Download SUPPORTING INFORMATION FULL LEGENDS Figure S1

SUPPORTING INFORMATION FULL LEGENDS
Figure S1. Expression levels of ERF-VII type transcription factors in public available database. Gene
expression analysis in public database shows transcript levels of RAP2.12 (At1g53910), RAP2.2
(At3g14230), RAP2.3 (At3g16770), HRE1 (At1g72360) and HRE2 (At2g47520) during developmental
stages of Arabidopsis plants (https://www.genevestigator.com/gv/plant.jsp) .
Figure S2. Stress regulation of ERF-VII type transcription factors. Gene expression changes of
RAP2.12 (At1g53910), RAP2.2 (At3g14230), RAP2.3 (At3g16770), HRE1 (At1g72360) and HRE2
(At2g47520) in abiotic stress conditions from the Genevestigator database.
(https://www.genevestigator.com/gv/plant.jsp) .
Figure S3. Transactivation of ADH1-LUC by estradiol induced RAP2.12, RAP2.2 and RAP2.3 in
independent lines. Independent transgenic lines were generated by transforming ADH-LUC plants
with XVE-RAP2.12, XVE-RAP2.2 or XVE-RAP2.3, respectively. Single-copy XVE-RAP2 insertion lines
were selected in T2 generation, based on their segregation of Hygromycin resistant growth. ADH1LUC activation of ten selected lines from each genotypes were tested. A line with high
transactivation ability from each genotype was selected for further studies.
Figure S4. Selected ABA and osmotic stress induced genes that were unaffected by RAP2.12
overexpression. For the qPCR experiment samples were harvested from two weeks old plants that
were treated with 5 μM estradiol -/+ 30 μM ABA for 9 hours. The following osmotic stress tolerance
genes were tested: DREB2A (DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2A), RAB18
(RESPONSIVE TO ABA 18), RD29B (RESPONSE TO DROUGHT 29), ABF3 (ABA RESPONSIVE ELEMENTSBINDING FACTOR 3), RAP2.6 (RELATED TO APETALA2.6). Expression of RAP2.12 was 65-70 times
higher in the AE-RAP2.12 line compared to ADH-LUC control line after 9 hours estradiol treatment.
Figure S5. Expression of RAP2.12 and RAP2.3 genes in single rap2.12-2, rap2.3-1 and the rap2.12-2
rap2.3-1 double mutant. RT-PCR analysis with RAP2 specific and Tubulin primers using total RNA
extracted from 1 week old plantlets of the wild type (Col-0) and mutant lines.
Figure S6. Comparison of wild type and rap2.2-4 insertion mutant line in osmotic stress growth
assay. Plants were germinated on ½ MS media and 4 day after germination were transferred to ½
MS or ½ MS complemented with 300 mM mannitol media. Relative rosette growth calculated 15
days later.
Figure S7. Gene expression changes of osmotic stress induced genes in wild type and rap2.12-2
rap2.3-1 double mutant line. For qPCR analysis samples were harvested from two weeks old plants
that were treated 300 μM mannitol for 9 hours.
Figure S8. Amino acid sequence of RAP2.12 and HA:RAP2.12 proteins. Construction of HA:RAP2.12
fusion protein resulted in the addition of 22 amino acids to the N-termini of RAP2.12 (see underlined
amino acids). HA:RAP2.12 therefore, is not substrate for Arg/N-end rule pathway.
Figure S9. HA:RAP2.12 protein is stabilised by the proteasome inhibitor MG132. Ten days old XVEHA:RAP2.12 plants were treated with 5 μM estradiol for 24 hours to induce the expression of
HA:RAP2.12 protein. Total protein was extracted with extraction buffer that did not contain MG132.
The protein extract was shared into two tubes and 100 μM MG132 or 1% DMSO solvent was added,
respectively. Then, the two tubes were incubated on ice, samples were taken at 0, 1 and 2 hours
time points. For western detection with anti-HA, 75 μg total protein was loaded.
Figure S10. Phylogenetic analysis of TRAF domain-like proteins of Arabidopsis.
An unrooted phylogenetic tree based on Maximum likelihood method with 1000 bootstrap
replicates was drawn to show the degree of relationship between amino acid sequences.
Figure S11. Co-expression of the Arabidopsis SINAT1 and SINAT2.
The Arabidopsis SINAT1 and SINAT2 show high level similarity in their transcriptional regulation
according to microarray data (http://genecat.mpg.de).
Figure S12. Co-silencing of SINAT1 and SINAT2 genes.
Artificial microRNAs (amiRNAs) were designed to silence the two homologous RING-finger type
ubiquitin ligases. A) cDNA sequences of SINAT1 and 2, the underlined nucleotides correspond to the
SINAT1/2 specific amiRNA sequence. B) Alignment of the SINAT1 and SINAT2 sequences showing the
amiRNA sequence (underlined). C) The over-expressed SINAT1/2 specific short hairpin RNAs are
derived from the miR-319a microRNA precursor and are able to silence both SINAT1 and 2 gene
expression.
Figure S13. Model of RAP2.12, RAP2.2 and RAP2.3 mediated transcriptional activation of the low
oxygen and ABA induced ADH1 gene.
During submergence the ERF-VII-type transcription factors are stabilised and function as
transcriptional activators of low oxygen induced genes including ADH1. After re-oxygenation
dehydration of tissues mediates ABA accumulation, ERF-VII-type transcription factors affect ADH1
and possibly other ABA induced genes.
Table S1. List of primers used in this study.
References for the primers listed in Table S1:
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