Fiber weight per seed (mg)
(a)
Fiber weight per seed (g)
0.09
90
60
0.06
30
0.03
1
2
3
4
28-4-1
28-4-3
61-9-1
61-9-2
28-4-2
28-8-2
61-1-1
61-6-1
28-2-1
28-8-1
61-1-2
61-6-2
00
WT
Seed weight (g)
Type 2
Type 3
180
0.18
Seed weight (mg)
(b)
Type 1
120
0.12
60
0.06
1
2
3
4
28-4-1
28-4-3
61-9-1
61-9-2
28-4-2
28-8-2
61-1-1
61-6-1
28-2-1
28-8-1
61-1-2
61-6-2
00
WT
Type 1
Type 2
Type 3
Figure S1. Fiber and seed weight of T3 mature seeds on T2 individuals. GhVIN1-RNAi
cotton exhibited reduced fiber yield per seed (a), but increased seed weight (b) as compared
to those in WT.
(a)
WT
Type 1
Type 2
Type 3
WT
Type 1
Type 2
Type 3
GhCWIN1
GhSusA
GhSUT1
Gh18srRNA
(b)
GhCWIN1
GhSusA
GhSUT1
GhMST1
Gh18srRNA
Figure S2. Representative results of RT-PCR analyses of the expression levels of GhCWIN1, GhSusA,
GhSUT1, and GhMST1 in WT and GhVIN1-RNAi cotton seed at -1DAA (a) and 3DAA (b).
WT
-18 hours
(a)
-6 hours
(b)
-3 hours
(c)
D
During flowering
WT
(d)
WT(-control)
(e)
Type 1
(f)
Figure S3. VIN activity became evident on the ovule surface , starting from the chalaza region
(arrows) at about 6 hours before anthesis.
Histochemical staining for Inv activity on the surface of WT ovules at ~18 (a), 6 (b) or 3 hours (c)
before anthesis, and WT (d) and type 1 transgenic (f) ovules during flowering. (e) represents a
background control of (d) with no added sucrose. Bars = 0.5mm.
Sugar content (mg/g FW)
a
20
WT
16
Type 1 (28-4-1)
Type 1 (61-9-1)
12
8
4
a
ab b
a
b b
b b
0
Glu
Fru
Suc
Figure S4. Glc and Fru levels were significantly reduced in 0 DAA type 1 transgenic seeds as
compared to that in WT. Different letters indicate a significant difference at p < 0.05, according to
randomization one-way ANOVA test.
100mM Glc + 20mM Fru
(a)
WT
(b)
Type 1
120mM Glc
(c)
120mM Fru
WT
(d)
WT
WT
Figure S5. Representative images of WT and type 1 GhVIN1-RNAi ovules cultured for 15
days on original BT medium (a and b, respectively) and WT ovules cultured on BT media
supplemented with 120mM Glc (c) or Fru (d) for 15 days. Bars = 5 mm.
(a)
myb25li-a
myb25li-d
agpaseS
amy
iso1-b
iso1-c
PI-II
ps20
sbeI
vsp
-1370
-572
-1132
-1375
-568
-514
-548
-172
-314
-759
--ACAACAATAATATAAAATAATGATAT-----AAACAAAATAAATAAAATTACTTAAT-----------TAAAATAAAAACAAAGG------------GCAGAAGATAAAAAAAACAA---------AATACCAAAAAATAATAATAAAA--------AAAAAATAAAGAAAATGAAATC-------ATGATAATTATTTAAAAAAACAAGCAAGT--------AATACTAATAAAGAATAGAAAAAG
------ACATAAAATAAAAAAAGG------------AAAGAAAATAAAAAATAAAG-------
-1345
-597
-1116
-1356
-546
-494
-520
-149
-297
-778
(b)
GhMYB25like-A
SURE
-1362
-1354
AATATAAAA
+1
+134
Start codon
TATA box
GhMYB25like-D
SURE
SP8b
SP8b
SP8b
SP8b-like -587
-580 -556 -550
-280 -246
-240
-681
-675 AATAAAAT-----TACTtTT -286
TACTtTT-----TACTtTT
TACTtTT
+1
TATA box
+178
Start codon
Figure S6. Sugar-responsive elements in GhMYB25like promoter.
(a) Alignment of SURE sequences in promoters of GhMYB25like and other plant genes
known to be subjected to sugar-induced expression. Conserved A nucleotides are in bold
and flanked by putative consensus core (AA/TAA) highlighted in yellow. myb25li-a and
myb25li-d refer to the SURE sequence of cotton MYB25like promoter from A- and Dgenome, respectively; agpaseS, barley gene for agpaseS (Thorbjørnsen et al., 1996); amy,
Arabidopsis gene for β-amylase (Mita et al., 1995); iso-b and iso-c, barley iso1 promoter
SURE-b and SURE-c respectively (Sun et al., 2003); PI-II, potato gene for proteinase
inhibitor II (Kim et al., 1991); ps20, potato patatin class I gene (Grierson et al., 1994); sbeI,
maize sbeI (Kim and Guiltinan, 1999); vsp, soybean gene for vegetative storage protein
(Rhee and Staswick., 1992).
(b) Schematic representation of the sugar-responsive elements SURE and SP8b-like in
GhMYB25like promoters from cotton A- and D-genome. The position of SURE and SP8b-
like are numbered relative to the putative transcription start site (+1). Core element of
SP8b, ACT, is highlighted in bold. Mismatches are indicated with lower case letter in
SP8b-like element.
(b)
WT
GhExp1
GhExp2
Gh18srRNA
Type 1
Type 2
Type 3
Relative expression level
intensity
Relative
(a)
2.5
2.5
WT
Type 2
a
2.02
Type 1
Type 3
1.5
1.5
1.01
b b
b
00
a
b b
0.5
0.5
GhExp1
GhAExp1
b
WT
Type 1
Type 2
Type 3
GhExp2
GhExp2
Figure S7. RT-PCR analyses of the expression levels of cotton genes encoding two expansins (a)
in WT and GhVIN1-RNAi transgenic 3 DAA seeds. Values in the according graph (b) are
presented as ratio of densitometric readings of samples to corresponding Gh18srRNA gene
expression levels.