0

1

2

3

C S3 S2 S1

1-seed C quantity (mg)

S1

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.

S2

S3

(a)

(b)

(c)

(d)

Ratios of SCN contents (S-starved versus control plants)

0

0.01

0.02

C S3 S2 S1

1-seed S quantity (mg)SCN quantities (mg) per seed(e)

SS

CC

N

0

0.25

0.5

C S3 S2 S1

1-seed N quantity (mg)

NN

***

**

***

***

Co S3 S2 S1

Co

**

**

**

**

****

*

****

****

**

**

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.

SOSO442-2-

APS

SO32-

PAPS

γ-Glu-Cys

S2-

Ser

Cys

Glu

Gly

GSH

Vacuole

SOSO442-2-

MtSULTR4;1ATP-

sulfurylase

5'-phosphosulfate reductase

Sulfite reductase

o-acetyl-serine-thiol-lyase

γ-glutamylcysteine (γ-Glu-Cys) synthetase

glutathione (GSH) synthetase

Co S2

*

OAS

Met

Co S2

Co S2

Co S2Co S2

Co S2

Co S2

Co S2 Co S2

GSSG

AdoMet

Co S2Co S2

a

Co S2

b

Co S2

c

Co S2

d

Co S2

Co S2

Co S2

Co S2

Co S2

f

Co S2

e

*

***

*

**

*

Co S2

µg/m

g

Sulfate content

Relative transcript levels (arbitrary units)

Relative metabolite levels (arbitrary units)

***

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).

******

(a) (c)

(d)Anion contents in mature seeds

µg/

mg

of s

eed

dry

wei

ght

Sulfate (µg/mg) % of S-sulfate

14 dap maturity

A A

B

C

A A

B

C

(b) S content (µg/mg)

Co S20

2

4

6

Co S2 Co S2

B

A

CA

97.4

66.2

45.0

31.0

21.5

14.4

4 5 6 7 8 10

pIMr (kDa)

52

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/

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.

26 2161

3

3

Down-regulated polypeptides

S1 S2

S3

(a)

30

43

3

1

1

S1

S2

S3

(b)

Up-regulated polypeptides