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University of Tokyo Research Internship Program 2012
Project Report – Fukuda Lab
Identification of factors involved in Xylem Cell
Differentiation
Aarush Mohit Mittal1, 2
1
Department of Biological Sciences and Bio-Engineering, Indian Institute of Technology, Kanpur, India
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan
2
The vascular system of plants is composed of mainly two types of cells – Xylem and Phloem.
Xylem are the water carriers and the secondary cell wall of these cells also provides mechanical
support to the plant body. This cell wall is primarily composed of cellulose which is deposited in
several distinct patterns regulated by a two-dimensional network of microtubules. With the aim of
finding which factors regulate this microtubule network, I tested 6 different inhibitors of cell
differentiation and characterized each of them based on their effect on the differentiation rate and
the pattern of cell wall formation. I then chose 2 inhibitors out of the 6 to further investigate their
role in the process of microtubule regulation. As each inhibitor affected a different factor in the
cell, these factors are possible candidates in the regulation of microtubule network and further
studies need to be performed to fully elucidate their exact role in the process.
1
Introduction
Xylem cells are the most important cells for
water transport in plants and they form a large
network of vessels throughout the plant body.
Primary xylem vessels have two kinds of cells –
Protoxylem and Metaxylem. Protoxylem are the
first cells to develop and they further
differentiate into metaxylem as the plant
matures. In the roots, the metaxylem are found
close to the center and the protoxylem are
arranged around the metaxylem cells. This
arrangement is known as exarch (see Figure 1).
The secondary cell wall pattern is determined by
the deposition of cellulose on a two-dimensional
network of microtubules. These microtubules
are in turn regulated by several different factors
in the cell which leads to such specific
networks. It has been seen that certain inhibitors
that affect cell differentiation also affect the
pattern of secondary cell wall pattern. As each
of these works in a different way, they would be
useful in identifying the factors involved in the
process.
The secondary cell wall of these cells is of
special interest as it provides mechanical
support to the plant organs. It is primarily
compose of cellulose, hemicellulose and lignin.
Xylem cells show different patterns of
secondary cell wall depending on their stage of
differentiation. Most common patterns are
annular, spiral, reticulate and pitted. Protoxylem
exhibit spiral patterns while mature Metaxylem
is pitted (see Figure 1).
2
Experimental Plan
The first experiment (Experiment 1) was to
check the optimum time at which most of the
cultured cells are differentiated. This was done
using an established cell culture protocol for
Arabidopsis thaliana cells. The differentiation
was induced in triplicate and the cells were
characterized at 23hrs, 25hrs and 43hrs after
induction.
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Then I checked (Experiment 2) the effect of 5
inhibitors, namely Orizine (Orz), Taxol,
Latrunculin B (LatB), RacI and Okadaic Acid
(OA), on cell differentiation using cultured
cells. All the inhibitors were at a concentration
of 10µM except OA. OA was tested at 3
different concentrations (0.005µM, 0.05µM and
0.5µM). This was done the optimum
concentration at which OA completely inhibits
differentiation was not known. The cells were
characterized at 24hrs after induction.
The next step was to check the effect of these
inhibitors on Arabidopsis plants grown in-vitro.
For this purpose I chose 2 inhibitors, Taxol and
OA as these two have different mechanisms by
which they inhibit differentiation. Taxol
stabilizes microtubules and thus prevents them
from elongating. In contrast, OA inhibits protein
phosphatases like PP1, PP2A etc. These
phosphatases are responsible for a variety of
functions in the cell including cell
differentiation. Also time constraints prevented
me from checking other inhibitors.
The plants were grown (Experiment 3) on
normal media for 10 days and then transferred
to different plates containing different
concentrations of Taxol (10µM and 20µM) and
OA (0.05µM, 0.5µM and 2µM). The plant roots
were observed under microscope on the 13th
day.
To verify the importance of protein
phosphatases in the process of cell
differentiation, I tested (Experiment 4) another
inhibitor, namely Calyculin A (CalA), as it has
the same modus operandi as OA. CalA was
tested at 7 different concentrations (0.05nM,
0.5nM, 0.005µM, 0.01 µM, 0.05µM, 0.1µM and
0.5µM). Cells were observed at 24hrs after
induction.
The last experiment (Experiment 5) was done to
check the localization of the identified factors
on the secondary cell wall. For this plasmids
were made for different subunits of PP2B,
tubulin and MAP70 (see Appendix). The cells
were transformed using these plasmids and
cultured. Differentiation was induced in these
cells and the observations were recorded at
24hrs after induction.
3
Materials and Methods
3.1
Arabidopsis Cell Culture
Arabidopsis thaliana suspension cells, strain
Columbia-0, were cultured in 27mL of modified
Murashige and Skoog (MS) medium, pH 5.8,
containing 4.33g·L–1 of MS inorganic salts,
4.1mM 2,4-D, 3.0% (w/v) sucrose, and
vitamins, including 8mg·L–1 nicotinic acid,
8mg·L–1 pyridoxine-HCl, 80mg·L–1 thiamineHCl, and 800mg·L–1 myoinositol. The cells
were agitated on a rotary shaker at 124 rpm at
23°C in the dark. At weekly intervals, 12mL
aliquots of the culture were transferred to 15mL
of fresh medium in a 100mL culture flask.
3.2
Induction of Differentiation
To induce differentiation, a 1mL aliquot of 7-dold cell culture was suspended in 9mL of 2,4-D–
free MS medium. After 3 min of settling, 5mL
supernatant was removed to adjust cell density.
The remaining cell suspension was supplied
with 2µM estrogen and 2µM brassinolide, and
cultured on a rotary shaker at 124 rpm at 23°C
in the dark. 1µL of inhibitor was added in each
of the wells.
3.3
In-vitro growth of Arabidopsis plants
Arabidopsis ecotype Columbia was used as the
wild-type plant. Seedlings were germinated on
half-strength MS phytogel plates in a Percival
incubator with 24hrs light for 7 to 10 d at 22°C.
The plants were transferred to half strength MS
phytogel
plates
containing
different
concentrations of inhibitor as mentioned above.
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3.4
Cell Transformation
Agrobacterium tumefaciens strain GV3101
(MP90) was first transformed using the 9
different plasmids (see Appendix) by
electroporation. The bacteria carrying the
expression constructs were grown in LuriaBertani media with Spectinomycin for 3 days at
3°C.
To establish a transformant strain, 3-d-old cells
were co-cultured with the transformed bacterial
cells in MS medium supplemented with
50mg·L–1 acetosyringone for 2 d. Then,
0.5g·L–1 claforan was added to the cell
suspension and the cells were cultured for a
further 5 d. Thereafter, the cell suspension was
transferred into 15mL fresh medium and
maintained as described above, and the
surviving cells were used for experiments.
3.5
Characterization of cells
To stain secondary cell walls, the cells were
incubated with 1mg·L–1 WGA–AlexaFluor 594
(Invitrogen) for 15 min
The plant roots were stained using 1% safranin
in 30% EtOH. This was followed by washing in
different concentrations of EtOH from 30% to
100% at 15 min intervals to remove the excess
stain. The roots were then fixed in clear solution
and the coverslip was sealed using manicure.
Differential Interference Contrast (DIC)
microscopy was used to visualize the cultured
cells and the plant roots. Fluorescence
microscopy was used to visualize the secondary
cell walls at 561nm and GFP tagged proteins in
the transformed cells at 488nm. Confocal
Microscope was used to visualize the
transformed cells and the cultured cells at high
resolution.
4
Results
4.1
Experiment 1
The rate of differentiation started to increase at
about 19hrs after induction and reached the
saturation point at about 25hrs. After this time
there was a slight reduction in the rate (Figure
2). Also under microscope, the control cells
showed no secondary cell wall pattern while a
spiral pattern was clearly visible in the
secondary walls of the induced cells (Figure 3).
4.2
Experiment 2
The different inhibitors showed different
characteristics
regarding
the
rate
of
differentiation (Figure 4) and pattern of
secondary cell wall (Figure 5). Orz completely
disrupted the pattern but had no effect on
differentiation rate. LatB highly affected the
differentiation rate and also affected the pattern.
RacI had a moderate effect on the differentiation
rate but the pattern appeared almost normal.
Taxol affected both differentiation rate and the
pattern of secondary cell walls which appeared
to be slightly lopsided as compared to the
control. OA had no effect on both differentiation
rate and pattern at low concentrations (0.005µM
and 0.05µM). But at 0.5µM it severely affected
the differentiation rate and completely disrupted
the pattern.
4.3
Experiment 3
OA at concentration 2µM showed disruption of
pattern in protoxylem. However the metaxylem
seems to be unaffected. It inhibits root tip
elongation and formation of root hair with
increasing concentrations showing increased
effect. Also at 0.05µM we can see that the
gravity sensing of the roots is also affected. This
is peculiar and needs further verification (Figure
6,7).
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University of Tokyo Research Internship Program 2012
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Taxol severely affected the protoxylem at the
root tip showing the lopsided spiral pattern
similar to that seen in the cultured cells before.
Also the pattern of secondary cell wall in
metaxylem seems to be very faint. It exhibits
reduced rate of elongation of root tip as well
(Figure 6,7).
4.4
Experiment 4
CalA shows an effect similar to that of OA, as
predicted by previous studies. At concentrations
below 5nM the differentiation rate is affected.
However the pattern is similar to normal
secondary cell wall pattern. At 5nM
concentration the differentiation rate is reduced.
The pattern is affected in most of the cells and
some cells are dead. At 10nM concentration,
there is very low differentiation rate. Only a few
cells show pattern and many cells are dead. At
concentrations above 10nM almost all the cells
are dead (Figure 8).
4.5
Experiment 5
2052, 2129, 2131 and 2404 appear to be
localized at the cytoplasm. 2051 and 2202 are
localized at the cytoplasm but they show no
differentiation. 2130 appears to localize along
the secondary wall. Tub6 and MAP70 localize
along the secondary cell wall pattern (see
Appendix for complete nomenclature) (Figure
9).
5
Discussion
The results from Experiment 1 suggested that
the best time to observe differentiated cells is
between 23-25hrs after induction. Thus for
further experiments, the time point of 24hrs
after induction was set to observe the cells.
The different inhibitors tested in Experiment 2
show that different factors affect the
differentiation pattern differently. Thus while
some inhibitors affected both the differentiation
rate as well as the type of pattern formed (Taxol,
OA and LatB), others only affected the
differentiation rate (RacI) and some only
disrupted the pattern (Orz) and had no effect on
the differentiation rate. Thus the factors
inhibited by each of these become possible
candidates for future studies to find out the
exact mechanism of the pattern formation in
secondary cell walls.
Also, higher concentrations of OA eventually
lead to cell death. This maybe because the
protein phosphatases it inhibits may be
responsible for other vital functions of the cell.
The role of phosphatases was validated by the
experiment using the inhibitor CalA. It showed
the same inhibition characteristics as OA. The
only difference was in the concentration
required to inhibit completely the differentiation
process. CalA was found to be 10 times more
potent than OA as it completely inhibited at
0.05µM while OA showed the same at 0.5µM.
Due to time constraints, I could only check for
the effect of 2 inhibitors (Taxol and OA) on the
WT plants grown in-vitro. As these have
different mechanisms of action, they provide
insight into the role of two different factors. As
plants grown in OA showed decreasing rate of
root tip elongation as the concentration
increased from 0.05µM to 2µM, it is possible
that due to reduced differentiation rate, the
xylem vessels lose their ability to elongate.
Similarly in the case of Taxol, the root tip
elongation is disrupted. However due to the very
different pattern of protoxylem in this case, the
root tips show a very bulbous appearance.
The root hairs are also reduced in the case of
OA but they seem to be increased in the case of
Taxol. The gravity sensing mechanism
disruption in the case of OA is also a curious
phenomenon. These need further studies to
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verify and find out the exact reason behind these
results.
The experiment with transformed cells showed
some interesting results. The transformed cells
2052, 2129, 2131 and 2404 don’t seem to be
associated with secondary cell wall patter n
formation as they localized at the cytoplasm.
2051 and 2202 are potential candidates as
inhibitor proteins because they showed no cell
differentiation.
2130 is the only tested phosphatase that shows
promise as being associated in the process of
secondary wall pattern formation. Thus it needs
to be investigated in future studies to figure out
how exactly it controls the microtubule network.
Tub6 and MAP70 are the two other proteins
which have been shown to be associated in the
pattern formation.
Thus, in the above study I succeeded in testing a
variety of inhibitors and found out 3 factors
which may be directly involved in the process of
pattern formation. These experimental results
need to be verified and studied in future
research. Also the other untested inhibitors
provide ample material for future research.
Knockout studies can be performed on the
factors identified to elucidate their exact role in
the cell. As we gain more information about
how this whole process takes place, we can then
modify different factors to invent novel patterns
or structures that may have beneficial functions.
6
7
References
Yoshihisa Oda, Hiroo Fukuda, Secondary cell wall
patterning during xylem differentiation, Current Opinion
in Plant Biology, 2012.
Kyoko Ohashi-Ito, Yoshihisa Oda, Hiroo Fukuda,
Arabidopsis VASCULAR-RELATED NAC-DOMAIN6
directly regulates the genes that Govern programmed cell
death and secondary wall formation during xylem
differentiation, Plant Cell, 2010.
Yoshihisa Oda, Seiichiro Hasezawa, Cytoskeletal
organization during xylem cell differentiation, Journal of
Plant Research, 2006.
8
Appendix
The protein phosphatase PP2B is a protein
composed of a number of subunits. Tubulin
(Tub) is the main component of microtubules
while MAP70 (Microtubule Associated Protein)
has been shown to bind to microtubules. The
plasmid constructs include the GFP (Green
Fluorescent Protein) gene which can be used to
visualize the translated protein. The plasmids
used are:









2051PP2A-B’-theta-GFP
2052PP2A-B’-zeta-GFP
2129PP2A-B’-eta-GFP
2130PP2A-B’-gamma-GFP
2131PP2A-B’-delta-GFP
2202PP2A-B’-beta-GFP
2404PP2A-B’-alpha-GFP
31GFP-MAP70-7
GFP-TUB6
Acknowledgements
I am extremely grateful to Professor Fukudasensei for offering me a place in his laboratory;
Assistant Professor Oda-san, Katayama-san and
Nakashima-san for their guidance and advice on
the practical and analytical aspects of my
project; Soeda-san for her support throughout
the program; and finally all the members of the
Fukuda Lab for making me feel so welcome.
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Figure 1: Exarch arrangement in Arabidopsis root. Note
the spiral pattern in protoxylem and the pitted pattern in
metaxylem.
Figure 2: The percentage of differentiation as a function of time. The optimum time for observation of differentiated cells is between
23h to 25h. The Average is over the 3 experiments conducted simultaneously.
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Figure 3: Extent of differentiation in cultured
cells. Red colour shows the presence of
secondary cell wall.
1.a,1.b,1.c: Control cells(uninduced) at 23h, 25h
and 43h respectively.
2.a,2.b,2.c: Induced cells at 23h, 25h and 43h
respectively.
3.a,3.b,3.c: Induced cells at 23h, 25h and 43h
respectively.
4.a,4.b,4.c: Induced cells at 23h, 25h and 43h
respectively.
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Figure 4: The effect of different inhibitors on the differentiation rate. Values taken at 24h after induction.
Figure 5: The effect of different inhibitors on the
secondary cell wall pattern. Values taken at 24h
after induction. The concentration of OA is in µM
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Figure 6: The effect of Taxol and OA on the roots of the WT Arabidopsis plants grown in-vitro.
Figure 7: The pattern of secondary cell walls in the roots of WT plants. a – Control(MS only); b – Protoxylem of plants grown in 2µM
OA; c – Metaxylem of plants grown in 2µM OA; d – Protoxylem of plants grown in 10µM Taxol; e – Metaxylem of plants grown in
10µM Taxol; Red colour shows the presence of secondary cell wall.
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Figure 8: The effect of Calyculin A on the rate of differentiation at different concentrations.
Figure 9: The localization of
different proteins on the xylem
cells. Green shows the presence of
protein of interest. Red shows the
presence of secondary cell wall.
a – 2051, b – 2051, c – 2129, d –
2130, e – 2131, f – 2202, g – 2404,
h – MAP70, i – Tub6
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