plant signaling pathways

New observation from Stacey lab could help advance plant engineering

May 25, 2021 By Lauren Hines in Research Tags: plant science, plant signaling pathways

Large amounts of the Arabidopsis plant are grown at Bond Life Sciences Center for multiple labs to experiment with and use. | photo by Mariah Cox, Bond LSC

By Lauren Hines | Bond LSC

Think about how a home alarm system alerts a person to a potential burglary with sensors detecting whether an intruder picked a lock, came through a window or came through a garage.

Plants are much like this, surviving with the help of their thousands of sensors that can send danger signals to the whole plant so it can react effectively.

“Plants have to have a whole variety of different mechanisms to respond to their environment because they’re stuck in one spot,” said Gary Stacey, principal investigator at Bond Life Sciences Center. “The way they do that is they have all these membrane-associated receptors, and they receive signals from the outside … so they can induce a defense response.”

Stacey’s lab recently made an observation that could lead to interesting future advancements in plant breeding and engineering. Its work was published May 12 in the journal Nature Communications.

The lab found when a lipid — a fatty molecule — is attached to a receptor, a cysteine amino acid within a protein is modified. This process, in turn, makes the receptor silent. The addition of the lipid is called acylation. The lab found that they could also reverse this process and reactivate the receptor.

In other words, this process can turn receptors on and off, which is what tells the plant that there’s danger.

In addition, the lab can use the acylation process to identify the binding protein and, therefore, identify the receptor that is binding to the protein.

“There’s a lot of receptors for which we don’t know the ligand,” said Stacey. “In other words, if you line up all the receptors, there are only a few of which we know what they’re binding to.”

Identifying these receptors’ functions and what these receptors bind to is a high priority in plant research and agriculture communities.

“If we knew all the receptors, say, that responded to drought or if we knew all the receptors that responded to high light or pathogens or whatever there might be in the ozone … then that would open up pathways for us to engineer plants that are more resistant to these stresses, which would make crop production more sustainable, especially in lieu of the changing climate that we have, and would really be a big step forward for agriculture,” Stacey said

According to Stacey, researchers have been able to identify the function of “a small fraction” of receptors out of 2,000 in the model plant, Arabidopsis. However, the Stacey lab is currently developing a way to find these receptors’ functions.

Developing this analysis has been technically difficult so far — needing more funding and manpower to do all the experiments — but the lab is currently in the proofing stage to verify what they know. So far, they can correctly identify the binding protein and the receptor it binds to for some receptors, but it’s less clear for other receptors. More experiments are needed to see if the analysis will work.

“There are 2,000 [receptors], and we only know the function of a handful,” Stacey said. “There’s a lot of stuff that remains to be discovered and a lot of potentials to do something useful.”’

More information about the study can be found in, “S-acylation of P2K1 mediates extracellular ATP-induced immune signaling in Arabidopsis.” Stacey’s lab collaborated with Dongqin Chen, Fengsheng Hao and Huiqi Mu of the State Key Laboratory of Agrobiotechnology in China, Nagib Ahsan of the University of Oklahoma and Jay Thelen at Bond Life Sciences Center.

Shining Light on Plant Reaction

November 5, 2020 By Lauren Hines in Research Tags: plant signaling pathways, reactive oxygen species

By Lauren Hines | Bond LSC

Daylight might not seem dangerous, but for plants, too much daylight can cause hazards similar to a nasty sunburn or a human scalding themselves.

When you jerk your hand back from a boiling pan or a hot faucet, in a millisecond electrical signals are sent up your arm to tell your brain to move away. Like this reaction, plants have their own protective reactions to too much light.

“I think what you need to understand is that plants can’t survive without light,” said Ron Mittler, principal investigator at the Bond Life Sciences Center. “Light is the master of the plant basically.”

Mittler’s lab discovered that light receptor phytochrome B is responsible for triggering the signals for light danger that reverberate from one cell to another and not chloroplasts as previously thought. Understanding how plants respond can help build a picture of a process called systemic signaling to help researchers create genetically modified crops to withstand these stresses. Their results were recently published in the Plant Physiology journal.

The scientific community thought if light was put on a plant, the organelles that conduct photosynthesis, called chloroplasts, would trigger systemic signaling and the ROS wave.

“It’s really about having this spreadsheet of knowledge and not knowing where the pieces of the puzzle from high light-reactive action to chloroplast or to stomatal closure, where all those pieces fit,” said Mannie Liscum, collaborator and Mizzou professor of biological sciences.

The ROS wave, named by Mittler in 2009, is the process where reactive oxygen triggers a cell reaction that subsequently triggers the cell next to it and so on creating a wave of activation. Reactive oxygen is a tricky molecule that reacts very easily. It can cause destruction in cells with these reactions or use its reactivity to send a message in a proactive way between many cells.

“What we found is that if we use a phytochrome mutant — phytochrome B in particular — we completely eliminate this ROS production by high light stress,” Mittler said. “To me, it was really amazing because everybody else thought that the light would impact the chloroplast.”

While chloroplasts absorb light to turn into energy, phytochromes can take certain types of light and turn genes on. Depending on the type of phytochrome, they can take in different wavelengths of light and help plants understand light intensity and light quality. In turn, this helps a leaf or a stem figure out where to grow and if light is coming through shade.

The Mittler and Liscum labs used the sensitivity of the light sensor to their advantage.

“By using different wavelengths, different colors of light, we started narrowing down which of the sensory systems in the plant were actually doing the job,” Liscum said.

Eventually, they found responses to red light. This meant that phytochromes, especially phytochrome B, was responsible.

However, just like how we all learn to not touch the hot stove after our first burn, researchers also found that the ROS wave triggered a systemic stress memory mechanism. That meant the plant became familiar with the stress and its response was stronger.

“Evolution has realized that these high light intensities don’t just show up and instantaneously leave, but that they might stay around for a while,” Liscum said. “Evolution is adapted to have some system for the chemistry in the plant to change for some period of time before it goes back to what it was before it saw the stress.”

While the paper revealed much about phytochrome B and the systemic response to light stress, the Mittler and Liscum labs want to further investigate the memory and mechanistic aspects of the process.

“The next stage is to figure out how the sensing of the light stress by the receptors and not by the chloroplast triggers this response,” Mittler said.

Learning From History

October 26, 2020 By Lauren Hines in Research Tags: extracellular ATP, plant signaling pathways

Sung-Hwan Cho, a research scientist in the Gary Stacey lab, checks in on the arabidopsis plants that he uses in his purinergic signaling experiments. | photo by Lauren Hines, Bond LSC.

By Lauren Hines | Bond LSC

Until the 1990s, the presence and significance of extracellular ATP, a nucleotide that normally provides energy to a cell, in animal cells was highly contested for decades. Now, the Gary Stacey lab at Bond Life Sciences Center is using that history lesson to explore ATP’s role in plant cells.

Let’s say you have $100 in your pocket. As you’re walking down the street, you throw dollar bills over your shoulder. Sounds ridiculous, right?

Much like these dollar bills, ATP is the currency of cells. When the idea of animal cells dispensing ATP outside the cell to trigger danger signals came out in the 1970s, the scientific community thought it was wasteful and therefore the idea was ridiculed.

“The idea of throwing money over your shoulder is a very good analogy because that’s a good way to influence people,” said Stacey, a principal investigator at Bond LSC. “Give them a financial incentive. It’s the same thing with ATP. The idea is that this is the currency of the cell, but they’re putting it outside the cell in order to signal and influence interactions with their environment or other cells.”

This process of ATP receptors on the cellular membrane bonding with molecules to send signals and communicate between cells is called purinergic signaling.

The term was coined by retired neurobiologist Geoffrey Burnstock in the 1970s when he discovered it in animals; however, Burnstock’s findings were met with skepticism. It wasn’t until 20 years later when the first ATP receptor in animal cells was discovered that his idea gained acceptance.

Today, animal cell purinergic signaling has been used to make drugs that target diseases and develop drug therapies. As for purinergic signaling in plant cells, the research is just beginning. Much like what Burnstock faced, this new research is met with skepticism.

“It was clear to me that if we really wanted to establish this so that people would believe it, we had to get the receptor,” Stacey said.

In 2014, the Stacey lab reported its first ATP receptor called P2K1. Earlier this year, they found another one called P2K2.

“I think more and more people are working on purinergic signaling in plants,” Stacey said. “I think we will see a big explosion of people working in this area. Just as is the case with the animal field, the plant field is really going to take off now that we actually know what the receptors are.”

Currently, the Stacey lab is trying to understand how these receptors work. Research scientist Sung-Hwan Cho from the Stacey lab took over the project regarding P2K1.

“The ATP study in animals is just half a piece of the puzzle,” Cho said. “You need to find the other half of the piece. For 40 years, many labs have already studied this, but in plants, there’s just been a few.”

Research done by the Stacey lab has shown that although not much is known about purinergic signaling in plants compared to in animals, purinergic signaling can impact plant growth, development, defense against diseases and stress tolerance. Cho is currently studying how P2K1 impacts certain metabolic pathways in plants.

In another experiment done by the lab, they found that mutants without the ATP receptor that experienced stress did not grow smaller while plants that did have the receptor and experienced stress grew smaller.

“That says the purinergic signaling is what’s mediating the stress response,” Stacey said. “That is affecting the development of the plant in response to stress… So, if we understand this better then maybe we can make plants that are more drought and stress-tolerant and grow better.”

Close encounters of the plant protein kind

October 25, 2016 By Jennifer Lu in Research Tags: BioID, plant signaling pathways, seed grant

Bond LSC researchers David Mendoza (left) and Scott Peck (right) are collaborating to develop a new method for studying plant signaling pathways as they happen inside the cell. | photo by Jennifer Lu, Bond LSC — Bond LSC researchers David Mendoza (left) and Scott Peck (right) are collaborating to develop a new method for studying protein signaling pathways inside plant cells. | photo by Jennifer Lu, Bond LSC

By Jennifer Lu | Bond LSC

Sometimes, timing is everything.

That was the case in what led to a new collaboration between the Mendoza and Peck laboratories. The two researchers were recently awarded $48,250 in seed money from the Bond Life Sciences Center to adapt a new technology to the study of signaling pathways in plant cells.

David Mendoza, a Bond LSC researcher and assistant professor of plant sciences who is interested in nutrient uptake in plants, got the idea for the project when he attended the Trace Elements in Biology and Medicine conference in June. There, he kept hearing about an enzyme called BioID used to identify protein interactions in mammalian cells.

“In plants, we have a hard time figuring out how proteins interact with each another to transfer information within the cell,” Mendoza said. BioID could be the key.

BioID works like a spy slipping a small tracker into the coat pocket of every person it encounters, but instead of a tracker, BioID transfers a unique molecular tag onto every protein that comes near. It’s a speedy process, no matter how brief the interaction between BioID and the incoming protein. But once the proteins are tagged, they can be rounded up and identified later, even if they’ve moved elsewhere in the cell.

Scientists can study which proteins interact with their protein of interest by linking BioID to their protein. This lets them track the signals being communicated to and through their protein without disrupting what’s happening inside the cell.

Although BioID has exclusively been used in animal systems, Mendoza talked to the scientist behind BioID to see if it could be used in plants.

Incidentally, BioID has been publicly available for several years but the enzyme was impracticable for plant experiments. It needed a lot of raw material on hand before it could start tagging proteins, much more material than what is normally found within plant cells.

However, research on a more suitable candidate called BioID2 was published just months before the conference. Unlike its predecessor, BioID2 required very little starting material to function in plants.

“Like a lot of things,” Mendoza said, “timing was key.”

When he approached Scott Peck, a colleague at the Bond LSC and professor of biochemistry specializing in plant proteomics, with the news, Peck saw immediate applications for BioID2.

With currently available methods, plant scientists have to look at protein interactions in artificial environments, such as in a test tube or in yeast systems. A real-time protein-tagging method would allow plant scientists to observe signaling pathways in their native environment–the cell–under a variety of conditions.

“It allows the contextual information within the plant to still be present,” Peck said.

For example, with BioID2 the Peck lab, which studies plant resistance to bacteria, could watch how incoming stimuli such as plant pathogens or stress from drought affect overall protein-to-protein interactions within plants, compare these protein interactions across different cell types, or even discover previously unknown protein interactions, he said.

“You know you have a good idea when the other person gets excited right away,” Mendoza said.

Peck also had a suitable model handy in which they could test BioID2 at work, but the two researchers first had to make sure plant cells could produce functional BioID2. Mission accomplished, the next step is to make plants produce BioID2 that is linked to their protein of interest.

“The nice part of this seed grant is it lets us get a jump on some new technology to develop here,” Peck said.

Using BioID2 in plants is an interesting and novel idea, Mendoza said. “For me, that’s enough to try.”

This seed funding is one of seven awarded this year at the Bond Life Sciences Center. These awards, which range from $40,000 to $100,000 in funding, foster inter-laboratory collaboration and make possible the development of pilot projects.