Uncovering the secrets of this curious process may benefit both farmers and the environment says Janine Sherrier, a plant molecular biologist at the Delaware Biotechnology Institute and assistant professor in the University of Delaware College of Agriculture and Natural Resources. Through protein biochemistry, Sherrier is striving to do just that.
“Because nitrogen is a limited nutrient in the soil, many crops and plants must be supplemented with nitrogen-rich fertilizers for optimal growth,” Sherrier explains. “Unlike most plants, however, soybeans and other legumes are hugely successful in utilizing their own supply of nitrogen from structures called root nodules.
As the mechanism for harnessing nitrogen becomes clearer, Sherrier says we can better predict and quantify the amount of nitrogen that may be utilized by a plant. We then can breed plants specifically to maximize this trait.
“By selecting for this trait, farmers can ensure very fruitful harvests without the tons of nitrogen they usually apply to their fields,” Sherrier says. “Not only that, but soils enriched with legumes actually will retain enough extra nitrogen — a couple of hundred pounds per acre — to nurture the next season’s crop.
“Farmer benefits economically, and the environment benefits from the application of less chemical fertilizers.”
Root nodules are formed from a symbiosis between plants and bacteria, a process that has interested scientists for years. Trying to understand this important relationship is nothing new. What is new, however, is Sherrier’s approach, which is proteomics.
“Proteomics is a very effective way to study the physiological state of the cell,” says Sherrier. “It is simply the study of a set of PROTEins encoded by the genOME — which is where it gets its name.”
In other words, proteomics is protein biochemistry, a tool of modern biotechnology. Proteomics has developed into a powerful tool because of genome sequencing projects and improvements in methods to sequence proteins.
Sherrier’s research in proteomics is targeted toward answering specific questions about the plant cell membrane that surrounds the soil bacterium, rhizobia, the naturally occurring bacterium that induces nitrogen-fixing root nodules to form on legume roots.
“I selected Medicago truncatula as my model system because there is a large genome project that was recently funded to sequence the genes involved in symbiosis from this plant,” she says. “In addition, the genome of the bacterial partner will be completely sequenced this year. That means there is lots of background information to support our work in biochemistry.
“There are populations of scientists in the world who work together, sharing resources, and the team working with Medicago is excellent. We’re competitive, but we are a friendly scientific community, which promotes the advancement of our scientific questions.”
According to Sherrier, the relationship between legumes and rhizobia is very specific, and that specificity is controlled by a complex exchange of signals between the plant and rhizobia.
In response to the correct bacterial signals, the plant actually promotes bacterial infection of its roots and the formation of a new plant structure – the root nodule. This nodule is both external and internal. Once infected by rhizobia, the root hair begins to curl up and bulge out as a narrow infection thread or tube begins growing within the root hair.
The internal cells continue dividing as the infected tube stretches into the cell, where rhizobia are released into the plant cell’s cytoplasm and modify atmospheric nitrogen into a form that the plant can use to grow.
“Rhizobia are special,” says Sherrier. “First of all, their genes encode proteins that are important for the symbiosis. In the nodule, they also are surrounded by a plant membrane that envelops and protects them immediately as they enter the plant cell cytoplasm.” Sherrier notes that this is somewhat peculiar, as plants ordinarily do not allow bacteria within their cells.
Within the cytoplasm, a bacterium will grow to 100 times its previous size. The large bacterium and the surrounding membrane are called a symbiosome. At this point, it actively harnesses nitrogen for the plant.
“The plant membrane is a unique biochemical structure,” Sherrier says. “It is very important for the exchange of nutrients between the plant and the microbe, and yet we know little about it. We want to understand what exact role this membrane is playing, and what important proteins are involved in this process.
“This is a tremendous amount of work, but once we can begin to find these answers, then we can use those proteins to enhance that symbiosis and to make symbiotic interactions more effective in soybeans, alfalfa and other legumes.”
To study the membrane, Sherrier and her team harvest the nodules from Medicago, then purify the membranes to isolate them from everything else inside the symbiosome. They then purify membranes from uninfected Medicago roots.
The next step is another procedure wherein more than 700 individual proteins from the plant membrane migrate into individual spots on a two-dimensional gel that looks like an 8-by-8-inch graph. The procedure is repeated with the uninfected Medicago roots. On each of these gels, the individual proteins separate both by isoelectric focusing (positive or negative charge) and by mass (weight).
“By comparing the gels from the infected and uninfected roots, we may recognize if there is a new protein present or if an existing protein becomes more abundant,” Sherrier says. “Or maybe a protein will disappear entirely. Then we can ask, ‘What was its role in the first place? Why did it disappear?’ ”
According to Sherrier, her work has application not only directly in helping farmers reduce or eliminate the amount of nitrogen they use, but also in trying to puzzle out how legumes encourage this particular beneficial bacterial infection while discouraging other disease-causing bacteria. Anything they can learn from this process may help biologists discover how to protect against potential pathogens in another plant.
Proteomics has many applications in agriculture, Sherrier says, but it has applications in human health as well – with rheumatoid arthritis, for example.
“If you check the fluid in the elbow, you might see that certain proteins disappear and new ones appear over time. Then you might ask, ‘What are those proteins? Do they exacerbate the disease?’
“At least you have something you can grasp onto to try to get to the root of the disease,” she says. “Many diseases can be studied in this manner. Pharmaceutical companies are really big into proteomics right now.”
One question Sherrier often is asked is whether what she is learning may one day enable biologists to develop corn and other plants that, like legumes, can harness nitrogen from the atmosphere.
“That’s like the Holy Grail,” she says. “It’s a long time off – but it may in fact be possible. Certainly, it’s one reason that the U.S. Department of Agriculture promotes this type of research.”