ROP GTPASE INVOLVEMENT WITH
MAIZE POLLEN TUBE FUNCTION
Nathan Snyder
Dr. John Fowler
Dept. Botany and Plant Pathology
10/07/07
Pollen Tube
• Pollen grain germination and
growth is important for:
Fertilization
Pollen
Grain
Seed production
Pollen
Tube
• Certain genes are involved in
germination and pollen tube function
Purpose
Explore pollen tube growth in maize plants to
learn more about:
• Regulation of plant cell signal transduction
• Cell morphogenesis
Stomata Cell
Trichome Cell
Pollen Grain
Maize plant
Advantage:
• Male and female
floral organs are
separate.
Disadvantage:
• Long life cycle
of 5 to 6 months.
Female Ear
Male Tassel
Maize Plant
• Technical advantages:
Pollen marker: waxy
Pollen Tube Growth
Pollen Tube Growth
Certain genes encode proteins to help regulate
pollen tube growth.
•
•
•
•
•
rop subfamily
GTP binding proteins
9 rop genes in maize
Known: rop2 is involved in pollen tube growth
My focus: rop9
rop9 and rop2
• Ancient duplicate gene
• 97% nucleotide
• 99% amino acid
• Is rop9 similar to rop2 in function?
Experimental Approach
• Use a mutant to determine gene function.
Broken
Not broken
Alternative Hypotheses
1. rop9 plays a crucial function in pollen germination
and tube growth.
2. rop9 has nothing to do with pollen germination and
tube growth.
3. rop9 provides a redundant function for rop2
(provides backup).
Methods
• PCR
• Microscopy
PCR: rop9 gene
rop9 Wild Type Product
rop9 Mutant Products
Genotyping by PCR
Electrophoresis Gel
rop9 Wild Type
rop9
heterozygote
Microscopy
(Pollen Marker)
Stained-black:
rop9 mutant
Genotypes of
rop9:
50 % Mutant:
rop9-mu
50% Wild
Type:
ROP9 +
Non-stained:
wild type rop9
Experimental Protocol
1 rop9 heterozygous plant
1 rop2 heterozygous (positive control) plant
Shedding
Plating
Iodine
Staining
Microscopy
Pollen Germination Experiment
rop9 Heterozygote
80
80
70
70
percent germinated
percent germinated
rop2 Heterozygote
(Postive Control)
60
50
40
30
20
10
ROP2 +
rop2-mu
0
60
50
40
30
20
10
0
Clear
Black
ROP9 +
rop9-mu
Combined Data: Germination
Relative Pollen Tube Germination Rate
1.2
(Mutant / Wild-type)
1
0.8
rop9-mu
0.6
0.4
0.2
0
rop2-mu
Pollen Tube Growth Experiment
rop9 Heterozygote Measurements
rop2 (Positive Control)
Heterozygote Measurements
120
100
80
60
ROP2 +
40
20
0
rop2-mu
average length (um)
average length (um)
120
100
80
60
40
20
0
ROP9 +
rop9-mu
Combined Data:
Pollen Tube Growth
Relative Pollen Tube Growth Rate
1.4
(Mutant / Wild-type)
1.2
1
0.8
0.6
rop2-mu
0.4
0.2
0
•No significant differences
rop9-mu
Conclusion: rop9 Experiment
1. rop9 plays a crucial function in pollen
germination and tube growth.
X
?
2. rop9 has nothing to do with pollen
germination and tube growth.
? 3.
rop9 provides a redundant function for
rop2 (provides backup).
Double Mutant Experiments
• Similar procedure using a
double mutant plant
Parent Plant
Pollen
Grains
Double Mutant Germination
Double Mutant Heterozygote
percentage germinated
80
70
60
50
40
rop9-mu
30
ROP2 +
20
10
0
rop9-mu
rop2-mu
Combined Data:
Double Mutant Germination
Double Mutant Germination
Relative Germination Rate
(Mutant / Wild-type)
1.2
1
0.8
rop9-mu
ROP2 +
0.6
0.4
0.2
0
ROP9 +
rop2-mu
rop9-mu
rop2-mu
Double Mutant Pollen Tube Growth
Double Mutant Heterozygote
100
average length (um)
90
80
70
60
50
40
30
20
10
0
rop9-mu
ROP2 +
rop9-mu
rop2-mu
Growth in Double Mutant?
• Waxy pollen marker has a recombination
rate of about 5%
• Therefore, the few black stained pollen
grains that grew tubes may not have been
double mutants
Double Mutant Pollen Tube Growth
Double Mutant Pollen Growth
Relative Growth Rate
(Mutant / Wild-type)
1.4
1.2
1
0.8
rop9-mu
0.6
ROP2 +
0.4
0.2
0
ROP9 +
rop2-mu
rop9-mu
rop2-mu
Conclusion: Double Mutant
X
1. rop9 plays a crucial function in pollen
germination and tube growth.
X 2.
rop9 has nothing to do with pollen
germination and tube growth.
3. rop9 provides a redundant function for
rop2 (provides backup).
Maize pollen with DAPI
• P = pollen grain pore
• Vn = vegetative nucleus
• Sn = sperm nuclei
Acknowledgements
• Howard Hughes Medical Institute
• Dr. Kevin Ahern
• Dr. John Fowler
Fowler Lab:
• Zuzana Vejlupkova
• Rex Cole
• Luisa Snyder
• Maria Ivanchenko
• Ann Ketter
• Viktor Zarsky
Thank You
Work Cited
Pictures:
1. http://plantbiology.stanford.edu/images/imagecollection9.jpg
2. http://www.grdc.com.au/growers/res_upd/north/n05/images/wilson7.jp
g
3. http://labspace.open.ac.uk/file.php/1944/S103_5_005i.jpg
4. http://www.nikonsmallworld.com/images/gallery2003/fourbythree/303
3_Arabidopsis-.jpg
5. http://images.businessweek.com/ss/06/09/consumer_prices/image/corn
.jpg
6. http://www.hpcgears.com/images/brokengear3.png
7. http://chip-art.com/Broken_gear_1.jpg
8. sps.k12.ar.us/massengale/plant_reproduction.htm