Supplementary data

Table S1 Putative stress-related cis-elements in the AtERF53 promoter.

Putative cis-element / Positiona / Sequence / Biotic stress related / Abiotic stress related
DRE/CRE / 208, / 5’-RCCGAC-3’ / +
MYB CORE / 1196 / 5’-CNGTTR-3’ / +
MYB1 LEPR / 484 / 5’-GTTAGTT-3’ / +
MYC ATERD1 / 669 / 5’-CATGTG-3’ / +
MYC CONSENSUSAT / 257 / 5’-CANNTG-3
RAV1AAT / 176, 64 / 5’-CAACA-3’
WBOX ATNPR1 / 328, 1193 / 5’-TTGAC-3’ / +
WBOX NTCHN48 / 334 / 5’-CTGACY-3’
WBOX NTERF3 / 1192, 335, 333 / 5’-TGACY-3’ / +
WRKY71 OS / 327, 333, 517, 665, 1192 / 5’-TGAC-3’ / +

a Positions of the cis-elements are from the translation start site.

Y=T/C, R=G/A and N=A/G/C/T

Table S2 Selected stress-related genes which were up- and downregulated by at least 2-fold in AtERF53 in leaves identified by GeneChip analyses

Agilent probe ID / Gene code a / Gene and description b / Fold-Change
Upregulated / ABA-related gene
A_84_P810688 / at5g15960 / KIN1 | KIN1 / 5.64
A_84_P18335 / at3g02480 / ABA-responsive protein-related / 2.93
A_84_P258350 / at5g15970 / KIN2, COR6.6 | KIN2 / 6.5
A_84_P19490 / at4g19170 / NCED4 | NCED4 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 4) / 2
Heat-responsed gene
A_84_P23899 / at2g29500 / 17.6-kDa class I small heat shock protein (HSP17.6B-CI) / 9.03
A_84_P504309 / at5g21430 / DNAJ heat shock N-terminal domain-containing protein / 6.13
A_84_P10011 / at4g09350 / DNAJ heat shock N-terminal domain-containing protein / 1.73
A_84_P248295 / at1g80030 / DNAJ heat shock protein / 3
A_84_P17108 / at1g71000 / heat shock protein binding / 2.79
A_84_P505660 / at2g33735 / DNAJ heat shock N-terminal domain-containing protein / 2.78
A_84_P20130 / at2g22360 / DNAJ heat shock family protein / 2.61
A_84_P267000 / at2g42750 / DNAJ heat shock N-terminal domain-containing protein / 2.42
A_84_P277620 / at5g54660 / heat shock protein-related / 2.24
A_84_P790663 / at3g17830 / DNAJ heat shock family protein / 2.13
A_84_P14085 / at5g59610 / DNAJ heat shock N-terminal domain-containing protein / 2.07
A_84_P21468 / at4g39960 / DNAJ heat shock family protein / 2.03
A_84_P21617 / at5g49910 / CPHSC70-2EAT SHOCK PROTEIN 70-2, ATP binding / unfolded protein binding / 2.35
A_84_P13836 / at4g24280 / CHLOROPLAST HEAT SHOCK PROTEIN 70-1, cpHSC70-1 / 1.84
A_84_P17787 / at5g37670 / Arabidopsis thaliana 15.7 kDa class I-related small heat shock protein-like / 1.84
Cold-related genes
A_84_P23852 / at2g42540 / COR15A, COR15 | COR15A (COLD-REGULATED 15A) / 29
A_84_P10613 / at2g42530 / COR15B | COR15B (COLD REGULATED 15B) / 9.25
A_84_P167173 / at2g39250 / DREB1A, CBF3, ATCBF3 | DREB1A (DEHYDRATION RESPONSE ELEMENT B1A); DNA binding / transcription activator/ transcription factor / 2.7
A_84_P16214 / at2g21040 / C2 domain-containing protein / 2.06
Drought- /salt-related genes
A_84_P10208 / at5g25610 / RD22 | RD22; nutrient reservoir / 2.53
A_84_P12921 / at4g30650 / hydrophobic protein, putative / low temperature- and salt-responsive protein / 2.28
A_84_P299330 / at1g13930 / Involved in response to salt stress. Knockout mutants are hypersensitive to salt stress. | chr1:4761042-4761725 FORWARD / 2.24
A_84_P19252 / At3g05030 / NHX2 (SODIUM HYDROGEN EXCHANGER 2), sodium ion transmembrane transporter/ sodium:hydrogen antiporter (NHX2) mRNA / 1.79
Others
A_84_P178774 / at2g41240 / Arabidopsis thaliana BHLH100 (BASIC HELIX-LOOP-HELIX PROTEIN 100); DNA binding / transcription factor (BHLH100) mRNA / 98.48
A_84_P761268 / at3g13405 / Arabidopsis thaliana (MIR169A) / 10.41
A_84_P10874 / at3g50970 / LTI30, XERO2 | LTI30 (LOW TEMPERATURE-INDUCED 30) / 5.93
A_84_P169313 / at2g36590 / Prot3, PROLINE TRANSPORTER 3 / 4.18
A_84_P16114 / at1g09350 / AtGolS3 | AtGolS3 (Arabidopsis thaliana galactinol synthase 3); transferase, transferring glycosyl groups / transferase, transferring hexosyl groups / 3.75
A_84_P11587 / at2g39800 / P5CS1 (drought tolerance) (1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1) / 3.31
A_84_P91089 / at5g39100 / GLP6 | GLP6 (GERMIN-LIKE PROTEIN 6); manganese ion binding / nutrient reservoir / 3.18
A_84_P15672 / at4g04450 / Arabidopsis thaliana WRKY42; transcription factor (WRKY42) mRNA / 2.9
A_84_P11342 / at1g01470 / LEA14, LSR3 | LEA14 (LATE EMBRYOGENESIS ABUNDANT 14) / 2.78
A_84_P807878 / at1g54410 / dehydrin family protein / 2.59
A_84_P17792 / at5g38910 / germin-like protein / 2.55
A_84_P12960 / at4g14630 / GLP9 | GLP9 (GERMIN-LIKE PROTEIN 9); manganese ion binding / nutrient reservoir / 2.48
A_84_P13135 / at4g38530 / Arabidopsis thaliana PLC1 (PHOSPHOLIPASE C 1); phospholipase C (PLC1) mRNA / 2.4
A_84_P803388 / at4g23670 / major latex protein-related / MLP-related / 2.35
Downregulated
ABA-related gene
A_84_P803506 / at5g45340 / CYP707A3 | CYP707A3; (+)-abscisic acid 8'-hydroxylase/ oxygen binding / -2.045
A_84_P11314 / at2g17770 / ATBZIP27, FDP | ATBZIP27; transcription factor / -2
A_84_P518129 / at2g44990 / CCD7, MAX3, ATCCD7 | CCD7 (CAROTENOID CLEAVAGE DIOXYGENASE 7); 9-cis-epoxycarotenoid dioxygenase / -1.77
A_84_P245685 / at3g44300 / NIT2 | NIT2 (nitrilase 2); indole-3-acetonitrile nitrilase/ indole-3-acetonitrile nitrile hydratase/ nitrilase / -1.7
A_84_P827923 / at4g32810 / CCD8, MAX4, ATCCD8 | CCD8 (CAROTENOID CLEAVAGE DIOXYGENASE 8); oxidoreductase, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen / -1.58
A_84_P17595 / at4g18350 / NCED2, ATNCED2 | NCED2 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 2); 9-cis-epoxycarotenoid dioxygenase / -1.55
A_84_P286120 / at4g19230 / CYP707A1 | CYP707A1; (+)-abscisic acid 8'-hydroxylase/ oxygen binding / -1.445
A_84_P10637 / at2g40170 / ATEM6, GEA6 | GEA6 (LATE EMBRYOGENESIS ABUNDANT 6) / -1.34
A_84_P23686 / at1g75700 / HVA22G | HVA22G (HVA22-LIKE PROTEIN G) / -1.215
Heat-related genes
A_84_P19109 / at1g44160 / DNAJ chaperone C-terminal domain-containing protein / -2.94
A_84_P173361 / at3g62570 / DNAJ heat shock N-terminal domain-containing protein / -2.405
A_84_P144479 / at5g47590 / heat shock protein-related / -2.015
A_84_P23875 / at2g20550 / DNAJ chaperone C-terminal domain-containing protein / -1.515
A_84_P11439 / at1g52560 / 26.5 kDa class I small heat shock protein-like (HSP26.5-P) / -1.46
A_84_P54420 / at4g02100 / DNAJ heat shock N-terminal domain-containing protein / -1.36
A_84_P860356 / at1g80920 / heat shock protein binding / unfolded protein binding / -1.28
A_84_P805572 / at3g44110 / ATJ3, ATJ | ATJ3; protein binding / -1.25
A_84_P18401 / at3g12580 / HSP70 | HSP70 (heat shock protein 70); ATP binding / -1.245
A_84_P21249 / at1g02650 / DNAJ heat shock N-terminal domain-containing protein / -1.21
A_84_P850593 / at4g36040 / DNAJ heat shock N-terminal domain-containing protein (J11) / -1.195
A_84_P825746 / at4g21320 / HSA32 | HSA32 (HEAT-STRESS-ASSOCIATED 32); catalytic / -1.04
A_84_P805582 / at5g22060 / ATJ2, J2 | ATJ2; protein binding / -1.03
A_84_P547662 / at4g29920 / heat shock protein-related / -1.025
Cold-related genes
A_84_P10441 / at1g05260 / RCI3, RCI3A | RCI3 (RARE COLD INDUCIBLE GENE 3); peroxidase / -1.105
Drought- /salt-related genes
A_84_P266650 / at3g54510 / early-responsive to dehydration protein-related / ERD protein-related / -3.36
A_84_P580474 / at4g22120 / early-responsive to dehydration protein-related / ERD protein-related / -1.18
A_84_P124252 / at2g41010 / ATCAMBP25 | ATCAMBP25 (Arabidopsis thaliana CALMODULIN (CAM)-BINDING PROTEIN OF 25 KDA); calmodulin binding / -1.15
Touch- /wounding-related genes
A_84_P17578 / at4g10270 / wound-responsive family protein / -2.745
A_84_P800816 / at3g10985 / SAG20, WI12, ATWI-12 | SAG20 (SENESCENCE ASSOCIATED GENE 20) / -1.815
Others
A_84_P22017 / at2g28790 / osmotin-like protein, putative | chr2:12354430-12355431 REVERSE / -3.75
A_84_P803506 / at3g30775 / Arabidopsis thaliana ERD5 (EARLY RESPONSIVE TO DEHYDRATION 5); proline dehydrogenase (ERD5) mRNA / -3.5193233
A_84_P812152 / at4g21850 / ATMSRB9 | methionine sulfoxide reductase domain-containing protein / SeIR domain-containing protein | / -2.365
A_84_P13801 / at4g11650 / ATOSM34 | ATOSM34 (osmotin 34) / -1.995
A_84_P17398 / at3g15620 / UVR3 | UVR3 (UV REPAIR DEFECTIVE 3); DNA (6-4) photolyase / -1.985
A_84_P557122 / at2g45130 / ATSPX3, SPX3 | SPX3 (SPX DOMAIN GENE 3) / -1.22
A_84_P22643 / at5g20150 / ATSPX1, SPX1 | SPX1 (SPX DOMAIN GENE 1) / -1.205

a AGI, Arabidopsis Genome Initiative.

b Description as given by The Institute for Genomic Research database.

Table S3 The primer sequences for ChIP-qPCR analysis.

Gene name / Primer sequence 5' → 3'
Actin2 F
Actin2 R
COR15A GCC F / CGTTTCGCTTTCCTTAGAGTTAGCT
AGCGAACGGATCTAGAGACTCACCTTG
CGTATGGATCAGTCGATCTCG
COR15A GCC R / CGATCCTTCGACATCCTTCAAG
COR15A DRE F / CTTGAAGGATGTCGAAGGATCG
COR15A DRE R / CTGTCCATGAAACTGAATAAACTCC
COR15B DRE1 F / CAAATATAACTATTTTGCTCAACAGC
COR15B DRE1 R / GCCACACCACATAATTAAGGAGC
COR15B DRE2 F / CGTTGGTTCGTACTAGTTACGTG
COR15B DRE2 R / GCTAACATGTAAATATCGTAGCCAA
COR15B DRE3 F / CATTACCGAGTTTCTGTTCGTTC
COR15B DRE3 R / CCCTCTTAAGTAGTACTGGAAATTG
COR15B DRE4 F / CCCTCTTAAGTAGTACTGGAAATTG
COR15B DRE4 R / CATTTAGGCTTGCAACCTTGTC
KIN1 DRE F / CTCAAAAAGAAAACACGGAG
KIN1 DRE R / GGTACGATCAGTTTTGCCTT
KIN2 DRE F / AAAACGACACGTGATGTCTT
KIN2 DRE R / TGGGCTTAAGGGTTTATATT
PLC DRE F / CTATATTATGCATCATTTCGCC
PLC DRE R / GCCGTAATATATCTAGTAAGAGACA
Prot3 DRE F / CGAAATCGCCATACAAAACC
Prot3 DRE R / AATCAAACGATTGTGGGTTC
Prot3 5'DRE F / GAAAGAAACTGTTCAACAACTTCTG
Prot3 5'DRE R / AAATGGTTATTGTGTCTCTGTAGCT
ERD5 DRE F / AAACTACGACATTATTTCAACTCCC
ERD5 DRE R / AGGTGATCGATGATACCAGTTG
P5CS1 5'GCC F / TGAAAAAATTGTTGTGGCGCC
P5CS1 5'GCC R / GGTGATGGGTATTTTGGGAAA
P5CS1 DRE F / TTGTTTTGGCTTAAACCTGAGA
P5CS1 DRE R / TGCGTTTTGTTCAAAGCCAAG
Gols3 DRE1 F / GGCTTTTGGCCAGTTAACAAA
Gols3 DRE1 R / AAAGCTAATTTGCTGCTCGTT
Gols3 DRE23 F / CTATTTTTTGTTTGGTCAGGATTAC
Gols3 DRE23 R / TGTTGGGTATTCTGATGTTGA
Gols3 DRE4 F / GTACACGAGAAAGAGACGGTCTG
Gols3 DRE4 R / TTCGGTTCGATCGGTTAATGG
LEA14 DRE1 F / AATCAAAAATCAAGCAGAAGACACG
LEA14 DRE1 R / GGTGTTTTAGGTTTGTGTGTATGAT
LEA14 DRE23 F / ATCAGAAACCCATACCAATACG
LEA14 DRE23 R / CCTTTTTAGATAGTCACACGAT
LEA14 DRE4 F / TTTGTAAAAGAAACACCGAACAAGG
LEA14 DRE4 R / AATCAGTGAATTAAGACATTC
COR47 DRE F / GCGGAGGTAACGCTGTAAAG
COR47 DRE R / GGTGATACTGATATCATGATATTCATC
RD29B GCC F / CAACACACAGTGCATTTGCC
RD29B GCC R / CCAGTTGCTCTCTACGTGGC
RD29B DRE F / CATCGATAGGCTTCTCTAAAGATCG
RD29B DRE R / CTGGCTTCTGTCTCTTTACTTCTG
ERF53 DRE1 F / CTTTGACGACAAATCGCTAACC
ERF53 DRE1 R / AGGACCGTACTTGTGTTTCTTTT
ERF53 DRE2 F / TATGGTGATACTGACATGGGTAGGG
ERF53 DRE2 R / CGATTTGTCGTCAAAGTCAGCTTT
ERF53 DRE3 F / GGAATAGCAAGTTTTATGATTGGAG
ERF53 DRE3 R / CGGCAGATAAAGATAAACAAAC

Supplementary Figure 1

Fig. S1 Comparison of the AtERF53 amino acid sequence and related gene products from different species. The AP2 domain was indicated by underline. Letters with a yellow background indicate identical amino acids, and those with a blue and green background indicate conserved and similar amino acids, respectively.

Supplementary Figure 2

Fig. S2 Predition of PEST sequence of AtERF53. The amino acid sequence of the AtERF53 assay is available on the website: https://emb1.bcc.univie.ac.at/toolbox/pestfind/pestfind-analysis-webtool.htm.

Supplementary Figure 3

Fig. S3 The protein-gel blot analysis of AtERF53-GFP protein level in WT, rglg1/2 and 35S:AtERF53/rglg1rglg2 transgenic plants using GFP antibody under normal condition. The pEarlerGate103 was used as a positive control, and WT as negative control. The AtERF53-overexpressed transgenic lines 3-4, 3-5, and 3-10 derived from OE3 were used for further experiments, and only 3-10 was used for ChIP assay.

Supplementary Figure 4

Fig. S4 Salt tolerance of WT, rglg1rglg2 double mutants, and 35S:AtERF53/rglg1rglg2 transgenic plants. a Three-week-old plants were watered for 12 days at 4-day intervals with increasing concentrations of NaCl of 100, 200, and 300 mM. Photographs were taken before salt treatment (upper panel) and after 21 days of incubation (lower panel). OE2-1 and OE2-5 were T6 generations of OE2. b. Survival rates of plants exposed to salt stress. Average survival rates and standard errors were calculated using results of three replicate experiments. Twenty plants (ten plants for each line/pot) were tested in each experiment. In all experiments, both rglg1rglg2 double mutants and 35S:AtERF53/rglg1rglg2 transgenic plants had significantly higher survival rates than WT plants. Significant differences from WT were indicated (** P＜0.01, ***, P＜0.001).

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