Supplementary Fig 1. The responses of ERF109 to different hormones and stresses. (a to k) The induced expression of ERF109 in 7-day-old Arabidopsis

Transcription

1 Supplementary Fig 1. The responses of ERF109 to different hormones and stresses. (a to k) The induced expression of ERF109 in 7-day-old Arabidopsis seedlings expressing ERF109pro-GUS. The GUS staining in the shoot and root tissues of 7-day-old seedlings treated by MS without any supplement as control (a), 10 μm MeJA (b), 10 μm SA (c), 10 μm ACC (d), 50 μm ABA (e), 100 μm GA (f), 1 μm IAA (g), 1 μm 6-BA (h), 200 mm NaCl (i), 300 mm Mannitol (j), and 10 μm PQ (k) for 0.5h were observed. The transgenic seedlings were grown on MS medium vertically for 7 days. Then, the seedlings were transferred to MS medium without (control) or with different hormones or compounds for 0.5h before GUS staining. For shoot tissues, bars=2 mm, for root tissues, bars=100 μm. (l) The expression levels of ERF109 were detected by quantitative RT-PCR using 7-day-old Arabidopsis wild type seedlings. The Col-0 seedlings were grown on MS medium vertically for 7 days. Then, the seedlings were transferred to MS medium without (control) or with different hormones or compounds for 0.5h before RNA extraction. Values are mean ±SD (n=3 experiments, P < 0.05, P < 0.01, P <

2 0.001). Asterisks indicate Student s t test significant differences. The result of quantitative RT-PCR analysis is consistent with the results of GUS staining. The MeJA-induced expression of ERF109 is intensively, which is great higher than the induced expression of ERF109 caused by other hormones and stresses. (m and n) The phenotype of wild type under NaCl treatment. The phenotype of wild type grown on MS without (m) or with NaCl was observed (n). Bars = 1cm. (o) The expression of ERF109 was analysis by quantitative RT-PCR during the salt-induced adventitious root (AR) formation. RNA was isolated from root tissues of wild type grown on MS without or with 120 mm NaCl. The transcript levels of ER109 were analysed. Values are mean ±SD (n=3 experiment).

3 Supplementary Fig 2. The expression pattern of ERF109. (a) ERF109 transcript level in different tissues of wild type. RNA was isolated from inflorescence stem, rosette leaves, cauline leaves, flowers and siliques of adult Arabidopsis as well as the roots and shoots of Arabidopsis seedlings. Expression of ERF109 was analysed by quantitative RT-PCR. Values are mean ±SD (n =3 experiments). (b to k) Promoter-GUS expression patterns of the ERF109. ERF109 was expressed in seedling (b) (Bar=5 mm), the edge of rosette leaves and cauline leaves of adult Arabidopsis (c and d) (Bars=5 mm), base of sliques (e) (Bar=2 mm), exclusively in the anthers of stamen (f) (Bar=2 mm), and LRPs at early developmental stages (g). ERF109 was also weakly expressed in the tip and base of old LRPs (h and i), and in the tip and base of LRs (j and k). Bars=100 μm for g to k.

4 Supplementary Fig 3. Protein localization of ERF109. (a to c) 35S-GFP was used as a control in transient expression assay. Bars=200 μm. (d to f) Subcellular localization of the ERF109-GFP protein in onion epidermal cell. 35S-ERF109-GFP was transiently expressed in onion epidermal cells. Bars=200 μm. (g to i) The transgenic plants expressing ERF109-GFP fusion construct were observed to confirm subcellular localization of ERF109. Bars = 100 μm. The ERF109-coding region was cloned into pgwb5 and the construct was transferred into Col-0 to create transgenic plants. The T1 population was used for GFP observation. Three independent lines were analysed and the same result was obtained. The photographs from left to right: white light (left), superimposed pictures of the same field (middle) and green fluorescent (right).

5 Supplementary Fig 4. Homozygous T-DNA insertion mutants of Salk_150614, and 35S-ERF109 were identified. (a) The schematic illustration of the chromosomal location of the T-DNA insertion in Salk_ The locations of T-DNA (inverted open triangle), UTR (black box), and exon (white box) were shown. (b) PCR analysis of homozygous T-DNA insertion mutants of Salk_ Genomic DNA extracted from leaves of Salk_ line and the wild type was used as templates for PCR. (c) The homozygous T-DNA insertion mutant of Salk_ was confirmed by RT-PCR. 10-day-old wild type and erf109 seedlings were treated without or with 1μM MeJA for 0.5h before the RNA extractions. TUB8 was used as a loading control. (d) The ERF109 transcript level was measured with quantitative RT-PCR for the shoot and root tissues of erf109, wild type, and 35S-ERF109-3 seedlings. Values are mean ±SD (n =3 experiments).

6 Supplementary Fig 5. The phenotypes of different 35S-ERF109 lines. (a) Identification of 35S-ERF109 plants. Quantitative RT-PCR analysis of the transcript levels of ERF109 in different 35S-ERF109 lines. Total RNA was isolated from rosette leaves of adult Arabidopsis. Values are mean ±SD (n=3 experiments). (b) PR elongation of wild type and different 35S-ERF109 lines grown on MS were measured at the indicated time points. Values are mean ±SE (n = 30 seedlings). (c) The numbers of LRs of wild type and different 35S-ERF109 lines grown on MS were counted at the indicated time points. Values are mean ±SE (n = 30 seedlings). (d) The phenotypes of different 35S-ERF109 lines. The seedlings of 35S-ERF109 were grown on MS for two weeks. The numbers of LR of 35S-ERF109 lines were increased in different degrees and the length of PR of 35S-ERF109 lines were shortened in different degrees. Bars=1 cm.

7 a b c d e f g h i j ASA1 TUB8 Supplementary Fig 6. PR elongation curve of erf109, wild type, 35S-ERF109-1, 35S-ERF109-2, and asa1 seedlings grown on medium containing indicated concentrations of MeJA. (a to h) PR elongation of asa1 (CS16398), wild type, erf109, 35S-ERF109-1 and 35S-ERF109-2 seedlings grown on MS without (a) or with MeJA (b to h) in different concentrations were measured at the indicated time points. Values are mean ±SE (n = 25 seedlings). The 35S-ERF109-1 and 35S-ERF109-2 data overlap so heavily that they are difficult to distinguish. Moreover, the erf109, Col-0 and asa1 data also have a lot of overlap. (i) MeJA dose response curve of 12-day-old erf109, wild type, 35S-ERF109-1, 35S-ERF109-2, and asa1 seedlings for PR elongation. PR length of 12-day-old erf109, wild type, 35S-ERF109-1, 35S-ERF109-2, and asa1 seedlings grown on medium

8 containing indicated concentrations of MeJA were measured. Values are mean ±SE (n = 25 seedlings). The 35S-ERF109-1 and 35S-ERF109-2 data overlap so heavily that they are difficult to distinguish. Moreover, the erf109, Col-0 and asa1 data also have a lot of overlap. (j) The asa1 mutant was identified by RT-PCR. RNA extracted from leaves of asa1 line and wild type was used as the templates for RT-PCR. TUB8 was used as a loading control.

9 a b c d e f g h i Supplementary Fig 7. LR formation curve of erf109, wild type, 35S-ERF109-1, 35S-ERF109-2, and asa1 seedlings grown on medium containing indicated concentrations of MeJA. (a to h) The numbers of LRs of asa1, wild type, erf109, 35S-ERF109-1 and 35S-ERF109-2 seedlings grown on MS without (a) or with MeJA (b to h) in different concentrations were counted at the indicated time points. Values are mean ±SE (n = 25 seedlings, P < 0.05, P < 0.01, P < 0.001). Asterisks indicate Student s t test significant differences. In Fig. a, the erf109 and Col-0 data overlap so heavily that they are difficult to distinguish. (i) MeJA dose response curve of 12-day-old erf109, wild type, 35S-ERF109-1, 35S-ERF109-2, and asa1 seedlings for LR formation. The numbers of LRs of erf109, wild type, 35S-ERF109-1, 35S-ERF109-2, and asa1 seedlings grown on medium containing indicated concentrations of MeJA were counted. Values are mean ±SE (n = 25 seedlings, P < 0.05, P < 0.01, P < 0.001). Asterisks indicate Student s t test significant difference.

10 a b c d e f g h i j Supplementary Fig 8. PR elongation and LR formation curve of erf109, wild type, and 35S-ERF109-1 seedlings grown on medium containing indicated concentrations of salt. (a to d) PR elongation of wild type, erf109, and 35S-ERF109-1 seedlings grown on MS without (a) or with NaCl (b to d) in different concentrations were measured at the indicated time points. Values are mean ±SE (n = 25 seedlings). The erf109 and Col-0 data overlap so heavily that they are difficult to distinguish. (e) Salt dose response curve of 12-day-old erf109, wild type, and 35S-ERF109-1 for PR elongation. PR length of 12-day-old erf109, wild type, and 35S-ERF109-1 seedlings grown on medium containing indicated concentrations of NaCl were measured. Values are mean ±SE (n = 25 seedlings). The erf109 and Col-0 data overlap so heavily that they are difficult to distinguish. (f to i) The numbers of LRs of wild type, erf109, and 35S-ERF109-1 seedlings grown on MS without (f) or with NaCl (g to i) in different concentrations were counted at the indicated time points. Values are mean ±SE (n = 25 seedlings, P < 0.05). Asterisk indicates Student s t test significant difference. In Fig. f, the erf109 and Col-0 data overlap so heavily that they are difficult to distinguish. (j) Salt dose response curve of 12-day-old erf109, wild type, and 35S-ERF109-1 for LR formation. The numbers of LRs of 12-day-old erf109, wild type, and 35S-ERF109-1 seedlings grown on medium containing indicated concentrations of NaCl were counted. Values are mean ±SE (n = 25 seedlings, P < 0.05). Asterisk indicates Student s t test significant differences.

11

12 Supplementary Fig 9. Functional complementation assay. (a) The expression level of ERF109 in FC line was analysed by quantitative RT-PCR. Values are mean ±SD (n =3 experiments). (b) The numbers of LRPs of 5-day-old wild type, erf109 and FC seedlings grown on MS were counted. Values are mean ±SD (n = 15 seedlings, P < 0.01). Asterisks indicate Student s t test significant differences. (c and d) The numbers of LRs of wild type, erf109, and FC seedlings grown on MS without (c) or with 10nM MeJA (d) were counted at the indicated time points. Values are mean ±SE (n = 20 seedlings, P < 0.05, P < 0.001). Asterisks indicate Student s t test significant differences.

13 Supplementary Fig 10. Analysis of different FC lines. (a) Quantitative RT-PCR analysis of the expression levels of ERF109 in different FC lines. RNA was isolated from the leaves of adult plants of FC lines. Quantitative RT-PCR analysis was performed using the primers listed in Supplementary Table 1. Values are mean ±SD (n=3 experiments). (b) The root phenotypes of FC lines. The seedlings of FC lines were grown on MS for two weeks. Bars=1 cm. (c and d) The FC lines with overexpression of ERF109 show the similar phenotypes of 35S-ERF109. The phenotype of FC-16 was observed at 3d (c) and 10 d (d). FC-16 has short PR and more LRs. Bars = 1 cm. (e) The numbers of LRPs of 4-day-old wild type, erf109, and FC-16 seedlings were counted. Values are mean ±SD (n = 10 seedlings, P < 0.05, P < 0.01). Asterisks

14 indicate Student s t test significant differences. The erf109 has less number of LRP and FC-16 has more number of LRP than wild type. (f) PR elongation of wild type, erf109, 35S-ERF109-1 and FC-16 seedlings grown on MS was measured at the indicated time points. Values are mean ±SE (n = 25 seedlings, P < 0.001). Asterisks indicate Student s t test significant differences. The erf109 and Col-0 data overlap so heavily that they are difficult to distinguish. (g) The numbers of LRs of wild type, erf109, 35S-ERF109-1 and FC-16 seedlings grown on MS were counted at the indicated time points. Values are mean ±SE (n = 25 seedlings, P < 0.05, P < 0.01, P < 0.001). Asterisks indicate Student s t test significant differences. The erf109 and Col-0 data overlap so heavily that they are difficult to distinguish. Moreover, overlap is also found between 35S-ERF109-1 and FC-16 data. Supplementary Table 1. The Primers Used in This Study. β-tubulin8 P1 β-tubulin8 P2 ERF109 RT P1 ERF109 RT P2 UBQ5(Q-PCR) P1 UBQ5(Q-PCR) P2 ERF109(Q-PCR) P1 ERF109(Q-PCR) P2 ASA1(Q-PCR) P1 ASA1(Q-PCR) P2 YUC2(Q-PCR) P1 YUC2(Q-PCR) P2 IAA14(Q-PCR) P1 IAA14(Q-PCR) P2 IAA 19(Q-PCR) P1 5 -CTTAAGCTCACCACTCCAAGCT-3 5 -GCACTTCCACTTCGTCTTCTTC-3 5 -ATGCATTATCCTAACAACAGAA-3 5 -TCACTGGAACATATCAGCAAT-3 5 -AGAAGATCAAGCACAAGCAT-3 5 -CAGATCAAGCTTCAACTCCT-3 5 -CTTATGATCGAGCCGCGATT-3 5 -TCCTCCGTTCCATTGCTCTG-3 5 -ATGTCTTCCTCTATGAACGTAGC-3 5 -ACAGCGGTAAATTGGTATAAGG-3 5 -GGTGACACGGATCGGTTAGGGT-3 5 -TGCCGAATAATGCATTACCCGT-3 5 -CCTTCTAAGCCTCCTGCTAAAGC-3 5 -CCGCTCTTCTGATTAGCCATAAC-3 5 -GTGGTGACGCTGAGAAGGTT-3

15 IAA 19(Q-PCR) P2 LBD16(Q-PCR) P1 LBD16(Q-PCR) P2 Y1H ERF109 P1 Y1H ERF109 P2 Y1H GCC P1 Y1H GCC P2 Y1H YUC2 P1 Y1H YUC2 P2 Y1H ASA1 P1 Y1H ASA1 P2 CHIP YUC2pro P1 CHIP YUC2pro P2 CHIP YUC2pro NC P1 CHIP YUC2pro NC P2 CHIP ASA1pro P1 CHIP ASA1pro P2 CHIP ASA1pro NC P1 CHIP ASA1pro NC P2 8E/ERF109 P1 5 -CGTGGTCGAAGCTTCCTTAC-3 5 -TGACCCTGTTTATGGATGTGTC-3 5 -TGATTGCAAGAAAGCCACCT-3 5 -CGGGATCCATGCATTATCCTAACAACAGAA-3 5 -GCTCTAGATCACTGGAACATATCAGCAAT-3 5 -CGCCGCC GCCGCCGCCGCCA-3 5 -CGCGTGGCGGC GGCGGCGGCGGCGAGCT-3 5 -CACATCTTAAAAGGCCGCCAGGAGCTATAACA-3 5 -CGCGTGTTATAGCTCCTGGCGGCCTTTTAAGATGTGAGCT-3 5 -CTCAAACATTCCGGCCGCCACCGAGTTCTTA A-3 5 -CGCGT TAAGAACTCGGTGGCGGCCGGAATGTTTGAGAGCT-3 5 -TGTACCTTACTTTGATCCGTCC-3, 5 -A ACCCACTAAAGAATCCAACTTG-3, 5 -TTAACCTGAGATTCGGGTTTCG-3 5 -TACAAATGTGGACCATGATCTAAT-3 5 -GTTCGCTGCCTTCATGGTTC-3, 5 -TCCAGTAACGGTCAC AGAAG A CTCATATCATTACACCGCACAAT-3 5 -TAGATTGACAATCCCACACGTAA-3 5 -CCCAAGCTTATGCATTATCCTAACAACAGAA-3 8E/ERF109 P2 5 -CGGGATCCCACTGGAACATATCAGCAATT- 3 GUS/ ERF109 P1 GUS/ ERF109 P2 Salk_ P1 Salk_ P2 LBb1 pcb2004/erf109 P1 pcb2004/erf109 P2 pgwb5/erf109 P1 5 -GGGGACAAGTTTGTACAAAAAAGCAGGCT-TTTATTTTCACATGGCGAACATT-3 5 -GGGGACCACTTTGTACAAGAAAGCTGGGT-GGGAAACTGAAAGTGAGAAAGA-3 5 -CGCGATGCTTTGTAGGAGTAG-3 5 -GATCTCAGGGGAAAACGAAAC-3 5 -GCGTGGACCGCTTGCTGCAACT GGGGACAAGTTTGTACAAAAAAGCAGGCT-ATGCATTATCCTAACAACAGAA GGGGACCACTTTGTACAAGAAAGCTGGGT-TCACTGGAACATATCAGCAAT-3 5 -GGGGACAAGTTTGTACAAAAAAGCAGGCT-

16 ATGCATTATCCTAACAACAGAAC-3 pgwb5/erf109 P2 5 -GGGGACCACTTTGTACAAGAAAGCTGGGT- CCTGGAACATATCAGCAATTGTAT-3 ASA1-EMSA-P1-DIG ASA1-EMSA-P2 ASA1-EMSA-P1 ASA1-EMSA-Mu-P1 ASA1- EMSA-Mu-P2 YUC2-EMSA-P1-DIG YUC2-EMSA-P2 YUC2-EMSA-P1 YUC2- EMSA-Mu-P1 YUC2- EMSA-Mu-P2 HA-ERF109 P1 5'-DIG-TCAAACATTCCGGCCGCCACCGAGTTCTTA-3 5 -TAAGAACTCGGTGGCGGCCGGAATGTTTGA-3 5'-TCAAACATTCCGGCCGCCACCGAGTTCTTA-3 5'-TCAAACATTCCGCATTGAACCGAGTTCTTA-3 5'-TAAGAACTCGGTTCAATGCGGAATGTTTGA-3 5'-DIG-ACATCTTAAAAGGCCGCCAGGAGCTATAAC-3 5'-GTTATAGCTCCTGGCGGCCTTTTAAGATGT-3 5'-ACATCTTAAAAGGCCGCCAGGAGCTATAAC-3 5'-ACATCTTAAAAGCATTGAAGGAGCTATAAC-3 5'-GTTATAGCTCCTTCAATGCTTTTAAGATGT-3 5 -GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTACCCATACGATGTTCCAGAT- TACGCTATGCATTATCCTAACAACAGAA -3 HA-ERF109 P2 asa1 (CS16398) P1 asa1 (CS16398) P2 5 -GGGGACCACTTTGTACAAGAA AGCTGGGTTCACTGGAACATATCAGCAAT-3 5 -GAGATCGTGGCAAAGGAGAAT-3 5 -AATGCAGATTCAGCCAAGTCG-3