Cell Signaling
Greg Clark, University of Texas at Austin, The
Stanley Roux
University of Texas at Austin
Location: Texas
Abstract
A recent exciting discovery in plants is that ATP is released into the extracellular matrix where it plays a major role in regulating growth and development. Extracellular ATP (eATP) has been confirmed to act as a hormone in animal cells, where it rapidly induces an increase in cytoplasmic calcium levels, a change that commonly leads to the activation of signaling pathways that greatly influence cell activities in both plants and animals. There is a biphasic growth response to applied ATP in growing plant cells and tissues suggesting that eATP functions like a hormone in plants as it does in animals. Students in this CURE carry out novel experiments on how eATP controls plant growth. They learn methods of experimental design, data gathering, data interpretation, and data presentation, and they learn principles of stimulus-response coupling that apply equally well to animals and plants. Students use the model plant, Arabidopsis. They to do their experiments on extracellular ATP signaling in root hairs, an agriculturally important model system for studying plant growth. Specifically, this CURE addresses the question of what are the early signaling steps that mediate the effects of eATP on the polarized growth of single-celled root hairs, structures that are crucial for plants' absorption of water and nutrients from the soil. Their experiments constitute a novel test of the signaling steps required for eATP-mediated changes in root hair growth.
Student Goals
- Perform an experiment comparing the root hair growth rate in Arabidopsis wild-type and mutant seedlings or wild-type seedlings with and without a treatment.
- Design a well-controlled experiment that builds on prior knowledge and makes progress toward answering a research question.
- Improve skills of scientific writing and oral communication of research results.
Research Goals
- Identify and characterize the receptor for eATP-mediated promotion and inhibition of root hair growth in the model plant Arabidopsis.
- Determine the role of auxin and auxin transporters in the signaling pathway for eATP-mediated promotion and inhibition of root hair growth in Arabidopsis.
Context
In the first semester of this CURE, there are typically 26 students enrolled. The students work in pairs and perform their experiments using a microscope/camera/computer set-up. The students meet in lab one afternoon a week. Before their lab time the students receive a lecture providing them background information on their research field so that they can understand the rationale for their experiments. The lab experimental time, data analysis, and preparation of oral and written reports requires 8-10hrs/week of student time. The students need a basic level of math competency before beginning the course (passing Aleks basic math test).
The second semester, students do independent research and must first learn new techniques to become independent. They work individually to perform experiments and spend 8-10 hrs/week in the course. There is also a focus on further development of their written and oral science communication skills during this second semester.
Target Audience:Introductory
CURE Duration:Multiple terms
CURE Design
The main theme of the research done in this CURE, is to better understand extracellular ATP signaling in plants. Each year a new research question/area is addressed which ensures that students are doing novel experiments and allows for continued advancement of the research project. The CURE is based on a relationship of science collaboration with each student and this and other strategies are used to create an open and inclusive learning environment for all students.
Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Constructing explanations (for science) and designing solutions (for engineering), Developing and using models, Planning and carrying out investigations, Using mathematics and computational thinking
Nature of Research: Basic Research, Wet Lab/Bench Research
Tasks that Align Student and Research Goals
Student Goals ↓
1) Describe the properties of a model organism in the context of why Arabidopsis is a model plant.
2) Perform statistical analysis of your data. Is the value for your standard deviation reasonable?
4) Create a bar graph using letters of significance to indicate statistically significant differences.
3) Compare your average root hair growth rate with those reported in the literature. Are your results reliable? If not, how can you improve your technique?
1) Describe the properties of a model organism in the context of why Arabidopsis is a model plant.
2) Describe a signal transduction pathway and three ways to test a hypothesized signaling step.
3) Explain what is meant by gain- and loss-of-function mutants.
4) Summarize current, general knowledge about the role of auxin in regulating plant growth.
1) Make and defend decisions made during your research.
2) Briefly summarize the current status of an experiment.
3) Make and defend a claim about the current status of an experiment. Is it "working"? How do you know?
4) Define what is meant by a biological repeat. How do you establish credibility in your data?
1) Make an outline for presentation or written report.
2) Incorporate elements in "excellent" example report.
3) Avoid mistakes in "poor" example report.
4) Proof-read oral or written report while cross-checking rubric for critical elements.
1) Distinguish between primary and secondary literature sources.
2) Create an annotated bibliography with citation of 6 articles from the primary literature.
Instructional Materials
Syllabus (Acrobat (PDF) 121kB Mar4 18)
Protocols (Acrobat (PDF) 296kB Mar4 18)
Experimental Proposal Form (Acrobat (PDF) 12kB Mar4 18)
Assessment
Example final exam (Acrobat (PDF) 114kB Mar4 18)
Final written paper rubric (Acrobat (PDF) 21kB Mar4 18)
Instructional Staffing
This CURE is part of the of Freshman Research Initiative at the University of Texas at Austin. The program provides for a graduate or undergraduate student teaching assistant for one of the two semesters. The graduate student teaching assistant for this CURE works the first semester and helps with assessment. The program also provides undergraduate mentors for both semesters. In this CURE the undergraduate mentors play three important roles during the first semester: (1) sterilize seeds, prepare the growth media and make the growth and treatment plates for student experiments, (2) teach the new students experimental techniques and data analysis skills, and (3) assist the students during their experiments. During the second semester, the undergraduate mentors teach the students additional techniques allowing the students to become independent in the lab.
Greg Clark, University of Texas Austin
This CURE was developed to provide freshman students the opportunity to learn through doing research. The experiments the students perform are both novel and relevant such that their results are potentially publishable in a peer-reviewed journal. Thus the experiments performed by the students contribute to the research goals of the principal investigator while providing the students' inquiry-based experiential learning in the classroom.
Advice for Implementation
The biggest challenge for this CURE is to teach freshman biology students the current status in their field of research so that each of them understand the rationale for their experiments and can participate in data interpretation and experimental design. This is a process and is achieved through lectures, student presentations of their results and one-on-one meetings to discuss proposed experiments. Each computer/microscope/camera systems cost approximately $2500.
Iteration
Students are taught the needed techniques to perform root hair experiments early in the first semester and then they perform four rounds of experiments with a partner. In each of these first four rounds they have the opportunity to improve their experimental techniques through trouble-shooting and problem-solving. Besides experiment days, they have the opportunity to learn what the key/critical steps in the procedures are through discussions about each group's data on presentation days. Typically, a student group will begin observing expected average root hair growth rates (1-2 micrometers/minute) in their second or third experiment.
When a student group obtains an interesting result, they are challenged to establish credibility in this result by obtaining 3-4 biological repeats including performing the experiment under double-blind conditions. This is an example of iteration which can result in accepting data as credible or rejecting data as inconsistent.
The structure for failure and unexpected outcomes is a part of the students doing novel research. The students are prepared for potential problems and failure in each round of experiment by the instructor's constant reminders that this is the nature of doing novel research.
Using CURE Data
Student results are placed on the course website so that all the results obtained in a particular semester are available to all students in the course. Data quality is ensured by the requirement that any student-made discoveries are confirmed in at least 4 biological replicates which include double-blind experiments. The students share their results with the CURE PI and instructor through oral and written reports. When credibility of novel results are established and there are enough of these results to be assembled into a manuscript for submission for possible publication, undergraduate student authors must meet the following criteria: (1) contributed to experimental design and obtained data to be included in the manuscript, (2) demonstrate familiarity with the published literature of the research field, (3) contribute to the writing describing their contribution to the manuscript.
Resources
Students read these articles in the first semester of the CURE:
Platt JR (1964) Strong Inference. Science 146: 347-353.
Khakh BS, Burnstock G (2009) The double life of ATP. Scientific American 301: 84-92.
Note: During the second semester of this CURE, students read recently published articles from the field of extracellular ATP signaling in plants and present data from these articles in a journal club format.
Faculty should read these articles:
Clark G, Wu M, Wat N, Onyirimba J, Pham T, Herz N, Ogoti J, Gomez D, Canales AA, Aranda G, Blizard M, Nyberg T, Terry A, Torres J, Wu J, Roux SJ (2010) Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species. Plant Molecular Biology 74: 423-435.
Clark G, Roux S (2011) Apyrases, extracellular ATP and the regulation of growth. Current Opinion in Plant Biology 14: 700-706.
Clark G, Morgan RO, Fernandez M-P, Salmi ML, Roux S (2014) Review: Breakthroughs spotlighting roles for extracellular nucleotides and apyrases in stress responses and growth and development. Plant Science, 225, 107-116.