SYLLABUS, BIO 328D: DISCOVERY LABORATORY IN PLANT BIOLOGY

SYLLABUS, BIO 328D: DISCOVERY LABORATORY IN PLANT BIOLOGY Instructor: Stan Roux; BIO 16; 471-4238; sroux@uts.cc.utexas.edu; Office hrs: TTh, 10-11:30 AM TA: Ashley Cannon; BIO 15; 471-1074; ashleycannon@utexas.edu; Office hrs: MW, 10-12 AM Brief Overview of Experiments As in past years, this year's Discovery Laboratory will emphasize learning methods of experimental design, data gathering, data interpretation, and data presentation. The novel experiments you will carry out this semester will constitute an original test of a relatively new hypothesis. This hypothesis predicts that Ceratopteris spores require a fixed amount of light in order to initiate germination but can then be moved to the dark in order to continue development until the growth of organs that are required for photosynthesis is initiated, at which time, unless more light is given, development will be arrested. Four rounds of original experiments will be carried out to test this hypothesis. This year's experiments will use fern spores as test plants to address the question of how much light is needed to initiate germination, and when a second light treatment is needed for development to proceed all the way to spore germination. This new information is crucial to understanding the process of light-induced germination and how this process differs from germination events that require only an initial light treatment. General Introduction on Signaling Changes Induced by Light Plants use many different environmental stimuli to regulate their development and morphology throughout their lifetime. Light, a common environmental stimulus, has a most profound effect on development. Plants are very sensitive to any illumination, sensing the quality, intensity, direction, and duration of the light and altering their development in relation to all of these factors. In seed plants, light responses occur throughout post-embryonic growth. Light affects germination, seedling development, shade escape, and the timing of flowering. Light signaling in lower plants that reproduce using spores (e.g. ferns and mosses), is not as well understood because fewer studies have been done on these systems. Based on studies largely focused on angiosperms, we know that plants primarily respond to UV light, blue light, red light, and far-red light. To respond to these various light qualities plants use photoreceptors, proteins responsible for converting light into signals that direct their development. Most of these photoreceptors consist of a protein bound to a light-absorbing pigment called a chromophore. Phytochromes, photoreceptors primarily responsible for perceiving red and far-red light, are involved in initiating germination in seeds of flowering plants and in spores of ferns. Phytochromes exist as two, interconvertible conformations that have distinct absorption spectra. Pr, the inactive conformation, absorbs in the red region, and Pfr, the active conformation absorbs most strongly in the far-red region. In the presence of red light, Pr is converted to Pfr; and far-red light converts Pfr back to Pr. Fern phytochromes control both red/far-red light, reversible spore germination and de-etiolation (Possart and Hiltbrunner, 2013). Light can also affect spore polarization, because the rhizoid will emerge in the direction opposite to the direction of unilateral light treatment. This directional response to light is induced by the blue-light photoreceptor, phototropin. The short-term red light sufficient to induce early development of the spore is apparently not sufficient for the spore to complete its first cell division and germinate. Why would the spore need more light to germinate? In addition to providing environmental information, light also generates for plants the energy that is utilized for photosynthesis. During photosynthesis, light energy is used to drive the synthesis of carbohydrates, and the energy stored in the carbohydrates produced is used to power cellular processes. Could it be that the additional light needed for spore germination was required for photosynthesis? Ceratopteris as a Model System for Studying Gravity Effects on Cell Polarization Growth of fern gametophytes begins when the haploid spores germinate and a single-celled rhizoid emerges from one end of the spore (see image on p. 5). Multiple studies have shown that gravity directs the developmental polarity of these haploid cells, and the most visible manifestation of this is the emergence of a 1

polarly-growing, single-celled rhizoid pointing downward in 80-90% of the germinated spores about 3 d after the spores are induced to germinate by light. Prior to this, at about 40 h after the light treatment, there is an asymmetric cell division, with the smaller of the two cells situated in the lower part of the cell and destined to develop into the rhizoid. This unequal cell division is set up by a prior migration of the nucleus from a central position to the lower part of the cell, which happens about 20 h after the cells are irradiated. All of these polarized events require a light-induced entry of Ca 2+ into the bottom of the cell, which happens within minutes after the irradiation. This initial entry of Ca 2+ induces a trans-cell calcium current that runs parallel to the vector of gravity and exits out of the top of the cell. Blocking the current with a calcium channel inhibitor, nifedipine, blocks the ability of gravity to orient the direction of rhizoid emergence; turning the cell upside down quickly reverses the direction of the current; and changing the magnitude of the g-force similarly changes the magnitude of the current. These data indicate that gravity rapidly modulates the magnitude and direction of the current, and the direction of the current predicts the direction of polarized development in spores. A key feature of the gravity response in the spores is that gravity fixes the polarity of the cells at about 10-14 h after irradiation; i.e., before the nucleus migrates downward. Proof of this is that if the spores are turned upside down after ~12 h, the nucleus in most cells migrates upward, and the rhizoid emerges pointing upward. That all the polarized development events are directed by gravity was demonstrated when spores were flown on Shuttle flight STS-93 and observed by live video microscopy after they were induced to germinate by light while the Shuttle was in orbit. In this micro-g environment, the spores germinated and developed normally, but the nucleus migrated in random directions, and the rhizoids emerged (also randomly) in the same direction as the nucleus had migrated (Roux et al., 2003). Data show that although gravity is the primary stimulus directing the gravity response in Ceratopteris spores, light is also a contributing factor that could alter the direction of rhizoid growth based on the direction of illumination. Preliminary studies have shown that when spores are exposed to 8 h of light, they can be moved to the dark during the remainder of polarization and will still germinate if, and only if, they are re-illuminated after 48 hours. Keeping the cells in darkness between 8 and 48 h makes it less likely that light could influence the direction of gravity-induced cell polarization, because gravity does not fix this direction until about 12 h after the initial light treatment starts. Understanding how much less light than 8 h could be used in order to initiate germination in Ceratopteris spores, and how much later than 48 h the re-illumination could be given to allow germination, are new pieces of information needed to allow scientists studying gravity-directed cell polarization in this system to eliminate light as an additional variable in their experiments. Moreover, learning when and why the second illumination is needed will help resolve what photoreceptor (is it chlorophyll??) is required for the completion of the germination process. In addition to improving current protocols that could lead to new discoveries, investigating the timing and quantity of light treatments necessary to irreversibly induce germination in Ceratopteris spores will provide novel data that could provide insight into light-signaling responses that are not well understood. Hypotheses To Be Tested This Semester The preliminary data discussed above suggest the hypothesis that two separate light sstimuli are needed to irreversibly induce germination in Ceratopteris spores. Recently, Ashley Cannon (TA for course) completed a set of preliminary experiments in which 8 hours of light was enough to initiate germination as long as the spores were returned to the light after 48 hours. This discovery raises the questions of how little light is needed to initiate germination and whether the molecular mechanism needed to continue germination after 48 hours is photosynthesis or another molecular process. Starting Point for Our Experiments This Semester Even though spores are relatively large cells (> 100 µm in diameter), in order to visualize the direction rhizoids emerge from them will require some practice. To achieve this, we will begin the laboratory with a relatively short microscope session in the first week of class. For this experiment you will need to 2

learn how to take clear digital images of both un-germinated spores and the rhizoids that emerge in germinating spores, so as to determine how changes in the amount of light is affecting germination and the gravity response. You will take digital images of at least 50 germinating cells, and you will be instructed on how to analyze these images to evaluate whether the amount of light used in your experiment was enough to initiate germination and how it affected the gravity response. The results of this test will be reported and discussed on February 1. Each "round" of experiments will consist of 3 lab sessions: two sessions of measuring the effects of treating germinating fern spores with either set amounts of light and/or photosynthetic inhibitors, and one session of data presentation and analysis, including statistical treatment to determine the significance of any differences noted. At the end of each round, students will react to the results obtained by refining the experimental design to get more detailed and/or more accurate data. References Possart A, Hiltbrunner A. (2013) An Evolutionarily Conserved Signaling Mechanism Mediates Far-Red Light Responses in Land Plants. Plant Cell. Vol 25: 102-114. Roux SJ, Chatterjee A, Hillier S, Cannon T. (2003) Early Development of Fern Gametophytes in Microgravity. Space Life Sciences: Missions to Mars, Radiation Biology, and Plants as a Foundation for Long-Term Life Support Systems in Space. Vol 31: 215-220. The lab schedule for Spring 2015 will be: Jan. 25 Introduction to the course and the experimental system; microscope training Feb. 1 Round 1 of experiments begins. Report preliminary imaging results from Jan. 25. Improve observation methods and record new data. After class, measure effects of treatments; tabulate results. 8 Carry out variations of Feb. 1 experiments. 15 Meet to discuss results. Propose second round of experiments to test alternative hypotheses that may account for results of Round 1 experiments. 22 Begin Round 2 of experiments, and continue them on February 29. March 7 Meet to discuss results. Propose third round of experiments to test alternative hypotheses that may account for results of second round. Half of your Final Report* is due. 21 Begin Round 3 of Experiments [three weeks, same procedure as first two rounds] April 11 Begin Round 4 of experiments [3 weeks, same procedure as first 3 rounds] May 2 Final Exam; Final Reports* due *The instructions for the Final Report will be discussed during class. A document with a description and an example report will be posted on Blackboard by February 1 st. INSTRUCTIONS FOR WRITTEN & ORAL REPORTS FOR BIO 328D Each team will present one paper and one oral report for each experiment. One member will write the written report and the other member will present the oral report, so each student will do two written and two oral reports during the semester. Teams should work together when preparing reports. Each written Lab Report should include: 1. A Cover page with date and names of team members (place an asterisk next to the author's name), a title for the report, and a VERY brief (less than 250 words) description of the experiment done. The description should focus on the hypothesis tested and results. 2. On p. 2 should be a bar graph to describe the results. Each bar in the graph should give the standard deviation around the mean value, with a different letter above bars that differ significantly from each other. 3

3. On p. 3 there should be two Tables: Table I should show the raw data in columns including: The treatment being measured The range of values measured The mean of the values measured The standard deviation of the values measured The number of samples measured Table II should describe the Student t-test-comparison being measured, the t-value measured for that comparison and a column stating the level of significance. Note that Level of Significance values above 0.05 are considered insignificant. Each Table and Graph should have a figure legend. Each Oral Report should: 1. Be 10 min or less. 2. Include an introduction that gives some background information & specifies the hypothesis being tested. 3. Show (as Powerpoint slides) and discuss the summarized table & graph results. Interpret the results and present what might be the next experiment. LAB COURSE GRADING* 2 ORAL REPORTS: 25 2 WRITTEN REPORTS: 25 Final exam 15 Final Report 20 Overall performance ** 15 *Note that this course carries the Independent Inquiry flag. Independent Inquiry courses are designed to engage you in the process of inquiry over the course of a semester, providing you with the opportunity for independent investigation of a question, problem, or project related to your major. Therefore, a substantial portion of your grade will come from the independent investigation and presentation of your own work. **The success of this laboratory experience, as is true for the success of all original research, will depend strongly on how much thought and consultation time you put into your project. Although Dr. Roux and the TA cannot predict the outcome of your experiments, we can surely help you think about your results, solve technical problems that you may encounter, and evaluate options for the new experiments you will plan. The research you will do this semester is original and could generate potentially exciting results. We ask you to take on both the joy and responsibility of discovering new insights about how plants grow and develop, and we hope you will enjoy this collaboration with us and with your student colleagues. On-time attendance is a key factor considered in determining the Overall Performance grade. Active participation in the design and execution of your experiments and in the discussion sessions at the end of each round will enhance your Overall Performance grade; and, of course, surfing the web, texting, or other signs of inattention during the labs or discussion sessions will significantly hurt your performance grade. The image above shows 2 germinating fern spores. Scale bar = 100 µm 4

Evaluation of Written and Oral Reports Each report will be graded based on the following criteria: Written Reports: Oral Reports: Paragraphs are organized logically Checked for grammatical and spelling errors Figures are labeled correctly Sentences are clearly written and easy to understand Text is written in proper tense, clear and concise Used the layout described in Dr. Roux s syllabus Cited sources using a standard format Slides are organized logically Presentation is clear and concise Gives a good rationale and provides evidence Attractive and balanced layout, legible font Facilitates discussion and is receptive to feedback Can identify what to do in the next experiment Cited sources using a standard format Example of Bar Graphs for P. 2 Figure 1: Ceratopteris spores growing in 96 hours of light (light control) or 8 hours of light, then moved to a dark growth chamber and returned to the light from 48-96 hours. After 96 hours, the direction of rhizoid growth (A) and germination (B) was assessed. An asterisk denotes a statistically significant difference from the light control where p < 0.05 using the Student s t-test of three technical replicates. Example of Table for P. 3 Light Treatment Range of Values Average Downward Growing Rhizoids (%) Standard Deviation n-value Light Control 85.43% - 89.5% 87.38 2.1 3 8h + 48-96h 93.9% - 96.5% 95.2 1.3 3 T-Test P-Value Light Control vs. 8h+48-96h 0.04 5