PtABI3 represses the age biomarker gene <i>PtDAL1</i> during male cone development in conifer

The age biomarker gene <i>PtDAL1</i> in conifers undergoes expression resetting during pollen maturation, but its regulatory mechanisms remain unclear. This study identifies <i>PtABI3</i>, a B3 transcription factor (TF) in <i>Pinus tabuliformis</i>, as a key repressor of <i>PtDAL1</i>. <i>PtABI3</i> exhibits strict spatiotemporal complementarity with <i>PtDAL1</i>. Phylogenetic and structural studies revealed <i>PtABI3</i> as a gymnosperm homolog of angiosperm <i>ABI3</i>, retaining conserved B3 domains critical for DNA binding and transcriptional repression. Ectopic expression of <i>PtABI3</i> in <i>Arabidopsis</i> recapitulated the classic <i>ABI3</i> late-flowering phenotype. Mechanistically, <i>PtABI3</i> directly binds the RY motif (CATGCA) within the <i>PtDAL1</i> 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 <i>PtABI3</i> is an evolutionary conserved transcriptional repressor that silences <i>PtDAL1</i> 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.