Abstract
The age biomarker gene PtDAL1 in conifers undergoes expression resetting during pollen maturation, but its regulatory mechanisms remain unclear. This study identifies PtABI3, a B3 transcription factor (TF) in Pinus tabuliformis, as a key repressor of PtDAL1. PtABI3 exhibits strict spatiotemporal complementarity with PtDAL1. Phylogenetic and structural studies revealed PtABI3 as a gymnosperm homolog of angiosperm ABI3, retaining conserved B3 domains critical for DNA binding and transcriptional repression. Ectopic expression of PtABI3 in Arabidopsis recapitulated the classic ABI3 late-flowering phenotype. Mechanistically, PtABI3 directly binds the RY motif (CATGCA) within the PtDAL1 promoter via its B3 domain, as demonstrated by dual-luciferase assays, yeast one-hybrid assays, and electrophoretic mobility shift assays (EMSA). These results revealed that PtABI3 is an evolutionary conserved transcriptional repressor that silences PtDAL1 during pollen maturation, providing the first evidence of B3 TFs mediating age timer gene resetting in conifers. This work bridges gymnosperm and angiosperm regulatory paradigms, highlighting ancient mechanisms for gene expression resetting in plants.