MtERF74/75 are orthologues of RAP2.2 and RAP2.12 ERF-VII genes.
Four genes were identified in M. truncatula as belonging to the ERF-VII TF family (Fig. 1a), all of which possess the conserved N-terminal MCGGAI(I/L) amino acid motif (Nakano et al. , 2006). A phylogenetic tree was constructed to show the evolutionary relationship in the ERF-VII family between M. truncatula , A. thaliana ,Glycine max and Oryza sativa. Phylogenetic analysis of the ERF-VII protein sequences from M. truncatula , soybean, Arabidopsis and rice was performed using the Neighbour-Joining method (Fig. 1b). The resulting phylogenetic tree showed a clear separation between rice ERF-VII (SUB1 or SK orthologs) and ERF-VII proteins from the three other species. ERF-VII members from M. truncatula and soybean were distributed into three distinct clades. In the first clade, the gene showing sequence similarity to AtRAP2.3 was namedMtERF72 while the putative Medicago orthologous ofAtHRE2 was named MtERF73 (Table S3). In both legume species, no sequences homologous to Arabidopsis Hypoxia Responsive ERF1 (HRE1 ) (Tamang et al. , 2014) were identified (Fig. 1b). In the third clade, the two closest homologous sequences have been named MtERF74 and MtERF75 , as their orthologous in Arabidopsis, AtRAP2.12 and AtRAP2.2 (Gibbs et al., 2011; Licausi et al., 2011)(Table S3).
In silico gene expression analysis using MtExpress, a gene expression atlas that compiles an exhaustive set of published M. truncatula RNAseq data (https://medicago.toulouse.inrae.fr/MtExpress), revealed that the four ERF-VII genes were expressed in stems, leaves, seeds and roots, and also in nodules at different developmental stages (Fig. S2). MtERF72 expression was higher in mature seeds, stems, and roots whereas MtERF73 expression was strongly up-regulated during nodule development. MtERF74 andMtERF75 were found to be constitutively expressed in these different organs. However, MtERF75 was expressed approximately 8-fold more strongly than MtERF74 (Fig. S2). These observations were confirmed by a qPCR experiment performed in the roots and nodules of M. truncatula . MtERF73 expression was increased 8-fold in nodules compared with non-inoculated roots (Fig. 2a) and was strongly induced (6-fold) in roots after 24 hours of hypoxia (Fig. 2b). Using the Symbimics database (https://iant.toulouse.inra.fr/symbimics(Roux et al. , 2014)) available on MtExpress database, we obtained also data on the expression of ERF-VII mRNAs in the different zones of M. truncatula nodules (Fig. S2). MtERF72 showed higher expression in the zone I and II of the nodule, whereasMtERF73 was found in the interzone II-III (IZ) and N2-fixing zone III (Fig. S2). As previously observed for the different organs, MtERF74 and MtERF75 were found identically expressed in the different zones of the nodule.