figure s1. fiber and seed weight of t3 mature seeds on t2 individuals. ghvin1-rnai cotton exhibited...
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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)
(b)
0
0.06
0.12
0.18
1 2 3 428
-4-1
28-4
-361
-9-1
61-9
-228
-4-2
28-8
-261
-1-1
61-6
-128
-2-1
28-8
-161
-1-2
61-6
-2
Seed
wei
ght
(g)
WT Type 1 Type 2 Type 3
0
0.03
0.06
0.09
1 2 3 428
-4-1
28-4
-361
-9-1
61-9
-228
-4-2
28-8
-261
-1-1
61-6
-128
-2-1
28-8
-161
-1-2
61-6
-2Fibe
r wei
ght
per s
eed
(g)
Fibe
r w
eigh
t per
see
d (m
g)Se
ed w
eigh
t (m
g) 180
120
60
0
WT Type 1 Type 2 Type 3
90
60
30
0
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).
(a)
Gh18srRNA
GhSusA
WT Type 1 Type 2 Type 3
GhCWIN1
GhSUT1
Gh18srRNA
GhSusA
WT Type 1 Type 2 Type 3
GhCWIN1
GhSUT1
GhMST1
(b)
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.
WT WT(-control) Type 1
During flowering
(d) (e) (f)
-18 hours -6 hours -3 hours
(b) D (c)(a)
WT
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.
0
4
8
12
16
20
Glu Fru Suc
Suga
r con
tent
(mg/
g FW
)
WT
Type 1 (28-4-1)
Type 1 (61-9-1)
a
b b
a
b b
a
bab
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.
100m
M G
lc +
20m
M F
ru (a)
(b)
120m
M G
lc12
0mM
Fru
WT
WT
Type 1
(c)
(d)
WT
WT
(b)
+1
TATA box
+134Start codon
AATATAAAA-1362 -1354
SURE
GhMYB25like-A
+178Start codon
-286 -280 -246 -240 TACTtTT-----TACTtTT
SP8b SP8b
+1
TATA box
TACTtTT-681 -675
SP8b-like
AATAAAAT-----TACTtTT
SURE SP8b-587 -580 -556 -550
GhMYB25like-D
myb25li-a -1370 --ACAACAATAATATAAAATAATGATAT---- -1345myb25li-d -572 --AAACAAAATAAATAAAATTACTTAAT---- -597 agpaseS -1132 --------TAAAATAAAAACAAAGG------- -1116 amy -1375 ------GCAGAAGATAAAAAAAACAA------ -1356 iso1-b -568 ----AATACCAAAAAATAATAATAAAA----- -546 iso1-c -514 ----AAAAAATAAAGAAAATGAAATC------ -494 PI-II -548 --ATGATAATTATTTAAAAAAACAAGCAAGT- -520 ps20 -172 --------AATACTAATAAAGAATAGAAAAAG -149 sbeI -314 ------ACATAAAATAAAAAAAGG-------- -297 vsp -759 -----AAAGAAAATAAAAAATAAAG------- -778
(a)
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 D-
genome, 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.
WT Type 1 Type 2 Type 3
GhExp1
GhExp2
Gh18srRNA
(a)
0
0.5
1
1.5
2
2.5
GhExp1 GhExp2
Rel
ativ
e exp
ress
ion
leve
l WT
Type 1
Type 2
Type 3
WT Type 1 Type 2 Type 3
2.5
2.0
1.5
1.0
0.5
0
(b)
GhAExp1 GhExp2
a
b bb
ab b
b
Rel
ativ
e in
tens
ity
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.