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

2. lazy-2 plants, auxin, and motivation
Introduction
lazy-2 plants, auxin, and motivation

3. lazy-2 and Wild Type Tomato plants
These are the varieties of tomatoes used in my summer’s research. They are the same species and variety of tomato, but the one on the left is a mutant in the lazy-2 gene, which causes a reversed gravitropic response.
lazy-2 tomato

4. 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
Lz tomato mutants exhibit reversed gravitropic responses in the presence of red light. They grow downwards instead of up.
Lz is a one-gene mutation. This means that one altered protein is responsible for the difference between lz and wild type behavior.
Shoot curvature is the only difference between the mutant and wild type plants.

5. Auxin OH 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
In the shoots high concentrations of auxin increase elongation, in the roots low concentrations increase elongation
Influx and efflux proteins are located in the plasma membranes of cells. They facilitate the active transport of auxin throughout the plant.

6. 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)
Plants sense gravity by the sedimentation of amyloplasts. This sedimentation triggers a signal pathway that sets up an auxin gradient which induces curvature in the shoot or root. Little is known about the signal pathway between the amyloplasts sedimenting and the plants curving.
If auxin is transported to the wrong side of the plant, the plant will curve the wrong direction.

7. Wild Type Auxin Transport and Differential Growth
Seedling placed
horizontally
Upright seedling
This diagram shows the gravity response of wild type tomato seedlings. When a seedling is placed on its side, auxin is transported to the bottom side of the plant, which induces upward curvature in the shoots and downward curvature in the roots.
Lateral auxin
transport
Differential
growth

8. Gravitropic Response in lz-2 plants
Seedling placed
horizontally
Upright seedling
When a lazy tomato seedling isplaced on its side, both the roots and the shoots curve downwards. The mechanism for this curvature was a point of study for this summer’s research. ?
Lateral auxin
transport
Differential
growth

9. 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
Our lab has been studying auxin placement in the wild type and lazy seedlings to detect any differences between the two. To detect the auxin in each of these plants, the coupled promoter and reporter DR5::GUS gene segment was inserted into both the wild type and lazy plants. DR5 is a promoter that responds to auxin and GUS is a gene that produces a product that can be stained blue. Blue staining indicates the presence of auxin in the plant.
These plants were gravity stimulated for 4 hours. As you can see, these pictures show auxin in the bottom side of the wild type plant and in the top side of the lazy plant. This suggests that auxin is transported to the wrong side of the plant, not that the plant’s responses to auxin are reversed.

10. 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?

11. Seedling Growth and Development
Hook to push
through dirt
Direction of
growth
Elongation zone
Root
Region of interest
This gives an example of a developing plant seedling. Notice the elongation zone, where the plant elongates and, when gravistimulated, the plant curves. Also, though the cotyledons here are pointing downwards, the top of the plant is actually the tip of the hook, so these plants are growing upwards from the hook.
Cotyledons
Wild Type plants responding to gravity

12. 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
I originally became interested in this research because of the lab’s connections to NASA and the importance of this type of knowledge to space exploration.

13. Methods

14. 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
Remember, the mutant plants do not exhibit their lazy responses in the dark. Without red light they grow like the wild type plants.
Note that the mutants only
show the lazy response in the
presence of red light

15. Methods 2 Seedling layout:
Plates are placed horizontally under red light
The seedlings are taken from the magenta boxes and place onto agar in square petri dishes as shown here. The petri dishes are placed upright under a red light.
gravity

16. 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.
If the hooks are oriented opposite to this, the plants grow in inconvenient curls, with the hook getting in the way.
These pictures were taken at time=0 hours, right after the plants were placed on these plates. They have not been gravistimulated yet.
gravity
Wild type
lazy-2
Controls at time=0, right after they were taken from the magenta boxes

17. Null treatment T=0 T=5 T=24 Wt null lz null gravity
This is a time course of a sample experiment. The plants shown here are untreated, or null plants. Notice at time=5 hours that the hooks of the wild type plants are beginning to curve upwards and the hooks of the lazy plants are beginning to curve downwards. By 24 hours most plants are growing directly up or directly down. Also, the plants at time=24 hours show that most of the elongation occurs between the area of curvature and the hook of the plant.
gravity
Null treatment

18. Measurements: Measure this angle Measure this length
Data is collected by measuring the plants from the pictures that were taken every hour. Measurements are done as shown. The angle measured is the angle the plant has curved compared to the stem section just before the curve. The length is measured from the root shoot junction to the end of the hook. For data analysis, the increase in length since time=0 hours is used.
Plot the length of increase vs. time and angle vs. time

19. 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
Without Hooks
To show the importance of the hooks and the cotyledons in the wild type and lazy plants, an experiment was run by removing those organs before gravistimulation. The plants were grown on null agar, without chemicals. These diagrams show the excision sites for these two treatments.
Cut
Here
Cut Here

20. Experiments and Results
Each experiment was formulated with the goal of determining the importance of a specific function or structure to the graviresponses of lazy and wild type plants. The results of these experiments will be used to gain a better understanding of the lazy pbenotype as well as the graviresponse of plants as a whole.

21. Cyclohexamide Cyclohexamide inhibits protein synthesis
Two members of the lab had a bet about how auxin could end up on the top side of the lazy plants. One bet that the mutant plants synthesized new auxin transport proteins upon gravistimulation and placed these proteins on the wrong sides of the cell membranes. Cyclohexamide, a protein synthesis inhibitor, was used to test this hypothesis. If wild type or lazy graviresponses depend on new protein synthesis, then one or both of lazy or wild type plants will be unable to curve. The graph on the left is angle of curvature vs. time and the graph on the right is elongation from initial length vs. time. As you can see, the cyclohexamide treated plants, shown in dark blue for the wild type and pink for the lazy, did not curve as much as the untreated plants, but still curved in the same direction as their untreated counterparts.
Cyclohexamide inhibits protein synthesis
When grown in the light, the chx treated plants curve less and grow less than the non-treated controls.

22. NPA (naphthylphthalamic acid)
blocks auxin efflux proteins (PIN proteins)
NPA treated plants didn’t curve at all, but they seem to grow longer
NPA has long been known to block auxin efflux proteins. This chemical blocks auxin transport so that no auxin gradients can be established. As shown in the angle of curvature vs. time graph on the left, plants treated with NPA did not curve at all. The length of increase vs. time graph on the right shows that the treated plants increased in length more than the untreated plants.

23. Brefeldin A
Auxin transport is facilitated by influx and efflux membrane proteins. It is possible that these proteins are relocated upon gravistimulation to send auxin in the desired direction. Brefeldin A, a chemical that disrupts the cycling from membrane to endosome to membrane, was used to test this idea. It appears to have no effect on the growth or curvature of either type of plant.
Disrupts cycling of auxin efflux proteins between plasma membrane and endosomes
No apparent effects

24. Latrunculin B 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.
Other labs have done experiments on the effects of cytoskeleton disruptors, such as Latrunculin B, on gravicurvature of Arabidopsis roots. Their data show that latrunculin B, in some concentrations, can actually increase the gravicurvature of roots. Our experiment with Latrunculin B on tomato shoots does not show a large difference between treated and non treated plants.

25. 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
Latrunculin B was of interest, though, because it caused the plants to grow in curls. Under visual inspection, the lazy plants curled more than the wild type plants.

26. Decapitations
Without Cotyledons
Without Hooks
Cut
Here
The most recent experiment I ran was to determine the importance of the hooks and the cotyledons on seedling growth and development in wild type and lazy plants. Once again, the left graph is angle of curvature vs. time and the right graph is length of increase vs. time. The interesting result of this experiment is that the lazy plants without hooks were unable to curve downwards, or curve at all for that matter, while all other combinations curved the direction and approximate intensity as the null treatments.
Cut Here
The lazy plants did not curve downwards without hooks, but the wild type did curve upwards

27. Decapitations in the Dark
Lz plants curved more than they did in the light
Removing hooks from plants impaired elongation a lot and curvature a little
The decapitation experiment was also run in the dark. Recall from the introduction that the reversed gravitropic response of the mutant plants only occurs in the presence of red light. This experiment shows that lazy plants without their hooks are able to curve in the dark, a sharp contrast to the same experiment run in the light.

28. 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

29. 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

30. 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

31. Special Thanks To: HHMI Fund Dr. Kevin Ahern Jaworski Fund
Kirk Findlay
Dr. Maria Ivanchenko
TJ White
Dr. Terri Lomax
Mariah Parker
Warren Coffeen