Pan-plant phylogenomics reveals ancestral origins, duplication-driven expansion, and conserved regulatory modules of the bZIP transcription factor family

Abstract Basic leucine zipper (bZIP) transcription factors represent one of the largest and most conserved transcription factor families in plants, yet their evolutionary origin, expansion dynamics, and regulatory diversification across the plant kingdom remain incompletely understood. Here, we identified 22,378 bZIP genes from 321 representative plant species spanning major lineages from red algae to angiosperms and integrated phylogenomic reconstruction, duplication-mode analysis, evolutionary constraint assessment, comparative transcriptomics, regulatory-network inference, pangenome analysis, and functional validation. Phylogenetic analyses revealed that plant bZIP proteins are organized into four major clades comprising 13 conserved subgroups, and that the core subgroup architecture was established before the emergence of land plants. Family expansion showed strong lineage dependence, with dispersed duplication predominating in early-diverging lineages and whole-genome/segmental duplication becoming the major expansion mode in angiosperms. Despite extensive expansion during plant evolution, pervasive purifying selection maintained the structural conservation of bZIP proteins, particularly within the conserved bZIP domain. Comparative expression and regulatory network analyses revealed substantial divergence in bZIP regulatory programs across plant lineages. AtbZIP27 and MsbZIP26 activated their corresponding MIOX promoters, whereas SlbZIP70 did not, indicating that the bZIP–MIOX regulatory relationship is conserved in some lineages but has diverged in others. Functional assays under aluminum stress further supported the biological relevance of this regulatory relationship, while tomato pan-genome analysis revealed that such regulatory divergence can occur against a largely conserved genomic background. Collectively, these findings reveal that plant bZIP evolution combines duplication-driven expansion and strong selective constraint with progressive diversification of regulatory relationships and functions.