1. Supplemental Figure 1 GhCWIN1 (1) Atßfruct2 (1) Lin6 (1) NtCWIN1 (1) OsCWIN1 (1) ZmCWIN1 (1) Atßfruct4 (1) GhVIN1 (1) GhCWIN1 (45) Atßfruct2 (45) Lin6 (50) NtCWIN1 (49) OsCWIN1 (47) ZmCWIN1 (52) Atßfruct4 (98) GhVIN1 (96) GhCWIN1 (130) Atßfruct2 (129) Lin6 (133) NtCWIN1 (132) OsCWIN1 (130) ZmCWIN1 (135) Atßfruct1 (198) GhVIN1 (190) GhCWIN1 (224) Atßfruct2 (224) Lin6 (229) NtCWIN1 (227) OsCWIN1 (226) ZmCWIN1 (232) Atßfruct1 (292) GhVIN1 (285) GhCWIN1 (317) Atßfruct2 (320) Lin6 (324) NtCWIN1 (322) OsCWIN1 (318) ZmCWIN1 (326) Atßfruct1 (389) GhVIN1 (380) GhCWIN1 (414) Atßfruct2 (417) Lin6 (421) NtCWIN1 (419) OsCWIN1 (415) ZmCWIN1 (426) Atßfruct1 (487) GhVIN1 (479) GhCWIN1 (499) Atßfruct2 (513) Lin6 (510) NtCWIN1 (508) OsCWIN1 (503) ZmCWIN1 (515) Atßfruct1 (575) GhVIN1 (558)

2. Supplemental Figure 1. Alignment of GhCWIN1 sequence with five plant CWINs (Atβfruct2, Lin6, NtCWIN1, OsCWIN1 and ZmCWIN1) and two VINs (Atβfruct4 and GhVIN1). Thirteen boxed sequences indicate the conserved regions of plant invertases. The β-fructosidase motif (NDPD/NG) and cysteine catalytic domain (WECP/VD) were underlined. Arrowsheads denote five consistently different amino acids between VINs and CWINs. Arrow show GhCWIN1 signal sequence cleavage site between residue 19 and residue 20.

3. Plant CWIN Plant VIN Bacterial invertase Yeast invertase Plant CIN 0.1 Supplemental Figure 2 Supplemental Figure 2. Phylogenetic analysis classifies GhCWIN1 as a plant CWIN. Genbank accession number for each invetrase listed and the corresponding species were listed in Supplemental Table 1.

4. GhCWIN1 GhINH1 GhMST1 GhSUT1 Gh18srRNA Seed Filial tissue 5 10 15 20 DAA Supplemental Figure 3. Transcript level of cotton cell wall invertase GhCWIN1, invertase inhibitor GhINH1, monosaccharide tranporter GhMST1, sucrose transporter GhSUT1 in cotton seed and filial tissue at different development stages. Gh18srRNA was used as an internal control. Supplemental Figure 3

5. a b b c a b b c Seed Seed coat Glucose content (mg g -1 FW) 25 20 15 10 5 0 5 10 15 20 Glucose content (mg g -1 FW) 25 20 15 10 5 0 Seed Filial tissue 5 10 15 20 A a ab b b a b bc c Fructose content (mg g -1 FW) 25 20 15 10 5 0 B Seed Seed coat 5 10 15 20 Seed Filial tissue 5 10 15 20 Fructose content (mg g -1 FW) 25 20 15 10 5 0 a bc ab c Seed Filial tissue 5 10 15 20 Sucrose content (mg g -1 FW) 25 20 15 10 5 0 Sucrose content (mg g -1 FW) 25 20 15 10 5 0 C Seed Seed coat 5 10 15 20 Supplemental Figure 4 Supplemental Figure 4. Glucose (A), fructose (B) and sucrose (C) level in cotton seed, seed coat and filial tissue during development. Each value is the mean ± SE of at least three biological replicates. Different letters indicate significant differences at P ≤.05 according to randomization one-way ANOVA test.