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Life in Bloom Module Experiment Duration: 9 days Difficulty: Moderate Seeds require the right conditions to germinate. Temperature, light, and water are the external forces that influence germination. The plant hormone Gibberellic Acid (GA) is a critical component of the internal control of the developmental processes, acting to promote germination, plant growth and development. This experiment will explore the effects of several mutations in genes responsible for GA synthesis and signaling on germination. You will be able to see the difference between mutations that affect the early (ga1) and late (ga5) stages of GA synthesis and compare these with a mutation involved in GA sensing and response (gai-1). These differences will be demonstrated by observing germination in water versus GA solution of seeds that are grown either in the dark or under continuous light. Experimental Objective: This experiment will give you an understanding of plant hormone biosynthetic pathways, hormone sensing and mutant analysis, using a model plant Arabidopsis thaliana (Arabidopsis). Materials: Below is a list of materials required for this experiment. Make sure that you have all materials ready before you start. • • • • • • • Seeds (Table 1) Petri Dishes Filter Paper Water GA Solution Toothpicks Wax Paper • Micropore Tape or Parafilm • Aluminum Foil • Microscope or Magnifying Glass • Masking Tape • Pen Table 1: Seeds Genotype Stock Number Ler (Wild type) CS20 ga1-2 CS3103 ga1-4 CS3105 ga5-1 CS62 gai-1 CS63 Experimental Protocol: Treatments and Experimental Design Order seeds from ABRC (abrcoutreach.org/educational-kits) well in advance to when you plan to start the experiment. You should receive around 200 seeds for each of 5 Arabidopsis genotypes in small tubes labeled with the ABRC stock number (Table 1 shows the corresponding genotypes for each of the ABRC stock numbers). Students will be testing how well each of the 5 Page 1 Arabidopsis genotypes germinate in petri dishes that contain either water or 200 µM (micro molar) GA solution and then are either kept in complete darkness or are placed under direct light. Separate students into 4 groups. Each group will perform one of four experimental treatments on 3 replicate petri dishes (Table 2). It is important for each group to replicate their experimental treatment at least 3 times so there are enough results to statistically analyze the data. Table 2: Experimental Treatments Light Dark Water Group 1, 3 replicates Group 3, 3 replicates GA 200 µM Group 2, 3 replicates Group 4, 3 replicates Experimental Timetable This experiment is designed to take nine days (Figure 1). It is best to have students start the experiment on a Thursday and then remove the petri dishes from cold treatment on Sunday. Students can then count the results of the Petri dishes in the Light treatments on Monday-Friday. Since groups 3 and 4 will only count the results of their Petri dishes on Friday, they can recount the Petri dishes of groups 1 and 2 on Monday-Thursday, to double check their results. Figure 1: Timetable Preparation of Petri Dishes 1. Print out a copy of the Petri dish template, provided as a link on the Life in Bloom kit detail page (abrcoutreach.org/educational-kits). 2. Cut the template as indicated and give each group the template that corresponds to their treatment (Table 2). 3. Give each group 3 Petri dishes and 12 pieces of filter paper. 4. Have each group label 3 separate pieces of filter paper exactly as is indicated by the template they receive, making sure to fill in the current date and the appropriate replicate number (Figure 2 gives an overview of how a final Petri-Dish will look). Page 2 Figure 2: Petri dish layout 5. Have students stack 2 blank pieces of filter paper together and place the stacked pieces into a Petri dish. 6. Students then need to use their finger to smooth the edges of the filter paper in the Petri dish (Figure 3A). 7. Take one of the labeled pieces of filter paper and stack this on top of another blank piece of filter paper and smooth these pieces into the Petri-dish on top of the 2 blank pieces that were already smoothed into the dish (Figure 3B, C). 8. Repeat this process for all three of their Petri dishes (all finished dishes should resemble Figure 3D). Each dish should contain 4 pieces of filter paper, 3 blank and 1 labeled. Figure 3: Putting filter paper in Petri Dishes 9. Let groups 1 and 3 prepare 30 ml of water. 10. Let groups 2 and 4 prepare 30 ml of 200 µM GA solution. 11. Have students soak the filter paper in each of their Petri dishes with 10 ml of the liquid they were given. 12. Pour off excess liquid from their Petri dishes. Excess liquid may cause seeds to move around and ruin the experiment. Page 3 13. Give each group at least 30 seeds of each genotype (Table 1). Be careful when transporting seeds as they are very small and can easily be blown away. It is best not to transport the seeds in an open container and they should never be carried around when poured on weighing paper. 14. Pour out the seeds from one genotype onto a piece of weighing paper. 15. Take a toothpick and wet its tip using the wet filter paper of a Petri dish prepared in Steps 11 and 12 . 16. Using the wet tip of the toothpick pick up one seed at a time from the weighing paper and put it in its corresponding place on the Petri dish. 17. Place 10 seeds in their appropriate sections for each genotype on each of the 3 Petri dishes for their group, as shown in Figure 2. 18. Place the lid on the Petri dishes and wrap the edges of each Petri dish with Parafilm or Micropore tape completely sealing them shut. 19. Each group should stack their 3 Petri dishes on top of one another and wrap their groups stack completely with aluminum foil so that no light reaches the seeds. 20. Each stack of wrapped Petri dishes should be labeled, using masking tape, with the corresponding treatment, group number and date. 21. Put each stack of wrapped Petri dishes in a cold room or refrigerator at 4°C-6°C. This process is called stratification. Stratification is important for breaking seed dormancy and synchronizing the germination of the seeds. Be extremely careful when preparing, transferring or working on Petri dishes with seeds. Rough handling can cause seeds to shift from their intended position and ruin the experiment. 22. All Petri dishes should be kept in cold treatment for a minimum of 2 days and a maximum of 6 days. Data Collection: • • • • • • After cold treatment is complete remove Petri dishes from refrigerator or cold room. Remove the aluminum foil for Group 1 and 2 Petri dishes (LIGHT treatments) and place these dishes unstacked under bright light (For best results use a light rack). Place the Group 3 and 4 wrapped Petri dishes (DARK treatments) in a drawer or cabinet away from any light source. The day Petri dishes are removed from cold treatment is considered Day 0. On Days 1-4 Groups 1 and 2 should count the number of seeds germinated for each of the genotypes on each of their Petri dishes (LIGHT treatments) and record the results each day. Groups 3 and 4 can recount Group 1 and 2 Petri dishes double checking the results. Counting germinated seeds can be challenging. Arabidopsis seeds are extremely small and the early stages of germination cannot be observed by the naked eye. Use a large magnifying glass or a dissecting microscope to examine the seeds. Page 4 • • • Condensation will often form inside the lid of the Petri dish. If you need to, remove the lid to count germinating seeds. Carefully close and reseal edges of opened Petri dishes with Parafilm or Micropore tape after you are done counting. On Day 5 remove the Group 3 and 4 Petri dishes (DARK treatments) from the dark storage location and remove the aluminum foil. Have each group count the germinated seeds for their own groups Petri dishes and record the results. Seed Germination: Figure 4: Arabidopsis seed germination stages A B C Germination is the process by which plants emerge from their seeds. Figure 4 represents what you should see when observing Arabidopsis seeds under a microscope during different stages of germination. To count germinating seeds look for the emergence of root radicles (root tips) from the seeds. You can see the difference between an intact seed that has not germinated yet (Figure 4A), and a germinating seed (Figure 4B). As the experiment progresses you will observe later stages of germination as the seedling elongates, begins to green and forms root hairs (Figure 4C). Only count seeds where you clearly observe the emergence of the root radicle. If you are not sure about a particular seed, do not count it and wait for the following day when its germination status should be clearer. Discuss your counts with the rest of your group and be consistent! Page 5 Germination Data Analysis: Figure 5: Germination data examples Light, GA 200 µM 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% B CS20 Ler CS3103 ga1-2 CS3105 ga1-4 CS62 ga5-1 Germination % Germination % A CS63 gai-1 Day 1 Day 2 Day 3 Day 4 Day 5 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% GA (200 µM) 5 Day Light 5 Day Dark CS20 CS3103 CS3105 CS62 Ler ga1-2 ga1-4 ga5-1 CS63 gai-1 Create X-Y Scatter plots and bar charts to compare the germination data you have collected. Figure 5 shows examples of a germination rate scatterplot for the five genotypes in the Light, GA treatment (Figure 5A) and of a bar chart for the five genotypes in the GA treatment (Figure 5B). A number of graphs will automatically be created for you if you enter your results into the data analysis spreadsheet available at abrcoutreach.osu.edu/educational-kits (choose the Life in Bloom education kit to get access to a variety of downloadable resources). Compare the rates of germination for the genotypes in the light treatments, and also compare the final germination values in both the light and dark treatments. Observe the differences in germination for the genotypes across the various treatment variables (Light, Dark, Water, and GA). Page 6 GA & Germination: Figure 6: GA Biosynthesis & Signaling While analyzing the data in this experiment a few things should become clear. You will first notice that without the addition of GA the ga1-4 and ga1-2 genotypes will not germinate. You may notice that ga5-1 germinates more slowly than wild-type on water. You should see that without the addition of GA nothing germinated in the dark (a few plants will occasionally germinate in the “dark, water” treatment depending on how long you take to prepare and cover your petri-dishes during plate preparation). You can also observe that adding GA in the dark treatment did not rescue the germination of gai-1 (gai stands for GA insensitive), while it did rescue the germination of all the other genotypes in the same conditions (Figure 5B). You can compare your results with a set of example results available at abrcoutreach.osu.edu/educationalkits. Figure 6 describes the GA synthesis and signaling pathway and shows which part is disrupted for the various genotypes in the experiment. The ga1-2 and ga1-4 mutations block the function of the same gene and demonstrate that both regions of the gene are necessary for the gene function. Notice that the ga5-1 mutation affects a later stage of GA synthesis reducing the production of several GA products. Discuss with your classmates the results obtained for the ga5-1 mutant. Based on the position of the GA20 oxidase enzyme encoded by the GA5 gene, did you expect to observe reduced germination rate for the ga5-1 mutant? Discuss different reasons for a mild reduction of the germination rate of this genotype (e.g. A) a role of some of the GA Page 7 precursors in stimulating germination or B) a nature of the ga5-1 mutation that might cause just a knock-down of the GA20 oxidase function). Figure 7: GA induces the degradation of DELLAs Figure from Davière and Achard: Gibberellin signaling in plants. Development, 2013. The DELLA proteins are a group of proteins involved in GA signaling with a role to repress GA-dependent responses, including germination. There are 5 different DELLA proteins in Arabidopsis: GAI, RGA, RGL1, RGL2, and RGL3. The binding of GA to its receptor initiates a process by which DELLA proteins are degraded (Figure 7). Once DELLA proteins are degraded, the genes previously blocked by DELLA become activated and germination can occur. Therefore GA works in combination with the DELLA proteins to signal the activation of genes responsible for germination. The gai-1 mutation acts to stabilize the GAI DELLA protein and either prevent or reduce the ability of GA to induce its degradation (Figure 6). From the experiment you should see that the gai-1 mutation prevents GA-induced germination in the dark. Adding GA will not rescue this phenotype because the mechanism that responds to the presence of GA is disrupted. What is the germination rate of gai-1 under light conditions? Is there any difference between water and GA under light? Discuss why you think gai-1 seedlings germinate in the light but not in the dark. Keep in mind what you observed for the other GA mutants. Is it possible that light stimulates the production of larger amounts of GA than what was provided in the experiment under dark conditions? Would adding more GA to the Petri dishes in the dark stimulate germination of the gai-1 mutant? You may test this hypothesis by performing additional experiments. Page 8 Helpful Resources: Web Resources available at abrcoutreach.osu.edu • 18 educational kits exploring a variety of topics and experiments • Information about the ABRC education and outreach program • Arabidopsis news and events • Links to other useful resources Specific Resources Available for this Experiment (Educational Tools Life in Bloom) • Protocol (PDF) • Petri Dish Template (PDF) • Data Collection Sheet (PDF) • Data Analysis Spreadsheet (XLS) • Example Results (XLS) • Life in Bloom Protocol Video • Button to order seeds for this experiment Page 9