Transcript Auxin-induced gravitropic responses in wild type and lazy-2 tomatoes (Lycopersicon esculentum) By Kathy VanWormer From the lab of Dr.

Auxin-induced gravitropic
responses in wild type and
lazy-2 tomatoes
(Lycopersicon esculentum)
By Kathy VanWormer
From the lab of
Dr. Terri Lomax
Introduction
lazy-2 plants, auxin, and
motivation
lazy-2 and Wild Type Tomato plants
Wild type
tomato
lazy-2 tomato
Why lazy?

lazy-2 tomato mutants exhibit reversed
gravitropic responses in the presence of red light

lz-2 is a one gene mutation

The mutants behave like wild type plants in all
other aspects including root gravity response
OH
Auxin
NH O
Indole-3-acetic acid

Auxins are plant hormones, one of which
is IAA (indole-3-acetic acid)

Auxin gradients direct plant shape

Auxin is transported to desired locations
by influx and efflux proteins
Auxin Continued

Reversed placement or function of auxin
transport proteins could result in reversed
gravicurvature.

Preliminary results show that the mutant
plants sense gravity normally (the
amyloplasts sediment to the bottoms of
cells) but their signal is reversed (auxin is
transported to the reversed side of the
plant)
Wild Type Auxin Transport
and Differential Growth
Seedling placed
horizontally
Upright seedling
Lateral auxin
transport
Differential
growth
Gravitropic Response in lz-2
plants
Seedling placed
horizontally
Upright seedling
?
Lateral auxin
transport
Differential
growth
DR5::GUS shows presence of
auxin
DR5 is a promoter that responds to auxin. The DR5::GUS
coupling makes it possible to stain auxin-containing
areas blue
Wildtype
lz-2
gravity
Hypothesis

Does the reversed gravitropic response in
lz-2 tomato mutants come from reversed
auxin transport or reversed responses to
auxin location? What are the phenotypic
differences between the mutant and wild
type seedlings under a variety of
conditions?
Seedling Growth and
Development
Elongation zone
Direction of
growth
Root
Hook to push
through dirt
Region of interest
Cotyledons
Wild Type plants responding to gravity
Motivation

Plants will be essential to NASA’s goal of
manned space exploration

We must fully understand plant growth,
development, and environmental
responses especially the reactions to
microgravity and space conditions, before
plants can be grown effectively in space
Methods
Methods

lazy-2 mutant tomatoes and wild type
tomatoes are sprouted in the dark and
grown for 3 to 5 days
The seedlings are sprouted in
containers like these
Note that the mutants only
show the lazy response in the
presence of red light
Methods 2
Seedling layout:
Plates are placed horizontally under red light
gravity
Methods 3
Pictures are taken of the seedlings every hour
Notice the wild type seedlings have been placed with their hooks
down and the lazy plants are placed with their hooks up to allow
comparable responses.
gravity
Wild type
lazy-2
Controls at time=0, right after they were taken from the magenta boxes
T=0
T=5
T=24
Wt null
lz null
gravity
Null treatment
Measurements:
Measure
this
angle
Measure
this
length
Plot the length of increase vs. time and angle vs. time
Decapitation Experiments


Hooks or cotyledons are removed from the
plants
Plants grown without chemicals to determine the
importance of the hooks and cotyledons
Without Cotyledons
Cut Here
Without Hooks
Cut
Here
Experiments and
Results
Cyclohexamide
CHX and Null Average Increase in Length
(cm)
CHX and Null Average Angles of Curvature
4.5
100.0
50.0
Wild Type CHX
lazy CHX
0.0
0
10
20
-50.0
-100.0
30
Wild Type Null
lz Null
Average Increase (cm)
Average angles of Curvature
150.0
4.0
3.5
3.0
Wild Type CHX
2.5
lz CHX
2.0
Wild Type Null
1.5
lz Null
1.0
0.5
0.0
0
-150.0
Time (Hours)


10
20
30
Time (Hours)
Cyclohexamide inhibits protein synthesis
When grown in the light, the chx treated plants curve
less and grow less than the non-treated controls.
NPA (naphthylphthalamic
acid)
NPA: average lenth of increase
150
5.0
120
4.5
90
wt null averages
60
30
lazy null averages
0
-30
0
5
10
15
30
wt NPA averages
4.0
wt null
3.5
3.0
lz null
2.5
2.0
wt NPA
1.5
1.0
lz NPA
0.5
-90
0.0
0
-120
time (hours)

25
lazy NPA averages
-60

20
lenght increased (cm)
degrees curvature
NPA: average angles of curvature
5
10
15
20
25
30
time (hours)
blocks auxin efflux proteins (PIN proteins)
NPA treated plants didn’t curve at all, but they
seem to grow longer
Brefeldin A
BFA and Null Average Increase in Length
(cm)
BFA and Null Average Angles of Curvature
4.5
100.0
50.0
Wild Type BFA
lazy BFA
0.0
0
5
10
15
-50.0
-100.0
lz Null
3.5
3.0
Wild Type BFA
2.5
lz BFA
2.0
Wild Type Null
1.5
lz Null
1.0
0.5
0
Time (Hours)

25
4.0
0.0
-150.0

20
Wild Type Null
Average Increase (cm)
Average angles of Curvature
150.0
10
20
30
Time (Hours)
Disrupts cycling of auxin efflux proteins
between plasma membrane and endosomes
No apparent effects
Latrunculin B
150.0
4.5
100.0
50.0
Wild Type LatB
lazy LatB
0.0
0
10
20
-50.0
-100.0


30
Wild Type Null
lz Null
4.0
3.5
3.0
Wild Type Lat B
2.5
lz LatB
2.0
Wild Type Null
1.5
lz Null
1.0
0.5
0.0
-150.0
0
Time (Hours)

LatB and Null Average Increase in Length
(cm)
Average Increase (cm)
Average angles of Curvature
Lat B and Null Average Angles of
Curvature
10
20
30
Time (Hours)
disrupts the actin cytoskeleton
latB has been found to increase the gravicurvature of
Arabidopsis roots
LatB treated plants, especially the lazy plants, grew in
random curls, a characteristic that is not detected by
our methods of measurement.
Latrunculin B cont.

LatB treated plants tend to curl and pull
themselves off of the plate, the lazy plants
more so than the wild type
Decapitations
Decapitated vs. Null Average Increase in
Length (cm)
Decapitated vs. Null Average Angles of
Curvature
2.5
100.0
50.0
0.0
0
5
10
15
20
25
-50.0
30
Wild Type No Cotyledon
lazy No Cotyledon
Wild Type Null
lz Null
Wild Type No Hook
lz No Hook
-100.0
Average Increase (cm)
Average angles of Curvature
150.0
Wild Type No
Cotyledon
2.0
lz No Cotyledon
1.5
Wild Type Null
1.0
lz Null
0.5
Wild Type No Hook
0.0
lz No Hook
0
-150.0
10
20
30
Time (Hours)
Time (Hours)
Without Cotyledons
Without Hooks
Cut
Here
Cut Here

The lazy plants did not curve downwards without
hooks, but the wild type did curve upwards
Decapitations in the Dark
Dark Average Increase in Length (cm)
3.5
120.0
Wild Type No
Cotyledon
100.0
lazy No Cotyledon
80.0
Wild Type Null
60.0
lz Null
40.0
Wild Type No Hook
20.0
lz No Hook
0.0
10
20
Time (Hours)

Wild Type No
Cotyledon
3.0
lz No Cotyledon
2.5
Wild Type Null
2.0
1.5
lz Null
1.0
Wild Type No Hook
0.5
lz No Hook
0.0
0

Average Increase (cm)
Average angles of Curvature
Dark Average Angles of Curvature
30
0
10
20
30
Time (Hours)
Lz plants curved more than they did in the
light
Removing hooks from plants impaired
elongation a lot and curvature a little
Summary of Results




The protein synthesis inhibitor
cyclohexamide non-specifically decreased
both elongation and curvature in both
Effects of cytoskeleton inhibitors and cell
cycling inhibitors are yet to be determined
Auxin transport blockers halt all curvature
Lazy plants respond more than wild type
plants to the removal of their hooks
Conclusions



Lateral transport of auxin is probably
reversed in lazy-2 mutant tomatoes
The Lz-2 gene product operates upstream
in the signal pathway from the lateral auxin
transport
The lz-2 plants require red light and an
attached hook to exhibit downward
curvature
Future work






Find new chemicals to explore
Run statistical analyses of all results
Determine auxin concentration in top and
bottom of elongation zone
Repeat GUS experiment for consistent
results
Repeat all experiments in the dark
Run microarrays to find differences in
genes activated under various conditions
for mutant and wild type plants
Special Thanks To:









HHMI Fund
Dr. Kevin Ahern
Jaworski Fund
Kirk Findlay
Dr. Maria Ivanchenko
TJ White
Dr. Terri Lomax
Mariah Parker
Warren Coffeen