Discovering the Roles of Myosin and Exocyst Proteins in Cell

Expansion of Arabidopsis thaliana

Danielle GoodrichDr. John Fowler’s LabHHMI Summer 2012

Botany and Plant PathologyOregon State University

Arabidopsis thaliana as a Genetic Model

Its genetics are well known

Many mutant lines

Grows quickly

Agricultural benefits• Closely related to mustard, kale,

broccoli, and cauliflower• Relevant to all crop plants

Use the Col-0 wild type (WT)

Hypocotyl of Arabidopsis thaliana

Diffuse cell expansion

Exo70a1-2 and Exo84b: known to have shorter hypocotyls

exo

84se

c15

sec 5 se

c3

sec1 0

sec6

exo

70

sec 8

exo70WT

 

hypo

cotyl

Root Hairs of Arabidopsis Root Hair cells grow at the tip

Measure the effects of the absence of a myosin or exocyst protein

Determine which proteins are important for tip growth

WT exo70

XI-K

*scale bars: 200 um

Myosins XI and Exocyst Function in Cell Expansion

It is already known that myosin and exocyst proteins function in cell expansion

Yet it is unknown whether they work together in diffuse cell expansion and tip growth

Hypothesis:Myosin XI and Exocyst interaction

vesicle

actin cytoskeleton

Vesicle transport

myosin XI

vesicle

exo

70se

c3

sec1

5se

c1 0ex

o84

sec 8

sec6

sec5

cell m

em

bra

ne

exocyst

com

ple

x

tetheringfusion

vesicle

Main Focus

Which, if any, myosin XI proteins and exocyst proteins play a role in growth of hypocotyls and root hairs?

Why are mutant hypocotyls shorter? Why are mutant root hairs shorter? Do myosin XI proteins and exocyst

proteins interact?

Interesting Mutant Hypocotyls

* These numbers were gathered from a series of three tests and compared to the Wild Type grown on the same day.

WT

exo7

0a1-

2

exo8

4b

XI-G XI-IXI-1 XI-2 XI-K

XI-12K

XI-12K

I0

20

40

60

80

100

120

140

Hypocotyl Lengths of Wild Type and Mutant Arabidopsis Plants

Series1

Exocyst Myosin Genotype

% o

f W

T H

yp

oco

tyl

Le

ng

th

Interesting Mutant Combinations

WT

exo7

0a1-

2

sec8

-6

exo7

0; X

I-1

sec8

-6; X

I-I

sec8

-6; X

I-K0

20

40

60

80

100

120Hypocotyl Lengths of Mutant Combination-

Arabidopsis Plants

Series1

Genotype

% o

f W

T H

yp

oco

tyl Len

gth

Hypocotyl Cell Length

Do mutants with shorter hypocotyls than WT have shorter cells in the hypocotyl or less cells?

Stain cell walls with toluidine blue solution

WT Exo70

Hypocotyl Cell Length

WT XI-12KI exo700

20

40

60

80

100

120

140

Hypocotyl Cell Lengths of Mutated Plants

Series1

GenotypeAvera

ge H

yp

oco

tyl C

ell L

en

gth

(u

m)

Root Hair Measurements

Exocyst affects root hairs and hypocotyls

Myosin has a stronger effect on root hairs than hypocotyls

WT sec8#exo70a1 xi-b xi-i xi-1 xi-2 xi-k0

400

350

300

250

200

150

100

50

Avera

ge R

oot

Hair

Len

gth

(µ

m)

*

*

*

*

* *

exocyst myosin XI

** Previous work done by Dr. Amy Klocko

Root Hair Lengths of Exocyst and Myosin XI Mutants

Exocyst and Myosin Double Mutants

Loss of both SEC8 and XI-I has strong effect on the root hairs and no effect on hypocotyls

WT sec8 xi-i sec8xi-i

0

300

250

200

150

100

50

Avera

ge R

oot

Hair

Len

gth

(µ

m)

*

400

350

WT sec8 xi-i sec8xi-i

Avera

ge H

yp

ocoty

l Len

gth

(%

W

T L

en

gth

)

0

20

40

60

80

100

Root Hair Lengths of a Combination

Mutant

Hypocotyl Lengths of a Combination

Mutant

Root Hair Time Lapse

Root hair cells are shorter in some mutants.

Exo70 and XI-K mutants

Do they grow slower or grow at same rate and stop at a certain point?

Exo70 root 30 min time lapse

exo70 movie 1 - Copy.avi

Root Hair Time Lapse

WT XI-K exo700

0.1

0.2

0.3

0.4

0.5

0.6

Root Hair Growth Rates of Wild Type and Protein Mutants

Series1

Genotype

Mean

Root

Hair

Gro

wth

Rate

(u

m/m

in)

Discussion

Exocyst and Myosin XI proteins are important for growth in the hypocotyl and root hairs.

May not work together in hypocotyl growth.

Hypocotyl cells of myosin XI mutants are shorter than WT.

Exo70 and XI-K root hairs grow at a slower rate than WT root hairs.

I would like to thank…

Professor:Dr. John FowlerMentor:Dr. Amy Klocko

Dr. Kevin AhernZuzana VejlupkovaDr. Rex ColeDr. Maria

Ivanchenko

Grant # IOS-0920747

References

Arabidopsis thaliana- first plant genome sequenced. The European Commission. Research. 13 December 2000. http://ec.europa.eu/research/quality-of-life/arabidopsis.html

Welcome to the Savaldi-Goldstein Lab. 2011. Photograph. Savaldi-Goldstein Plant Biology Lab, Haifa, Israel. Web. 3 March 2012. <http://biology.technion.ac.il/~sigal/>.

National Science Foundation. Web. 27 June 2012. Nsf.gov