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 .