Supplementary Tables
Supplementary Table S1. Results of the suppression subtractive hybridization (SSH) analysis for in vitro ABA treatments.
Construction of SSH library and differential screening were performed as previously reported by Ikegami et al., (2007). Each three A- and NA-type individual of the Atf-line used in in vitro
ABA-treatment (Fig. 3) was subjected to the analysis. To reduce false-positive clones, we performed mirror orientation selection (Rebrikov et al. 2000) in the SSH analysis. We aimed to isolate
cDNA fragments expressed specifically in ABA-treatments which strongly induct DkMyb4 expression. Thus, we performed two sets of SSH analyses: (i) for A-type using the ABA-treated (0.1
µM ABA) bulk as the tester and non-treated (0 µM ABA) bulk as the driver, and (ii) for NA-type the ABA-treated (10 µM ABA) bulk as the tester and non-treated (0 µM ABA) bulk as the
driver. We isolated approx. 1,000 recombinant clones each in the SSH library for the A-type and NA-type samples, and differential screening allowed the identification of 34 clones distinctly
expressed in the ABA-treated fruit. From the results of quantitative PCR analysis, it was suggested that five of them were highly expressed in ABA-treated fruit (above four ranks), but others
(below ranks) didn’t show significant expression changes.
Highest or near highest hit by Blast X with annotated function to each sequence
Annotation (homolog)
No. clones
BLAST Score
(e-value)
Fold induction of
expression by ABA
Fold induction of
expression by ABA
in A-type* (P value)
in NA-type** (P value)
species
accession
2
1
1
1
Diospyros kaki
Gypsophila elegans
Diospyros kaki
Diospyros kaki
A4PB65
Q84QS6
D0VYJ9
C6L1M3
193 (1e-47)
248 (2e-64)
172 (2e-41)
180 (7e-44)
2.78 (< 0.05)
3.12 (< 0.01)
5.94 (< 0.001)
3.12 (< 0.05)
10.98 (< 0.001)
12.14 (< 0.001)
34.69 (< 0.001)
6.12 (< 0.01)
8
4
3
3
2
1
1
1
6
Vitis riparia
Citrus hybrid
Oryza sativa
Vitis vinifera
Capsicum annuum
Glycine max
Vigna unguiculata
Arabidopsis lyrata
Q9SPB3
Q8W3U7
A6N0B2
A9CSJ8
Q4G3H5
Q2PMD4
Q9AYM8
D7KH17
257 (2e-67)
50 (3e-06)
60 (4e-08)
362 (2e-98)
223 (9e-57)
95 (2e-18)
85 (4e-15)
101 (4e-20)
0.75 (0.24)
1.22 (0.27)
1.67 (0.14)
0.55 (0.26)
1.14 (0.28)
1.06 (0.37)
1.41 (0.30)
2.12 (0.23)
1.41 (0.32)
0.87 (0.21)
1.26 (0.26)
0.74 (0.26)
0.81 (0.29)
1.86 (0.33)
1.29 (0.28)
0.74 (0.19)
Genes giving significant expression changes
anthocyanidin reductase (ANR)
anthocyanidin synthase (ANS)
Myb transcription factor (DkMyb4)
flavanone 3ʹ5ʹ-hydroxylase (F3'5'H)
Genes with no significant expression changes
QM-protein homolog
polygalacturonase-like protein
formate dehydrogenase
plastid lipid-associated protein
RAV transcription factor
flavin adenine dinucleotide-linked oxidoreductase
flavin adenine dinucleotide-binding CPRD2
Ceramidase family protein
unknown
*The expression levels in 0.1 µM ABA treatments relative to those in 0 µM ABA treatments
** The expression levels in 10 µM ABA treatments relative to those in 0 µM ABA treatments
1
Supplementary Table S2. Sequences of primers for promoter analysis
The primers used for promoter analysis in transient reporter assay. All are given 5’ to 3’, and orientation is shown as F (forward)
or R (reverse). Primer sequences were derived from the genomic sequences in A-type cv. Kuramitsu.
DkMyb4 promoter regions for each
reporter construct (see figure 8A)
pWA(WNA)-LUC2
Primer name
DkMyb4-promWF-T OPO
DkMyb4-prom1R
pR1-LUC2
DkMyb4-prom1F-T OPO
DkMyb4-prom1R
**
pR1mut-LUC2
DkMyb4-prom1mutF-T OPO
DkMyb4-prom1R
pR2-LUC2
DkMyb4-prom2F-T OPO
pR3-LUC2
DkMyb4-prom3F-T OPO
DkMyb4-prom1R
DkMyb4-prom2R
Primers sequences *
F: CACCAAATCAGTGGTGGTGATTTAGTCG
R: CTCTTCTTCTGATATTTGTGACGCAC
F: CACCATGTAAAAAGACAACACATGTACGTGTGTG
R: CTCTTCTTCTGATATTTGTGACGCAC
F: CACCATGTAAAAAGACAACACATGTAGGATTGTG
R: CTCTTCTTCTGATATTTGTGACGCAC
F: CACCGGGAAAAGTATTGGGGCGATA
R: CTCTTCTTCTGATATTTGTGACGCAC
F: CACCAAATCAGTGGTGGTGATTTAGTCG
R: ATATAAAAACCAAATTTGATACGATGC
* The sequence shown in boldface, CACC, is an additional sequence necessary for directional cloning in Gateway system
** Underlined nucleotides (AGGAT) indicate mutated sites on the ABRE given in Fig. 8A
2
Supplementary Figures
Supplementary Figure S1. ABA concentration in fruit flesh of a A-type (cv. Kurokuma) and two NA-type (cvs. Jiro and
Fuyu) sampled at 1, 7, and 13 WAB in 2009.
ABA concentration (ug/100mg DW)
ABA concentration in fruit flesh were analyzed by LC-MS/MS in The Institute of Physical and Chemical Research (RIKEN) Plant
Science Center (thank to Dr. Hitoshi Sakakibara, Group Director of the Plant Productivity Systems Research Group).
1600
Jiro (NA-type)
1400
1200
1000
Fuyu (NA-type)
Kurokuma (A-type)
800
600
400
200
0
3
Supplementary Figure S2. Expression patterns of DkMyb1, 2, 3 and DkbZIP5 in ABA/abamine treatments.
qRT-PCR analysis for gene expression in control fruit, and fruits treated with abamine and S-ABA; A, DkMyb1; B, DkMyb2; C
DkMyb3; D DkbZIP5 expression. Each treatment is shown as a black point and solid line (control), white point and dotted line
(Abamine), and gray point and semi-dotted line (S-ABA) in cvs. Kuramitsu (A-type; square), Suruga (NA-type; circle), and
Okugosho (NA-type; diamond). Each three fruit was sampled at 1, 3, 5, 7, 9 and 13 WAB, and subjected to qRT-PCR analysis
independently. The expression levels of each gene are given as relative values of those in cv. Kuramitsu at 1 WAB, defining the
expression levels as ‘100’. All error bars indicate standard deviations (SDs) (n = 3). Note that expression levels of DkMyb1 (A)
and DkMyb3 (C) at 13 WAB were under the detection in the qRT-PCR analyses.
A-type
cv. Kuramitsu
A
NA-type
cv. Suruga
1000
relative expression level
relative expression
Cont.
Cont.
Abamine
Abmine
100
100
10
10
10
1
2
4
6
8
10
12
relative expression
1000
6
8
10
12
14
0
100
100
10
4
6
8
10
12
1
14
0
2
4
100
6
8
10
12
14
0
12
14
14
14
S-ABA
100
10
1
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
1000
Cont.
Abamine
S-ABA
100
10
Cont.
Abmine
S-ABA
10
Cont.
Abamine
S-ABA
1
1
1
6
12
Abamine
100
4
10
S-ABA
100
2
8
S-ABA
1000
0
6
Cont.
1000
10
4
Abamine
1
10
2
Cont.
1
8
12
Abmine
10
6
10
Cont.
10
4
8
1000
100
2
6
Cont.
Abamine
S-ABA
S-ABA
1000
0
4
10
Cont.
1
2
2
Abamine
1000
relative expression
4
1000
Cont.
Abmine
S-ABA
0
C
2
1000
100
10
S-ABA
1
0
14
1
relative expression
Abamine
S-ABA
S-ABA
0
D
Cont.
100
1
B
cv. Okugosho
1000
1000
8
10
12
14
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
weeks after bloom (WAB)
4
Supplementary Figure S3. Sequence alighment and conserved subdomains of bZIP-TFs classified to AREB.
Sequence alighment analysis was performed by Clustal X using 16 full-length amino acid sequences of plant AREB-like
bZIP-TFs (Suzuki et al., 2003) and DkbZIP5 and visualized by BioEdit. Three conserved subdomains (C1-C3) and a bZIP motif
were shown by solid and dotted frames respectively. The serine/threonine residues subjected to phosphorylation involved in the
ability of transcript activation (Wasilewska et al., 2008) were shown by two triangle arrows. The origin species and accession
numbers of each AREB is given below.
ABF1: Arabidopsis, NM_001198254; ABF2: Arabidopsis, NM_001198228; ABF3: Arabidopsis, NM_001036708; ABF4:
Arabidopsis, NM_001203005; ABI5: Arabidopsis, NM_129185; AREB3: Arabidopsis, NM_115544; At2g17770: Arabidopsis,
NM_001202616; At3g44460: Arabidopsis, BT004342; At5g42910: Arabidopsis, NM_123656; AtZIP13: Arabidopsis,
NM_123775; DkbZIP5: persimmon (Diospyros kaki), AB670685; EEL: Arabidopsis, NM_201926; GBF3: Arabidopsis,
NM_180118; GBF4: Arabidopsis, NM_100278; HvABI5: barley (Hordeum vulgare subsp. vulgare), AY150676; OsABI5: rice
(Oryza sativa), EF199630; TaABF: wheat (Triticum aestivum), AF519804.
C1-subdomain
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
AtZIP13
GBF4
ABF1
ABF4
ABF3
HvABI5
ABF2
At5g42910
AREB3
EEL
DkbZIP5
OsABI5
TaABF
ABI5
At3g44460
At2g17770
GBF3
Clustal Co
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
------MTSFQVMRSSNSRNSDLSRRISS-----------ASTSSSSIRPQQQFRRDLTSVGYGGRNDGLYSSNSMTVEGILHDTFASDPPAPTESSLLDASIN-----------------------------LMDASPAPMEITTTTAS
------MASFKLMSSSN---SDLSRRNSS-----------SASSSPSIRSSHHLR--------------------------------PNPHADHSRISFAYGGG-----------------------------VNDYTFASDSKPFEMAI
------MGTHIDINNLGGDTSRGNESK-----------PLARQSSLYSLTFDELQSTLGE--------PGKDFGSMNMDELLKNIWTAEDTQAFMTTTS-----------------------------------SVAAPGPSGFVPGGNG
------MGTHINFNNLGGGGHPGGEGSSNQMKPTGSVMPLARQSSVYSLTFDELQNTLGG--------PGKDFGSMNMDELLKSIWTAEEAQAMAMTSAPAA--------------------------------TAVAQPGAGIPPPGGN
------MGSRLNFKSFVDGVSEQQPTVG-------TSLPLTRQNSVFSLTFDEFQNSWGGG-------IGKDFGSMNMDELLKNIWTAEESHSMMGNNTSYTN----------ISNGNSGNTVINGGGNNIGGLAVGVGGESGGFFTGGS
----------MDFRSSNGGSSSERRPAAEG-------ASLTRQGSIYSLTFEEFQSTLGGGAGVGGGDLGKDFSSMNMDELLRSIWTAEESQAMAASASG-----------------------------------------AGAGAPPMS
------MDGSMNLGNEPPGDGGGGGG-------------LTRQGSIYSLTFDEFQSSVGK-----------DFGSMNMDELLKNIWSAEETQAMASGVVPVL----------------------------------------GGGQEGLQ
------MDSYWRLKN-LVNDLPVSTS-------------LSRQGSIYSWTVDQFQTSLGL-----------DCGSMNMDELVKHISSAEETQEGS------------------------------------------------------------MDSQRGIVEQAKSQS------------------LNRQSSLYSLTLDEVQNHLGSS--------GKALGSMNLDELLKSVCSVEANQPSSMAVNG-------------------------------------------GAAAQEG
------MGSIRGNIEEPISQS------------------LTRQNSLYSLKLHEVQTHLGSS--------GKPLGSMNLDELLKTVL----------------------------------------------------------PPAEEG
------MGFQTMGSNGSGQQPHLQP--------------VARQDSWFSLTLGEIENQLGELG--------KPLGSLNLDELLKNVWTAEANQINGMIMDS---------------------------------------------SSVSS
MASEMSKNVKVTDDQEVTSQERDQSGGTKVGGE-EEIAPLARQSSILSLTLEELQNSLCEP--------GRNFGSMNMDEFVANIWNAEEFQATTG---------------------------GCKGAMEEAKVVDSGSGSG--DAGGSG
MASEMSKDVKFSE-------EEVSSHPRVLEGE-ELTVAPARQSSIFALTLDELQYSVCEA--------GRNFGSMNMDEFMSNIWNAEEFQAATG---------------------------GGLVGMEVAPVVGAGAGGGGADAGGSN
---MVTRETKLTSEREVESSMAQARHNGGGGGENHPFTSLGRQSSIYSLTLDEFQHALCEN--------GKNFGSMNMDEFLVSIWNAEENNNNQQQAAAAAGSHSVPANHNGFNNNNNNGGEGGVGVFSGGSRGNEDANNKRGIANESS
-----MSVFESETSNFHVYNNHEIQTQPQMQTFLSEEEPVGRQNSILSLTLDEIQMKSGKS-----------FGAMNMDEFLANLWTTVEENDNEGGGAHND------------------------------------------GEKPAV
-----------------------------------------------------------------------------MEEVWKEINLG----------------------------------------------------------SLHY
------MGNSSEEPKPPTKSDKPSSPP------------VDQTNVHVYPDWAAMQAYYGPR-----VAMPPYYNSAMAASGHPPPPYMWNPQHMMS---------------------------------------------PYGAPYAAV
C2-subdomain
C3-subdomain
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
AtZIP13
GBF4
ABF1
ABF4
ABF3
HvABI5
ABF2
At5g42910
AREB3
EEL
DkbZIP5
OsABI5
TaABF
ABI5
At3g44460
At2g17770
GBF3
Clustal Co
105
70
91
105
121
93
81
64
76
61
78
113
108
140
93
16
83
DVVDHGGGTETTRGGKSVDEIWREMVSGEGK--GMKEETSEE----------------------IMTLEDFLAKAAVEDETAVTASAEDLDVKIPVTN---------------------------------------------------DVDRSIGDRNSVNNGKSVDDVWKEIVSGEQKTIMMKEEEPED----------------------IMTLEDFLAKAEMDEG-----ASDEIDVKIPTER---------------------------------------------------LQRQGSLTLPRTLSQKTVDEVWKYLNSKEGSNGNTGTDALE-----------------RQQTLGEMTLEDFLLRAGVVKEDNTQQNE-----NSSSGFYANNGAAG-LEFGFGQPNQNSISFNGN------NSSMIMNQAPGLGLKVGGT
LQRQGSLTLPRTISQKTVDEVWKCLITKDGNMEGSSGGGGES----NVPP-------GRQQTLGEMTLEEFLFRAGVVREDNCVQQMGQVNGNNNNGFYGNSTAAGGLGFGFGQPNQNSITFNGT------NDSMILNQPPGLGLKMGGT
LQRQGSLTLPRTISQKRVDDVWKELMKEDDIGNGVVNGGTSG------IP-------QRQQTLGEMTLEEFLVRAGVVREEPQPVESVTN-FNGGFYGFGSNGGLGTASNGFVANQPQDLSGNGV------AVRQDLLTAQTQPLQMQQP
LQGQGSLTLPRTLSAKTVDEVWRNLVRDDPLPVGAEGAEPQP---------------HRQATLGEMTLEEFLVKAGVVREIPTAPAVPPP---------------------PMQPRPVPVAPKGA------TFYGNFPSANDVGTAALGLQRQGSLTLPRTLSQKTVDQVWKDLSKVGSSGVGGSNLSQVAQAQSQSQS-------QRQQTLGEVTLEEFLVRAGVVREEAQVAARAQI-AENNKGGYFGNDANTGFSVEFQQPSPRVVAAGVMGNLGAETANSLQVQGSSLPLNVNGA
-QRQGSTTLPPTLSKQNVGEVWKSITEEKHTNNNGG-------VTNITHL-------QGQQTLGEITLEEFFIRAGARGGNTNGGSIHDS-SSSISGNPHTSLG--------VQIQPKAMVSDFMNNMVPRSHDSYLHQN------VNGS
LSRQGSLTLPRDLSKKTVDEVWKDIQQNKNG-GSAHERRDKQ------------------PTLGEMTLEDLLLKAGVVTETIPGSNHDGP---------------------------------------VGGGSAGSGAGLGQNITQVGP
LVRQGSLTLPRDLSKKTVDEVWRDIQQDKNGNGTSTTTTHKQ------------------PTLGEITLEDLLLRAGVVTETVVPQ------------------------------------------------------ENVVNIASNGQ
DEHQASQTLAKAFNGKTVDEVWREIQQGQKMKNVGEIKGQER-----------------QPTLGDITLEQFLIKAGIFAEASSGPIVGVN-----------------------------------------------------------LCRQGSFSLPLPLCQKTVEEVWTEINQ---APAHTSAP---ASALQPHAGSGGVAANDRQVTLGEMTLEDFLVKAGVVRGSFTG-QAAMGSGMVNGP--VNPMQQGQG-GPMMFPVGPVNAMYPVMG-----DGMGYPG-GYNGMAIVPP
LARQESFSLPPPLCRKTVEEVWAEINR---EPRQVHAQPQGARASQQPPVQPPVAANDRQGTLGEMTLEQFLVKAGVVRGSGAGGQAPVPVGMVHAQ--MNPVQQGQQPGPMMYPMAPANGMFQVMG-----DGMGFVPNGYAGMAVVPP
LPRQGSLTLPAPLCRKTVDEVWSEIHRGGGSGNGGDSNGRSSSSNGQNNAQNGGETAARQPTFGEMTLEDFLVKAGVVREHPTNPKPNPNPNQNQNPSSVIPAAAQQQLYGVFQGTGDPSFPGQAMG-----VGDPSGYAKRTGGGGYQQ
LPRQGSLSLPVPLCKKTVDEVWLEIQNGVQQHPPSSNSGQNSAEN-----------IRRQQTLGEITLEDFLVKAGVVQEPLKTTMRMSS--------------------------------------------SDFGYNPEFGVGLHCQ
HRQLNIGHEPMLKNQNPNNSIFQDFLNMPLNQPPPPPPPPSS-----------------------STIVTALYGSLPLPPPATVLSLNSG-----------------------------------------------------------YPHGGGVYAHPGIPMGSLPQGQKDPPLTTPGTLLSIDTPTKS-----------------TGNTDNGLMKKLKEFDGLAMSLGNGNPENGADEHKRSRN----------------------------------SSETDGSTDGSDGNTTGA
.
:
bZIP motif
310
320
330
340
350
360
370
380
390
400
410
420
430
440
450
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
AtZIP13
GBF4
ABF1
ABF4
ABF3
HvABI5
ABF2
At5g42910
AREB3
EEL
DkbZIP5
OsABI5
TaABF
ABI5
At3g44460
At2g17770
GBF3
Clustal Co
178
140
212
238
251
199
223
185
168
139
150
247
248
285
188
82
182
AtZIP13
GBF4
ABF1
ABF4
ABF3
HvABI5
ABF2
At5g42910
AREB3
EEL
DkbZIP5
OsABI5
TaABF
ABI5
At3g44460
At2g17770
GBF3
Clustal Co
240
196
320
360
381
282
345
302
234
199
202
311
313
364
256
133
245
5
------------------------------------------------------------------------------------YGFDHSAPPHNPFQMIDKVEGSIVAFGNGLDVYGGGARGKRAR-----VMVEPLDKAAAQRQRRMI
------------------------------------------------------------------------------------LNNDGSYTFDFPMQRHSSFQMVEGSMG------GGVTRGKRGR-----VMMEAMDKAAAQRQKRMI
MQQQQQP-----------HQQQLQQPHQRLPPTIFPKQANVTFAAPVNMVNRG------------LFETSADGPANSN----MGGAGGTVTATSPG-------TSSAENNTWSSPVPYVFGRGRRSN--------TGLEKVVERRQKRMI
MQQQQQQQQLLQQQQQQMQQLNQPHPQQRLPQTIFPKQANVAFSAPVNITNKG------------FAGAANNSINNNNGLASYGGTGVTVAATSPG-------TSSAENNSLS-PVPYVLNRGRRSN--------TGLEKVIERRQRRMI
QMVQQPQ------------MVQQPQQLIQTQERPFPKQTTIAFSNTVDVVNRSQPATQCQEVKPSILGIHNHPMNNNLLQAVDFKTGVTVAAVSPGSQMSPDLTPKSALDASLSPVPYMFGRVRKTG--------AVLEKVIERRQKRMI
----------------------------------FPPVAMGDLALGNGLMPRALG--------------------------MGGAPLVVQTAVKPVDSGSKGSEDLSSPSEPMPYSFEGIVRGRRTG--------GGVEKVVERRQRRMI
RTTYQQS---------------------QQQQPIMPKQPGFGYGTQMGQLNSPGIRGGGLVGLGDQSLTNNVGFVQGASAAIPGALGVGAVSPVTPLSSE-GIGKSNGDSSSLSPSPYMFNGGVRGR------KSGTVEKVVERRQRRMI
MSTYQ------------------------PQQSIMSMPNGYSYGKQIRFSN-------GSLGSGNQSLQDTKRSLVPSVATIP--SEAITCSPVTPFPTLNGKQKINGESSLLSPSPYISNGSTSTRGGKINSEITAEKQFVDKKLRRKI
WIQYHQLPS----------------------------------------------------------------------------MPQPQAFMPYPVSDMQAMVSQSSLMGGLSDTQTPGRKRVASG--------EVVEKTVERRQKRMI
WVEYHHQP------------------------------------------------------------------------------QQQQGFMTYPVCEMQDMVMMG----GLSDTPQAPGRKRVAG--------EIVEKTVERRQKRMI
------------------------------------------------------------------------------------------NVATPEKRLPQMGLSLNPSFHSISDTSAPGQKRDAAD---------AIEKILDRRLRRKI
PPPAQG----------------------------------------------------------------------------------AMVVVSPGSSDGMSAMTHADMMNCIGNGMMIENG-TRKRPHRED---GCAEKTVERRQRRMI
PPPPQG----------------------------------------------------------------------------------GMGIVSPGSSDGRSAMTQADMMNCMGDGAMMENGGARKRGAPED---QSCERSIERRHRRMI
APPVQAGVC-------------------------------------------------------------------YGGGVGFGAGGQQMGMVGPLSPVSSDGLGHG-QVDNIGGQYGVDMGGLRGRKRVVD---GPVEKVVERRQRRMI
NQNNYGD-------------------------------------------------------------------------------NRSVYSENRPFYSVLGESSSCMTGNGRSNQYLTGLDAFRIKKRIID---GPPEILMERRQRRMI
--------------------------------------------------------------------------------------------VGFEFLDTTENLLASNPRSFEESAKFGCLGKKRGQ--------DSDDTRGDRRYKRMI
DEPKLKR------------------------------------------------------------------------------SREGTPTKDGKQLVQASSFHSVSPSSGDTGVKLIQGSGAILS---------PGNERELKRERRKQ
.
:: :*
460
470
480
490
500
510
520
530
540
550
560
570
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.
KNRESAARSRER-----------KQAYQVELEALAAKLEEENELLSKEIEDKRKERYQKLMEFVIPVVEKPKQQPPRFLRRIRSLEW--------------------------------------KNRESAARSRER-----------KQAYQVELETLAAKLEEENEQLLKEIEESTKERYKKLMEVLIPVDEKPRP-PSRPLSRSHSLEW--------------------------------------KNRESAARSRAR-----------KQAYTLELEAEIESLKLVNQDLQKKQAEIMKTHNSEVITFFLYLSKGIFEAASIAGQKTMLEKNPYRSVVRR------------------------------KNRESAARSRAR-----------KQAYTLELEAEIEKLKKTNQELQKKQAEMVEMQKNEQLKETSKRPWGSKRQCLRRTLTGPW-----------------------------------------KNRESAARSRAR-----------KQAYTMELEAEIAQLKELNEELQKKQVCLASSLSQLRISRFSYFLEVVFTDQMFHAG---------------------------------------------KNRESAARSRAR-----------KQAYTMELEAEVQKLKDLNEELVKKQTEILKMQKREAPEMKDQFGRK-KRQCLRRTLTGPW-----------------------------------------KNRESAARSRARKQIFTADQTIYWNAYTVELEAEVAKLKEENDELQRKQARIMEMQKNQETEMRNLLQGGPKKKLRRTESGPW------------------------------------------KNRESAARSRAR-----------KQAQTMEVEVELENLKKDYEELLK---QHVELRKRQMEPGMISLHERPERKLRRTKSDIK------------------------------------------KNRESAARSRAR-----------KQAYTHELEIKVSRLEEENERLRKQKEVEKILPSVPPPDPKRQLRRTSSAPF--------------------------------------------------KNRESAARSRAR-----------KQAYTHELEIKVSRLEEENEKLRRLKEVEKILPSEPPPDPKWKLRRTNSASL--------------------------------------------------KNRESAARSRAR-----------KQAYHNELVSKISHLEEENMKLKKEKDLERILPWDLSPEPRYQLRRTTSF----------------------------------------------------KNRESAARSRAR-----------KQAYTVELEAELNYLKQENARLK----------EAELVEKMMEQSKEKMNANRG-GSQLRRSGSCMW-----------------------------------KNRESAARSRAR-----------KQAYTVELEAELNHLKEENARLKAEEKTILLTKKQMLVEKMIEQSKENVNVKKG-GTLSRRCGSCIW-----------------------------------KNRESAARSRAR-----------KQAYTVELEAELNQLKEENAQLKHALAELERKRKQQYFESLKSRAQPKLPKSNGRLRTLMRNPSCPL-----------------------------------KNRESAARSRAR-----------RQAYTVELELELNNLTEENTKLKEIVEENEKKRRQEIISRSKQVTKEKSGDKLRKIRRMASAGW--------------------------------------KNRESAARSRAR-----------KQAYTNELELEIAHLQTENARLKIQQEQLKIAEATQNQVKKTLQRSSTAPF---------------------------------------------------SNRESARRSRLR-----------KQAETEELARKVEALTAENMALRSELNQLNEKSDKLRGANATLLDKLKCSEPEKRVPANMLSRVKNSGAGDKNKNQGDNDSNSTSKLHQLLDTKPRAKAVAAG
.***** *** *
:*
*:
*
*
5
Supplementary Figure S4. Expression levels of PA pathway genes in the SbZ5-lines.
Analysis for gene expression in the DkbZIP5-overexpressed SbZ5-lines and control calluses by qRT-PCR. Six structural genes of
the PA biosynthetic pathway, PAL, CHS, F3′H, DFR, LAR, and ANS, are given. The expression level is shown as the value relative
to expression in Cont. defining the expression levels as ‘1’. We analyzed each four individual for all transgenic lines with three
technical replications in qRT-PCR. Error bars indicated standard deviations (SD). In all figures, one asterisk (*) or two asterisks
(**) indicated significant differences (P < 0.05, and P < 0.01 rerspectively) according to Student’s t-test, in comparison to the
control lines.
Genes under the regulation
by DkMyb4
Genes not affected by
DkMyb4 expression
7
SbZ5-2
DkbZIP5
overexpression lines
SbZ5-3
**
**
**
**
*
4
SbZ5-1
**
5
**
6
**
**
**
3
**
relative expression level
to Cont. callus
**
Cont.
2
1
0
N.D.
DkPAL
DkCHS
DkDFR
DkANS
DkF3'H
DkLAR
6
LITERATURE CITED
Ikegami A, Eguchi S, Kitajima A, Inoue K, Yonemori K (2007) Identification of genes involved in proanthocyanidin
biosynthesis of persimmon (Diospyros kaki) fruit. Plant Sci 172: 1037-1047
Rebrikov DV, Britanova OV, Gurskaya NG, Lukyanov KA, Tarabykin VS, Lukyanov SA (2000) Mirror orientation selection
(MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization.
Nucleic Acids Res 28: No.20 e90
Suzuki M, Ketterling MG, McCarty DR (2003) Viviparous1 alters global gene expression patterns through regulation of
abscisic acid signaling. Plant Physiol 132: 1664-1677
Wasilewska A, Vlad F, Sirichandra C, Redko Y, Jammes F, Valon C, Frei dit Frey N, Leung J (2008) An update on abscisic
acid signaling in plants and more… Mol Plant 1: 198-217
7