CIN VIN CWIN (A) (B) Xu142 Xu142 fuzzless-lintless mutant -1 0 1 -1 0 1 DAA Control Activity in seed surface Magnified view (C) 2.0 1.5 1.0 0.5 0 Enzyme activity (μmol mg protein-1 min-1) -1 0 1 -1 0 1 DAA Xu-142 Xu-142 fl mutant Supplemental Figure 1
Supplymental Figure1. In situ localization and assay of invertase activity in a fiberless cotton seed mutant and its wild-type. (A) Histochemical staining of invertase activity in the wild type cotton (G. hirsutum L. cv Xu-142) ovule at -1 DAA and seed at 0- and 1-DAA. Note, strong invertase activity signals indicated by the dark blue color in the seed surface at 0 and 1 DAA but its absence at -1 DAA before fiber initiation. A magnified view was presented from the boxed region. Also note the absence of the signals in the negative controls. (B) Histochemical staining of invertase activity in a fiberless mutant from the G. hirsutum L. cv. Xu-142 background. Note, no invertase activity was localized at the seed surface. Bars = 1 mm in (A) and (B). (C) Measurement of invertase activity from extract of ovule and seed described in (A) and (B). Note the significantly higher activity of VIN but not CWIN and CIN in the wild-type seed than that of the fiberless mutant. Also note, in the wild type the VIN activity was higher at 0 and 1 DAA than that in ovule at -1DAA, consistent with observation from the histochemical staining in (A). Each value is the mean of six biological replicates with SE.
Supplemental Figure 2. Alignment of GhVIN1 sequence with three plant VINs (LeVIN1, OsINV3 and ZmIvr1 and two CWINs (OsCWI1 and NtCWIN1). Thirteen boxed sequences indicate the conserved regions of plant invertases. The β-fructosidase motif (NDPD/NG) and cysteine catalytic domain (WECP/VD) were underlined. Arrows denote five consistently different amino acids between VINs and CWINs. The “LL” or “PLP” site (double underline), strongly basic region (dot line) hydrophobic transmembrane segment (dash line) and a dashed-box representing motifs characteristic of VINs were marked on the N-terminus of the VINs including GhVIN1.
Plant CIN Plant VIN Bacterial invertase Plant CWIN Yeast invertase 0.1 Supplemental Figure3. Phylogenetic analysis classifies GhVIN1 as a VIN. Genbank accession number for each invetrase listed and the corresponding species were listed in Supplemental Table 1. Calculated pIvalues are indicated in brackets.
(A) A OE A WT B null 2-10 5-8 50 B Root hair 45 Root length (mm) length (μm) 40 > 400 (C) 201-400 35 A WT null OE 2-10 OE 5-8 1-200 190 B 100% 170 Root cell length (μm) BC C 80% 150 60% 130 WT 40% OE 5-8 OE 2-10 OE null 20% WT: 149.03±2.62 μm (116) OE null: 146.99 ±2.58 μm(113) OE 2-10: 158.71 ±6.5 μm (118) OE 5-8: 179.17 ±4.22 μm (116) 0% WT WT inv-5 inv-7 OE 2-10 OE null OE 5-8 C14 2-1 C7 3-2 OE 2-10 C5 null OE 5-8 OE null (E) (F) VIN CWIN A A 2.5 2.5 A A A A B B 2.2 2.2 Enzyme activity (μmol mg protein-1 min-1) (G) 1.9 1.9 1.6 1.6 WT WT OE 5-8 OE 5-8 OE null OE null OE 2-10 OE 2-10 (B) Atβfruct4 GhVIN1 AtTUB (D) (H) Supplemental Figure 4
Supplemental Figure 4. Expression of GhVIN1 in wild-type Arabidopsis promotes root cell elongation. (A) Semi-quantitative RT-PCR analysis confirmed the over-expression (OE) of GhVIN1 mRNA in line 2-10 and 5-8. AtTUB was used as an internal control. (B) GhVIN1 OE lines 2-10 and 5-8 displayed a longer-root phenotype, in comparison with that of WT and OE null. (C) Increased cell length in GhVIN1 OE lines of 2-10 and 5-8, as compared with WT and OE null plants. (D) Percentage of different sized root hairs in Arabidopsis Atβfruct4 T-DNA insertion mutants (inv-5 and -7), GhVIN1 complementation (C) lines (null, C7 3-2 and C14 2-1), wild type (WT) and GhVIN1 over-expression (OE) lines (OE null, OE 2-10 and OE 5-8). Each value is the mean of 120 root hairs derived from 10 seedlings. (E) Histochemical staining of invertase activity in a root hair from GhVIN1 over-expression line 5-8, showing stronger signals (arrow) (F) A negative control for (B) where sucrose was omitted from the reaction solution. (G) A magnified view of a root hair from (B), showing invertase activity in vacuole (arrow) but not in cell wall and cytoplasm (arrow head). (H) Over-expression of GhVIN1 (line 2-10 and 5-8) increased VIN, but not CWIN, activities in comparison with that of WT and OE null. Different letters in (B), (C) and (H) indicate a significant difference at p < 0.01, according to randomization one-way ANOVA test. Bars = 1 mm in (B), 50 µm in (E) and 20 µm in (G). The scale in (F) is the same as that in (E).
Glucose Fructose Sucrose A 4.0 4.0 4.0 A A A 3.0 3.0 3.0 B B B Sugar level (mg g-1 dry weight) 2.0 2.0 2.0 C C 1.0 1.0 1.0 0 0 0 WT WT WT inv-7 inv-7 inv-7 OE 5-8 OE 5-8 OE 5-8 Supplemental Figure 5 Sugar levels in mature leaves of Arabidopsis inv-7 mutant, wild-type and over-expression line OE 5-8. Each value is the mean ± SE of three biological replicates Different letters indicate a significant difference at p < 0.01 according to randomization one-way ANOVA test. Supplemental Figure 5