Peering Into the Gray Cloud Using Spaceflown RNA

colinkruseColin Kruse, Wyatt Lab manager and researcher, is looking for genes not yet identified as related to gravity response in plants. To do this, Colin and Dr. Sarah Wyatt enlisted the help of NASA to germinate seedlings in microgravity conditions aboard the International Space Station. The spaceflown samples have since returned to earth and Colin will soon complete the next milestone in the NASA-sponsored BRIC-20 microgravity experiment. Using RNA extracted from spaceflown seedlings and ground controls, Colin hopes to  identify the genes involved in the plant signalling biochemical pathway.

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Modeling Arabidopsis Responses to Microgravity

Dr. Sarah Wyatt and graduate student researcher Marilyn Hayden are converging their knowledge of genetic research and computer-assisted network analysis to build a novel computer model. The model will collectively present International Space Station (ISS) plant experiment data from the past 10 years. The meta-analysis will provide plant geneticists with a visual, interactive network model of the genes acting and interacting across the genome in response to microgravity conditions.NODEs

The network model is part of the Wyatt Lab’s current space-based research experiment, known as BRIC-20, and will include 47 space-based plant research projects conducted by international researchers aboard the ISS. Many international scientific journals have published some of the data, but a large portion of it has never been publicly available.

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gps2 Mutant Candidate Gene Identified

The Wyatt Lab researchers studying the gps2 mutant genome have identified a gene that appears to play a significant role in plant signal transduction. The candidate gene was identified through deep sequencing of the gps2 mutant genome and then comparing the results to the wild type (WT) genome. The analysis revealed a difference of a single gene in one area of the genome associated with plant signaling which had been disrupted by a semi-random T-DNA insertion that silenced (or shut off) the gene in the mutant. In the WT genome, the gps2 gene is intact and expresses normally during GPS treatment. Continue reading

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.

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.