Presentation on theme: "The role of the phytohormone gibberellic acid in maize embryo maturation Sarah Hill-Skinner Mentor: Dr. Carol Rivin."— Presentation transcript:
The role of the phytohormone gibberellic acid in maize embryo maturation Sarah Hill-Skinner Mentor: Dr. Carol Rivin
Maize: An Economically Important Grain Annual grasses like maize, wheat, & rice are critically important for: Food Feed Biofuel (Ethanol) 2008 US maize production worth $47.4 billion Maize http://www.flickr.com/photos/craftybohemian/1607735114
Maize Embryo Maturation Embryo maturation: Cell growth Sequestering of nutrients Desiccation tolerance Seed dormancy 1 2 3 4 5 6 Morphological Stage mature embryo Embryo maturation From poster “Interplay of ABA and GA in maize embryos”
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.99/99 0903-images.html
Maize Embryo Maturation: Hormones Abscisic acid (ABA): Promotes embryo maturation via storage molecule creation Gibberellins (GA’s): Promote seed germination in mature embryos via storage molecule breakdown Unknown function during maturation GA concentration ABA concentration Embryo hormone levels From poster “Interplay of ABA and GA in maize embryos”
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
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.
Experimental Strategy 1. Compare gene expression in GA - v. WT embryos – Microarray 2. Assign protein functions to GA-regulated genes – Gene Ontology (GO) 3. Identify GA-affected metabolic processes – GO & Gramene’s Omics Viewer 4. Summarize GA’s impact: integrate GA- affected metabolic processes – KEGG charts
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/Mic roarrayanalysis/Introduction/index.html
Comparing Gene Expression Two-color Microarrays – how to use them: Collect cDNA from two samples of interest Label cDNA with fluorescent molecules Hybridization of labeled cDNA to probes http://biomath.stanford.edu/oligo_bar_code_ta g_sequences.html −cDNA 1 = red, cDNA2 = green Wash microarray chip with combined labeled cDNA −cDNA hybridizes to complementary sequences on chip Scan chip with machine
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”
Comparing Gene Expression Our comparisons of 4 genotypes: Identified genes significantly differentially expressed (SDE) in/between our mutant embryos ABA - = ABA deficient GA - = GA deficient ABA - /GA - = ABA & GA deficient WT = wild-type GA - v. WT SDE genes annotated with Gene IDs = 3906 Microarray comparisons From poster “Interplay of ABA and GA in maize embryos”
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. “Galactokinase activity,” shortened to the GO term GO:0004335 Gene products & their assoc. GO’s From own analysis
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/
# 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 Assigning Protein Functions & Identifying Affected Processes
Identifying Affected Processes Gramene’s Omics 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.
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!
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
Integrating Affected Processes For each chart: 1. Determined connections to other pathways 2. Identified GA-regulated enzymes in the pathway (encoded by GA-regulated genes) 3. Estimated overall impact of GA’s presence on pathway Created final visual
GA Final Pathway Chart Glycolysis 10 /10 Tyrosine, Phenylalanine, Tryptophan Pyruvate SerineGlycine Threonine Isoleucine THF Cysteine Glutamate Proline Glutamine Fumarate Arginine AspartateLysine Methionine Histidine Valine, Leucine Abbreviations: TCA = Citrate Cycle PRPP = Phosphoribosyl pyrophosphate G3P = Glycerate-3P DAHP = 2-Dehydro-3-deoxy-D- arabino-heptonate 7- phosphate TCA 6 /10 DAHP PRPP Glyoxylate Cycle 0/2 G3P Calvin Cycle 5 /8 Urea Cycle 2 /3 - 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 - 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
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.co m/2009/11/blaze_tca_cycle.jpg
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.
Acknowledgments HHMI Program Dr. Carol Rivin Dr. Kevin Ahern Dr. Pankaj Jaiswal & the Jaiswal lab Fowler lab