Subcellular localization of MtERF75 in M. truncatulaprotoplast
Subcellular localization of MtERF75 was investigated in M. truncatula protoplasts using a MtERF75 protein fused to the green fluorescent protein (GFP). GFP signal of the pAVA control vector (von Arnim et al., 1998) was observed mainly in cytoplasm by confocal microscopy. In contrast, M. truncatula protoplasts transformed with MC_ERF75:GFP fusion protein present a clear fluorescent signal located in a single point in the cell (Fig. 8) suggesting the presence of the protein in the nucleus even in absence of hypoxic stress. A second construction, MA_ERF75:GFP fusion, where the Cys residue was replaced by an Ala, was used to determine the role of the N-terminal Cys residues in the subcellular localization of ERF75 fused to GFP. A similar subcellular localization of the fluorescence was observed with MA_ERF75:GFP fusion to that with the MC_ERF75:GFP (Fig. S6A). However, we counted much more fluorescent protoplast after transformation with MA_ERF75:GFP construction (Fig. S6B). A statistical difference which could be due to a higher stability of the protein when the regulation by the N-end rule pathway is blocked.