Online resource 1
Title: Genome wide identification of C1-2i zinc finger proteins and their response to abiotic stress in hexaploid
wheat
Journal name: Molecular Genetics and Genomics
Author names: Arnaud Cheuk, Mario Houde
Affiliation : Centre TOXEN, Département des sciences biologiques, Université du Québec à Montréal, Montréal,
QC, Canada H3C 3P8
E-mail address of corresponding author: houde.mario@uqam.ca
Figure S1 Effect of water spraying on TaZFPs expression in wheat leaves. Wheat plants (cv. Atlas66) were
sprayed with distilled water every 15 min (4 sprays/hour) or 5 min (12 sprays/ hour) for 6 h. Expression levels were
determined for the original 16 TaZFPs previously published in Ali-Benali et al (2012) by qRT-PCR and calculated
relative to the respective time 0 values set to 1. The 18S rRNA was used to normalize the expression level. Error
bars represent the standard deviation for 4 biological replicates. Statistical analysis was performed by one-way
ANOVA followed by Tukey’s test (p<0.05). An “a” indicates a significant difference between samples treated every
15 min (4 times per hour white columns) or every 5 min (12 times per hour black columns) and time 0. An asterisk
with a bracket indicates a significant difference between samples treated every 15 min (black columns) and every 5
min (white columns). Only TaZFP transcripts showing a significant difference in expression are shown.
Figure S2 Effect of H2O2 spraying on TaZFPs expression in wheat leaves. Wheat plants (cv. Atlas66) were
sprayed with 10 mM H2O2 or water (controls) every 15 min for 12 h. Expression levels were determined for the
original 16 TaZFPs previously published in Ali-Benali et al (2012) by qRT-PCR and calculated relative to time 0
values set to 1. The 18S rRNA was used to normalize the expression level. Error bars represent the standard
deviation for 4 biological replicates. Statistical analysis was performed by one-way ANOVA followed by Tukey’s
test (p<0.05). An “a” indicates a significant difference between treated samples (black columns) or controls (white
columns) at each time point and time 0. An asterisk with a bracket indicates a significant difference between treated
samples (black columns) and controls (white columns). Only TaZFP transcripts showing a significant difference in
expression are shown.
Figure S3 Effect of high light on TaZFPs expression in wheat leaves. Wheat plants (cv. Atlas66) were exposed to
high light (800 μmol m-2 s-1) for 12 h. Expression levels were determined for the original 16 TaZFPs previously
published in Ali-Benali et al (2012) by qRT-PCR and calculated relative to the respective time 0 values set to 1. The
18S rRNA was used to normalize the expression level. Error bars represent the standard deviation for 4 biological
replicates. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test (p<0.05). An “a”
indicates a significant difference between treated samples (black columns) or control (white columns) at each time
point and time 0. An asterisk with a bracket indicates a significant difference between treated samples (black
columns) and controls (white columns). Only TaZFP transcripts showing a significant difference in expression are
shown.
Figure S4 Effect of flooding on TaZFPs expression in leaves of wheat cv. Wheat plants (cv. Atlas66) were
submerged in distilled water to simulate the effect of flooding for 6 h. Expression levels were determined for the
original 16 TaZFPs previously published in Ali-Benali et al (2012) by qRT-PCR and calculated relative to the
respective time 0 values set to 1. The 18S rRNA was used to normalize the expression level. Error bars represent the
standard deviation for 4 biological replicates. Statistical analysis was performed by one-way ANOVA followed by
Tukey’s test (p<0.05). An “a” indicates a significant difference between treated samples (black columns) or controls
(white columns) at each time point and time 0. An asterisk with a bracket indicates a significant difference between
treated samples (black columns) and controls (white columns). Only TaZFP transcripts showing a significant
difference in expression are shown.
Figure S5 Effect of drought stress on TaZFPs expression in wheat leaves. Wheat plants were exposed to drought
stress during 6 days. Expression levels were determined for the original 16 TaZFPs previously published in AliBenali et al (2012) by qRT-PCR and calculated relative to the respective time 0 values set to 1. The 18S rRNA was
used to normalize the expression level. Error bars represent the standard deviation for 4 biological replicates.
Statistical analysis was performed by one-way ANOVA followed by Tukey’s test (p<0.05). An “a” indicates a
significant difference between treated samples (black columns) or controls (white columns) at each time point and
time 0. An asterisk with a bracket indicates a significant difference between treated samples (black columns) and
controls (white columns). Only TaZFP transcripts showing a significant difference in expression are shown.
Figure S6 Validation of amplicons obtained for 53 TaZFPs in qRT-PCR analyses. qRT-PCR products were run
on an ethidium bromide-stained 2.5% TBE agarose gel. M: 100 bp markers (Bio Basic); Lane 1, Time 0; Lane 2,
plants sprayed with water for 12 h; Lane 3, plants exposed to H 2O2 for 12 h. Since TaZFP12B and TaZFP19B are
not induced by H2O2 exposure, samples were taken from the dehydration experiment (Lane 1, Time 0; Lane 2, plants
watered daily for 4 days; Lane 3, plants dehydrated for 4 days).
Figure S7 Effect of developmental stage on TaZFPs expression in wheat leaves. TaZFP expressions in the first,
second and third leaves of 14 day-old wheat plants (cv. Atlas66) were separately determined for the original 16
TaZFPs previously published in Ali-Benali et al (2012) by qRT-PCR and calculated relative to the respective time 0
values set to 1. The 18S rRNA was used to normalize the expression level. Error bars represent the standard
deviation for 4 biological replicates. Statistical analysis was performed by one-way ANOVA followed by Tukey’s
test (p<0.05). An “a” indicates a significant difference between first leaf-samples (black columns) or second leafsamples (dashed columns) and third leaf samples (white columns) which is used as reference.
Figure S8 A phylogenetic view of the wheat, rice and Arabidopsis zinc finger protein family. The neighborjoining tree was generated using aligned full-length amino-acid sequences. Bootstrap values from 10,000 replicates
were used to assess the robustness of the tree. The proteins are named to their gene name. Protein sequences
retrieved from Oryza sativa (rice) and Arabidopsis thaliana have already been characterized and are identified by
(Os) and (At), respectively.
10
8
15
TaZFP3
6
a
4
*
TaZFP7
a
10
10
5
5
a a
a
a
a
2
( F o ld c h a n g e )
R e la t iv e e x p r e s s io n le v e l
15
TaZFP6
0
0
0
15
10
10
TaZFP9
8
10
8
6
a
5
0
10
8
TaZFP 10
*
4
4
2
2
0
0
a
6
*
4
8
10
TaZFP 13
8
*
4
2
2
2
0
0
0
1
3
6
0
1
3
T im e (h )
Figure S1
6
a
*
6
4
0
TaZFP 15
a
6
a
a
6
a
10
TaZFP 12
TaZFP 11
a a
0
1
3
6
30
15
TaZFP1
a
*
20
TaZFP2
a
10
*
a
*
10
5
0
0
30
40
TaZFP4
TaZFP3
a
20
10
*
a
a
10
0
0
25
30
a
TaZFP5
*
TaZFP6
20
b
15
a
*
10
a
*
a
a a
10
5
( F o ld c h a n g e )
R e la t iv e e x p r e s s io n le v e l
*
a
a
*
a
a
30
20
20
*
0
0
50
50
40
TaZFP7
40
a
30
*
20
*
a
a
*
30
20
a
10
10
0
0
*
a
a
40
50
40
TaZFP9
a
TaZFP 11
TaZFP 10
a
30
*
*
30
*
20
a
a
20
10
10
a
0
0
20
40
TaZFP 13
TaZFP 12
15
a
*
10
30
a
*
20
10
5
0
0
30
40
TaZFP 15
TaZFP 14
a
20
a
*
10
*
a
30
*
20
10
b
a
0
0
0
1
3
6
12
0
T im e (h )
Figure S2
1
3
6
12
15
20
TaZFP2
TaZFP1
15
a
a
*
10
5
*
a
*
a
*
a
*
5
0
0
30
15
TaZFP3
TaZFP4
a
20
a
*
10
10
*
*
10
*
a
*
a
5
0
0
15
15
TaZFP5
( F o ld c h a n g e )
R e la t iv e e x p r e s s io n le v e l
a
a
a
*
10
0
10
20
TaZFP8
15
*
a
a
*
a
*
10
*
*
5
2
0
0
20
15
TaZFP 10
a
*
a
*
a
a
a
*
*
*
5
0
0
15
15
TaZFP 12
a
*
a
*
TaZFP 13
a
*
*
*
a
a
*
5
a
*
0
0
15
15
TaZFP 14
*
a
*
a
a
a
10
*
5
5
0
0
1
*
TaZFP 15
10
0
a
10
a
*
5
a
a
10
5
10
*
TaZFP 11
15
10
a
TaZFP9
a
4
*
5
a
6
a
*
0
8
a
*
*
*
a
*
TaZFP6
10
5
*
a
a
3
6 12
*
0
T im e (h )
Figure S3
1
3
a
*
a
a
*
6 12
10
25
20
TaZFP1
8
a
15
10
a
*
a
*
2
0
0
15
TaZFP3
TaZFP6
10
a
a
*
10
*
a
a
a
0
0
15
15
*
*
5
*
a
TaZFP9
TaZFP7
10
( F o ld c h a n g e )
R e la t iv e e x p r e s s io n le v e l
a
4
15
5
a
*
6
*
5
TaZFP2
a
a
*
a
10
*
*
5
*
a
5
0
0
15
15
TaZFP 10
a
TaZFP 11
a
10
*
a
*
a
10
*
5
*
5
0
0
15
10
TaZFP 12
8
10
a
a
TaZFP 13
a
6
a
*
4
5
2
0
0
10
15
8
TaZFP 14
TaZFP 15
a
6
a
*
a
a
*
*
*
*
4
a
10
5
2
0
0
0
1
3
6
0
T im e (h )
Figure S4
1
3
6
10
8
80
TaZFP1
TaZFP2
6
*
a
*
a
4
*
40
0
0
15
10
TaZFP3
a
10
a
a
a
8
a
TaZFP4
a
6
*
a
*
a
20
2
*
*
a
5
4
2
( F o ld c h a n g e )
R e la t iv e e x p r e s s io n le v e l
a
60
0
0
10
10
8
TaZFP6
8
a
a
*
6
4
2
2
0
0
10
8
a
6
a
4
TaZFP8
15
TaZFP 10
TaZFP 12
10
6
a
a
*
*
4
5
2
0
0
10
10
8
TaZFP 13
8
a
6
*
4
6
*
a
2
0
0
2
4
a
4
2
0
TaZFP 14
6
0
T im e ( D a y s )
Figure S5
2
4
6
Figure S6
10
10
8
TaZFP1
8
6
6
4
4
2
2
0
0
A
B
TaZFP3
4
a
a
a
a
2
a
0
B
D
10
8
6
TaZFP2
D
B
8
TaZFP4
TaZFP5
TaZFP6
6
a
D
8
6
6
a
4
4
4
2
2
0
0
A
B
0
D
A
8
D
A
TaZFP7
8
a
TaZFP8
a
TaZFP9
4
a
2
a
a
a
a
2
0
0
B
D
A
10
B
TaZFP10
a
a
B
D
10
TaZFP11
a
A
D
15
6
4
a
2
0
8
D
4
6
4
B
6
10
6
( F o ld c h a n g e )
R e la t iv e e x p r e s s io n le v e l
2
8
10
TaZFP 12
6
a
4
5
a
2
2
0
0
A
B
D
0
A
B
D
6
8
TaZFP14
TaZFP13
6
TaZFP15
a
4
a
2
2
B
0
B
D
15
D
3
TaZFP16
TaZFP18
4
1
a
0
2
0
B
D
0
B
D
8
8
a
6
4
4
2
2
a
0
0
B
D
D
TaZFP21
6
a
a
2
A
B
8
TaZFP20
TaZFP19
6
a
D
6
2
a
5
4
B
8
TaZFP17
a
10
A
4
2
0
0
D
6
4
A
B
8
0
A
D
T a Z F P g e n e c o p ie s
Figure S7
A
B
D
TaZFP1A
TaZFP1D
96
TaZFP1B
TaZFP2B
TaZFP2D
49
61
99 TaZFP3B
TaZFP3D
TaZFP4A
68
TaZFP4B
51
TaZFP4D
69
71
TaZFP5A
53
TaZFP5D
TaZFP6A
43
TaZFP6B
88
TaZFP6D
51
ZFP15 (Os)
ZFP182 (Os)
91
TaZFP7B
45
TaZFP7D
86
TaZFP8D
TaZFP8A
65
TaZFP8B
54
62
TaZFP9A
TaZFP9B
94
TaZFP9D
76
54
TaZFP10A
TaZFP10B
99
TaZFP10D
ZFP179(Os)
ZOS11-10 (Os)
99
ZAT7 (At)
ZAT8 (At)
ZAT12 (At)
ZAT11 (At)
99
TaZFP20A
TaZFP20D
TaZFP11A
94
TaZFP11B
TaZFP11D
74
58 TaZFP12B
TaZFP12D
70
74 TaZFP13D
TaZFP13A
93
TaZFP13B
99
TaZFP14B
76
TaZFP14D
ZFP252 (Os)
TaZFP15B
TaZFP15D
99
AZF2 (At)
SCOF1 (Os)
ZAT10 (At)
ZAT6 (At)
AZF1 (At)
TaZFP17D
TaZFP17B
99
99
TaZFP18B
TaZFP18D
ZOS3-12 (Os)
99 ZFP36 (Os)
99
TaZFP16A
98
TaZFP16B
TaZFP16D
ZFP245 (Os)
76 TaZFP21A
TaZFP21D
99 TaZFP21B
87
TaZFP19A
TaZFP19D
99
TaZFP19B
STOP1 (At)
ART1 (Os)
41
49
78
87
69
71
67
45
99
37
20
50
99
52
79
43
27
33
31
52
26
37
53
64
53
38
99
0.05
Figure S8