Testing a Role for Rop9
GTPase in Maize Pollen
Tube Growth
Lauren A. Osborn
Dr. John Fowler
Botany and
Plant Pathology
Summer 2005
Pollen Tube Growth

Pollination and pollen tube growth is
essential



fertilization
seed production in plants
Two phases for pollen tube growth in
Maize


Germination
Growth in the silk
Germination
Pollen Tubes
Pollen
Grains
Mature Adult
Silk
and
Egg
Fertilization
Seedlings
Seed
Rop Genes



Nine different Rop genes in maize
ROP GTPases are proteins that act as a signaling
molecule (example: pollen tube growth)
Rop2 mutant
pollen has a
reduced ability
to fertilize
My Research: Rop9 gene
ATG
Unusual Inheritance


Previous analysis on a
Rop9 mutant corn ear
had more wild type
kernels
Also the kernels were
distributed with more
mutants at the top
m
+
Mutant
Pollen
m/+
m/+
Wild type
+/+
Corn ear
+/+
Less mutants expected if mutation affects pollen
+
+
Hypothesis

Rop9 affects pollen tube growth in silk

Mutant pollen at a disadvantage
competing with wild type to fertilize
Predictions


Pollen tubes from Rop9
mutant pollen grains will
grow more slowly
If pollen tube growth is
affected Rop9 mutants
would have a disadvantage
at reaching kernels at the
bottom of the ear
Short silks
Long Silks
Experiments

There are two ways to test for the role of
Rop9 in pollen tube growth

Indirectly and Directly
The Indirect Test


Test through genotyping kernels
by PCR
Test top and bottom of maize ears

Top
Genotype kernels at top and bottom
Rop9
Wild type
band
Rop9 Mutant
bands
Bottom
Rop9 Result Totals
Number of samples
WildComparison
Type vs. Mutant
40
35
30
25
20
15
10
5
0
mutant
Ear 1
wildtype
Ear 2
Ear 3
m2
m2
m2
Mutations
Ear 4
Ear 5
m1
m6
m1
m6
Top and Bottom Comparison
Percentage Totals
60
50
40
30
20
10
Ear 1
Ear 2
m2
m2
Ear 3
Ear 4
Ear 5
m2
m1
m1
m6
m6
0
Mutants
% mutant top
% mutant bottom
Results do not
confirm top and
bottom prediction
The Direct Test



Use fresh pollen
Microscope imaging to
observing pollen tube
growth
See directly whether
mutant pollen grains
germinate or grow
pollen tubes more slowly
than wild-type pollen
grains
Pollen Germination on Media
Light: wild type
Dark: mutant
Results from Direct Test I
Percent Germination
Percent
Germination
40
Percent Germination
35
30
25
Light
Dark
20
15
10
5
0
Mutant 1
Mutant 2
Wild-type
Results from Direct Test II
Pollen
Length
PollenTube
Tube Length
1 Hour
450
Pollen Tube Length in Microns
400
350
300
250
Light
Dark
200
150
100
50
0
Mutant 1
Mutant 2
Summary


Rop9 has an effect on
pollen’s ability to carry
out fertilization
However my results do
not confirm a role for
Rop9 in pollen tube
growth
Future Research
Initial hypothesis:
Pollen Tube Growth
Possibly may have an
effect on late events
Fertilization?
Acknowledgements








Ernest and Pauline Jaworski Fund
Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. John Fowler
Zuzana Vejlupkova
Rex Cole
Kirsten Carol
Darin Olson