1. Testing a Role for Rop9 GTPase in Maize Pollen Tube Growth Lauren A. Osborn Dr. John Fowler Botany and Plant Pathology Summer 2005

2. Pollen Tubes Pollen Tube Growth Pollination and pollen tube growth is essential Pollination and pollen tube growth is essential fertilization fertilization seed production in plants seed production in plants Two phases for pollen tube growth in Maize Two phases for pollen tube growth in Maize Germination Germination Growth in the silk Growth in the silk Germination

3. Seed Seedlings Fertilization Pollen Grains Silk and Egg Mature Adult

4. Rop Genes Nine different Rop genes in maize Nine different Rop genes in maize ROP GTPases are proteins that act as a signaling molecule (example: pollen tube growth) ROP GTPases are proteins that act as a signaling molecule (example: pollen tube growth) Rop2 mutant pollen has a reduced ability to fertilize Rop2 mutant pollen has a reduced ability to fertilize

5. ATG My Research: Rop9 gene

6. Unusual Inheritance Previous analysis on a Rop9 mutant corn ear had more wild type kernels Previous analysis on a Rop9 mutant corn ear had more wild type kernels Also the kernels were distributed with more mutants at the top Also the kernels were distributed with more mutants at the top

7. m + + + Less mutants expected if mutation affects pollen

8. Hypothesis Rop9 affects pollen tube growth in silk Rop9 affects pollen tube growth in silk Mutant pollen at a disadvantage competing with wild type to fertilize Mutant pollen at a disadvantage competing with wild type to fertilize

9. Predictions Pollen tubes from Rop9 mutant pollen grains will grow more slowly Pollen tubes from Rop9 mutant pollen grains will grow more slowly If pollen tube growth is affected Rop9 mutants would have a disadvantage at reaching kernels at the bottom of the ear If pollen tube growth is affected Rop9 mutants would have a disadvantage at reaching kernels at the bottom of the ear Short silks Long Silks

10. Experiments There are two ways to test for the role of Rop9 in pollen tube growth There are two ways to test for the role of Rop9 in pollen tube growth Indirectly and Directly Indirectly and Directly

11. The Indirect Test Test through genotyping kernels by PCR Test through genotyping kernels by PCR Test top and bottom of maize ears Test top and bottom of maize ears Genotype kernels at top and bottom Genotype kernels at top and bottom Top Bottom Rop9 Wild type band Rop9 Mutant bands

12. Rop9 Result Totals Ear 5 Ear 2 Ear 3 Ear 4 Ear 1 m1m6 Wild Type vs. Mutant

13. m1m6 Results do not confirm top and bottom prediction Ear 1Ear 2 Ear 3 Ear 4Ear 5

14. The Direct Test Use fresh pollen Microscope imaging to observing pollen tube growth See directly whether mutant pollen grains germinate or grow pollen tubes more slowly than wild-type pollen grains

15. Pollen Germination on Media Light: wild type Dark: mutant

16. Results from Direct Test I Percent Germination

17. Results from Direct Test II Pollen Tube Length 1 Hour

18. Summary Rop9 has an effect on pollen’s ability to carry out fertilization Rop9 has an effect on pollen’s ability to carry out fertilization However my results do not confirm a role for Rop9 in pollen tube growth However my results do not confirm a role for Rop9 in pollen tube growth

19. Future Research Initial hypothesis: Pollen Tube Growth Possibly may have an effect on late events Fertilization?

20. Acknowledgements Ernest and Pauline Jaworski Fund Ernest and Pauline Jaworski Fund Howard Hughes Medical Institute Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Kevin Ahern Dr. John Fowler Dr. John Fowler Zuzana Vejlupkova Zuzana Vejlupkova Rex Cole Rex Cole Kirsten Carol Kirsten Carol Darin Olson Darin Olson