Title: Enzyme activity
1 (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
2Supplymental 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.
3 Supplemental Figure 2
4Supplemental 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 ß-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.
5Supplemental 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 pI values are indicated in
brackets.
6(B)
Atßfruct4 GhVIN1 AtTUB
(D)
(H)
Supplemental Figure 4
7Supplemental 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 Atßfruct4 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 lt
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).
8Supplemental 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 lt 0.01 according to randomization
one-way ANOVA test.
Supplemental Figure 5