Arabidopsis

Unmasking the unknown

Scientists explore genetic similarities between plants and mice

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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.

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A series of Arabidopsis plants show the differences between the plants, from left, without SRFR1, with MmSRFR1 and with SRFR1. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

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.

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University of Missouri PhD Candidate Daniel L. Leuchtman poses for a portrait in a Bond Life Sciences Center greenhouse. Leuchtman has been experimenting with replacing a gene in Arabidopsis plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

“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

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.

“Mutant seeds” blossom in the pollen research field

  • A mutant arabidopsis model nearing pollination.

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 MPK6signaling” 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.

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

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.

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

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

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.