Expression and Characterization of MdERFs with Roles in Apple Softening

A rapid increase in endogenous ethylene production during ripening is related to fruit softening in the apple (Malus domestica Borkh.) cultivar ‘Taishanzaoxia’. Two genes involved in the ethylene response, MdERF1.1 and MdERF2.1, were isolated and characterized. MdERF1.1 and MdERF2.1 were predicted to encode proteins of 252 and 334 amino acids, respectively. MdERF1.1 and MdERF2.1 were expressed in Escherichia coli, and their molecular weights were determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and western blotting analyses (28 and 37 kD, respectively). Sequence comparisons and phylogenetic and secondary structure indicated that MdERF1.1 and MdERF2.1 are members of the ERF family. Both of them include a typical GCC‑box binding domain consisting of an α-helix and a three-stranded anti-parallel β-sheet. Sequence comparisons showed that MdERF1.1 is highly identity to MdERF1 from ‘Golden Delicious’, but Gly replaces Ala at position 110 in β-strand 2. This amino acid variation may be involved in fruit softening. Similarly, MdERF2.1 is highly identity to MdERF2 from ‘Golden Delicious’ but differs at three amino acids (Ala, Thr and Glu replace Pro, Ala and Asp at positions 97, 139, and 269, respectively). These three amino acid differences in MdERF2.1 might also be related to fruit softening. In ripening fruit, MdERF1.1 transcript levels rapidly increased during fruit softening in ‘Taishanzaoxia’, and this increase was associated with ethylene production. MdERF2.1 was predominantly expressed in softening ‘Taishanzaoxia’ fruit. The results indicate that ethylene signaling and MdERF1.1 and MdERF2.1 are involved in fruit softening in ‘Taishanzaoxia’.