MtERF74/75 regulates hypoxia response genes in Medicago roots
Constitutive expression of M. truncatula MtERF74 andERF75 recalls the expression profile of RAP2.2 andRAP2.12 in Arabidopsis. Both constitutively synthesized TFs are described to play redundantly as main activators of core hypoxia-responsive genes in Arabidopsis (Gasch et al., 2016). To determine if MtERF74 and MtERF75 can also induce the expression of hypoxic stress in M. truncatula , MtERF74/75 knock-down roots were generated by RNA interference, through targeting a specific region of their N-terminal part (Fig. S1). The construction of the double mutant was preferred due to the technical difficulty of performing the two single mutants in view of the level of identity between the two sequences (85% identities). We assessed the effectiveness and specificity of the MtERF74/75 RNAi constructs by qPCR analysis. MtERF74 and MtERF75 transcript levels measured in independent non stressed MtERF74/75 RNAi transgenic roots were 37 % and 47 % reduced when compared to control transgenic roots (Fig. S3). Furthermore, MtERF72 and MtERF73expression were not affected in MtERF74/75 RNAi construct showing the specificity of this construction for MtERF74 andMtERF75 . Concerning, MtERF74/75 RNAi transgenic roots phenotype, root growth and secondary roots number showed respectively 29% and 18% reduction as compared to the control transgenic roots in aerobic condition (Fig. S4). Transgenic roots were then analyzed under hypoxic stress conditions. We exposed our transgenic roots knock-down for MtERF74 and MtERF75 to 24 hours of hypoxic stress in hypoxic chamber. The expression analysis of several HRGs , namelyADH1 , PDC1 , Pgb1.1 , AlaAT , and ERF73(Mustroph et al., 2010) showed a reduction in transcript levels in theMtERF74/75 RNAi transgenic roots, with a repression ranging from 50% (ADH1 ) to 24% (AlaAT ) compared to the GUS RNAi control (Fig. 3 and S5). Thus, the results showed that ERF74 and/or ERF75 contribute to hypoxia-dependent gene activation in M. truncatula .