Download Seven Zinc Finger transcription factors are novel regulators of the

Supplementary Materials for
Title: Seven Zinc Finger transcription factors are novel regulators of the stress responsive gene
OsDREB1B
Authors: Duarte D. Figueiredo, Pedro M. Barros, André M. Cordeiro, Tânia S. Serra, Tiago Lourenço,
Subhash Chander, M. Margarida Oliveira, Nelson J. M. Saibo
Supplementary Figure S1
Fig. S1. Analysis of the OsDREB1B gene expression under abiotic stress conditions. The intensity of the
bands obtained by semi-quantitative RT-PCR (Fig. 1A) was quantified using the ImageJ software and
normalized to the internal control.
Majority YKCTVCGKAFSSYQALGGHKASHRKP---SLSGG
10
AZF1
AZF2
AZF3
STZ
ZOS3-12
ZOS11-10
ZOS12-7
Supplementary Figure S2
A
20
Majority QALGGHKRCH
Majority YKCTVCGKAFSSYQALGGHKASHRKP---SLSGGGDDLSTSSATTVLAVTI--------G-A-SGKIHVCSICGKSFATG
10
AZF1
AZF2
AZF3
STZ
ZOS3-12
ZOS11-10
ZOS12-7
20
30
40
50
60
70
90
80
AZF1
QALGGHKRCH
YKCTVCGKSFSSYQALGGHKTSHRKPTNTSITSGNQELSNNSHSNSGSVVINVTVNTGNGVSQSGKIHTCSICFKSFASG
80
AZF2
QALGGHKRCH
--CNVCEKAFPSYQALGGHKASHRIKPPTVISTTADD--------STAPTISIVAGEKHPIAASGKIHECSICHKVFPTG
70
AZF3
QALGGHKRCH
YKCGVCYKTFSSYQALGGHKASHR-----SLYGGGENDKSTPSTAV-------------------KSHVCSVCGKSFATG
56
STZ
QALGGHKRCH
YKCSVCDKTFSSYQALGGHKASHRKNLSQTLSGGGDDHSTSSATTTSAVTTG-----------SGKSHVCTICNKSFPSG
69
ZOS3-12 QALGGHKRCH
FRCTVCGKAFASYQALGGHKSSHRKPPS-----PGDHYGAAAAAQQLASAGDSKEDSASSAAGSTGPHRCTICRRSFATG
75
ZOS11-10 QALGGHMRRH
FQCRTCGRRFSTFQALGGHRTSHKRP---RVRADGLDLLLGARPGKLGA----------GGASTPVVHRCDMCGKVFATG
67
ZOS12-7 GSLREHMMMH
HCCNKCDKSFRSGNALGGHMSCHRS-------VGNQPKSTSSPPTVVDLHMPLLSSCDDNLLLLPPETQCQMCSKVFSTS
73
Zn Finger
Zn Finger
Majority QALGGHKRCH
90
B
AZF1
QALGGHKRCH
Majority QALGGHKRCH
KKRFRTKFTQEQKEKMLAFAERVGWRIQKQDDAAVQRFCEEIGVKRHVLKVWMHNNKH
AZF2
AZF3
QALGGHKRCH
20
30
40
50
STZ
QALGGHKRCH 10
ZOS3-12 QALGGHKRCH
OsZHD1
KKRFRTKFTQEQKDKMLAFAERVGWRIQKHDEAAVQQFCDEVGVKRHVLKVWMHNNKH
ZOS11-10 QALGGHMRRH
OsZHD2
KKRFRTKFTQEQKDKMLAFAERLGWRIQKHDEAAVQQFCEEVCVKRHVLKVWMHNNKH
ZOS12-7 GSLREHMMMH
OsZHD4
OsZHD8
AtZHD1
AtZHD4
AtZHD5
AtZHD8
KKRFRTKFTAEQKARMLEFAERVGWRLQKLDDAMVHHFCQEIGVKRRVLKHAVGDVTV
RKRFRTKFTPEQKERMREFAEKQGWRINRNDDGALDRFCVEIGVKRHVLKVWMHNHKN
RKRHRTKFTAEQKERMLALAERIGWRIQRQDDEVIQRFCQETGVPRQVLKVWLHNNKH
KKRFRTKFTPEQKEKMLSFAEKVGWKIQRQEDCVVQRFCEEIGVKRRVLKVWMHNNKI
KKRFRTKFTTDQKERMMDFAEKLGWRMNKQDEEELKRFCGEIGVKRQVFKVWMHNNKN
RKRFRTKFSQYQKEKMFEFSERVGWRMPKADDVVVKEFCREIGVDKSVFKVWMHNNKI
90
80
66
79
85
77
83
Homeodomain
Fig. S2. Alignment of conserved DNA-binding domains of the Zn Finger TFs. (A) Alignment of the
C2H2-type Zn Finger TFs identified in this study with the Arabidopsis TFs described by Sakamoto et al.
(2004). In that study, those TFs were shown to bind to the cis-element A(G/C)T-X3-4-A(G/C)T. For
ZOS12-7, only the two first Zn Finger domains were considered in this analysis. Amino-acid residues
conserved in all the proteins are shown in dark grey, while residues conserved in all proteins except one
are shown in light grey. (B) Alignment of ZF-HD TFs identified in this study with the closest Arabidopsis
homologues: AtZHD1 is the closest homologue to OsZHD1 and OsZHD2, while AtZHD4 is to OsZHD4
and AtZHD8 to OsZHD8. AtZHD5/ATHB33 was also included in the alignment since it was shown to
bind to the TAATTA cis-element in the study by Tan and Irish (2006).
30
YKCTVCGKSFSSYQALGGHKTSHRKPTNTSITSG
--CNVCEKAFPSYQALGGHKASHRIKPPTVISTT
YKCGVCYKTFSSYQALGGHKASHR-----SLYGG
YKCSVCDKTFSSYQALGGHKASHRKNLSQTLSGG
FRCTVCGKAFASYQALGGHKSSHRKPPS-----P
FQCRTCGRRFSTFQALGGHRTSHKRP---RVRAD
HCCNKCDKSFRSGNALGGHMSCHRS-------VG
58
58
58
58
58
58
58
58
Supplementary Figure S3
6
Putative C2H2
DNA-binding elements
5
4
3
2
1
0
Putative ZF-HD
DNA-binding elements
2,5
2
1,5
1
0,5
0
Fig. S3. Putative Zn Finger binding elements present in the OsDREB1B promoter. The plots show the
number of copies of the cis-elements in each of the four promoter fragments used in the Y1H screen, as
well as in the OsDREB1B coding region (cds; used for comparison purposes). Top panel shows the
enrichement in the A(G/C)T-X3-4-A(G/C)T cis-element. The promoter fragment ranging from -1028 to 388bp is particularly enriched in this cis-element, which correlates with the fact that ZOS3-12, ZOS11-10
and ZOS12-7 all bind to it in the Y1H assay (see Fig. 3). Bottom panel shows the enrichment in the ZFHD binding element (TAATTA), which is more abundant in the OsDREB1B promoter fragment ranging
from -1527 to -961bp. This also correlates with the fact that all the ZF-HD identified in our study bind to
this region of the promoter.
Supplementary Figure S4
Fig. S4. Expression analysis of the TF encoding genes under abiotic stress conditions. The intensity of the
bands obtained by semi-quantitative RT-PCR (Fig. 4) was quantified using the ImageJ software and the
expression of selected genes was normalized to the internal control.
Supplementary Figure S5
GFP
DAPI
Bright
field
GFP:OsZHD1
GFP:OsZHD2
GFP:OsZHD4
GFP:OsZHD8
GFP:ZOS3-12
GFP:ZOS10-11
GFP:ZOS12-7
GFP
Fig. S5. Cellular localization of the Zn Finger TFs. The TFs were expressed in a translational fusion with
GFP in Arabidopsis protoplasts. DAPI staining shows the localization of the nucleus in each cell.
Supplementary Table S1 – Primers used to isolate the OsDREB1B promoter fragments
Fragment
Primer Sequence1
5’-3’
Fragment
size (bp)
Promoter
region
498
-1945 to
-1447 bp
566
-1527 to
-961bp
6402
-1028 to
-388bp
485
-488 to
-3bp
GCTCTAGATGAATTTGGCTTGAAGGATG
1
GGACTAGTTCCGTCATTAACAAATGTGAGTT
GCTCTAGATCACGAGATGAATCTTTTGAGC
2
GGACTAGTGCACCGTCAATTCTTCGATA
ATGCGGCCGCCAAAGTTTTGGCTTCTAATTGGT
3
GGACTAGTTGACTCTCTCTGGTTCACTTCG
ATGCGGCCGCTCGGAGTAACACTCGTGCAG
4
GGACTAGTTGACTCTCTCTGGTTCACTTCG
1
Adaptors with restriction enzyme sites are underlined.
Fragment obtained by enzymatic digestion with XbaI of a 1000bp fragment obtained with the primers
indicated.
2
Supplementary Table S2 – Primers used for semi-quantitative RT-PCR
Gene
Primer Sequence
5’-3’
Number of
PCR cycles
Annealing
temperature
31
58ºC
30
58ºC
28
58ºC
35
63ºC
33
63ºC
32
58ºC
32
58ºC
30
58ºC
20
58ºC
22
58ºC
23
58ºC
CGCGAGGGGGGTCAGGGA
OsDREB1B
TAGTAGCTCCAGAGCGGCAT
CAGCAGTTCTGTGACGAGGT
OsZHD1
TAGAGCCGAGAACGAATGCT
GGATGCACAACAACAAGCAC
OsZHD2
AGAACCCAACCACAATGGAA
AGCCATTGTTGCTGTGTCTG
OsZHD4
CCCATTGTTACCCACATGCT
CTGAGCCTGGTGCCCTAC
OsZHD8
ACGGACGACGGGAGGAAG
AGGAGGAGGAGGTGCAGAGT
ZOS3-12
TTGAGCAGATTGAGTACATTAATTTTT
GGTGAGTCGTAATGGCACAA
ZOS11-10
TGAACATTACAAAATCAAAGACGAA
GTGGTGTGTCACGAGCAATC
ZOS12-7
GCATGCAACATGGAAATCAG
GTCGCACTTCATGATGGAGTTG
ACTIN1
CATGCTATCCCTCATCTCGAC
ACCCTCCTCTTGGTCGTTTT
eEF1α
AAATACCCGCATTCCACAAC
CAAAATTTTCCACCCGAATG
UBC2
ATCACATGAATCAGCCATGC
Supplementary Table S3 – cis-acting elements in the promoter region of OsDREB1B as predicted
by the PlantCARE and PlantPAN softwares (continues on the next page)
Position(s)
(from ATG)
Putative TF binding sites
ABI4 coupling element
1140
AGAMOUS and
AGAMOUS-like
275, 297, 307, 319, 411, 416, 949, 1319,
1365, 1473 1624, 1657
678, 725, 766, 1073, 1428, 1446, 1489,
1675, 1798, 1814, 1917, 1970
ARR1
Sequence(s)
(5’->3’)
Species
CTGCCCTT
maize
CCA(A/T)A
Arabidopsis
(T/A)AGATT
Arabidopsis
GCTTT
maize
ACACTCG
Arabidopsis,
carrot
DOF1-3
427, 667, 780, 889, 1406
DPBF1-2 and ABI5
474
GT-1
(involved in light signaling)
599, 784, 926, 1092, 1215, 1567, 1990
G(A/T)AAA(T/A)
Arabidopsis
Homeodomain proteins
672, 719, 948, 980, 982, 1067, 1964,
1383, 1483, 1792, 1909, 1961, 1966
ATTAT, TTATT,
ATGAT
Arabidopsis
HvGAMYB
46, 336, 936, 1807
CAACC
barley
MYB
(involved in water stress)
268, 422, 571, 1084, 1809
AAACCA,
TAACGG
Arabidopsis
MYB1
49
CTCCAACC
Arabidopsis
MYC
1459
CATTTG
Arabidopsis
OsBIHD1
342
TGTCA
rice
OsMYBS1-3
1855
TATCCA
rice
RAV1
271, 984, 1769
CAACA
Arabidopsis
WRKY
(involved in biotic stress)
517, 688, 882, 967, 1350, 1979
TTGAC
Arabidopsis,
rice, barley,
tobacco
ZF-HD
720, 1441, 1499, 1545
TAATTA
Arabidopsis
Supplementary Table S3 (continued)
Position(s)
(from ATG)
Other cis-elements
Sequence(s)
(5’->3’)
Species
CCGCGTG, GACCGTA
Arabidopsis
ABRE-related
174, 1685
Anaerobic response
element (ARE)
1083
TGGTTT
maize
DRE1 core
335
ACCGAGA
maize
Elicitor response element
881
TTGACC
parsley,
tobacco
695
ATTTCAAA
tomato
AGAAATTTCT, AAAAAATTTC
Arabidopsis
529
ACTCAT
Arabidopsis
62
CAACTC
rice
GCCAC, GGGCC,
AATCTAATCT, ATAGATAA,
GATAGGG
Arabidopsis,
maize
Ethylene responsive
element
Heat Shock Element
(HSE)
Hypoosmolarity response
element
Involved in gibberellinrelated expression
1039, 1320
Involved in light signaling
171, 178, 1567, 1601,
1884
Involved in MeJa signaling
359, 965, 1450
TGACG
barley,
tobacco,
Arabidopsis
937, 1667,
AACCAA
duckweed
1361
CCGAC
Arabidopsis
GCTGCT
Arabidopsis,
maize, pea,
barley, rice
CC(G/A)CCC
maize
Involved in phytochrome
regulation
Low-temperature
Responsive Element
(LTRE; portion of CRT)
Present in promoters of
anaerobic-responsive
genes
Sp1 element (involved in
light signaling)
218, 221, 224, 227
308, 1231, 1235