1. Testing the Role of Conserved Genes in Pollen Development Luisa Snyder Mentor: Dr. John Fowler Lab Mentor: Dr. Rex Cole Dept. Botany and Plant Pathology Summer 2008

2. Pollen The way that flowering plants undergo sexual reproduction Allows exchange of gametes among plants sols.unlv.edu/Schulte/Anatomy/Repro/Repro.html specialcomment.wordpress.com/2007/04/

3. en.wikivisual.com/index.php/Sporopollenin Pollen Shape Diversity

4. www.umanitoba.ca/.../Lab8/biolab8_1.html www.yugatech.com/photos/?a=flowers&i=672 www.simple- expressions.org/2007_12_01_archive... 90ways.com/sciarchive/sci25.php www.answers.com/topic/maize Despite diversity there are many common pollen characteristics across species

5. Project Purpose To identify genes involved in the development and function of pollen. Potentially may lead to: - Improved crops - Containment for genetically engineered plants staffwww.fullcoll.edu/tmorris/an_inconvenient... www.coextra.eu/country_reports/news1018_en.html

6. Quartet Pollen +/m Heterozygous +/+ Wild Type Male Gametophyte Life Cycle Mitosis

7. Arabidopsis thaliana http://www.prep.biotech.vt.edu/expinfo/expinfo_anatomy.html

8. Hypothesis A core set of pollen genes provide important functions in all angiosperm pollen. www.physorg.com/news117381801.html www.gramene.org/species/zea/maize_intro.html www.botanical.com/botanical/mgmh/r/rice--15.html Arabidopsis Rice Maize

9. Predictions 1.The genes in the pollen core set should be: - Conserved and highly expressed in pollen across Arabidopsis, rice, and maize. 2. Mutation in the genes in the pollen core set should cause a pollen defect. Arabidopsis Pollen Rice PollenMaize Pollen epigenome.eu/en/3,41,0www.lec.lancs.ac.uk/ccm/ed.htm www.biomedcentral.com/1471-2229/5/22/figure/F1

10. Ancient Gene Arabidopsis gene Rice gene Maize gene Orthologous genes Pollen Genes Criteria Putative Orthologous Group (POG) Highly expressed in Arabidopsis, maize, and rice pollen Mutant gene is available No duplicate genes in Arabidopsis

11. Candidate Pollen Genes GenesFunction At1g64110ATPase family protein At5g17290Autophagy protein ATG5 At5g20690Transmembrane protein kinase At1g23540Protein kinase At1g20080C2 domain-containing protein At3g03900Adenylylsulfate kinase At1g47380Protein phosphatase 2C-related At4g39080Vacuolar proton ATPase subunit VHA-a isoform

12. Experimental Approach and Methods Determine if there is a phenotypic defect due to mutation in the candidate genes. Determine if there is a transmission defect due to a mutation in the candidate genes. www.sparknotes.com/.../lifecycle/section2.rhtml Microscopy PCR www.molecularstation.com/molecular-biology-im... Gel Electrophoresis www.territorioscuola.com/wikipedia

13. At5g17290- Autophagy protein ATG5 Testing for Phenotypic Defect At5g20690- Transmembrane protein kinase

14. Results of Pollen Phenotypic Defect GenesFunctionDefect in Pollen Grain Defect in Pollen tube At1g64110ATPase family proteinNone observed Occasional branching At5g17290Autophagy protein ATG5None observed Occasional Branching At5g20690Transmembrane protein kinase None observed None observed At1g23540Protein kinaseNone observed None observed

15. Reciprocal Crosses : +/m stigma+/+ pollen +/m pollen +/+ stigma Testing for Transmission Defect

16. Transmission Defect +/+ +/m + m ++ Stigma Pollen Half wild type plants Half heterozygous plants Mostly wild type plants No Transmission Defect Prediction of the Results

17. Genotyping by PCR At1g64110- ATPase family protein At1g64110 Wild Type Heterozygous

18. Example: Example: PCR Test for Transmission Defect Frequency: 12 Heterozygous 12 Wild type 1:1 = No transmission defect At1g64110- ATPase family protein Controls

19. GenesFunctionDefect in Pollen At1g64110ATPase family proteinNone observed At5g17290Autophagy protein ATG5None observed At5g20690Transmembrane protein kinase None observed At1g23540Protein kinaseNone observed Summary: Transmission Defect Experiments* * At least 18 plants tested for each gene

20. Hypothesis A core set of pollen genes provide important functions in all angiosperm pollen. Prediction Mutation in the genes in the pollen core set should cause a pollen defect. Possible Explanations - Unrecognized duplicate genes - 3 out of 4 have likely duplicates Conclusions-So Far Observation - Not enough candidate genes tested None of my four candidate genes showed a defect - Transmission defect might be mild - Not enough plants

22. Reciprocal Crosses : +/m stigma +/+ pollen +/m pollen +/+ stigma Testing Transmission Defect Female Gametophyte

23. (An Unexpected) Female-Specific Transmission Defect ! At5g17290- Autophagy protein ATG5 Using heterozygous pollen 9:9 Using heterozygous stigma 9:20 WT Band Mut Band   = 4.17 p<0.05Statistically Significant! Controls Het : WT

24. Future Research Currently working with four more candidate genes Follow up on pollen tube branching phenotype in the genes At1g64110 and At5g17290 Test the next generation for the female specific transmission defect in the gene At5g17290 - If confirmed, determine the phenotypic defect in the female gametophyte

25. Acknowledgements Howard Hughes Medical Institute Dr. Kevin Ahern Dr. John Fowler Dr. Rex Cole Fowler Lab: Zuzana Vejlupkova Nathan Snyder Dr. Maria Ivanchenko Dr. Lol Cooper Sierra Wolfenbarger