Supporting Information Methods S1, Notes S1, Figs S1–S4
Jian Liu et al.
Methods S1
Cell viability assay
Viability of suspension cells was determined by fluorescein diacetate
(FDA)-propidium iodide (PI) staining. FDA can pass through living cell membranes
and is converted to a green fluorescent dye by intracellular esterases found
exclusively in living cells. By contrast, PI passes through dead cell membranes and
generates a red fluorescence by forming a PI–nucleic acid conjugate. The FDA-PI
solution was made fresh each time by adding 1 μl PI and 1 μl FDA to 98 ml ultra high
purity water. Ten μl FDA-PI solution was added to 90 μl solution containing cells and
incubated for 2 min at room temperature. Stained cells with FDA-PI were observed
with a light/fluorescence microscope (Olympus BX51, Japan). The experiment was
conducted three times.
Transmission electron microscopy (TEM) characterization
TEM was performed using JEM-1200EX (JEOL, Japan). Cell specimens were fixed
with gluteraldehyde (2.5 %), OsO4, and were dehydrated in ethanol/acetone. Then
they were embedded in Epon812/Araldite M resin and were ultrathin sectioned with a
Reichert ultratome (Zeiss, Germany).
Notes S1 Details of viability of cells
To identify the role of Si in Cd tolerance on individual cells, we determined the
viability of cells following a 2-month culture. The results showed that the viability of
cells in culture medium without Cd in the presence and absence of 1 mM silicic acid,
was 65 ± 7% (n = 220) and 40 ± 2% (n = 200), respectively. Upon shifting to higher
Cd concentration to 30 μM, the viability for Si-limiting cells (-Si) dropped to 29 ± 3%
(n = 160), whereas the viability for Si-accumulating cells (+Si) was 50 ± 5% (n = 130).
When the Cd concentration was increased to 60 μM, only about 20% of all cells,
regardless of the presence or absence of Si in the medium, could survive (Fig. S3A);
the -Si cells were much more susceptible to the elevated Cd concentration than +Si
cells. A consistent result of cell viability at a Cd concentration of 30 μM was achieved
by using a two-color fluorescent probe (Fig. S3B, green corresponds to viable cells,
red to dead ones).
Fig. S1 Effect of Cd2+ on the uptake of Ca2+ in suspension cells of rice cultivated in
the absence (-Si) and presence (+Si) of 1 mM silicic acid for 2 months after
transplantion to solution medium from solid culture medium. (A) Net Ca2+ fluxes in
cells before and after exposure to 30 μM Cd2+. (B) The mean rate of Ca2+ fluxes
before and after exposure to 30 μM Cd2+. Five individual cells were measured.
Fig. S2 Effect of Cd2+ on the uptake of Ca2+ in root cells of rice (500 μm from root tip)
grown in the absence and presence of 1.0 mM silicic acid for 1 month in a hydroponic
culture of whole plants. (A) Net Ca2+ fluxes in roots before and after exposure to 30
μM Cd2+. (B) The mean rate of Ca2+ fluxes before and after exposure to 30 μM Cd2+.
Three individual roots were measured.
Fig. S3 (A) Viability of suspension cells following 5 d exposure to different Cd
concentrations ranging from 5 to 60 μM (n = 7). (B) In situ fluorescent microscopy of
suspension cells following 5 d exposure to the free Cd concentration of 30 μM in the
aqueous medium, showing those that remained viable (green) or died (red).
Fig. S4 TEM images of the ultra microstructure of suspension rice cells cultivated in
the absence and presence of 1 mM silicic acid and/or 30–60 μM Cd2+. In the absence
of Cd in culture medium, (A, B) organelle structures of individual cells including
mitochondria and Golgi were relatively normal regardless of the absence or presence
of Si. However, In the presence of Cd in medium, (C, D) -Si cells experienced
obvious structural damage after a 5-d culture and no intact organelles were present.
Precipitates (a typical Cd toxic symptom) with high electron density formed in
vesicles (shown by an arrow in D), whereas the structure of organelles remained
relatively complete for +Si cells, although small amounts of precipitate were also
present in vesicle.
80
60
Adding 30 μM Cd2+
-2
-1
Net Ca flux (pmol cm s )
A
+Si cells
40
2+
20
0
-20
-40
80
0
200
400
600
800
1000
1200
Time (seconds)
-Si cells
40
20
0
2+
-2
-1
Net Ca flux (pmol cm s )
60
-20
-40
-60
0
200
400
600
800
1000
1200
Time (seconds)
B
a
14
12
2+
-2
-1
Net Ca flux (pmol cm s )
+Si Cells
-Si Cells
a
13
11
a
10
9
8
a
7
6
5
4
3
2
1
0
Before adding Cd ions
Fig. S1
After adding Cd ions
A
-Si root cells (500 m from the tip)
+Si root cells (500 m from the tip)
60
30 μM Cd2+
2+
-2
-1
Net Ca flux (pmol cm s )
50
40
30
20
10
0
-10
-20
-30
0
200
400
600
800
1000
Time (seconds)
B
28
+Si Roots
-Si Roots
20
16
a
a
12
2+
-2
a
a
-1
Net Ca flux (pmol cm s )
24
8
4
0
Before adding Cd ions
After adding Cd ions
Fig. S2
1200
A
B +Si
+Si + Cd
80
Cell viability (%)
70
-Si cells
+Si cells
60
50
20 μm
20 μm
40
30
-Si + Cd
-Si
20
10
0
10
20
30
40
50
60
Cadmium in medium (M)
20 μm
Fig. S3
+Si, A
-Si, B
+Si+Cd, C
-Si+Cd, D
Fig. S4