Apple rootstock dwarfing and dense planting are common practices in apple farming. However, the dwarfing mechanisms are not understood. In our study, the expression of MdARF3 in the root system of dwarfing rootstock ‘M9’ was lower than in the vigorous rootstock from Malus micromalus due to the deletion of the WUSATAg element in the promoter of the ‘M9’ genotype. Notably, this deletion variation was significantly associated with dwarfing rootstocks. Subsequently, transgenic tobacco (Nicotiana tabacum) cv. Xanthi was generated with the ARF3 promoter from ‘M9’ and M. micromalus genotypes. The transgenic apple with 35S::MdARF3 was also obtained. The transgenic tobacco and apple with the highly expressed ARF3 had a longer root system and a higher plant height phenotype. Furthermore, the yeast one-hybrid, luciferase, electrophoretic mobility shift assays, and Chip-qPCR identified MdWOX4-1 in apples that interacted with the pMm-ARF3 promoter but not the pM9-ARF3 promoter. Notably, MdWOX4-1 significantly increased the transcriptional activity of MdARF3 and MdLBD16-2. However, MdARF3 significantly decreased the transcriptional activity of MdLBD16-2. Further analysis revealed that MdARF3 and MdLBD16-2 were temporally expressed during different stages of lateral root development. pMdLBD16-2 was mainly expressed during the early stage of lateral root development, which promoted lateral root production. On the contrary, pMmARF3 was expressed during the late stage of lateral root development to promote elongation. The findings in our study will shed light on the genetic causes of apple plant dwarfism and provide strategies for molecular breeding of dwarfing apple rootstocks.

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