Testing the Role of Conserved
Genes in Pollen Development
Luisa Snyder
Mentor: Dr. John Fowler
Lab Mentor: Dr. Rex Cole
Dept. Botany and Plant Pathology
Summer 2008
Pollen
The way that flowering plants undergo
sexual reproduction
 Allows exchange of gametes among
plants

sols.unlv.edu/Schulte/Anatomy/Repro/Repro.html
specialcomment.wordpress.com/2007/04/
Pollen Shape Diversity
en.wikivisual.com/index.php/Sporopollenin
www.umanitoba.ca/.../Lab8/biolab8_1.html
www.yugatech.com/photos/?a=flowers&i=672
90ways.com/sciarchive/sci25.php
www.answers.com/topic/maize
Despite diversity there are
many common pollen
characteristics across
species
www.simpleexpressions.org/2007_12_01_archive...
Project Purpose

To identify genes involved
in the development and
function of pollen.

Potentially may lead to:
- Improved crops
- Containment for
genetically engineered
plants
staffwww.fullcoll.edu/tmorris/an_inconvenient...
www.coextra.eu/country_reports/news1018_en.html
Male Gametophyte Life Cycle
+/m Heterozygous
+/+ Wild Type
Quartet Pollen
Arabidopsis thaliana
http://www.prep.biotech.vt.edu/expinfo/expinfo_anatomy.html
Hypothesis
A core set of pollen genes provide
important functions in all angiosperm
pollen.
Rice
Maize
Arabidopsis
www.physorg.com/news117381801.html
www.gramene.org/species/zea/maize_intro.html
www.botanical.com/botanical/mgmh/r/rice--15.html
Predictions
1. The genes in the pollen core set should be:
- Conserved and highly expressed in pollen
across Arabidopsis, rice, and maize.
2. Mutation in the genes in the pollen core set
should cause a pollen defect.
Arabidopsis Pollen
www.biomedcentral.com/1471-2229/5/22/figure/F1
Rice Pollen
epigenome.eu/en/3,41,0
Maize Pollen
www.lec.lancs.ac.uk/ccm/ed.htm
Pollen Genes Criteria

Putative Orthologous Group
(POG)
Ancient Gene
Highly expressed in
Arabidopsis, maize, and
rice pollen

Arabidopsis
gene
Rice
gene
Mutant gene is available
No duplicate genes in
Arabidopsis
Orthologous
genes
Maize
gene
Candidate Pollen Genes
Genes
Function
At1g64110
ATPase family protein
At5g17290
Autophagy protein ATG5
At5g20690
Transmembrane protein kinase
At1g23540
Protein kinase
At1g20080
C2 domain-containing protein
At3g03900
Adenylylsulfate kinase
At1g47380
Protein phosphatase 2C-related
At4g39080
Vacuolar proton ATPase subunit
VHA-a isoform
Experimental Approach and Methods
Microscopy
Determine if there is a
phenotypic defect due to
mutation in the candidate
genes.
www.sparknotes.com/.../lifecycle/section2.rhtml
Determine if there is
a transmission
defect due to a
mutation in the
candidate genes.
PCR
Gel
Electrophoresis
www.molecularstation.com/molecular-biology-im...
www.territorioscuola.com/wikipedia
Testing for Phenotypic Defect
At5g17290- Autophagy protein ATG5
At5g20690- Transmembrane protein kinase
Results of Pollen Phenotypic Defect
Genes
Function
Defect in
Pollen
Grain
Defect in
Pollen tube
At1g64110
ATPase family protein
None
observed
Occasional
branching
At5g17290
Autophagy protein ATG5
None
observed
Occasional
Branching
At5g20690
Transmembrane protein
kinase
None
observed
None
observed
At1g23540
Protein kinase
None
observed
None
observed
Testing for Transmission Defect
Reciprocal Crosses:
+/+ stigma
+/m pollen
+/m stigma
+/+ pollen
Prediction of the Results
Defect
NoTransmission
Transmission
Defect
+
Pollen
Stigma
+
+
+/+
+/+
m
+/m
+/m
Half wild type plants
Mostly wild type
plants
Half heterozygous
plants
Genotyping by PCR
At1g64110- ATPase family protein
Wild Type
Heterozygous
At1g64110
Example: PCR Test for Transmission Defect
At1g64110- ATPase family protein
Controls
Frequency:
12 Heterozygous
12 Wild type
1:1 = No
transmission
defect
Summary: Transmission
Defect Experiments*
Genes
Function
Defect in
Pollen
At1g64110
ATPase family protein
None
observed
At5g17290
Autophagy protein ATG5
None
observed
At5g20690
Transmembrane protein
kinase
None
observed
At1g23540
Protein kinase
None
observed
* At least 18 plants tested for each gene
Conclusions-So Far
Hypothesis
A core set of pollen genes provide important functions in all
angiosperm pollen.
Prediction
Mutation in the genes in the pollen core set should
cause a pollen defect.
Observation
None of my four candidate genes showed a defect
Possible Explanations
- Transmission defect might be mild
- Not enough plants
- Unrecognized duplicate genes
- 3 out of 4 have likely duplicates
- Not enough candidate genes tested
Testing Transmission Defect
Reciprocal Crosses:
+/+ stigma
+/m pollen
+/m stigma
+/+ pollen
Female
Gametophyte
(An Unexpected) Female-Specific
Transmission Defect !
At5g17290- Autophagy protein ATG5
Controls
Using
heterozygous
pollen
9:9
Het : WT
WT Band
Mut Band
Controls
WT Band
Mut Band
c2 = 4.17
p<0.05
Using
heterozygous
stigma
9:20
Het : WT
Statistically Significant!
Future Research
Currently working with four more candidate genes
Follow up on pollen tube branching phenotype in
the genes At1g64110 and At5g17290
Test the next generation for the female specific
transmission defect in the gene At5g17290
- If confirmed, determine the phenotypic
defect in the female gametophyte
Acknowledgements
Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. John Fowler
Dr. Rex Cole
Fowler Lab:
Zuzana Vejlupkova
Nathan Snyder
Dr. Maria Ivanchenko
Dr. Lol Cooper
Sierra Wolfenbarger