Auxin-induced gravitropic Auxin-induced gravitropic responses in wild type and responses in wild type and

lazy-2lazy-2 tomatoes tomatoes ((Lycopersicon Lycopersicon esculentumesculentum))By Kathy VanWormerBy Kathy VanWormer

From the lab of From the lab of

Dr. Terri LomaxDr. Terri Lomax

IntroductionIntroduction

lazy-2lazy-2 plants, auxin, and plants, auxin, and motivationmotivation

lazy-2lazy-2 and Wild Type Tomato and Wild Type Tomato plantsplants

lazy-2lazy-2 tomato tomato

Wild typeWild typetomatotomato

Why lazy?Why lazy?

lazy-2lazy-2 tomato mutants exhibit reversed tomato mutants exhibit reversed gravitropic responses in the presence of gravitropic responses in the presence of red lightred light

lz-2lz-2 is a one gene mutation is a one gene mutation

The mutants behave like wild type plants The mutants behave like wild type plants in all other aspects including root gravity in all other aspects including root gravity responseresponse

AuxinAuxin

Auxins are plant hormones, one of Auxins are plant hormones, one of which is IAA (indole-3-acetic acid)which is IAA (indole-3-acetic acid)

Auxin gradients direct plant shapeAuxin gradients direct plant shape

Auxin is transported to desired Auxin is transported to desired locations by influx and efflux locations by influx and efflux proteins proteins

NH O

OH

Indole-3-acetic acid

Auxin ContinuedAuxin Continued Reversed placement or function of Reversed placement or function of

auxin transport proteins could result auxin transport proteins could result in reversed gravicurvature.in reversed gravicurvature.

Preliminary results show that the Preliminary results show that the mutant plants sense gravity normally mutant plants sense gravity normally (the amyloplasts sediment to the (the amyloplasts sediment to the bottoms of cells) but their signal is bottoms of cells) but their signal is reversed (auxin is transported to the reversed (auxin is transported to the reversed side of the plant)reversed side of the plant)

Wild Type Auxin Wild Type Auxin Transport and Transport and

Differential GrowthDifferential Growth

Seedling placedSeedling placedhorizontallyhorizontally

Lateral auxin Lateral auxin transporttransport Differential Differential

growthgrowth

Upright seedlingUpright seedling

Gravitropic Response in Gravitropic Response in lz-2lz-2 plants plants

Seedling placedSeedling placedhorizontallyhorizontally

Lateral auxin Lateral auxin transporttransport

Differential Differential growthgrowth

Upright seedlingUpright seedling

?

DR5::GUS shows DR5::GUS shows presence of auxinpresence of auxin

Wildtype lz-2

DR5 is a promoter that responds to auxin. The DR5::GUS coupling makes it possible to stain auxin-containing

areas blue

gravity

HypothesisHypothesis

Does the reversed gravitropic Does the reversed gravitropic response in response in lz-2lz-2 tomato mutants tomato mutants come from reversed auxin transport come from reversed auxin transport or reversed responses to auxin or reversed responses to auxin location? What are the phenotypic location? What are the phenotypic differences between the mutant and differences between the mutant and wild type seedlings under a variety wild type seedlings under a variety of conditions? of conditions?

Seedling Growth and Seedling Growth and DevelopmentDevelopment

Hook to push Hook to push through dirtthrough dirtElongation zoneElongation zone

RootRoot

CotyledonsCotyledons

Region of interestRegion of interest

Wild Type plants responding to gravity

Direction ofDirection ofgrowthgrowth

MotivationMotivation

Plants will be essential to NASA’s Plants will be essential to NASA’s goal of manned space explorationgoal of manned space exploration

We must fully understand plant We must fully understand plant growth, development, and growth, development, and environmental responses especially environmental responses especially the reactions to microgravity and the reactions to microgravity and space conditions, before plants can space conditions, before plants can be grown effectively in spacebe grown effectively in space

MethodsMethods

MethodsMethods

lazy-2lazy-2 mutant tomatoes and wild mutant tomatoes and wild type tomatoes are sprouted type tomatoes are sprouted in the in the darkdark and grown for 3 to 5 days 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 thepresence of red light

Methods 2Methods 2

Seedling layout:Seedling layout:

gravity

Plates are placed horizontally under red lightPlates are placed horizontally under red light

Methods 3Methods 3

Notice the wild type seedlings have been placed with their hooks Notice the wild type seedlings have been placed with their hooks down and the lazy plants are placed with their hooks up to allow down and the lazy plants are placed with their hooks up to allow comparable responses.comparable responses.

Pictures are taken of the seedlings Pictures are taken of the seedlings every hourevery hour

Wild type lazy-2

Controls at time=0, right after they were taken from the magenta boxesControls at time=0, right after they were taken from the magenta boxes

gravity

Wt null

lz null

T=0 T=5 T=24

Null treatmentNull treatment

gravity

MeasureMeasurethis this angleangle

MeasureMeasurethis this lengthlength

Measurements:Measurements:

Plot the length of increase vs. time and angle vs. timePlot the length of increase vs. time and angle vs. time

Decapitation Decapitation ExperimentsExperiments

Hooks or cotyledons are removed from the Hooks or cotyledons are removed from the plantsplants

Plants grown without chemicals to determine Plants grown without chemicals to determine the importance of the hooks and cotyledons the importance of the hooks and cotyledons

Without CotyledonsWithout Cotyledons

Cut HereCut Here

Without HooksWithout Hooks CutCutHereHere

Experiments and Experiments and ResultsResults

CyclohexamideCyclohexamide

Cyclohexamide inhibits protein synthesis When grown in the light, the chx treated plants curve

less and grow less than the non-treated controls.

CHX and Null Average Increase in Length (cm)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 10 20 30

Time (Hours)

Average Increase (cm)

Wild Type CHX

lz CHX

Wild Type Null

lz Null

CHX and Null Average Angles of Curvature

-150.0

-100.0

-50.0

0.0

50.0

100.0

150.0

0 10 20 30

Time (Hours)

Average angles of Curvature

Wild Type CHX

lazy CHX

Wild Type Null

lz Null

NPA NPA (naphthylphthalamic (naphthylphthalamic

acid)acid)

blocks auxin efflux proteins (PIN proteins)blocks auxin efflux proteins (PIN proteins) NPA treated plants didn’t curve at all, but they NPA treated plants didn’t curve at all, but they

seem to grow longerseem to grow longer

NPA: average angles of curvature

-120

-90

-60

-30

0

30

60

90

120

150

0 5 10 15 20 25 30

time (hours)

degrees curvature

wt null averages

lazy null averages

wt NPA averages

lazy NPA averages

NPA: average lenth of increase

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 5 10 15 20 25 30

time (hours)

lenght increased (cm)

wt null

lz null

wt NPA

lz NPA

Brefeldin ABrefeldin A

Disrupts cycling of auxin efflux proteins Disrupts cycling of auxin efflux proteins between plasma membrane and endosomesbetween plasma membrane and endosomes

No apparent effects No apparent effects

BFA and Null Average Increase in Length (cm)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 10 20 30

Time (Hours)

Average Increase (cm)

Wild Type BFA

lz BFA

Wild Type Null

lz Null

BFA and Null Average Angles of Curvature

-150.0

-100.0

-50.0

0.0

50.0

100.0

150.0

0 5 10 15 20 25

Time (Hours)

Average angles of Curvature

Wild Type BFA

lazy BFA

Wild Type Null

lz Null

Latrunculin BLatrunculin 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.

LatB and Null Average Increase in Length (cm)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 10 20 30

Time (Hours)

Average Increase (cm)

Wild Type Lat B

lz LatB

Wild Type Null

lz Null

Lat B and Null Average Angles of Curvature

-150.0

-100.0

-50.0

0.0

50.0

100.0

150.0

0 10 20 30

Time (Hours)

Average angles of Curvature

Wild Type LatB

lazy LatB

Wild Type Null

lz Null

Latrunculin B cont.Latrunculin B cont.

LatB treated plants tend to curl and LatB treated plants tend to curl and pull themselves off of the plate, the pull themselves off of the plate, the lazy plants more so than the wild lazy plants more so than the wild typetype

DecapitationsDecapitations

The lazy plants did not curve downwards The lazy plants did not curve downwards without hooks, but the wild type did curve without hooks, but the wild type did curve upwardsupwards

Decapitated vs. Null Average Increase in Length (cm)

0.0

0.5

1.0

1.5

2.0

2.5

0 10 20 30

Time (Hours)

Average Increase (cm)

Wild Type NoCotyledon

lz No Cotyledon

Wild Type Null

lz Null

Wild Type No Hook

lz No Hook

Decapitated vs. Null Average Angles of Curvature

-150.0

-100.0

-50.0

0.0

50.0

100.0

150.0

0 5 10 15 20 25 30

Time (Hours)

Average angles of Curvature

Wild Type No Cotyledon lazy No CotyledonWild Type Nulllz NullWild Type No Hooklz No Hook

Without CotyledonsWithout Cotyledons

Cut HereCut Here

Without HooksWithout HooksCutCutHereHere

Decapitations in the Decapitations in the DarkDark

Lz plants curved more than they did in Lz plants curved more than they did in the lightthe light

Removing hooks from plants impaired Removing hooks from plants impaired elongation a lot and curvature a littleelongation a lot and curvature a little

Dark Average Increase in Length (cm)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 10 20 30

Time (Hours)

Average Increase (cm)

Wild Type NoCotyledon

lz No Cotyledon

Wild Type Null

lz Null

Wild Type No Hook

lz No Hook

Dark Average Angles of Curvature

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 10 20 30

Time (Hours)

Average angles of Curvature

Wild Type NoCotyledon

lazy No Cotyledon

Wild Type Null

lz Null

Wild Type No Hook

lz No Hook

Summary of ResultsSummary of Results

The protein synthesis inhibitor The protein synthesis inhibitor cyclohexamide non-specifically decreased cyclohexamide non-specifically decreased both elongation and curvature in bothboth elongation and curvature in both

Effects of cytoskeleton inhibitors and cell Effects of cytoskeleton inhibitors and cell cycling inhibitors are yet to be determinedcycling inhibitors are yet to be determined

Auxin transport blockers halt all curvatureAuxin transport blockers halt all curvature Lazy plants respond more than wild type Lazy plants respond more than wild type

plants to the removal of their hooksplants to the removal of their hooks

ConclusionsConclusions

Lateral transport of auxin is probably Lateral transport of auxin is probably reversed in reversed in lazy-2lazy-2 mutant tomatoes mutant tomatoes

The The Lz-2Lz-2 gene product operates gene product operates upstream in the signal pathway from upstream in the signal pathway from the lateral auxin transportthe lateral auxin transport

The The lz-2lz-2 plants require red light and plants require red light and an attached hook to exhibit an attached hook to exhibit downward curvaturedownward curvature

Future workFuture work Find new chemicals to exploreFind new chemicals to explore Run statistical analyses of all resultsRun statistical analyses of all results Determine auxin concentration in top Determine auxin concentration in top

and bottom of elongation zoneand bottom of elongation zone Repeat GUS experiment for Repeat GUS experiment for

consistent resultsconsistent results Repeat all experiments in the darkRepeat all experiments in the dark Run microarrays to find differences in Run microarrays to find differences in

genes activated under various genes activated under various conditions for mutant and wild type conditions for mutant and wild type plantsplants

Special Thanks To:Special Thanks To: HHMI FundHHMI Fund Dr. Kevin Ahern Dr. Kevin Ahern Jaworski FundJaworski Fund Kirk FindlayKirk Findlay Dr. Maria IvanchenkoDr. Maria Ivanchenko TJ WhiteTJ White Dr. Terri LomaxDr. Terri Lomax Mariah ParkerMariah Parker Warren CoffeenWarren Coffeen