The Interactions of
Plant Hormones
and
rop Genes
By
Malem Gutema
Mentor
Dr. John Fowler
Summer 2004
Rop Proteins

G proteins or GTPases play a crucial role in the control of
growth and shape of eukaryotic cells

Plants possess a special
subfamily of GTPases
denoted by Rop (Rho of
Plants) proteins

Rop proteins have been
first identified in pea
(Yang and Watson),
where they contribute to
control tip growth in
pollen tubes
GTPase protein
How does all it work?
Signal
Receptor
GDP
ROP
inactive
ROP GTP
Active
- Control of growth and development
- Stress responses
- Disease resistance
The plant hormone
Abscisic Acid (ABA)

ABA’s main roles
- Regulating transpiration
- Stress response
- Slow down growth
- Germination of seeds.
The Plant hormone Auxin
Auxin’s promotes initiation of
- lateral roots
- new leaves
Auxin regulates elongation of
- stem
- root cells
Part I - Purpose

Abscisic acid and auxin restrain root growth.

Rop proteins are
thought to interact with
plant hormone signals,
particularly during root
growth.
Hypothesis and Prediction
We know
ABA,
Auxin
Root growth
We Hypothesize
ABA,
Auxin
Rop
Root growth
Our prediction
Rop
ABA,
Auxin
EVEN SHORTER
Root growth
a symbol to
show inhibition
Part I - Experiments

The experiments are designed to measure
the effects of
- Abscisic acid (ABA)
- Auxin

Samples are
- Wild-types
- mutated at rop genes
Maize rop mutants

There are nine rop genes in maize

I used samples with mutation of the rop 2,
rop 6 and rop 7 genes

How do you get the mutation?
rop mutation Maps
Transposable
element
Exon
Intron
Root growth assay

Question?
Would additional ABA and Auxin affect
root growth?
How are the wild-type and
mutated samples affected?
Experiment set up
Germinate seedlings in a water soaked
paper towel
 Measure root length
before treatment
 Add auxin or ABA
 Measure after 24 and 48hrs
 Average the length

Auxin – rop 6/7
Day One Comparison
Mutant Effect !!
100
rop 6/7 Double mutant
80
60
Wildtype grow th
40
Mutant grow th
SLOWER root growth
20
0
0
0 1
0.12µM
Auxin
1.0 3µM
4
Day Two Comparison
120
% of root growh
% of root growth
120
100
80
Wildtype grow th
60
Mutant grow th
40
20
0
0
0
1
2
0.1 µM
Auxin
3
1.0 µM
4
Auxin rop 2
Day One Compaison
No Mutant Effect !!!
100
rop 2 mutant
80
60
Wildtype grow th
40
Mutant grow th
SAME as wild-type
20
0
0
01
0.1 µM
2
Auxin
1.03 µM
4
Day Two comparison
120
100
% of root growth
% of root growth
120
80
60
Wildtype growth
40
Mutant growth
20
0
0
01
0.1
2 µM
Auxin
31.0
µM
4
ABA rop 2
Day One Comparison
Mutant Effect !!
100
80
60
Wildtype grow th
40
Mutant grow th
rop 2 mutant
SLOWER root growth
20
0
0
01
2 µM
100
ABA
3
500µM
4
Day Two Comparison
120
% of root growth
% of root growth
120
100
80
60
Wildtype grow th
40
Mutant grow th
20
0
0
0
1
2
100 µM
ABA
5003µM
4
ABA- rop 6/7
Day One Comparison
No Mutant Effect !!!
100
rop 6/7 mutant
80
60
Wild-type grow th
40
Mutant grow th
SAME as wild-type
20
0
0
01
100 2µM
ABA
3 µM
500
4
Day Two Comparison
120
100
% root growth
% of root growth
120
80
60
Wild-type grow th
40
Mutant grow th
20
0
0
1
0
ABA
2
100 µM
3
500 µM
4
Summary of Results

ABA, Auxin
Root growth
Rop

Auxin
Auxin
rop 6/7
rop 2
Supports hypothesis
No effect

ABA
ABA
rop 2
rop 6/7
Supports hypothesis
No effect
Working Model
ABA
Auxin
rop 6/7
rop 2
Root Growth
Part II

Do same root assay on a segregating
population of rop2-m1 mutants

Take samples from an ear that has both
homozygote and heterozygote
genotypes

Do genotyping
What does segregating mean?
rop2-m1/+
Χ
rop2-m1 / rop2-m1
rop2-m1 / rop2-m1 a homozygote
rop2-m1 / +
a heterozygote
Population is 50:50 homozygotes:heterozygotes
+ is a symbol for wild-type
Procedure

Collect leaf samples

Extract DNA

Do a PCR reaction
with rop 2 primers

Analyze the PCR product on an agarose gel to
check the fragment sizes
Detecting rop 2 mutations
rop 2 primers
Transposable element primer
Wild type
band
Mutation
bands
Summary Conclusion

Tested hypothesis how rop genes inhibit
plant hormones
ABA
Auxin
-Some data support rop 2
rop 6/7
-Some data don’t
Root Growth

A more conclusive result will be obtained
from the segregating experiment that is
Coming soon………..
Acknowledgments
Ernest and Pauline Jaworski Fund
 Howard Hughes Medical Institute
 Dr. John Fowler
 Dr. Kevin Ahern
 Everybody at the lab
- Zuzana Vejlupkova
- Kirstin Arthur
- Rex Cole
