Nitrate Reductase Gene Research Articles

CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE.

The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh.

Regulation of nitrate and methylamine metabolism by multiple nitrogen sources in the methylotrophic yeastCandida boidinii

The methylotrophic yeast Candida boidinii, which is capable of growth on methanol as a sole carbon source, can proliferate on the leaf surface of Arabidopsis thaliana. Previously, we demonstrated that adaptation to a change in the major available nitrogen source from nitrate to methylamine during the host plant aging was crucial for yeast survival on the leaf environment. In this report, we investigated the regulatory profile of nitrate and methylamine metabolism in the presence of multiple nitrogen sources in C. boidinii. The transcript level of nitrate reductase (Ynr1) gene was induced by nitrate and nitrite, and was not repressed by the coexistence with other nitrogen sources. In contrast, the transcript level of amine oxidase (Amo1) gene, which was induced by methylamine, was significantly repressed by the coexistence with ammonium or glutamine. In addition, we investigated the intracellular dynamics of Ynr1 during the nitrogen source shift from nitrate to other compounds. Under these tested conditions, Ynr1 was effectively transported to the vacuole via selective autophagy only during the shift from nitrate to methylamine. Moreover, Ynr1 was subject to degradation after the shift from nitrate to nitrate plus methylamine medium even though nitrate was still available. These regulatory profiles may reflect life style of nitrogen utilization in this yeast living in the phyllosphere.

Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium.

Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness at low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. We discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp. The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB.

Effects of NaCl stress on nitrogen metabolism of cucumber seedlings

To investigate the effects of NaCl stress on plant growth, nitrogen assimilation, proline and soluble protein contents, and gene expression of enzymes of nitrogen metabolism, a hydroponic experiment using cucumber (Cucumis sativus L.) seedlings was performed. The seedlings were grown in nutrient solution supplemented with 0 or 84 mM NaCl for up to 9 days. Plant biomass, especially root biomass, was significantly decreased under NaCl stress. Salinity significantly increased ammonium content, but decreased nitrate and soluble protein contents in leaves and roots. Salt stress caused a significant increase in proline content, which peaked on day 3 of NaCl treatment. Moreover, salt stress significantly decreased activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the roots and leaves on the 3rd, 6th, and 9th day of NaCl treatment. A semiquantitative RT-PCR approach showed that changes in NR, GS and GOGAT gene expression were consistent with the salt-induced changes in enzyme activities. These results suggest that salt stress-induced growth inhibition in cucumber seedlings may involve disruption of nitrogen absorption and decreased activities of enzymes associated with nitrogen assimilation.

Arabidopsis CMT3 activity is positively regulated by AtSIZ1-mediated sumoylation

The activities of mammalian DNA and histone methyltransferases are regulated by post-translational modifications such as phosphorylation and sumoylation; however, it is unclear how the activities of these enzymes are regulated at the post-translational level in plants. Here, we demonstrate that the DNA methylation activity of Arabidopsis CHROMOMETHYLASE 3 (CMT3) is positively regulated by the E3 SUMO ligase AtSIZ1. The methylation level of the Arabidopsis genome, including transposons, was significantly lower in the siz1-2 mutant than in wild-type plants. CMT3 was sumoylated by the E3 ligase activity of AtSIZ1 through a direct interaction, and the DNA methyltransferase activity of CMT3 was enhanced by this modification. In addition, the methylation levels of a large number of genes, including the nitrate reductase gene NIA2, were lower in siz1-2 and cmt3 plants than in wild-type plants. Furthermore, the CHG methylation activity of CMT3 was specific for NIA2and not NIA1 (the other nitrate reductase gene in Arabidopsis), indicating that CMT3 selectively regulates the CHG methylation levels of target genes. Taken together, our results indicate that the sumoylation of CMT3 is critical for its role in the control of gene expression and that AtSIZ1 positively controls the epigenetic repression of CMT3-mediated gene expression.

Nitrogen Removal Characteristics of a Newly Isolated Indigenous Aerobic Denitrifier from Oligotrophic Drinking Water Reservoir, Zoogloea sp. N299.

Nitrogen is considered to be one of the most widespread pollutants leading to eutrophication of freshwater ecosystems, especially in drinking water reservoirs. In this study, an oligotrophic aerobic denitrifier was isolated from drinking water reservoir sediment. Nitrogen removal performance was explored. The strain was identified by 16S rRNA gene sequence analysis as Zoogloea sp. N299. This species exhibits a periplasmic nitrate reductase gene (napA). Its specific growth rate was 0.22 h−1. Obvious denitrification and perfect nitrogen removal performances occurred when cultured in nitrate and nitrite mediums, at rates of 75.53% ± 1.69% and 58.65% ± 0.61%, respectively. The ammonia removal rate reached 44.12% ± 1.61% in ammonia medium. Zoogloea sp. N299 was inoculated into sterilized and unsterilized reservoir source waters with a dissolved oxygen level of 5–9 mg/L, pH 8–9, and C/N 1.14:1. The total nitrogen removal rate reached 46.41% ± 3.17% (sterilized) and 44.88% ± 4.31% (unsterilized). The cell optical density suggested the strain could survive in oligotrophic drinking water reservoir water conditions and perform nitrogen removal. Sodium acetate was the most favorable carbon source for nitrogen removal by strain N299 (p < 0.05). High C/N was beneficial for nitrate reduction (p < 0.05). The nitrate removal efficiencies showed no significant differences among the tested inoculums dosage (p > 0.05). Furthermore, strain N299 could efficiently remove nitrate at neutral and slightly alkaline and low temperature conditions. These results, therefore, demonstrate that Zoogloea sp. N299 has high removal characteristics, and can be used as a nitrogen removal microbial inoculum with simultaneous aerobic nitrification and denitrification in a micro-polluted reservoir water ecosystem.

Tepidicaulis marinus gen. nov., sp. nov., a marine bacterium that reduces nitrate to nitrous oxide under strictly microaerobic conditions.

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium,Parvibaculum, and Rhodoligotrophos (92-93 % similarity) within the class Alphaproteobacteria. Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16-48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

The inducible nitA promoter provides a powerful molecular switch for transgene expression in Volvox carteri.

BackgroundThe multicellular green alga Volvox carteri represents an attractive model system to study various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ECM biogenesis. However, functional and molecular analyses of such processes require a wide array of molecular tools for genetic engineering. So far there are only a limited number of molecular tools available in Volvox.ResultsHere, we show that the promoter of the V. carteri nitrate reductase gene (nitA) is a powerful molecular switch for induction of transgene expression. Strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. We also show that the luciferase (g-luc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of the unicellular alga Chlamydomonas reinhardtii, is a suitable reporter gene in V. carteri. Emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer. Long-term stability of inducible expression of the chimeric nitA/g-luc transgenes after stable nuclear transformation was demonstrated by transcription analysis and bioluminescence assays.ConclusionTwo novel molecular tools for genetic engineering of Volvox are now available: the nitrate-inducible nitA promoter of V. carteri and the codon-adapted luciferase reporter gene of G. princeps. These novel tools will be useful for future molecular research in V. carteri.

Nitrate reductase-dependent nitric oxide production is required for regulation alternative oxidase pathway involved in the resistance to Cucumber mosaic virus infection in Arabidopsis

Nitrate reductase (NR), a key enzyme of nitrogen metabolism, catalyzes the reduction of nitrite to produce nitric oxide (NO). In this study, we investigated the role of NR-dependent NO in alleviating the cucumber mosaic virus (CMV)-caused damage in Arabidopsis thaliana (Arabidopsis). Quantitative real-time polymerase chain reaction analysis indicated that NR genes NIA1 and NIA2 transcripts were significantly increased by CMV infection in Arabidopsis. Further evidence showed that the nia1nia2 mutant and cPTIO-pretreated plants exhibited more serious symptoms (more reactive oxygen species accumulation, higher H2O2 content, malonaldehyde content, electrolyte leakage and transcription of CMV coat protein gene) compared with the wild-type (WT) after CMV infection. Analysis of putative molecular mechanism revealed that the expression of genes encoding pathogenesis-related 1 protein, pathogenesis-related 2 protein and alternative oxidase1a protein in nia1nia2 mutant were significantly decreased compared to the WT, and this phenomenon could be reversed by pretreatment with sodium nitroprusside (SNP). However, pretreatment with SNP could not effectively decrease the damage in aox1a mutant caused by CMV. Taken together, our results indicated that the NR-dependent NO was required for regulation the salicylic acid-mediated defense response and cyanide-resistant respiration pathway involved in the resistance to CMV infection, and alternative oxidase pathway played an important role in the defense response mediated by NO.

Ubiquity and diversity of heterotrophic bacterial nasA genes in diverse marine environments.

Nitrate uptake by heterotrophic bacteria plays an important role in marine N cycling. However, few studies have investigated the diversity of environmental nitrate assimilating bacteria (NAB). In this study, the diversity and biogeographical distribution of NAB in several global oceans and particularly in the western Pacific marginal seas were investigated using both cultivation and culture-independent molecular approaches. Phylogenetic analyses based on 16S rRNA and nasA (encoding the large subunit of the assimilatory nitrate reductase) gene sequences indicated that the cultivable NAB in South China Sea belonged to the α-Proteobacteria, γ-Proteobacteria and CFB (Cytophaga-Flavobacteria-Bacteroides) bacterial groups. In all the environmental samples of the present study, α-Proteobacteria, γ-Proteobacteria and Bacteroidetes were found to be the dominant nasA-harboring bacteria. Almost all of the α-Proteobacteria OTUs were classified into three Roseobacter-like groups (I to III). Clone library analysis revealed previously underestimated nasA diversity; e.g. the nasA gene sequences affiliated with β-Proteobacteria, ε-Proteobacteria and Lentisphaerae were observed in the field investigation for the first time, to the best of our knowledge. The geographical and vertical distributions of seawater nasA-harboring bacteria indicated that NAB were highly diverse and ubiquitously distributed in the studied marginal seas and world oceans. Niche adaptation and separation and/or limited dispersal might mediate the NAB composition and community structure in different water bodies. In the shallow-water Kueishantao hydrothermal vent environment, chemolithoautotrophic sulfur-oxidizing bacteria were the primary NAB, indicating a unique nitrate-assimilating community in this extreme environment. In the coastal water of the East China Sea, the relative abundance of Alteromonas and Roseobacter-like nasA gene sequences responded closely to algal blooms, indicating that NAB may be active participants contributing to the bloom dynamics. Our statistical results suggested that salinity, temperature and nitrate may be some of the key environmental factors controlling the composition and dynamics of the marine NAB communities.

AtTGA4, a bZIP transcription factor, confers drought resistance by enhancing nitrate transport and assimilation in Arabidopsis thaliana

To cope with environmental stress caused by global climate change and excessive nitrogen application, it is important to improve water and nitrogen use efficiencies in crop plants. It has been reported that higher nitrogen uptake could alleviate the damaging impact of drought stress. However, there is scant evidence to explain how nitrogen uptake affects drought resistance. In this study we observed that bZIP transcription factor AtTGA4 (TGACG motif-binding factor 4) was induced by both drought and low nitrogen stresses, and that overexpression of AtTGA4 simultaneously improved drought resistance and reduced nitrogen starvation in Arabidopsis. Following drought stress there were higher nitrogen and proline contents in transgenic AtTGA4 plants than in wild type controls, and activity of the key enzyme nitrite reductase (NIR) involved in nitrate assimilation processes was also higher. Expressions of the high-affinity nitrate transporter genes NRT2.1 and NRT2.2 and nitrate reductase genes NIA1 and NIA2 in transgenic plants were all higher than in wild type indicating that higher levels of nitrate transport and assimilation activity contributed to enhanced drought resistance of AtTGA4 transgenic plants. Thus genetic transformation with AtTGA4 may provide a new approach to simultaneously improve crop tolerance to drought and low nitrogen stresses.

Effects of root restriction on nitrogen and gene expression levels in nitrogen metabolism in Jumeigui grapevines (Vitis vinifera L.×Vitis labrusca L.)

To decipher the relationship between the inhibited shoot growth and expression pattern of key enzymes in nitrogen metabolism under root restriction, the effects of root restriction on diurnal variation of expression of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS1-1, GS1-2, GS2) and glutamate synthase (Fd-GOGAT, NADH-GOGAT) genes and nitrogen levels were evaluated in two-year-old Jumeigui grapevines (Vitis vinifera L.×Vitis labrusca L.) when significant differences in shoot growth were observed between treatments at expansion stage (22 days after anthesis). Grapevines were planted in root-restricting pits as root restriction and in an unrestricted field as the control. Results showed that root restriction significantly reduced shoot growth, but promoted the growth of white roots and fibrous brown roots and improved the fruit quality. (NO3−+NO2−)-N concentration in all plant parts, NH4+-N concentration in white roots and total N concentration in leaves and brown roots were significantly reduced under root restriction. Gene expression analysis revealed that mRNA levels of genes related to the GS1/NADH-GOGAT pathway were lower in root-restricted than in control petioles, whereas genes involved in the GS2/Fd-GOGAT pathway were up-regulated under root restriction. Root restriction also resulted in down-regulation of genes involved in nitrogen metabolism in leaves, especially at 10:00, while transcript levels of all these genes were enhanced in root-restricted white and brown roots at most time points. This organ-dependent response contributed to the alteration in NO3− reduction and NH4+ assimilation under root restriction, leading to less NO3− transported from roots and then assimilated in root-restricted leaves. Therefore, this study implied that shoot growth inhibition in grapevines under root restriction is closely associated with down-regulation of gene expression in nitrogen metabolism in leaves.

Effects of NaCl Stress on Nitrogen Metabolism of Cucumber Seedlings

To investigate the effects of NaCl stress on plant growth, nitrogen assimilation, proline and sol uble protein contents, and gene expression of enzymes of nitrogen metabolism, a hydroponic experiment using cucumber (Cucumis sativus L.) seedlings was performed. The seedlings were grown in nutrient solu tion supplemented with 0 or 84 mM NaCl for up to 9 days. Plant biomass, especially root biomass, was sig nificantly decreased under NaCl stress. Salinity significantly increased ammonium content, but decreased nitrate and soluble protein contents in leaves and roots. Salt stress caused a significant increase in proline content, which peaked on day 3 of NaCl treatment. Moreover, salt stress significantly decreased activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the roots and leaves on the 3rd, 6th, and 9th day of NaCl treatment. A semi quantitative RT PCR approach showed that changes in NR, GS and GOGAT gene expression were consis tent with the salt induced changes in enzyme activities. These results suggest that salt stress induced growth inhibition in cucumber seedlings may involve disruption of nitrogen absorption and decreased activities of enzymes associated with nitrogen assimilation.

Induction of disease resistance against Botrytis cinerea in tomato (Solanum lycopersicum L.) by using electrostatic atomized water particles

This study examined the effect of electrostatic atomized water particles (EAWP) on the disease resistance of tomato against Botrytis cinerea. Disease development of B. cinerea was suppressed by EAWP pretreatment prior to pathogen inoculation. Treatment of tomato plants with EAWP resulted in the upregulation of the nitrate reductase (NR) gene, which led to the accumulation of nitric oxide (NO) and S-nitrosylated proteins in the cells. In addition, the salicylic acid (SA)-dependent chitinase 3 (CHI3) gene was rapidly induced in leaves pretreated with EAWP after the leaves were inoculated with B. cinerea. Light microscopy showed browning of the epidermal cells surrounding the inoculation sites and inhibition of fungal hyphal development in the cells. These results suggest that pretreatment of tomato leaves with EAWP inhibits the development of B. cinerea in the early stages of infection by regulating the redox flux via the NO- and SA-mediated signal transduction pathways.

Identification and monitoring of nitrification and denitrification genes in Klebsiella pneumoniae EGD-HP19-C for its ability to perform heterotrophic nitrification and aerobic denitrification.

Microbes capable of performing heterotrophic nitrification and aerobic denitrification simultaneously have application in nitrogen level management in effluent treatment plants. Klebsiella pneumoniae EGD-HP19-C is a metabolically versatile bacterium capable of utilising NH3-N, NO2-N and NO3-N as sole sources of nitrogen. The annotation was done for the genes involved in N-assimilation and N-dissimilation pathways from the draft genome sequences of this bacterium (NCBI GenBank accession no. AUTW02000000.1). The sequence data also suggested possible existence of plasmid associated with this bacterium. Multiple gene sequence alignments of glutamine synthetase (gln), hydroxylamine reductase (har), nitrite reductase (nir), nitric oxide reductase (nor), assimilatory nitrate reductase (nas) and respiratory nitrate reductase (nar) genes from EGD-HP19-C genome were performed to compare sequence identities with that of closely related bacterial species. The metabolic pathways were mapped using KAAS and 3D structures for representative enzyme sub-units were also elucidated. The study suggested that the organism, though it has incomplete nitrification and denitrification pathways still removes the inorganic nitrogen content from the system via ammonification reaction.

A stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis.

Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 10(8) recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami-Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.

Effect of Nitrogen Starvation on the Responses of Two Rice Cultivars to Nitrate Uptake and Utilization

Ammonium (NH+4) is the main nitrogen (N) form for rice crops, while NH+4 near the root surface can be oxidized to nitrate (NO−3) by NH+4 oxidizing bacteria. Nitrate can be accumulated within rice tissues and reused when N supply is insufficient. We compared the remobilization of NO−3 stored in the tissue and vacuolar between two rice (Oryza sativa L.) cultivars, Yangdao 6 (YD6, indica) with a high N use efficiency (NUE) and Wuyujing 3 (WYJ3, japonica) with a low NUE and measured the uptake of NO−3, expression of nitrate reductase (NR), NO−3 transporter genes (NRTs), and NR activity after 4 d of N starvation following 7-d cultivation in a solution containing 2.86 mmol L−1 NO−3. The results showed that both tissue NO−3 concentration and vacuolar NO−3 activity were higher in YD6 than WYJ3 under N starvation. YD6 showed a 2- to 3-fold higher expression of OsNRT2.1 in roots on the 1st and 4th day of N starvation and had significantly higher values of NO−3 uptake (maximum uptake velocity, Vmax) than the cultivar WYJ3. Furthermore, YD6 had significantly higher leaf and root maximum NR activity (NRAmax) and actual NR activity (NRAact) as well as stronger root expression of the two NR genes after the 1st day of N starvation. There were no significant differences in NRAmax and NRAact between the two rice cultivars on the 4th day of N starvation. The results suggested that YD6 had stronger NRA under N starvation, which might result in better NO−3 re-utilization from the vacuole, and higher capacity for NO−3 uptake and use, potentially explaining the higher NUE of YD6 compared with WYJ3.

Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii.

The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA- and siRNA-based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one-step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G-Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon-counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids1) and the flagellar protein CDPK3 (calcium-dependent protein kinase3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.

Nitric oxide production and scavenging in waterlogged roots of rape seedlings

When plant roots are waterlogged, plants can experience hypoxic stress. Large quantities of nitric oxide (NO) can be generated under hypoxic conditions as a result of nitrate reduction. Our objective was to investigate the molecular mechanisms behind NO production and scavenging (turnover) in waterlogged roots of rape (‘Tammi’ variety) seedlings by surveying waterlogging-responsive genes. Waterlogging for up to 72 h enhanced NO production rapidly in the roots. Of 53,107 genes assayed, 9,692 showed a twofold change in expression within 36 h of waterlogging. Two nitrate reductase (NaR) genes (TC201891, TC161540) and four nitrite reductase (NiR) genes (TC168889, TC164215, TC163914, TC185634) were potentially involved in NO production in response to waterlogging stress. Strong hypoxic induction of non-symbiotic hemoglobin (Hb) gene (TC165566), which increased 656- and 645-fold at 36 and 72 h of waterlogging, respectively, could oxidize the NO overproduced in the roots. Our results suggested that reduction of nitrate to NO by NaR and NiR and subsequent NO turnover by Hb provide a mechanism for maintaining bioenergetics in waterlogged roots. The up-regulation of many additional waterlogging-responsive genes with potential roles in the anaerobic respiration, sucrose and starch degradation, glycolysis, and pyruvate metabolism, may acclimate the plant to waterlogging-induced hypoxic condition.

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 ‰.