A chance encounter brought Katalin Toth to Mizzou.
The postdoctoral fellow, first heard about MU when Gary Stacey visited University of Munich. Toth heard of a position opening up in his lab. She has now been in the Stacey Lab for six years.
“I knew his work was important and well known,” Toth said. “You can almost directly apply what you find on soybeans to agriculture.”
Science has led Toth from the mountains of Slovakia where she did her masters to Hungary for her Ph.D. and Germany for research. Missouri ended up being the next step in her journey.
Being a foreign postdoc in the lab gives Toth a unique perspective. She has seen two different research environments — one in Europe and the other here —that highlight cultural and experimental differences.
“Here you have a facility core and you have to pay for it,” Toth said. “You have to really consider what you are doing and how you design your experiment. It is actually good because you really have to think about how you use your time in your experiment.”
Toth has always been given opportunities in the labs she has worked at, but as a postdoc she now has a chance to express her love for science to others.
“As a postdoc here, I get the opportunity to be involved in tasks not related to the everyday life of a researcher,” Toth said. “From organizing outreach activities to promoting plant science, it is important to me that people understand the importance of science.”
Toth’s love for science sprouts from the exploration of soybean research and how it will affect our world and other cultures. Currently, she is studying soybean association with beneficial soil bacteria that help soybeans to better access nutrients such as nitrogen, vital for a crop’s growth.
“There are different bacteria in the soil and I am looking at how the plant’s immune system is responding to the beneficial bacteria.”
Toth’s travels have also opened up her perspective on different culture’s needs.
“I care about the environment and doing something that is useful like this for agriculture, hopefully will help the environment,” Toth said. “After I saw how local people take care of Amazon while traveling in Peru, for example, I want my research to help improve crops and make an impact on the environment in a different way.”
Toth has done research for 10 years, and doesn’t want to stop anytime soon. From long hours at the lab to even weekends of research, Toth is always eager.
“Everyday there is something more exciting,” Toth said.
As busy as she is, Toth has always found time to enjoy her favorite place: outdoors. However, there is only one slight fall back from her move to Missouri.
“I just do not like the hot summers here,” Toth said.
8,124 miles. That’s how far Ph.D. student Ha Duong traveled from home to work in the Stacey Lab at Bond LSC.
Duong came from her home in Vietnam where she studied plant sciences at Hanoi University of Agriculture. A chance encounter brought her to MU.
“Back in my last year of undergrad, a professor from MU came and gave a talk,” Duong said. “I thought about MU. I then received a fellowship then chose here. I got it so it is destiny.”
With some questionable looks from her mother when she first heard of the idea, Duong went for it anyways. Despite a significant culture change from Vietnam to Missouri, Duong is embracing the change as an opportunity to get to know herself better.
“I get to compare these two countries and see the differences,” Duong said. “Which will always be good for me.”
Duong’s love of science stems from hanging out in her father’s material physics lab growing up. Duong did not realize the impact this would have on her until looking back on all the times of being in his lab.
“When you grow up, certain things you do not realize, get to you,” Duong said. “I quite liked the environment, it was quiet and you have your own creativity.”
Years later, Duong is now going into her fifth year as a Ph.D. student. She is trying to find the missing components in the extracellular ATP signaling pathway in plants. ATP is a high-energy molecule typically found inside cells where it stores and supplies the plant with fuel, so it is somewhat surprising that it also has a signaling role outside of the cell.
The Stacey Lab discovered the first extracellular ATP receptor in plants, so now the research is digging more into their discovery. Duong is happy about being around pioneers in plant science and wishes to be a pioneer as well.
“The moment I realized I am into science is thinking about how today I can discover a new thing,” Duong said. “But while it starts with the theories, later it can turn into an even bigger thing and have applications throughout life.”
To Duong, science can be applied from the lab to her home.
“Science means daily life to me,” Duong said. “Science influences the way I am thinking and how I do the simplest thing most effectively. Almost everything around us, we can criticize it using science. I am a practical person so anything you can apply to life is what I like.”
However, Duong emphasizes that science isn’t always as serious as one thinks. She has flexibility and creativity when it comes to her work and being half the equator away from home while studying what she loves makes missing home a little easier.
“I miss home, but not miss miss it,” Duong said. “I have work to do every day, and you need to do what you need to do and finish it. I do miss the food a lot, though.”
A soybean plant grows in the Bond Life Sciences Center’s greenhouse. | Photo by Samantha Kummerer, Bond LSC.
By Samantha Kummerer | Bond Life Sciences Center
Every summer, MU Bond Life scientists Gary and Bing Stacey plant soybeans. In the summer of 2016, they were testing mutant crops’ tolerance to different herbicides. Among the multiple weed killers tested was one called dicamba.
The researchers knew this particular chemical was tricky so they turned to an expert to apply it, MU herbicide researcher Kevin Bradley.
The next morning, a soybean breeder with a neighboring plot discovered his soybeans were damaged.
“These were plots where some of his graduate students experimented so they basically couldn’t use any of their data and we felt terrible, but we explained to them we took every precaution we could possibly take but it was this vaporization that took place,” Gary Stacey explained.
What Gary Stacey didn’t understand at the time was dicamba has an ability to travel even after it is sprayed. The herbicide doesn’t just kill weeds, it kills or damages everything not engineered to be resistant to it.
“So let’s say I spray it in this spot right here. You would think its localized but if the temperature and humidity conditions are right it will vaporize and come up and then go into the air,” Gary Stacey said.
Just how far it can travel and how much damage it can achieve was realized all too well by farmers throughout the country this year.
An estimated 3.5 million acres of soybeans were damaged this summer.
One obvious solution may be to simply stop using the weed killer. But the issue is not that simple.
“This is the hardest issue I can remember because there are good responsible farmers on either side of the issue,” said Missouri Farm Bureau president Blake Hurst.
With so much on the line for all sides, dicamba has tangled farmers, corporations and researchers together in a controversial issue.
Bradley is right in the middle. He’s received calls from farmers who just lost 10 percent of their income for nothing they did wrong.
He’s also received calls from people who are upset by any suggestion that anything about the chemical is wrong. These are the farmers who need dicamba to control weeds that are no longer responding to the traditional weed killer Roundup.
“I’ve had the farmers who planted the traits saying ‘These are my highest yields ever how can you say these things?’ And their neighbor across the road just lost 20 bushels an acre because of your highest yields ever. It’s just a very personal issue for each person involved,” Bradley explained.
One case got so personal that a farmer in Arkansas allegedly shot his neighbor.
“I’ve been here for 14 years and I’ve been doing this kind of work for 20, never seen anything like this is agriculture. Period. Never seen this level of controversy between farmer to farmer and farmer to company or between company and university people. I’ve never seen anything like this,” Bradley said.
Dicamba is not a new formulation, but its use is. Monsanto developed genetically modified soybeans and cotton seeds that are resistant to dicamba. One of the problems farmers are pointing to is that Monsanto released the new seeds while still in the process of developing a better formula of dicamba. The new formula aimed to reduce volatilization, a tendency to vaporize after being sprayed on fields and then drift to neighboring areas. Monsanto claims the new formula reduces volatility by 90 percent, but Bradley said 90 percent is not 100 percent.
Bradley’s work has been consumed by this single herbicide as he tries to find the truth of what aspect of dicamba is causing the damage.
In Bradley’s eyes, there are four factors contributing to the widespread damage: physical drift mistakes (spraying with the wind, nozzle not attached correctly), tank contamination, temperature inversion, and volatility.
These factors are recognized by other researchers and Monsanto. The disagreement is over which factor is most at fault.
“Monsanto has a pretty high number for the farmer fault percentage,” Bradley said explaining the blame game. “ I don’t know when they’ll ever really say, ‘yeah, volatility could be contributing to this problem, too’ and that’s the difference between university weed science.”
This contributes to the confusion among users.
“You don’t know who to believe,” Gary Stacey said.
But Gary Stacey thinks this is where researchers are able to help. By acting as an objective third party, scientists can sort the fact from the fiction.
“We’re just trying to get out the truth and what science says, that’s my job,” Bradley explained. “I don’t care necessarily what amount of money a company has invested in something. Our job is to call it like we see it and show the science.”
With a controversial issue like this, sometimes the truth comes with some risk.
MU has been conducting experiments that test the air for the volatility of the chemical. The research is detecting dicamba in the air up to four days after initial application of the chemical. Bradley explained this is not something the companies want to be made public and there’s been considerable pushback.
In addition to research, Bradley is working with the Missouri Department of Agriculture to create training courses for farmers wanting to use the chemical next season.
Despite millions of damaged acres, dicamba is not going away anytime soon.
Gary and Bing Stacey haven’t used dicamba again, but many farmers making their money off crops have no choice. Bradley said Monsanto is planning on doubling the amount of dicamba-resistant soybeans in 2018 and many of the farmers who have been continuously hit by their neighbors’ chemical plan to plant the new seeds.
Bradley said part of the issue is soybeans are not a crop people directly consume. In general, soybeans yields were considerably high this year, so the damaged acres didn’t make as big of an impact on overall production.
“I think the only thing that is going to make a difference next year is if we have an off-target movement that is hitting more high-value crops, more high-value plant species throughout a wider geography,” Bradley said.
If this same type of damage was affecting produce people directly consume or trees, Bradley thinks dicamba would have been off the market by now.
EPA will reevaluate the use of the herbicide next November. This is one of the first times Bradley can remember that the industry granted only a two-year registration.
“I am absolutely convinced that if we have a summer in 2018 like we had in 2017, it will not be renewed,” Hurst said.
Bradley is not so certain. He said he has heard mixed reviews about how the future of this controversial weed killer could go.
“It is an unique situation for sure, hopefully it ends soon,” Bradley said.
Sterling Evans is a sophomore plant sciences major conducting research in Gary Stacey’s lab in Bond LSC. | photo by Allison Scott
By Allison Scott | Bond LSC
“#IAmScience because I want to focus my research on problems that exist in agriculture in undeveloped and third world countries.”
Sterling Evans’ mind wasn’t focused on research when he started college, but that would soon change.
The sophomore plant sciences major uncovered his interest thanks to Freshman Research in Plant Sciences (FRIPS) — a program dedicated to introducing research to freshman students from plant-related degree programs.
“I was interested in plant sciences-related fields when I started here, but I had no intention of getting involved in undergraduate research,” Evans said. “Being selected for FRIPS was instrumental in getting me involved with research.”
Along with a handful of students selected for FRIPS each year, Evans got to interact with various professors and mentors around campus on a weekly basis. Because of that exposure, Evans found a place in the lab of Bond Life Sciences Center’s Gary Stacey.
After a year working in Stacey’s lab, Evans just joined a new project that aims to improve the nutritional value of soybeans.
“They’re used as a main source of protein for a lot of countries, so improving their nutritional content would have a huge impact,” Evans said.
The team is applies CRISPR, a gene-editing tool, to model plants called Arabidopsis as a first step.
“We are working on Arabidopsis right now as a proof of concept, because it can be done in a relatively short period of time, before investing as much as a two additional years in soybeans,” Evans said.
While he only spends 15 hours in the lab each week, Evans noticed the lab’s impact on his approach to academics in other ways.
“Research gives me more motivation to think about how to apply information I’ve learned in class to work in the lab,” Evans said. “It has made me more analytical in classes because I have more of a desire to understand things.”
Evans plans to earn a Ph.D. in a plant sciences field and wants to continue research in his career. He’s most interested in helping ensure small communities throughout the world have enough to eat, and he hopes to contribute by studying orphan crops.
“I think they’re cool because they’re really important to small people groups. No one studies them because they aren’t a big deal in the United States or other countries,” Evans said. “If we work on them we won’t have a huge impact on hundreds of millions of people, but we will have a huge impact on small communities.”
That impact all started in a lab. Had he not stepped out of his comfort zone he might never have discovered this path, and he highly encourages other students to give research a chance.
“There are labs for almost everything and there’s an area for everyone,” Evans said. “I didn’t know I wanted to do research until I did it.”
Beverly Agtuca, a Ph.D. candidate that works in Dr. Gary Stacey’s lab in Bond LSC. | photo by MJ Rogers
Beverly Agtuca was born in New York, but has family in the Philippines, a country that struggles with malnutrition and undernourishment. Her overall goal for her research is to help countries that struggle with undernourishment by increasing the agricultural productivity in those countries.
“When I was little, I went on summer vacation to visit my family, which included my grandmother in the Philippines,” she said. “Everyday my grandmother wanted me to go out to the rice fields from 5 a.m. to 10 p.m. with the other children to get rice for our meals. That was not an easy task and that moment changed my life. That’s when I decided that I wanted to be a plant scientist.”
Agtuca graduated in 2014 with honors in Biotechnology and a minor of Microscopy from the State University of New York College of Environmental Science and Forestry (SUNY ESF) in Syracuse, NY. She’s currently a Ph.D. candidate in plant breeding, genetics, and genomics at MU. She chose to come to Bond LSC because of the community and Dr. Stacey, her supervisor and mentor.
“If you ever need help, there’s always help here,” she said. “Everyone at Bond LSC is so kind, including the staff. I love to make small talk with the custodians and they are always supporting me and say I should never give up when I have a bad day.”
Ever since coming to MU in 2014, Agtuca has been keeping busy. In June, she received a travel award to go to the American Society of Plant Biologists (ASPB) in Hawaii. The International Society for Molecular Plant-Microbe Interactions (IS-MPMI) also awarded her a travel award to attend the 2016 meeting in Portland, Oregon, where she gave an oral and poster presentation. She also has two original research publications under her belt and is currently working in Dr. Gary Stacey’s lab at Bond LSC.
The research for her dissertation is focused on the relationship between rhizobia and soybeans. She collaborates with scientists at George Washington University (GWU) in Washington, D.C. and the Pacific Northwest National Laboratory (PNNL) in Richland, Washington to enhance the capabilities of the 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T FTICR) through application of laser ablation – electrospray ionization mass spectrometry (LAESI-MS) technology that can analyze the contents of single plant cells. This 21T FTICR machine was recently installed at PNNL and represents one of only two such machines in the world.
This is revolutionary because few people do single cell analysis. Usually, scientists deal with the law of averages, which dilutes the final measurements. But this technology gives an in-depth glimpse into a single cell so scientists can obtain a more comprehensive bigger picture.
“After we finish building this technology, we want to spread the technique to different research groups so they can answer these research questions on their own,” said Agtuca. “It can help people outside of plant sciences too, and hopefully will help with cancer treatment and disease prevention.”
Bond LSC researchers showed for the first time ever that a grass, Setaria viridis, can receive 100 percent of its nitrogen needs from bacteria when associated with plant root surfaces. This grass will now serve as model for research into biological nitrogen fixation that could benefit crop development. | Photo by Roger Meissen, Bond LSC
By Roger Meissen | MU Bond Life Sciences Center
As farmers spend billions of dollars spreading nitrogen on their fields this spring, researchers at the University of Missouri are working toward less reliance on the fertilizer.
Less dependence on nitrogen could start with a simple type of grass, Setaria viridis, and its relationship with bacteria. The plant promises to lay groundwork for scientists exploring the relationship between crops and the fixing nitrogen bacteria that provide them the nitrogen amount plants need daily.
“In science sometimes you have to believe because we often work with such small microorganisms and DNA that you cannot see,” said Fernanda Amaral, coauthor and MU postdoctoral fellow at Bond Life Sciences Center. “Before this research no one had actually proved such evidence that nitrogen excreted by bacteria could be incorporated into plants like this.”
Fernanda Amaral, coauthor and MU postdoctoral fellow at Bond Life Sciences Center. | Photo by Roger Meissen, Bond LSC
Biological Nitrogen fixation — where diazotrophic bacteria fix atmospheric nitrogen and convert it to ammonium — provides a free way for plants to alter and absorb the nutrient. Farmers have long known that legumes like soybean fix nitrogen due to the symbiosis with bacteria in the soil through development of nodules on their roots, but since grasses like corn and rice don’t form this specialized structures that relationship has been trickier to explore.
Yet in fact, this team’s experiments showed the grass Setaria viridis received 100 percent of its nitrogen needs from the bacteria Azospirillum brasilense when associated with plant root surfaces.
“I believed in these bacteria’s ability, but I was really surprised that the amount of nitrogen fixed by the bacteria was 100 percent,” Amaral said. “That’s really cool, and that nitrogen can make so much of a difference in the plant.”
Worldwide farmers used more than 100 million tons of nitrogen on fields in 2011, according to the United Nations Food and Agriculture Organization. In the same year, the U.S. alone produced and imported more than $37 billion in nitrogen.
This grass can serve as a simple model for research, standing in for grass relatives such as corn, rice and sugarcane to explore a similar relationship in those crops. This research, “Robust biological nitrogen fixation in a model grass–bacterial association,” was published in the March 2015 issue of The Plant Journal.
A nutrient, a nuclear reactor and a model plant
Proving that this grass actually uses nitrogen excreted from the bacteria took some clever experiments, a global collaboration and a nuclear reactor.
MU researchers in the lab of Gary Stacey, a Bond LSC investigator, partnered with scientists in Brazil and at Brookhaven National Laboratory in New York to find a robust plant model system.
They screened more than 30 genotypes of Setaria viridis grass, looking for a strong nitrogen fixing response when colonized with three different bacteria strains. They germinated the seeds in Petri dishes and inoculated those three days after germination with a bacterial solution. Then plants were transplanted into soil containing no nutrients. By eliminating nitrogen in the soil, the scientists were able to make sure that the bacteria was the only source of nitrogen for plant.
The team settled on Azospirillum brasilense bacteria, which has been used commercially in South America to improve crop plant growth. It colonizes the surface of the roots and showed the greatest amount of plant growth when associated with plant roots.
Proving that the bacteria truly fixed the nitrogen used by the plant, required exposing plants to radioactive isotopes at Brookhaven National Laboratory. That began with Nitrogen 13, an unstable radio isotope that showed exactly where and how quickly this nutrient was taken up from the bacteria.
A radio tracer chamber at Brookhaven National Laboratory was needed to test if Setaria viridis actually used nitrogen produced by the bacteria. The scientists allowed only one leaf to contact the radioactive nitrogen, so they could truly tell if it was being used. | Photo provided by Fernanda Amaral
“Nitrogen 13 is really sensitive matter with a half-life of less than 10 minutes, and we first thought there wouldn’t be that much nitrogen fixed by the plant,” Amaral said. “We administered Nitrogen 13 only on the roots, quickly scanned the samples and calculated how much of the nitrogen the plants assimilated based on the decay analysis of the tracer.”
This experiment, paired with several others, showed that this model grass truly incorporated the nitrogen released by the bacteria and metabolizes it in several components.
Model (plant) citizen
But why does a type of grass that doesn’t produce food matter so much?
The answer is time and simplicity.
“Corn is really good at responding to bacterial inoculation, but it’s very big and takes a long time to produce seeds and also the genome is complex,” said Beverly Agtuca, an MU Ph.D. student who worked on the study. “Setaria viridis is a small plant that can produce a lot of seeds faster, has a pretty simple genome and can serve as a model for research.”
That makes it perfect to explore how the plant actually uses its bacterial partners, and labs around the world are already using this plant model for research.
For the Stacey lab, the next step is to pinpoint the gene in the model grass that makes this possible.
“We want to identify the genes responsible for the interaction between plant and bacteria and meanly the ones involved with the nitrogen uptake,” Fernanda said. “We hope that will allow us to improve plant growth based on the gene to further study.” We believe that our findings can stimulate others studies at this area, which seems to be a promise plant friendly way to apply for promoting a sustainable agriculture, especially to crop systems including bioenergy grass.
Amaral and Agtuca work in the lab of Gary Stacey at Bond LSC. Stacey is a Bond LSC investigator and a Curators Professor of Plant Sciences in the College of Agriculture, Food and Natural Resources at the University of Missouri. Collaborators included researchers at Brookhaven National Laboratory, State University of New York, Federal University of Paraná in Brazil and Federal University of Santa Catarina in Brazil.
Funding for this project came from the National Institute of Science and Technology- Biological Nitrogen Fixation, INCT-FBN, the Brazilian Research Council, Ciência Sem Fronteiras Program, The Department of Energy and SUNY School of Environmental Science and Forestry Honors Internship Program.
Yaya Cui, an investigator in plant sciences at the Bond Life Sciences Center examines data on fast neuron soybean mutants that are represented on the SoyKB database.
The most puzzling scientific mysteries may be solved at the same machine you’re likely reading this sentence.
In the era of “Big Data” many significant scientific discoveries — the development of new drugs to fight diseases, strategies of agricultural breeding to solve world-hunger problems and figuring out why the world exists — are being made without ever stepping foot in a lab.
Developed by researchers at the Bond Life Sciences Center, SoyKB.org allows international researchers, scientists and farmers to chart the unknown territory of soybean genomics together — sometimes continents away from one another — through that data.
Digital solutions to real-world questions
As part of the Obama Administration’s $200 million “Big Data” Initiative, SoyKB (Soy Knowledge Base) was born.
The digital infrastructure changes the way researchers conduct their experiments dramatically, according to plant scientists like Gary Stacey, Bond LSC researcher, endowed professor of soybean biotechnology and professor of plant sciences and biochemistry.
“It’s very powerful,” Stacey said. “Humans can only look at so many lines in an excel spreadsheet — then it just kind of blurs. So we need these kinds of tools to be able to deal with this high-throughput data.”
The website, managed by Trupti Joshi, an assistant research professor in computer science at MU’s College of Engineering, enables researchers to develop important scientific questions and theories.
“There are people that during their entire career, don’t do any bench work or wet science, they just look at the data,” Stacey said.
The Gene Pathway Viewer available on SoyKB, shows different signaling pathways and points to the function of specific genes so that researchers can develop improvements for badly performing soybean lines.
“It’s much easier to grasp this whole data and narrow it down to basically what you want to focus on,” Joshi said.
A 3D-protein modeling tool lends itself especially to drug design. A pharmaceutical company could test the hypothesis and in some situations, the proposed drug turns out to yield the expected results — formulated solely by data analysis.
The Big Data initiative drives a blending of “wet science” — conducting experiments in the lab and gathering original data — and “dry science” — using computational methods.
Testament of the times?
“Oh, absolutely,” Joshi said.
Collaboration between the “wet” and “dry” sciences
Before SoyKB, data from numerous experiments would be gathered and disregarded, with only the desired results analyzed. The website makes it easy to dump all of the data gathered to then be repurposed by other researchers.
“With these kinds of databases now, all the data is put there so something that’s not valuable to me may be valuable to somebody else,” Stacey said,
Joshi said infrastructure like SoyKB is becoming more necessary in all realms of scientific discovery.
“(SoyKB) has turned out to be a very good public resource for the soybean community to cross reference that and check the details of their findings,” she said.
Computer science prevents researchers having to reinvent the wheel with their own digital platforms. SoyKB has a translational infrastructure with computational methods and tools that can be used for many disciplines like health sciences, animal sciences, physics and genetic research.
“I think there’s more and more need for these types of collaborations,” Joshi said. “It can be really difficult for biologists to handle the large scope of data by themselves and you really don’t want to spend time just dealing with files — You want to focus more on the biology, so these types of collaborations work really well.
It’s a win-win situation for everyone,” she said.
The success of SoyKB was perhaps catalyzed by Joshi. She adopted the website and the compilation of data in its infant stages as her PhD dissertation.
Joshi is unique because she has both a biology degree and a computer science background. Stacey said Joshi, who has “had a foot in each camp,” serves as an irreplaceable translator.
Most recently, the progress of SoyKB as part of the Big Data Initiative was presented at the International Conference on Bioinformatics and Biomedicine Dec. 2013 in Shanghai. The ongoing project is funded by NSF grants.
Jeongmin Choi (left), Gary Stacey (center) and Kiwamu Tanaka recently discovered the first plant receptor for extracellular ATP using Arabidopsis plants. Roger Meissen/Bond LSC
It’s the genetic equivalent to discovering a new sensory organ in plants.
A team at the University of Missouri Bond Life Sciences Center found a key gene that sniffs out extracellular ATP.
Scientists believe this is a vital way plants respond to dangers, such as insects chewing on its leaves. The journal Science published their research Jan. 17.
“Plants don’t have ears to hear, fingers to feel or eyes to see. They recognize these chemical signals as a way to tell themselves they are being preyed upon or there’s an environmental change that could be possibly detrimental, and they have ways to defend themselves,” said Gary Stacey, a Bond LSC biologist. “We have evidence that extracellular ATP is probably a central signal that controls the ability of plants to respond to a whole variety of stresses.”
ATP (adenosine triphosphate) is the main energy source inside any cell. All food converts to it before being used in a cell, and ATP is necessary to power many of the cell processes that create more energy. Its value as an energy reserve is squandered outside the cell.
Scientists spent years trying to figure out what this compound did while floating outside cell walls. Animal researchers found that answer in the 1990s. They identified the first ATP receptors, now seen to play roles in muscle control, neurotransmission, inflammation and development.
Plant scientists observe similar extracellular ATP responses in plant biochemistry, but until now could not identify the exact receptor for it or what it did.
“We call this new receptor P2K, and it’s unique to plants,” Stacey said. “Even though animals and plants hold some responses in common, they have evolved totally different mechanisms to recognize extracellular ATP.”
Led by Stacey, MU graduate student Jeongmin Choi and postdoc Kiwamu Tanaka screened 50,000 mutant Arabidopsis plants to find ones that didn’t respond to extracellular ATP. Using a protein called aequorin – which causes jellyfish to glow – the two-year process boiled down to whether a plant would produce light when ATP was added. Since aequorin only luminesces when it binds to calcium, those plants without extracellular ATP receptors stayed dark.
“If you add ATP to wild-type plants, calcium concentrations go up and the plants produce more blue light,” Choi said. “We found nine mutant plants with no increase in calcium and, therefore, no increase in light emission.”
These Arabidopsis plants contain aequorin. Aequorin produces blue light as a result of oxidation of the substrate, in a calcium-dependent manner. When wild-type seedlings were treated with 500 uM of ATP, intracellular calcium concentration is rapidly elevated. Increased calcium were bound to aequorin, which leads to blue light emission. Courtesy Jeongmin Choi
By comparing the genetic sequences of these nine mutants, Stacey’s lab pinpointed the gene to chromosome 5 and labeled it DORN 1, since it doesn’t respond to the nucleotide ATP.
This discovery casts a different light on previous research.
“What we think is happening is that when you wound a plant, ATP comes out in the wound and that ATP triggers gene expression, not the wounding in and of itself,” Stacey said. “We think ATP is central to this kind of wound response and probably plays a role in development, in a lot of other kinds of things.”
Future research will focus on exactly how this receptor works with ATP. Tanaka plans to study its protein structure, how it reacts to pests in lab situations and possible co-receptors that could also play a role in recognizing ATP.
Grants from the U.S. Department of Energy Office of Basic Energy Sciences and the Republic of Korea supported this research.
Yan Liang and Gary Stacey research the symbiosis between legumes, like these soybeans, and nitrogen-fixing bacteria at the Bond Life Sciences Center.
A silent partnership exists deep in the roots of legumes.
In small, bump-like nodules on roots in crops like soybeans and alfalfa, rhizobia bacteria thrive, receiving food from these plants and, in turn, producing the nitrogen that most plants need to grow green and healthy.
Scientists have wondered for years exactly how this mutually beneficial relationship works. Understanding it could be the first step toward engineering other crops to use less nitrogen, benefitting both the bottom line and the environment.
University of Missouri Bond Life Sciences Center researchers recently identified what keeps crops like corn and tomatoes from the sort of symbiotic relationship enjoyed by legumes. Science published this discovery online Thursday, September 5, 2013.
“Our work uniquely shows that all flowering plants, not just legumes, actually do recognize the chemical signal given off by rhizobia bacteria,” said Gary Stacey, Bond LSC investigator and plant sciences professor.
His lab identified the most likely receptor for this chemical and showed that the signal suppresses the plant immune response, which normally protects plants from pathogens. This allows rhizobia a better chance to infect and live inside the plant.
Grounded
Chemical signals are extremely important to plants.
Since plants can’t walk around to explore or avoid danger, receptors in cells of each plant act as its eyes and ears. They gather information about insects, bacteria and other threats and stresses from chemicals. These signals allow the plant to respond and adapt to its environment, such as resisting stresses like drought and infection by pathogens.
With rhizobia, the bacteria produce lipo-chitin, a sugar polymer with a fatty acid attached. This molecule is similar to chitin normally found in the cell walls of fungi, the exoskeletons of crustacea or insects.
Legumes, such as soybean, sense this signal – called a NOD factor since it triggers nodulation – and create the nodules where the bacteria fix atmospheric nitrogen into the soil.
That doesn’t happen in other plants.
Two possibilities
Scientists once gravitated toward thinking that non-legumes, which are not infected by rhizobia, just weren’t capable of receiving the NOD factor signal. But, a less popular theory guessed that plants like corn do receive the NOD factor signal but interpret it differently or have a problem with the mechanistic pathway.
Stacey said figuring out which is happening is like fixing a motion-detecting light.
“If you walk into a room and the light doesn’t turn on, either the motion detector is broken or there’s a breakdown of the electrical circuit between the detector and the light bulb,” Stacey said. “The analogy in a corn plant would be that it either doesn’t recognize the signal or recognizes the signal but lacks the ability to couple it with downstream developmental effects.”
Stacey’s lab set out to determine which was true.
Corn, soybean, tomato and Arabidopsis were treated with bacterial flagellin, a protein known to cause a strong immune response, and also received doses of the NOD factor. These represent a diverse spectrum of plants to ensure the results were wide ranging. Results showed the NOD factor suppressed the immune response by 60 percent.
“After these results, what allowed us to take the next step forward is that we were able to make mutant plants with changes in what we think is the receptor for the NOD factor,” Stacey said. “That step showed that when plants lack the ability to recognize the NOD factor, you don’t see the suppression of the immune system.”
Since all plants seem to respond to the NOD factor, scientists think this immunity suppression ability could be evolutionarily ancient and part of how rhizobia bacteria changed from foe to a symbiotic partner.
“There’s this back and forth battle between a plant and a pathogen,” said Yan Liang, an MU post doc in Stacey’s lab. “Rhizobia eventually developed this chemical to inhibit the defense response and make the plant recognize it as a friend.”
A future in the field
Nitrogen has both an environmental impact and high price tag.
Fossil fuels are combined with nitrogen from the atmosphere to create the fertilizer.
When applied to fields, the excess fertilizer also ends up in rivers and streams, contributing to nitrification and hypoxia in waterways.
“The dead zone in the Gulf of Mexico is generally attributed to agricultural runoff and producing nitrogen fertilizer increases dependence on fossil fuels,” Stacey said. U.S. farms used almost 13 million tons in 2011, according to the USDA, with almost half of it being applied to corn. Nitrogen prices ranged from $400-$850 per ton in the U.S. in 2013.
Nitrogen fixed in the soil by rhizobia is the closest thing to a free lunch a plant can get. For farmers, that’s one less input needed and is part of why legumes remain a staple of crop rotations.
While it’s a long way off, Stacey’s research is another step to improving crops.
“Since the discovery in 1888 of this nitrogen-fixing symbiosis between this bacteria and plants like soybean, the dream has always been to transfer this technology into other plants like corn, wheat or rice,” Stacey said. “Once we understand exactly what’s mechanistically unique in a legume, then we hope to be able to transfer that trait into corn many years down the road.”
A grant from the Office of Basic Energy Sciences of the U.S. Department of Energy funded this research. Gary Stacey is a Bond Life Sciences Center investigator, a professor of plant science in the University of Missouri College of Agriculture, Food and Natural Resources and has an adjunct appointment in MU’s biochemistry department.
