically quantify biological responses on an individual cell basis rather than averaged responses from bulk populations, we performed single cell-based high content analysis (HCA) to examine MCF-7 breast cancer cells in terms of simultaneous expression of several type-1 nuclear receptors (e.g., Estrogen, Androgen and Glucocorticoid receptors - ER, AR and GR) and coregulators (e.g., SRC-2 and SRC-3). Quantitative immunofluorescence reveals a wide range of heterogeneity in the expression and ratios of ER, AR, and GR. Multi-parametric analyses describing NR expression and spatial distribution were used to evaluate ligand dose responses and time pattern of the three nuclear receptors, simultaneously. Next, we are expanding our analysis to endogenous target gene expression via mRNA fluorescence in situ hybridization (RNA FISH) and to cell cycle marker analysis to link heterogeneity of NRs/coregulators expression with transcriptional output and cell cycle phases. We are also implementing robust analysis pipelines to describe and quantify NRs and coregulators patterns of phenotypic heterogeneity to create “biological response fingerprints,” which we are using to classify the effects of individual or mixed ligand treatments with particular focus on chemotherapeutic drugs and endocrine disruptors. We are also starting to expand our HCA approach to other breast cancer cell lines moving towards primary tumors microarrays analysis to evaluate the prognostic/diagnostic significance of NR heterogeneity. Collectively, these high throughput-amenable approaches are poised to greatly aid in the understanding the role of heterogeneity in breast cancer, and serve as a robust model for next-generation drug screening and endocrine disruptor assays moving toward personalized medicine. Citation Format: Fabio Stossi, Alexander Renwick, Radhika D. Dandekar, Justin Y. Newberg, Arvind Rao, Michael A. Mancini. Single cell-based image analysis of nuclear receptor levels and activity in breast cancer cells reveals heterogeneous subpopulations: Development of biological response fingerprints for ligands and endocrine disruptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2013-3558

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

FC29-03 - Effects of chronic oral treatment with aripiprazole on expression of nmda receptor subunits and binding sites in rat brain

IntroductionThe glutamatergic theory of schizophrenia proposes a dysfunction of ionotropic N-Methyl-D-aspartate (NMDA)-receptors (NR). Several therapeutic strategies address NR function and effects of antipsychotic agents on NR expression have been described.ObjectivesThe partial dopaminergic and serotonergic agonist aripiprazole (APZ) was able to counteract behavioural effects of NR antagonists, but effects of APZ on NR expression have not been investigated.AimsTo evaluate effects of APZ on NR mRNA and protein expressionMethodsWe treated Sprague-Dawley rats for 4 weeks or 4 months with APZ in daily oral doses of 10 and 40 mg per kg body weight. Expression of the NR subunits NR1, NR2A, NR2B, NR2C and NR2D was assessed by semiquantitative radioactive in situ-hybridization, and in parallel receptor binding using 3H-MK-801 receptor autoradiography.ResultsIncreased expression levels of NR1 (4 weeks), NR2A (4 weeks), NR2C (4 weeks and 4 months) and NR2D (4 months) were observed in several hippocampal and cortical brain regions. The parallel reduced expression of NR2B mRNAs (4 months) resulted in a relative increase of the NR2A/NR2B ratio. Marked differences between specific brain regions, the doses and time points of assessment became obvious. On the receptor level, increased MK-801-binding was found after 4 weeks in the 40 mg-group and after 4 months in the 10 mg-group.ConclusionsThe effects of APZ converge in enhanced NMDA-receptor expression and a shift of subunit-composition towards adult-type receptors. Our results confirm regulatory connections between dopaminergic, serotonergic and glutamatergic neurotransmission with relevance for cognitive and negative symptoms of schizophrenia.

Nuclear receptors from the ctenophore Mnemiopsis leidyi lack a zinc-finger DNA-binding domain: lineage-specific loss or ancestral condition in the emergence of the nuclear receptor superfamily?

BackgroundNuclear receptors (NRs) are an ancient superfamily of metazoan transcription factors that play critical roles in regulation of reproduction, development, and energetic homeostasis. Although the evolutionary relationships among NRs are well-described in two prominent clades of animals (deuterostomes and protostomes), comparatively little information has been reported on the diversity of NRs in early diverging metazoans. Here, we identified NRs from the phylum Ctenophora and used a phylogenomic approach to explore the emergence of the NR superfamily in the animal kingdom. In addition, to gain insight into conserved or novel functions, we examined NR expression during ctenophore development.ResultsWe report the first described NRs from the phylum Ctenophora: two from Mnemiopsis leidyi and one from Pleurobrachia pileus. All ctenophore NRs contained a ligand-binding domain and grouped with NRs from the subfamily NR2A (HNF4). Surprisingly, all the ctenophore NRs lacked the highly conserved DNA-binding domain (DBD). NRs from Mnemiopsis were expressed in different regions of developing ctenophores. One was broadly expressed in the endoderm during gastrulation. The second was initially expressed in the ectoderm during gastrulation, in regions corresponding to the future tentacles; subsequent expression was restricted to the apical organ. Phylogenetic analyses of NRs from ctenophores, sponges, cnidarians, and a placozoan support the hypothesis that expansion of the superfamily occurred in a step-wise fashion, with initial radiations in NR family 2, followed by representatives of NR families 3, 6, and 1/4 originating prior to the appearance of the bilaterian ancestor.ConclusionsOur study provides the first description of NRs from ctenophores, including the full complement from Mnemiopsis. Ctenophores have the least diverse NR complement of any animal phylum with representatives that cluster with only one subfamily (NR2A). Ctenophores and sponges have a similarly restricted NR complement supporting the hypothesis that the original NR was HNF4-like and that these lineages are the first two branches from the animal tree. The absence of a zinc-finger DNA-binding domain in the two ctenophore species suggests two hypotheses: this domain may have been secondarily lost within the ctenophore lineage or, if ctenophores are the first branch off the animal tree, the original NR may have lacked the canonical DBD. Phylogenomic analyses and categorization of NRs from all four early diverging animal phyla compared with the complement from bilaterians suggest the rate of NR diversification prior to the cnidarian-bilaterian split was relatively modest, with independent radiations of several NR subfamilies within the cnidarian lineage.

Thermoperiod affects the diurnal cycle of nitrate reductase expression and activity in pineapple plants by modulating the endogenous levels of cytokinins

Nitrate reductase (NR, EC 1.6.6.1) activity in higher plants is regulated by a variety of environmental factors and oscillates with a characteristic diurnal rhythm. In this study, we have demonstrated that the diurnal cycle of NR expression and activity in pineapple (Ananas comosus, cv. Smooth Cayenne) can be strongly modified by changes in the day/night temperature regime. Plants grown under constant temperature (28 degrees C light/dark) showed a marked increase in the shoot NR activity (NRA) during the first half of the light period, whereas under thermoperiodic conditions (28 degrees C light/15 degrees C dark) significant elevations in the NRA were detected only in the root tissues at night. Under both conditions, increases in NR transcript levels occurred synchronically about 4 h prior to the corresponding elevation of the NRA. Diurnal analysis of endogenous cytokinins indicated that transitory increases in the levels of zeatin, zeatin riboside and isopentenyladenine riboside coincided with the accumulation of NR transcripts and preceded the rise of NRA in the shoot during the day and in the root at night, suggesting these hormones as mediators of the temperature-induced modifications of the NR cycle. Moreover, these cytokinins also induced NRA in pineapple when applied exogenously. Altogether, these results provide evidence that thermoperiodism can modify the diurnal cycle of NR expression and activity in pineapple both temporally and spatially, possibly by modulating the day/night changes in the cytokinin levels. A potential relationship between the day/night NR cycle and the photosynthetic pathway performed by the pineapple plants (C(3) or CAM) is also discussed.

Lipoteichoic acid modulates hepatic drug metabolizing enzymes by activating the Toll‐like receptor 2 signaling pathway

Background:Altered expression of drug metabolizing enzymes (DMEs) in inflammation can affect the metabolism and toxicity of drugs. It is known that inflammatory responses are induced in immune cells by Lipopolysaccahride (LPS) and Lipoteichoic acid (LTA), derived from gram −ve and gram +ve bacteria, respectively. We reported that LPS modulates gene expression of hepatic DMEs and their regulatory nuclear receptors (NRs: PXR, CAR and RXRα) by activating Toll‐like receptor (TLR) 4 signaling pathway. We hypothesize that LTA can modulate NR and DME gene expression by activating the TLR2 signaling pathway. Methods: Adult male C57BL/6 mice were i.p. injected with 6 μg/g bw LTA or saline. Livers were harvested and RNA and proteins were analyzed by real‐time PCR and western blotting, respectively.Results:LTA treatment for 2h resulted in ∼80% suppression of CAR and target DME genes, Cyp2b10, Cyp2a4 and Sultn. The NR, PXR, and the main phase 1 DME, Cyp3a11 were reduced ∼70% by 4–8 h of LTA treatment. Nuclear protein levels of RXRα were reduced in LTA‐treated mice. LTA injection induced cytokine production and cell‐signaling components in the liver. The effects of LTA on NRs, DMEs, cytokines and cell‐signaling pathways were blocked in TLR2−/− mice, indicating that the effects of LTA on hepatic genes were mediated by TLR2.Conclusions:TLR2 is involved in regulating DME gene expression during induction of inflammation by LTA.

HY5 and HYH are positive regulators of nitrate reductase in seedlings and rosette stage plants

HY5 and HYH are bZIP transcription factors well known to be involved in photomorphogenesis and light signalling. Loss-of-function mutants of HY5 and HYH revealed that these genes are essential for induction of a key enzyme in nitrogen assimilation, nitrate reductase (EC 1.7.1.1). In Arabidopsis thaliana seedlings nitrate reductase was expressed under low irradiance far-red or red light at the same level as under higher irradiance, photosynthetic active, white light. However, high NR expression at low light levels occurred only in the presence of sucrose in the growth medium. Sucrose did not promote expression in darkness. Whereas HY5 was necessary for high nitrate reductase expression in far-red light, HYH was important in red light. COP1 is known to promote degradation of HY5 and HYH, and in the cop1 mutant, nitrate reductase activity was relatively high also in darkness. PhyA and PhyB mutants were tested, and confirmed the phytochrome dependency for far-red and red light induction of nitrate reductase in seedlings. In rosette leaves of 3-week-old green plants the daily increase in nitrate reductase expression in response to light-on was abolished in the hyh and hy5 hyh double mutant. The hy5 hyh double mutant had lower nitrate reductase activity than any of the single mutants in photosynthetic active light in both seedlings and rosette leaves.

Impact of atmospheric CO 2 on growth, photosynthesis and nitrogen metabolism in cucumber ( Cucumis sativus L.) plants

Expression and activity of nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2) were analysed in relation to the rate of CO(2) assimilation in cucumber (Cucumis sativus L.) leaves. Intact plants were exposed to different atmospheric CO(2) concentrations (100, 400 and 1200microLL(-1)) for 14 days. A correlation between the in vivo rates of net CO(2) assimilation and the atmospheric CO(2) concentrations was observed. Transpiration rate and stomatal conductance remained unaffected by CO(2) levels. The exposure of the cucumber plants to rising CO(2) concentrations led to a concomitant increase in the contents of starch and soluble sugars, and a decrease in the nitrate content in leaves. At very low CO(2), NR and GS expression decreased, in spite of high nitrate contents, whereas at normal and elevated CO(2) expression and activity were high although the nitrate content was very low. Thus, in cucumber, NR and GS expression appear to be dominated by sugar levels, rather than by nitrate contents.

Effect of Light Quality on the Circadian Expression of Nitrate Reductase in the Red Macroalga Gracilaria tenuistipitata

A circadian rhythm in the activity of nitrate reductase (NR: EC 1.6.6.1) isolated from the marine red algae Gracilaria tenuistipitata is shown to be attributable to the daily oscillation of protein levels. The experiments reported here indicate that light quality has differential effects over NR expression. In extracts of algae grown under white light : dark, red light : dark and blue light : dark cycle, the activity of NR peaks during photophase, as does photosynthesis. Staining with a monoclonal antibody (NR10), raised against NR purified from Porphyra yezoensis, shows that the amount of protein changes by a factor of about 20, with a maximum occurring during photophase when algae are submitted to white and blue light. Red light changes the circadian rhythm of NR protein levels and also inhibits its night degradation. Illumination with blue light is able to restore the NR activity as well as its protein levels only when the light irradiance was the same of the white light. Surprisingly, the red light promoted 40% induction on NR activity under the same conditions.

Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers.

Twenty transformed Solanum tuberosum plants issued from five different varieties and carrying a chimeric tobacco nitrate reductase gene (a truncated tobacco Nia2 coding sequence fused to the CaMV 35S promoter) were cultivated in field conditions at INRA Ploudaniel in 1999 and 2000. In 60% of the transgenic plants, the presence of the tobacco Nia2 transcript was detected by RT-PCR. These clones exhibited a drastic decrease in the nitrate content in tubers. Indeed the nitrate content decreased by about 95% in the tubers of transformed plants compared to nontransformed potato plants from the same variety. This decrease was correlated with a modified regulation of NR expression as revealed by a higher chlorate sensitivity of these transgenic lines. Two methods of nitrate content determination in tubers were also compared and were found to give similar results.

Differential expression of glutamate and GABA-A receptor subunit mRNA in cortical dysplasia

Focal cortical dysplasia is characterized by disorganized cortical lamination, dysplastic and heterotopic neurons, and an association with epilepsy. The contribution that dysplastic and heterotopic neurons make to epileptogenesis in focal cortical dysplasia is unknown and the phenotype of these cells may be distinct. The authors hypothesized that the expression of genes encoding glutamatergic (glutamate [GluR] and N-methyl-D-aspartate NMDA receptors [NR]) and gamma-aminobutyric acid A receptor (GABA(A)R) subunits is distinct in dysplastic and heterotopic neurons and that changes in receptor gene expression could be defined in a cell-specific pattern. Single immunohistochemically labeled dysplastic and heterotopic neurons were microdissected from human focal cortical dysplasia specimens obtained during epilepsy surgery. Pyramidal neurons were microdissected from postmortem control cortex and from temporal cortex without dysplasia resected during temporal lobectomy. Poly (A) messenger RNA (mRNA) from single neurons was amplified, radiolabeled, and used to probe complementary DNA (cDNA) arrays containing GluR(1-6), NR(1A,1B), NR(2A-D), and GABA(A)Ralpha(1-6), and -Rbeta(1-3) subunit cDNAS: The relative hybridization intensities of each mRNA-cDNA hybrid were quantified by phosphorimaging. GluR, NR, and GABA(A)R subunit mRNA expression did not differ between control neurons and nondysplastic epilepsy specimens. Expression of GluR(4), NR(2B), and NR(2C) subunit mRNA was increased, and NR(2A) and GABA(A)Rbeta(1) subunit mRNA was decreased in dysplastic compared with pyramidal and heterotopic neurons. In contrast, GABA(A)Ralpha(1), -Ralpha(2), and -Rbeta(2) as well as GluR(1) mRNA levels were reduced in both dysplastic and heterotopic neurons. Differential expression of GluR, NR, and GABA(A)R mRNA in dysplastic and heterotopic neurons demonstrates cell specific gene transcription changes in focal cortical dysplasia. These results suggest that dysplastic and heterotopic neurons may be pharmacologically distinct and make differential contributions epileptogenesis in focal cortical dysplasia.

A short-term exposure of cucumber plants to rising atmospheric CO2 increases leaf carbohydrate content and enhances nitrate reductase expression and activity.

Nitrate reductase (NR; EC 1.6.6.1) is the first enzyme of the nitrate-assimilatory pathway and is regulated transcriptionally and post-translationally by several metabolic and environmental signals. To investigate whether NR is controlled by the rate of photosynthetic CO2 assimilation in cucumber (Cucumis sastivus L.), intact plants were exposed, after the dark period, to light under different atmospheric CO2 concentrations (100, 400 and 2,000 microL x L(-1)) for 2 h. The in-vivo rates of net CO2 assimilation correlated with atmospheric CO2 concentrations. The CO2-fixation rate under 2,000 microL x L(-1) CO2 was 2.4- and 5.4-fold higher than under 400 and 100 microL x L(-1), respectively. Stomatal conductances and transpiration rates were almost identical after the 2-h light period under the various CO2 concentrations tested. Increasing atmospheric CO2 concentrations caused concomitant increases in the contents of starch and soluble sugars in the leaves and a decrease in the nitrate content. The activity and activation state of NR were both higher under elevated CO2 than under low CO2. High CO2 also enhanced NR-gene expression in the leaves. Sugars were supplied via roots to intact carbohydrate-starved plants and NR mRNA levels were analysed after 7 h. Fructose markedly stimulated NR-gene transcription in both leaves and roots. It is concluded that, in cucumber plants, the rate of CO2 assimilation controls the rate of nitrate assimilation by modulation of NR expression and activity, and that sugars are presumably involved as regulatory metabolites.

Integration of photosynthetic carbon and nitrogen metabolism in higher plants

Concomitant assimilation of C and N in illuminated leaves requires the regulated partitioning of reductant and photosynthate to sustain the demands of amino acid and carbohydrate biosynthesis. The short-term responses of photosynthesis and photosynthate partitioning to N enrichment in wheat (Triticum aestivum, L.) and maize (Zea mays L.) leaves were studied in order to understand the regulatory strategy employed in higher plants. Transgenic tobacco plants (Tobacco plumbaginifolia) over-expressing NR or with poor NR expression were used to compare plants differing in their capacities for NO3 (-) assimilation. Similar regulatory responses to NO3 (-) were observed in leaves having C4- and C3-type photosynthesis. It was shown that the extra- C needed in the short-term to sustain amino acid synthesis was not provided by an increase in photosynthetic CO2 fixation but rather by a rapid shift in the partitioning of photosynthetic C to amino acid at the expense of sucrose biosynthesis. The modulation of three enzymes was shown to be important in this C and N interaction, namely PEPCase (EC 4.1.1.31), SPS (EC 2.4.1.14) and NADH/NR (EC 1.6.6.1). The first two enzymes were shown to share the common feature of regulatory post-transcriptional NO3 (-)-dependent phosphorylation of their proteins on a seryl-residue. While PEPCase is activated, SPS activity is decreased. In contrast the NR phosphorylation state is unchanged and all N-dependent control of NR activity is regulated at the protein level. A number of arguments support the hypothesis that Gln, the primary product of NO3 (-) assimilation, is the metabolite effector for short-term modulation of PEPCase, and SPS in response to N enrichment. Since a major effect of NO3 (-) on the PEPCase-protein kinase activity in concentrated wheat leaf extracts was demonstrated, the hypothesis is put forward that protein phosphorylation is the primary event allowing the short-term adaptation of leaf C metabolism to changes in N supply.

Control of Nitrate Reductase and Nitrite Reductase Gene Expression by Light, Nitrate and a Plastidic Factor

AbstractIn the present review we consider regulation of the build‐up of the nitrate reducing apparatus (NO−3 → NH+4) in the course of light‐mediated plastidogenesis. In particular, the regulation of the appearance of nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) will be treated. While both enzymes are nuclear‐encoded, NR is a cytosolic and NIR a plastidic protein. Substrate induction by nitrate, control by light (phytochrome) and dependence on a ‘plastidic factor’ are the main features of the control system.In mustard (Sinapis alba L.) seedling cotyledons the NR‐mRNA level was determined by a strong synergistic coaction of nitrate and light. Synthesis of NR (a cytosolic protein with two isoforms) correlated with the level of NR mRNA. A positive control of NR gene expression by the plastidic factor was observed. It appeared that the plant cell regulates NR gene expression as if NR would be a plastidic protein.Control of gene expression in the case of plastidic NIR in the same mustard cotyledons was conspicuously different. Whereas, in case of NR, the transcript level was determined by a synergism between light and nitrate, in the case of NIR the transcript level was controlled by light alone, with nitrate being ineffective. However, in both cases actual enzyme synthesis was controlled by nitrate, and the dependence on positive control by the plastidic factor was the same. Thus, the difference between NR and NIR gene expression appears to be a matter of transcriptional control. From a teleonomic viewpoint, the control system to establish the nitrate‐reducing apparatus appears (almost) perfect.In spinach (Spinacia oleracea L.) seedling cotyledons the NIR transcript level was unaffected by light but determined by nitrate. The strong action of light on NIR synthesis was multiplicatively superimposed on the action of nitrate, consistent with the conclusion that nitrate affects the transcript level while light controls enzyme synthesis.In tobacco (Nicotiana tabacum L., cv. Coker 176) seedlings the NIR transcript level was determined by a synergistic action between nitrate and light, i.e. both factors coact at the level of transcription. There was no effect of light on NIR synthesis in the absence of nitrate while a strong action of light on protein synthesis was observed in the presence of nitrate. The specific effect of blue light on enzyme appearance, i.e. an effect which cannot be attributed to phytochrome, was not seen at the transcript level.Thus, in tobacco a coaction of nitrate and light (phytochrome) is required to bring about a high NIR transcript level, while in mustard the NIR transcript level was determined by phytochrome alone and in spinach by nitrate alone. It is obvious that in different plants phytochrome and nitrate control NIR gene expression differently. Only the dependence on the plastidic factor appears to be the same in all cases studied so far.The conspicuous regulatory differences between spinach and tobacco offer a chance to address the crucial question of whether the NIR‐promoter from spinach fused to a reporter gene and introduced in tobacco, responds to nitrate and phytochrome in accordance with spinach (donor) or in accordance with tobacco (host). Experiments are in progress.