A 32-bp deletion in LiBBX16 underlies the dominant purple leaf trait in crape myrtle.

Structural variations that generate novel alleles are fundamental drivers of phenotypic diversity. In crape myrtle (Lagerstroemia indica), an important summer-flowering woody species, the purple leaf trait is prized for its visual appeal and stress resistance, representing a key breeding objective. Although anthocyanin accumulation underlies purple leaf formation, its key genetic locus has remained unclear. Here, we constructed five segregated populations of crape myrtle and confirmed that the purple leaf trait is a single-gene dominant trait. Combining bulked segregant analysis with metabolomics and RNA-seq led to the identification of LiBBX16, a B-box zinc-finger transcription factor. This gene has two alleles in purple-leafed (LiBBX16p and LiBBX16a) and green-leafed (LiBBX16g and LiBBX16a) plants. LiBBX16p contains a unique 32-bp deletion causing a frameshift, premature termination, and nucleo-cytoplasmic localization. An InDel marker derived from this deletion effectively distinguished purple-leafed from green-leafed plants and identified that all plants homozygous for LiBBX16p are seedling lethal. Heterologous expression of LiBBX16p in Arabidopsis resulted in purple leaves and stems, with anthocyanin content ~9.5-fold higher than in controls. Conversely, silencing LiBBX16 in purple-leafed L. indica reduced anthocyanin content, turning leaves green. We further found that the 32-bp deletion enhances LiBBX16's activation of anthocyanin synthesis-related genes, particularly LiF3GT and LiMYB75. Mechanistically, unlike LiBBX16g, which directly binds DNA, LiBBX16p likely acts as a coactivator recruited via protein-protein interactions. Overall, our results identified a neomorphic LiBBX16 allele that coregulates anthocyanin accumulation and seedling development, providing valuable insights into purple leaf formation and a target gene for molecular breeding.