Figure legends
Figure 1: Comparison of N-terminus and phylogenetic analysis of the ERF protein sequences of M. truncatula, A. thaliana, Glycine max and flooding related SK and SUB1 genes ofOryza sativa. (a) Multi-alignment with motives at the N-terminus of the ERF-VII protein sequences. The alignment quality and the consensus sequence were inferred using JalView (http://www.jalview.org/). The alignment file was obtained from Clustal Omega online tool (http://www.ebi.ac.uk). (b) A phylogenetic tree to highlight the evolutionary relationship in the ERF-VII families were inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates and the Evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA7 (https://www.megasoftware.net/).
Figure 2: Expression patterns of M. truncatula ERF-VII TFs performed by RTqPCR. (a) Expression analysis of ERF72 ,ERF73 , ERF74 and ERF75 in roots and nodules of 5 week-old plants. Nodules were collected 3 weeks post-inoculation. (b) Transcriptional regulation of ERF73 in roots of 5 week-old plants growing in aerobic conditions and after 24 hours of hypoxia-treatment (mean ± SE, **p-value <0.01, ***p-value < 0.001, one-way ANOVA, n=4).
Figure 3: Effect of MtERF74/75 RNAi on transcripts level of some hypoxia-responsive genes in M. truncatula root. Relative expression measured by RTqPCR of transgenic hairy roots submitted to 24 hours of hypoxia stress in hypoxic chamber. Expression level of each gene in RNAi 74/75 was compared to control plants normalized to 100%. Results are the mean (± SE (*P<0.05, **P < 0.01, one-way ANOVA) of three independent experiments composed by, at least, 10 plants for each condition.
Figure 4: Effect of MtERF74/75 RNAi during the symbiotic interaction between M. truncatula and S. meliloti . (a) Nodules number per plant at 14 days post-inoculation (dpi) in RNAi composite plants (RNAi 74/75) compared to control (Ctrl). Three independent series of 100 plants were used to calculate the average nodule number for each condition. (b) The nitrogen fixing capacity was measured by the acetylene reduction assay (ARA) expressed in nmol of ethylene (C2H4) reduced per mg of nodules and per hour, from plant nodules at 5 weeks post-inoculation (wpi). Results are the mean ± SE of three independent experiments composed by 15 plants each. (c) Nodules were collected, photographed and measured in size using ImageJ imaging software (https://imagej.nih.gov/ij/). For each independent experiment, at least 100 nodules were collected. Results are mean ± SE (*** p-value < 0.001, ** p-value < 0.01, one-way ANOVA)
Figure 5: Effect of MtERF74/75 RNAi on transcripts level of some hypoxia-responsive genes in M. truncatula nodules. Relative expression measured by RTqPCR in transgenic nodules (RNAi 74/75) in comparison to control nodules at 5 weeks post-inoculation (wpi) (Ctrl). Expression level of each gene in RNAi 74/75 was compared to control condition normalized to 100%. The results shown are the mean ± SE (*P<0.05, **P < 0.01, one-way ANOVA,) of five independent experiments with, at least, 10 plants for each condition.
Figure 6: Effect of MtERF74/75 RNAi on NO production in3 wpi-old M. truncatula mature nodules. The fluorescence intensity of the NO production was measured using the DAF-2 fluorescent probe. The results are the mean ± SE (*P<0.05, **P < 0.01, one-way ANOVA,) of the five independent experiments.
Figure 7: Measurement of luciferase activity on A. thaliana protoplast with MC80-ERF75::LUC and MA80-ERF75::LUC construction. (a) Luciferase activity of MC80-MtERF75::LUC and MA80-MtERF75::LUC in protoplasts of A. thaliana (col-0); (b) Luciferase activity of MC80-MtERF75::LUC in control protoplast (col-0) and in protoplast obtained from transgenic lines lacking in E3 ligase enzyme (prt6 ) (mean ± SE, *p-value <0.05, **p-value < 0.01, t-student test, n=4)
Figure 8: Subcellular localization of MtERF75 transcription factor in M. truncatula leaf protoplasts. pAVA vector was used as a 35S:GFP control. pMC-ERF75::GFP vector, C-terminal GFP fusion with full length ERF75 protein, was used to follow the subcellular localisation of ERF75 protein. Autofluorescence of chloroplasts was visualized in red under TRITC filter. Merged is the superimposition of the Bright field, GFP and TRICT picture. Scale bar 5 µm.
Figure 9: Post-translational modifications (S-nitrosylation, dimerized peptide) of MR21 and CR20 peptides after DEA-NONOate treatment . Peptides were treated for 5 min with various concentrations of DEA-NONOate and subsequently analyzed by MS. NM : not modified, NO : nitric oxide. Dimer: dimerized peptide. N=3.