Mutations in Arabidopsis Exocyst Gene AtSEC8
Jennie Hines
Mentor: John Fowler
The Big Picture
• Plant morphology
• Cells have the ability to grow in specialized ways
• What the Fowler lab examines is the mechanism behind the way that cells grow
Arabidopsis leaf cells
The Exocyst• Thought to direct the way in which
cells grow
The exocyst in yeast
• Polarized cell growth in yeast
• An 8 protein complex
Pollen Tubes
• Pollen tubes exhibit polarized growth.
• One question is whether or not the exocyst is important for the pollen tube polarized growth.
Picture thanks to Rex Cole
pollen grain
pollen tube
How does this apply to what I’m doing?
My hypothesis is that the gene that codes for a part of the exocyst in Arabidopsis, AtSEC8, plays a role in the way pollen tubes grow.
I have several different mutant plants, and the mutations may adversely affect AtSEC8.
Prediction: The AtSEC8 mutant plants will have poorly
functioning pollen.
Transmission Defects
A mutation that causes pollen to function poorly also causes a transmission defect.
Pollen grain
Pollen tube
Egg sac
The mutation will be present in the next generation at a lower than expected frequency.
The wild-type gene
T-DNA
T-DNA
Bam!
F
Mutations
• F and G
G
My mutations No defectTransmission defect
5’ 3’
AtSEC8
• Other mutations previously studied• Transmission defects• F and G are in between
Part One:Initial Genotyping
DNA was extracted from the plants and PCR was done to find out which plants had the insert
Primers
From http://signal.salk.edu/tdnaprimers.html
LBb1
R F
T-DNA
Genome
~900 bp
~600 bp
900 bp
600 bp
Heterozygote Homozygous mutant
Homozygous wildtype
Results of Initial Genotyping
Homozygous mutants
Heterozygous mutants
Homozygous wild-type
F Plants 5 4 5
G Plants 1 6 10
Part Two:Crosses
The plants were crossed to see if the next generation had the mutation
Two Types of Crosses
• Self pollination• Backcrosses with
wildtypeTeeny tiny seeds that were planted one by one
Pretty Wild
From the AtSec8 “G” mutation self-cross
A Transmission Defect in the “G” Mutant
Homozygous mutants
Heterozygous mutants
Homozygous wildtype
F expected 5.5 11 5.5F Self-cross 3 11 8G expected 12 24 12
G Self-cross 4 15 29
SIGNIFICANTLY DIFFERENT - p < 0.001
F
Location Matters
G
No defectTransmission defect
5’ 3’
AtSEC8
5’ end have 0% transmission.3’ end have 100% transmission.G is located between with 33% transmission.
Conclusions
Since there was a transmission defect, it can be concluded that my “G” mutation in AtSEC8 likely causes problems with pollen tube growth.
A New Hypothesis:The 3’ end of the gene (encoding the protein
C-terminal end) is not critical for exocyst function.
What next?
• Genotype the backcrosses
• Study the pollen in media
• Look for phenotypic differences – Roots
– Size of plants
Preliminary Results
Small homozygous mutantG mutant roots 0.3 cm longer
Special Thanks to…
John Fowler, Rex Cole, Zuzana Vejlupkova and Kirstin Arthur.
And to the HHMI program for giving me the opportunity to be
a part of this.
Fin