Testing a Role for Testing a Role for Rop9Rop9 GTPase in Maize GTPase in Maize Pollen Tube GrowthPollen Tube Growth

Lauren A. OsbornLauren A. OsbornDr. John FowlerDr. John Fowler

Botany and Botany and Plant PathologyPlant Pathology

Summer 2005Summer 2005

Pollen Tubes

Pollen Tube GrowthPollen Tube Growth Pollination and pollen tube growth is Pollination and pollen tube growth is

essential essential fertilization fertilization seed production in plantsseed production in plants

Two phases for pollen tube growth in MaizeTwo phases for pollen tube growth in Maize Germination Germination Growth in the silkGrowth in the silk

Germination

Seed

Seedlings

Fertilization

Pollen Grains

SilkandEgg

Mature Adult

Rop GenesRop Genes Nine different Nine different RopRop genes in maize genes in maize ROP GTPases are proteins that act as a ROP GTPases are proteins that act as a

signaling molecule (example: pollen signaling molecule (example: pollen tube growth)tube growth)

Rop2Rop2 mutant mutant pollen has pollen has a reduced a reduced ability to ability to fertilizefertilize

ATG

My Research: My Research: Rop9Rop9 gene gene

Unusual InheritanceUnusual Inheritance

Previous analysis Previous analysis on a on a Rop9Rop9 mutant mutant corn ear had more corn ear had more wild type kernelswild type kernels

Also the kernels Also the kernels were distributed were distributed with more mutants with more mutants at the topat the top

MutantPollenm/+

m/+ +/+

Wild type+/+

Corn ear

m

+

+

+

Less mutants expected if mutation affects pollen

HypothesisHypothesis Rop9Rop9 affects pollen tube affects pollen tube

growth in silkgrowth in silk Mutant pollen at a Mutant pollen at a

disadvantage competing with disadvantage competing with wild type to fertilizewild type to fertilize

PredictionsPredictions

Pollen tubes from Pollen tubes from Rop9Rop9 mutant pollen mutant pollen grains will grow grains will grow more slowlymore slowly

If pollen tube growth If pollen tube growth is affected is affected Rop9Rop9 mutants would have mutants would have a disadvantage at a disadvantage at reaching kernels at reaching kernels at the bottom of the the bottom of the earear

Short silks

Long Silks

ExperimentsExperiments There are two ways to test for the There are two ways to test for the

role of role of Rop9Rop9 in pollen tube growth in pollen tube growth Indirectly and DirectlyIndirectly and Directly

The Indirect TestThe Indirect Test

Test through genotyping Test through genotyping kernels by PCR kernels by PCR

Test top and bottom of Test top and bottom of maize earsmaize ears Genotype kernels at top and bottom Genotype kernels at top and bottom

Top

Bottom

Rop9 Wild type

bandRop9

Mutant bands

Rop9Rop9 Result Totals Result TotalsComparison

05

10152025303540

m2 m2 m2 m1 m6

Mutations

Number of samples

mutant

wildtype

Comparison

05

10152025303540

m2 m2 m2 m1 m6

Mutations

Number of samples

mutant

wildtype

Ear 5

Ear 2 Ear

3

Ear 4

Ear 1

m1 m6

Wild Type vs. Mutant

Top and Bottom Comparison

0

10

20

30

40

50

60

m2 m2 m2 m1 m6

Mutants

Percentage Totals

% mutant top % mutant bottom

m1 m6

Results do not confirm top and bottom prediction

Results do not confirm top and bottom prediction

Ear 1 Ear 2Ear 3 Ear 4 Ear 5

The Direct TestThe Direct Test Use fresh pollenUse fresh pollen Microscope Microscope

imaging to imaging to observing pollen observing pollen tube growth tube growth

See directly See directly whether mutant whether mutant pollen grains pollen grains germinate or grow germinate or grow pollen tubes more pollen tubes more slowly than wild-slowly than wild-type pollen grainstype pollen grains

Pollen Germination on Pollen Germination on MediaMedia

Light: wild type

Dark: mutant

Results from Direct Test Results from Direct Test II

Percent Germination

0

5

10

15

20

25

30

35

40

Mutant 1 Mutant 2 Wild-type

Percent Germination

Light

Dark

Percent Germination

Results from Direct Test Results from Direct Test IIII

Pollen Tube Length

0

50

100

150

200

250

300

350

400

450

Mutant 1 Mutant 2

Pollen Tube Length in Microns

Light

Dark

Pollen Tube Length1

Hour

SummarySummary

Rop9Rop9 has an effect has an effect on pollen’s ability on pollen’s ability to carry out to carry out fertilizationfertilization

However my However my results do not results do not confirm a role for confirm a role for RRop9op9 in pollen in pollen tube growthtube growth

Future ResearchFuture Research

Initial hypothesis: Initial hypothesis: Pollen Tube Pollen Tube GrowthGrowth

Possibly may have Possibly may have an effect on late an effect on late eventsevents

Fertilization?Fertilization?

AcknowledgementsAcknowledgements

Ernest and Pauline Jaworski FundErnest and Pauline Jaworski Fund Howard Hughes Medical InstituteHoward Hughes Medical Institute Dr. Kevin AhernDr. Kevin Ahern Dr. John FowlerDr. John Fowler Zuzana Zuzana VejlupkovaVejlupkova Rex ColeRex Cole Kirsten CarolKirsten Carol Darin OlsonDarin Olson