RNAi Dicer Helps Confirm gps3 Candidate Gene

BiancaCorreiaBianca Corriea, visiting exchange-student researcher working in the Wyatt Lab, is using antisense RNA (asRNA) to verify the candidate gene responsible for the unique phenotype of the gps3 mutant in response to GPS treatment.

To test the gps3 candidate gene, Corriea must silence the expression of this single gene and verify that this targeted transformation results in the gps 3 phenotype. The gene silencing method that Corriea will deploy is to sequence the candidate gene and then splice a copy of the gene into agrobacterium. Agrobacterium is used in plant biology as a method of introducing genes into the genome of test plants through a process known as agrobacterium-mediated transformation mechanism. This mechanism takes advantage of the agrobacterias natural process of invading host organisms and semi-randomly inserting its DNA into the host genome.

Continue reading

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.

Continue reading

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.