plant sciences

Planting a seed for sciences

Jinghong Chen | Bond Life Sciences Center

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Plants on the left grow with rhizobia bacteria, one type of fixing nitrogen bacteria, in the greenhouse, while the plants on the right grow without the bacteria. | photo by Jinghong Chen, Bond LSC

Since eight years old, Beverly Agtuca knew she wanted to be a scientist.

A trip to Philippines changed Agtuca, an American-born Filipino, and inspired her passion on plants.

“My grandma always told me to work in the field all day so that they can have enough food for us to eat,” Agtuca said. “The life [in Philippines] is so different from here…I want to not just provide food but be that scientist trying to figuring something out, and hopefully saving the world.”

Agtuca is on her way to her dream. She is now a third year doctoral student in Gary Stacey’s lab at Bond Life Sciences Center with a focus on nitrogen-fixing bacteria.

Although she has been involved in research since high school, Agtuca recently faced a new challenge of telling people about her work. The Preparing Tomorrow’s Leaders of Science class tasked her with making a 90-second video to explain her two-year study to the general public.

Her team, “The A Team,” chose to go with the benefits of having nitrogen-fixing bacteria.

For decades, people have been adding nitrogen fertilizers to plants to improve yields, but this can lead to pollution in water systems and ecosystems. Scientists need to enhance plant productivity to meet a huge food demand by the year of 2050.

One little bacteria might make this possible and save the world. Rhizobia, a type of natural bacteria in soil, are able to fix nitrogen via biological nitrogen fixation. These bacteria can convert nitrogen gas into ammonia as a plant nutrient source, while the plants give all the carbon sources back to the bacteria.

“It is like a walky-talky,” Agtuca said. “They are communicating with each other.”

Yet before speaking to the public, Agtuca needs to explain the plant-bacteria interaction to her teammates. Students less well versed in science like Jessica Kaiser, a strategic communication student, thinks of science differently.

“The biggest issue we ran into is jargon, like basic science words that [my teammates] are so comfortable with,” Kaiser said. “We need to focus on what people care about instead of the technical sides, to focus on why it matters to anybody rather than just to a science person.”

Within two weeks, they produced the video “Good Microbes: reducing pollution one farm at a time.” Along with two other teams, their videos will be commented and judged by representatives from Monsanto.

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“The A Team” stands together at Bond Life Sciences Center. From left to right: Jessica Kaiser, Sven Nelson, Anna Glowinski, Eleni Galata and Beverly Agtuca. | photo by Jinghong Chen, Bond LSC

The 90-second video is just a glimpse of Agtuca’s study. In the last two years she has been focusing on the use of a new technique — laser ablation electrospray ionization mass spectrometry (LAESI-MS) — that does in situ metabolic profiling of tissues. The lab is using LAESI-MS to investigate the metabolites in a well-characterized model plant-rhizobium system, specifically nitrogen-fixing soybean nodules resulting from root infection by the symbiotic bacterium Bradyrhizobium japonicum.

This work includes a huge collaboration that was developed through a Department of Energy (DOE) grant involving the George Washington University, Washington D.C. and the Environmental Molecular Science Laboratory (EMSL), Pacific Northwest National Laboratory, Richland, WA.

LAESI-MS works like a superhero’s laser-like beams. You first aim the laser on the sample, which then heats it and causes neutral particles to be released into the air. This plume of neutrals is then captured and ionized by the electrospray, and finally analyzed by the spectrometer to figure out the exactly what metabolites in nodules are involved in biological nitrogen fixation.

“It takes about three seconds to analyze one sample using this LAESI-MS technique,” Agtuca said. Other metabolic techniques require extensive pre-treatment of the sample before analysis.

By analyzing the data collected via LAESI-MS, the lab is able to confirm that future plant studies could apply this new approach to understand the interactions between plant and bacteria.

Agtuca’s research is a long way from her first experiences with plants. She still remembers the moment she found her plants in her own garden died. She was less than 10 years old, yet devoted to taking care of her plants with water and fertilizers.

“I was really sad. I could not get my tomatoes, peppers and eggplants to live.…That makes me think that I want to answer why they didn’t grow,” Agtuca said.

More than ever, her future is helping her answer those question for herself.

 

Gary Stacey is a Bond LSC investigator and MU curators’ professor of plant science and MSMC endowed professor of soybean biotechnology. Read more here about Stacey lab.

Sven Nelson is a USDA/ARS postdoctoral research scientist at the University of Missouri. Anna Glowinski is a Ph.D. student in the USDA/ARS lab. Jessica Kaiser is a graduate student in strategic communication. Eleni Galata works as the team mentor and she is a Ph.D. student in agricultural and applied economics at MU.

Understanding spit

Scientists find how nematodes use key hormones to take over root cells

Roger Meissen | Bond Life Sciences Center
This Arabidopsis root shows how the beet cyst nematode activates cytokinin signaling in syncytium 10 days after infection. The root fluoresces green when the TCSn gene associated with cytokinin activation is turned on because it is fused with a jellyfish protein that acts as a reporter signal. (N=nematode; S=Syncytium). Contributed by Carola De La Torre

This Arabidopsis root shows how the beet cyst nematode activates cytokinin signaling in the syncytium 10 days after infection. The root fluoresces green when the TCSn gene associated with cytokinin activation is turned on because it is fused with a jellyfish protein that acts as a reporter signal. (N=nematode; S=Syncytium). Contributed by Carola De La Torre

This is a story about spit.

Not just any spit, but the saliva of cyst nematodes, a parasite that literally sucks away billions in profits from soybean and other crops every year.

Researchers are working to uncover exactly how these tiny worms trick plant root cells into feeding them for life.

A team at the University of Missouri Bond Life Sciences Center collaborated with scientists at the University of Bonn in Germany to discover genetic evidence that the parasite uses its own version of a key plant hormone and that of the plants to make root cells vulnerable to feeding. Their research recently appeared in Proceedings of the National Academy of Sciences.

Melissa Mitchum

Melissa Mitchum

Cytokinin is normally produced in plants, but these researchers determined that this growth hormone is also produced by nematode parasites that use it to take over plant root cells.

“While it’s well-known that certain bacteria and some fungi can produce and secrete cytokinin to cause disease, it’s not normal for an animal to do this,” said Melissa Mitchum, an MU plant scientist and co-author on the study. “This is the first study to demonstrate the ability of an animal to synthesize and secrete cytokinin for parasitism.”

 

 

Not Science Fiction

Reprogramming another organism might sound like a far out concept, but it’s a reality for plants susceptible to nematodes.

Cyst nematodes hatch from eggs laid in fields and quickly migrate to the roots of nearby plants. They inject nematode spit into a single host cell of soybean, beet and other crop roots.

Carola De La Torre

Carola De La Torre

“Imagine a hollow needle at the head of the nematode that the parasite uses to penetrate into the plant cell wall and secrete pathogenic proteins and hormone mimics,” said Carola De La Torre, a co-author of the study and plant sciences PhD student with Mitchum’s lab. “Nematodes use the spit to transform the host cell into a nutrient sink from which they feed on during their entire life cycle. This de novo differentiation process greatly depends on nematode–derived plant hormone mimics or manipulation of plant hormonal pathways caused by effector proteins present in the nematode spit.”

These effector proteins and other small molecules in their spit cause the root cell to forego normal processes and create a huge feeding site called a syncytium. In a short period of time, this causes hundreds of root cells to combine into a large nutrient storage unit that the nematode feeds from for its entire life.

Being able to convince a root cell to do the nematode’s bidding starts with a takeover of the plant host cell cycle — which regulates DNA replication and division. This implies that a plant hormone like cytokinin is involved, says Mitchum. Cytokinin normally regulates a plant’s shoot growth, leaf aging, and other cell processes.

 

Proving the relationship

While Mitchum’s lab had a hunch that cytokinin was key to this takeover, proving it took some creative science.

De La Torre and Demosthenis Chronis, a postdoctoral fellow MU at the Bond LSC depended on mutant Arabidopsis plants to explore the relationship. “One of the great things about using Arabidopsis as our host plant is the vast genetic resources of cytokinin and hormone mutants that are available through the scientific community,” De La Torre said.

She infected Arabidopsis that contained a reporter gene called TCSn/GFP with nematodes. This gene is associated with cytokinin responses within the plant cells and is fused with a jellyfish protein that glows green when turned on. So, De La Torre saw nematodes activated cytokinin responses in the plant early after infection when her plants emitted a green fluorescent glow under the microscope.

Next, she infected plants missing the majority of their cytokinin receptors with nematodes. Then she started counting nematodes present.

“After a careful evaluation of nematode infection, we observed less female nematodes developing in the receptor mutants compared to the wild type” De La Torre said. “The nematodes could not infect well, and that was a clear piece of evidence suggesting that cytokinin plays a main role in plant–nematode interactions.”

Another experiment looked at Arabidopsis containing a reporter gene called GUS that was fused to the regulatory sequences of the cytokinin receptor genes. All three cytokinin receptor genes were activated where the nematode was feeding.

A final experiment used a mutant that created an excess of an enzyme that degrades cytokinin, finding that a base level of plant cytokinin was also necessary for nematode growth.

“The simple statement is that the cytokinin receptors were activated in response to nematode infection and the mutants did not support growth and development of the nematodes,” Mitchum said. “This shows that if you take away the ability of the plant to recognize cytokinin the worms are unable to fully develop.”

 

An international collaboration

Mitchum’s team did not work alone.

The lab of Florian Grundler at Rheinische Friedrich-Wilhelms-University of Bonn, Germany, was also on a mission to uncover if genes in the nematode controlled cytokinin activation. They had identified a key gene in the beet cyst nematode that makes the cytokinin hormone. When they took away the ability of the nematode to secrete cytokinin certain cell cycle genes were not activated at the feeding site and the nematodes did not develop. Now we know that the nematode is also secreting cytokinin to modulate the pathways.

De La Torre took that information and found the same gene in the soybean cyst nematode.

Now, Mitchum’s team is trying to find how this key gene might work differently in other nematode types, like root-knot nematode as part of a new National Science Foundation grant. They hope this will help lead to better resistance in future crops.

“Understanding how the nematode modulates its host is going to help us exploit new technologies to engineer plants with enhanced resistance to this terribly devastating pathogen,” Mitchum said. “Technology is changing all the time, we’re gaining new tools constantly, so you never know when something new is going to allow us to do something specific at the site of nematode feeding that will lead to a breakthrough.”

Mitchum is a Bond LSC investigator and an associate professor of Plant Sciences in the College of Agriculture, Food and Natural Resources. The study “A Plant Parasitic Nematode Releases Cytokinin that Control Cell Division and Orchestrate Feeding-Site Formation in Host Plants” recently was published by the Proceedings of the National Academy of Sciences and was supported by the National Science Foundation (Grant #IOS-1456047 to Mitchum). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Frogs help researchers find genetic mechanism for mildew susceptibility in grapevine

Powdery mildew on a cabernet sauvignon grapevine leaf. | USDA Grape genetics publications and research

Powdery mildew on a cabernet sauvignon grapevine leaf. | USDA Grape genetics publications and research

A princess kisses a frog and it turns into a prince, but when a scientist uses a frog to find out more information about a grapevine disease, it turns into the perfect tool narrowing in on the cause of crop loss of Vitis vinifera, the world’s favorite connoisseur wine-producing varietal.

MU researchers recently published a study that uncovered a specific gene in the Vitis vinifera varietal Cabernet Sauvingon, that contributes to its susceptibility to a widespread plant disease, powdery mildew. They studied the biological role of the gene by “incubating” it in unfertilized frog eggs.

The study, funded by USDA National Institute of Food and Agriculture grants, was lead by Walter Gassmann, an investigator at the Bond Life Sciences Center and University of Missouri professor in the division of plant sciences.

The findings show one way that Vitis vinifera is genetically unable to combat the pathogen that causes powdery mildew.

Gassmann said isolating the genes that determine susceptibility could lead to developing immunities for different varietals and other crop plants and contribute to general scientific knowledge of grapevine, which has not been studied on the molecular level to the extent of many other plants.

The grapevine genome is largely unknown.

“Not much is known about the way grapevine supports the growth of the powdery mildew disease, but what we’ve provided is a reasonable hypothesis for what’s going on here and why Cabernet Sauvingon could be susceptible to this pathogen,” Gassmann said.

The research opens the door for discussion on genetically modifying grapevine varietals.

Theoretically, Gassmann said, the grapevine could be modified to prevent susceptibility and would keep the character of the wine intact — a benefit of genetic modification over crossbreeding, which increases immunity over a lengthy process but can diminish character and affect taste of the wine.

Grapevine under attack

Gassmann’s recent research found a link between nitrate transporters and susceptibility through a genetic process going on in grapevine infected with the powdery mildew disease.

Infected grapevine expressed an upregulation of a gene that encodes a nitrate transporter, a protein that regulates the makes it possible for the protein to enter the plant cell.

Once the pathogen is attracted to this varietal of grapevine, it tricks grapevine into providing nutrients, allowing the mildew to grow and devastate the plant.

As leaves mature, they go through a transition where they’re no longer taking a lot of nutrients for themselves. Instead, they become “sources” and send nutrients to new “sink” leaves and tissues. The exchange enables plants to grow.

The powdery mildew pathogen, which requires a living host, tricks the grapevine into using its nutrient transfer against itself. Leaves turn into a “sink” for the pathogens, and nutrients that would have gone to new leaves, go instead, to the pathogen, Gassmann said.

“We think that what this fungus has to do is make this leaf a sink for nitrate so that nitrate goes to the pathogen instead of going to the rest of the plant,” Gassmann said.

Walter Gassmann, of the Bond Life Sciences Center at the University of Missouri was the lead investogator on the research. Much of his work has been on grapevine susceptibility to pathogens.

Walter Gassmann, of the Bond Life Sciences Center at the University of Missouri was the lead investogator on the research. Much of his work has been on grapevine susceptibility to pathogens. | Roger Meissen, Bond Life Sciences Center

According to a report by the USDA, powdery mildew can cause “major yield losses if infection occurs early in the crop cycle and conditions remain favorable for development.”

Powdery mildew appears as white to pale gray “fuzzy” blotches on the upper surfaces of leaves and thrives in “cool, humid and semiarid areas,” according to the report.

Gassmann said powdery mildew affects grapevine leaves, stems and berries and contributes to significant crop loss of the Vitas vinifera, which is cultivated for most commercial wine varietals.

“The leaves that are attacked lose their chlorophyll and they can’t produce much sugar,” Gassmann said. “Plus the grape berries get infected directly, so quality and yield are reduced in multiple ways.”

Pinpointing a cause

Solutions to problems start with finding the reason why something is happening, so Gassmann and his team looked at a list of genes activated by the pathogen to find transporters that allowed compounds like peptides, amino acids, and nitrate to pass.

Genes for nitrate transporters, Gassmann said, pointed to a cause for vulnerability to the mildew pathogen.

Over-fertilization of nitrate increases the severity of mildew in many crop plants, according to previous studies sited in Gassmann’s article in the journal of Plant Cell Physiology.

The testing system for isolating and analyzing the genes began with female frogs.

Gassmann used frog oocytes (unfertilized eggs), to verify the similar functions of nitrate transporters in Arabidopsis thaliana, a plant used as a baseline for comparison.

A nitrate transporter, he hypothesized, would increase the grapevine’s susceptibility to mildew.

“The genes that were upregulated in grapevine showed similarity to genes in Arabidopsis that are known to transport nitrate,” Gassmann said. “We felt the first thing we had to do was verify that what we have in grapevine actually does that.”

The eggs are very large relative to other testing systems and act as “an incubating system” for developing a protein. Gassmann and his team of researchers injected the oocyte with RNA, a messenger molecule that contains the information from a gene to produce a protein. The egg thinks it’s being fertilized and protein reproduces and is studied.

“The oocyte is like a machine to crank out protein,” Gassmann said. “We use that technique to establish what we have is actually a nitrate transporter.”

The system confirmed that the gene isolated from grapevine encodes a nitrate transporter.

“We contributed to the general knowledge of the nitrate transporter family,” Gassmann said. “It turned out to be the first member of one branch of nitrate transporters that, even in Arabidopsis haven’t been characterized before.”

The mounting knowledge of Vitis vinifera genes could make genetically modifying the strain to prevent the susceptibility easier.

“Resistance is determined sometimes by a single gene,” Gassmann said. “Until people are willing to have the conversation of genetic modification, the only way to save your grapevines is to be spraying a lot.”

Sharon Pike, Gassmann, other investigators from the MU Christopher S. Bond Life Sciences Center and post-doctoral student, Min Jung Kim from Daniel Schachtman’s lab at the Donald Danforth Plant Science Center in Saint Louis, Mo. contributed to the report.

The article was accepted November 2013 into the Plant Cell Physiology journal.