Out of Thin Air

Air is a big tease. Nothing against oxygen, of course, but air is 78 percent nitrogen. Nitrogen is often the deal-breaker for life on Earth, the nutrient that sets the limit for how much of what grows where. Yet even a bonanza of airborne nitrogen passing through lung or leaf does neither animal nor plant a bit of good: One of life’s most precious resources just blows away unused with every breath.

Nitrogen wafts around in the air as paired atoms (N2) locked together chemically with a robust triple bond. Despite a great need for the element, the bodies of living things complex enough to have cells with a nucleus—paramecia and potatoes and people alike—have no natural way to break that bond. Here’s where humanity and their kin are routinely humbled by green slime. A roster of “simple” life forms, such as cyanobacteria floating in water or the rhizobia group of bacteria lurking in soil, breaks that bond. This feat, called nitrogen fixation, turns N2 into user-friendly ammonia.

Since 1920, the Haber-Bosch industrial process has let people sunder nitrogen’s triple bond as long as there’s energy available to raise temperatures to 400° to 500° Celsius and pressures to 200 atmospheres. Your basic pond scum fixes nitrogen at room temperature and everyday atmospheric pressure.

Certain plants have come up with a tidier solution. By themselves, soybeans, peas, alder trees and others can’t fix nitrogen any better than a person could. Instead they lure immigrant microbes to move in, and do the job for them.

In a border-crossing as delicate as any in human society, the microbes and the plants exchange signals and test chemical bona fides until the immigrants settle down, often in specialized lumps or pockets within the plant, and start fixing nitrogen. With help from their new friends, those plants get fertilizer out of thin air.

It’s enough to turn a person soybean-green with envy. Making fertilizer via the Haber-Bosch process in order to grow crops takes an enormous amount of energy. And energy costs are soaring, not to mention that burning fossil fuels is increasing concentrations of greenhouse gases, and that a growing global population means more and more demand for food. For more than a third of the world’s people, more food means more artificial fertilizer. If only food crops could use the nitrogen from N2 in the air.

“People are always asking me when we’re going to get nitrogen-fixing wheat,” says Allan Downie of the John Innes Centre in Norwich, England, who authored a recent article about plant-microbe signaling in the Annual Review of Plant Biology. It’s not that easy, notes Downie, who started studying nitrogen fixation during the 1980s and sees a long way left to go.

The good news is that science is picking up the pace. Explorations of both plants and their microbes have found new, unsuspected diversity in nitrogen fixing and given scientists more partnerships to study for clues on how to engineer the process. Researchers are also refining their knowledge of how legumes use a chemical Craigslist to find and negotiate with potential microbe workers. Science is apprenticing itself to the masters, crowding in to watch each nuance of the process. Even if the masters are just dots in the dirt.

Fixers

There’s power in those dots, says David Dalton of Reed College in Portland, Ore. Some of them, such as the cyanobacteria, drift in the sea and process so much nitrogen they are now recognized as a major force in ocean chemistry.

Much of the nitrogen in the old-growth Douglas fir forests of the Pacific Northwest could be coming from Nostoc cyanobacteria, Dalton says. Several Lobaria lichens include Nostoc in their shaggy, green forms, which after some 80 years can establish abundant colonies high in the trees. Dalton, a tree-climber, says, “It’s like somebody dumped a trainload of exploding lettuce.”

Other nitrogen fixers form loose associations with plants, nestling near the roots or moving into tissues without any obvious specialized accommodations. One of the most famous of these, now called Gluconacetobacter diazotrophicus, turned up inside sugarcane plants in Brazil in 1988. It belongs to a bacterial group known for producing acetic acid, but under the right circumstances, this species makes enough nitrogen to boost sugarcane growth.

The richest partnerships, though, involve more specialized structures, such as separate tissues inside the plant. Cycads, which Dalton describes as looking “like squatty palms,” grow little bumps as cyanobacteria condos. And one oddball genus of flowering plants, Gunnera, accepts pockets of cyanobacteria in its stems. Cut a Gunnera stem just below one of its umbrella-sized leaves and look for the green blobs.

School textbooks may feature bean plants in the diagram of nitrogen fixation, but the Frankia genus of bacteria evoke nodules in un-beanish plants, such as alder trees and bayberries. The nitrogen fixers, looking “extremely skinny,” live in clusters of nodules on the roots, Dalton says.

The most celebrated microbe-plant arrangements arise between bacteria and the legumes. Each plant recruits its labor force anew, and bacteria enter tiny root hairs that end up bulging into nitrogen-factory nodules that look like faintly pink peas. The pink comes from botanical hemoglobin, cousin to the oxygen-carrying molecules in mammal blood.

“This great explosion” is how John Howieson of Murdoch University in Australia describes the abundance of new discoveries of nitrogen-fixing bacteria found in legume nodules in recent years. Biologists knew that lots of microorganisms turned up inside the nodules but had no good way to separate the fixers from the slackers.

For more than 100 years, biologists had reported nodules forming only with bacteria in the alpha branch of group Proteobacteria, especially those in the Rhizobiaceae family. Starting in 2000, though, researchers have found legume nodulators in an entirely different branch, called beta. The first, a member of the Burkholderia genus, was found fixing for mimosa trees in Brazil.

“We were used to boring gray colonies, milky white colonies, and up come these pink things,” says Howieson. His collection of new nitrogen-fixing bacteria includes “strange, pink, fast-growing, slimy things” as well as an unpublished prize: “an orange, slimy, yet-to-be-named thing.”

Another specialist in nitrogen-fixing nodules, Janet Sprent of the University of Dundee in Scotland, remembers simpler times for systematists. “From the orderly situation of a century ago,” she says, “we now have something approaching chaos.”

And, Sprent points out, scientists have barely even begun to survey the many species of tropical plants, especially trees in the legume family, that could easily harbor new species of nitrogen-fixing bacteria.

Eavesdropping

For the plant, it’s a perilous undertaking to permit bacteria to move in. The guests have to behave themselves without multiplying out of control, trashing plant structures or disrupting the local chemistry. And the microbes take a chance that their new host, which provides them with food, won’t go berserk and unleash its defenses on them. So researchers are exploring the complex back-and-forth signals that create the arrangements.

“We have a really eloquent conversation that we can’t quite translate,” says Bruce Hungate of Northern Arizona University in Flagstaff.

Ann M. Hirsch of the University of California, Los Angeles says, “I think of it as a dance, but maybe that’s because I studied ballet for so long.” She and colleague Angie Lee, now at the University of California, San Diego, described nodulation in terms of ballet in a 2006 paper in Plant Signaling & Behavior.

The process begins, they say, with a pas de deux between the legume root hairs, which release flavonoid compounds into the soil, and hang-about bacteria that, in turn, secrete molecules called Nod factors. Even faint traces of these substances prompt dramatic calcium movements within the root hairs. (“Allegro,” says Hirsch.) Often within seconds of the whiff of Nod factor, calcium floods into root hair cells. In a few more minutes, calcium concentrations begin to spike repeatedly, continuing for an hour. The calcium frenzy may activate the genes for building the nodule, Hirsch speculates.

If all goes well, the little root hairs kink into hooks and eventually curl around the bacteria. In many of the legumes, the curled root cells open an internal tunnel, or infection thread, that guides incoming bacteria to the microbes’ new home-internal tissue that eventually bulges out into a nodule.

The basic ballet still holds surprises. Last summer the genomes of two bacterial strains, ORS278 and BTAi1, turned out to have no Nod factors. Yet the bacteria still can trigger nodulation in certain jointvetch legumes in a respectable fashion.

Sharon Long of Stanford University welcomes the counterintuitive finding. “It’s quite important,” she says. “It doesn’t answer anything yet, but really opens up some questions.”

Plants can be quite picky when choosing a microbial dance partner. Howieson’s current work, for example, finds that two clover species take up specific strains of Rhizobium leguminosarum bacteria even when those strains are rare in the surrounding soil. A particularly efficient strain ends up as the clover’s preferred partner even when its population is outnumbered 100-fold by a crowd of ineffective potential partners that form nodules but don’t fix N2, Howieson and his colleagues report in the March Soil Biology and Biochemistry.

Other teams are investigating genes that plants use during negotiations with their partners. The gene SymRK encodes a protein involved in picking up Nod signals—the bacterial answer to the plant’s call for partners. SymRK does other jobs in legumes though, says Didier Bogusz of the Institute of Research for Development in Montpellier, France. Earlier work found SymRK active in an ancient partnership in which the legumes, like some three-quarters of plant species, allow intimate connections between their roots and fungi. The network of root-cuddling fungi, called arbuscular mycorrhizae, carries nutrients such as phosphate from the soil to the plant.

Australia’s casuarina trees with their feathery foliage aren’t legumes, and they don’t get personal with legume-type bacteria. Now Bogusz has found that, like legumes, the trees rely on SymRK when they team up with their nitrogen-fixing bacteria, the Frankia. Also the trees use SymRK to connect with their version of the fungal network, Bogusz and his colleagues report in the March 25 Proceedings of the National Academy of Sciences. The finding supports a notion that plants using nitrogen-fixing nodules evolved the powers by borrowing components of the ancient, widespread system for forming partnerships with fungi.

Lunch costs

Finding the nitrogen-fixer genes and conferring the ability to fix nitrogen on non-bean crops seems “very likely in the long term,” says Bogusz.

Now might be a good time for a major push toward re-engineering crops, says Eric Triplett of the University of Florida in Gainesville. Early efforts in the 1970s didn’t get far but didn’t have a lot of sustained funding or the tools available today, he says. Last year he made a presentation to the National Research Council on the prospects for such a feat.

Triplett rejects the notion of trying to move the full legume ballet-recruiting the right partner and growing nodules for it—into a radically different species like corn. That would take a suite of specialized plant genes that need to be tuned to particular bacterial partners. “My feeling is, it’s just too hard,” he says. “It seems to me the only way to go is to engineer plants directly with nitrogen-fixing genes.”

Bacteria do their magic with some 20 genes, but plants already have versions of some of them. He proposes putting the machinery into one of the metabolic workshops already in business in a plant cell, such as the energy producing mitochondria or the light-catching chloroplast. “I don’t think there’s anything more important you could do for feeding sub-Saharan Africa,” he says.

Even if putting nitrogen-fixing genes directly into plants offers the easier approach, it’s hardly easy. For example, Downie cautions there would be costs and trade-offs even if the complex machinery could get into a novel plant. Fancy biochemistry aside, plants would still need a whopping amount of energy to crack the nitrogen triple bond. Calculations based on bacterial enzymes have estimated that processing a molecule of N2 takes at least eight times as many molecules of ATP, the cell’s energy currency, than does processing a carbon dioxide molecule during photosynthesis. That’s energy that wouldn’t go to other plant projects, such as building leaves or soybeans or peas.

Legume crops that support their own nitrogen factories tend to have lower yields than corn and wheat that farmers fertilize. So adding nitrogen-fixing power might cost a species some of its farm efficiency. “You won’t get anything for nothing,” Downie says. “How much of a yield penalty would you accept?”

The possible yield penalty is a drawback, but there’s another way to look at the issue of nitrogen supply, says environmental scientist Vaclav Smil of the University of Manitoba in Canada. He tracks nitrogen use worldwide, and he’s not expecting a new fertilizer-making crop from genetic engineers any time soon. “They’ve been promising that for years,” he says.

“With diet as it is today, about 40 percent of all food is produced thanks to artificial fertilizer,” he says. But that dependence comes from a food system he calls “all mismanaged.”

Smil reels off statistics of excess and waste from field to table. For example, fertilizer budgets differ considerably depending on food choice, particularly on how much meat and dairy products a nation consumes. The American diet, depending about 50 percent on artificial fertilizer, features nearly five times the meat consumption per capita of Asian diets.

The challenges of providing nitrogen for the world’s current food habits are quite real, Smil says, but he thinks it’s a mistake to wait for nitrogen-fixing wheat. “Decrease food losses,” he says. “Before you go sticking genes into anything, reform the diet.”