Planta, The BdGAMYB protein from Brachypodium distachyon interacts
with BdDOF24 and regulates transcription of the BdCathB gene upon seed
germination
Virginia González-Calle, Raquel Iglesias-Fernández, Pilar Carbonero, Cristina
Barrero-Sicilia
Supplementary data:
Supplementary Fig. S1 Sequence analysis and comparison of PBF proteins of barley (BPBFHvDOF24), wheat (WPBF), maize (MPBF), rice (RPBF) and Brachypodium (BdDOF24). a
Alignment of complete deduced PBF amino-acid sequences by Clustal Omega. The DOF DNA
binding domain is underlined and the four cysteine residues (C), putatively responsible of the
zinc-finger structure, are highlighted in grey. Polar amino-acid residues, aspartic acid (D),
glutamic acid (E), arginine (R) and histidine (H) in the C- terminal region of the BPBF and
WPBF proteins are highlighted in pink and the asparagine (N) enriched stretch of BPBF, WPBF
and MPBF is colored in blue. Highly conserved residues are indicated as follows: asterisks
indicate an entirely conserved column; colons show columns where all the residues have
roughly the same size and the same hydropathy; and periods indicate columns where the size or
the hydropathy has been preserved. b Rooted phylogenetic tree with Arabidopsis orthologous
AtDOF25 (Lijavetzky et al. 2003) and schematic distribution of conserved amino-acid motives
(MEME; Bailey et al. 2006) among the deduced protein sequences encoded by Poaceae PBF
genes of Brachypodium distachyon, Oryza sativa, Triticum aestivum, Hordeum vulgare, and
Zea mays. Bootstrapping values are indicated in the branches of the tree. The gene loci, protein
size (amino-acids and kilo Daltons), pI and the identity (%) of PBFs proteins relative to BPBFHvDOF24 are summarized in the table (Nt, N-terminal; Ct, C-terminal)
Supplementary Fig. S2 Rooted phylogenetic tree with Arabidopsis orthologous AtMYB65
(Gocal et al. 2001) and schematic distribution of conserved amino-acid motives (MEME; Bailey
et al. 2006) among the deduced protein sequences encoded by the GAMYB genes of Hordeum
vulgare, Triticum monococum, Brachypodium distachyon, Oryza sativa and Zea mays.
Bootstrapping values are indicated in the branches of the tree. The gene loci, protein size
(amino-acid and kilo Daltons) and pI are indicated in the table. Motives 1 and 2 span MYB
DNA binding domain
Supplementary Fig. S3 Germination time course of Brachypodium distachyon seeds in the
presence of different concentrations of abscisic acid (ABA) and paclobutrazol (PAC). Time
necessary to get the 50% of coleorhize emergence beyond the husk is indicated (see insets).
Data are means ± SE of three independent experiments. Significant differences between values
are shown as different letters (P ≤ 0.05)
Supplementary Fig. S4 Comparison of HvGAMYB and BdGAMYB proteins. a Alignment of
complete deduced HvGAMYB and BdGAMYB amino-acid sequences by Clustal Omega. The
DNA binding domain residues are indicated in bold. Tanscriptional activation domains and
negative regulatory domains are shaded in light grey and dark grey, respectively. Highly
conserved residues are indicated as follows: asterisks indicate an entirely conserved column;
colons show columns where all the residues have roughly the same size and the same
hydropathy; and periods indicate columns where the size or the hydropathy has been preserved.
b Schematic representation of HvGAMYB protein according to Gubler et al., 1999; and % of
identity in respect to the equivalent regions in BdGAMYB. DNA binding domain (DBD, white
box), tanscriptional activation domains (TAD, light grey boxes) and negative regulatory
domains (NRD, dark grey)
Supplementary Fig. S5 Polarity profiles of PBF proteins from barley (a), wheat (b), maize (c),
Brachypodium (d) and rice (e) based on Zimmerman scale (Zimmerman et al. 1968; Gasteiger
et al. 2005). Residue scores are computed considering its neighboring residues. The DOF
domain (white box), polar regions (pink box; enriched in aspartic acid, D; glutamic acid E;
arginine, R; and histidine, H; and conserved asparagine region (blue box) are indicated
Table S1 Oligonucleotide sequences of primers used for RT-qPCR analyses, amplicon length
and PCR efficiency
Gene
Primer sense
BdGAPDH CTCCCGCTATTTCGTTTGTC
Primer antisense
TGAAGATGTTGGAGCTGACG
Amplicon Tm
Slope
size (bp) (⁰C)
E*
76
71.5 -3.07
2.11
BdCathB
TGAGTGTGGCATCGAAGAAG CGGCGCTGTTCAAAGTATAG
100
75.3 -3.66
1.88
BdCathB*
GGTCCGTAATCGCAACACTT
TATCGACCCTCAACCCTCAT
155
75.4 -3.98
1.80
BdGAMYB GCCCAATAAAGTTCGTGGAG CAACATAATCCTGGCCACAG
100
68.3 -3.11
2.09
ATATGAGAGCAGCAGCATGG GCAAATGCAAGGTAGAGCAAC 110
71.4 -3.50
1.93
BdDof24
*E=efficiency=10
(-1/slope)
Table S2 List of primers used for cloning
Primer name
Sequence 5'-3'
BdGamyb-attB1
AAAAAGCAGGCTTCATGTACCGGGTTAAGAGCGAG
BdGamyb-attB2
CAAGAAAGCTGGGTCTTTGAATTCTTCCGACATTTGAC
BdDof24-attB1
AAAAAGCAGGCTTCATGGCTCCCGATGCAGCAGCC
BdDof24-attB2
CAAGAAAGCTGGGTCGTTGCCGGACGCTGGCGGCCAG
attB1
GGGGACAAGTTTGTACAAAAAAGCAGGCTTC
attB2
GGGGACCACTTTGTACAAGAAAGCTGGGTC
BdCatB promoter -463 s
CTGATTGGACAAACAGGCGC
BdCatB promoter -264 s
CGTCACTCCCAACTTCCAAG
BdCatB promoter as
CGGGATCCCTTCCTCGATCGGTAG
BdGamyb-s
AGGGATCCATGTACCGGGTTAAG
BdGamyb-as
TCGGATCCATTTGAATTCTTCCG
BdDof24-s
GTAGATCTATGGCTCCCGATGC
BdDof24-as
GTAGATCTCGTTGCCGGACG
Table S3 Sequences of conserved amino-acid motives (MEME; Bailey et al. 2006) of the
Catehsine B-like proteins from Hordeum vulgare, Triticum aestivum, Brachypodium
distachyon, Oryza sativa and Zea mays in Fig. 1a
Motif
E-value
Consensus sequences
1
2.5E-213
TDECDPYFDQVGCKHPGCEPAYPTP[VK]CEKKCKVQNQVWQEKKHFS[IV][DN]AY[RQ]
2
6.6E-211
LPKEFDARS[KA]WSGCSTIG[TK]ILDQGHCGSCWAFGAVECLQDRFCIH[LH]NMNI
3
2.5E-209
MGGHAVKLIGWGTSDAGEDYWLLANQWNRGWGDDGYFKIIRGKNECGIEE
4
9.7E-170
VNSDPHDIMAEVYKNGPVEVAFTVYEDFAHYKSGVYKHITG
5
1.1E-167
[DG][NH]SL[GR]IIQ[KN]DII[EQ]T[IV]N[NK]HPNAGWTAG[HR]NPY[FL]ANYTI
[AE]QFKH[IM]LGVKPTPP
6
5.2E-103
LS[VA]NDL[VL]ACCGFMCGDGCDGGYPISAW[QR]Y
7
4.4E-036
DV[VT]AGMPSTKN[MI][AV][RG][NS]
8
1.1E-025
LLAGV[PR][TV]KT[YH][SP]RS[EL]Q
9
2.4E-013
[LP]L[LA][LA][LV][VL]LS[AS][AV]AAAPQL[AV]GA
Table S4 Sequences of conserved amino-acid motives (MEME; Bailey et al. 2006) of the PBF
proteins from Brachypodium distachyon, Oryza sativa, Triticum aestivum, Hordeum vulgare
and Zea mays in Fig. S1b
Motif
E-value
Consensus sequences
1
2.7E-148
2
4.6E-011
3
7.0E-010
4
2.3E-007
T[TP]SF[LM][DE][MV]LR
5
7.0E-007
DD[DN][DN]GGSSRDCYWINNGGSNPWQSLLNS[ST]SLM
6
2.0E-006
[MEEVF[PS]SNSKSKAGQMAGEA[AI]A[AG]AEKK[PS]R
7
5.1E-002
[YS]YY[GA][GP][HLP][AI][INT]G[SA][GS][NI]GMLM[TP]PP
8
1.9E-001
[HK]H[LM][GM][TM][GV][NP]N[VM]TM
CPRC[KDGN]S[GIN][NK]TKFCYYNNYS[MT][SA]QPR[YH]FC[KR][AE]CRRYWT[HQ]GG[ST]L
RNVP[IV]GG[GA]CR[KR]
IP[SV]PMQQHG[DG]LVVGGNGIG[AG][AT]T[AS]S[IT]FQG[AS][AT][GS]EEGDDG[MT]G[GS][I
V]MGLQWQP[HQ]V
DAHKLG[MV]ASS[PS]EPT[GT]VVPPS[NT]CTGMNFANVLPTFMS[GV]GF[DE]I[PQ]SSLSLT[
AT]F
Table S5 Sequences of conserved amino-acid motives (MEME; Bailey et al. 2006) of the
GAMYB proteins from Hordeum vulgare, Triticum monococum, Brachypodium distachyon,
Oryza sativa and Zea mays in Fig. S2
Motif
1
2
3
E-value
5.6E-180
1.2E-170
1.4E-133
4
2.1E-123
5
6.9E-105
6
1.5E-094
7
7.4E-094
8
2.5E-056
9
10
11
12
13
14
1.9E-049
2.5E-048
5.4E-036
1.4E-034
1.7E-021
4.2E-021
Consensus sequences
EERLIIQLH[SA]KMGNKWARMA[AG]HLPGRTDNEIKNYWNTRIKRCQRA[GS]LPIY
KKGPWTSAEDAILVDYVKK[HN]GEGNWNAVQKNTGLFRCGKSCRLRWANHLR
LKMELPSLQDTESDPNSWLKYTVAPA[ML]QPTEL[VI]DPY[LM]QSP[TAS][AT]TPSVKSEC
LYLPDFT[CS]DNFI[AG]N[SP]EAL[SP]YAP[QH]LSAVSIS[NS]LLGQSFASK[NS]C[GS]FMDQV
[ND]Q
SGNT[AT][DG][SP]S[VI]FNNAIAMLLGN[DG][MIL][NDS][ITA][ED][CY][KR][PS]V[LF]GDG[IT][
VM][FL][DN][STP]S[SPV]W[SN]N[ML][PQ]HA[CF][QE]M
[ED][YC]A[PH]FSGNS[LF]T[EG][SP]T[AP]P[VM]SAASPD[VI]FQLSK[IV]SPAQS[PT]S[LM]GS
GEQ[AEV][ML][EGQ]P[AK][YH]E
MLKQS[DG][GPC][LV]LP[GA]LSD[TA]I[ND][GS][TVA][LI]SS[VA]D[QH]FSNDS[EQ][KN]L[KR][
QK][AT][LV]GFDYL[HN]EAN[SA][ST]SK
L[EL]E[LE][LIV][HL][EG]A[QE][GATV]L[RK]SGKN[QK]Q[LQST]SV[RI]SSSSS[VA][SG]TP[CY][
DEN]TT
[IMSV][IA][AP][PF]F[GE][GV]AL[TN]GSHAFLNG[NT]FS[TA]SR[PTA][TI][NS]GP
P[AT]S[VI]CNQS[SAT]NEDQQ[GCL]S[SG][DN]F[ND][CG]G[ED]N[LI]S[SN]DL
V[VGL][SC]PEFD[LMI][CG]Q[ED]YWEE
MYRVKSE[SG][DE][CG][ED][MG][MEI][HG][QD]
[DA][TA]S[PS][HRY]PE[NI][FL]RPDA[LF]F
[DE]QMDS[PL]V[AG]DD[GV]SSGG