Testing the Role of Testing the Role of Conserved Genes in Conserved Genes in Pollen DevelopmentPollen Development

Luisa SnyderMentor: Dr. John FowlerLab Mentor: Dr. Rex Cole

Dept. Botany and Plant PathologySummer 2008

PollenPollenThe way that flowering plants

undergo sexual reproductionAllows exchange of gametes

among plants

sols.unlv.edu/Schulte/Anatomy/Repro/Repro.html

specialcomment.wordpress.com/2007/04/

en.wikivisual.com/index.php/Sporopollenin

Pollen Shape Diversity

www.umanitoba.ca/.../Lab8/biolab8_1.htmlwww.yugatech.com/photos/?a=flowers&i=672

www.simple-expressions.org/2007_12_01_archive...

90ways.com/sciarchive/sci25.php

www.answers.com/topic/maize

Despite diversity there are many common pollen characteristics across

species

Project PurposeProject 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

Quartet Pollen+/m Heterozygous+/+ Wild Type

Male Gametophyte Life CycleMale Gametophyte Life Cycle

Mitosis

Arabidopsis thalianaArabidopsis 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.

www.physorg.com/news117381801.html www.gramene.org/species/zea/maize_intro.html

www.botanical.com/botanical/mgmh/r/rice--15.html

Arabidopsis

RiceMaize

Predictions1. 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 Rice Pollen Maize Pollen

epigenome.eu/en/3,41,0 www.lec.lancs.ac.uk/ccm/ed.htm www.biomedcentral.com/1471-2229/5/22/figure/F1

Ancient Gene

Arabidopsis gene

Rice gene

Maize gene

Orthologous genes

Pollen Genes Criteria

Putative Orthologous Group (POG)

Highly expressed in Arabidopsis, maize, and rice pollen

Mutant gene is available

No duplicate genes in Arabidopsis

Candidate Pollen GenesCandidate 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

Determine if there is a phenotypic defect due to mutation in the candidate genes.

Determine if there is a transmission defect due to a mutation in the candidate genes.

www.sparknotes.com/.../lifecycle/section2.rhtml

Microscopy

PCR

www.molecularstation.com/molecular-biology-im...

Gel Electrophoresis

www.territorioscuola.com/wikipedia

At5g17290- Autophagy protein ATG5

Testing for Phenotypic Defect

At5g20690- Transmembrane protein kinase

Results of Pollen Phenotypic DefectGenes 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

Noneobserved

Noneobserved

At1g23540 Protein kinase Noneobserved

Noneobserved

Reciprocal Crosses:

+/m stigma +/+ pollen

+/m pollen+/+ stigma

Testing for Transmission Defect

Transmission DefectTransmission Defect

+/+ +/+

+/m +/m

+

m

+ +Stigma

Pollen

Half wild type plants

Half heterozygous plants

Mostly wild type plants

No Transmission Defect

Prediction of the Results

Genotyping by PCRGenotyping by PCRAt1g64110- ATPase family protein

At1g64110

Wild Type Heterozygous

ExampleExample: : PCR Test for Transmission Defect

Frequency:12 Heterozygous12 Wild type

1:1 = No transmission

defect

At1g64110- ATPase family protein

Controls

Genes Function Defect in Pollen

At1g64110 ATPase family protein

Noneobserved

At5g17290 Autophagy protein ATG5

Noneobserved

At5g20690 Transmembrane protein kinase

Noneobserved

At1g23540 Protein kinase None observed

Summary: Transmission Defect Experiments*

* At least 18 plants tested for each gene

HypothesisHypothesisA core set of pollen genes provide important functions in all angiosperm pollen.

PredictionPrediction Mutation in the genes in the pollen core set should

cause a pollen defect.

Possible ExplanationsPossible Explanations

- Unrecognized duplicate genes

- 3 out of 4 have likely duplicates

Conclusions-So Far

ObservationObservation

- Not enough candidate genes tested

None of my four candidate genes showed a defect

- Transmission defect might be mild

- Not enough plants

Reciprocal Crosses:

+/m stigma +/+ pollen

+/m pollen+/+ stigma

Testing Transmission Defect

Female Gametophyte

(An Unexpected) Female-Specific Transmission Defect !

At5g17290- Autophagy protein ATG5

Using heterozygous

pollen 9:9

Using heterozygous

stigma9:20

WT Band

WT Band

Mut Band

Mut Band

= 4.17

p<0.05 Statistically Significant!

Controls

Controls

Het : WT

Het : WT

Future ResearchFuture ResearchCurrently 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