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Supplementary Material
1. Primers used in this research
Table S1 RT-PCR primers used in this research. OsABF6 appears not to have been
adequately annotated at this time. Its genomic annotation is not yet supported by a fulllength cDNA sequence. The first reverse primer (*), derived from a predicted exon, failed
to produce an RT-PCR product (Fig. 2), whereas the second reverse primer (**), derived
from a predicted intron, produced an RT-PCR product (result not shown).
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Gene Name#
Locus
Forward Primer
Reverse Primer
OsVP1
Os01g0911700
5' GCAGGAGGGTGATTTCATTGTG 3'
5' CAGGACGACGACACACTACAG 3'
OsABF1
Os01g0859300
5' GCGGTCCTATGATGTTCCCAG 3'
5' CTAGTGCCACACCAGAAGCAG 3'
OsABF2
Os02g0766700
5' GTTGGAGCAGCAAAAGAATGAG 3'
5' ATACAGGTAGCAGGCACCATG 3'
OsABF3
Os06g0211200
5' GCCTTACCCATTCGACACCG 3'
5' GCAACGATCTCGCAGCTAAAC 3'
OsABF4
Os09g0456200
5' GGCAGATATCATGGAAATGCAG 3'
5' ATACTTCAGGAAGCAGGTACAG 3'
OsABF5/TRAB1
Os08g0472000
5' GCGGTGGTAACGTGGAGAAGG 3'
5' GGTGACAGGGCAAGTTAAGCAC 3'
OsABF6
Os01g0813100
5' CGGTTGGAAGAGGTGTTACAG 3'
5' AAATGCTTGACGGGACATGCC 3'*
OsABF6
5' GCAGAAGACAATTCCTACACAC 3'**
OsABF7
Os05g0489700
5' GAGGAGAATGAGAGGCTAAAG 3'
5' ACCACAAGAAACAGGCAAAGG 3'
OsABF8
Os07g0686100
5' GAAGCGCATGATCAAGAACCG 3'
5' CTCTTGTTCACAACAGCCAGC 3'
OsEm
Os05g0349800
5' CTAGTGTTTGGCAATGGCGTC 3'
5' GCACGGTTACAAAGGACACAC 3'
WSI18
Os01g0705200
5' GGGAGCTACATTGGACAGACC 3'
5' AAACATTGACCTCCACACTGGC 3'
OsLEA3
Os05g0542500
5' AAGGACAAGACCTCCAGCAC 3'
5' TGCGACGACCACCACTTCAT 3'
Os01g0750300
OsCesA4
5’ CACCTCTCTTCCTCTTATTGC 3’
5' TCCTGGAGCACAAATGTCGC 3'
Os10g0467800
OsCesA7
5' CTACCCCTTCACCTCCATTCC 3'
5' CAGAGACACAGAGAAGTAGCAG 3'
Os09g0422500
OsCesA9
5' CTCACCGGCAAGTTCATCATG 3'
5' GAAATCGCTGACGCCCTCTC 3'
#
Names in italics derive from publications mentioned in the text. Other names are given for the first time here.
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Table S2 Primers for amplification and cloning of probes for RNA in situ hybridization
Gene
OsVP1
OsABF1
TRAB1
OsEm
WSI18
OsLEA3
Forward primer
5' GCGGTTAGCATCTGAAGAAAC 3'
5' TCTCAAGCAGGAGAACGCTCG 3'
5' GAGGCGGTCAACAATCCTTACG 3'
5' TGTAGGTCTTGACGGATGCAC 3'
5' AGGACACCAGCACCTACAAG 3'
5' CTAGACGCCGTGAATGATTTCC 3'
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Reverse primer
5' TACAGGACGACGACACACTAC 3'
5' CGGTGAACACGATGGCAAGAC 3'
5' GGTGACAGGGCAAGTTAAGCAC 3'
5' GCACGGTTACAAAGGACACAC 3'
5' ACATTGACCTCCACACTGGC 3'
5' AACCACAAATGCGGGCTTTAGG 3'
2. Identification of the structural orthologues of ABI3 and ABI5 in rice
Orthologues and paralogues were originally defined by Fitch (1970), but controversy
surrounds several aspects of the use of these terms, as noted by Fitch (2000). Although
the original definition of orthology emphasized common ancestry, it has often been
assumed that orthologues should have a common function and that declaration of
orthology should be contingent on demonstration of common function. Fitch (2000) and
others (Koonin, 2005; Hulsen et al., 2006) disagreed with this tendency, noting that while
common function may provide evidence for orthology, it was not a suitable criterion and
indeed may often be lost as species diverge. However, Fitch (2000) suggested that the
state of confusion might be dealt with better by qualifying “orthologue” with a suitable
adjective rather than insisting on an invariant definition. The adjective could indicate the
source of evidence for orthology. We have followed this suggestion and use the term
“structural orthologue” to indicate that orthology is supported by sequence data. Through
the use of BLAST analysis we identified the rice orthologues of ABI3 and ABI5. We
confirmed that OsVP1 is the orthologue of ABI3 and established that OsABF1, a protein
previously known only from the annotation of the rice genome, is the orthologue of
ABI5.
OsVP1 is the structural orthologue of the B3-domain protein ABI3 in rice
The notion that VP1, OsVP1 and ABI3 are orthologous transcription factors was put
forward by Hattori et al. (1992, 1995). Since then, the genomes of both Arabidopsis and
rice have been sequenced, revealing that both of these genomes encode 52 B3-domain
proteins (Riano-Panchon et al., 2007). However, ABI3 and OsVP1 are the only members
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of these families with the three additional conserved domains found in VP1: A1, B1 and
B2 (Fig. S1a). TBLASTN analysis shows that the next closest homologues of ABI3 in
Arabidopsis and rice are much shorter proteins containing only the B3 domain (Fig. S1b).
These data confirm the structural orthology of ABI3 and OsVP1.
OsABF1 is the structural orthologue of the bZIP protein ABI5 in rice
The genomes of Arabidopsis, japonica rice and indica rice encode 70, 85 and 109 bZIP
domain proteins, respectively (Riano-Panchon et al., 2007; http://drtf.cbi.pku.edu.cn).
Protein alignment shows that at least 8 proteins in both Arabidopsis and rice contain six
domains found in ABI5: DNA-binding domain, leucine zipper domain, and C1–C4
domains (Fig. S2a). The cladogram in Fig. S2b compares the whole protein sequence of
ABI5 with those of the seven most similar Arabidopsis proteins and the top eight rice
proteins (designated as OsABF1–OsABF8), together with three related proteins studied
by others (TaABFA, TaABFB and HvABI5). The key result is that ABI5 and AtDPBF2
of Arabidopsis cluster with OsABF1 of rice and TaABFA and TaABFB of wheat.
TRAB1, the only OsABF protein to have been studied in detail (Hobo et al., 1999) and
often regarded as the orthologue of ABI5, is only fifth in terms of relatedness to ABI5
and is in a different clade.
More detailed sequence analysis supports the orthology of ABI5 and OsABF1
(Fig. S3). First, OsABF1, ABI5, AtDPBF2, TaABFA and TaABFB contain a cysteine in
domain C2, at a position where most other bZIP proteins in Fig. S3 contain a serine. This
serine is predicted to be part of a PRXXS phosphorylation site for a SnRK2 protein
kinase (Huang & Huber, 2001). Indeed, in TRAB1, this serine (S102) is phosphorylated
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as part of a mechanism to activate the transcription factor in response to ABA (Kagaya et
al., 2002). The kinases that phosphorylate the site include three members of the SnRK2
protein kinase family, SAPK8, SAPK9 and SAPK10 (Kobayashi et al., 2005). ABI5 is
known to be regulated by phosphorylation elsewhere in the polypeptide (Lopez-Molina et
al., 2001) and OsABF1 is presumably similar.
Second, there is a question as to whether OsABF1 is orthologous to ABI5 or
AtDPBF2. These two Arabidopsis proteins appear to have arisen through gene
duplication since divergence from the last common ancestor of rice and Arabidopsis (Fig.
S2b). As the dimerizing properties of bZIP proteins are determined principally by the
DNA-binding domain and the leucine zipper domain (Fujii et al., 2000; Vinson et al.,
2006), we compared these contiguous domains in OsABF1, ABI5 and AtDPBF2 over 69
amino acids (Fig. S3). The DNA-binding and leucine zipper domains of the three proteins
are identical at 38 positions and different at 15 positions. At the remaining 16 out of 69
positions, two of the proteins differ from the third. ABI5 and AtDPBF2 differ from
OsABF1 at 6 positions, OsABF1 and ABI5 differ from AtDPBF2 at 9 positions, and
OsABF1 and AtDPBF2 differ from ABI5 at only one position. We conclude that, in these
domains, OsABF1 shows greater homology to ABI5 than to AtDPBF2, strengthening the
case for the orthology of ABI5 and OsABF1.
Fitch WM. 1970. Distinguishing homologous from analogous proteins. Systematic
Zoology 19: 99–113.
Fitch WM. 2000. Homology: a personal view on some of the problems. Trends in
Genetics 16: 227–231.
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Fujii Y, Shimizu T, Toda T, Yanagida M, Hakoshima T. 2000. Structural basis for the
diversity of DNA recognition by bZIP transcription factors. Nature Structural &
Molecular Biology 7: 889 -893.
Hattori T, Terada T, Hamasuna S. 1995. Regulation of the Osem gene by abscisic acid
and the transcriptional activator VP1: analysis of cis-acting promoter elements required
for regulation by abscisic acid and VP1. Plant Journal. 7: 913–925.
Hattori T, Vasil V, Rosenkrans L, Hannah LC, McCarty DR, Vasil IK. 1992. The
Viviparous-1 gene and abscisic acid activate the C1 regulatory gene for anthocyanin
biosynthesis during seed maturation in maize. Genes and Development 6: 609-618.
Hobo T, Kowyama Y, Hattori T. 1999. A bZIP factor, TRAB1, interacts with VP1 and
mediates abscisic acid-induced transcription. Proceedings of the National Academy of
Sciences of the USA 96: 15348-15353.
Huang JZ, Huber SC. 2001. Phosphorylation of synthetic peptides by a CDPK and plant
SNF1-related protein kinase. Influence of proline and basic amino acid residues at
selected positions. Plant and Cell Physiology 42:1079-1087.
Hulsen T, Huynen MA, de Vlieg J, Groenen PMA. 2006. Benchmarking ortholog
identification methods using functional genomics data. Genome Biology 7(4): R31.
Kagaya Y, Hobo T, Murata M, Ban A, Hattori T. 2002. Abscisic acid-induced
transcription is mediated by phosphorylation of an abscisic acid response element binding
factor, TRAB1. Plant Cell 14: 3177-3189.
Kobayashi Y, Murata M, Minami H, Yamamoto S, Kagaya Y, Hobo T, Yamamoto
A, Hattori T. 2005. Abscisic acid-activated SNRK2 protein kinases function in the gene-
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regulation pathway of ABA signal transduction by phosphorylating ABA response
element-binding factors. Plant Journal 44:939-949.
Koonin EV. 2005. Orthologs, paralogs and evolutionary genomics. Annual Reviews of
Genetics 39: 309-338.
Lopez-Molina L, Mongrand S, Chua NH. 2001. A postgermination developmental
arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor
in Arabidopsis. Proceedings of the National Academy of Sciences of the USA 98: 47824787.
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integrative plant transcription factor database. BMC Bioinformatics 8: 42
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Fig. S1 OsVP1 is the rice orthologue of ABI3. (a) Conserved domain structure of ABI3,
VP1 and OsVP1. (b) Cladogram of ABI3, VP1, OsVP1 and four closely related B3
proteins from Arabidopsis (FUSCA3 and LEAFY COTYLEDON2) and rice
(NP_001044053 and NP_001054257). Accession numbers for ABI3, VP1, OsVP1,
FUSCA3 and LEC2 are CAA48241, AAA33506, BAA04066, AAC35247 and
AAL12005. Numbers of amino acids in polypeptide chains are shown on the right.
Fig. S2 OsABI5 is the rice orthologue of ABI5. (a) Conserved domain structure of ABI5
and related bZIP proteins. DB, DNA-binding domain. LZ, leucine zipper domain. (b)
Cladogram of ABI5 and related proteins from Arabidopsis, rice, wheat and barley. Box:
ABI5 and orthologues. See Table S1 for locus numbers for OsABF1-OsABF8. The
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following synonyms (and accession numbers) are currently in use in the literature:
ABI5/AtDPBF-1 (AAD21438), AtABF2/AREB1 (BAB12404), AtDPBF2/AtbZIP67
(AAK19600), AtDPBF3/AREB3 (BAB12406), AtDPBF4/AbZIP12/EEL (AAK19602),
ABRE/AtABF1 (BAE99050), AtDPBF5/AtABF3 (NP_567949), AtABF4 (NP_566629),
OsABF5/TRAB1 (BAD09357), TaABFA (AAM75354), TaABFB (AAM75355),
HvABI5 (AAO06115).
Fig. S3 Alignment of C2 domains, basic DNA-binding domains and leucine zipper
domains of ABI5 and similar Arabidopsis, rice, wheat and barley proteins. The
downward-pointing arrow marks the conventional boundary between the DNA-binding
domain and the leucine zipper domain. PRXXS: putative phosphorylation motif. Shading
in C2 domain highlights cysteine conserved among ABFs clustered in box in Fig. S2b.
Shading in DNA-binding domain and leucine zipper domain indicates amino acids
conserved between two or three of ABI5, AtDPBF2 and OsABF1.
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Fig. S1
(a)
N
C
A1
(b)
B1
B2
B3
NP:001044053 (289aa)
FUSCA3 (313 aa)
OsVP1 (727 aa)
ZmVP1 (723 aa)
AtABI3 (720 aa)
LEC2 (362 aa)
NP:001054257 (433 aa)
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Fig. S2
(a)
C
N
C1
C2 C3
DB LZ C4
(b)
TaABFA
TaABFB
OsABF1
ABI5
AtDPBF2
OsABF2
OsABF3
AtABF2
OsABF4
HvABI5
OsABF5/TRAB1
AtABF1
AtABF4
AtABF3
OsABF6
OsABF7
OsABF8
AtDPBF3
AtDPBF4
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Fig. S3
Domain C2
AtDPBF2:LPRQGSLSLPVPLCKKTVDEVWLEI
AtABI5 :LPRQGSLTLPAPLCRKTVDEVWSEI
TaABFA :LARQESFSLPPPLCRKTVEEVWAEI
TaABFB :LARQESFSLPPPLCRKTVDEVWAEI
OsABF1 :LCRQGSFSLPLPLCQKTVEEVWTEI
OsABF2 :VQRQGSLTLPRTLSQKTVDEVWRDM
OsABF3 :IQRQGSLTLPRTLSQKTVDEVWRDI
OsABF4 :LQRQGSLTLPRTLSAKTVDEVWRNL
OsABF5 :LQRQGSLTLPRTLSVKTVDEVWRDL
OsABF6 :LLRQGSITMPPELSKKTVDEVWKGI
OsABF7 :LQRQGSITMPPELSKKTVDEVWKGI
OsABF8 :----AAAAAAETAGRKTVDEVWRDI
HvABI5 :LQGQGSLTLPRTLSAKTVDEVWRNL
AtABF1 :LQRQGSLTLPRTLSQKTVDEVWKYL
AtABF2 :LQRQGSLTLPRTLSQKTVDQVWKDL
AtABF3 :LQRQGSLTLPRTISQKRVDDVWKEL
AtABF4 :LQRQGSLTLPRTISQKTVDEVWKCL
AtDPBF3:LSRQGSLTLPRDLSKKTVDEVWKDI
AtDPBF4:LVRQGSLTLPRDLSKKTVDEVWRDI
PRXXS
Basic DNA-binding domain
Leucine zipper domain
TaABFA :CERSIERRHRRMIKNRESAARSRARKQAYTVELEAELNHLKEENARLKAEEKTILLTKKQMLVEKMIEQ
TaABFB :CERSIERRHRRMIKNRESAARSRARKQAYTVELEAELNHLKEENARLKAEEKTILLTKKQMLVEKMIEQ
AtDPBF2:PEILMERRQRRMIKNRESAARSRARRQAYTVELELELNNLTEENTKLKEIVEENEKKRRQEIISRSKQV
AtABI5 :VEKVVERRQRRMIKNRESAARSRARKQAYTVELEAELNQLKEENAQLKHALAELERKRKQQYFESLKSR
OsABF1 :AEKTVERRQRRMIKNRESAARSRARKQAYTVELEAELNYLKQENARLKEAEKTVLLTKKQMLVEKMMEQ
OsABF2 :IEKVVERRQRRMIKNRESAARSRQRKQAYMMELEAEVAKLKELNDELQKKQDEMLEQQKNEVLERMSRQ
OsABF3 :VEKVVERRQRRMIKNRESAARSRARKQAYIMELEAEVAKLKEQKAELQKKQVEMIQKQNDEVMERITQQ
OsABF4 :VEKVVERRQRRMIKNRESAARSRARKQAYTLELEAEVQKLKEMNKELERKQADIMEMQKNEVEEMIKDP
OsABF5 :VEKVVERRQRRMIKNRESAARSRARKQAYTMELEAEVQKLKEQNMELQKKQEEIMEMQKNFFPEMQKNQ
OsABF6 :ADKLMERRQKRMIKNRESAARSRARKQAYTNELENKVSRLEEENVRLKRQKESDYLHYTRSNLVMENIE
OsABF7 :ADKLVERRQKRMIKNRESAARSRARKQAYTNELENKVLRLEEENERLKKQKELDEILNSAPPPEPKYQL
OsABF8 :VEKTVERRQKRMIKNRESAARSRARKQAYTNELENKISRLEEENQRLREHKAVADFSTFPSCVDFLKAF
HvABI5 :VEKVVERRQRRMIKNRESAARSRARKQAYTMELEAEVQKLKDLNEELVKKQTEILKMQKREAPE-MKDQ
AtABF1 :LEKVVERRQKRMIKNRESAARSRARKQAYTLELEAEIESLKLVNQDLQKKQAEIMKTHNSELKEFSKQP
AtABF2 :VEKVVERRQRRMIKNRESAARSRARKQAYTVELEAEVAKLKEENDELQRKQARIMEMQKNQETEMRNLL
AtABF3 :LEKVIERRQKRMIKNRESAARSRARKQAYTMELEAEIAQLKELNEELQKKQVCLASSLSQLRISRFSYF
AtABF4 :LEKVIERRQRRMIKNRESAARSRARKQAYTLELEAEIEKLKKTNQELQKKQAEMVEMQKNELKETSKRP
AtDPBF3:VEKTVERRQKRMIKNRESAARSRARKQAYTHELEIKVSRLEEENERLRKQKEVEKILPSVPPPDPKRQL
AtDPBF4:VEKTVERRQKRMIKNRESAARSRARKQAYTHELEIKVSRLEEENEKLRRLKEVEKILPSEPPPDPKWKL
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