T6P nmol/g FW

0 20 40 60 80

nmol

/g F

W

0

50

100

150

200

250

300

Glucose 1-phosphateFructose 6-phosphate

B

Glucose 6-phosphate

T6P (nmol/g FW)0 20 40 60 80

nm

ol/g

FW

0

100

200

300

400

500

ASupplemental Figure S1. Correlation between T6P, and sugar phosphates.A, T6P and glucose 6-phosphate. B, T6P and glucose 1-phosphate and fructose 6-phosphate.

µm

ol g

-1 F

Wµ

mol

g-1 F

W

T6P (nmol g-1 FW)

T6P (nmol g-1 FW)

days after anthesis

0 10 20 30 40 50

S

nRK

1 ac

tivity

nmol

/min

/mg

prot

ein

0

2

4

6

8

AMARA

SPS

days after anthesis

0 10 20 30 40 50

SnR

K1

activ

ity

T6P

inhi

bitio

n (%

)

0

20

40

60

80

100

AMARA

SPS

A

B

Supplemental Figure S2. SnRK1 activity in most recently fully expanded flag leaves. A, Between 1 and 45 DAA using AMARA and SPS as substrates. B, Between 1 and 45 DAA using AMARA and SPS peptide as substrate in the presence of 1 mM T6P in the assay.

SnR

K1

activ

itynm

ol m

in-1 m

g-1 p

rote

inS

nRK

1 ac

tivity

T6P

inhi

bitio

n (%

)

AT

1G06410

AT

1 G1 70 00

0.2

TPS (consensus tree)

TPS1

TPS4TPS3TPS2

TPS5

TPS8TPS9TPS10

TPS11

TPS7TPS6

0.05

TPP (consensus tree)

TPPD

TPPC

TPPBTPPETPPH

TPPI

TPPJ

TPPATPPFTPPG

A B

Supplemental Figure S3. Phylogenetic trees of Arabidopsis (TAIR9) and Brachypodium distachyon (Bd21_v1 gene models) protein sequences for the TPs and TPP gene families. Trees were created with the PHYML program (Guindon et al., 2005) implementing the WAG model of amino-acid substitution (Whelan and Goldman, 2001) from a full-length alignment (excluding gaps). Consensus trees from 100 bootstrap runs shown. A, trehalose phosphate synthase. B, trehalose phosphate phosphatase.

days after anthesis

0 10 20 30 40 50

Tra

nsci

pt a

bund

ance

0

200

400

600

800

1000

C

days after anthesis

0 10 20 30 40 50

Tra

nscr

ipt a

bund

ance

0

200

400

600

800

1000

days after anthesis

0 10 20 30 40 50

Tra

nscr

iopt

abu

ndan

ce

0

200

400

600

800

1000

B E

days after anthesis

0 10 20 30 40 50

Tra

nscr

ipt a

bund

ance

0

1000

2000

3000

4000

5000

days after anthesis

0 10 20 30 40 50

Tra

nscr

ipt a

bund

ance

0

200

400

600

800

1000

A DAKIN10 β1

β3

βγ

γ

Supplemental Figure S4. SnRK1 gene transcript abundance during grain development. Multiple wheat Affymetrix probesets which can match to more than one Arabidopsis gene are plotted. A, AKIN10 ●, Ta.2525.1.S1; ○, TaAffx.92935.1.A1;▼, Ta.1312.1.S1; ■, Ta.27319.1.S1. B, β3 subunit. ●, Ta.19103.1.S1_x; ○, Ta.19103.1.S1C, βγ subunit. Ta.9920.1.S1. D, β1 subunit. ●, Ta.13624.1.S1; ○, Ta.13624.2.S1; ▼, Ta.3840.1.S1_at; ∆, Ta.3840.2.S1_at.E, γ subunit. ●, Ta.6899.3.S1_a; ○, Ta.6899.1.S1

0

2

4

6

8

CPS

x10

5

2

4

6

8

10 12 14 16 18 20 22 24 26

Minutes

0

0

T6PL1P

2

4

6

8

CPS

x10

5CP

S x1

0 5

M6P

S6P

A

C

B

Supplemental Figure S5. Separation of phospho-disaccharides with identical mass as T6P in wheat extracts using the LC/MS method described by Delatte et al. (2009). Counts per second (CPS) of ions of a specific mass. A, Internal standard lactose-1-phosphate (L1P) added prior to extraction. B, With maltose-6-phosphate (M6P) added before injection. C, With sucrose-6-phosphate (S6P) added before injection.