Cold stress is one of the adverse effects on apple distribution and production.However,the molecular mechanisms underlying apple cold stress tolerance are not clear.In this study,we cloned a novel R2R3 MYB transcription factor,MdMYB5523,and its close homolog MdMYB0970 from Golden Delicious (Malus × domestica).Expression of MdMYB5523 and MdMYBO970 was slightly induced by cold stress.Sub-cellular localization revealed that both proteins are localized in the nucleus.With Affinity Purification-Mass Spectrometry (AP-MASS) and yeast two-hybrid screening,we obtained two interacting partners of MdMYB5523 and MdMYB0970,MdHYL1 (HYPONASTIC LEAVES 1) and MdSERRATE.Chromatin Immunoprecipitation-PCR showed that MdMYB5523 and MdMYB0970 target genes involved in cold stress,including MdSIZ1 and MdCSP3 (COLD SHOCK DOMAIN PROTEIN 3).RNAi lines of MdMYB5523 and MdMYB0970 in Gala-3 (Malus × domestica) showed increased sensitivity to cold stress,while over-expression lines of MdMYB5523 or MdMYB0970 showed increased tolerance to cold stress,indicating that MdMYB5523 and MdMYB0970 are two positive regulators of cold stress in apple.Taken together,our results indicate that MdMYB5523 and MdMYB0970 played important roles in cold stress tolerance of apple.