Although we take this biological process for granted, it’s such an essential process that human society might not exist without it. Really, there’s nothing modest about it at all.
This story has two amazing parts. The first part is the general process of how plants “fix” nitrogen – which means capturing nitrogen from the atmosphere. The second part of the story is how certain bacteria infect the roots of those plants and create a highly specialized nitrogen-fixing factory in those roots.
Pulling nitrogen from the air is no trivial matter. Although nitrogen comprises 78 percent of our atmosphere, this nitrogen occurs as a stable N2 molecule. The two nitrogen atoms are bound together with a very strong triple bond – so strong that if we want to break this bond ourselves, we must use the industrial “Haber process,” a heavy-duty manufacturing procedure involving very high temperatures, high pressures and metal catalysts. In fact, each year we rely on the Haber process to produce more than 100 million tons of synthetic nitrogen fertilizer that is used on farms.
In contrast, legume plants quietly fix nitrogen in their roots. No loud industrial clanging, no risk of high-pressure explosions or toxic fumes. Instead, in the tiny dark spaces of the soil, specialized gram-negative rod bacteria called “rhizobia” (there are a few related genera) infect the plant roots during early root development. These bacteria combine with plant tissue to form a highly organized root mass called a nodule.
The bacteria contain an enzyme known as nitrogenase, which is the actual molecular complex that uses plant energy (originally captured in the leaves during photosynthesis) to accomplish its tasks. The nitrogenase enzyme splits the N2 triple bond and adds hydrogen atoms to convert the free N to ammonia (NH3). The ammonia is then quickly converted to other biologically useful nitrogen compounds like amino acids and proteins.
As we all learned in school, this is a classic symbiotic arrangement, where two independent species form a partnership in which both gain. The bacteria gain nutrients from the plant and a secure place to flourish and reproduce, while the host legume plant gains nitrogen in a useful form, which gives the plant a competitive edge in a harsh world where nitrogen is often in short supply.
The nitrogenase enzyme, however, deserves closer inspection. This is the guts of the fixation machinery. Nitrogenase consists of two large metalloproteins (proteins that contain metal atoms): “dinitrogenase reductase,” which contains iron, and “dinitrogenase,” which contains iron and molybdenum. These metalloproteins are two of the most complex metal-protein arrangements known, and we can see why: They intimately work in tandem to fix nitrogen. These two molecules routinely do something that the industrial Haber process only achieves with high pressures and high temperatures.
But here’s a practical tip: notice that nitrogenase contains molybdenum, which means that legumes require molybdenum to capture nitrogen. Therefore, if they are expected to add nitrogen to the soil, fields of clover or alfalfa must have more molybdenum than fields of grass. But molybdenum is a micronutrient, so only a little is needed – perhaps only a few grams per acre. The application rate depends on specific soil characteristics. The lesson here is to be aware of the needs, and then check with your local agronomist.
One peculiar characteristic of the nitrogenase enzyme is that it is irreversibly inactivated by oxygen. This poses an interesting metabolic conundrum. The plant’s root cells and the rhizobia bacteria all require oxygen to survive, yet oxygen will also shut down the nitrogen-fixing molecules that are their reason for existing.
What’s a fellow to do? Well, legumes have evolved an elegant solution: “leghemoglobin.” This molecule is designed much like the hemoglobin in our own blood, and it does much the same thing – holds oxygen and transports it. The root nodule cells synthesize leghemoglobin. When atmospheric oxygen permeates through the nodule’s outside shell, the leghemoglobin captures these oxygen atoms, holds them and keeps them away from the nitrogenase enzyme. But at the same time, it transports enough oxygen to root cells and bacteria to allow them to respire properly.
And like our own hemoglobin, leghemoglobin is red when it’s loaded with oxygen. If you take a healthy nodule and carefully split it open (with a scalpel or fine tweezers), you can actually see a distinct pinkish color. That’s the leghemoglobin. Which brings us to a time-honored maxim. I’m sure you’ve heard the old phrase, “You can’t get blood from a turnip.” Well, yes, that’s true, but if you look closely at a legume root …
Nodulation
Now for the rest of the story: nodulation – the formation of root nodules. If anything, this is even more remarkable than the chemistry of nitrogen fixation.
The nodulation story starts with a young legume seedling just beginning to send out roots. These roots release flavonoid compounds into the surrounding soil. If the right species of rhizobia is present and detects these flavonoids, the bacterial “nod” gene (probably for nodulation, get it?) goes into action and produces a species-specific “nod factor,” which then binds to surface receptors on the root epidermis cell. Then things really begin to happen.
The root epidermis cell begins to bulge outwards, forming a microscopic root hair that extends outwards and pushes into the soil. Calcium ions stream from the interior of the epidermis cell to the tip of the lengthening root hair. The rhizobia bacterium cell then attaches itself tightly to the side of the root hair. As the root hair continues to grow, something very peculiar happens: The bacterial “nod factor” causes the root hair to change its direction of growth.
Instead of continuing to grow outward, the tip of the root hair does a 180-degree turn and grows back on itself, forming a tight clamp that looks a little like a bobby pin – trapping the bacterium between the two parts of this clamp. Now sandwiched between two root hair cell walls, the bacterium reproduces and grows into a tiny microcolony. This bacterial colony then projects a living microtubule – called an infection thread – down through the center of the root hair.
This infection thread extends downward, working its way into deeper layers of cells. Parts of the infection thread fuse with the cell walls of some root cells. Bacteria populate the infection thread and then move into the nodule cells.
This is the beginning of a “proto-nodule” – an intermingled blend of bacterial and legume material. The rhizobia then differentiate into bacteroids, which lie inside the nodule cells, surrounded by plant cell cytoplasm. These bacteroids synthesize nitrogenase, and the plant synthesizes leghemoglobin and other molecules and also provides nutrients to the bacteroids. The proto-nodule grows larger and differentiates into a well-organized protuberance on the root. We now have liftoff – a fully functioning nodule.
Communication … recognition … attachment … growth … involvement … fusion … mutual benefit … nitrogen – a system for capturing atmospheric nitrogen that evolved piece by piece over millions of years. It’s the epitome of species cooperation. The legume and the rhizobia engage in an intricate duet, move for move, increasing complexity, almost like a dance.
On our farms, however, we hardly give it a thought. During the growing season, we go to the seed store, buy some legume seeds and inoculant, mix them together, dump the seed into our seeder, plant it and hope for the best. Two or three months later, we may walk through the field, dig up a few plants and look for nodules by carefully teasing the soil away from the roots. If we find nodules, we are happy. If we don’t, we scratch our heads.
But if we recognize the marvelous complexity of legume nodules and the intricacies of nitrogen fixation, we might strive to do a better job. The next time we buy legume inoculant, we’ll make sure that it is fresh – not more than six months on the shelves at room temperature or one year in the refrigerator. We’ll also check the expiration date. And of course, we’ll be extra careful to choose the right inoculant to match our legume species. We really want that dance to go well. 
PHOTO: Nitrogen-fixing nodules have formed on the root of this berseem clover plant. Photo by Lynn Jaynes.
Woody Lane is a certified forage and grassland professional with AFGC and teaches forage/grazing and nutrition courses in Oregon, with an affiliate appointment with the crop and soil science department at Oregon State. His book, From The Feed Trough: Essays and Insights on Livestock Nutrition in a Complex World, is available through Woody Lane.
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Woody Lane, Ph.D.
- Lane Livestock Services
- Roseburg, Oregon
