The Interactions of The Interactions of Plant Hormones Plant Hormones

and and roprop Genes Genes

By By Malem GutemaMalem Gutema

MentorMentor Dr. John FowlerDr. John Fowler

Summer 2004Summer 2004

Rop ProteinsRop Proteins

Plants possess a special Plants possess a special subfamily of GTPases subfamily of GTPases denoted by Rop (Rho of denoted by Rop (Rho of Plants) proteins Plants) proteins

Rop proteins have been Rop proteins have been first identified in pea first identified in pea (Yang and Watson), (Yang and Watson), where they contribute where they contribute to control tip growth in to control tip growth in pollen tubes pollen tubes

G proteins or GTPases play a crucial role in the G proteins or GTPases play a crucial role in the control of growth and shape of eukaryotic cellscontrol of growth and shape of eukaryotic cells

             

GTPase protein

Signal

Receptor

ROPGDPActive

GTP

inactive

- Control of growth and development

- Stress responses

- Disease resistance

How does all it work?How does all it work?

ROP

The plant hormone The plant hormone Abscisic Acid (ABA)Abscisic Acid (ABA)

ABA’s main roles- Regulating transpiration - Stress response- Slow down growthSlow down growth

- - Germination of seeds.

The Plant hormone AuxinThe Plant hormone Auxin

Auxin’s promotes initiation ofAuxin’s promotes initiation of

- lateral roots- lateral roots

- new leaves- new leaves

Auxin regulates elongation ofAuxin regulates elongation of

- stem - stem

- root cells - root cells

Part I - PurposePart I - Purpose

Rop proteins are Rop proteins are thought to interact thought to interact with plant hormone with plant hormone signals, particularly signals, particularly during root growth.during root growth.

Abscisic acid and auxin restrain root Abscisic acid and auxin restrain root growth.growth.

Hypothesis and PredictionHypothesis and Prediction

We knowWe know

ABA, ABA, AuxinAuxin

We HypothesizeWe Hypothesize

ABA, ABA, AuxinAuxin

Our predictionOur prediction

ABA, ABA, AuxinAuxin

Root growth

Root growthRop

Rop

a symbol to show inhibition

EVEN SHORTERRoot growth

Part I - ExperimentsPart I - Experiments

The experiments are designed to measure The experiments are designed to measure the effects of the effects of

- - Abscisic acid (ABA) (ABA)

- Auxin- Auxin

Samples areSamples are

- Wild-types - Wild-types

- mutated at - mutated at roprop genes genes

Maize Maize roprop mutants mutants

There are nine There are nine roprop genes in maize genes in maize

I used samples with mutation of the I used samples with mutation of the rop rop 2, 2, roprop 6 and 6 and roprop 7 genes 7 genes

How do you get the mutation?How do you get the mutation?

roprop mutation Maps mutation Maps

Exon

IntronTransposable element

Root growth assayRoot growth assay

Question?Question?

Would additional ABA and Auxin Would additional ABA and Auxin affect root growth?affect root growth?

How are the wild-type and How are the wild-type and mutated samples mutated samples

affected?affected?

Experiment set upExperiment set up

Germinate seedlings in a water Germinate seedlings in a water soaked paper towelsoaked paper towel

Measure root length Measure root length

before treatmentbefore treatment Add auxin or ABA Add auxin or ABA Measure after 24 and 48hrs Measure after 24 and 48hrs Average the length Average the length

Auxin – Auxin – roprop 6/7 6/7

Day Two Comparison

0

20

40

60

80

100

120

0 1 2 3 4

% o

f ro

ot

gro

wh

Wildtype grow th

Mutant grow th

Day One Comparison

0

20

40

60

80

100

120

0 1 2 3 4

% o

f ro

ot

gro

wth

Wildtype grow th

Mutant grow th

Mutant Effect !!

rop 6/7 Double mutant

SLOWER root growth

0 0.1 µM 1.0 µM Auxin

0 0.1 µM 1.0 µM Auxin

Auxin Auxin roprop 2 2Day One Compaison

0

20

40

60

80

100

120

0 1 2 3 4

% o

f roo

t gro

wth

Wildtype grow th

Mutant grow th

Day Two comparison

0

20

40

60

80

100

120

0 1 2 3 4

% o

f ro

ot

gro

wth

Wildtype growth

Mutant growth

No Mutant Effect !!!

rop 2 mutant

SAME as wild-type

0 0.1 µM 1.0 µM Auxin

0 0.1 µM 1.0 µM Auxin

ABA ABA roprop 2 2 Day One Comparison

0

20

40

60

80

100

120

0 1 2 3 4

% o

f ro

ot

gro

wth

Wildtype grow th

Mutant grow th

Day Two Comparison

0

20

40

60

80

100

120

0 1 2 3 4

% o

f ro

ot

gro

wth

Wildtype grow th

Mutant grow th

Mutant Effect !!

rop 2 mutant

SLOWER root growth

0 100 µM 500µM ABA

0 100 µM 500 µM ABA

ABA- ABA- roprop 6/7 6/7Day One Comparison

0

20

40

60

80

100

120

0 1 2 3 4

% o

f roo

t gro

wth

Wild-type grow th

Mutant grow th

0 100 µM 500 µM ABA

Day Two Comparison

0

20

40

60

80

100

120

0 1 2 3 4

% r

oo

t g

row

th

Wild-type grow th

Mutant grow th

0 100 µM 500 µM ABA

No Mutant Effect !!!

rop 6/7 mutant

SAME as wild-type

Summary of ResultsSummary of Results

ABA, AuxinABA, Auxin

AuxinAuxin rop rop 6/76/7 Supports Supports hypothesis hypothesis

AuxinAuxin rop rop 22 No effectNo effect

ABAABA roprop 2 2 Supports hypothesis Supports hypothesis ABAABA roprop 6/7 6/7 No effectNo effect

Root growth

Rop

Working ModelWorking Model

ABA

Root Growth

Auxin rop

6/7rop 2

Part IIPart II

Do same root assay on a segregating Do same root assay on a segregating population of population of rop2-m1rop2-m1 mutants mutants

Take samples from an ear that has Take samples from an ear that has both homozygote and heterozygote both homozygote and heterozygote

genotypesgenotypes Do genotypingDo genotyping

What does segregating What does segregating mean?mean?

rop2-m1rop2-m1/+/+ ΧΧ rop2-m1rop2-m1 / / rop2-m1rop2-m1

rop2-m1rop2-m1 / / rop2-m1rop2-m1 a homozygote a homozygote

rop2-m1rop2-m1 / + a heterozygote / + a heterozygote

Population is 50:50 homozygotes:heterozygotesPopulation is 50:50 homozygotes:heterozygotes

+ is a symbol for wild-type

ProcedureProcedure

Collect leaf samplesCollect leaf samples

Extract DNAExtract DNA

Do a PCR reactionDo a PCR reaction with with rop rop 2 primers 2 primers

Analyze the PCR product on an agarose Analyze the PCR product on an agarose gel to check the fragment sizesgel to check the fragment sizes

Detecting Detecting roprop 2 mutations 2 mutationsrop 2 primers

Transposable element primer

Mutation bands

Wild type band

Tested hypothesis how Tested hypothesis how roprop genes inhibit genes inhibit plant hormonesplant hormones

-Some data support-Some data support

-Some data don’t-Some data don’t

A more conclusive result will be A more conclusive result will be obtained from the segregating obtained from the segregating experiment that is experiment that is

Coming soon………..Coming soon………..

Summary ConclusionSummary Conclusion

ABArop 2

Auxin

rop 6/7

Root Growth

AcknowledgmentsAcknowledgments

Ernest and Pauline Jaworski FundErnest and Pauline Jaworski Fund Howard Hughes Medical InstituteHoward Hughes Medical Institute Dr. John FowlerDr. John Fowler Dr. Kevin AhernDr. Kevin Ahern Everybody at the labEverybody at the lab

- Zuzana Vejlupkova- Zuzana Vejlupkova- Kirstin Arthur- Kirstin Arthur- Rex Cole- Rex Cole