Putting down roots

Plant scientist Ruthie Angelovici joins the Bond Life Sciences Center

By Jennifer Lu | MU Bond Life Sciences Center

Ruthie Angelovici

Ruthie Angelovici

Ruthie Angelovici clearly remembers her big eureka moment in science thus far. It didn’t happen in a laboratory. It wasn’t even her experiment.

At the time, Angelovici was in college studying marine biology. She had spent a year going on diving trips to figure out whether two visibly different corals were polymorphs of the same species, or two separate species.

A simple DNA test told her the answer in one afternoon.

“That’s the day I decided that there was a lot to be discovered, just in the lab,” Angelovici said. She switched majors and hasn’t looked back.

Better Nutrition in Crops

Angelovici studies the molecular biology of plants.

As a newly minted assistant professor in biological sciences at the Bond Life Sciences Center, her goal is to increase the nutritional quality of staple crops like corn, rice, and wheat.

Although these crops make up 70 percent of people’s diet across the world, Angelovici said, they aren’t very nourishing.

Corn, rice, and wheat are deficient in several key nutrients called essential amino acids. For example, if a person lived on wheat alone, they would have to eat anywhere from three to 17 pounds of the grain per day to reach the daily recommended amount for these nutrients.

Moreover, harsh growing conditions cause amino acids levels in plants to plummet—an increasingly grave problem as the earth’s climate gets warmer.

“If you think about the future, we’re going to face more droughts, more heat,” Angelovici said. “We need to figure out how we can maintain quality under those circumstances.”

Scientists have been trying to improve the nutritional quality of crops for years, whether through classical breeding or genetic engineering. The latter requires knowing which genes to alter.

Angelovici uses a technique called genome-wide association mapping. This allows her to link the natural variations within a particular trait — say, a special type of amino acids that are branched in structure — with the genes that affect this trait.

In previous studies, Angelovici chose Arabidopsis thaliana, which is popular among plant scientists for its simple genome and short life cycle, as her model plant.

She collected seeds from 313 varieties and burst them open, one seed type at a time, to release their contents. After separating the free amino acids from the rest of the seed pulp, she measured the branched amino acid levels — as a ratio to each other and to other amino acids — to build a nutritional profile that acts like a fingerprint for each plant.

Angelovici used this fingerprint to identify plants that shared similar traits. Then she scanned their DNA for any small genetic variations, or mutations, that plants had in common.

When she tallied up the frequency of each mutation in what is called a Manhattan plot, she found one particular variation that outstripped the others, standing out like a skyscraper over a city: a small section on chromosome 1 close to a gene called bcat2.

Angelovici then switched this gene off. When branched amino acid levels changed, it suggested that this trait was linked to the bcat2 gene.

However, Angelovici warned that often plants resist genetic tinkering. They lose viability, or cannot germinate seeds.

“We get yield penalty,” Angelovici says, “and the question is why?”

Metabolism, she explains, is like a network. “If you pull one way, something else is going to be affected.”

That’s where bioinformatics comes in handy. Angelovici uses an approach called network analysis to look at many pathways within the plant at once. This allows her to see the big picture, as well as the fine detail.

Moving to Missouri

Angelovici has being studying plant metabolism for ten years. Originally from Israel, she earned her PhD in 2009 under Gad Galili at the Weizmann Institute of Science in Rehovot, Israel. Then, she continued her research as a postdoctoral fellow at Michigan State University.

She prefers working with plants to animals because plants are relatively easy to manipulate and breed. Also, she loves animals and at one point wanted to be a veterinarian.

Angelovici says she was immediately drawn to the University of Missouri, and is looking forward to collaborating with researchers at Bond LSC.

“There is a great program here, great plant people here,” she said. “So, Mizzou is spot on.”

Although she has found an undergraduate and a post-doctoral researcher to help her so far, the benchtops in her laboratory remain uncluttered save for some equipment, like glassware and a few gel boxes. Three pristine white lab coats hang neatly from hooks on the wall.

But Angelovici is not fazed by the enormous task of getting her lab up and running.

“I just love doing this. It’s like climbing a mountain,” Angelovici said, about the research process. “You do it slowly and then you feel like you’re going up and you are achieving more and you can see more. It’s really fulfilling.”

As for that big eureka moment, Angelovici says she doesn’t put much stock in it.

Then she laughs. “But maybe I will experience one, and then I’ll change my mind.”

Maze Runners

Female rats struggle to find their way in BPA study from MU and the NCTR/FDA

Cheryl Rosenfeld is one of 12 researchers partnering with the NCTR/FDA to study BPA

Cheryl Rosenfeld is one of 12 researchers partnering with the NCTR/FDA to study BPA

Despite concerns about bisphenol A (BPA), academic and regulatory scientists have yet to reach a consensus on BPA’s safety.

The National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP), the Food and Drug Administration and independent university researchers are working together to change that.

Five years after the Consortium Linking Academic and Regulatory Insights on BPA Toxicity, or CLARITY-BPA for short, launched, results are beginning to come in. This new information will allow researchers to better compare the effects of fixed doses of BPA on the brain, various cognitive behaviors, reproduction and fertility, accumulation of fat tissue, heart disease, the immune system, and several types of cancers.

“The idea of this Consortium is to examine the potential systems that have been previously suggested to be affected by BPA,” said Cheryl Rosenfeld, an associate professor of biomedical sciences at the University of Missouri and one of twelve researchers involved in the project.

Rosenfeld’s group looked at spatial navigation learning and memory. They found that prenatal exposure to BPA could potentially hinder the ability of female rats to learn to find their way through a maze. This effect was not seen in male rats.

Approved by the FDA in the early 1960s, BPA can be found in a wide variety of products, including plastic food and drink containers with recycle codes 3 or 7, water and baby bottles, toys, the linings of metal cans and water pipes, even patient blood and urine samples.

BPA has structural similarities to estrogen and can potentially act as a weak estrogen in the body.

In Rosenfeld’s experiment, researchers at the National Center for Toxicology Research gave pregnant rats a fixed dose of BPA every day: a low, medium, or high dose.

After the baby rats were born, researchers continued to dose the babies, both male and female, according to what their mothers had received.

When these rats reached three months old, they were tested in a circular maze with twenty possible exit holes, one of which was designated as the correct escape hole. Every day for seven days, researchers tested the rats’ abilities to solve the maze in five minutes and timed them as they ran.

Rats solve mazes in three ways, Rosenfeld said.

They can run through the labyrinth in a spiral pattern, hugging the outer walls, and work their way in until they find the correct exit hole in what is called a serial search strategy.

Or they might move aimlessly in the maze using an indirect search strategy, Rosenfeld said. “In this case, the rats seemingly find the correct escape hole by random chance.”

Lastly, they can travel directly from the center of the maze to the correct escape hole. The third strategy is considered the most efficient method because the rats find their way swiftly, Rosenfeld said.

Sarah Johnson, a graduate student and first author on the paper, assessed each rat’s performance in the maze using a three-point tracking program that recognizes the rat’s nose, body, and tail.

Using the program, Johnson measured their performances in terms of the total distance traveled, the speed at which the rat ran the maze, how long it took the rats to solve the maze (latency), and how often the rat sniffed at an incorrect hole.

The last two parameters are considered the best gauges of spatial navigation learning and memory.

“What you expect to see is that they should start learning where that correct escape hole is,” Rosenfeld said. “Thus, their latency and sniffing incorrect holes should decrease over time.”

Rosenfeld’s group found that female rats that had been exposed to the highest dose of BPA since fetal development were less likely to find the escape hole than rats that hadn’t been exposed to BPA.

As for how this study may translate to people, Rosenfeld said, “the same brain regions control identical behaviors in rodents and humans.”

She considers it a starting point for setting up future experiments that take into consideration sex differences in cognitive behaviors and neurological responses to BPA.

Immediate next steps for the Rosenfeld group include analyzing tissue collected from the brains of rats that had undergone maze testing. Rosenfeld’s team of researchers will measure DNA methylation and RNA expression in the brain to determine which genes might be involved in navigational learning and memory. Their overarching goal is to determine how changes in observed sex- and dose-dependent behaviors occur on the molecular level.

NIEHS grant U01 ES020929 supported this research. Additional coauthors include Mark Ellersieck and Angela Javurek of the University of Missouri, Thomas H. Welsh Jr. of Texas A&M University, and Sherry Ferguson, Sherry Lewis, and Michelle Vanlandingham of the National Center of Toxicological Research/Food and Drug Administration. Read the full study on the Hormones and Behavior website and browse the supplementary data for this work.

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.