1. Welcome! McWilliams, 2013

2. Clover Lab Using Trifolium repens to understand natural variations in cyanogenesis pages.wustl.edu/cloverproject McWilliams, 2013

3. What do the following have in common? McWilliams, 2013

4. All of these plants are cyanogenic! McWilliams, 2013

5. Cassava is cyanogenic! MAJOR food source (tuber) in poor regions of Africa Problem: low nutritional value; cyanogenic! Geneticists are developing hybrids to increase protein value, plant disease resistance, and vitamin content, etc. Danida Research Danforth Research McWilliams, 2013

6. Essential Questions What adaptations have evolved in plants to provide protection from herbivory? What is the genetic basis of the protective adaptations? What factors might cause the genes for those adaptations to vary within a species? McWilliams, 2013

7. What is White clover (Trifolium repens) Life cycle: A ”mat-forming” perennial. Leaves: Alternate, compound with three stalkless, egg-shaped leaflets. Leaflets are smooth with small teeth along the margins, a slight notch at the tip and usually a whitish V-shaped watermark. McWilliams, 2013

8. What is White clover (Trifolium repens) Stems: Grows close to the ground. Plants spread by creeping stolons that root at the nodes and are generally smooth. Flowers and fruit: White or pinkish white, globe-shaped flower heads occur at the ends of long flower stalks. Each head may contain up to 85 individual flowers. Fruit are small, three- to six-seeded pods. Reproduction: Seeds and stolons. McWilliams, 2013

9. What is Clover Cyanogenesis? Together with your lab partner, brainstorm, then list, different types of protective defenses that plants have evolved to prevent herbivory (being eaten by herbivores). McWilliams, 2013

10. What is Clover Cyanogenesis? McWilliams, 2013

11. What is Clover Cyanogenesis? Based on the appearance of clover, which of the protective adaptations from your previous list do you think clover would use to avoid being eaten by herbivores? McWilliams, 2013

12. What is cyanide? When inhaled or absorbed, it binds to the cytochrome oxidase McWilliams, 2013

13. What is cyanogenesis? Linamarin Lotaustralin Cyanide Released by Linamarase (enzyme) McWilliams, 2013

14. What’s an enzyme? (REVIEW) Enzymes are biological catalysts (specialized proteins) They accelerate chemical reactions (by lowering activation energy) They are unchanged during the reaction Catabolic Enzymes Anabolic Enzymes McWilliams, 2013

15. Where does cyanide come from? E C Enzyme (E) is stored in the cell wall Cyanide-sugar (C) is stored in the central vacuole McWilliams, 2013 Cyanide

16. What does cyanide do? 3.Do you think cyanide will kill the plant cells? Why or why not? 3.Why would having two genes to control the release of the poisonous cyanide be advantageous to the plant? 3.Recall what you learned about plant cell structure. How might a clover plant keep the two compounds separate within the cell? 3.How would grazing on clover cause the release of the cyanide? McWilliams, 2013

17. The Genes C = cyanide-sugar compound (Linamarin/Lotaustralin) E = enzyme (Linamarase) McWilliams, 2013

18. The Genes C = cyanide-sugar compound (Linamarin/Lotaustralin) E = enzyme (Linamarase) McWilliams, 2013

19. The Genes C = cyanide-sugar compound (Linamarin/Lotaustralin) E = enzyme (Linamarase) McWilliams, 2013

20. Biosynthesis is the production of biological compounds by organisms. Clover plants, in order to be cyanogenic, must synthesize both compounds – the cyanide-sugar and the enzyme. Synthesis is an energy consuming process. 9. Based on the fact that synthesis requires energy, do you think it would be an advantage to a clover plant to produce the linamarase enzyme if the cyanide containing sugar is not present or to produce the sugar if the enzyme is not present? Remember… Cell Energy is a Limited Resource McWilliams, 2013 Metabolism

21. 10. Based on your answer, make a prediction about the relative frequency of the following genotypes. 11. Cyanogenic glucosides can be used as a way of storing energy and so can serve a function besides cyanogenesis. Linamarase, on the other hand, is only useful in cyanogenesis and is energetically especially expensive to produce. Based on this information, would you expect to find more C_ee plants or more ccE_ plants in nature? Explain why? McWilliams, 2013 Making Predictions

22. 98%15% 10% McWilliams, 2013 The Environment

23. McWilliams, 2013 Add W OnlyAdd W Only Add C OnlyAdd C Only Add E OnlyAdd E Only

24. Blue dots indicate the presence of cyanide Identifying Results McWilliams, 2013

25. Individual/Team Results

26. McWilliams, 2013 Class Results

27. Cyanogenesis genes Cyanogenesis polymorphism Adaptive significance Herbivore defense Energetic tradeoffs Ac/ac Li/li Climatic adaptation What is the genetic basis of adaptation? Genes Trait s Fitness Olsen, 2012

28. 1. Cyanogenesis deters grazing herbivores (snails, slugs, voles…) ( e.g., Stahl 1888… … Majumdar et al. 2004) Kooyers, Caulkins & Olsen, J. Chem. Ecol, in review Olsen, 2012 Adaptation

29. 2.Climate-associated cyanogenesis clines have evolved in native and introduced regions worldwide. Nic Kooyers v Percentage AcLi genotypes. N = 137 293 135 147 143 146 144 Kooyers & Olsen, 2012. Mol Ecol 21: 2455-2468 Adaptation Olsen, 2012

30. 3. Predominance of acyanogenic plants in colder climates may reflect energetic tradeoffs, lower herbivore abundance … Acyanogenic plants reportedly show greater reproductive output: Floral mass (g) Kakes 1989, Theor. Appl. Genet. 77: 111-118. AcLiacli Olsen, 2012 Adaptation

31. Sheryl Silverberg Lindbergh HS Classrooms nationwide survey local clover populations, contribute to online database Clover cyanogenesis in the Biology classroom… Connects genetics, natural selection, ecology Chuck McWilliams Maplewood HS pages.wustl.edu/cloverproject Olsen, 2012

32. McWilliams, 2013 Sample Results

33. McWilliams, 2013 Sample Results

34. Questions and Answers McWilliams, 2013

35. Collecting Clover 1 leaf = 3 leaflets 1.Be sure to get 2 separate individual plants 2.Collect enough leaves for 6 tests for each plant (18 leaves/plant) 3.Place samples in bag 4.Label w/ location and name 5.Store in refrigerator. McWilliams, 2013