The Interactions of The Interactions of Plant Hormones Plant Hormones and and rop rop Genes Genes By By Malem Gutema Malem Gutema Mentor Mentor Dr. John Fowler Dr. John Fowler Summer 2004 Summer 2004
Dec 21, 2015
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