The basic helix-loop-helix (bHLH) transcription factor DTT1 is part of a paired key that unlocks the tapetum...

The production of viable pollen is essential for effective fertilization and optimal crop yields; however, our understanding of the underlying mechanisms remains limited. Here, we characterize a barley (Hordeum vulgare) anther basic helix-loop-helix (bHLH) gene, DEFECTIVE TAPETUM TRANSITION1 (DTT1), a gatekeeper that regulates tapetum development. The dtt1 mutant is male sterile, failing to acquire tapetum cell fate identity with overproliferation of indeterminate tapetal precursor cells, a lack of tapetum endomitosis, and cell wall degeneration. DTT1 forms heterodimers with DYSFUNCTIONAL TAPETUM1 (HvDYT1) through bHLH and ACT-like(BIF) domains, with the ACT-like(BIF) domain and the IKL motif being critical in partner selection. These heterodimers may subsequently interact with each other through the bHLH-ACT-like(BIF) domain to activate expression. Transcriptome analysis confirmed that anther development transition from stages 6 to 7 fails in dtt1. We show that HvTDF1-related pathways are downstream of DTT1 and work in independent and overlapping networks with other conserved tapetum regulators. SELEX-seq analysis indicates that DTT1 can bind to DNA with a chimeric or canonical E-box motif only when it forms a complex with HvDYT1. In vivo dual-luciferase assays confirmed that the DTT1-HvDYT1 complex directly regulates the expression of several stage 7-specific transcription factors, such as HvTDF1, HvEAT1, and the identified GAMYB target genes. Therefore, the paired DTT1-HvDYT1 complex appears crucial in orchestrating the transition of tapetum cell fate by modulating genes involved in diverse biological pathways. This work uncovers detailed relationships in barley tapetum regulation and male fertility.