Strategy and tactics of disarming GHG at the source: N2O reductase crops

Nitrous oxide (N(2)O), the third most abundant greenhouse gas (GHG), is highly stable and plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N(2)O stems from use of nitrogen fertilizers in soil. The bacterial enzyme nitrous oxide reductase (N(2)OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N(2)O to N(2). In this opinion, we discuss potential biology-based strategies to reduce N(2)O by amplifying the amount of available enzyme catalyst in agri-system environments during crop growth and in post-harvest detritus. N(2)OR from Pseudomonas stutzeri has been tested in transgenic plants with promising results. Such seed-borne phytoremediation systems targeted towards GHGs merit field testing.     

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes

The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 μmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 μmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century.     

Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria

Nitric oxide (NO) and nitrous oxide (N2O) are climatically important trace gases that are produced by both nitrifying and denitrifying bacteria. In the denitrification pathway, N2O is produced from nitric oxide (NO) by the enzyme nitric oxide reductase (NOR). The ammonia-oxidizing bacterium Nitrosomonas europaea also possesses a functional nitric oxide reductase, which was shown recently to serve a unique function. In this study, sequences homologous to the large subunit of nitric oxide reductase (norB) were obtained from eight additional strains of ammonia-oxidizing bacteria, including Nitrosomonas and Nitrosococcus species (i.e., both beta- and gamma-Proteobacterial ammonia oxidizers), showing widespread occurrence of a norB homologue in ammonia-oxidizing bacteria. However, despite efforts to detect norB homologues from Nitrosospira strains, sequences have not yet been obtained. Phylogenetic analysis placed nitrifier norB homologues in a subcluster, distinct from denitrifier sequences. The similarities and differences of these sequences highlight the need to understand the variety of metabolisms represented within a "functional group" defined by the presence of a single homologous gene. These results expand the database of norB homologue sequences in nitrifying bacteria.     

Effects of Elevated Cytosolic Glutathione Reductase Activity on the Cellular Glutathione Pool and Photosynthesis in Leaves under Normal and Stress Conditions

Tobacco (Nicotiana tabacum var Samsun) was transformed using the bacterial gor gene coding for the enzyme glutathione reductase. Transgenic plants were selected by their kanamycin resistence and expression of the bacterial gor gene. After separation by isoelectric focusing techniques, leaf extracts from transgenic plants having both native and bacterial glutathione reductase activity gave, in addition to the six bands of the native enzyme, two further closely running isoenzymes. These additional bands originating from the expression of the bacterial gor gene were nonchloroplastic. Leaves from transgenic plants had two- to 10-fold higher glutathione reductase activity than non-transgenic controls. The amount of extractable glutathione reductase activity obtained in transgenic plants was dependent on leaf age and the conditions to which leaves were exposed. Both light and exposure to methylviologen increased leaf glutathione reductase activity. Elevated levels of cytosolic glutathione reductase activity in transgenic plants had no effect on the amount or reduction state of the reduced glutathione/oxidized glutathione pool under optimal conditions or oxidative conditions induced by methylviologen. The glutathione pool was unaltered despite the oxidation-dependent loss of CO₂ assimilation and oxidation of enzymes involved in photosynthesis. However, the reduction state of the ascorbate pool was greater in transgenic plants relative to nontransgenic controls following illumination of methylviologen-treated leaf discs. Therefore, we conclude that in the natural state glutathione reductase is present in tobacco at levels above those required for maximal operation of the ascorbate-glutathione pathway.     

A novel aldose/aldehyde reductase protects transgenic plants against lipid peroxidation under chemical and drought stresses

Rapid accumulation of toxic products from reactions of reactive oxygen species (ROS) with lipids and proteins significantly contributes to the damage of crop plants under biotic and abiotic stresses. Here we have identified a stress-activated alfalfa gene encoding a novel plant NADPH-dependent aldose/aldehyde reductase that also exhibited characteristics of the homologous human enzyme. The recombinant alfalfa enzyme is active on 4-hydroxynon-2-enal, a known cytotoxic lipid peroxide degradation product. Ectopic synthesis of this enzyme in transgenic tobacco plants provided considerable tolerance against oxidative damage caused by paraquat and heavy metal treatment. These transformants could also resist a long period of water deficiency and exhibited improved recovery after rehydration. We found a reduced production of lipid peroxidation-derived reactive aldehydes in these transformed plants under different stresses. These studies reveal a new and efficient detoxification pathway in plants.     

Reduction in denitrification activity in field soils exposed to long term contamination by 2,4,6-trinitrotoluene (TNT)

Terrestrial sites contaminated with 2,4,6-trinitrotoluene (TNT) are a widespread and persistent problem and often contain non-vegetated areas with TNT concentrations well in excess of 1000 mg kg(-1). In this study, we examined the effect of TNT on denitrification activity in field soils, and compared the sensitivity of denitrifying enzymes to TNT. DNA probes assessed the prevalence of nirS, nirK and nosZ (encoding cd(1) or copper nitrite reductase and nitrous oxide reductase, respectively), denitrifying genotypes in the culturable and total microbial community. The nitrate (NaR), nitrite (NiR) and nitrous oxide (N(2)OR) reductase activities in field soil and in isolates were assessed by gas chromatography. The relative occurrence of the nirK, nirS or nosZ genotypes increased in the cultured community and in total uncultured community DNA as nitroaromatic concentrations increased. However, denitrifying activity decreased in response to increasing TNT concentrations, with an IC(50) for NaR+NiR+nitric oxide reductase (NOR) of 400 mg TNT kg(-1) soil and for N(2)OR of 26 mg TNT kg(-1) soil. The denitrifying activity of four soil isolates also decreased in response to TNT, with N(2)OR activity being three times more sensitive to TNT than NaR+NiR+NOR activity. Interestingly, there were 118 times more nirK isolates than nirS isolates in uncontaminated soil but only 1.5 times more in soil containing 17400 mg kg(-1) TNT. The results from this study indicated that TNT reduced denitrification activity in field soils, and N(2)OR was much more sensitive to TNT than NaR+NiR+NOR.     

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.     

Expression of bacterial biphenyl-chlorobiphenyl dioxygenase genes in tobacco plants

Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.     

In vivo emission of dinitrogen by earthworms via denitrifying bacteria in the gut

Earthworms emit the greenhouse gas nitrous oxide (N2O), and ingested denitrifiers in the gut appear to be the main source of this N2O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N2), the end product of complete denitrification. When [15N]nitrate was injected into the gut, the earthworms Aporrectodea caliginosa and Lumbricus terrestris emitted labeled N2 (and also labeled N2O) under in vivo conditions; emission of N2 by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N2 per h per g (fresh weight), respectively. Isolated gut contents also produced [15N]nitrate-derived N2 and N2O under anoxic conditions. N2 is formed by N2O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [15N]nitrate-derived N2 by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N2O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N2 (i.e., without added nitrate) by A. caliginosa and L. terrestris were 1.1 and 1.5 nmol N2 per h per g (fresh weight), respectively; these emission rates approximated that of N2O. These collective observations indicate that (i) earthworms emit N2 concomitant with the emission of N2O via the in situ activity of denitrifying bacteria in the gut and (ii) N2O is quantitatively an important denitrification-derived end product under in situ conditions.     

Anaerobic purification, characterization and preliminary mechanistic study of recombinant nitrous oxide reductase from Achromobacter cycloclastes

An overexpression system for nitrous oxide reductase (N(2)OR), an enzyme that catalyzes the conversion of N(2)O to N(2) and H(2)O, has been developed in Achromobacter cycloclastes. Anaerobically purified A. cycloclastes recombinant N(2)OR (AcN(2)OR) has on average 4.5 Cu and 1.2 S per monomer. Upon reduction by methyl viologen, AcN(2)OR displays a high specific activity: 124 U/mg at 25 degrees C. Anaerobically purified AcN(2)OR displays a unique absorption spectrum. UV-visible and EPR spectra, combined with kinetics studies, indicate that the as-purified form of the enzyme is predominately a mixture of the fully-reduced Cu(Z)=[4Cu(I)] state and the Cu(Z)=[3Cu(I).Cu(II)] state, with the latter readily reducible by reduced forms of viologens. CD spectra of the as-purified AcN(2)OR over a range of pH values reveal perturbations of the protein conformation induced by pH variations, although the principal secondary structure elements are largely unaltered. Further, the activity of AcN(2)OR in D(2)O is significantly decreased compared with that in H(2)O, indicative of a significant solvent isotope effect on N(2)O reduction. These data are in good agreement with conclusions reached in recent studies on the effect of pH on catalysis by N(2)OR [K. Fujita, D.M. Dooley, Inorg. Chem. 46 (2007) 613-615].     

In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.     

Nitrous oxide reductase genes (nosZ) of denitrifying microbial populations in soil and the earthworm gut are phylogenetically similar

Earthworms emit nitrous oxide (N2O) and dinitrogen (N2). It has been hypothesized that the in situ conditions of the earthworm gut activates ingested soil denitrifiers during gut passage and leads to these in vivo emissions (M. A. Horn, A. Schramm, and H. L. Drake, Appl. Environ. Microbiol. 69:1662-1669, 2003). This hypothesis implies that the denitrifiers in the earthworm gut are not endemic to the gut but rather are regular members of the soil denitrifier population. To test this hypothesis, the denitrifier populations of gut and soil from three different sites were comparatively assessed by sequence analysis of nosZ, the gene for the terminal enzyme in denitrification, N2O reductase. A total of 182 and 180 nosZ sequences were retrieved from gut and soil, respectively; coverage of gene libraries was 79 to 100%. Many of the nosZ sequences were heretofore unknown, clustered with known soil-derived sequences, or were related to N2O reductases of the genera Bradyrhizobium, Brucella, Dechloromonas, Flavobacterium, Pseudomonas, Ralstonia, and Sinorhizobium. Although the numbers of estimators for genotype richness of sequence data from the gut were higher than those of soil, only one gut-derived nosZ sequence did not group phylogenetically with any of the soil-derived nosZ sequences. Thus, the phylogenies of nosZ from gut and soil were not dissimilar, indicating that gut denitrifiers are soil derived.     

A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.     

Role of the Tat ransport system in nitrous oxide reductase translocation and cytochrome cd1 biosynthesis in Pseudomonas stutzeri

By transforming N2O to N2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N(2)O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N2O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.     

Phenotyping shows improved physiological traits and seed yield of transgenic wheat plants expressing the alfalfa aldose reductase under permanent drought stress

Members of the aldo–keto reductase family including aldose reductases are involved in antioxidant defense by metabolizing a wide range of lipid peroxidation-derived cytotoxic compounds. Therefore, we produced transgenic wheat genotypes over-expressing the cDNA of alfalfa aldose reductase gene. These plants consequently exhibit 1.5–4.3 times higher detoxification activity for the aldehyde substrate. Permanent drought stress was generated in the greenhouse by growing wheat plants in soil with 20 % water capacity. The control and stressed plants were monitored by a semi automatic phenotyping platform providing computer-controlled watering, digital and thermal imaging. Calculation of biomass values was based on the correlation (R ² = 0.7556) between fresh weight and green pixel-based shoot surface area. The green biomass production by plants of the three transgenic lines was 12–26–41 % higher than the non-transgenic plants’ grown under water limitation. Thermal imaging of stressed non-transgenic plants indicated an elevation in the leaf temperature. The thermal status of transformants was similar at both normal and suboptimal water regime. In drought, the transgenic plants used more water during the growing season. The described phenotyping platform provided a comprehensive data set demonstrating the improved physiological condition of the drought stressed transgenic wheat plants in the vegetative growth phase. In soil with reduced water capacity two transgenic genotypes showed higher seed weight per plant than the control non-transgenic one. Limitation of greenhouse-based phenotyping in analysis of yield potential is discussed.     

采用玉米Ubi-1启动子获得低拷贝转基因玉米植株

通过基因枪粒子轰击和草丁膦（PPT）选择获得可育的玉米转基因植株,并分析了外源基因在转化体中的拷贝数与启动子之间的关系。用玉米Ubi-1启动子驱动外源基因,玉米转化体中外源基因的拷贝数较低;可能的原因为Ubi-1启动子通过与其内部同源序列发生重组而被定点整合进玉米基因组,共转化的两种质粒DNA在整合至玉米染色体DNA之前已重构成为一个整体。结果显示使用某一植物自身基因的启动子可以降低外源基因在该物种转基因个体中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因玉米种子。