Meet the gps Mutants

In the search for genes regulating plant signaling responses to changes in gravity, the Wyatt Lab has focused several research projects on a series of mutant Arabidopsis thaliana plants known as gps mutants. The gps mutants react to gravity differently than wild type (WT) plants. For example, when gps-2 mutants are exposed to the GPS treatment they bend the opposite way from WT plants. The predictable direction each gps mutant strain bends in response to the GPS treatment denotes a phenotype, and these physical differences are important because they are attributable to underlying genetic differences between the plants (that is, differences in genotypes).

gps_types

Figure 1 – Wild type (here labeled Ws) and gps mutant phenotypes shown at room temperature “remembering” the gravity vector from an earlier GPS treatment. The wild type phenotype (panel A) demonstrates the expected gravitropic reaction; the three gps mutants show aberrant reactions to the GPS treatment characteristic of each phenotype.

One method of determining the role individual genes play in a plant genome is to randomly disrupt the WT genome and observe the effects on the plant. When the WT genome was disrupted through DNA insertion the resulting mutants reacted differently to changes in gravity (gravity persistent signaling) than WT plants. The discovery of gps mutant phenotypes allowed the Wyatt Lab to isolate early signaling events and identify genes controlling plant signal transduction by focusing on genetic differences between WT and gps mutant genotypes in response to the GPS treatment.

WT and gps2 phenotypes

Figure 2 – gps2 mutant created from WT through T-DNA insertion of Feldman Tag.

Seven gps mutants have been identified to date, and three gps mutant phenotypes are currently under study at the Wyatt Lab: gps-2, gps-3, and gps-6.

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Project Profile: Microarray Analysis

Why Do the Shoots Go Up?

Remember the little seed in the styrofoam cup: The roots go down and the plant goes up and nobody really knows how or why but we are all kind of like that. (Fulghum, 1990).

To understand why the “roots go down and the plant goes up,” researchers in the Wyatt Lab are literally turning the “styrofoam cup” on its side. When you place a plant on its side, it responds by bending upward and toward the light, but if this same plant is set on its side at 4°C, it will not respond – not until it is returned to room temperature. Interestingly, if you return the plant to an upright position at room temperature, the plant will “remember” the pull of gravity (as if it were still on its side) and the stem will begin to curve to the side even though it is upright. [Click here to see the response.] This method of interrupting the signaling response to gravity is called gravitropic persistence signal (GPS) treatment and is an important part of how the microarray analysis project team is trying to define why “the plant goes up.”

Figure 1: Gravity response pathway

In 1928 two scientists working independently, Nikolai Cholodny and Frits Warmolt Went, proposed an explanation for why shoots grow upward toward the light (phototropism) and roots grow downward (gravitropism). In the model that would become known as the Cholodny-Went model, both scientists attributed the ability of plants to bend in response to light and gravity to the growth hormone auxin. Although widely accepted at the time, scientists working in recent decades have shown that auxin is not the only agent responsible for plant tropism. The work of these scientists has resulted in a revised picture of the gravity response pathway that includes a “cloud model” of signal transduction (the cloud representing numerous, yet-undefined elements) in place of a simplified model where auxin played the central role in driving a plant’s response to gravity. The question the Wyatt Lab microarray analyses project seeks to answer is: what are the genes and transduction factors in the “cloud model” of signal transduction that help plants respond to changes in gravity?

The gravity response pathway through which a plant perceives and then reacts to gravistimulation is today explained with four main steps:

  • Perception – a biophysical process whereby the plant perceives changes in the gravity vector.
  • Signal Transduction – the “cloud model” process in which the plant mobilizes auxin in preparation for transport.
  • Signal Transmission – a biochemical process by which auxin is distributed causing asymmetrical growth according to the concentration and location of auxin.
  • Reaction – the bending or straightening of the plant in response to gravistimulation.

The microarray analyses project team is focused on developing a detailed understanding what happens at the signal transduction step in response to perception. To do this, they first had to sift through the entire Arabidopsis genome to identify candidate genes to test.

Methodology

To identify candidate genes involved in transduction, The Wyatt Lab employed microarray analyses of RNA extracted from tissue samples from Arabidopsis thaliana plants that underwent gravity persistent signaling (GPS) treatment.

The standard GPS treatment consists of (1) placing a plant on its side for 1 hour at 4°C, (2) returning it to upright at room temperature, and then (3) observing the plant response. Using the GPS treatment allowed the team to design an experiment which effectively isolated and slowed down the events of signal transduction for close examination. To do this, the microarray project team collected samples at intervals of 2, 4, 10, and 30 minutes during the GPS cold phase, extracted RNA from these samples, and then probed each RNA sample using a microarray chip.

Results

The Magic Eight – Through microarray analyses the team was able to test the entire Arabidopsis genome (~27,000 genes) to search for genes that showed both a large difference in RNA transcript relative to the control sample and a high degree of confidence (p < 0.05). After testing samples collected at the two-minute interval, eight candidate genes suspected to play an important role in the early signaling stages of the gravitropic signal transduction pathway were identified and collectively labeled the “Magic Eight.”

Transcription Factors – A second set of eight genes, all transcription factors, were collected at the four-minute mark (from a set of more than 2400 originally identified). Transcription factors are genes promoting or inhibiting the transcription of DNA into mRNA which is then translated into protein supporting the biochemical reactions driving the gravitropic response of the plants.

Next Steps

Narrowing the focus to 16 candidate genes in their search for genes controlling the “cloud model” processes of signal transduction between plant perception and signal transmission was the important first step in defining the project. Now that candidate genes have been defined, the team will isolate and test each gene (by silencing or “knocking out” the gene) to determine what role each gene plays in signal transduction. From there, the team will work to define the complex relationships between these genes as they interact in response to plant perception and in turn mobilize auxin and the plant’s response.

References:

Fulgham, R. (1990). All I Really Needed to Know I Learned in Kindergarten. New York, NY: Villard Books.

My Plant Story – Proma Basu

Proma Basu – Graduate StudentPatabahar
Proma Basu

During the early years of my life, I lived in an apartment with a huge verandah. My mother is fond of gardening and she started collecting and growing ornamental plants in pots. Soon the plants grew and made our verandah a favorite amongst my friends for evening playtime. We used to spend most of our evenings and summer vacations playing amidst the beautiful plants. These plants are called ‘patabahar’ locally. Later while studying plants I came to know that these plants belonged to the Euphorbiaceae family.

My Plant Story – Marilyn Hayden

My (not so) Secret Garden
Marilyn Hayden

marilyn-haydenI was 5 years old when my family moved to Athens. Before we had lived in Galloway, Ohio, so I did not get to see my grandparents often. Once we moved, I spent almost every weekend in the summer with my great grandmother Marilyn in her garden. My grandmother loves a full garden of flowers. She especially loves sunflowers; she even makes snacks with their seeds. I loved going to her house on the weekends. We spent hours baking and gardening. Because she grew up in the 1930’s, she thought that every little girl should learn proper housework and baking.

My most favorite memory was when she gave me a neon green spade, hand-rake and matching watering can to use. That was the beginning of summer after 5th grade. With my new tools in hand, I really wanted to grow my own little patch of sunflowers to surprise my great grandmother. I tried to be sneaky and planted a handful of sunflower seeds closer to the woods. While I took excellent care of the plants on the weekends, no flowers sprouted. I was so disappointed and felt badly for stealing some seeds that did not flower.

When I went to apologize and tell my great-grandmother what had happened, she told me that she knew I had been trying to grow a secret patch of sunflowers. She then told me that if I wanted my plants to grow I had to take care of them more than just on the weekends. She reminded me that plants are living things just like people, who need love and care around the clock. She was so excited that I was into gardening she started an aloe plant off her bigger aloe plant for me to take home and care for on my own.

Currently, I still have a part of that aloe plant growing in my apartment and I frequently use it for sunburn. My mother and I mostly plant the garden now since my great grandmother is 87 years old and my great-grandfather, Bernie, is 90 years old. While the garden has shrunk in size, it is still packed full of color and sunflowers, especially around the edges towards the woods.

Meet Marilyn Hayden, Graduate Student Researcher

marilyn-hayden

M.S. Molecular and Cellular Biology, (Anticipated May 2016) B.S. Biology, Ohio University  2013

Marilyn is currently a Masters Student in the  Molecular and Cellular Biology program. Her current research involves assembly and analysis of RNAseq data from  in the BRIC-20 experiment and development of a microgravity gene network.  From her analysis of the RNAseq data,  she will identify differentially expressed mRNA transcripts specific to germination and early development of Arabidopsis seedlings in space. The RNAseq data will also provide an additional expression profile for the microgravity gene network. Marilyn is also working to identify the mutation responsible for the Arabidopsis mutant gps6.  A typical day in the lab for Marilyn includes compiling publically available spaceflight expression data, creating pipelines for next generation sequencing data,  phenotype analysis of gps6 and RNA extractions. When she is not in the lab, Marilyn enjoys hiking around Athens with her dog and making jewelry for her friends.

Fun Science – Ice Custard Friday

You might not be surprised that just a five minute walk from the Wyatt Lab you can enjoy Whit’s Frozen Custard a one-of-a-kind frozen custard experience that originated in Granville, Ohio. But you may be surprised when you show up to the lab on an extra-hot Friday afternoon to find the lab empty …

At least that’s what happened last Friday!

wyatt-whits

Fun Facts – Why Arabidopsis?

If NASA is going to spend all the time and trouble to send plants into space aboard the International Space Station (ISS), you might wonder why Arabidopsis thaliana?

Arabidopsis thaliana (commonly called Arabidopsis)  is a small flowering plant native to Eurasia. Arabidopsis has enjoyed a long history as a model organism for scientists interested in plant biology and genomic research. Model plants are selected for their traits with the understanding that insights into the research model plant will yield insights to other plant species. One key aspect that makes Arabidopsis a good model organism is its genome. Arabidopsis has one of the smallest genomes of any plant, and as a result of its reduced genetic complexity, it was the first plant genome to be sequenced in full.

Another important quality that makes Arabidopsis so attractive to gene hunters is its size. The Arabidopsis seeds are so tiny Wyatt Lab researchers will plate 800 seeds to a single petri dish, and this is mission critical for two reasons. First, space is limited aboard the ISS, and as a result, the Wyatt Lab is only allotted 20 petri dishes total. And since the researchers need plant tissue to extract both protein and RNA, the more seeds that can fit on a dish, the better.

In summary, by sending a model organism (in this case, Arabidopsis) into space, Wyatt Lab researchers are able to tap into and leverage the enormous amount of genomic research already compiled on this amazing little space traveller. And after all, why reinvent the allele?

IMG_8260-SKETCH

Fun Facts – The Eagle has landed …

You might not be surprised to learn that on July 21st, 1969 when Neil Armstrong took that first big step onto the surface of the moon, Sarah Wyatt was back on Earth watching events unfold on the television with millions of other American youngsters. Like many other people who witnessed the first moonwalk (the one before Michael Jackson made it infamous), it made a big impact on the young Wyatt’s future.

You might be surprised to learn that since that day Wyatt has amassed a remarkable collection of NASA spaceflight paraphernalia that includes is sure to impress even the most avid space collector. So, if you ever find yourself at one of Dr. Wyatt’s famous backyard cookouts, make sure you ask to see the collection!

apollo-11

Learn more about the Apollo 11 mission here:

My Plant Story – Adam Cook

Adam Cook – Student ResearcherThe Fuzzybush
Adam Cook

My grandmother and grandfather, better known as Mimi and Pappy, own a house and a surrounding three acres of land. As a child my mother would drop off my sister and me at their house before she went to work. We were accompanied by our two cousins most of the time. Upon turning onto Holly Drive a full view of Mimi and Pappy’s glorious property presents itself at the end of a cul-de-sac and my mind would race with all the possible activities to be done that day.

On Mimi and Pappy’s property were a wide variety of plants: apple trees that seemed to rain down fruit, lavender under the hammock, and all the climbing trees a six-year-old boy could desire. Most noteably, there was the fuzzybush. The fuzzybush was a large smokebush growing next to a pond in the backyard that was transplanted from my great-grandmother’s property years ago. We always started off the day by walking in to find our grandparents pretending to be asleep—an antic still heavily practiced in our family—and racing off the back patio into the yard. The fuzzybush was our summertime bowery. 

Its tantilizing tan flowers and the shade it provided always kept us hornery kids occupied. We were wont to spend the better part of our day under the bush playing house or taking advantage of its low sprawling limbs to nap on. The smokey flowers of the fuzzybush became a sacred backyard relic. They became the main ingrediants in mudpies and imaginary boats floating on the surface of the water. We were entertained by their graceful descent when a gust of wind jostled them and challenged to swipe them out of the air before they touched ground. The fuzzybush still stands in Mimi and Pappy’s backyard and is perhaps the most evocative image of my childhood nostalgia. 

Meet Anna Brito, Student Researcher

Anna Brito – Wyatt LabAnna Brito is an exchange student from Brazil where she studied biotechnology for two years as an undergraduate student. In Fall 2013, Brito  came to Ohio University to major in Biology and joined the Wyatt Lab during the Spring 2014 semester. Brito’s research involves plant gravitropism mutants. She is working to identify the role of genes involved in the signaling process to figure out which gene is causing gps3 mutant phenotype in Arabidopsis thaliana.