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Are there any plants that fix their own nitrogen?

Are there any plants that fix their own nitrogen?


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I know that most nitrogen is fixed through industrial processes and bacterial symbiotic relationships. However, are there any plants that can fix their own atmospheric nitrogen?


There are no known plants that fix their own nitrogen. However, there soon may be!

Because N is the major limiting factor in agricultural productivity, there is huge interest in plant systems which can fix their own. It's important enough that the Bill & Melinda Gates foundation have started a project aimed at reducing dependence on fertilisers by giving plants the ability to fix their own nitrogen. There are several potential strategies, the most obvious being encouraging more species to form symbioses with nitrogen fixing bacteria.

However, there's another proposal which builds on recent discoveries about the mechanism of nitrogen fixing in bacteria: we now know how the crucial enzyme complex is made (Rubio & Ludden, 2008). As a result, there are many people calling for efforts to engineer the system directly into a plant organelle (e.g. Beatty & Good, 2011; Godfray et al., 2010).

So, in 10-15 years time you can check back and the answer to this question might have changed! The most likely answer then will be "just the usual suspects: rice, maize, wheat".

References:

  • Beatty, P.H. & Good, A.G. (2011) Future Prospects for Cereals That Fix Nitrogen. Science. [Online] 333 (6041), 416 -417. Available from: doi:10.1126/science.1209467 [Accessed: 2 February 2012].
  • Godfray, H.C.J., Beddington, J.R., Crute, I.R., Haddad, L., Lawrence, D., Muir, J.F., Pretty, J., Robinson, S., Thomas, S.M. & Toulmin, C. (2010) Food Security: The Challenge of Feeding 9 Billion People. Science. [Online] 327 (5967), 812 -818. Available from: doi:10.1126/science.1185383 [Accessed: 2 February 2012].
  • Rubio, L.M. & Ludden, P.W. (2008) Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase. Annual Review of Microbiology. [Online] 62 (1), 93-111. Available from: doi:10.1146/annurev.micro.62.081307.162737 [Accessed: 2 February 2012].

As far as I know, all biotic nitrogen fixation is performed by prokaryotic organisms such as Rhizobium. I don't know of any plants which can carry out this function on their own.

Plants can't use atmospheric N2 because it is held essentially inert by the nitrogen triple bond. The process of reducing N2 to NH3 which is usable by plants can be summarized:

N2 + 8e- + 8 H+ + 16 ATP -> 2 NH3 + H2 + 16 ADP + 16 Pi

(where Pi is a phosphate group)

Nitrogenase catalyses the reaction reducing N2 to NH3 by adding H+ and electrons. The whole process requires 8 ATP and is therefore energy intense.

In order to perform this coversion, bacteria require sufficent carbohydrates from decaying matter or plant vascular tissues (this is how Rhizobium derives energy from the host plant).

However, I should add that bacteria often have a mutualistic relation with the plant to perform this function, so in this sense you could say that plants can fix their own nitrogen.

There are also "free living" ammonifying bacteria in soils.

Ref

Science and the Garden, eds. Ingram, D.S., Gregory, P.J., Blackwell, 2008


Corn that acquires its own nitrogen identified, reducing need for fertilizer

A public-private collaboration of researchers at the University of Wisconsin–Madison, the University of California, Davis, and Mars Inc., have identified varieties of tropical corn from Oaxaca, Mexico, that can acquire a significant amount of the nitrogen they need from the air by cooperating with bacteria.

To do so, the corn secretes copious globs of mucus-like gel out of arrays of aerial roots along its stalk. This gel harbors bacteria that convert atmospheric nitrogen into a form usable by the plant, a process called nitrogen fixation. The corn can acquire 30 to 80 percent of its nitrogen in this way, but the effectiveness depends on environmental factors like humidity and rain.

Scientists have long sought corn that could fix nitrogen, with the goal of reducing the crop’s high demand for artificial fertilizers, which are energy intensive, expensive and polluting. Further research is required to determine if the trait can be bred into commercial cultivars of corn, the world’s most productive cereal crop.

The findings are reported Aug. 7 in the journal PLOS Biology.


How Do Plants Fix Nitrogen?

Nitrogen fixing plants don’t pull nitrogen from the air on their own. They actually need help from a common bacteria called Rhizobium. The bacteria infects legume plants such as peas and beans and uses the plant to help it draw nitrogen from the air. The bacteria converts this nitrogen gas and stores it in the roots of the plant.

When the plant stores the nitrogen in the roots, it produces a lump on the root called a nitrogen nodule. This is harmless to the plant but very beneficial to your garden.


Nitrogen fixation engineering in cereal crops moves a step closer

Credit: CC0 Public Domain

A new way of engineering nitrogen fixation has been discovered by a UK-China research team, bringing us one step closer to realising the goal of engineering a range of crops to fix their own nitrogen.

One of the major factors that limit crop growth is the availability of nitrogen, but only bacteria and other single-celled microbes called archaea can take nitrogen from the air and fix it into a form that can be used by plants. The process carried out by these microbes is known as biological nitrogen fixation.

Legumes obtain nitrogen from symbiotic nitrogen-fixing bacteria, but cereal crops including wheat and maize, rely on the availability of fixed nitrogen in the soil. In many cases the addition of chemical fertilisers is the only way to provide crops with enough nitrogen to ensure a good harvest.

The use of nitrogen fertilisers releases nitrous oxide, a greenhouse gas which is 300 times more powerful than carbon dioxide. By engineering crops to fix their own nitrogen, we hope to reduce the use of nitrogen fertilisers, thus mitigating their impact on the environment. A break though like this could also have worldwide implications for cereal crop productivity.

In this paper, the research team has been able to engineer nitrogen fixation by employing a novel strategy, which simplifies the process of engineering multiple genes to make sure their expression is balanced in their new host. Nitrogen fixation is an intricate and delicate process which requires a balance of numerous key components. Until now, achieving the right balance of these components has been a major challenge to the engineering of nitrogen fixation in cereal crops.

The new method works by organising large numbers of genes which are required for nitrogen fixation into a smaller number of "giant genes". These are then expressed in the host cell as huge proteins known as "polyproteins" which are subsequently cut by a specific protease enzyme to release the individual nitrogen fixation components. One innovative part of this method is how the group identified the amount of each component required, and then grouped these together. This step ensures that the right balance is produced.

Professor Ray Dixon project leader in molecular microbiology at the John Innes Centre said: "This is a really exciting development for synthetic biology because it brings nearer the aim of engineering nitrogen fixation in cereals."

The collaborative Peking University - John Innes Centre team say that this exciting method will be useful for transforming complex systems from prokaryotes such as bacteria to eukaryotic hosts such as plants.

Professor Dixon continues, "In the future this method may also be applied to engineering metabolic pathways in plants to produce antifungal and antibacterial secondary metabolites that provide resistance to pathogens."

Key findings from the study which appeared in the journal PNAS include:

  • a post- translational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system to optimise the stoichiometry of nitrogen fixation (nif) gene expression
  • genes were grouped together on the basis of their expression levels and tolerance of their protein products to a C-terminal "tail" that remains after TEVp protease cleavage
  • after multiple rounds of test-regroup cycles 14 essential genes were selectively assembled into 5 giant genes that enable growth on dinitrogen

Stefan Burén et al. Extreme bioengineering to meet the nitrogen challenge, Proceedings of the National Academy of Sciences (2018). DOI: 10.1073/pnas.1812247115


Creating plants that make their own fertilizer

Nancy Duan (left), Michelle Liberton and Lingxia Zhao are members of a team that has taken the first proof-of-principle steps toward inserting the genes needed to fix nitrogen — otherwise found only in bacteria and the bacteria-like Archae — into the cells of crop plants. Credit: JAMES BYARD/WUSTL

Scientists at Washington University are undertaking an ambitious project to engineer tiny nitrogen-fixing devices within photosynthetic cells.

Since the dawn of agriculture, people have exercised great ingenuity to pump more nitrogen into crop fields. Farmers have planted legumes and plowed the entire crop under, strewn night soil or manure on the fields, shipped in bat dung from islands in the Pacific or saltpeter from Chilean mines and plowed in glistening granules of synthetic fertilizer made in chemical plants.

No wonder Himadri Pakrasi's team is excited by the project they are undertaking. If they succeed, the chemical apparatus for nitrogen fixation will be miniaturized, automated and relocated within the plant so nitrogen is available when and where it is needed—and only then and there.

"That would really revolutionize agriculture," said Pakrasi, PhD, the Myron and Sonya Glassberg/Albert and Blanche Greensfelder Distinguished University Professor, in Arts & Sciences, and director of the International Center for Advanced Renewable Energy and Sustainability (I-CARES) at Washington University in St. Louis.

Engineering with biological parts

Although there is plenty of nitrogen in the atmosphere, atmospheric nitrogen is not in a form plants can use. Atmospheric nitrogen must be "fixed," or converted into compounds that make the nitrogen available to plants.

Much of modern agriculture relies on biologically available nitrogenous compounds made by an industrial process, developed by German chemist Fritz Haber in 1909. The importance of the Haber-Bosch process, as it eventually was called, can hardly be overstated today, the fertilizer it produces allows us to feed a population roughly a third larger than the planet could sustain without synthetic fertilizer.

On the other hand, the Haber-Bosch process is energy-intensive, and the reactive nitrogen released into the atmosphere and water as runoff from agricultural fields causes a host of problems, including respiratory illness, cancer and cardiac disease.

Pakrasi thinks it should be possible to design a better nitrogen-fixing system. His idea is to put the apparatus for fixing nitrogen into plant cells, the same cells that hold the apparatus for capturing the energy in sunlight.

The National Science Foundation just awarded Pakrasi and his team more than $3.87 million to explore this idea further. The grant will be administered out of I-CARES, a university-wide center that supports collaborative research regionally, nationally, and internationally in the areas of energy, the environment and sustainability.

This award is one of four funded by the National Science Foundation jointly with awards funded by the Biotechnology and Biological Sciences Research Council in the United Kingdom. The teams will collaborate with one another and meet regularly to share progress and successes. The NSF release is available here.

As a proof of principle, Pakrasi and his colleagues plan to develop the synthetic biology tools needed to excise the nitrogen fixation system in one species of cyanobacterium (a phylum of green bacteria formerly considered to be algae) and paste it into a second cyanobacterium that does not fix nitrogen.

The team includes: Tae Seok Moon, PhD, and Fuzhong Zhang, PhD, both assistant professors of energy, environmental and chemical engineering in the School of Engineering & Applied Science at Washington University and Costas D. Maranas, the Donald B. Broughton Professor of Chemical Engineering at Pennsylvania State University.

"Ultimately what we want to do is take this entire nitrogen-fixation apparatus—which evolved once and only once—and put it in plants," Pakrasi said. "Because of the energy requirements of nitrogen fixation, we want to put it in chloroplasts, because that's where the energy-storing ATP molecules are produced." In effect, the goal is to convert all crop plants, not just the legumes, into nitrogen fixers.

Amazing cycling chemistry

All cyanobacteria photosynthesize, storing the energy of sunlight temporarily in ATP molecules and eventually in carbon-based molecules, but only some of them fix nitrogen. Studies of the evolutionary history of 49 strains of cyanobacteria suggest that their common ancestor was capable of fixing nitrogen and that this ability was then repeatedly lost over the course of evolution.

The big hurdle to redesigning nitrogen fixation, however, is that photosynthesis and nitrogen fixation are incompatible processes. Photosynthesis produces oxygen as a byproduct and oxygen is toxic to nitrogenase, the enzyme needed to fix nitrogen. This is why most organisms that fix nitrogen work in an anaerobic (oxygenless) environment.

Cyanobacteria that both photosynthesize and fix nitrogen separate the two activities either in space or in time. Cyanothece 51142, a cyanobacterium Pakrasi's lab has studied for more than 10 years, does it through timing.

Cyanothece 51142 has a biological clock that allows it to photosynthesize during the day and fix nitrogen at night. During the day, the cells photosynthesize as fast as they can, storing the carbon molecules they create in granules. Then, during the night, they burn the carbon molecules as fast as they can. This uses up all the oxygen in the cell, creating the anaerobic conditions needed for nitrogen fixation.

Thus, the environment within the cell oscillates daily between the aerobic conditions needed for capturing the energy in sunlight and the anaerobic conditions needed for fixing nitrogen.

A single mega transfer

The scientists have chosen their proof-of-principle project very carefully to maximize the odds it will work.

Cyanothece 51142 is particularly attractive as a parts source for the project because it has the largest contiguous cluster of genes related to nitrogen fixation of any cyanobacterium. Roughly 30 genes are part of the same functional unit under the control of a single operating signal, or promoter.

The scientists hope this cluster of genes can be moved to another cyanobacterial strain in a single mega-transfer. The one they've picked as the host, Synechocystis 6803, is the best-studied strain of cyanobacteria. Not only has its genome been sequenced, it is naturally "transformable" and able to integrate foreign DNA into its genome by swapping it with similar native strands of DNA.

But it's actually the next step in the project that will provide the greater challenge for Pakrasi and his team. The scientists will need to figure out how to connect the transplanted nitrogen-fixing gene cluster to Synechocystis' clock. "Like every cyanobacterium," Pakrasi said, " Synechocystis has a diurnal rhythm. But how to tap into that rhythm we don't know yet. We have some ideas we're going to test, but that's where the challenge lies."

Overcoming the challenge of sustainably producing food for a world population of more than 7 billion while reducing pollution and greenhouse gases will require more than luck. Odds are it will take a daring, "out of the box" idea like this one.


Why Incorporate Nitrogen Fixing Plants in Your Garden?

Incorporating nitrogen fixing plants in your garden can help maintain a natural balance.

It can help prevent soil from becoming depleted of this vital plant nutrient.

If you do not pay attention to the nitrogen cycle, you may find that productivity decreases over time. You may even find that plants develop deficiencies and fail to thrive.

Yellowing leaves can be a sign of nitrogen deficiency

Many gardeners and growers who experience nitrogen deficiency turn to synthetic or commercial nitrate fertilizers. But these fertilizers can harmful.

They can harm the environment during their manufacture.

What is more, when too much nitrogen is added, this can harm the garden and the wider local environment.

It can cause instability in the soil ecosystem.

An excess of nitrogen can over-stimulate green growth. Plants may put on leafy growth at the expense of flowers and fruits.

Over-use of nitrogen fertilizers also causes nutrient leaching, and can damage local rivers, waterways and marine environments.

One of the very best ways to make sure that your growing areas always has sufficient nitrogen (but not too much) is to judiciously utilise nitrogen fixing plants.

It is important to note that you should be careful about how and where you use them. Nitrogen fixing plants can cause excess nitrogen in the same way as other sources of nitrogen fertilizer.

However, using nitrogen fixers well is a wonderful idea.

Not only will the plants provide a source of nitrogen, they will also improve soil structure when chopped and dropped over time.


Are there any plants that fix their own nitrogen? - Biology

Since man discovered agriculture, farmers have used ingenious ways to pump more nitrogen into crop fields farmers have planted legumes and plowed the entire crop under, strewn night soil or manure on the fields, shipped in bat dung from islands in the Pacific or saltpeter from Chilean mines and plowed in glistening granules of synthetic fertilizer made in chemical plants.

A new Washington University in St. Louis project seeks to miniaturize, automate and relocate the chemical apparatus for nitrogen fixation within the plant so nitrogen is available when and where it is needed — and only then and there.

“That would really revolutionize agriculture,” said Himadri Pakrasi, PhD, the Myron and Sonya Glassberg/Albert and Blanche Greensfelder Distinguished University Professor, in Arts&Sciences, and director of the International Center for Advanced Renewable Energy and Sustainability (I-CARES) at Washington University in St. Louis.

Smart Engineering With Biological Parts

Although there is plenty of nitrogen in the atmosphere, atmospheric nitrogen is not in a form plants can use. Atmospheric nitrogen must be “fixed,” or converted into compounds that make the nitrogen available to plants.

Much of modern agriculture relies on biologically available nitrogenous compounds made by an industrial process, developed by German chemist Fritz Haber in 1909. The importance of the Haber-Bosch process, as it eventually was called, can hardly be overstated today, the fertilizer it produces allows us to feed a population roughly a third larger than the planet could sustain without synthetic fertilizer.

On the other hand, the Haber-Bosch process is energy-intensive, and the reactive nitrogen released into the atmosphere and water as runoff from agricultural fields causes a host of problems, including respiratory illness, cancer and cardiac disease.

Pakrasi thinks it should be possible to design a better nitrogen-fixing system. His idea is to put the apparatus for fixing nitrogen into plant cells, the same cells that hold the apparatus for capturing the energy in sunlight.

The National Science Foundation just awarded Pakrasi and his team more than $3.87 million to explore this idea further. The grant will be administered out of I-CARES, a university-wide center that supports collaborative research regionally, nationally, and internationally in the areas of energy, the environment and sustainability.


Nancy Duan (left), Michelle Liberton and Lingxia Zhao are members of a team that has taken the first proof-of-principle steps toward inserting the genes needed to fix nitrogen — otherwise found only in bacteria and the bacteria-like Archae — into the cells of crop plants. James Byard/WUSTL

Proof of Principle

As a proof of principle, Pakrasi and his colleagues plan to develop the synthetic biology tools needed to excise the nitrogen fixation system in one species of cyanobacterium (a phylum of green bacteria formerly considered to be algae) and paste it into a second cyanobacterium that does not fix nitrogen.

The team includes: Tae Seok Moon, PhD, and Fuzhong Zhang, PhD, both assistant professors of energy, environmental and chemical engineering in the School of Engineering & Applied Science at Washington University and Costas D. Maranas, the Donald B. Broughton Professor of Chemical Engineering at Pennsylvania State University.

“Ultimately what we want to do is take this entire nitrogen-fixation apparatus — which evolved once and only once — and put it in plants,” Pakrasi said. “Because of the energy requirements of nitrogen fixation, we want to put it in chloroplasts, because that’s where the energy-storing ATP molecules are produced.” In effect, the goal is to convert all crop plants, not just the legumes, into nitrogen fixers.

Amazing cycling chemistry

All cyanobacteria photosynthesize, storing the energy of sunlight temporarily in ATP molecules and eventually in carbon-based molecules, but only some of them fix nitrogen. Studies of the evolutionary history of 49 strains of cyanobacteria suggest that their common ancestor was capable of fixing nitrogen and that this ability was then repeatedly lost over the course of evolution.

The big hurdle to redesigning nitrogen fixation, however, is that photosynthesis and nitrogen fixation are incompatible processes. Photosynthesis produces oxygen as a byproduct and oxygen is toxic to nitrogenase, the enzyme needed to fix nitrogen. This is why most organisms that fix nitrogen work in an anaerobic (oxygenless) environment.

Cyanobacteria that both photosynthesize and fix nitrogen separate the two activities either in space or in time. Cyanothece 51142, a cyanobacterium Pakrasi’s lab has studied for more than 10 years, does it through timing.

Cyanothece 51142 has a biological clock that allows it to photosynthesize during the day and fix nitrogen at night. During the day, the cells photosynthesize as fast as they can, storing the carbon molecules they create in granules. Then, during the night, they burn the carbon molecules as fast as they can. This uses up all the oxygen in the cell, creating the anaerobic conditions needed for nitrogen fixation.

Thus, the environment within the cell oscillates daily between the aerobic conditions needed for capturing the energy in sunlight and the anaerobic conditions needed for fixing nitrogen.

A single mega transfer

The scientists have chosen their proof-of-principle project very carefully to maximize the odds it will work.

Cyanothece 51142 is particularly attractive as a parts source for the project because it has the largest contiguous cluster of genes related to nitrogen fixation of any cyanobacterium. Roughly 30 genes are part of the same functional unit under the control of a single operating signal, or promoter.

The scientists hope this cluster of genes can be moved to another cyanobacterial strain in a single mega-transfer. The one they’ve picked as the host, Synechocystis 6803, is the best-studied strain of cyanobacteria. Not only has its genome been sequenced, it is naturally “transformable” and able to integrate foreign DNA into its genome by swapping it with similar native strands of DNA.

But it’s actually the next step in the project that will provide the greater challenge for Pakrasi and his team. The scientists will need to figure out how to connect the transplanted nitrogen-fixing gene cluster to Synechocystis’ clock. “Like every cyanobacterium,” Pakrasi said, “ Synechocystis has a diurnal rhythm. But how to tap into that rhythm we don’t know yet. We have some ideas we’re going to test, but that’s where the challenge lies.”

Overcoming the challenge of sustainably producing food for a world population of more than 7 billion while reducing pollution and greenhouse gases will require more than luck. Odds are it will take a daring, “out of the box” idea like this one.


GMO sustainability frontier: Helping crops acquire their own nitrogen

GMO sustainability frontier: Helping crops acquire their own nitrogen

It comprises 78 percent of our atmosphere, yet it enters and leaves your lungs unchanged. That’s partly because the triple bond holding its two atoms together is really hard to break up, and that’s why most plants don’t do anything with it either.

Molecular nitrogen (N2) has loads of chemical energy packed into that triple bond, and nature has ways to break it up. In a process called nitrogen fixation, energy is consumed to convert atmospheric N2 into compounds like ammonia (NH3), ammonium (NH4 + ), and nitrate (NO3 – ), which plants are equipped to utilize in their growth. Physical forces that can do this include volcanism and lightning strike, but certain bacteria and certain archaea also can fix nitrogen using an the enzyme nitrogenase, which breaks up N2 and adds hydrogen, producing ammonia. In another process, called nitrification, soil bacteria can convert ammonia to nitrate, which plants love, though plants also can utilize ammonium, the ionic form of ammonia.

Despite these mechanism, however, nitrate is not concentrated enough in most soils to produce the kind of agricultural yields needed to support the human population —plus the livestock that many humans prefer as a source of protein. Consequently, farmers must enrich soils with nitrogen fertilizers, which go into the roots of plants where they’re needed, but also end up in places where they’re not wanted. Furthermore, their production is costly and energy consuming.

So this raises a question: Might we instead use our biotechnology to enable food crops to fix their own nitrogen?

The answer is yes people are working on it with potential benefits not only in terms of cost, but also in terms of environmental stewardship. But, somewhat analogous new generation nuclear power, advocates of nitrogen fixation biotech are also tasked to win over the public in a political and cultural landscape that has been plagued with an intense distrust of any biotechnology.

Climate change issues

Fertilizers can be synthetic or, for lack of a better term, natural, and use of either category of fertilizer entails tradeoffs of pluses and minuses. When it comes to synthetic fertilizers, for instance, they have enabled dramatic yields of staple crops in large-scale agriculture, but the quantity of energy that goes into producing them and putting them into the soil is staggering. It’s roughly two percent of all of the energy that human civilization consumes and that’s with a high carbon footprint, since the machinery for putting them into soil is powered by fossil fuels. For this reason, biotechnology aimed at making staple crops like cereals capable of nitrogen fixation, thereby eliminating the need for fertilizer, is emerging a pathway to a green, more sustainable future.

Two basic strategies

The direct approach for making plants self-fertilizing is a transgenic strategy. You give them the nitrogenase gene from bacteria. It’s not as simple as just banging the gene into the plant genome though, nitrogenase is a complex enzyme whose activity is obstructed by oxygen, which is plentiful in plant cells and must be kept away from the enzyme. There are tricks that can be adapted from cyanobacteria and other organisms that produce nitrogenase in high-oxygen environments, but researchers need to work out which trick is optimal, and this includes deciding where exactly to put the gene—in the cell’s nuclear genome, or in the chromosomes of the cells mitochondria (the energy organelles) or chloroplasts (organelles of photosynthesis)?

The indirect approach is to leave nitrogen fixation to microorganisms, but make that happen inside the plant, instead of the soil. Certain plants, particularly legumes (includes soybeans, peas, beans, alfalfa, clover, peanuts, beans, lentils, and carob), already fix nitrogen, not within their own cells, but through a symbiosis with nitrogen-fixing bacteria. But imagine if this capability could be transferred into cereal crops, such as wheat. Doing this is a major goal of the John Inness Centre in Norwich, UK, where researchers are using the indirect approach with the goal of producing wheat that can outgrow any wheat, and without fertilizer to boot.

Cereal crops actually do have their own symbiotic bacteria that live in various nooks and crannies in the plant, so some researchers are working on transferring the nitrogenase gene into those bacteria. However the thrust of the Inness Centre strategy for nitrogen fixing wheat is to make the wheat attract bacteria from soil that have nitrogen fixation already, the kind of bacteria that live symbiotically inside legumes. The tactic involves manipulation of the wheat’s gene expression causing the plant to sense the presence of the nitorgen-fixing bacteria and take them inside nodules in the roots. Within a few years, it’s anticipated that this could lead to nitrogen fixation, in wheat, and also corn and canola.

Advantages for big and small agriculture

Nitrogen fixing cereals could be extremely valuable for big farms, since they would avoid the need for massive amounts of fertilizer and that entails, but economics is just one factor.

“Through combining scientific excellence with strategic relevance that we can address the major societal and environmental challenges that lie ahead,” said Professor Dale Sanders, the John Inness Centre Director.

The carbon footprint of growing these staple crops would be reduced dramatically, along with environmental issue, such as those connected with nitrate runoff. Potentially, the technology could also help small farmers to compete, since they also could avoid the expensive fertilizers as well as the needed safety measures to contain them. As for organic farms that already avoid synthetic fertilizers but use manure, compost, or other methods, they also would stand to benefit from nitrogen fixing wheat. The risk of coliform bacterial infection associated with manure fertilization is not exclusive to organic farms, since manure is also used in some conventional farming. But the infection risk is nevertheless statistically higher among organic farmers, and it’s plausible that some organic farms would consider replacing their cereals with engineered nitrogen fixing varieties. This is true, particularly for nitrogen fixing wheat developed with GE methods, rather than through transgenic approaches, since farmers could still be organic-certified, even in areas, such as in the EU, with strong restrictions on GM crops. To be sure, the distinction between transgenic and GE may not be rational in terms of safety , but it can affect how a product develops by affecting the legal and political environment. And as the technology advances it could end up being a win-win scenario for parties with differing perspectives on biotech policy.

David Warmflash is an astrobiologist, physician and science writer. Follow @CosmicEvolution to read what he is saying on Twitter.


Contents

Biological nitrogen fixation was discovered by Jean-Baptiste Boussingault in 1838. [9] Later, in 1880, the process by which it happens was discovered by German agronomist Hermann Hellriegel and Hermann Wilfarth [de] [10] and was fully described by Dutch microbiologist Martinus Beijerinck. [11]

"The protracted investigations of the relation of plants to the acquisition of nitrogen begun by Saussure, Ville, Lawes and Gilbert and others culminated in the discover of symbiotic fixation by Hellriegel and Wilfarth in 1887." [12]

"Experiments by Bossingault in 1855 and Pugh, Gilbert & Lawes in 1887 had shown that nitrogen did not enter the plant directly. The discovery of the role of nitrogen fixing bacteria by Herman Hellriegel and Herman Wilfarth in 1886-8 would open a new era of soil science." [13]

In 1901 Beijerinck showed that azotobacter chroococcum was able to fix atmospheric nitrogen. This was the first species of the azotobacter genus, so-named by him. It is also the first known diazotroph, the species that use diatomic nitrogen as a step in the complete nitrogen cycle.

Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by a nitrogenase enzyme. [1] The overall reaction for BNF is:

The process is coupled to the hydrolysis of 16 equivalents of ATP and is accompanied by the co-formation of one equivalent of H
2 . [14] The conversion of N
2 into ammonia occurs at a metal cluster called FeMoco, an abbreviation for the iron-molybdenum cofactor. The mechanism proceeds via a series of protonation and reduction steps wherein the FeMoco active site hydrogenates the N
2 substrate. [15] In free-living diazotrophs, nitrogenase-generated ammonia is assimilated into glutamate through the glutamine synthetase/glutamate synthase pathway. The microbial nif genes required for nitrogen fixation are widely distributed in diverse environments. [16]

For example decomposing wood which has generally low content of nitrogen was shown to host diazotrophic community. [17] [18] Bacteria through fixation enrich wood substrate with nitrogen thus enabling deadwood decomposition by fungi. [19]

Nitrogenases are rapidly degraded by oxygen. For this reason, many bacteria cease production of the enzyme in the presence of oxygen. Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with a protein such as leghemoglobin. [1]

Importance of nitrogen Edit

Atmospheric nitrogen is inaccessible to most organisms, [20] because its triple covalent bond is very strong. The nitrogen requirements for life is highly variable. [ clarification needed ] Considering atom acquisition, for every 100 atoms of carbon, roughly 2 to 20 atoms of nitrogen are assimilated. The atomic ratio of Carbon(C): Nitrogen(N): Phosphorus(P) observed on average in planktonic biomass was originally described by Alfred Redfield. [21] The Redfield Ratio, the stoichiometric relationship between C:N:P atoms, is 106:16:1. [21]

Nitrogenase Edit

The protein complex nitrogenase is responsible for catalyzing the reduction of nitrogen gas (N2) to ammonia (NH3). [22] In Cyanobacteria, this enzyme system is housed in a specialized cell called the heterocyst. [23] The production of the nitrogenase complex is genetically regulated, and the activity of the protein complex is dependent on ambient oxygen concentrations, and intra- and extracellular concentrations of ammonia and oxidized nitrogen species (nitrate and nitrite). [24] [25] [26] Additionally, the combined concentrations of both ammonium and nitrate are thought to inhibit NFix, specifically when intracellular concentrations of 2-oxoglutarate (2-OG) exceed a critical threshold. [27] The specialized heterocyst cell is necessary for the performance of nitrogenase as a result of its sensitivity to ambient oxygen. [28]

Nitrogenase consist of two proteins, a catalytic iron-dependent protein, commonly referred to as MoFe protein and a reducing iron-only protein (Fe protein). There are three different iron dependent proteins, molybdenum-dependent, vanadium-dependent, and iron-only with all three nitrogenase proteins variations containing an iron protein component. Molybdenum-dependent nitrogenase is the most commonly present nitrogenase. [22] The different types of nitrogenase can be determined by the specific iron protein component. [29] Nitrogenase is highly conserved, gene expression through DNA sequencing can distinguish which protein complex is present in the microorganism and potentially being express. Most frequently, the nifH gene is used to identify the presence of molybdenum-dependent nitrogenase followed by closely related nitrogenase reductases (component II) vnfH and anfH representing vanadium-dependent and iron-only nitrogenase, respectively. [30] In studying the ecology and evolution of nitrogen-fixing bacteria, the nifH gene is the biomarker most widely used. [31] nifH has two similar genes anfH and vnfH that also encode for the nitrogenase reductase component of the nitrogenase complex [32]

Microorganisms Edit

Diazotrophs are widespread within domain Bacteria including cyanobacteria (e.g. the highly significant Trichodesmium and Cyanothece), as well as green sulfur bacteria, Azotobacteraceae, rhizobia and Frankia. Several obligately anaerobic bacteria fix nitrogen including many (but not all) Clostridium spp. Some archaea also fix nitrogen, including several methanogenic taxa, which are significant contributors to nitrogen fixation in oxygen-deficient soils. [33]

Cyanobacteria, commonly known as blue-green algae, inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. In general, cyanobacteria can use various inorganic and organic sources of combined nitrogen, such as nitrate, nitrite, ammonium, urea, or some amino acids. Several cyanobacteria strains are also capable of diazotrophic growth, an ability that may have been present in their last common ancestor in the Archean eon. [34] Nitrogen fixation not only naturally occurs in soils but also aquatic systems, including both freshwater and marine. Nitrogen fixation by cyanobacteria in coral reefs can fix twice as much nitrogen as on land—around 660 kg/ha/year. The colonial marine cyanobacterium Trichodesmium is thought to fix nitrogen on such a scale that it accounts for almost half of the nitrogen fixation in marine systems globally. [35]

Marine surface lichens and non-photosynthetic bacteria belonging in Proteobacteria and Planctomycetes fixate significant atmospheric nitrogen. [36]

Species of nitrogen fixing cyanobacteria in fresh waters include: Aphanizomenon and Dolichospermum (previously Anabaena). [37] Such species have specialized cells called heterocytes, in which nitrogen fixation occurs via the nitrogenase enzyme. [38] [39]

Root nodule symbioses Edit

Legume family Edit

Plants that contribute to nitrogen fixation include those of the legume family—Fabaceae— with taxa such as kudzu, clover, soybean, alfalfa, lupin, peanut and rooibos. They contain symbiotic rhizobia bacteria within nodules in their root systems, producing nitrogen compounds that help the plant to grow and compete with other plants. [40] When the plant dies, the fixed nitrogen is released, making it available to other plants this helps to fertilize the soil. [1] [41] The great majority of legumes have this association, but a few genera (e.g., Styphnolobium) do not. In many traditional farming practices, fields are rotated through various types of crops, which usually include one consisting mainly or entirely of clover. [ citation needed ]

Fixation efficiency in soil is dependent on many factors, including the legume and air and soil conditions. For example, nitrogen fixation by red clover can range from 50 to 200 lb./acre. [42]

Non-leguminous Edit

Other nitrogen fixing families include:

  • Some cycads. [citation needed]
  • Parasponia, a tropical genus in the family Cannabaceae, which are able to interact with rhizobia and form nitrogen-fixing nodules [43] such as alder and bayberry can form nitrogen-fixing nodules, thanks to a symbiotic association with Frankia bacteria. These plants belong to 25 genera [44] distributed across eight families.

The ability to fix nitrogen is present in other families that belong to the orders Cucurbitales, Fagales and Rosales, which together with the Fabales form a clade of eurosids. The ability to fix nitrogen is not universally present in these families. For example, of 122 Rosaceae genera, only four fix nitrogen. Fabales were the first lineage to branch off this nitrogen-fixing clade thus, the ability to fix nitrogen may be plesiomorphic and subsequently lost in most descendants of the original nitrogen-fixing plant however, it may be that the basic genetic and physiological requirements were present in an incipient state in the most recent common ancestors of all these plants, but only evolved to full function in some of them.

Several nitrogen-fixing symbiotic associations involve cyanobacteria (such as Nostoc):

Endosymbiosis in diatoms Edit

Rhopalodia gibba, a diatom alga, is a eukaryote with cyanobacterial N
2 -fixing endosymbiont organelles. The spheroid bodies reside in the cytoplasm of the diatoms and are inseparable from their hosts. [46] [47]

Eukaryotic Nitrogenase Engineering Edit

Some scientists are working towards introducing the genes responsible for nitrogen fixation directly into plant DNA. As all known examples of nitrogen fixation takes place in prokaryotes, transferring the functionality to eukaryotes such as plant is a challenge one team is using yeast as their eukaryotic test organism. A major problem to overcome is the oxygen-sensitivity of the produced enzymes, as well as the energy requirements. Having the process taking place inside of mitocondria or chloroplasts is being considered. [48]

The possibility that atmospheric nitrogen reacts with certain chemicals was first observed by Desfosses in 1828. He observed that mixtures of alkali metal oxides and carbon react at high temperatures with nitrogen. With the use of barium carbonate as starting material, the first commercial process became available in the 1860s, developed by Margueritte and Sourdeval. The resulting barium cyanide reacts with steam, yielding ammonia. A method for nitrogen fixation was first described by Henry Cavendish in 1784 using electric arcs reacting nitrogen and oxygen in air. This method was implemented in the Birkeland–Eyde process. [49] The fixation of nitrogen by lightning is very similar natural occurring process.

Frank-Caro process Edit

In 1898 Frank and Caro developed a way to fix nitrogen in the form of calcium cyanamide. The Frank-Caro and Ostwald processes dominated industrial fixation until the discovery of the Haber process in 1909. [50] [51]

Haber process Edit

The most common ammonia production method is the Haber process. The Haber-Bosch nitrogen reduction process for industrial fertilizer production revolutionized modern day technology. [52] Fertilizer production is now the largest source of human-produced fixed nitrogen in the terrestrial ecosystem. Ammonia is a required precursor to fertilizers, explosives, and other products. The Haber process requires high pressures (around 200 atm) and high temperatures (at least 400 °C), which are routine conditions for industrial catalysis. This process uses natural gas as a hydrogen source and air as a nitrogen source. The ammonia byproduct has resulted in an intensification of nitrogen fertilizer globally [53] and is accredited with supporting the expansion of human population from roughly 2 billion in the early 20th century to roughly 7 billion people presently. [54]

Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of reducing energy requirements. However, such research has thus far failed to approach the efficiency and ease of the Haber process. Many compounds react with atmospheric nitrogen to give dinitrogen complexes. The first dinitrogen complex to be reported was Ru(NH 3)5(N2) 2+ .

Homogeneous catalysis Edit

Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of reducing energy requirements. However, such research has thus far failed to approach the efficiency and ease of the Haber process. Many compounds react with atmospheric nitrogen to give dinitrogen complexes. The first dinitrogen complex to be reported was Ru(NH
3 )
5 ( N
2 ) 2+ . [55] Some soluble complexes do catalyze nitrogen fixation. [56]

Nitrogen can be fixed by lightning that converts nitrogen gas ( N
2 ) and oxygen gas ( O
2 ) present in the atmosphere into NO
x (nitrogen oxides). NO
x may react with water to make nitrous acid or nitric acid, which seeps into the soil, where it makes nitrate, which is of use to plants. Nitrogen in the atmosphere is highly stable and nonreactive due to the triple bond between atoms in the N
2 molecule. [57] Lightning produces enough energy and heat to break this bond [57] allowing nitrogen atoms to react with oxygen, forming NO
x . These compounds cannot be used by plants, but as this molecule cools, it reacts with oxygen to form NO
2 . [58] This molecule in turn reacts with water to produce HNO
3 (nitric acid), or its ion NO −
3 (nitrate), which is usable by plants. [59] [57]


The Wonder Plant That Could Slash Fertilizer Use

An indigenous Mexican corn gets its nitrogen from the air.

For thousands of years, people from Sierra Mixe, a mountainous region in southern Mexico, have been cultivating an unusual variety of giant corn. They grow the crop on soils that are poor in nitrogen—an essential nutrient—and they barely use any additional fertilizer. And yet, their corn towers over conventional varieties, reaching heights of more than 16 feet.

A team of researchers led by Alan Bennett from UC Davis has shown that the secret of the corn’s success lies in its aerial roots—necklaces of finger-sized, rhubarb-red tubes that encircle the stem. These roots drip with a thick, clear, glistening mucus that’s loaded with bacteria. Thanks to these microbes, the corn can fertilize itself by pulling nitrogen directly from the surrounding air.

The Sierra Mixe corn takes eight months to mature—too long to make it commercially useful. But if its remarkable ability could be bred into conventional corn, which matures in just three months, it would be an agricultural game changer.

All plants depend on nitrogen to grow, and while there’s plenty of the element in the air around us, it’s too inert to be of use. But bacteria can convert this atmospheric nitrogen into more usable forms such as ammonia—a process known as fixation. Legumes, like beans and peas, house these nitrogen-fixing bacteria in their roots. But cereals, like corn and rice, largely don’t. That’s why American farmers need to apply more than 6.6 million tons of nitrogen to their corn crops every year, in the form of chemical sprays and manure.

“All that fertilizer takes a lot of energy to produce, and the excess ends up in places where it distorts the nutrient balance, creating algae blooms and dead zones in waterways,” says Jeremy Yoder, an evolutionary biologist at California State University, Northridge who was not involved in the study. “So self-fertilizing [corn] could substantially cut the cost and environmental impact of a staple crop.” It could also make it easier to grow the crop in developing countries where fertilizer is unaffordable or in areas where soils are poorer.

Crucially, the Davis team involved the Sierra Mixe community throughout their research. They also established legal agreements with the Mexican government to ensure that any benefits from their research—and its subsequent commercialization—would be shared with the community, under the auspices of the Nagoya Protocol, an international framework intended to thwart bio-piracy. Alejandra Barrios, the director of biosafety and biodiversity at Mexico’s environmental agency, repeatedly praised the approach on Twitter, calling it “great work” and a “win-win solution.”

The certificate that ratifies the agreement is the first of its kind to be “issued by the Mexican government, and the first issued to any entity in the U.S. by any country,” says Howard-Yana Shapiro, an agricultural scientist from Mars, Incorporated who initiated the project. “That is as important as the discovery. We’re showing the way of the future.”

Corn, or maize, originated in southern Mexico when it was domesticated from a wild cereal called teosinte. The region is still home to the greatest diversity of the crop, with thousands of unique varieties or “landraces.” In 1980, Shapiro (who was then an independent researcher) was busily collecting these landraces on behalf of the Mexican government when he heard about the giant, mucus-covered corn.

From the start, he suspected that the plant might fix its own nitrogen, and that the shining mucus was somehow involved. But with the technology of the time, he had no way of testing his hunch. And without that evidence, other scientists were rightfully skeptical, including the team at Davis. “I would talk about this maize, and Alan would say, ‘It’s not possible,’” Shapiro recalls. “I said it is possible but I just don’t know how to prove it. And I took him to Sierra Mixe for a visit in 2008. He was gobsmacked. He said, ‘I take it all back. There’s something going on here.’”

To find out what was really happening, the team used modern DNA-sequencing techniques to show that the mucus contains microbes that belong to nitrogen-fixing families, and that carry nitrogen-fixing genes. They also chemically analyzed the mucus to show that it provides its resident microbes with exactly the conditions they need to thrive—an all-you-can-eat buffet of sugar, and protection from oxygen.

Next, they used five different tests to confirm that the microbes really are fixing nitrogen, that the nitrogen moves into the corn, and that the corn gets a lot of its nitrogen—anywhere from 30 to 80 percent—in this way. All five techniques have their own shortcomings, but together “they all pointed to the same conclusion,” Bennett says. “We’ve been working on this for 10 years and we have a high degree of confidence that the results we report are correct.”

Others agree: “It was a very ambitious study that was really well done, and the results should be believed,” says Michael Kantar, a botanist at the University of Hawai’i at Mānoa. Yoder points out that the team hasn’t identified the specific nitrogen-fixing microbes, but beyond that, “I think it’s pretty convincing,” he told me. “Aerial-root mucilage that hosts nitrogen-fixing microbes is, quite honestly, a thing I’d have called a little far-fetched if I saw it on an episode of Star Trek,” he added on Twitter.

Scientists have spent years trying to create nitrogen-fixing cereal crops through genetic engineering, with little progress to show for it. But since we now know that at least one type of corn can fix nitrogen naturally, the ability could potentially be moved into conventional varieties through classical crossbreeding, mucus transplants, or both. These methods might make the final produce more publicly acceptable than a genetically edited crop.

For now, Bennett and his colleagues want to identify the genes that allow the Sierra Mixe corn to produce its mucus-coated aerial roots, and attract the right bacteria. They also want to take a closer look at the microbes themselves. “We’ve isolated thousands, but of those, we don’t know if there are two species that are really important or a hundred,” Bennett says.

And Shapiro, with the blessing of the Sierra Mixe community, is trying to find a company to take charge of commercialization. “It probably won’t be Mars Inc., ’cause we’re not a maize company,” he says, “but I’m trying to find the right partner.”

Kantar cautions that it’s too early to say if there are any big implications for food security, because the team hasn’t shown that the resulting corn can fix enough nitrogen to grow at commercially useful scales. It’s also unclear if the genes behind the ability come with any drawbacks. But “if these questions can be resolved, this may provide a way to significantly reduce fertilizer use worldwide, which would have hugely beneficial environmental effects,” he says.

Kristin Mercer from Ohio State University is similarly cautious. ‘This corn has been likely doing a very good job ensuring some level of food security for families in the region for a long time,” she says. “If one were to think about capturing that beneficial diversity and distributing it more widely, a number of potential issues arise.” Are there intellectual property issues around dispersing the genetic variation underlying this trait into public or private breeding programs? Would it be a good approach to create varieties of nitrogen-fixing corn for other areas where poor farmers live and asking them to buy those varieties?

“It is easy to jump from describing the amazing biology of these genetic resources stewarded by farmers in the region for millennia to trying to solve the world’s massive, intractable problems—but that is stickier than it may seem,” Mercer adds.


Literature Cited

Aranjuelo, I., J.J. Irigoyen, S. Nogués, and M. Sánchez-Díaz. 2009. Elevated CO2 and water-availability effect on gas exchange and nodule development in N2-fixing alfalfa plants. Environmental and Experimental Botany, 65, 18x26.

Burton, J.C. 1972. Nodulation and symbiotic nitrogen fixation. In C.H. Hanson (Ed.), Alfalfa Science and Technology (Monograph 15 pp. 229�). Madison, WI: American Society of Agronomy.

Cash, D., B. Melton, J. Gregory, and L. Cihacek. 1981. Rhizobium inoculants for alfalfa in New Mexico [Research Report 461]. Las Cruces: New Mexico State University Agricultural Experiment Station.

Delwiche, C.C., and J. Wijler. 1956. Non-symbiotic nitrogen fixation in soil. Plant and Soil, 7, 113�.

Frankow-Lindberg, B.E., and A.S. Dahlin. 2013. N2 fixation, N transfer, and yield in grassland communities including a deep-rooted legume or non-legume species. Plant and Soil, 370, 567�.

Guldan, S.J., C.A. Martin, J. Cueto-Wong, and R.L. Steiner. 1996. Interseeding legumes into chile: Legume productivity and effect on chile yield. HortScience, 31, 1126�.

Lamb, J.F.S., D.K. Barnes, M.P. Russelle, C.P. Vance, G.H. Heichel, and K.I. Henjum. 1995. Ineffectively and effectively nodulated alfalfas demonstrate biological nitrogen fixation continues with high nitrogen fertilization. Crop Science, 35, 153�.

Layzell, D.B., R.M. Rainbird, C.A. Atkins, and J.S. Pate. 1979. Economy of photosynthate use in nitrogen-fixing legume nodules. Plant Physiology, 64, 888�.

Russelle, M.P., J.F.S. Lamb, N.B. Turyk, B.H. Shaw, and B. Pearson. 2007. Managing nitrogen contaminated soils. Agronomy Journal, 99, 738�.

Sørensen, J., and A. Sessitsch. 2007. Plant-associated bacteria—Lifestyle and molecular interactions.
In J.D. van Elsas, J.K. Jansson, and J.T. Trevors (Eds.), Modern Soil Microbiology, 2nd ed. (pp. 211�). Boca Raton, FL: CRC Press, Taylor and Francis Group.

Unkovich, M.J., J. Baldock, and M.B. Peoples. 2010. Prospects and problems of simple linear models for estimating symbiotic N2 fixation by crop and pasture legumes. Plant and Soil, 329, 75󈟅.

Walley, F.L., G.O. Tomm, A. Matus, A.E. Slinkard, and C. van Kessel. 1996. Allocation and cycling of nitrogen in an alfalfa-bromegrass sward. Agronomy Journal, 88, 834�.

Warembourg, F.R., D. Montange, and R. Bardin. 1982. The simultaneous use of CO2 and N2 labelling techniques to study the carbon and nitrogen economy of legumes grown under natural conditions. Physiologia Plantarum, 56, 46󈞣.

For more on this topic, see the following publications:

A-148: Understanding Soil Health for Production Agriculture in New Mexico
http://aces.nmsu.edu/pubs/_a/A148/welcome.html

A-150: Principles of Cover Cropping for Arid and Semi-arid Farming Systems
http://aces.nmsu.edu/pubs/_a/A150/welcome.html

Original authors: W.C. Lindemann, soil microbiologist and C.R. Glover, Extension agronomist.

Robert Flynn is an Associate Professor of Agronomy and Soils and an Extension Agronomist at New Mexico State University. He earned his Ph.D. at Auburn University. His research and Extension efforts aim to improve grower options that lead to sustainable production through improved soil quality, water use efficiency, and crop performance.

To find more resources for your business, home, or family, visit the College of Agricultural, Consumer and Environmental Sciences on the World Wide Web at aces.nmsu.edu

Contents of publications may be freely reproduced for educational purposes. All other rights reserved. For permission to use publications for other purposes, contact [email protected] or the authors listed on the publication.

New Mexico State University is an equal opportunity/affirmative action employer and educator. NMSU and the U.S. Department of Agriculture cooperating.

Revised June 2015

We seek to improve the lives of New Mexicans, the nation, and the world through research, teaching, and extension.



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