Nitrite Reductase Gene Research Articles

Profiling gene expression to distinguish the likely active diazotrophs from a sea of genetic potential in marine sediments

Nitrogen (N) cycling microbial communities in marine sediments are extremely diverse, and it is unknown whether this diversity reflects extensive functional redundancy. Sedimentary denitrifiers remove significant amounts of N from the coastal ocean and diazotrophs are typically regarded as inconsequential. Recently, N fixation has been shown to be a potentially important source of N in estuarine and continental shelf sediments. Analysis of expressed genes for nitrite reductase (nirS) and a nitrogenase subunit (nifH) was used to identify the likely active denitrifiers and nitrogen fixers in surface sediments from different seasons in Narragansett Bay (Rhode Island, USA). The overall diversity of diazotrophs expressing nifH decreased along the estuarine gradient from the estuarine head to an offshore continental shelf site. Two groups of sequences related to anaerobic sulphur/iron reducers and sulphate reducers dominated libraries of expressed nifH genes. Quantitative polymerase chain reaction (qPCR) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) data shows the highest abundance of both groups at a mid bay site, and the highest nifH expression at the head of the estuary, regardless of season. Several potential environmental factors, including water temperature, oxygen concentration and metal contamination, may influence the abundance and nifH expression of these two bacterial groups.

Effect of biochar on nitrous oxide emission and its potential mechanisms

Extensive use of biochar to mitigate nitrous oxide (N2O) emission is limited by the lack of understanding on the exact mechanisms altering N2O emission from biochar-amended soil. Biochars produced from rice straw and dairy manure at 350 and 500 °C by oxygen-limited pyrolysis were used to investigate their influence on N2O emission. A quadratic effect of biochar levels was observed on the N2O emissions. The potential mechanisms were explored by terminal restriction fragment length polymorphism (T-RFLP) and real-time polymerase chain reaction (qPCR). A lower relative abundance of bacteria, which included ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), was observed at 4% biochar application rate. Reduced copy numbers of the ammonia monooxygenase gene amoA and the nitrite reductase gene nirS coincided with decreased N2O emissions. Therefore, biochar may potentially alter N2O emission by affecting ammonia-oxidizing and denitrification bacteria, which is determined by the application rate of biochar in soil.Implications:Biochar research has received increased interest in recent years because of the potential beneficial effects of biochar on soil properties. Recent research shows that biochar can alter the rates of nitrogen cycling in soil systems by influencing nitrification and denitrification, which are key sources of the greenhouse gas nitrous oxide (N2O). However, there are still some controversial data. The purpose of this research was to (1) examine how applications of different dose of biochar to soil affect emission of N2O and (2) improve the understanding of the underlying mechanisms.

Biochar does not affect soil N-transformations or microbial community structure under ruminant urine patches but does alter relative proportions of nitrogen cycling bacteria

Nitrogen (N) cycling, especially denitrification, can be significantly altered when biochar is used as a soil conditioner. These alterations in N-cycling have been attributed to a combination of physicochemical change, alterations in microbial community ecology and pervading climatic conditions. This study investigated seasonal bacterial community change over two years in combination with a short-term winter study of N-transformations under bovine urine patches. A silt-loam pastoral soil in Canterbury, New Zealand was amended with either 0, 15 or 30tha−1 of Pinus radiata biochar (pyrolysed at ∼450°C) and bovine urine was added to patches within the 0 and 30tha−1 biochar amended plots (designated as 0U and 30U treatments, where U indicates ‘urine’).No discernible differences in bacterial community structure were observed during the two year study or the short term N-transformation study when comparing non-amended and biochar-amended soil. Differences in bacterial community structure were only evident when comparing seasons, with data pertaining to each season from successive years clustering together. During the short-term N-transformation study, bacterial communities formed 3 distinct clusters corresponding to elevated levels of urine derived NH4+-N (days 0–10), increases in NO3−-N and N2O (days 10–22) and a decline in NO3−-N and N2O (day 20 onward). Biochar amendment did increase the relative abundance of up to 50% of individual operational taxonomic units (OTUs or ‘species’), including key nitrite oxidisers and nitrate reducers. Biochar amendment did not affect the concentrations of inorganic-N compounds.The nirS (nitrite reductase) gene became elevated in the 30U treatment relative to the 0U treatment ∼10 days after the initial urine application. The nosZ (nitrous oxide reductase) gene became elevated in the 30U plots during the latter part of the experiment.Conclusions:•Biochar did not have a significant impact on the microbial community structure in pastoral soil over the course of two years.•The relative proportion of nitrifiers and denitrifiers increased in biochar amended soils subjected to large influxes of urine derived N.•Differences in N-transformation dynamics in the presence of biochar during the winter months were not statistically significant.

Nitrogen fixation, hydrogen production and N2O emissions

Flynn, B., Scott, N. and Dong, Z. 2014. Nitrogen fixation, hydrogen production and N2O emissions. Can. J. Plant Sci. 94: 1037-1041. H2 is a by-product of the nitrogenase reaction. Exposure to H2 is linked to increased N2O production, increased CO2 fixation and plant growth promotion in soil. The effects of H2 exposure on soil were observed using controlled H2 gas treatments and field trials with legumes. In field trials, increased N2O production was observed in soil adjacent to legume nodules and inoculation of H2-oxidizing isolates led to increased N2O emissions in corn fields. Many H2-oxidizing isolates tested positive for key denitrification genes, indicating a connection between H2 uptake and N2O emissions. H2 treatment significantly increased copy number of the nitrite reductase (nirK) gene suggesting increased denitrification as the source of N2O. There was also a significant increase in copy number and expression of the RubisCO (cbbL) gene in soil. H2-oxidizing bacterial isolates (JM63 and JM162a) ...

Nitrite reductase genes as functional markers to investigate diversity of denitrifying bacteria during agricultural waste composting

The purpose of this study was to investigate the diversity of denitrifier community during agricultural waste composting. The diversity and dynamics of the denitrifying genes (nirK and nirS) were determined using polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE). Relationships between physico-chemical parameters and denitrifying genes structures were simultaneously evaluated by redundancy analysis (RDA). Phylogenetic analysis indicated that nirK clones grouped into six clusters and nirS clones into two major clusters, respectively. The results showed a very high diversity of nir gene sequences within composting samples. RDA showed that the nirK and nirS gene structures were significantly related to pH and pile temperature (P < 0.05). Significant amounts of the variation (49.2 and 38.3% for nirK and nirS genes, respectively) were explained by pH and pile temperature, suggesting that those two parameters were the most likely ones to influence, or be influenced by the denitrifiers harboring nirK and nirS genes.

Combinatorial function of velvet and AreA in transcriptional regulation of nitrate utilization and secondary metabolism

Velvet is a conserved protein complex that functions as a regulator of fungal development and secondary metabolism. In the soil-inhabiting pathogen Fusarium oxysporum, velvet governs mycotoxin production and virulence on plant and mammalian hosts. Here we report a previously unrecognized role of the velvet complex in regulation of nitrate metabolism. F. oxysporum mutants lacking VeA or LaeA, two key components of the complex, were impaired in growth on the non-preferred nitrogen sources nitrate and nitrite. Both velvet and the general nitrogen response GATA factor AreA were required for transcriptional activation of nitrate (nit1) and nitrite (nii1) reductase genes under de-repressing conditions, as well as for the nitrate-triggered increase in chromatin accessibility at the nit1 locus. AreA also contributed to chromatin accessibility and expression of two velvet-regulated gene clusters, encoding biosynthesis of the mycotoxin beauvericin and of the siderophore ferricrocin. Thus, velvet and AreA coordinately orchestrate primary and secondary metabolism as well as virulence functions in F. oxysporum.

Activity, Abundance, and Diversity of Nitrifying Archaea and Denitrifying Bacteria in Sediments of a Subtropical Estuary: Bahía del Tóbari, Mexico

Fixed nitrogen (N) removal from estuaries via coupled nitrification–denitrification plays a significant role in the global N cycle and the biogeochemistry of individual estuaries. Much of our understanding of these processes is drawn from temperate estuaries, yet tropical and subtropical estuaries may respond differently to N inputs. I tested the hypothesis that nitrification is limited within subtropical estuaries by comparing nitrification and denitrification potentials, and the abundance of archaeal ammonia monooxygenase (amoA) and bacterial nitrite reductase (nirS) genes, across five sites in Bahia del Tobari, Mexico. Sampling was conducted when agricultural runoff supplied substantial quantities of N (ca. 20–80 μM ammonium), yet nitrification was detected at a single site. Denitrification was measured at four sites, and three displayed nitrate uptake rather than net nitrification—indicating a N sink within these sediments. Bacterial nirS genes uniformly outnumbered archaeal amoA genes (3- to 49-fold) and were more abundant in the northern part of the estuary. Patterns of community similarity among different sites were also different for nirS and archaeal amoA: similarities between sites based on nirS were often greater than for amoA, and sites were more rarely statistically different from each other. While amoA abundance was inversely related to temperature, neither amoA nor nirS was correlated with nitrification or denitrification potentials. My results are broadly consistent with known and proposed patterns of nitrification and denitrification in subtropical estuarine sediments, including the idea that nitrification is limited within subtropical estuarine sediments.

Denitrification in restored and unrestored Danish streams

Stream restoration often aims at mitigating nutrient pollution in aquatic ecosystems. However, despite recent research efforts, effects of restoration practices on in-stream nitrogen removal remain unclear. In this study, denitrification rates as well as factors controlling denitrification in unrestored and restored sections of two Danish streams (S1 and S2) were compared. The 15N isotope pairing technique was used to measure denitrification in situ. Denitrifier presence was analyzed by denaturing gradient gel electrophoresis (DGGE) and quantitative PCR of nitrite reductase (nirK and nirS) and nitrous oxide reductase (nosZ) genes. Denitrification rates were highly variable, with denitrification rates of 3106μmolNm−2h−1 in the unrestored section of S1, but no detectable denitrification in the restored section of S1, whereas in S2 restored and unrestored sections had similar denitrification rates of around 250μmolNm−2h−1. These large differences in denitrification rates were mainly due to differences in hydrologic conditions and sediment characteristics. High nitrate fluxes from upwelling groundwater created denitrification hotspots in the unrestored section of S1. Moreover, a lack of organic matter in the restored section of S1 likely caused a low abundance of denitrifiers and consequently no detectable denitrification. Our results indicate the importance of hydrology and sediment organic matter for stream nitrogen dynamics, which should be considered in restoration design.

An NLP-binding site in the 3' flanking region of the nitrate reductase gene confers nitrate-inducible expression in Arabidopsis thaliana (L.) Heynh.

As a signaling molecule, nitrate induces the expression of multiple genes involved in nitrate assimilation in plants. We previously showed that the expression of the Arabidopsis thaliana (L.) Heynh. nitrite reductase gene (NIR1) was activated by nitrate signaling through an interaction between NODULE INCEPETION-like protein (NLP) transcription factors (NLP1-9) and a nitrate-responsive cis-element (NRE) in the NIR1 promoter. By in vitro binding assays, we have also demonstrated the binding of NLP6 to an NRE-like sequence, designated NIA1-NRE1, in the 3' flanking region of an A. thaliana nitrate reductase gene, NIA1. In our current study, we characterize the role of NIA1-NRE1 in NIA1 expression. Deletion analysis of the 3' flanking region of NIA1 revealed that a 345-bp sequence containing NIA1-NRE1 has a dominant role in nitrate-inducible NIA1 expression. We also found that the 35S minimal promoter fused to the 345-bp sequence, or to NIA1-NRE1, can confer nitrate-inducible expression. Furthermore, the NIA1-NRE1-fused 35S minimal promoter is transactivated by NLP6 in vivo. These results suggest that NIA1-NRE1 alone is sufficient to direct nitrate-responsive gene expression and that nitrate-inducible NIA1 expression is regulated through the interaction between NLPs and NIA1-NRE1. Additionally, we show that the distance between the NRE and the gene under the control of the NRE is not critical for this process.

Denitrifying bacterial community in manure compost pellets applied to an Andosol upland field

A large amount of nitrous oxide (N2O), one of the most important greenhouse gases, was emitted after the application of manure compost pellets (MCPs) compared to treatments with ordinary manure composts or chemical fertilizers. Emission of N2O was found to be due to denitrification in the pellets. To study the relationship between N2O emission and denitrifiers, the denitrifying bacterial communities in the MCPs were investigated by comparing clone libraries of nitrite reductase genes (nirK and nirS) fragments. Pellet samples were obtained three days (P3) and 26 days (P26) after pellet application to the field. The P3 samples were emitting high levels of N2O, and the P26 samples had ceased N2O emission. Polymerase chain reaction (PCR) amplicons of both nirK and nirS were obtained from pellet samples. The diversity index (Shannon–Weiner index) of clone libraries was higher for the nirS genes than for the nirK genes for the same pellet samples and higher in P3 than in P26 for both nir genes. The clone libraries showed that the compositions of nirK denitrifiers in the pellets were relatively stable, but those of the nirS denitrifiers were variable within a month after pellet application; nirS clones that showed a lower similarity with nirS of known denitrifiers were dominant in P3 and those related to nirS of Cupriavidus spp. and Herbaspirillum spp. were dominant in P26. Most of the nirK clones in this study were related to the nirK genes of Rhizobiales denitrifying bacterial isolates. The nirS clones were divided into three groups; (1) those related to nirS of Bradyrhizobium spp., and Rhodanobacter sp. D206a, (2) those related to nirS of Cupriavidus spp., Herbaspirillum spp., and (3) those that had a lower similarity with nirS of known denitrifiers. This study showed a different response of nirK and nirS denitrifiers in the applied MCPs. In order to understand the relation between the denitrifying diversity and the N2O emissions, transcriptional and metabolic analysis will be also needed.

Bacterial community structure of nirK-bearing denitrifiers and the development of properties of soils in created mitigation wetlands

We investigated the abundance and genetic heterogeneity of bacterial nitrite reductase genes (nir) and soil structural properties in created and natural freshwater wetlands in the Virginia piedmont. Soil attributes included soil organic matter (SOM), total organic carbon (TOC), total nitrogen (TN), pH, gravimetric soil moisture (GSM), and bulk density (Db). A subset of soil attributes were analyzed across the sites, using euclidean cluster analysis, resulting in three soil condition (SC) groups of increasing wetland soil development (i.e., SC1<SC2<SC3; less to more developed or matured) as measured by accumulation of TOC, TN, the increase of GSM, and the decrease of Db. There were no difference found in the bacterial community diversity between the groups (p=0.4). NirK gene copies detected ranged between 3.6×104 and 3.4×107copiesg−1 soil and were significantly higher in the most developed soil group, SC3, than in the least developed soil group, SC1. However, the gene copies were lowest in SC2 that had a significantly higher soil pH (~6.6) than the other two SC groups (~5.3). The same pattern was found in denitrifying enzyme activity (DEA) on a companion study where DEA was found negatively correlated with soil pH. Gene fragments were amplified and products were screened by terminal restriction fragment length polymorphism (T-RFLP) analysis. Among 146 different T-RFs identified, fourteen were dominant and together made up more than 65% of all detected fragments. While SC groups did not relate to whole nirK communities, most soil properties that identified SC groups did significantly correlate to dominant members of the community.

Predominance of ammonia-oxidizing archaea andnirK-gene-bearing denitrifiers among ammonia-oxidizing and denitrifying populations in sediments of a large urban eutrophic lake (Lake Donghu)

The coupled nitrification-denitrification process plays a pivotal role in cycling and removal of nitrogen in aquatic ecosystems. In the present study, the communities of ammonia oxidizers and denitrifiers in the sediments of 2 basins (Guozhenghu Basin and Tuanhu Basin) of a large urban eutrophic lake (Lake Donghu) were determined using the ammonia monooxygenase subunit A (amoA) gene and the nitrite reductase gene. At all sites of this study, the archaeal amoA gene predominated over the bacterial amoA gene, whereas the functional gene for denitrification nirK gene far outnumbered the nirS gene. Spatially, compared with the Tuanhu Basin, the Guozhenghu Basin showed a significantly greater abundance of the archaeal amoA gene but less abundance of the nirK and nirS genes, while there was no significant difference of bacterial amoA gene copy numbers between the 2 basins. Unlike the archaeal amoA gene, the nirK gene showed a significant difference in community structure between the 2 basins. Archaeal amoA diversity was limited to the water-sediment cluster of Crenarchaeota, in sharp contrast with nirK for which 22 distinct operational taxonomic units were found. Accumulation of organic substances were found to be positively related to nirK and nirS gene copy numbers but negatively related to archaeal amoA gene copy numbers, whereas the abundance of the bacterial amoA gene was related to ammonia concentration.

Unexpected Phytostimulatory Behavior for Escherichia coli and Agrobacterium tumefaciens Model Strains

Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.

The Ammonia Oxidizing and Denitrifying Prokaryotes Associated with Sponges from Different Sea Areas

Marine sponges have been suggested to play an important role in the marine nitrogen cycling. However, the role of sponge microbes in the nitrogen transformation remains limited, especially on the bacterial ammonia oxidization and denitrification. Hence, in the present study, using functional genes (amoA, nirS, nirK, and nxrA) involved in ammonia oxidization and denitrification and 16S rRNA genes for specific bacterial groups as markers, phylogenetically diverse prokaryotes including bacteria and archaea, which may be involved in the ammonia oxidization and denitrification processes in sponges, were revealed in seven sponge species. Ammonia oxidizers were found in all species, whereas three sponges (Placospongia sp., Acanthella sp., and Pericharax heteroraphis) harbor only ammonia-oxidizing bacteria (AOB), two sponges (Spirastrellidae diplastrella and Mycale fibrexilis) host only ammonia-oxidizing archaea (AOA), while the remaining two sponges (Haliclona sp. and Lamellomorpha sp.) harbor both AOB and AOA. S. diplastrella and Lamellomorpha sp. also harbor denitrifying bacteria. Nitrite reductase gene nirK was detected only in Lamellomorpha sp. with higher phylogenetic diversity than nirS gene observed only in S. diplastrella. The detected functional genes related to the ammonia oxidization and nitrite reduction in deep-sea and shallow-water sponges highlighted the potential ecological roles of prokaryotes in sponge-related nitrogen transformation.

Phenotypic variation and morphological changes in starved denitrifying Aeromonas hydrophila

In this study, an Aeromonas hydrophila identified as a denitrifying bacterium by PCR detection of nitrate reductase (narG) and nitrite reductase (nirK) genes was incubated in seawater microcosms for 8 months at room temperature and at 4 °C. A study of the phenotypic variation demonstrated that A. hydrophila becomes gelatinase-positive after the incubation in sea water. We noted that starved A. hydrophila becomes unable to produce leucine arylamidase, and that the starved strain appears to grow more slowly. Indeed, we also observed a severe decrease in cellular aggregation of Aeromonas after incubation. In addition, atomic force micrographs revealed a reduction in cell size.

Elevated Atmospheric CO2 Impacts Abundance and Diversity of Nitrogen Cycling Functional Genes in Soil

The concentration of CO(2) in the Earth's atmosphere has increased over the last century. Although this increase is unlikely to have direct effects on soil microbial communities, increased atmospheric CO(2) may impact soil ecosystems indirectly through plant responses. This study tested the hypothesis that exposure of plants to elevated CO(2) would impact soil microorganisms responsible for key nitrogen cycling processes, specifically denitrification and nitrification. We grew trembling aspen (Populus tremuloides) trees in outdoor chambers under ambient (360 ppm) or elevated (720 ppm) levels of CO(2) for 5 years and analyzed the microbial communities in the soils below the trees using quantitative polymerase chain reaction and clone library sequencing targeting the nitrite reductase (nirK) and ammonia monooxygenase (amoA) genes. We observed a more than twofold increase in copy numbers of nirK and a decrease in nirK diversity with CO(2) enrichment, with an increased predominance of Bradyrhizobia-like nirK sequences. We suggest that this dramatic increase in nirK-containing bacteria may have contributed to the significant loss of soil N in the CO(2)-treated chambers. Elevated CO(2) also resulted in a significant decrease in copy numbers of bacterial amoA, but no change in archaeal amoA copy numbers. The decrease in abundance of bacterial amoA was likely a result of the loss of soil N in the CO(2)-treated chambers, while the lack of response for archaeal amoA supports the hypothesis that physiological differences in these two groups of ammonia oxidizers may enable them to occupy distinct ecological niches and respond differently to environmental change.

Nitrite reductase (nii2) gene has a positive role in nitrogen metabolism in tobacco

Nitrite reductase (NiR) is an enzyme that can reduce the concentration of nitrite. Nitrite is one of the precursors of tobacco-specific nitrosamines (TSNA), which are potentially carcinogenic products and exist only in tobacco and tobacco products. In this study, we analyzed the over expression of ferredoxin-nitrite reductase nii2 gene (EC 1.7.7.1) in tobacco to determine the reduction of nitrate and nitrite in response to its increase in the plants. Accordingly, the Ntnii2 gene was cloned from tobacco based on the information from its wild-type (WT) homologous genes and it contained 1764 bp encoding the polypeptides of 587 amino acids. This gene was inserted into plant expression vector pBI121 and Agrobacterium-mediated transformation results showed 23 transgenic tobacco lines through kanamycin resistance screening. After PCR-based screening, Ntnii2 gene was confirmed to be successfully transcript and inherited 16 offspring’s and 8 lines were selected for further generation. The NiR mRNA levels, in vivo and in vitro NR activities were significantly higher in those selected eight transgenic lines than its counterpart WT and approximately 30% increase NiR activity in some transgenic lines. The nitrate content showed nearly 46% reductions. Although the nitrite contents were not considerably differed from that of non-transformed plants, most of the transgenic lines showed low nitrite level than wild-type plants. Therefore, transgenic plants with low level of nitrate and nitrite were produced. Key words: Nitrite reductase, over expression, nitrate, nitrite, tobacco-specific nitrosamines (TSNA).

Prevalence of internal parasites in sheep/goats and effective economic de-worming plan at upland Balochistan,Pakistan

A nitrite reductase gene related to somatic embryogenesis was first cloned from Gossypium hirsutum. The cDNA sequence of the gene, named GhNiR, is 2,257 bp in length, with 254 bp of the 5’ untranslated region and 236 bp of the 3’ untranslated region. The open reading frame is 1,767 bp in length, encoding a deduced amino acid sequence of 588 residues with a molecular weight of 65.722 kDa and an isoelectric point of 7.07. Semi-quantitative RT-PCR analysis showed that the expression level of GhNiR was higher in embryogenic calli and somatic embryoids than in nonembryogenic calli among different somatic embryogenesis stages, and that the level of GhNiR mRNA was also higher in the cultivar with higher somatic embryogenesis ability. The catalytic GhNiR was verified by transformation in E. coli BL21 (DE3) strain with the recombinant expression vector pET-28A-GhNiR. NiR activity assay showed that the crude GhNiR protein had obvious activity to NaNO2 substrate.

Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components

A method for Agrobacterium-mediated co-transformation of rice (Oryza sativa L.) was developed using rice-derived selection markers. Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP) (CSP:mALS) and the ferredoxin nitrite reductase gene (NiR) under the control of its own promoter (NiR P:NiR). The CSP:mALS gene cassette confers sulfonylurea herbicide resistance to transgenic rice callus. The NiR P:NiR construct complements NiR-deficient mutant cultivars such as 'Koshihikari', which are defective in the regulation of nitrogen metabolism. In the present study, the CaMV35S:GUS and CaMV35S:GFP gene cassettes were co-introduced into the 'Koshihikari' genome using our system. Approximately 5-10 independent transgenic lines expressing both the GUS and GFP reporters were obtained from 100 Agrobacterium co-inoculated calli. Furthermore, transgenic 'Koshihikari' rice lines with reduced content of two major seed allergen proteins, the 33 and 14-16kDa allergens, were generated by this co-transformation system. The present results indicate that the generation of selectable antibiotic resistance marker gene-free transgenic rice is possible using our rice-derived selection marker co-transformation system. Key message An improved rice transformation method was developed based on Agrobacterium-mediated co-transformation using two rice genome-derived selectable marker gene cassettes.

Soil Property Control of Biogeochemical Processes beneath Two Subtropical Stormwater Infiltration Basins

Substantially different biogeochemical processes affecting nitrogen fate and transport were observed beneath two stormwater infiltration basins in north-central Florida. Differences are related to soil textural properties that deeply link hydroclimatic conditions with soil moisture variations in a humid, subtropical climate. During 2008, shallow groundwater beneath the basin with predominantly clayey soils (median, 41% silt+clay) exhibited decreases in dissolved oxygen from 3.8 to 0.1 mg L and decreases in nitrate nitrogen (NO-N) from 2.7 mg L to <0.016 mg L, followed by manganese and iron reduction, sulfate reduction, and methanogenesis. In contrast, beneath the basin with predominantly sandy soils (median, 2% silt+clay), aerobic conditions persisted from 2007 through 2009 (dissolved oxygen, 5.0-7.8 mg L), resulting in NO-N of 1.3 to 3.3 mg L in shallow groundwater. Enrichment of δN and δO of NO combined with water chemistry data indicates denitrification beneath the clayey basin and relatively conservative NO transport beneath the sandy basin. Soil-extractable NO-N was significantly lower and the copper-containing nitrite reductase gene density was significantly higher beneath the clayey basin. Differences in moisture retention capacity between fine- and coarse-textured soils resulted in median volumetric gas-phase contents of 0.04 beneath the clayey basin and 0.19 beneath the sandy basin, inhibiting surface/subsurface oxygen exchange beneath the clayey basin. Results can inform development of soil amendments to maintain elevated moisture content in shallow soils of stormwater infiltration basins, which can be incorporated in improved best management practices to mitigate NO impacts.