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
• An 8 protein complex
• Polarized cell growth in yeast
The exocyst in yeast
Pollen Tubes
• Pollen tubes exhibit
polarized growth.
• One question is
whether or not the
exocyst is important
for the pollen tube
polarized growth.
pollen grain
pollen tube
Picture thanks to Rex Cole
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
Pollen grain
Pollen tube
A mutation that causes pollen to
function poorly also causes a
transmission defect.
The mutation will be present in
the next generation at a lower
than expected frequency.
Egg sac
T-DNA
T-DNA
Bam!
The wild-type gene
Mutations
•
•
•
•
F and G
Other mutations previously studied
Transmission defects
F and G are in between
AtSEC8
G
5’
3’
F
Transmission defect
My mutations No defect
Part One:
Initial Genotyping
DNA was extracted from the plants and PCR
was done to find out which plants had the insert
Primers
T-DNA
LBb1
~600 bp
Genome
R
F
~900 bp
From http://signal.salk.edu/tdnaprimers.html
900 bp
600 bp
Heterozygote
HomozygousHomozygous
mutant
wildtype
Results of Initial Genotyping
Homozygous
mutants
Heterozygous Homozygous
mutants
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
Teeny tiny seeds that were planted one by one
• Self pollination
• Backcrosses with
wildtype
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
5.5
F Self-cross
3
11
8
G expected
12
24
12
G Self-cross
4
15
29
SIGNIFICANTLY DIFFERENT -
p < 0.001
Location Matters
5’ end have 0% transmission.
3’ end have 100% transmission.
G is located between with 33%
transmission.
AtSEC8
G
5’
3’
F
Transmission defect
No defect
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
G mutant roots 0.3 cm longer
Small homozygous mutant
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