Swiss Youth in Science
Study week Biology and Medicine, 14. - 21. 3.
2016
12: Tissue distribution of a plant protein shown with two
different methods
Zenhäusern, Livia, Kollegium Spiritus Sanctus, Brig
Supervision: Lorenzo, Laura and Wild, Rebekka
Laboratory of Professor Michael Hothorn
Background: TTM3 is a plant protein with an unknown function in vivo
Results: TTM3 can be detected in different plant organs by western-blot and GUS
histochemical assay
Conclusion: TTM3 is localized in specific plant tissues. To know more about the
role that TTM3 can play in plants, we can study the processes that take place on
that tissues.
Proteins are biological molecules which take over many different functions in all
living organisms. Many protein domains are already characterized, meaning that
we know their function and structure.
Michael Hothorn’s laboratory focusses on the characterization of plant proteins
using structural biology methods and the model plant Arabidopsis thaliana. One
plant protein with unknown function is TTM3. The protein Vtc4p has the same
tunnel domain as TTM3. While Vtc4p produces polyphosphates, long chains of
phosphates, out of ATP, TTM3 is cleaving triphosphate into pyrophosphate and
phosphate (PPP -> PP) in vitro. (Martinez et al. Journal of Biological Chemistry
2015)
We want to find out if the TTM3 has in vivo the same function as in the yeast.
Therefore, we need to know if polyphosphates exist in plants.
That is why we started to look at the location of TTM3 in plants. To localize the
TTM3 in the plant we used two different methods: a GUS reporter system and a
western blot with an specific TTM3 antibody.
The GUS reporter system
This system analyses the activity of the TTM3 promoter. In our experiment, the
activity in different tissues of Arabidopsis thaliana plants was visualized with blue
colour.
The promoter of a gene determines the time and the place where a gene is
expressed. GUS is an enzyme that metabolizes a molecule (X-Gluc) into a blue
product. Therefore this assay can be used to investigate where and when the
Department for Botany and Plant Biology,
University of Geneva,
Switzerland
TTM3 is expressed by adding X-Gluc to different plant tissues. Tissues in which
TTM3 is expressed, will become blue.
The GUS reporter system was performed on leaves, flowers and seedlings.
Experimental procedure for GUS staining of leaves (in adult plants) and
seedlings
First the leaves and the seedlings were harvested and put into eppis. Then a 2%
formaldehyde (in 50 mM sodium phosphate buffer pH7) was added to fix the
plant tissue. A resting time of 30 minutes was given to them. With this step the
water was removed from the tissue to preserve the leaves and the seedlings. In
order to remove the formaldehyde (toxic) and to create proper conditions for the
GUS enzyme to act, the plant material was washed twice with 50 mM sodium
phosphate buffer. The plant material was suspended in a 10ml staining solution
(10 ml 50 mM sodium phosphate buffer, 0.50 mM K-Ferrocyanide,0.5 mM KFerricyanide, 1 mM X-GlcA (substrate). After adding the staining solution, the
samples were vacuumed for 15 minutes and then incubated at 37°C for 2 hours.
The staining solution was removed and the plant material washed with 96% EtOH
for 1 hour, 60% EtOH for 1 hour and 20% EtOH for 1h respectively. The aim was
to remove the chlorophyll (the green colour) with 96% EtOH and then to increase
the water content by lowering the % of EtOH.
Experimental procedure for GUS staining of flowers
Flowers were harvested and put into eppis, a 90% acetone solution was added to
fix the flower for 20 minutes. The acetone was removed and the flowers were
washed twice with 50 mM sodium phosphate buffer. The plant material was
suspended in a 10ml staining solution (50 mM sodium phosphate buffer, 0.05 mM
K-Ferrocyanide, 0.5 mM K-Ferricyanide, 1mM X-GlcA (substrate)). The flowers in
the solution were vacuumed for 15 min and then incubinated at 37°C for 1 hour.
The staining solution was removed and washed with 96% EtOH for 1 hour, 60%
EtOH for 1 hour, 20% EtOH for 1h, repetitively. The aim was to remove the
chlorophyll (the green colour) with 96% EtOH and then to increase the water
content by lowering the % of EtOH.
The Western Blot system
Western blotting is an analytical technique used to specifically detect proteins.
Gel electrophoresis is used to separate the proteins. The proteins are then
transferred to a membrane (in our case nitrocellulose), where they are stained
with antibodies specific to the target protein.
The Western Blot assay was performed on the wild-type plant Columbia and on a
ttm3 mutant (which does not express the TTM3 protein), as well as seedlings,
leaves and flowers expressing a TTM3-GUS fusion protein.
Experimental procedure for the detection of Arabidopsis thaliana
proteins with the Western Blot system
First, plant material was harvested and put into aluminium foil. Little packets
were formed and put in nitrogen liquid to freeze them. All the materials used
(mortar, spoon, eppis) were pre-chilled in liquid nitrogen. After the bubbling
Department for Botany and Plant Biology,
University of Geneva,
Switzerland
stopped they were taken out. The aluminium foil was unfolded and the plant
material was put into the mortar to crush it. The crushed plant material was filled
into the eppis and again frozen in liquid nitrogen. Buffer was added to the
crushed plants and samples were vortexed for 2 minutes. To separate the tissue
from the supernatant the eppis were centrifuged at 13000 rpms for 20 minutes.
The supernatant was piped into a new eppi. The protein concentration of the
solution was measured with the Bradford assay. In order to get a standard curve,
BSA (protein) was added to Bradford reagent. To calculate the concentration of
the solution, the absorption was measured and extrapolated using a standard
curve (comparison with standard measures). 15ug of total protein were added
(15ug /concentration= ul of the sample). The value of the sample needed was
calculated and SDS (sodium dodecyl sulphate) was added to break down the
structure of the protein. The solution was filled up with water to a total volume of
18uL. The samples were boiled in 95 °C for 7 minutes. The polyacrylamide gel
electrophoresis (SDS-PAGE) was prepared. The gel was put in MES buffer and
then the samples were loaded onto the gel. The SDS-PAGE was started at 100V
for 2 hours. The gel was taken out and put in between 1 sponge, 3 layers of
paper, gel, a nitrocellulose membrane and another 3 layers of paper and 1
sponge. The blotting was performed using a transfer buffer (25 Mm Trisbase, 100
Mm Glycine, 20% methanol). This step transfers the proteins of the gel onto the
nitrocellulose membrane. The blotting was run for 1 ½ hours at 110V in the coldroom. The membrane was taken out and put in a blocking solution for 2 hours.
The blocking solution was removed and a primary antibody (0.5ul antibody (anti
TTM3 made in rabbit), 1ml blocking solution, filled up with TBST buffer to a total
volume of 10ml) was added and left in a cold room overnight. The membrane was
washed 3x with TBST buffer (20mM Trisbase, 150 mM NaCl, 0.1% Tween 20) with
a waiting time of 5 minutes in between. The secondary antibody (2ul secondary
antibody (anti rabbit made in goat), 1ml blocking solution, filled up with TBST
buffer to a total volume of 10ml) was added. The secondary antibody which is
coupled to the HRP enzymes carrying out a chemical reaction which produces
photons, will bind to the primary TTM3 antibody. The membrane was left in the
secondary antibody solution for 1 hour. The membrane was washed 3x with TBST
buffer (20mM Trisbase, 150 mM NaCl, 0.1% Tween 20) with a waiting time of 5
minutes in between. The Western Blot was developed with a Chemiluminescence
Western Blotting Kit.
GUS results
The blue colour indicates where the TTM3 is expressed in the plant.
Our GUS experiment shows that TTM3 is expressed in:

Seedlings: cotyledons (specially in vascular bundles), hypocotyls and shoot
apical meristem.

Leaves: specially in vascular bundles.

Flowers: stigma and stamen.
Department for Botany and Plant Biology,
University of Geneva,
Switzerland
1.Leave
2.Stigma
3.Seedling
3.
Western Blot results
1.Columbia
2.TTM3 Mutant
3.Marker
4.TTM3-GUS leaves
5.TTM3-GUS flowers
6.TTM3-GUS seedlings
Size of TTM3 25kDa,
size of TTM3-GUS 100kDa
At the 25kDa line we can see that the TTM3 is
expressed in all the plant material except the
mutant. This shows us that the mutant contains
no TTM3.
At the 100kDa line we find the TTM3 coupled with
the GUS enzyme. We find this TTM3-GUS only in
the GUS plants.
Department for Botany and Plant Biology,
University of Geneva,
Switzerland
The nitrocellulose membrane was stained with
Ponceau. This solution stains proteins and allows
us to check, if similar amounts of protein sample
were loaded onto the SDS-Gel.
Our Western Blot experiment showed that TTM3 is expressed in flowers, leaves
and whole seedlings.
References
Martinez J., Truffault V. and Hothorn M. (2015) Structural Determinants for
Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes, JOURNAL
OF BIOLOGICAL CHEMISTRY, VOLUME 290, NR 38
Acknowledgements and affiliations
I would like to thank the Structural Plant Biology Laboratory, Department of
Botany and Plant Biology of the University of Geneva who let me have a look into
their lab and taught me a lot about Plant Biology and its importance. I would also
like to thank my two tutors Laura Lorenzo and Rebekka Wild for supervising me
during the week. Laura has carried out the two experiments with me and was
very competent and happy to explain me all the things whereas Rebekka has
helped me a lot to write my report and to finalize my poster.
I would also like to thank Michael Hothorn for taking me into his laboratory and
also supporting me during the week. You made this week to an unforgettable
memory.
I would also like to thank “Schweizer Jugend Forscht” to give me the opportunity
for this week.
Department for Botany and Plant Biology,
University of Geneva,
Switzerland