Supporting information
Additional Supporting information may be found in the online version of this article:
Supplementary fig. 1 Heat map of OSIPK, OSbHLH and OSFboxgenes drawn using microarray data
from leaf, root, seedling (SDL), six panicle developmental stages (P1-P6), premeiotic anther (PMA),
meiotic anther (MA), single cell pollen (SCP), trinucleate pollen (TPA), mature pollen (MP), five stages
of seed development (S1-S5) and three abiotic stress conditions (cold, desiccation and salt). Color scale
shown at the bottom of map represents average signal intensity values in log2.
Supplementary fig. 2 Nucleotide sequence of the upstream regulatory region of OSbHLH and
OSFboxgene. Translational initiation codon is shown in bold and blue colour. The 5’ UTR sequence is in
red colour. The intron in 5’UTR of OSbHLH gene is shown in pink colour. The first nucleotide of the 5’
UTR region is assigned +1 position. Previously identified cis-acting elements are underlined and shown
in bold letters. The names of the corresponding cis elements are mentioned below the respective motif.
The forward and the reverse primers used to amplify the promoter are in green.
Supplementary fig.3 Schematic representation of OSIPK, OSbHLH and OSFbox binary vector constructs
used for rice and Arabidopsis transformation. OSIPK, OSbHLH and OSFbox upstream regulatory regions
were cloned in binary vector pMDC164 for driving GUS expression or pMDC110 vector for driving GFP
by recombination mediated cloning. The arrows depict the direction of genes or promoters.
Supplementary fig. 4 Confirmation of the transgenic nature of riceplants carrying OSIPK (a),
OSbHLH(b) and OSFbox(c) upstream regulatory fragment in the binary vectors pMDC164 and
pMDC110. Binary vector construct for each construct was used as a positive (+) control and wild-type
rice DNA was used as a negative (-) control.
Supplementary fig. 5 Confirmation of the transgenic nature of Arabidopsisthaliana plants carrying
OSbHLH(a) and OSFbox (b) upstream regulatory fragment in the binary vector pMDC164 and
pMDC110. Binary vector construct for each was used as a positive (+) control and wild-type Arabidopsis
DNA was used as a negative (-) control.
Supplementary fig. 6 GUS histochemical assay of root (a), stem (b), leaf (c), flower (d), sepal (e), petal
(f), anther (g), gynoecium (h) and silique (i) from wild-type and transgenic Arabidopsis plants harboring
OSbHLH::pMDC164and OSFbox::pMDC164. The scale bar is equal to 0.1 mm.
Supplementary fig. 7 GFP expression pattern of root (a), stem (b), leaf (c), sepal (d), petal (e), anther (f)
and gynoecium (g) from wild-type and transgenic Arabidopsis plants carrying upstream regulatory
fragments of OSbHLHand OSFbox gene in the binary vector pMDC110. Untransformed wild-type plants
were used as a control. The scale bar is equal to 0.1 mm.
Supplementary fig. 8 Activity of OSIPK, OSbHLH and OSFbox during different anther developmental
stages. Different shades of red colour represent different stages in which the URRs are active and the
level of activity. Dark red colour indicates stage of maximum activity. (PMA, premeiotic anther stage;
MA, meiotic anther stage; SCP, single cell pollen stage; TPA, trinucleate pollen stage and DA, dehisced
anther stage).
Supplementary Table 1 Differences in the upstream regulatory sequences of OSbHLH and
OSFbox between indica (IR64) and japonica (Nipponbare) rice.
Name of the gene
OSbHLH URR
OSFbox URR
Position (bp)
Japonica
Indica
-345
-362
-850
-851
-1061
-1211
-1361
-1428
-1433
-1462
-1554
-1596
-1614
-1672
-1682
+17
-83
-231
-290
-438
-441
-564
-595
-664
-689
-690
-792
-815
-838
-839
-861
-912
-945
-981
-994
-1034
-1108
-1211
C
T
C
T
T
T
T
C
T
A
C
A
TATTATA
C
T
A
G
T
T
C
T
C
G
C
T
A
C
C
C
C
C
A
G
G
A
A
T
T
T
A
A
A
C
T
A
G
T
T
GTCGTCGTCGTC
T
C
A
C
C
A
C
T
A
T
C
G
T
T
A
T
T
CAAAA
C
A
Supplementary fig. 2
COLD
DES
SALT
DES
SALT
S4
S5
S5
COLD
S2
S3
DES
SALT
S3
S4
MP
S5
S1
S1
COLD
S2
S4
TPA
MP
MA
S2
SCP
SCP
S3
TPA
P5
TPA
P6
PMA
P6
MP
PMA
S1
MA
P3
MA
P4
SCP
P4
P5
P1III
P1
P5
P2
P1
P6
P2
PMA
P3
P1I
P1II
P3
P1II
P1III
P4
SAM
P1
SDL
P2
P1I
SAM
LY
ROOT
P1II
ROOT
P1III
SDL
SDL
LEAF
SAM
LEAF
LY
P1I
ROOT
LY
LEAF
Supplementary fig. 1
OSIPK
OSbHLH
OSFbox
B
kb
0.4
0.3kb
B
0.4
0.3
+
-
+
OSFbox :: pMDC110
OSFbox::pMDC110
OSFbox :: pMDC110
R30
A
kb
kb
0.5
0.4
0.3
0.3
A24
R38
R37
A23
A22
R36
R35
A21
A20
R33
A30
R30
A27
R29
R28
A25
A24
R27
A22
R26
A21
R25
R24
A21
R24
A29
R26 R26
A30
A29 R38
R24
R23
R23
A27
R21
R20
R18
R17
R25 R25
A27
R20
A22
A21
R18
R17
A20
R16
R15
R14
R12
R11
R10
A27 R37
OSFbox :: pMDC164
R22
0.7
0.3
R22
A26
OSbHLH::pMDC110
OSbHLH::pMDC110
OSbHLH::pMDC110
R21 R21
A25
kb
A26 R36
R20 R20
A22
R18 R18
0.3
A24
R21
R35
R33
0.2
A24
A24
A22
A21
A20
R17 R17
A19
R16
A17
R15
R14
A16
A15
R12
A14
R11
R9
R8
R9
R8
R26 R26
R25 R25
R21 R21
R20 R20
R18 R18
R17 R17
R16 R16
R14 R14
R13 R13
R12 R12
R11 R11
R10 R10
R9
R8
R7
R7
R7
R9
R24 R24
R23 R23
R22 R22
R21 R21
R20 R20
R18 R18
R17 R17
R16 R16
R15 R15
R14 R14
R12 R12
R11 R11
R10 R10
R9
R8
R5
R7
R8
R4
R5
nos T
R29 A23
R28 A22
R27 A21
R26 A20
R25 A19
A20
R32
A19
R16 R16
A19
A18
R14 R14
R13 R13
R12 R12
A16
A15
R11 R11
A13
A12
R10
A10
R9
R6
R6
R3
R4
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20R21
GUSA/mGFP6
R24 A18
A20 R31
R32
R30
A19
A17 R29
A16 R28
A15 R27
A12
R9
R10 R10
R9
A9
OSbHLH::pMDC110
R19
R18
A19
OSFbox :: pMDC164
OSFbox::pMDC164
R31
A18
OSFbox :: pMDC164
A18
0.3
R30
A17
R17
A16
R16
A15
R15
A14 R25
A12 R24
R8
R8
A8
R8
R5
R5
OSbHLH::pMDC110
OSIPK::pMDC110
OSbHLH::pMDC110
R19 A17
R14
A13
R20
OSbHLH::pMDC164
OSbHLH::pMDC164
A10 R22
R7A8
R7
R7
OSbHLH::pMDC164
OSbHLH::pMDC164
OSbHLH::pMDC164
R7
A7
R5
R3
R3
R2
R3
OSIPK::pMDC164
R29
A16
R28
A15
A14
R27
R25
A13
A12
R24
R12
A12
A9R9
R6A7
R6
R5
A6
R4
R2
R2
R2
OSbHLH::pMDC164
R18 A16
R17 A15
R16 A14
R15 A13
R14 A12
R12 A11
R20
R19
R5A6
R5
R4
A5
R3
R1
R1
+
A8
A7
R3A5
+
R3
R3
A4
R1
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20R21
R1
-
A11
R22
R8
A6 R12
A5
R2A3
-
R2
R2
R1
+
A8R8
A7R7
A6R6
A5R5
R6
R2
A2
R1
A1
-
A10
A9
R19
A8
R12
R8
A4
R5
+
R1A2
+
R9 A10
+
A2
-
R1
-
R8 A9
+
R7 A8
-
+
A3R4
+
R6 A7
-
+
R2
-
+
A6
+
A3
R6
-
+
R5 A6
A
-
+
R3
Mass ruler
0.9
0.7
R4 A5
BB
kb
kb
0.4
0.3
0.4
-
+
0.2
kb
0.9
0.7
A2
R5
(c)
-
A1
0.3
Mass ruler
+
R2 A3
kb
A
0.5
0.3
kb
-
+
R1
A
-
A1
Supplementary fig. 4
Mass ruler
ruler ruler
Mass Mass
Supplementary
fig. 3
kb
A2
0.3
-
+
R3
kb
-
A1
0.7
0.4
kb
+
R1 A2
kb
B
-
+
B
ruler
Massruler
ruler
Mass Mass
kb
A
-
A1
0.3
ruler ruler
Mass Mass
OSIPK/OSbHLH/OSFbox
kb
-
A1
B
Mass ruler
kb
A
A
0.2
kb
Mass ruler
A
ruler ruler
Mass Mass
0.2
kb
Mass ruler
0.9
0.7
kb
0.7
Mass ruler
A
Mass ruler
kb
B
kb
Mass ruler
(b)
B
Mass
ruler
Massruler
(a)
Mass ruler
RB
Mass ruler
attR1
OSIPK::pMDC164
R1 R2 R3 R5 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R19 R20 R21 R25 R26 R3 R38
4
attR2
OSIPK::pMDC110
0.9
0.7
Hygromycin
OSbHLH::pMDC164
R1 R2 R3 R5 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R19 R20 R21 R25 R26 R3 R38
4
0.7
0.3
OSbHLH::pMDC164
0.3
OSFbox
:: pMDC110
OSbHLH::pMDC110
OSbHLH::pMDC110
-
LB
Mass ruler
B
kb
0.4
0.3
+
A10
R9
A14
A24
R30
R29 A23
R28 A22
A22
R25 A19
R24 A18
R19 A17
R18 A16
R17 A15
R16
R15 A13
R14 A12
R12 A11
R27 A21
OSFbox::pMDC164
R26 A20
OSFbox :: pMDC164
A21
kb
0.4
0.3
A20
A19
A23
A
A23
A22
A21
A20
A19
A18
A9
R8
OSFbox :: pMDC110
OSFbox::pMDC110
OSFbox :: pMDC110
A18
A8
R7
R25
R24
R38
A24
A30 R30
A27 R29
A25 R28
R36
A22
R37
A23
A24 R27
A22 R26
A21
R19
R18
R17
R16
R15
A21
R35
A20
R33
A19
R32
A20
R31
A18
A16
A15
A13 R14
A12 R12
A9 R9
A8 R8
A7 R7
A6 R6
A5 R5
A19
OSFbox::pMDC164
A18
OSFbox:: ::
pMDC164
OSFbox
pMDC164
R30
A17
0.4
0.3
R29
A16
R28
A15
R27
A14
R25
A13
A12
R24
A11
R22
A10
R20
A9
R19
A8
R12
A6
R8
R4
OSFbox :: pMDC110
OSbHLH::pMDC110
OSbHLH::pMDC110
A17
A7
R6
A3
R2
R1
A29
A27
A26
A24
A22
A21
A20
A19
A17
A16
A15
A14
A12
A10
A8
A7
A6
A5
A4
A2
A1
R38
R37
R36
R35
R33
R32
R31
R30
R29
R28
R27
R25
R24
R22
R20
R19
R12
R8
R6
R5
R3
OSbHLH::pMDC164
OSbHLH::pMDC164
A17
A16
A15
A14
A13
A6
R5
A3
R6
A2
R5
R6
R7 A8
R8
R5
R6
R7
R26 R26
R25 R25
R21 R21
R20 R20
R18 R18
R17 R17
R16 R16
R14 R14
R13 R13
R12 R12
R11 R11
R10 R10
R9
R8
R9
R2 A3
R3 A5
R2
R3
R1 A2
R1
A30
A27
A25
A24
A22
A21
A20
A19
A18
A16
A15
A13
A12
A9
A7
A6
OSbHLH::pMDC110
A16
A15
A14
A13
A5
R4
A2
R5
A1
R3
+
A12
A3
+
-
A1
+
A12
A11
A10
A9
A8
A6
A2
+
Mass ruler
ruler ruler
Mass Mass
-
A11
A10
A9
A8
+
A7
-
+
A6
+
+
A3
+
+
R2
--
A1
-
A5
0.4
B
0.3
-
+
A2
B
kb
+
R1
AA
kbkb
0.4
0.5
0.3
0.3
-
A1
B
-
A3
Bkb
0.4
kb
0.3
Mass ruler
0.3
A1
(a)
Mass ruler
Supplementary fig. 5
Mass ruler
kb
A
0.5
0.3
Mass ruler
A
ruler
Mass ruler
Mass
0.4
0.7
0.3
kb
-
A2
(b)
Mass ruler
B
Mass ruler
kb
B
kb
A1
Mass ruler
B
OSbHLH::pMDC110
OSbHLH::pMDC110
OSbHLH::pMDC110
0.7
0.3
OSFbox :: pMDC164
0.3
kb
-
kb
0.4
0.3
OSFbox :: pMDC110
OSFbox::pMDC110
-
Supplementary fig. 6
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Wild-type
OSbHLH::pMDC164
OSFbox::pMDC164
Supplementary fig. 7
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Wild-type
OSbHLH::pMDC110
OSFbox::pMDC110
Supplementary fig. 8
Promoter
OSIPK
OSbHLH
OSFbox
PMA MA
SCP TPA DA