Ratios of SCN contents
(S-starved versus control plants)
(e)
1-seed
S quantity
SCN
quantities
(mg) (mg)
per seed
(a)
S
1
0.02
0.5
**
**
***
0.01
***
1-seed C quantity (mg)
0
0
(b)
C
S3
S2
S1
3
1
*
0.5
C
2
**
**
**
1
1-seed N quantity (mg)
**
0
C
0
S3
S2
0.5
(c)
S1
N
**
1.5
***
0.25
N
1
0
0.5
0
**
**
Co
C
S3
S3
S2
S2
S1
S1
**
(d)
**
1.5
S1
S2
1
S3
0.5
0
Co
****
S
C
N
Figure S1: Effect of S-deficiency applied at different growth stages on SCN contents in plant
parts of M. truncatula. SCN contents (% on a dry weight basis) were measured in different
compartments, i.e. mature seeds (a), empty mature pods (b), straws (c), and roots (d). These data are
presented under ratio (versus control). Bars represent mean ratio +/- standard deviation. (e) Quantity
(mg) of CNS per seed in the different plant samples. An analysis of variance (ANOVA) followed by
a Tukey test was performed to reveal significant variations (n=6). ** and * indicate a P<0.01 and
0.05, respectively. See Figure 1 for details on the plant samples.
SO426
ATPsulfurylase
*
4
2
2
1
S2
Co
APS
S2
Vacuole
5'-phosphosulfate
reductase
1
1
Ser
0
Co
S2
2
4
1
2
0
0
Co
S2
2
c
1
1
OAS
Co
0
Co
Co
S2
S2
6
d
4
S2
2
2
1
1
0
0
Co
Co
2
*
S2
S2
1
0
0
Co
Cys
γ-glutamylcysteine
(γ-Glu-Cys) synthetase
S2
S2-
2
0
**
2
o-acetyl-serine-thiol-lyase
b
S2
4
2
0
6
S2
*
2
SO32-
a
Co
2
3
Co
3
***
PAPS
0
Sulfite
reductase
1
0
0
0
Co
2
µg/mg
3
MtSULTR4;1
Sulfate content
SO42-
Co
S2
Co
S2
AdoMet
Met
2
2
Glu
1
1
0
S2
Co
glutathione (GSH)
synthetase
3
e
2
γ-Glu-Cys
2
f
Co
***
1
0
0
Co
S2
S2
2
Gly
1
1
0
Co
0
S2
GSH
Co
GSSG
S2
Relative transcript levels (arbitrary units)
2
2
1
1
*
0
0
Co
S2
Co
S2
Relative metabolite levels (arbitrary units)
Figure S2: Effect of S depletion from the flowering stage on the relative levels of transcripts, anions and metabolites
related to S metabolism and transport in 14 dap seeds. All sequences were retrieved from the 3.5 M. truncatula
genome version and/or from the M. truncatula Gene Index (http://compbio.dfci.harvard.edu/tgi/): 1) MtSULTR4;1
(Medtr7g022870), similar to the Arabidopsis sulfate transporter AtSULTR4;1; 2) ATP-sulfurylase (TC146218); 3) 5'phosphosulfate reductase (TC155557); 4) Sulfite reductase (TC154269); 5) O-acetyl-serine-thiol-lyase (a:
Medtr4g087520; b: Medtr1g081620; c: Medtr5g006340; d: Medtr4te087510); 6) γ-glutamylcysteine (γ-Glu-Cys)
synthetase (Medtr8g098350); 7) Glutathione (GSH) synthetase (e: Medtr7g113880; f: Medtr7g113890). Relative transcript
levels were determined by RT-qPCR in 14 dap seeds from non-starved (Co) and sulfur-starved (S2) plants. The relative
levels of transcripts (black bars) and metabolites (grey bars) are shown relative to the levels in control plants, which was
set to 1. The data are mean ± standard error of at least three biological replicates of plants grown independently.*, **
indicate P<0.05 and <0.01, respectively, between S2 and Co plants. TC, Tentative consensus sequence from the M.
truncatula Gene Index. AdoMet, S-adenosylmethionine; APS, adenosine 5'-phosphosulfate; GSH, reduced glutathione;
GSSG, oxidized glutathione; PAPS, 3'-Phosphoadenosine-5'-phosphosulfate; OAS, O-acetylserine.
(a)
Sulfate (µg/mg)
2
(b)
6
AA
B
C
20
A
C
2
S2
(d)
AA
B
10
C
0
Co
14 dap
µg/mg of seed dry weight
A
0
0
Co
(c) % of S-sulfate
B
4
1
S content (µg/mg)
S2
Co
S2
maturity
Anion contents in mature seeds
6
5
4
Co
S2
3
2
1
***
***
0
CHLORIDE
NITRATE
PHOSPHATE
SULFATE
Figure S3: S and anion contents in 14 dap and mature seeds of plants subjected (S2)
or not (Co) to S deficiency at flowering. Sulfate and total S contents (µg/mg of seed dry
weight) were determined (a, b) and the proportion of S under sulfate form (S-sulphate)
calculated (c). In (d) are compared chloride, nitrate, phosphate and sulfate contents
(µg/mg of seed dry weight) of mature seeds. The bars represent the mean ± standard error
of at least 4 biological replicates. Different letters indicate statistical differences with
P<0.05 and *** indicates P<0.001 (Tukey test).
pI
Mr
4
(kDa)
5
6
7
10
8
97.4
66.2
45.0
31.0
52
21.5
14.4
Figure S4: Proteome map of mature M. truncatula seeds analyzed in the present study. The figure shows the
position and number of the protein spots, which were focused in 24cm IPG strips (non linear pH 3-10) and separated
according to their molecular weight in 10% SDS-PAGE in the second dimension. The image represents a typical 2D gel
obtained
from
mature
seeds
of
control
plants.
A
clickable
map
is
available
at
http://www.thelegumeportal.net/medicago-seed-proteome/
(a)
(b)
Down-regulated polypeptides
S1
S2
Up-regulated polypeptides
S1
30
26
61
21
43
3
3 S3
1
1
S2
3
S3
Figure S5: Venn diagram of protein distribution in seeds from plants subjected to
S-deficiency at different growth stages. (a) Polypeptides whose abundance decreased
significantly in the S-starved plants compared to control plants. (b) Polypeptides whose
abundance increased significantly in the S-starved plants as compared to control plants.
These proteins showed significant variations in their normalized volumes with P<0.05.
S1, S depletion when tertiary branches appear and secondary branches start to elongate;
S2, S depletion at flowering; S3, S depletion at the onset of seed filling. See Table S1 for
protein abundance variations.