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.