In Class Research

January 30, 2014 at 12:53 pm

How Do They Know Which Way Is Up?

Marilyn Hayden and Megan Moore explore gravity signaling in plants.

Marilyn Hayden and Megan Moore explore gravity signaling in plants.

Gravity. Actors Sandra Bullock and George Clooney provided a theatrical look at how humans might experience the lack of gravity in space. But what about plants?

Humans have touched the moon and sequenced the human genome. But answers to how plants respond to gravity are just now being unlocked—by students in the Wyatt Lab at Ohio University.

While the Gravity actors dealt with a nightmare scenario, undergraduate students are processing through what would have been a nightmare amount of data thanks to Bioinformatics classes and the Genomics Lab.

In the genes of mutant plants, they are identifying how plants signal which way is up.

But unlike the movie, plants from the Wyatt lab really are going into space—on a NASA flight.

Gravity or Sunlight? Which Way Is Up?

Undergraduate student Marilyn Hayden ’13 dispelled the myth right away with a group of visiting Logan High School students. Plants do not grow “up” because they are attracted to sunlight. They grow up because they are responding to gravity.

Hayden picked up a spindly plant growing in a little black plastic container and turned it on its side, explaining that their research begins in the refrigerator, with plants on their side perpendicular to the gravitational pull of the Earth.

The plants are too cold to respond, but they “remember” which way is up, and when they are taken out of the refrigerator and returned to upright, they bend. They “remember” being on their side in the fridge.

Up in Space, in the Dark

When the Wyatt Lab experiment makes its space flight, the seeds will germinate and grow in the dark. A control lab on Earth will mirror the space conditions—with gravity as the only variable.

When seedlings germinate in the soil, gravitational signaling tells them which way to grow, and it was that gravitropic signaling that Dr. Sarah Wyatt began exploring about 15 years ago. At that time, scientists knew almost nothing about how plants “signaled” gravity.  Now hers is one of only a few labs in the world—including one in Wisconsin and one in Japan—studying how plants respond to gravity.

“If I touch you, you know you’ve been touched, and it’s electrical signals. Plants don’t have electrical signals. So I’m interested in how they transfer information, and the thing that I work on most is gravity because gravity affects how plants grow, it affects limb position, root position, everything,” says Wyatt, Professor of Environmental & Plant Biology and College of Arts & Sciences Coordinator of Undergraduate Research.

Wyatt’s student researchers—undergraduates and graduate students—work with a fast-growing plant called Arabidopsis.

Mutants and “Gypsis”

The “gypsy” mutants are the stars of the show.

It just sounds like science fiction. The plants referred to as “gypsis” are gravity persistent signal (gps) mutants. The mutants are Arabidopsis plants with genetic mutations that allow the researchers to help isolate the genes controlling the gravitropic signaling. Gravity is a fundamental stimulus governing plant growth and development. Normally, plant shoots bend up in response to being on their side (gravistimulation). However, if plants are put on their side in the cold, they don’t bend. But if you return them to room temperature, they bend in response to the cold signal. They “remember.” Plants can sense gravity in the cold but cannot respond. The hormone (auxin) that causes bending is not transported properly and, therefore, the plants don’t bend.

“We used this phenomenon to find mutants that were defective in the mechanisms responsible for the signal transduction events linking perception of gravity and the transport of auxin,” Wyatt says.

Once they had the mutants identified, it was time for the genetic work—both microarrays that are done in the Genomics Lab adjacent to Wyatt’s lab, but starting last year, deep-sequencing of the plant genes.

Dr. Sarah Wyatt demonstrates making an agarose gel on a lightbox that allows researchers to visualize the DNA band.

Dr. Sarah Wyatt demonstrates making an agarose gel on a lightbox that allows researchers to visualize the DNA band.

Bioinformatics and the ‘Magic 8’

Studying how genes are “expressing” during the cold and warm-up segments of the experiment produces an enormous amount of data that has to be analyzed. That’s where bioinformatics comes in.

“Mutants are great,” Wyatt says. “They’re defective in something. The problem is you have to figure out what the gene is. We’ve had a really hard time figuring out what some of these genes are. Now that sequencing has come down in cost so much, we took all the mutants that we didn’t have a gene for, and we sequenced them all, which turns into a bioinformatics nightmare, because now we have 125,000 base pairs and wild type plus four mutants worth of data.”

Sampling at time points across the cold and warm-up treatment, Wyatt and her students—almost all of them undergrads— looked at a gene expression microarray of what genes were coming up and down, using computer programs to help sort the huge amounts of data.

“When we did that, we wound up with 349 genes that were expressed in one way or the other.” She says. Venn diagrams then helped show how gene expression overlapped during the treatment.

They refer to a key result as “the magic 8.”

“The interesting part about this whole Venn diagram is there are 8 genes that were turned on at 2 minutes that were not up- or down- regulated significantly at any other time points. So we refer to these as the magic 8. That early time point (the time point everybody said I couldn’t get) I think is going to be the most interesting,” Wyatt says.

Each of the undergrad students in Wyatt’s lab gets a mutant to work on.

Meet the Undergraduate Research Team

Sarah Acomb ’13—She has been working on gravitropism in Arabidopsis thaliana. In the lab, she has been learning procedures and protocols planting, DNA extractions, PCRs, and gel electrophoresis.  Her project specifically focuses on mutant analysis. She majored in Environmental and Plant Biology with a minor in Geography and an Environmental Studies Certificate. Read more about Acomb.

Sean Clay ’16—His older sister, Shannon, also worked in Wyatt’s lab. “He started out washing dishes, making media, growing plants and learning the basics of the lab, and then he moved on to start doing some research,” says Wyatt. Now he is a Research Assistant in her lab. He started working in the lab his freshman year. He is majoring in Biological Sciences and pursuing the Bioinformatics Certificate. He received a Choose Ohio First for Bioinformatics Scholarship. Read more about Clay.

Sean Fenstemaker ’13 —He majored in Applied Plant Ecology and earned a Bioinformatics Certificate. He received a Choose Ohio First for Bioinformatics Scholarship. Read more about Fenstemaker.

Marilyn Hayden ’13—She is the lab manager in the Wyatt lab although she anticipates starting work on a MS in Molecular and Cell Biology in the Spring semester of 2014. Marilyn joined Dr. Wyatt’s lab in the summer of 2012, working on identifying and confirming the gene defective in gps6 in Arabidopsis thaliana. She majored in Biological Sciences, with a minor in Computer Sciences and the Binformatics Certificate. She received a Choose Ohio First for Bioinformatics Scholarship and Ohio University Undergraduate Research & Creative Activity Grant. She presented at the Great Lakes Bioinformatics Conference in Ann Arbor, MI.

Megan Moore ’15—She is working on identifying and confirming the gene defective in gps6 in Arabidopsis thaliana. To do so, she will use deep sequencing technology, then confirm the idetnifty of GPS6 through analysis of publically available mutant alleles and genetic rescue. She is majoring in Plant Biology. Read more about Moore.

Megan Osika ’16— She is working on a joint project with Dr. David Rosenthal. She is trying to alter expression of SBPase Arabidopsis thaliana. Once she successfully completed the cloning, she will characterize the effects of the gene under a variety of environmental conditions. She is majoring in Plant Biology. Read more about Osika.

Avery Tucker ’13—He is currently working from a set of microarray data to identify putative genes involved with the gravitropic signal transduction pathway. A typical day in the lab involves running PCR and RT-PCR or gel electrophoresis, tending to plants, and reading relevant literature. He has made presentations at the American Society of Plant Biologists Midwest Meeting in Chicago and the Sigma Xi Annual Meeting and International Research Conference in Raleigh, NC.

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