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.