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The “Spring-red-leaf” crabapple cultivar has young red leaves and mature green leaves. However, the mechanism of anthocyanin biosynthesis in crabapple leaves in spring remains unknown. In this study, Illumina RNA sequencing (RNA-Seq) was performed on  Malus  ‘Radiant’ leaf tissues in different stages of development. Twenty-two genes in the anthocyanin biosynthesis pathway and 44 MYB transcription factors (TFs) were significantly enriched among differentially expressed genes (DEGs). Three R2R3-MYB TFs in subgroup 22 of the MYB TF family,  MrMYB44-like1 ,  MrMYB44-like2 , and  MrMYB44-like3 , were highly expressed in green leaves according to RNA-Seq and quantitative real-time quantitative PCR results. Their expression levels were negatively correlated with anthocyanin content. In transient assays, overexpression of  MrMYB44-like1 ,  MrMYB44-like2 , or  MrMYB44-like3  inhibited anthocyanin accumulation and reduced pigment in leaf disks of  M . ‘Radiant’ and fruit peels of  M. domestica  ‘Fuji.’ When the conserved region of the three  MrMYB44-like s was silenced, the anthocyanin biosynthesis pathway was activated and pigments increased in both tissues. Moreover, bimolecular fluorescence complementation assays showed  MrMYB44-like s interacted with  MrWRKY6  to form protein complexes that regulated anthocyanin biosynthesis.

MrMYB44-Like Negatively Regulates Anthocyanin Biosynthesis and Causes Spring Leaf Color of Malus ‘Radiant’ to Fade From Red to Green