New Breeding Techniques for Greenhouse Gas ( GHG ) Mitigation : Plants May Express Nitrous Oxide Reductase

Nitrous oxide (N2O) is a potent greenhouse gas (GHG). Although it comprises only 0.03% of total GHGs produced, N2O makes a marked contribution to global warming. Much of the N2O in the atmosphere issues from incomplete bacterial denitrification processes acting on high levels of nitrogen (N) in the soil due to fertilizer usage. Using less fertilizer is the obvious solution for denitrification mitigation, but there is a significant drawback (especially where not enough N is available for the crop via N deposition, irrigation water, mineral soil N, or mineralization of organic matter): some crops require high-N fertilizer to produce the yields necessary to help feed the world’s increasing population. Alternatives for denitrification have considerable caveats. The long-standing promise of genetic modification for N fixation may be expanded now to enhance dissimilatory denitrification via genetic engineering. Biotechnology may solve what is thought to be a pivotal environmental challenge of the 21st century, reducing GHGs. Current approaches towards N2O mitigation are examined here, revealing an innovative solution for producing staple crops that can ‘crack’ N2O. The transfer of the bacterial nitrous oxide reductase gene (nosZ) into plants may herald the development of plants that express the nitrous oxide reductase enzyme (N2OR). This tactic would parallel the precedents of using the molecular toolkit innately offered by the soil microflora to reduce the environmental footprint of agriculture.


Introduction-Nitrous Oxide Continues to Bloom Unabated
Atmospheric nitrogen (N) deposition is a pressing matter for climate change scientists concerned with the increasing danger that nitrous oxide (N 2 O), a noxious greenhouse gas (GHG), poses.Reactive nitrogen (Nr)-ammonia (NH 3 ), nitrogen oxides (NO x ), nitrates (NO 3 − ), and N 2 O-enters the biosphere from its original form of atmospheric N as at least three derivatives: gas, dry deposit, and precipitation (wet deposition) [1,2].The sources of N 2 O are largely anthropogenic [3].Many crops must receive N-based fertilizer to reach yield targets, which is supplied by inorganic fertilizers and animal manure [4].In an effort to boost the yield in crop staples like wheat, corn, and soybeans, farmers apply N fertilizers at rates and times that are not always properly synchronized with crop demand [5].While crops thrive when fertilized, experimental analysis has demonstrated that up to 40% of fertilizer N can be lost via leaching [6,7].Other routes of N loss include soil erosion, NH 3 volatilization and oxidation, and bacterial/fungal denitrification [8], although N losses through NH 3 volatilization are higher than those via N leaching [9].Around 62% of total global N 2 O issues from natural and agricultural soils, and the bulk of this production, mainly results from the processes of bacterial nitrification and denitrification [10].
Nr compounds enter the atmosphere through biological processes, but the invention of the Haber-Bosch process in 1908 was a critical moment for the sudden increase in Nr and GHG production globally [11].This process of artificial N-fixation allowed for the large-scale reduction of N 2 to NH 3 , producing massive amounts of synthetic N-based fertilizers that supported dramatic increases in high-yield farming [12].This process now accounts for 80% of anthropogenic N-fixation (the remaining 20% resulting from combustion [13], with anthropogenic N-fixation in turn accounting for 60% of global N-fixation [14]).Haber-Bosch remains the industry standard synthetic N fertilizer today and as a result, has contributed to the ~2% increase in atmospheric levels of N 2 O [15,16].This effect is also magnified by the global emissions of N 2 O produced by fossil fuel combustion [17] and the natural ability of legumes to fix N through symbiotic relationships with soil bacteria [18].
N 2 O is the third most prevalent GHG, behind carbon dioxide (CO 2 ) and methane (CH 4 ) [19].The concentration of this gas in the atmosphere has been steadily increasing since the early 1900s (Figure 1), and it is 265 times more radiative than CO 2 [19].N 2 O also has an atmospheric lifetime of 121 years; by comparison, CH 4 has an atmospheric lifetime of only 12 years, but CO 2 also has a long half-life and can take anywhere from 20-200 years to be absorbed by the ocean [19], compounding the 'greenhouse gas' effect.Since chlorofluorocarbons (CFCs) were banned in 1989, N 2 O has become the leading cause of ozone layer depletion [20].
Climate 2018, 6, x FOR PEER REVIEW 2 of 16 40% of fertilizer N can be lost via leaching [6,7].Other routes of N loss include soil erosion, NH3 volatilization and oxidation, and bacterial/fungal denitrification [8], although N losses through NH3 volatilization are higher than those via N leaching [9].Around 62% of total global N2O issues from natural and agricultural soils, and the bulk of this production, mainly results from the processes of bacterial nitrification and denitrification [10].
Nr compounds enter the atmosphere through biological processes, but the invention of the Haber-Bosch process in 1908 was a critical moment for the sudden increase in Nr and GHG production globally [11].This process of artificial N-fixation allowed for the large-scale reduction of N2 to NH3, producing massive amounts of synthetic N-based fertilizers that supported dramatic increases in high-yield farming [12].This process now accounts for 80% of anthropogenic N-fixation (the remaining 20% resulting from combustion [13], with anthropogenic N-fixation in turn accounting for 60% of global N-fixation [14]).Haber-Bosch remains the industry standard synthetic N fertilizer today and as a result, has contributed to the ~2% increase in atmospheric levels of N2O [15,16].This effect is also magnified by the global emissions of N2O produced by fossil fuel combustion [17] and the natural ability of legumes to fix N through symbiotic relationships with soil bacteria [18].
N2O is the third most prevalent GHG, behind carbon dioxide (CO2) and methane (CH4) [19].The concentration of this gas in the atmosphere has been steadily increasing since the early 1900s (Figure 1), and it is 265 times more radiative than CO2 [19].N2O also has an atmospheric lifetime of 121 years; by comparison, CH4 has an atmospheric lifetime of only 12 years, but CO2 also has a long half-life and can take anywhere from 20-200 years to be absorbed by the ocean [19], compounding the 'greenhouse gas' effect.Since chlorofluorocarbons (CFCs) were banned in 1989, N2O has become the leading cause of ozone layer depletion [20].N2O emission results from the coupled oxidation and reduction of N performed by heterotrophic [21] (and some autotrophic) soil proteobacteria: (1) the nitrification pathway is catalyzed by autotrophs (Nitrosomonas spp.and other genera [22]) and also heterotrophs, and involves the oxidation of NH3/ammonium(NH4 + ) to nitrite (NO2 − ) [23] and nitric oxide (NO) [24]), which is followed by the oxidation of NO2 − to NO3 − by Nitrobacter spp.[25]; and (2) the denitrification pathway, whereby NO3 − is reduced to N2O and ultimately inert N2 gas [26].As many as a third of soil bacterial species [27] lack the nosZ gene that reduces N2O to inert N2 [28], which leads to a sizeable amount of incomplete denitrification reactions and the subsequent buildup of N2O since it is an obligate intermediate [29].This N2O diffuses out of the soil and into the atmosphere, contributing to the greenhouse effect, contaminating water, and leading to serious human health implications [30,31].N 2 O emission results from the coupled oxidation and reduction of N performed by heterotrophic [21] (and some autotrophic) soil proteobacteria: (1) the nitrification pathway is catalyzed by autotrophs (Nitrosomonas spp.and other genera [22]) and also heterotrophs, and involves the oxidation of NH 3 /ammonium(NH 4 + ) to nitrite (NO 2 − ) [23] and nitric oxide (NO) [24]), which is followed by the oxidation of NO 2 − to NO 3 − by Nitrobacter spp.[25]; and (2) the denitrification pathway, whereby NO 3 − is reduced to N 2 O and ultimately inert N 2 gas [26].As many as a third of soil bacterial species [27] lack the nosZ gene that reduces N 2 O to inert N 2 [28], which leads to a sizeable amount of incomplete denitrification reactions and the subsequent buildup of N 2 O since it is an obligate intermediate [29].This N 2 O diffuses out of the soil and into the atmosphere, contributing to the greenhouse effect, contaminating water, and leading to serious human health implications [30,31].

Combating GHGs: Current N 2 O Mitigation Strategies and Limitations
Demands for crop-borne food must be met, and so researchers must address the hazards of N-based fertilizers [32].There are multiple N 2 O mitigation strategies either currently in commercial use or in development (summarized in Table 1).

Mechanism of Action Pros Cons
(1) Conservation tillage and crop rotation [33] Tillage, rotation of N-fixing crops, cover cropping [33] Prevent NH 3 volatilization and eventual N 2 O emissions [34,35] Unreliable N 2 O mitigation [36,37].Yield reduction [38].Not effective at scrubbing N 2 O from the air (2) Best management practices (BMPs) [39] Correct source, placement, time, and rate of fertilization [40].Proper irrigation (fertigation) [41] Proven to reduce N 2 O emissions [41] and other N losses [42] Technical constraints [43] (3) EENFs [44] Multiple types: stable, short-release (SRFs), and constant-release (CRFs); rely on enrichment of chemical inhibitors or coated N-compounds that are released into the soil over a period of time [45]; urease inhibitors (UIs) [46] Proven to reduce N 2 O emissions [47,48] Inconsistent yields from year to year [48].More expensive than standard N fertilizers [49].Long lifetime of N-compounds in soil can lead to NH 3 volatilization [50,51].(1) Conservation tillage and crop rotation.Mechanical incorporation (tillage) of N-based fertilizer into the soil may also be effective [68], but this is affected by many other parameters, such as the method of N application (i.e., broadcast vs surface urea ammonium nitrate).These techniques also result in a reduced yield [38]. Conservation tillage increases N 2 O emissions compared with no-till and conventional tillage techniques using broadcast application, while tillage in general does not reduce N 2 O emissions produced from surface urea ammonium nitrate-treated fields [69].Other studies have shown that conservation tillage reduces N 2 O emissions [70], underscoring the lack of reliability of this N management technique [36,37].Crop rotation with N-acquisitive plant species can also reduce N 2 O emissions following the application of high N-fertilizer treatment [33]; cover cropping can also control N 2 O emissions, but the results are often variable and in some cases can increase N 2 O emissions [71]; (2) Best management practices (BMP) [39].Such nitrogen use efficiency techniques are myriad and involve simple steps such as proper fertilizer placement, timing of fertilizer application, the right type of N-compound, and so on.Others involve the proper incorporation of N-compounds into the soil so that they may be taken up by the plant more effectively and will be less likely to volatilize [72].Fertigation, a technique involving careful irrigation of fields following the application of N fertilizer, is effective at mitigating N 2 O emissions [41].Such knowledge-based N management practices have been shown to be effective at both increasing crop yield and reducing immediate N 2 O emissions [73], but some approaches may also increase N 2 O production in the long term [55].Their effectiveness also depends heavily on proper practices put in place by the farmers themselves, which requires proper training [43]; (3) Fertilizer management using enhanced efficiency nitrogen fertilizers (EENFs).These fertilizer cocktails are concocted in such a way that they prevent the volatilization of NH 3 and inhibit nitrification/denitrification [46].EENFs generally fall into one of three categories: (a) stabilized fertilizers, which contain nitrification and/or urease inhibitors; (b) slow-release fertilizers (SRFs), whereby the N source in the fertilizer is released over time from encapsulated granules, although the release rates can be variable; and (c) controlled-release fertilizers (CRFs), where the release rate is constant [45].Urease inhibitors (UIs) are also a common EENF component.N-(n-butyl) thiophosphoric triamide (NBPT), phenylphosphorodiamidate (PPD), and hydroquinone are used worldwide and act by inhibiting the bacterial hydrolysis of urea into NH 3 in fertilizer [46,74,75].UIs are typically used in conjunction with nitrification inhibitor (NIs) for maximum effectiveness [76,77], but NBPT alone can reduce N 2 O emissions from N-treated soil [78].There is controversy regarding the effectiveness of EENFs; while reductions in N 2 O emissions from the soil have been recorded [47,48], recent studies have shown that crop yields are only marginally higher when EENFs are used in place of standard N fertilizers [79].
Those studies that demonstrated reduced N 2 O emissions also reported inconsistent results from year to year [50].Questionable effectiveness notwithstanding, EENFs are more expensive than conventional N-containing fertilizers and require special handling and storage [49,80], which are all features that make these fertilizers less attractive to farmers; (4) Synthetic N 2 O mitigators.Synthetic nitrification inhibitors (SNIs) and UIs are both used in EENFs and can be applied to crops in conjunction with standard N fertilizer.NIs inhibit the activity of Nitrosomonas to block the nitrification of N in fertilizer (the oxidation of NH 3 to hydroxylamine via ammonia monooxygenase (AMO)) [23,52].The efficacy of the inhibitors is also dependent on environmental conditions, as they are unstable; 3,4-dimethylpyrazole phosphate (DMPP), for example, exhibited reduced activity in hot, dry conditions [81].The use of these inhibitors can also lead to less than desirable results: DMPP and 3-methylpyrazole 1,2,4-triazole (3MP + TZ) have been shown to increase N 2 O emissions in vegetable crop systems, as the inhibitors promote the buildup of N in the fraction of the soil most available to bacteria during the breakdown of vegetative matter.Synthetic denitrification inhibitors (SDIs) suppress denitrification via unknown mechanisms [82], although some are known to inhibit the activity of fungal copper reductase [83].
SDIs nitrapyrin [84], toluidine [54], and acetylene [44] all effectively mitigate N 2 O emission, albeit with toxic side-effects [55], and they do not technically inhibit nitric oxide reductase; (5) Biological N 2 O mitigators.This category is comprised of compounds produced by plants that inhibit enzymes in either the bacterial nitrification or denitrification pathway.The exploitation of such inhibiting root exudates is another intriguing approach towards N 2 O mitigation [82].Biological nitrification inhibitors (BNIs) are compounds that block the activity of NO 2 − producing enzymes.
The roots of the tropical grass Brachiaria humidicola exude brachialactone, a compound that can mitigate N 2 O emission from soil [85].Attempts at developing BNI-producing cultivated wheat by crossing Triticum aestivum with BNI-producer Leymus racemosus, a wild wheat, have imparted some BNI activity, but also made the lines susceptible to rust infection [86].The use of BNIs as an effective N 2 O mitigator is also severely limited by the fact that the enactor of nitrification is a plant itself and cannot be applied to growing crops, although growing B. humidicola in rotation with maize saw a four-fold increase in yield [87]. Biological denitrification inhibitors (BDIs) are a relatively new discovery.Currently, the only example of such an inhibitor is the procyanidin produced by the invasive Fallopia spp.(Asian knotweed).This compound has been demonstrated to be an allosteric inhibitor of Pseudomonas brassicacearum nitrate reductase and while it does reduce denitrification in the soil, it has not yet been proven to mitigate N 2 O levels [57]; (6) Microbial bioremediation [88].The success of N fertilizer management techniques and proper irrigation is largely due to the creation of a microsphere conducive to denitrifying bacteria flourishing [89].Proper water table management techniques can promote the growth of N 2 O-cracking bacteria in the soil and reduce N 2 O emissions from the managed soil regions [59].
Another type of microbial bioremediation takes advantage of the ability of certain bacterial species to inhabit the root nodules of leguminous crops.Field peas [62], broad beans [90], and soybean [63] house bacteria (or rhizobia) that fix N and, unfortunately, also produce N 2 O gas.While maintaining the rhizosphere, N 2 O emissions can be mitigated by inoculating the roots of leguminous plants with rhizobia modified to express higher levels of a bacterial N 2 O-cracking enzyme [60].Genetically engineered strains of Bradyrhizobium japonicum have been used to inoculate the roots of soybean and reduced N 2 O emissions [61].Needless to say, this method is far more effective on crops that naturally cultivate a rhizosphere of N 2 O-reducing microorganisms.
It is also another technique that cannot target atmospheric N 2 O; (7) Rhizosecretion.This is a biotechnology-based approach, involving the transformation of amenable crop plants with genes expressing recombinant bacterial proteins that reduce N 2 O by secreting N 2 O-cracking enzymes [64,91].Plants can be engineered to express proteins under the control of promoters that induce hairy root formation in plants.This rooting response results from the presence of the rolABCD genes from Agrobacterium rhizogenes, the bacterium that induces hairy root disease [92].The rhizosecretion expression system harnesses the ability of A. rhizogenes to both target gene expression to the roots and to increase root biomass, subsequently increasing the amount of recombinant protein secreted into the soil [91].Tobacco plants expressing a bacterial N 2 O-cracking enzyme tagged for secretion under the control of the A. rhizogenes rolD promoter have been successful in demonstrating reducing activity [64,93].Gas analysis was not performed to confirm that these plants mitigated N 2 O emission.Ultimately, this approach arrives at a similar problem as other 'rhizoremediative' techniques: the N 2 O-reducing ability of such a transgenic plant would be limited to the rhizosphere.This system would not have access to the bulk of N 2 O gas, much of which comes from other sources; (8) Atmospheric phytoremediation using genetically engineered plants.The potential of transgenic plants for environmental phytoremediation is well-documented: several fungal and bacterial oxidoreductases have been functionally expressed in plants as phytoremediation strategies including pentaerythritol tetranitrate reductase [94], mercuric reductase [95], and arsenate reductase [96].This type of plant-based decontamination strategy provides advantages, such as stable cultivation and control of the remediant organism and atmospheric exposure of the gas-cracking enzyme [97].
Atmospheric phytoremediation may ameliorate problems created by the other N 2 O mitigation strategies described.The concept here is to develop crops with the ability to "crack" N 2 O in both the soil and the atmosphere by incorporating the bacterial nosZ gene into their genomes.This gene encodes the nitrous oxide reductase enzyme (N 2 OR), an oxidoreductase that catalyzes the removal of N 2 O from the atmosphere, a process performed naturally by both denitrifying and non-denitrifying bacteria in the soil [98].While conventional N 2 O mitigation strategies aim to control N 2 O production at earlier stages in the nitrification/denitrification pathway, this approach will target the atmospheric sum of N 2 O emitted by all sources (Figure 2).Atmospheric phytoremediation may ameliorate problems created by the other N2O mitigation strategies described.The concept here is to develop crops with the ability to "crack" N2O in both the soil and the atmosphere by incorporating the bacterial nosZ gene into their genomes.This gene encodes the nitrous oxide reductase enzyme (N2OR), an oxidoreductase that catalyzes the removal of N2O from the atmosphere, a process performed naturally by both denitrifying and non-denitrifying bacteria in the soil [98].While conventional N2O mitigation strategies aim to control N2O production at earlier stages in the nitrification/denitrification pathway, this approach will target the atmospheric sum of N2O emitted by all sources (Figure 2).

Nitrous Oxide Reductase-An Orphaned Soil Protein?
The nosZ gene can be categorized as either 'clade I' or 'clade II' based on sequence and nos operon organization, including the lack of an accessory nosR gene in the clade II members [99].Clade II nosZ genes are also known as 'atypical' nos genes since they are found in non-denitrifying bacterial species.The N2OR enzyme that the clade II gene encodes catalyzes the same reaction performed by the clade I-encoded enzyme, but has a higher affinity for N2O [100], an important factor to consider when conceptualizing the development of an nosZ-expressing plant.
N2OR is a multi-copper protein encoded by the nosZ gene (which is accompanied by an operon cluster of additional genes (nosRDFYL) [101]) and is the only enzyme that can catalyze the conversion of N2O into N2.The first active N2OR was characterized from the soil bacterium Pseudomonas stutzeri and similar enzyme structures were resolved in bacterial species Marinobacter hydrocarbonoclasticus (formerly Pseudomonas nautica) (Figure 3), Achromobacter cyclocastes, and Paracoccus denitrificans.N2OR is a head-to-tail homodimer and each monomer contains two domains: an electron transferring domain (binuclear CuA centre) and a catalytic domain (tetranuclear CuZ centre) [102].There is some variability between the species regarding CuZ bridging and cupric coordination in the catalytic centre, suggesting that N2OR substrate binding is species-specific.Regardless, the catalytic mechanism of N2O reduction in N2OR is still unclear [103].1. Green arrows show nitrification and purple arrows represent denitrification reactions.BDI, biological denitrification inhibitor; BMPs, best management practices; BNI, biological nitrification inhibitor; EENFs, enhanced efficiency nitrogen fertilizers; SDI, synthetic denitrification inhibitor; SNI, synthetic nitrification inhibitor; UI, urease inhibitor.O Encircled numbers refer to Table 1 strategies.

Nitrous Oxide Reductase-An Orphaned Soil Protein?
The nosZ gene can be categorized as either 'clade I' or 'clade II' based on sequence and nos operon organization, including the lack of an accessory nosR gene in the clade II members [99].Clade II nosZ genes are also known as 'atypical' nos genes since they are found in non-denitrifying bacterial species.The N 2 OR enzyme that the clade II gene encodes catalyzes the same reaction performed by the clade I-encoded enzyme, but has a higher affinity for N 2 O [100], an important factor to consider when conceptualizing the development of an nosZ-expressing plant.
N 2 OR is a multi-copper protein encoded by the nosZ gene (which is accompanied by an operon cluster of additional genes (nosRDFYL) [101]) and is the only enzyme that can catalyze the conversion of N 2 O into N 2 .The first active N 2 OR was characterized from the soil bacterium Pseudomonas stutzeri and similar enzyme structures were resolved in bacterial species Marinobacter hydrocarbonoclasticus (formerly Pseudomonas nautica) (Figure 3), Achromobacter cyclocastes, and Paracoccus denitrificans.N 2 OR is a head-to-tail homodimer and each monomer contains two domains: an electron transferring domain (binuclear Cu A centre) and a catalytic domain (tetranuclear Cu Z centre) [102].There is some variability between the species regarding Cu Z bridging and cupric coordination in the catalytic centre, suggesting that N 2 OR substrate binding is species-specific.Regardless, the catalytic mechanism of N 2 O reduction in N 2 OR is still unclear [103].N2OR is organized as a head-to-tail homodimer.Monomers are coloured differently so that they can be distinguished.In both monomers, the N-terminal domain is dark-coloured.The N-terminal domain forms a seven-bladed β-propeller fold that coordinates the catalytic tetranuclear active site CuZ through seven histidine residues at its hub.The C-terminal domain forms a cupredoxin fold and binds the dinuclear mixed-valent CuA centre [104].
The proven ability of N2OR to "crack" the N2O molecule raises the question of why the protein has not yet been incorporated into a commercially available transgenic cropping choice for environmentally motivated producers and small-plot farmers.Work has been done on this gene and its potential role in plant biotechnology since it was originally isolated in 1998 from the anaerobic soil bacterium A. cyclocastes [105,106], but it has yet to be converted into a commercially valuable tool.In this sense, N2OR may be considered an "orphaned" protein, neglected among a veritable molecular toolkit of genes in the soil microflora [107,108].Such forays into integrating soil and air sciences are demonstrative of the possibilities of what the soil microbiome offers biotechnologists [27]; it has already been discussed regarding the N-management possibilities offered by the microbiome and the current practice of 'bioprospecting' is also revealing a plethora of beneficial bacterial products, which is only accelerating thanks to whole-system approaches involving computational analyses [109].
Web of Science reports that between 1900 and 1991, there are no records binned under the combined topics "nitrous oxide reductase" and "microb*".The scientific literature blossomed from its first occurrence of 1992 to the present day, witnessing at least 175 publications dealing with the science of this important enzyme in our total environment.The scientific community waited until 1996 to start discussing denitrification in a plant context, according to these same search terms.With the search terms "nitrous oxide reductase" and "plant", the scientific record shows that soil microbiologists have taken a growing interest in the movement of N into the atmosphere (Figure 4).It is encouraging to note that in the same time period, the linkage between N2OR and climate began its nascent phase.N 2 OR is organized as a head-to-tail homodimer.Monomers are coloured differently so that they can be distinguished.In both monomers, the N-terminal domain is dark-coloured.The N-terminal domain forms a seven-bladed β-propeller fold that coordinates the catalytic tetranuclear active site Cu Z through seven histidine residues at its hub.The C-terminal domain forms a cupredoxin fold and binds the dinuclear mixed-valent Cu A centre [104].
The proven ability of N 2 OR to "crack" the N 2 O molecule raises the question of why the protein has not yet been incorporated into a commercially available transgenic cropping choice for environmentally motivated producers and small-plot farmers.Work has been done on this gene and its potential role in plant biotechnology since it was originally isolated in 1998 from the anaerobic soil bacterium A. cyclocastes [105,106], but it has yet to be converted into a commercially valuable tool.In this sense, N 2 OR may be considered an "orphaned" protein, neglected among a veritable molecular toolkit of genes in the soil microflora [107,108].Such forays into integrating soil and air sciences are demonstrative of the possibilities of what the soil microbiome offers biotechnologists [27]; it has already been discussed regarding the N-management possibilities offered by the microbiome and the current practice of 'bioprospecting' is also revealing a plethora of beneficial bacterial products, which is only accelerating thanks to whole-system approaches involving computational analyses [109].
Web of Science reports that between 1900 and 1991, there are no records binned under the combined topics "nitrous oxide reductase" and "microb*".The scientific literature blossomed from its first occurrence of 1992 to the present day, witnessing at least 175 publications dealing with the science of this important enzyme in our total environment.The scientific community waited until 1996 to start discussing denitrification in a plant context, according to these same search terms.With the search terms "nitrous oxide reductase" and "plant", the scientific record shows that soil microbiologists have taken a growing interest in the movement of N into the atmosphere (Figure 4).It is encouraging to note that in the same time period, the linkage between N 2 OR and climate began its nascent phase.

Catch Me If You Can: Can Plants Catalytically Convert N2O in planta?
Rather than a 'cat and robin redbreast' conundrum, we are confronted with an opportunity to deploy protein engineering to ensure that more N2OR molecules are attracted to the substrate binding site of the copper enzyme.Protein engineering offers ways to sidestep the challenges of expressing a complex bacterial protein in a plant [110].There are potential issues with a recombinant metalloprotein like N2OR, such as whether the ABC transporter can assemble within a plant cell, or the plant can incorporate copper into the electron transferring and catalytic domains [111,112].It is possible to re-engineer N2OR and produce a functional product [66], so there is precedent for designing an artificial metalloenzyme through rational protein design.This approach may be key to engineering a plant-compatible N2OR protein.
A principle challenge associated with imparting N2OR functionality to plants is that transforming the nosZ sequence alone may not be effective [113]; in P. stutzeri, the transcription of nosZ was dependent on the nosDFY genes being expressed, as they encode components of a putative ABC transporter system for the biogenesis of the CuZ centre [114].Therefore, catalytically active N2OR may not be produced when only nosZ is expressed in a heterologous host [28].Nevertheless, a model N2O-expressing plant has been engineered [64,93].The clade I nosZ gene from soil bacterium Pseudomonas stutzeri was successfully expressed in a heterologous system-in this case, the tobacco plant (Nicotiana tabacum).In those proof-of-concept experiments the nosZ-expressing tobacco plants reduced 826 μg N2O/min/gram of leaf tissue [115].Assuming the tobacco yield to be 0.50 tonne/ha [116], the calculated N2O-cracking ability of the nosZ-expressing tobacco could be as high as 600 kg of N2O/ha/day [115], or 60 tonnes/ha/year (100 day growing season).This value surpasses the calculated N2O flux of 0.05-1.98kg N2O/ha/year [117].In other words, if every tobacco plant in the world produced N2OR, this industrial crop (6.6 million tonnes were produced worldwide in 2016 [118]) could conceivably crack 785 Tg of N2O (1 Tg = 1 million metric tonnes) during an average growing season of 100 days, far surpassing the estimated ~30 Tg of N2O emitted per year [119].Such catalytic capacity would give the 'Stop Smoking' campaigns a whole new flavour.Analytics).Publications by key word vs "nitrous oxide reductase" from 1990 to 2018.The orange line indicates "nitrous oxide reductase" + "microb*"; green: "nitrous oxide reductase" + "plant"; blue: "nitrous oxide reductase" + "climat*".

Catch Me If You Can: Can Plants Catalytically Convert N 2 O in planta?
Rather than a 'cat and robin redbreast' conundrum, we are confronted with an opportunity to deploy protein engineering to ensure that more N 2 OR molecules are attracted to the substrate binding site of the copper enzyme.Protein engineering offers ways to sidestep the challenges of expressing a complex bacterial protein in a plant [110].There are potential issues with a recombinant metalloprotein like N 2 OR, such as whether the ABC transporter can assemble within a plant cell, or the plant can incorporate copper into the electron transferring and catalytic domains [111,112].It is possible to re-engineer N 2 OR and produce a functional product [66], so there is precedent for designing an artificial metalloenzyme through rational protein design.This approach may be key to engineering a plant-compatible N 2 OR protein.
A principle challenge associated with imparting N 2 OR functionality to plants is that transforming the nosZ sequence alone may not be effective [113]; in P. stutzeri, the transcription of nosZ was dependent on the nosDFY genes being expressed, as they encode components of a putative ABC transporter system for the biogenesis of the Cu Z centre [114].Therefore, catalytically active N 2 OR may not be produced when only nosZ is expressed in a heterologous host [28].Nevertheless, a model N 2 O-expressing plant has been engineered [64,93].The clade I nosZ gene from soil bacterium Pseudomonas stutzeri was successfully expressed in a heterologous system-in this case, the tobacco plant (Nicotiana tabacum).In those proof-of-concept experiments the nosZ-expressing tobacco plants reduced 826 µg N 2 O/min/gram of leaf tissue [115].Assuming the tobacco yield to be 0.50 tonne/ha [116], the calculated N 2 O-cracking ability of the nosZ-expressing tobacco could be as high as 600 kg of N 2 O/ha/day [115], or 60 tonnes/ha/year (100 day growing season).This value surpasses the calculated N 2 O flux of 0.05-1.98kg N 2 O/ha/year [117].In other words, if every tobacco plant in the world produced N 2 OR, this industrial crop (6.6 million tonnes were produced worldwide in 2016 [118]) could conceivably crack 785 Tg of N 2 O (1 Tg = 1 million metric tonnes) during an average growing season of 100 days, far surpassing the estimated ~30 Tg of N 2 O emitted per year [119].Such catalytic capacity would give the 'Stop Smoking' campaigns a whole new flavour.
Although these transgenic plants produced a functional N 2 OR enzyme, no gas analysis was performed to quantifiably ensure that these plants could reduce N 2 O to N 2 using a recombinant N 2 OR.In the future, it is imperative that such analyses be performed to properly judge the efficacy of such a gene-stacking trait system for atmospheric phytoremediation.
An associated issue rests with P. stutzeri being an anaerobic species that produces enzymes that function optimally in a low-oxygen environment.While expressing nosZ in plants to reduce N 2 O appears to be an elegant solution, the N 2 OR enzyme was not evolutionarily engineered to be functional in the presence of oxygen.Most soil bacteria that produce N 2 OR do so in an anaerobic environment [102].
In the past five years, studies have identified several prokaryotic species that may express an oxygen-compatible N OR.Aerobic N 2 O reducers may be undertaking an important role in mitigating the amounts of N 2 O emitted to the atmosphere in events of oxic-to-anoxic transitions, but these systems have not yet been validated in plants.Here, we discuss two candidates for an oxygen-compatible nosZ expression system: clade II-nosZ member Gemmatimonas aurantiaca gen nov., spp.nov.strain T-27, a polyphosphate-accumulating soil aerobe that is strongly represented in many oxygen-rich soil samples [120]; and Azospira oryzae, another clade II N-fixing bacterium originally isolated from the roots of rice (Oryza sativa) [121].N 2 O reduction by the G. aurantiaca strain T-27 was observed in both the absence and presence of oxygen [120].The inability of this organism to consume N 2 O in the complete absence of oxygen and the observed oxygen-induced activation of nosZ expression compels one to consider in planta overexpression, whereby the diurnal fluctuation of photosynthetic oxygen production may offer an egress for N 2 O accumulation.The A. oryzae strains I09 and I13 also show more rapid N 2 OR recovery rates and tolerance against oxygen inhibition than P. stutzeri [121] and so may be appropriate candidates for crop plant transformation and N 2 OR expression.
If the ideal nosZ sequence were to be identified and transformed into commercially important crop plants, the benefits would be numerous and profound: seed-borne GHG technology foresees the transgenic cassette passed on from generation to generation, meaning that constant application of the beneficial catalyst would not be required (as with NI application and rhizoremediation); the expression of nosZ in the aerial tissues of the plants allows the reducing enzyme to confront N 2 O much more easily than when the enzyme is expressed in the soil.

Novel Breeding Task: "Gas Cracking" Plants
The challenge of expressing heterologous bacterial proteins in plants necessitates codon optimization due to differences in GC content and codon bias with eukaryotes [122].Altering the codon bias (or applying 'directed evolution' [123]) of a bacterial gene to be expressed in plants has been highly successful: P. stutzeri nosZ in tobacco [115], 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from Agrobacterium tumefaciens in Roundup Ready crops [124], and Bacillus thuringiensis Cry genes in maize [125] and rice [126].Indeed, the global advance promulgating engineered crops is pillared on today's artificial intelligence-guided plant codon optimization rules offered by both large and small boutique DNA houses.However, there has been success expressing native bacterial sequences in plants, i.e., in the case of cotton expressing the native sequence of the P. stutzeri gene ptxd (PHOSPHONATE DEHYDROGENASE) [127,128].One can dare to fathom how a universally-functional nosZ expression system could conceivably redirect some aspects of GHG mitigation research.Such a plant transformation cassette could theoretically be applied to any plant-wheat, rice, soybean, peat moss [129]-recruiting these species for the purpose of denitrification mitigation.
Even with an effective nosZ expression system, there are additional challenges in developing nosZ-expressing plant lines.There are relatively few powerful monocot-optimized expression systems available [130] (although Bt corn, LibertyLink wheat, and Roundup Ready wheat can attest to the effectiveness of the 35S promoter system in monocots), and there is difficulty in transforming monocots [65].With the advent of new plant transformation technologies like the soil bacterium Ochrobactrum haywardense [131] and the BABYBOOM/WUSCHEL2 system [132], the production of genetically modified crops with stacked or pyramided GHG genes may be expedited in the near future.

Conclusions-Challenges to the Future Success of nosZ
We must address what may be the greatest challenge of all for the modern molecular plant breeder: convincing the general public that transgenic crops may be beneficial for all the plant-planet's denizens, as modified crops that enter the food stream may appear unpopular in some boroughs.Regardless, there is a clear, urgent need to control soil N 2 O losses due to the detrimental effects of this potent GHG in the atmosphere.Climate-smart crops should be given a crack at directly addressing this issue and tackling climate change.Such GHG-reducing plant lines, endowed with the ability to catalytically "crack" N 2 O in the air, could be vital in the battle to shift public perception towards the acceptance of "GMOs" in agricultural research.
Involvement of N 2 O in climate change and global warming has been the subject of increasing investigations due to its potential heat-trapping properties [3].N 2 O emission from soil is primarily the result of an incomplete enzymatic reaction which is mediated by the bacterial enzyme, N 2 OR [98].Therefore, in the late 1990s [105,106], the development of N 2 OR-positive transgenic plants was proposed as an environmental phytoremediation strategy with promise to remove N 2 O from soil and the atmosphere (Figure 2).However, producing a foreign protein in a plant cell is often a serious challenge.For example, different codon usage [133] and cellular properties between eukaryotic and prokaryotic cells are considered as unknown aspects of this strategy.At least two key questions need to be addressed in future studies to probe the probability for success of this green gene de-toxic tactic for accelerating the destruction of nitrous oxide via canopy catalysis: (1) Which candidate is the best source-organism to donate nosZ sequence for plant transformation?Activity of bacterial N 2 OR is associated with the anaerobic conditions in soil [101], whereas the plant cell is mostly an aerobic environment.Photosynthesis and respiration cause different levels of oxygen content in plant cells in a diurnal cycle which is not consistent with the enzymatic activity of N 2 OR in anaerobic soil bacteria.Therefore, selecting obligate or facultative aerobic bacteria containing active N 2 OR enzymes as 'the source code' would be pivotal; (2) Which plant cell compartment is the best destination for targeting N 2 OR accumulation?The native enzyme N 2 OR in bacteria is directed to the periplasm, where Cu chaperones provide enough Cu for the assembly of metal centres [134].The absence of periplasmic space in plant cells reinforces the notion that subcellular localization of N 2 OR may influence its enzymatic activity in planta.Moreover, the important role of Cu in the functional assembly of N 2 OR posits whether the transformation of bacterial nosDFY, along with nosZ, is essential for a functional enzyme.Urgent exploration of how the cellular pool of metal nutrients and proteins (pseudo chaperones) in eukaryotic cells may suffice to activate N 2 OR in planta may compel the use of such climate-smart plants.
Author Contributions: J.J.D. constructed the initial paper draft; Q.-y.S., J.J.D., S.W., A.E.H., M.N., and I.A. contributed to study design and data analysis; J.J.D., S.W., and I.A. wrote the paper with input from A.E.H., and M.N.; M.N. constructed Figure 2; Q.-y.S. conceptualized the original approach to amplifying soil denitrification in planta; I.A. coordinated and supervised the project; all authors contributed to and approved the final draft.
Funding: This research was funded by a grant from the Government of Ontario in 2018.Q.-Y.S. is a grateful recipient of The Rockefeller Foundation's Career Biotechnology Fellowship.

Figure 1 .
Figure 1.GHG levels since 1850.The green line represents the increase in CO2 concentration since 1850; the orange line represents the increase in CH4 concentration since 1850; lastly, the red line represents the increase in N2O since 1850 [19].

Figure 1 .
Figure 1.GHG levels since 1850.The green line represents the increase in CO 2 concentration since 1850; the orange line represents the increase in CH 4 concentration since 1850; lastly, the red line represents the increase in N 2 O since 1850 [19].

Figure 2 .
Figure 2. Nitrification-denitrification pathway and overview of current N2O mitigation strategies.Orange arrows and lines show eight N2O mitigation strategies described in Table1.Green arrows show nitrification and purple arrows represent denitrification reactions.BDI, biological denitrification inhibitor; BMPs, best management practices; BNI, biological nitrification inhibitor; EENFs, enhanced efficiency nitrogen fertilizers; SDI, synthetic denitrification inhibitor; SNI, synthetic nitrification inhibitor; UI, urease inhibitor.O Encircled numbers refer to Table1strategies.

Figure 2 .
Figure 2. Nitrification-denitrification pathway and overview of current N 2 O mitigation strategies.Orange arrows and lines show eight N 2 O mitigation strategies described inTable 1. Green arrows show nitrification and purple arrows represent denitrification reactions.BDI, biological denitrification inhibitor; BMPs, best management practices; BNI, biological nitrification inhibitor; EENFs, enhanced efficiency nitrogen fertilizers; SDI, synthetic denitrification inhibitor; SNI, synthetic nitrification inhibitor; UI, urease inhibitor.O Encircled numbers refer to Table 1 strategies.

Figure 3 .
Figure 3. Structure of Marinobacter hydrocarbonoclasticus nitrous oxide reductase (N2OR) homodimer.N2OR is organized as a head-to-tail homodimer.Monomers are coloured differently so that they can be distinguished.In both monomers, the N-terminal domain is dark-coloured.The N-terminal domain forms a seven-bladed β-propeller fold that coordinates the catalytic tetranuclear active site CuZ through seven histidine residues at its hub.The C-terminal domain forms a cupredoxin fold and binds the dinuclear mixed-valent CuA centre[104].

Figure 3 .
Figure 3. Structure of Marinobacter hydrocarbonoclasticus nitrous oxide reductase (N 2 OR) homodimer.N 2 OR is organized as a head-to-tail homodimer.Monomers are coloured differently so that they can be distinguished.In both monomers, the N-terminal domain is dark-coloured.The N-terminal domain forms a seven-bladed β-propeller fold that coordinates the catalytic tetranuclear active site Cu Z through seven histidine residues at its hub.The C-terminal domain forms a cupredoxin fold and binds the dinuclear mixed-valent Cu A centre[104].

Table 1 .
Summary of current N 2 O mitigation strategies.