Arabidopsis

#IAmScience: Mengran Yang

April 29, 2022 By Penquite, Cara (MU-Student) in #IAmScience Tags: Arabidopsis, plant immune system, plant science

By Cara Penquite | Bond LSC

Mengran Yang sat perched on a stool too tall for the cart of lush green tobacco plants in front of her. Behind towering shelves of lab equipment, she hunched over the plants and steadily pricked each leaf with a syringe.

Yang works with Arabidopsis and tobacco plants to learn about plant immune systems as a postdoctoral fellow in the Gary Stacey lab. Her research focuses on signals plant cells send to coordinate a fight against pathogens.

“I think it’s very interesting to see how plants can defend against the pathogens,” Yang said.

20220421_YangIAmScience_1 — Mengran Yang infiltrates the leaves of tobacco plants with a syringe. While her work mostly focuses on Arabidopsis plants, Yang also uses tobacco plants as a tool in her research since it is convenient to express the correct target proteins in their leaves. Photo by Cara Penquite | Bond LSC

Just like humans have immune cells that fight germs, plants have a network of cells and signals to deal with harmful microbes. This system is particularly important for plants since they are rooted in place and cannot avoid the attackers in their environment.

The plant immune system protects it both inside and outside its cells. Proteins inside the cell can recognize molecules from invaders and trigger an immune response, and the second — the part Yang’s research focuses on — uses receptors on the surface of the cells to recognize molecular patterns from microbes to signal for the plant to get rid of the pathogen.

“Our lab is focused on the extracellular ATP signaling pathway,” Yang said. “ATP, as maybe everyone knows, is an energy source, but we found it can also be a signal when it is released to the extracellular matrix, where it is referred to as extracellular ATP.”

Yang’s work with the lab started when she moved from China to Columbia for her postdoctoral research at Mizzou. Although originally drawn to the lab because of Stacey’s prominence in the field of plant immunity and signaling, Yang stays because of the community within the lab.

“Our lab is very international — some people are from China, Korea, Brazil, Vietnam and India,” Yang said. “A lot of the same-aged girls will go out for fun.”

This community stems from Stacey’s emphasis on the team’s collective success.

“You try to develop an esprit de corps in the lab where people care about each other’s success,” Stacey said. “They’re taking care of themselves, but at the same time, they care about other people’s success.”

Becoming a good researcher started with strong academics, and Yang remembers being the top of her class in biology, physics and math in middle school and high school. When she started her studies at the university level she followed her growing interest in biology.

Growing up with her twin sister in China, Yang recalls societal pressures to fill any spare time with more study. Her parents thought her teachers gave too much homework and pushed back on this expectation.

“My father thinks this kind of education is not good, he thinks you need to promote your efficiency and not just work hard,” Yang said. “You need to study smart, not just hard, and I think my dad influenced us a lot.”

Yang applies this mentality to her research and is supported by Stacey who encourages her to plan experiments and life goals.

“While I was a Ph.D. student, I spent the most time in the lab for fun,” Yang said. “But now I’m good at scheduling stuff, so I schedule my experiments at least a week ahead.”

DSC_9057 copy — Mengran Yang shows a dish with Arabidopsis plants. Yang’s work primarily focuses on studying extracellular ATP in Arabidopsis plants. Photo by Cara Penquite | Bond LSC

Stacey works with Yang to ensure her future impact as a researcher. When picking a project, Stacey guides his researchers towards projects with lots of potential.

“That’s the kind of discussion I have with people, what’s the best project for them where they have the chance to make an impact,” Stacey said.

With her lab mates and other research labs, Yang collaborates to strengthen each other’s weaknesses. If she does not have experience with an experiment, Yang asks other postdoc students with more experience for help with her project. When she publishes her research, she makes sure to give credit to the other students who helped her.

“In Chinese, ‘shuangying’ means ‘good to both sides,” Yang said. “That’s very common. You will read a paper, and you can also see most [papers] have a lot of authors there. That’s the cooperation.”

After finishing her research in the U.S., Yang plans to return to China to continue doing what she loves — research.

“I want to have my own lab in China,” Yang said. “It’s difficult, I know, but it’s my dream.”

A NEET Connection

March 2, 2020 By Becca Wolf in Research Tags: Arabidopsis, cancer, reactive oxygen species

Protein is important in balancing iron and reactive oxygen in plant and cancer cells

Arabidopsis growing in Ron Mittler’s lab. | photo by Becca Wolf, Bond LSC

By Becca Wolf | Bond LSC

You might tend to think durability is more of an issue in building a car or engineering a building, but environmental stress makes resilience vital for plants, too.

In the world of understanding and engineering more durable crops, scientists recently identified a protein that’s key to some of that resilience in a neat way. Actually, it’s literally a NEET protein.

Ron Mittler, a Bond Life Sciences Center principal investigator and professor of plant sciences and surgery, is probing the use of the NEET protein to balance iron and reactive oxygen species (ROS) in the chloroplast to make plants stronger and healthier, and it plays a similar role in cancer cells. These proteins belong to a unique family of iron-sulfur (Fe-S) proteins ideally maintaining the proper balance of the heavy metal in and outside of plant cells. Along with several other scientists, Mittler published his most recent plant findings in The Plant Journal last October.

Since NEET proteins regulate both iron and ROS, Mittler teamed up with heavy metal expert David Mendoza, fellow Bond LSC primary investigator and associate professor of plant sciences. Through a Bond LSC Grant for Innovative Collaborative Research, the two started work at the intersection of two labs.

“The main philosophy of Bond LSC is to establish collaborations between researchers to advance the field of whatever sciences that you do,” Mendoza said.

Mittler and Mendoza looked at how NEET impacts iron and ROS, working together to figure out how to make plants more durable. Doing so, they have made a lot of progress in the field and believe that their collaboration has been a good one. Says Mendoza, “It is a very productive collaboration.”

Trays of Arabidopsis in Ron Mittler’s lab. | photo by Becca Wolf, Bond LSC

A Balancing Act

But the work doesn’t stop with plants. Mittler found NEET proteins also play a similar role in cancer cells, regulating the level of iron and potentially toxic ROS compounds. Humans and plants need iron and ROS, but too much of either can be destructive. NEET proteins allow cells to grow and spread quickly which is beneficial for plants, but detrimental for humans when cancers grow out of control. In humans, NEET proteins also play a significant role metabolism related to diabetes and progression of neurodegenerative diseases.

Despite needing both iron and ROS to function, there can be too much of a good thing. To keep iron levels balanced, oxygen is needed. While oxygen on its own is a fairly stable molecule, adding electrons or exciting the present electrons can make the molecule want to react with things around it creating oxidants. Electrons are the negatively charged particles of an atom that keep balance within the cell. When balanced, iron and ROS helps cells grow, but when placed together, the iron turns ROS into hydroxyl radical, an ion that reacts with anything and destroys everything. If not controlled, this desire to bond with things nearby can damage cells and lead to cancer.

Cancer cells like ROS and iron because it enables them to spread quickly and take over more of the body. To stay balanced, NEET proteins are needed.

“These proteins apparently enable the cells to keep high levels of both iron and ROS and to survive,” says Mittler, “So it looks like cancer needs these proteins because if you suppress these proteins or if you target them with drugs, the cancer cells die.”

Sara Zandalinas holding a dying Arabidopsis seedling and a living seedling in Ron Mittler’s lab. | photo by Becca Wolf, Bond LSC

Poisoning the cell

Knowing NEET proteins make cancer and plant cells stronger, Mittler wanted to look at “what happens if they’re not functioning well,” in hopes to understand their function. To test this, Mittler and his collaborators found a way to make the NEET proteins not function. Mittler called this “poisoning the cell.”

“If there is no protein, there’s no life,” he states. “We were able to control when we introduce this protein, and being able to introduce this protein enabled us to actually find out what happened to the cell if this protein goes bad.”

It was found that if the function of this protein is disrupted, the chloroplast is destroyed. Mittler described it as “completely blown to pieces.” The destruction of the chloroplast is caused by too much iron due to a broken NEET protein. Similarly in cancer cells, if there are no NEET proteins, the cells do not spread as quickly and die.

Mittler elaborated, “What we found is that we can target those proteins in cancer, so then we basically destroy the cancer cells.” Currently, NEET proteins are used as markers in the cancer field. Doctors and scientists look for them in order to help diagnose patients and to figure out life expectancy rates. But in the future, doctors hope to use targets these proteins to kill off cancer cells, a treatment that would be less toxic than chemotherapy.

In plants, Mittler has concluded that the NEET protein would make them grow bigger and faster, leading plants to be “more tolerant to stresses because it has the ability to modulate the levels of iron and reactive oxygen.” Findings like these may one day help plants deal with stress like drought and temperature extremes. With humans continuing to contaminate the environment and climate change escalating conditions, Mittler hopes his findings on iron, ROS, and NEET proteins will make plants able to withstand changes better.

“If I can improve the way plants balance reactive oxygen and iron, I can make plants tolerate much more of these bad combinations of stresses even, and maybe even make bigger, stronger, healthier plants.”

Read more about this research in the October 2019 edition of The Plant Journal article “Expression of a dominant‐negative AtNEET‐H89C protein disrupts iron–sulfur metabolism and iron homeostasis in Arabidopsis.”

The Bond LSC Grant for Innovative Collaborative Research program aims to foster inter-laboratory collaboration to make pilot projects that have potential for later federal funding.

Nature’s cinema: New technique allows plant imaging in real-time

October 23, 2019 By Mariah Cox in Research Tags: Arabidopsis, Ron Mittler

The September issue of Molecular Plant depicts a plant fluorescing in response to reactive oxygen species propogation taken by Yosef Fichman of the Mittler Lab.

By Mariah Cox | Bond LSC

Plant biologists across the country opened their mailboxes last month to the glowing leaves of Arabidopsis on the cover of the latest issue of Molecular Plant.

That cover taken by post-doctoral researcher Yosef Fichman of Bond Life Sciences Center depicts plants fluorescing in response to reactive oxygen species (ROS) propagation, a technique that allows researchers to track plant response to certain stressors.

The novel approach is the first of its kind to image the response of an entire plant, rather than small samples. To make this technique work, Fichman — who works in the lab of Bond LSC’s Ron Mittler — and his collaborators came up with the idea to fumigate the plant with a dye which enters the plant, reacts with ROS inside the plant and fluoresces under an in vivo imaging system (IVIS). From there, the researchers could watch the stress signals of the plant as they evolve in real-time following the application of different stresses.

Soil ‘cookies’ are used to grow Arabidopsis in the lab for use in Fichman’s ROS imaging research. | Photo by Mariah Cox, Bond LSC

ROS are essential regulators of cellular and systemic signals in plants as well as animals. Additionally, they play an important role in hormonal, physiological, and developmental reactions in plants such as growth and development as well as defense and acclimation to environmental stress.

ROS are both beneficial and detrimental to plants. On one hand, they regulate basic biological processes. On the other, if overproduced, they are toxic to cells and can cause cell stress or death. However, recent research by the Mittler lab supports the idea that, at certain levels, ROS can be more beneficial than harmful because they have the power to signal defense mechanisms against stressors in the environment. In essence, it is a way for cells to communicate with each other, creating a domino effect, or wave that sends a message to nearby cells and eventually over long distances within the plant.

Now that they have a technique to detect the fast-moving ROS signal in plants, the researchers can identify the proteins that are involved in the plants’ response to stress. The signal is important because it causes fast adjustments to stress within the environment, which means that other parts of the plant that are not subject to stress can build up protection mechanisms.

“In order to understand plants’ response to stress, we need to know the mechanism of that’s going on in plants,” Fichman said. “Basically, it’s a balance of resources within the plant.”

Currently, the researchers are working with Arabidopsis, but this technique applies to other plant and crop models. Previous methods of tracking biological responses in plants included the arduous process of gene manipulation and expression. The new method allows for a straight-forward approach to tracking and monitoring the biological reactions in plants.

Moving forward the researchers plan to use Arabidopsis mutants and other plants to track the response to different stresses compared to the wild type to see if there are changes in the genes involved. Fichman is particularly interested in the effect of abiotic stresses, such as high-intensity light and leaf destruction by insects.

Arabidopsis under high light — The Mittler lab uses high-intensity light shone directly on the plant’s leaves to imitate light stress in natural environments. | Photo by Mariah Cox, Bond LSC

“Whether you like to call it weather extremes or global climate change, there are changes. Plants are a main food source, so we need to be prepared to create better plants for the future that allows crops to grow in stressful conditions such as high light, less water and heat,” Fichman said. “If plants invest more resources into protecting themselves they will be able to survive these changes and continue to produce food for our future. I’m studying this mechanism in ROS waves and in different pathways to create better plants in the future.”

By understanding the proteins that are involved in plants’ response to stress, researchers can begin to re-engineer plants to survive harsher conditions. Fichman’s technique makes it easier to recognize plants’ responses to stress moving forward.

For Yosef, when it comes to research, imagination is the only limitation.

“In research, we can come up with the craziest ideas and science allows us to check those ideas and discover new things,” Fichman said. “This is why I wake up in the morning.”

This research was published in the September 2019 edition of Molecular Plant.

#IAmScience Brittany Cruzan

September 7, 2018 By Erica Overfelt in #IAmScience Tags: Arabidopsis

By Erica Overfelt | Bond LSC

When Brittany Cruzan does her laundry, she can’t stop thinking about science.

“Science controls our everyday life,” Cruzan said. “Coffee, tea, the way you cut your hair, it’s all science. I’ve liked science since I was a kid. I am obsessed with knowing how things work. I don’t care why but I care for how.

Cruzan applies that desire to know how things work to work in the lab. From her first semester of freshman year to now as a junior, Cruzan been in Mannie Liscum’s lab chasing the how behind the science.

“Freshman year I was sterilizing water and this year I am creating mutations in plants” Cruzan said. “I went from making water and dirt to now making a living organism. I am definitely getting a lot of guidance from grad students but it is exciting to feel independent.”

The lab uses Arabidopsis as their model plant to study its growth and how the plant responds directionally to light. This research can aid scientist in enhancing plant growth in drought and other circumstances, and they use mutations to explore which genes might create characteristics that are more resilient to stress in the environment. Now that Cruzan has her own project it’s easier for her to realize the potential impact this research has on others.

“I am researching to find the maximum potential for plant growth and maximize the output for agriculture,” Cruzan said. “Once we figure this out it could mean more corn, more cows and more full bellies.”

After college Cruzan wants to continue impacting lives. She plans to volunteer before she applies to med school, and she believes life is full of endless possibilities.

“I don’t even know what I’m supposed to do, but I want to make people happy,” Cruzan said. “Anything I can do for the greater good makes me sleep better, whether that is volunteering or doing research to feed the world.”

For now, you can find Cruzan in the lab focused on a way to improve plant growth.

“I don’t think I personally will get far enough to solve world hunger, but if we can figure out a plants growth then we can add to all the research being done,” Cruzan said. “I believe that research creates the world of tomorrow, and while it may seem small today, in 5 years it could change a life.”

Unmasking the unknown

March 1, 2016 By Justin L. Stewart in Research Tags: Arabidopsis, Bond Life Sciences Center, Dan Leuchtman, decoding science, genetics, Immune System, Mannie Liscum, mice, mizzou, MmSRFR1, plant science, research, SRFR1, Walter Gassmann

Scientists explore genetic similarities between plants and mice

University of Missouri PhD Candidate Daniel L. Leuchtman peers through an Arabidopsis plant. Leuchtman has been experimenting with replacing a gene in the plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

By Justin L. Stewart | MU Bond Life Sciences Center

Almost two-thirds of what makes a human a human and a fly a fly are the same, according to the NIH genome research institute.

If recent research at the University of Missouri’s Bond Life Sciences Center is verified, we’ll soon see that plants and mice aren’t all that different, either.

Dan Leuchtman studies a gene in Arabidopsis plants called SRFR1, or “Surfer One.” SRFR1 regulates plant immune systems and tell them when they are infected with diseases or illnesses. Leuchtman studies this model plant as a Ph.D. candidate at MU, splitting time between the labs of Walter Gassmann and Mannie Liscum.

His research involves breeding Arabidopsis plants missing the SRFR1 gene and then replacing it with the MmSRFR1 gene.

So, what is MmSRFR1? Leuchtman and company believe it’s the animal equivalent of SRFR1, though they aren’t fully aware of all of its’ functions.

“We’re actually one of the first groups to characterize it,” Leuchtman said.

Arabidopsis plants missing the SRFR1 gene struggle to grow at all, appearing vastly different from normal plants. Leuchtman says that a plant missing the SRFR1 gene is a mangled little ball of leaves curled in on itself. “It’s really strange looking.”

While his experiments haven’t created statuesque plants equal to those with natural SRFR1 genes present, the Arabidopsis plants with MmSRFR1 show a notable difference from those completely lacking SRFR1. Leuchtman says the plants with MmSRFR1 lie somewhere in between a normal plant and one lacking SRFR1.

“At its’ core, it’s more understanding fundamental biology. How do we work? How do organisms tick? How do you go from DNA in a little bag of salts to a walking, talking organism?” Leuchtman said. “The more you know about how an organism functions, the more opportunities you have to find something that makes an impact.”

Understanding spit

October 8, 2015 By Roger Meissen in Research 1,960 Comments Tags: Arabidopsis, genetics, nematodes, plant sciences, soybean

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

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. Contributed by Carola De La Torre

Roger Meissen | Bond Life Sciences Center

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.

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.

“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.

“Mutant seeds” blossom in the pollen research field

November 20, 2014 By Paige Blankenbuehler in Research Tags: Arabidopsis, Bond Life Sciences Center, decoding science, jack c schultz, mapk3, mapk6, mizzou, MU decoding science, plant science, pollen, shuqun zhang, University of Missouri, university of missouri science, wrky34, zhang pollen

A mutant arabidopsis model nearing pollination.
Mutant arabidopsis models under lamps in Shuqun Zhang's lab.
Three-month-old mutant arabidopsis models are used to study the function of pollen.

The thought of pollen dispersed throughout the air might trigger horrific memories of allergies, but the drifting dander is absolutely essential to all life.

Science has long linked this element of reproduction with environmental conditions, but the reasons why and how pollen functions were less understood. Now lingering questions about the nuanced control of plants are being answered.

“Pollen is a very important part of the reproductive process and if we understand how pollen develops and how environmental stresses impinge on this process, we might be able to prevent crop loss due to high temperature or drought stress etc.,” said Shuqun Zhang, a Bond Life Sciences Center investigator.

Zhang has developed a new line of seeds that helped him and his lab identify an influential signaling pathway that triggers a chain reaction associated with normal pollen formation and function.

This research could lead to improvement to a plant’s response to disastrous environmental variables like drought to optimize pollen production and increase the production of food crops.

Left: Pollen grains with MAPK3/4 genotypes are illuminated using a fluorescent microscope. RIGHT: Normally developed pollen grains shown by an electronic microscope scan. | Credit: Shuqun Zhang — Left: Pollen grains genotypes MAPK3 and MAPK6 are illuminated by red and yellow dye using a fluorescent microscope. RIGHT: Normally developed pollen grains shown by an electronic microscope scan. | Credit: Shuqun Zhang

Seeds of success

Mutant seeds are the key to this work.

Instead of glowing green in the soil like you might see in a science fiction movie, they are providing important insight on plant reproduction and stress tolerance.

Zhang developed these plants from a mutant strain of Arabidopsis, a model plant used in scientific research. Certain genes were “switched off”to pinpoint where important pollen functions were signaled.

Using this mutant plant and seed system, Zhang found that WRKY34and WRKY2, two proteins that turn on/off genes, are regulated by MPK3and MPK6 “signaling” enzymes. These enzymes basically transform proteins from a non-functional state to a functional state, turning on specific duties or functions. Zhang, a professor of biochemistry at MU, began tinkering with the MPK3 and MKP6 pathways more than twenty years ago during his post-doc at Rutgers University.

Zhang’s research shows the newly identified MPK3/MPK6-WRKY34/WRKY2 pathway is a key switch in the hierarchy of the signaling system in pollen formation.

The research showed that the plant’s defense/stress response and reproductive process are linked, and the influential proteins MPK3 and MPK6 were part of the bigger WRKY34/WRKY2control pathway, which is activated in early pollen production.

The system is so useful that researchers across the country won’t stop asking for the seeds, Zhang said.

“We have a lot of requests for seeds,” Zhang said. “This is a very nice system to study pollen formation and function.”

The cascade of control

The functions of MPK3/MPK6 in plants can be compared to a “mother board” switch. The pathway — MPK3 and MPK6 —are part of a hierarchy of response, turning functions on or off. In other words, it’s a switch that controls a lot of different things. Controlling WRKY34/WRKY2 is one of the many roles played by MPK3 and MPK6.

Shuqun Zhang, University of Missouri Bond Life Sciences investigator.

“Whatever is plugged into it is what comes on,” Zhang said. “We are actually very, very interested in the evolutionarily context, how this came to be.”

This signaling process is just one of many in plants. MPK3 and MPK6 are two out the 20 MPKs, or MAPKs (abbreviated from Mitogen-Activated Protein Kinases) in Arabidopsis. They control plant defense, stress tolerance, growth, and development including pollen formation and functions.

“We determined that this MAPK-WRKY signaling module functions at the early stage of pollen development,” Zhang said.

The “loss of function of this pathway reduces pollen viability, and the surviving pollen has poor germination and reduced pollen tube growth, all of which reduce the transmission rate of the mutant pollen,” according to the research.

Zhang and his lab worked with the MU Division of Biochemistry and Interdisciplinary Plant Group on the research, which published in PLoS Genetics in June of this year.

A world without pollen production and defense

Without pollen, plants would not reproduce — there aren’t any Single Bars in the plant world (that we know of) — and if plant generations don’t propagate, there would be no air or food for human life to sustain.

“The factors such as heat and drought stresses cause problems to the plant’s normal developmental process and that’s how pollen fails to develop,” Zhang said. “If we understand the process, and know how environmental factors impact negatively the process, we can then make plants that can handle environmental stress better.”

Zhang and his lab continue to research the complexities of these pathways. Next on the quest is to answer how MPK3/MPK6 are involved in pollen functions such as guiding the pollen tube growth towards ovule to complete the sexual reproduction process in plants.

“It is possible that MPK3 and MPK6 are activated quickly in response to the guidance signals,” he said. “There’s still a long way to go because very few players in this process have been identified, we try to understand the biological process how they work together.” This research is in collaboration with Dr. Bruce McClure, also professor of Division of Biochemistry.

Read more:

1. PLoS Genetics (May 2014): Phosphorylation of a WRKY Transcription Factor by MAPKs is Required for Pollen Development and Function in Arabidopsis — Funded by a Hughes Research Fellowship and grants from the National Science Foundation.

2. Plant Physiology (June 2014): Two Mitogen-Activated Protein Kinases, MPK3 and MPK6, are required for Funicular Guidance of Pollen Tubes in Arabidopsis — Funded by a National Science Foundation grant and a NSF Young Investigator Award.

Hearing danger: predator vibrations trigger plant chemical defenses

July 1, 2014 By Roger Meissen in Research Tags: anthocyanins, Arabidopsis, Bond Life Sciences Center, cabbage butterfly caterpillar, chemical defenses, glucosinolates, hearing, Heidi Appel, plants, Rex Cocroft, sound, University of Missouri, vibration

Experiments show chewing vibrations, but not wind or insect song, cause response

As the cabbage butterfly caterpillar takes one crescent-shaped bite at a time from the edge of a leaf, it doesn’t go unnoticed.

This tiny Arabidopsis mustard plant hears its predator loud and clear as chewing vibrations reverberate through leaves and stems, and it reacts with chemical defenses. Plants have long been known to detect sound, but why they have this ability has remained a mystery.

University of Missouri experiments mark the first time scientists have shown that a plant responds to an ecologically relevant sound in its environment.

“What is surprising and cool is that these plants only create defense responses to feeding vibrations and not to wind or other vibrations in the same frequency as the chewing caterpillar,” said Heidi Appel, an investigator at MU’s Bond Life Sciences Center and senior research scientist in the Division of Plant Sciences in the College of Agriculture, Food and Natural Resources.

Heidi Appel, investigator at MU’s Bond Life Sciences Center and senior research scientist in the Division of Plant Sciences in the College of Agriculture, Food and Natural Resources, and Rex Cocroft, a professor of Biological Sciences in MU’s College of Arts and Science, found that plants create chemical responses specifically to predator chewing vibrations.

Appel partnered with Rex Cocroft, an MU animal communication expert who studies how plant-feeding insects produce and detect vibrations traveling through their host plants.

“It is an ideal collaboration, that grew out of conversations between two people working in different fields that turned out to have an important area of overlap,” said Cocroft, a professor of Biological Sciences in MU’s College of Arts and Science. “At one point we began to wonder whether plants might be able to monitor the mechanical vibrations produced by their herbivores.”

While Appel focused on quantifying “how plants care and in what ways,” Cocroft worked to capture inaudible caterpillar chewing vibrations, analyze them and play them back to plants in experiments that mimic the acoustic signature of insect feeding, but without any other cues such as leaf damage.

Cocroft used specialized lasers to listen to and record what the plant hears.

“Most methods of detecting vibrations use a contact microphone, but that wasn’t possible with these tiny leaves because the weight of the sensor would change the signal completely,” said Cocroft.

This cabbage butterfly caterpillar munches on an Arabidopsis leaf adjacent to a leaf where a piece of reflective tape bounces back a laser beam used to detect the vibrations created by its chewing. Roger Meissen/Bond LSC

The laser beam reflects off a small piece of reflective tape on the leaf’s surface to measure its deflection, minimizing contact with the plant. The laser’s output can also be played back through an audio speaker, allowing human ears to hear the vibrations produced by the caterpillar.

Moved by the sound

Recording the sound is just the start.

You can’t put headphones on a leaf, so tiny piezoelectric actuators – essentially a tiny speaker that plays back vibrations instead of airborne sound – is required.

“It’s a delicate process to vibrate leaves the way a caterpillar does while feeding, because the leaf surface is only vibrated up and down by about 1/10,000 of an inch,” Cocroft said. “But we can attach an actuator to the leaf with wax and very precisely play back a segment of caterpillar feeding to recreate a typical 2-hour feeding session.”

Appel and Cocroft tested whether these chewing sounds could create more chemical defenses in the plants and whether these feeding recordings primed defenses when played before an actual caterpillar ate part of a leaf.

“We looked at glucosinolates that make mustards spicy and have anticancer properties and anthocyanins that give red wine its color and provide some of the health benefits to chocolate,” Appel said. “When the levels of these are higher, the insects walk away or just don’t start feeding.”

The researchers played 2 hours of silence to some Arabidopsis plants and 2 hours of caterpillar-chewing noises to others. They then chose three leaves around the plant, and allowed caterpillars to eat about a third of each leaf. After giving the plants 24 to 48 hours to respond to the caterpillar attack, they harvested the leaves for chemical analysis.

When they found higher levels of glucosinolates in the plants that were exposed to chewing vibrations, they knew they were on the right track.

A similar second experiment went further, testing whether the plants would simply respond to any vibration, or whether their response was specific to chewing vibrations. In this case Appel analyzed anthocyanins, which again were elevated – but only when plants had been exposed to chewing vibrations but not to vibrations created by wind or the sounds of a non-harmful insect.

Past echoes and future promise

While the past is littered with suggestions that people talk to their plants, Appel and Cocroft hope their work is shifting the focus on plant acoustics towards a better understanding of why plants can detect and respond to vibrations.

“The field is somewhat haunted by its history of playing music to plants. That sort of stimulus is so divorced from the natural ecology of plants that it’s very difficult to interpret any plant responses,” Cocroft said. “We’re trying to think about the plant’s acoustical environment and what it might be listening for, then use those vibrational sounds to figure out what makes a difference.”

The National Science Foundation seems to agree with the merit of their endeavor, awarding a grant to extend this project.

The next step includes looking at how other types of plants respond to insect predator sounds and pinpointing precisely what features of the sounds trigger the change in plant defenses.

These questions aim to further basic research understanding of how plants know what’s going on to respond appropriately to their environment. This could one day lead to ways to create better plants.

“Once you understand these things you can mess around with it in plant breeding through conventional methods or biotech approaches to modify plants so they are more responsive in the ways you want to make them more resistant against pests,” Appel said. “That’s the practical application one day.”

This research was published online in the journal Oecologia July 1, 2014 and will appear in print in its August issue.

Chemical beacons: LSC scientist discovers how plants beckon bacteria to attack

April 24, 2014 By Roger Meissen in Research Tags: Arabidopsis, bacteria, Bond Life Sciences Center, chemical signals, Scott Peck

Scott Peck studies Arabidopsis and how bacteria perceive it before initiating an infection. Roger Meissen/ Bond LSC — Scott Peck, Bond LSC scientist and associate professor of biochemistry, studies Arabidopsis and how bacteria perceive it before initiating an infection. Roger Meissen/ Bond LSC

Sometimes plants inadvertently roll out the red carpet for bacteria.

Researchers at the University of Missouri Bond Life Sciences Center recently discovered how a plant’s own chemicals act as a beacon to bacteria, triggering an infection. Proceedings of the National Academy of Sciences published their study April 21.

“When bacteria recognize these plant chemicals it builds a needle-like syringe that injects 20-30 proteins into its host, shutting down the plant’s immune system,” said Scott Peck, Bond LSC plant scientist and lead investigator on the study. “Without a proper defense response, bacteria can grow and continue to infect the plant. It looks like these chemical signals play a very large role in mediating these initial steps of infection.”

The question of how bacteria actually know they are in the presence of a plant has puzzled scientists for years. Being able to identify the difference between a plant cell and, say, a rock or a piece of dirt, means the bacteria saves energy by only turning on its infection machinery when near a plant cell.

“Our results show the bacteria needs to see both a sugar – which plants produce quite a bit of from photosynthesis – and five particular acids at the same time,” Peck said. “It’s sort of a fail-safe mechanism to be sure it’s around a host before it turns on this infection apparatus.”

Peck’s work started with one mutant plant called Arabidopsis mkp1.

Discovered several years ago by Peck’s lab, this little mustard plant acts differently than others by rebuffing the advances of bacteria. Lab tests confirmed that this mutant didn’t get infected by Pseudomonas syringae pv. tomato DC3000, a bacterial pathogen that causes brown spots on tomatoes and hurts the model plant Arabidopsis. Along with MU biochemistry research scientist Jeffrey Anderson and post doc Ying Wan, they showed that this mutant didn’t trigger the bacteria’s Type III Secretion System, the needle-like syringe and associated proteins that lead to infection.

Pacific Northwest National Laboratory (PNNL) worked with Peck’s team to compare levels of metabolites between the mutant Arabidopsis and normal plants. This comparison helped Peck identify a few of these chemicals – created from regular plant processes – that existed in much lower levels in their special little mutant.

Using the PNNL work as a guide, the team found five acids collectively had the biggest effect in turning on a bacteria’s infection: aspartic, citric, pyroglutamic, 4-hydrobenzoic and shikimic acid.

“The key experiment involved us simply adding these acids back into the mutant,” Peck said. “Suddenly we saw the mutant plant wasn’t resistant anymore and the bacteria were once again capable of injecting proteins to turn off the plant’s immune system.”

First contact and recognition means all the difference, whether bacteria or plant. Just a slight jump out of the starting blocks by one or the other could change who will win a battle of health or infection.

While low concentrations of these five acids trigger the bacteria’s attack, high levels blind it to the plant’s presence, leading Peck to believe it could be used to hinder bacterial growth. If this actually thwarts the bacteria’s head start, it could mean stopping disease in crops and could lead to a different approach in the field.

“A lot of the winning and losing occurs within the first 2-6 hours and it seems to be that if the microbe is too slow to turn off the immune system, the plant can actually fight off the infection,” Peck said. “In the future we could possibly make a new generation of anti-microbial compounds that don’t try to kill the bacteria, but rather just make them no longer virulent by blocking these chemical signals so the natural plant immune system can basically take over.”

Peck’s team believes at least some other bacteria will respond to these chemical signals, and he plans to test other bacterial pathogens to make certain. They also want to test bacteria to see if they are more virulent in humans once primed for attack by these plant chemical signals.

“In the long run the question is how far this extends. A lot of people get salmonella or listeria infections through a food source,” Peck said. “The question is do other bacteria that come in through plant food sources have similar perception systems and end up being more infectious in humans because they are already primed for infection.”

A $500,000 grant from the National Science Foundation supported this research.

Bond LSC team identifies first plant receptor for extracellular ATP

January 16, 2014 By Roger Meissen in Research Tags: Arabidopsis, extracellular ATP

Jeongmin Choi (left), Gary Stacey (center) and postdoc Kiwamu Tanaka recently discovered the first plant receptor for extracellular ATP. — 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.