<scp>EIL</scp> (ethylene‐insensitive 3‐like) transcription factors in apple affect both ethylene‐ and cold response‐dependent fruit ripening

EIN3/EIL (ethylene-insensitive 3/EIN3-like) transcription factors are positive downstream transcriptional regulators of ethylene signalling. In apple (Malus × domestica), a small family of MdEIL genes was identified, with four expressed in fruit. Transgenic lines were generated to manipulate MdEIL1 expression, and fruits were sampled at harvest maturity and after cold treatment. Their fruit ripening behaviour was compared with control lines and contrasted to a ACC OXIDASE 1 antisense line (ACO1as) which produced no ripening associated ethylene. Two transgenic lines showed strong co-suppression of MdEIL1-4 expression as well as reduced ethylene production, softening and aroma production, while one overexpressing line showed enhanced ripening. Key genes involved in ethylene biosynthesis and ethylene-dependent genes involved in cell wall modification (MdXTH1, MdβGAL) and aroma biosynthesis (MdAFS1, MdoOMT1) were downregulated in the co-suppressed lines. Co-suppressed lines showed reduced softening/volatile production after cold treatment and in contrast to the ACO1as line, expression of cold response-dependent genes (MdCBF2, dehydrins MdDHN2, -14, -16 and MdNAC29a) remained cold-repressed. The action of MdEILs was shown using dual-luciferase reporter assays to occur through direct activation of MdAFS1, MdXTH1 and MdβGAL promoters. Exogenous ethylene was unable to further stimulate ripening promoter activation, but cold treatment could. Promoter deletion analysis identified potential EIL binding sites in the MdAFS1 and MdβGAL promoters and electrophoretic mobility shift assays showed that MdEIL1-3 could all bind to a 32 bp fragment in the MdAFS1 promoter. Together these results indicate that MdEILs contribute to a suite of apple fruit ripening attributes via activation of genes in an ethylene-dependent manner, but also in response to cold.