Stable 1. Primers Used for RT-PCR and RACE Analyses*
STable 1. Primers used for RT-PCR and RACE analyses*
Locus ID / Primers / AnnealingTm (℃)**At1g12630 / GCCAGATTCTCGTGGTTTTCCTCGG / 71
TGCCTTTTTCTCCTCCTCCCCTTCC / 71
At4g16750 / ATGCAAGACTCTTCCTCTCACGAA / 58
GTAATTTTCCAAGAAGAAAGGATC
At1g24590 / ATGGAAGAAGCAATCATGAGACTC / 58
ATAATCATCATGAAAGCAATACTG
At1g25470 / ATGAAGTCCTTTGTGAAACCTGAG / 58
TTTAACCAAACCGAGAGGCGGTGT
**At1g28370 / CATATCTAACGACATCCATCACCACCGA / 70
CTAACAGAGTCAGATCCGAGGTTGAGAT / 65
At1g33760 / ATGGAAAACACTTACGTTGGCCAA / 58
ATTATTAGAATTCCATATGGACTG
**At1g50680 / GCTTGTTTTGGTCCCCCGATGTTG / 70
CCTTTTGTGGTCTGCGTAAATCTGGGC / 72
At1g51190 / ATGGCTTCTTCACATCAACAACAG / 58
AAATGCAATCAGTACACTTTCATC
**At2g25820 / CATTGTTTTCAAGACTCGGCAACTCCAC / 70
AGGATTGCGGAACTTCCTTTGATACTC / 67
At2g38340 / ATGGAAAAGGAAGATAACGGATCG / 58
GAATCTGAAATACTCAAAATATGA
At2g40340 / ATGCCGTCGGAGATTGTTGACAGG / 58
TGTAGATCCATGAACATCTTTGTC
**At3g16280 / GGTGACAAAAGTGCCGAGCCAAATACG / 72
TGCCCCAACTTCTCATCCGCACAC / 72
**At3g23220 / CTCGAACTCAATAACTTCCCTCCCATCA / 70
TGCCATTTGGACCGTCCTTCATCATT / 71
At3g25730 / ATGGATGCCATGAGTAGCGTAGAC / 58
CAAAACTCGTTGCTTCTTACACCT
At3g54990 / ATGTTGGATCTTAACCTAAAGATC / 58
AAAGATTACGTGTCTAACAAAATG
**At5g07580 / TCGCCTCACTCCTCGGTAATGTCTAGC / 70
TCGCCTCACTCCTCGGTAATGTCTAGC / 68
**At5g10510 / CGGTAGATCCACCGTGAAACCCTAGC / 70
GCGGAAGAAGCGTCATAGGAGACAAACTG / 72
At5g13910 / ATGAACACAACATCATCAAAGAGC / 58
GGAGCCAAAGTAGTTGAAACCTTG
At5g52020 / ATGTCGAATAATAATAATTCTCCG / 58
TTTATAACTCCAAAGATTATCTCC
**At5g67000 / CCGCCGTTTAATTCCTTCTCCGTTTG / 72
TGTCAAGTACGAAGAAGAAGGGTGTGGTG / 70
At2g25820 / GTCTCGCTCAGCCAACAGG / 58
TCGGCAACTCCACAATATCTC
At1g22985 / TTCAAGCAGCGAAGACGAG / 57
CCACCGCAGAATCAACATC
AT1G16060 / CCGTTATCTGAAACTCCCG / 56
GCTGTAGAAATCAGGCGTAAGG
At4g06746 / CTAATGAGAAGCGATACAAAGGA / 57
AGCCGCCTTCTTCCGTAT
At3g20310 / CCTCTCACCTACCTCCACAATCA / 60
CGAATAAGTCAACGCCATCCA
At4g25470 / CGGTGATTACAGTCCGAAGC / 58
TTTGGATTTCCTTGGCACAG
At4g25480 / CGGCGGGTCGTAAGAAGT / 58
ATCCGTCGTCGCATCACAC
At5g18450 / TGAGCCTAACCGTGGGACT / 58
CGCCTTCACCGCCATAAC
At5g18560 / AACTCATCTTCTAACGGCTCG / 57
AATCTTGCGGCTCCATCA
At5g67010 / GGCGTCACCAGTTGCTAC / 54
TGAATGCTTCCTTCTCCCTT
At1g72570 / GCACTCCAGCATTCCCACTCT / 60
CCTTGTCTGTCCCTCCTTCTTAG
At2g40220 / CGTGGGACCTCTATGTTATGC / 58
CCAACGGCGGTGGATGA
At5g64750 / ATGTGTGTCTTAAAAGTGGCAAAT / 58
GGAGGATGGACTATTATTGTAGTT
At1g68840 / ATGGATTCTAGTTGCATAGACGAG / 58
CAAAGCATTGATTATCGCCTGCTT
UBQ / GGTGCTAAGAAGAGGAAGAAT / 58
(At3g62250) / CTCCTTCTTTCTGGTAAACGT
*All RT-PCRs were carried out with followingparameters: Initiated with a 5min denaturation at 94℃, followed by 30 cycles of amplification with 30sec denaturation at 94℃, 30sec annealing at 58℃, 1min extension at 72℃. The reactions were all ended up with a final extension at 72℃for 10min. Forward primers are in upper lines and reverse primers in lower lines.
**Those primers were designed for RACE of the 9 AP2/EREBP TFs. In these cases, gene-specific reverse primers are in upper lines and nested reverse gene-specific primers in lower lines.
STable 2. Analysis of absolute signal intensities for genes used as internal controls*.
Treatment / Locus IDAt5g09810
(Actin2) / At3g04120#
(GAPDH) / At4g05320#
(UBQ10) / At5g62690
(BTub2) / At3g13920
(eIF-4A-1) / At1g02780
(RPL19A) / At2g37270
(RPS5A)
Cold / 30.3±3.7 / 64.9±0.03 / 64.8±0.1 / 52.3±10.3 / 33.5±1.0 / 23.2±1.6 / 34.7±2.2
NaCl / 31.0±2.9 / 64.8±0.3 / 64.7±0.2 / 64.5±0.4 / 37.8±4.4 / 31.7±2.7 / 51.3±6.3
ABA / 29.7±3.8 / 65.0±0.1 / 64.6±0.2 / 62.1±3.0 / 34.7±4.3 / 32.7±2.5 / 38.8±3.0
Wound / 28.1±5.0 / 65.2±0.3 / 65.2±0.3 / 57.6±8.1 / 32.7±8.2 / 31.4±7.4 / 52.3±11.5
Drought / 32.0±10.6 / 60.5±4.3 / 64.8±0.5 / 50.2±10.8 / 37.1±8.4 / 30.2±5.9 / 47.4±8.2
Ethylene / 25.9±3.7 / 56.7±9.1 / 65.2±0.4 / 49.6±15.6 / 35.6±8.6 / 31.0±8.8 / 53.2±12.9
SA / 33.4±2.0 / 65.1±0.2 / 65.1±0.3 / 62.9±3.2 / 35.7±2.3 / 37.1±2.2 / 59.8±5.5
Heat / 34.6±1.9 / 64.9±0.4 / 64.9±0.4 / 63.3±2.9 / 24.1±2.3 / 33.0±2.4 / 51.2±3.7
UV / 27.8±0.6 / 64.9±0.4 / 65.0±0.4 / 47.1±3.9 / 31.4±3.1 / 24.3±2.4 / 35.5±4.2
CK / 30.8±4.9 / 63.3±3.7 / 64.8±0.4 / 55.9±7.2 / 30.8±5.1 / 29.2±5.1 / 39.8±6.0
Average / 30.3±5.1 / 63.5±4.2 / 64.9±0.4 / 56.6±9.8 / 33.6±6.4 / 30.5±6.0 / 47.1±10.7
*Signal intensities reported for each gene are divided by a factor of 103 to save space.
#Absolute signal intensities for these two genes appear to be saturated on most or even all of the arrays. The high setting used for scanning is necessarily for analysis of the TFs.
STable 3.Previously characterized AP2/EREBP TFs
Locus ID / Gene name / Description / ReferencesAt1g12610 / DDF1
DDF2 / High-salinity stress, flowering pathway. / Magome et al. (2004), Plant J.37: 720-729
At1g63030
At4g37750 / ANT / Cell proliferation, integuments, floral organ and ovule development. / Mizukami and Fischer (2000), Proc. Natl. Acad. Sci.97: 942-947, Krizek et al. (1999), Dev. Genet.25: 224-236, (2000), Plant Cell12: 1357-1366, Elliott et al. (1996), Plant Cell8: 155-168, Liu et al. (2000), Plant Cell12: 1879-1891, Schneitz et al. (1998), Development125: 2555-2563, Baker et al. (1997), Plant Physiol.133: 231-242, Klucher et al. (1996),Plant Cell 8: 137-156
At2g40220 / ABI4 / Seed germination, root branching and
sugar responses. / Dekkers et al. (2003), Planta. 218：579-588, Price et al. (2003), Plant Physiol.132: 1424-1438, Brocard- Gifford et al. (2003), Plant Physiol.131: 78-92, Arroyo, et al. (2003), Genes & Dev.14: 2085-2096, Signora et al. (2001), Plant J.28: 655-662, Riechmann et al. (2000), Science290: 2105-2110, Arenas-Huertero et al. (2000), Genes & Dev.14: 2085-2096, Soderman et al. (2000) Plant Physiol.124: 1752-1765, Huijser et al. (2000), Plant J.23: 577-585, Finkelstein et al. (1998), Plant Cell.10: 1043-1054
At5g25810 / TINY / Suppress cell proliferation during flower development. / Wilson et al. (1996), Plant Cell8: 659-671
At1g12980 / ESR1 / Control of meristem cell fate and lateral organ development. / Kirch et al. (2003), Plant Cell 15: 694-705, Banno et al. (2001), Plant Cell. 13: 2609-2618
At1g15360 / SHN1/WIN1 / Drought responses, wax biosynthesis and altered cuticle properties. / Aharoni et al.(2004), Plant Cell. 16: 2463-80
At1g13260 / RAV1/EDF4 / Related to ABI3/VP1. / Kagaya et al. (1999), Nucleic Acids Res.27: 470-478
At1g68840 / RAV2/EDF2
At3g54990 / SMZ / Flower development. / Schmid et al. (2003), Development130: 6001-6012, Okamuro et al. (1997), Proc. Natl. Acad. Sci.94: 7076-7081, Ohme-Takagi et al. (1995), Plant Cell7: 173-182
At2g39250 / SNZ / Flower development. / Schmid et al. (2003), Development130: 6001-6012, Ohme-Takagi et al. (1995), Plant Cell7: 173-182
At1g46768 / RAP2.1 / Flower development or universal expression. / Okamuro et al.(1997), Proc. Natl. Acad. Sci.94: 7076-7081
At3g14230 / RAP2.2
At1g78080 / RAP2.4
At1g53910 / RAP2.12
At3g16770 / RAP2.3
/AtEBP / Flower development and defense response. / Kang et al. (1999), Plant J.20: 127-133, Büttner et al. (1997), Proc. Natl. Acad. Sci.94: 5961 -5966, Okamuro et al. (1997) Proc. Natl. Acad. Sci.94: 7076-7081
At2g28550 / RAP2.7
/TOE1 / Plant defense response and floral organogenesis. / Aukerman and Sakai (2003), Plant cell15: 2730-2741, Krizek et al. (2000), Schneitz et al., 1998, Okamuro et al. (1997), Proc. Natl. Acad. Sci.94: 7076-7081, Klucher et al. (1996), Plant Cell8: 137-156
At4g25480 / DREB1A
/CBF3 / Osmotic and cold stress. / Chinnusamy et al. (2003), Genes & Dev.17: 1043-1054, Boyce et al. (2003), Plant J.34: 395-406, Guo et al. (2002), Proc. Natl. Acad. Sci.99: 7786-7791, Fowler et al. (2002), Plant Cell14: 1675-1690, Sakuma et al. (2002), Biochem. Biophys. Res. Commun.290: 998-1009, Gong et al. (2002), Proc. Natl. Acad. Sci.99: 11507-11512,Hao et al. (2002), Biochemistry41: 4202-4208, Seki et al. (2001), Plant Cell 13: 61-72, (2002), Plant J. 31: 279-292,Kasuga et al. (1999), Nat. Biotech.17: 287-291, Gilmour et al. (1998), Plant J. 16: 433-442, (2000), Plant Physiol.124: 1854-1865, Medina et al. (1999), Plant Physiol.119: 463-469, Knight et al. (1999), Plant Cell11: 875-886, Shinwari et al. (1998), Biochem. Biophys. Res. Commun. 250: 161-170, Liu, et al. (1998), Plant Cell10: 1391-1406
At5g05410 / DREB2A / Osmotic and cold stress. / Boyce et al. (2003), Plant J.34: 395-406, Sakuma et al. (2002), Biochem. Biophys. Res. Commun.290: 998-1009, Hao et al. (2002), Biochemistry41: 4202-4208, Nakashima et al. (2000), Plant. Mol. Biol.42: 657-665, Liu, et al. (1998), Plant Cell10: 1391-1406
At4g25490 / CBF1
/DREB1B / Cold, ABA and dehydration stress. / Shen et al. (2003), Theor. Appl. Genet.106: 923-930, Boyce et al. (2003), Plant J.34: 395-406, Guo et al. (2002), Proc. Natl. Acad. Sci.99: 7786-7791, Fowler et al. (2002), Plant Cell14: 1675-1690, Gong et al. (2002), Proc. Natl. Acad. Sci.99: 11507-11512, Stockinger et al. (1997), Proc. Natl. Acad. Sci.94: 1035-1040, (2001) Nucleic Acids Res. 29: 1524-1533, Medina et al. (1999), Plant Physiol.119: 463-469, Knight et al. (1999), Plant Cell11: 875-886, Shinwari et al. (1998), Biochem. Biophys. Res. Commun. 250: 161-170, Jaglo-Ottosen et al. (1998), Science280: 104-106, Gilmour et al. (1998), Plant J. 16: 433-442, (2000), Plant Physiol.124: 1854-1865, Liu et al. (1998), Plant Cell10: 1391-1406
At4g25470 / CBF2
/DREB1C / Cold stress. / Medina et al. (1999), Plant Physiol.119: 463-469, Knight et al. (1999), Plant Cell11: 875-886, Gilmour et al. (1998), Plant J. 16: 433-442
At3g11020 / DREB2B / Dehydration and high salinity stress. / Boyce et al. (2003), Plant J.34: 395-406, Sakuma et al. (2002), Biochem. Biophys. Res. Commun.290: 998-1009, Hao et al. (2002), Biochemistry41: 4202-4208, Nakashima et al. (2000), Plant. Mol. Biol.42: 657-665, Liu et al. (1998),Plant Cell10: 1391-1406
At5g51990 / CBF4 / Drought stress. / Haake et al. (2002), Plant Physiol. 130: 639-648
At3g23240 / ERF1 / Ethylene response and defense response. / Lorenzo et al. (2003), Plant Cell 15: 165-178, Berrocal-Lobo et al. (2002), Plant J. 29: 23-32, Fujimoto et al. (2000), Plant Cell12: 393-404, Solano et al. (1998), Genes & Dev.12: 3703-3714
At5g44210 / ERF9 / Repressors of cold and/or drought inducible gene. / Ohta et al. (2001), Plant Cell13: 1959-1968
At1g28370 / ERF11
At1g28360 / ERF12
At1g03800 / ERF10 / Repressors of cold and/or drought inducible or defense response genes. / Ohta et al., (2001), Plant Cell13: 1959-1968, Onate-Sanchez et al. (2002), Plant Physiol.128: 1313-1322
At4g17500 / ATERF1 / Transcription activators (1,2,5) or repressors (3,4), recognizes GCC-box, response to ethylene signaling and disease resistance. / Fujimoto et al., (2000), Plant Cell12: 393-404, Gutterson et al. (2004), Curr. Opin. Plant. Biol. 7: 465-471
At5g47220 / ATERF2 / Fujimoto et al. (2000), Plant Cell12: 393-404, Brown, et al. (2003), Plant Physiol.132: 1020–1032
At1g50640 / ATERF3 / Brown et al. (2003), Plant Physiol.132: 1020–1032, Ohta et al. (2001), Plant Cell13: 1959-1968, Fujimoto et al. (2000), Plant Cell12: 393-404
At3g15210 / AtERF4
/RAP2.5
At5g47230 / ATERF5 / Fujimoto et al. (2000), Plant Cell12: 393-404
At1g53170 / ATERF8 / Repressor of cold, and/or drought inducible genes. / Ohta et al. (2001), Plant Cell13: 1959-1968
At2g44840 / AtERF13 / Defense through ethylene signaling pathway. / Onate-Sanchez et al. (2002) Plant Physiol.128: 1313-1322
At1g04370 / AtERF14
At2g31230 / AtERF15
At5g60120 / TOE2 / Flower development. / Aukerman and Sakai(2003) Plant cell15: 2730-2741
At5g67180 / TOE3
SFig. 1
TACATACAAAGGTAGCCTTTCCGTTTTTATAATCACATTCATGTGACTAATAATTTAG
ACGAATGGTAGCAAAATGTCAGGCCACATAAACGCTAAAAAAAGCATCACAGTTATGTTTCCATTCATTGGCTATAAAAACCATGCTCACTCCCTTTTGTTCTTCACACCATTTGCAGGAAAAAATGAATAGTTGTCAGTCTAATCCCACCAAAATGGATAATTCAGAAAATGTTCTATTTAATGATCAAAACGAAAATTTCACACTTGTTGCACCACACCCTTCTTCTTCGTACTTGACAAGAGATCAAGAGCACGAGATCATGGTCTCTGCTCTGCGACAAGTGATATCTAACTCCGGAGCTGACGACGCGTCATCATCAAACTTGATCATCACAAGCGTTCCGCCTCCAGACGCTGGCCCTTGTCCTCTCTGTGGCGTCGCCGGTTGCTACGGCTGCACATTACAACGGCCGCACCGAGAGGTAAAGAAGGAGAAGAAATACAAAGGAGTAAGGAAAAAACCATCGGGTAAATGGGCGGCGGAGATATGGGATCCGAGATCGAAATCAAGGAGGTGGCTTGGAACGTTTCTTACGGCGGAGATGGCGGCACAATCTTACAATGATGCGGCGGCTGAGTATCGAGCAAGACGTGGTAAAACAAACGGAGAAGGAATTAAACGGCGGTGGAGATGA
SFig. 1 (continued)
GTTCCTCATATTTCTTCATCGTTTTGTTTCGCTTCTCTTTCTCTGTCTCTCGTATCTTTCTACTACTCTGTTTCTTGAATTCTAATGAACAACATCGACGACGCAAAGACGGAGACTTCAGTGTCTTCAGGTTCAAGCGACTCTTTCTTGCCTCTCAAGAAACGCATGAGACTTGATGACGAACCAGAAAACGCCCTAGTGGTTTCGTCTTCACCAAAGACGGTTGTGGCTTCTGGCAATGTCAAGTACAAAGGAGTCGTTCAGCAACAGAACGGTCATTGGGGTGCCCAGATTTACGCAGACCACAAAAGGATTTGGCTTGGAACTTTCAAATCCGCTGATGAAGCCGCCACGGCTTACGATAGTGCATCTATCAAACTCCGAAGCTTTGACGCTAACTCGCACCGGAACTTCCCTTGGTCTACAATCACTCTCAACGAACCAGACTTTCAAAATTGCTACACAACAGAGACTGTGTTGAACATGATCAGAGACGGTTCGTACCAACACAAATTCAGAGATTTTCTCAGAATCAGATCTCAGATTGTTGCGAGTATCAACATCGGGGGACCAAAACAAGCCCGAGGAGAAGTGAATCAAGAATCAGACAAGTGTTTTTCTTGCACACAGCTTTTTCAGAAGGAATTGACACCGAGCGATGTAGGGAAACTAAATAGGCTTGTGATACCTAAAAAGTATGCAGTGAAGTATATGCCTTTCATAAGCGCTGATCAAAGCGAGAAAGAAGAGGGTGAAATAGTAGGATCTGTGGAAGATGTGGAGGTTGTGTTTTACGACAGAGCAATGAGACAATGGAAGTTTAGGTATTGTTACTGGAAAAGTAGCCAGAGCTTTGTCTTCACCAGAGGATGGAATAGTTTCGTGAAGGAGAAGAATCTCAAGGAGAAGGATGTTATTGCCTTCTACACTTGCGATGTCCCGAACAATGTGAAGACATTAGAAGGTCAAAGAAAGAACTTCTTGATGATCGATGTTCATTGCTTTTCAGACAACGGTTCCGTGGTAGCTGAGGAAGTAAGTATGACGGTTCATGACAGTTCAGTGCAAGTAAAGAAAACAGAAAACTTGGTTAGCTCCATGTTAGAAGATAAAGAAACCAAATCAGAGGAGAACAAAGGAGGGTTTATGCTGTTTGGTGTAAGGATCGAATGTCCTTAG
SFig. 1 (continued)
agaacacatatctatatactgtttacctttcagttctatatatatgtggctgatggtcaccatataaatcataccaaatgtccctgatcttaacccaaaacttcttcatcttctttaattagtttgccacctcgcacgtgtgacaaatccttcttcgccacgtgtgaaaacccttctccggcttgctactaatatacgactaatagtatgaatagttcaATGGCTTCTGCCGGCTTAGGTAGCCGGAGAAAGGATCCGGTGTACAGAGGAATCCGGTGCCGAAGTGGGAAATGGGTCTCCGAGATTCGTGAGCCGAGGAAAACCACGAGAATCTGGCTTGGAACTTACCCCATGGCAGAGATGGCAGCAGCCGCCTATGATGTGGCTGCTATGGCTCTTAAAGGAAGAGAAGCTGTCTTGAACTTCCCTGGATCCGTCGGGTCATACCCGGTTCCTGAATCAACATCCGCAGCAGATATACGAGCCGCTGCGGCAGCCGCAGCAGCAATGAAGGGATGTGAGGAAGGGGAGGAGGAGAAAAAGGCAAAGGAGAAGAAGAGTAGTAGTTCGAAGTCGAGAGCGCGTGAGTGCCACGTAGATAATGATGTTGGATCTTCGTCGTGGTGTGGGACAGAGTTCATGGACGAAGAAGAAGTCTTGAATATGCCTAATCTGCTGGCTAATATGGCAGAAGGGATGATGGTTGCGCCGCCGTCGTGGATGGGTTCTCGGCCGTCGGATGACTCTCCGGAGAATTCAAATGATGAGGACTTGTGGGGCTATTGA
SFig. 1. RT-PCR analysis of three TFs with potential upstream ORFs other than the ones annotated in NCBI, but failed to be amplified after RACE experiments. For each gene, RP1 (Reverse primer 1) is located upstream of the new ORF and RP2 is located around the original translation initiation codon. FP (Forward primer) is the complimentary sequence located close to the 3’ end of the cDNA.
1