The role of the phytohormone gibberellic acid in maize embryo maturation Sarah Hill-Skinner Mentor: Dr. Carol Rivin

1. The role of the phytohormonegibberellic acid in maize embryo maturation Sarah Hill-Skinner Mentor: Dr. Carol Rivin

2. Maize: An Economically Important Grain Annual grasses like maize,wheat, & rice are critically important for: • Food • Feed • Biofuel (Ethanol) 2008 US maize productionworth $47.4 billion Maize http://www.flickr.com/photos/craftybohemian/1607735114

3. Maize Embryo Maturation Embryo maturation: • Cell growth • Sequestering of nutrients • Desiccation tolerance • Seed dormancy Embryo maturation From poster “Interplay of ABA and GA in maize embryos” 2 3 4 5 6Morphological Stage mature embryo

4. Maize Embryo Maturation Without embryo maturation: • Desiccation intolerant = dries up • No food reserves • Seed ≠ viable Yield lost! Desiccated corn kernels http://www.agry.purdue.edu/ext/corn/news/articles.99/990903-images.html

5. Maize Embryo Maturation:Hormones GA concentration ABA concentration Abscisic acid (ABA): • Promotes embryo maturation viastorage molecule creation Gibberellins (GA’s): • Promote seed germination inmature embryos via storage molecule breakdown • Unknown function during maturation Embryo hormone levels From poster “Interplay of ABA and GA in maize embryos”

6. Maize Embryo Maturation:Hormone Mutants Embryo deficient in ABA: • Germinates (no maturation) • Desiccation intolerant Embryo deficient in ABA & GA: • Matures normally Embryo deficient in GA: • Matures normally • What does GA do? Premature germination http://www.maizegdb.org/cgi-bin/locusvarimages.cgi?id=12731

7. What is GA’s role in embryo development? • What genes are modulated by GA during embryo development? • How do GA & ABA signaling interact? Hypothesis: GA regulates breakdown of storage molecules created through energy metabolism. Therefore, GA may exert control over energy metabolism by regulating key enzymes.

8. Experimental Strategy Compare gene expression in GA- v. WT embryos – Microarray Assign protein functions to GA-regulated genes – Gene Ontology (GO) Identify GA-affected metabolic processes – GO & Gramene’sOmics Viewer Summarize GA’s impact: integrate GA-affected metabolic processes – KEGG charts

9. Comparing Gene Expression Microarray – what is it? • Chip embedded with 100’s of 1000’s of short DNA sequences (“probes”) to which cDNA hybridizes • Each probe sequence represents a different gene • Gene names provided separately • Determines which genes expressed in a cell type Arrangement of probes on a microarray http://www.charite.de/molbiol/bioinf/tumbiol/Microarrayanalysis/Introduction/index.html

10. Comparing Gene Expression Two-color Microarrays – how to use them: • Collect cDNA from two samples of interest • Label cDNA with fluorescent molecules • cDNA 1 = red, cDNA2 = green • Wash microarray chip with combined labeled cDNA • cDNA hybridizes to complementary sequences on chip • Scan chip with machine Hybridization of labeled cDNA to probes http://biomath.stanford.edu/oligo_bar_code_tag_sequences.html

11. Comparing Gene Expression • Yellow dots = genes expressed equally in both samples • Dot intensity – Brighter dots = higher gene expression • Software measures color/intensity & estimates expression levels • Microarray – interpreting the scan: • Red or green dots = genes expressed almost exclusively in one sample Microarray chip scan From poster “Interplay of ABA and GA in maize embryos: modulation of transcriptome profiles and developmental fate”

12. Comparing Gene Expression Our comparisons of 4 genotypes: • Identified genes significantly differentially expressed (SDE) in/between our mutant embryos GA-v. WT SDE genes annotated with Gene IDs = 3906 ABA- = ABA deficient GA- = GA deficient ABA- /GA- = ABA & GAdeficient WT = wild-type Microarray comparisons From poster “Interplay of ABA and GA in maize embryos”

13. Assigning Protein Functions Gene Ontology – what is it? • From GeneOntology.org: “… a controlled vocabulary to describe gene and gene product attributes in any organism.” • Composed of “GO terms” • Shorthand for gene product (protein) characteristics • Ex. “Galactokinaseactivity,” shortened to the GO term GO:0004335 Gene products & their assoc. GO’s From own analysis

14. Assigning Protein Functions & Identifying Affected Processes • Search tool: • Accepts list of gene identifiers (ID’s) • Annotates ID’s with protein function • GO term enrichment: • Accepts list of gene ID’s • Annotates ID’s with GO terms & definitions • Identifies “enriched” GO terms • Interested in enriched terms defined as pathways http://bioinfo.cau.edu.cn/agriGO/

15. Assigning Protein Functions & Identifying Affected Processes # of GA-regulated genes annotated with protein function: • 1587 – about 2/5 of total 3906 Processes impacted by presence of GA in WT: • Glycolysis • Amino acid synthesis/metabolism

16. Identifying Affected Processes Gramene’sOmics Viewer: • Closed Beta • Maintained by OSU’s Jaiswal lab To Use: • Submit tab-delimited file of gene ID’s & associated microarray estimates • Returns special metabolic pathway charts.

17. Identifying Affected Processes Omics Viewer Output http://pathway‐dev.gramene.org/MAIZEFILTERSET/overview-expression-map

18. Identifying Affected Processes Found many enzymes affected by GA in: • Glycolysis & synthesis/metabolism of a variety of amino acids • The TCA cycle, glyoxylate cycle, & Calvin cycle Now we know which processes are affected by GA! But how do these impacts form a bigger picture of GA’s role in maturation? • To find out, constructed a chart integrating all the pathways above!

19. Integrating Affected Processes To create this chart, used KEGG pathway charts of: • Glycolysis • Amino acid synthesis/metabolism pathways • TCA cycle • Glyoxylate cycle • Calvin cycle Example KEGG Chart Modified from http://www.genome.jp/kegg-bin/show_pathway?map00010

20. Integrating Affected Processes For each chart: Determined connections to other pathways Identified GA-regulated enzymes in the pathway (encoded by GA-regulated genes) Estimated overall impact of GA’s presence on pathway Created final visual

21. - Most or all affected enzymes in pathway have down-regulated gene expression • - Most or all affected enzymes in pathway have up-regulated gene expression • - Pathway contains affected enzymes that have both up or down-regulated gene expression • - No enzymes affected in this pathway - Leads to or from a direct product of a cycle (no additional enzymes involved) -number of enzymes affected/number of enzymes detectable by the microarray 1/1, 2/2, 3/3, 4/4 GAFinal Pathway Chart Cysteine THF Glyoxylate Cycle 0/2 Glutamate Glutamine Glycine Serine Proline 6/10 TCA G3P Threonine Fumarate 2/3 Urea Cycle Arginine Isoleucine 10/10 Glycolysis Pyruvate Aspartate Lysine Valine, Leucine G3P Methionine DAHP 5/8 Calvin Cycle Abbreviations: TCA = Citrate Cycle PRPP = Phosphoribosyl pyrophosphate G3P = Glycerate-3P DAHP = 2-Dehydro-3-deoxy-D- arabino-heptonate 7-phosphate Tyrosine, Phenylalanine, Tryptophan PRPP Histidine

22. Interpretations In maturing WT embryos, GA: • Promotes: • Glycolysis • Entry of pyruvate into the TCA cycle • Last ¾ of the TCA cycle • Synthesis of many amino acids • Depresses: • First ¼ of the TCA cycle • Funneling of amino acids into the TCA cycle • Synthesis of glycine and tryptophan from serine TCA Cycle http://thealchemistkitten.files.wordpress.com/2009/11/blaze_tca_cycle.jpg

23. Conclusions In general, via enzymes, GA appears to: • Promote central energy metabolism • Promote amino acid synthesis/metabolism Supports hypothesis! What is the overall impact on embryo physiology? • Possible increase in cell division & growth • Opportunity for future research Original hypothesis: GA may exert control over energy metabolism by regulating key enzymes.

24. Acknowledgments HHMI Program Dr. Carol Rivin Dr. Kevin Ahern Dr. PankajJaiswal & the Jaiswallab Fowler lab