Transcript The role of the phytohormone gibberellic acid in maize embryo maturation Sarah Hill-Skinner

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
Embryo maturation
From poster “Interplay of ABA and GA in maize embryos”
5
6
Morphological Stage
mature embryo
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
GA concentration
ABA concentration
Embryo hormone levels
From poster “Interplay of ABA
and GA in maize embryos”
mature embryos via storage
molecule breakdown
• Unknown function during maturation
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/cgibin/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 GAaffected 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
− 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_ta
g_sequences.html
Comparing Gene Expression
Microarray – interpreting the scan:
• Red or green dots = genes expressed almost
exclusively in one sample
• Yellow dots = genes expressed
equally in both samples
• Dot intensity – Brighter dots =
higher gene expression
• Software measures color/intensity
& estimates expression levels
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
GA- v. WT SDE genes annotated
with Gene IDs = 3906
ABA- = ABA deficient
GA- = GA deficient
ABA- /GA- = ABA & GA
deficient
WT = wild-type
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
http://bioinfo.cau.edu.cn/agriGO/
• 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
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
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.
Identifying Affected Processes
Omics Viewer Output
http://pathway‐dev.gramene.org/MAIZEFILTERSET/overviewexpression-map
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!
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/keggbin/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
- Most or all affected enzymes in pathway have downregulated gene expression
- No enzymes affected in this pathway
- Leads to or from a direct product of a cycle (no additional
enzymes involved)
- Most or all affected enzymes in pathway have upregulated gene expression
1/1, 2/2,
- number of enzymes affected/number of enzymes
3/3, 4/4 detectable by the microarray
- Pathway contains affected enzymes that have both up
or down-regulated gene expression
GA Final Pathway Chart
THF
Cysteine
Glycine
Serine
Threonine
G3P
Isoleucine
Glyoxylate
0/2 Cycle
6/10
10/10 Glycolysis
TCA
Fumarate
5/8
Tyrosine,
Phenylalanine,
Tryptophan
Aspartate
Valine, Leucine
Calvin Cycle
PRPP
2/3 Urea Cycle
Arginine
Pyruvate
G3P
DAHP
Glutamate
Glutamine
Proline
Histidine
Lysine
Methionine
Abbreviations:
TCA = Citrate Cycle
PRPP = Phosphoribosyl
pyrophosphate
G3P = Glycerate-3P
DAHP = 2-Dehydro-3-deoxy-Darabino-heptonate 7phosphate
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
TCA Cycle
http://thealchemistkitten.files.wordpress.co
m/2009/11/blaze_tca_cycle.jpg
• Depresses:
– First ¼ of the TCA cycle
– Funneling of amino acids into the TCA cycle
– Synthesis of glycine and tryptophan from serine
Conclusions
In general, via enzymes, GA appears to:
• Promote central energy metabolism
• Promote amino acid synthesis/metabolism
Supports hypothesis!
Original hypothesis: GA may exert control
over energy metabolism by regulating key
enzymes.
What is the overall impact on embryo physiology?
• Possible increase in cell division & growth
– Opportunity for future research
Acknowledgments
HHMI Program
Dr. Carol Rivin
Dr. Kevin Ahern
Dr. Pankaj Jaiswal & the Jaiswal lab
Fowler lab