1. Supplemental Figure S1. Correlation between T6P, and sugar phosphates. A, T6P and glucose 6-phosphate. B, T6P and glucose 1-phosphate and fructose 6-phosphate. µmol g -1 FW T6P (nmol g -1 FW)

2. 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. SnRK1 activity nmol min -1 mg -1 protein SnRK1 activity T6P inhibition (%)

3. TPS (consensus tree) TPS1 TPS4 TPS3 TPS2 TPS5 TPS8 TPS9 TPS10 TPS11 TPS7 TPS6 TPP (consensus tree) TPPD TPPC TPPB TPPE TPPH TPPI TPPJ TPPA TPPF TPPG AB 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.

4. AKIN10 β1β1 β3β3 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.S1 C, 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

5. 0 2 4 6 8 CPS x10 5 2 4 6 8 101214161820222426 Minutes 0 0 T6P L1P 2 4 6 8 CPS x10 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.