Genome-Wide Characterization of the PbeDof Gene Family Reveals PbeDof9.1 as a Key Regulator of Salt Tolerance via Enhancing Antioxidant Capacity in Pyrus betulifolia

Soil salinization severely restricts the sustainable development of the pear industry. <i>Pyrus betulifolia</i>, a vital native salt-tolerant rootstock in China, holds great significance for investigating stress resistance mechanisms. Plant-specific DNA-binding One Zinc Finger (Dof) transcription factors act as pivotal regulators in stress adaptation. However, their functions in <i>P. betulifolia</i> remain largely unexplored. In this study, we identified 43 <i>PbeDof</i> members within the <i>P. betulifolia</i> genome and classified them into eight subfamilies via phylogenetic analysis. Gene structure and conserved motif analyses revealed that <i>PbeDof</i> members within the same subfamily share similar exon-intron organizations and protein architecture, suggesting evolutionary conservation. Promoter analysis indicated that <i>PbeDof</i> genes are rich in cis-acting elements related to light, phytohormones (especially ABA and MeJA), and stress responses, implying their potential roles in diverse biological processes. Chromosomal localization and collinearity analyses revealed that segmental duplication was the primary driver of this family's expansion. Combined transcriptomic profiling and qRT-PCR assays demonstrated that <i>PbeDof9.1</i> is predominantly expressed in roots and is strongly induced by salt stress. Subcellular localization confirmed that <i>PbeDof9.1</i> targets the nucleus. Functional characterization indicated that heterologous overexpression of <i>PbeDof9.1</i> in <i>Arabidopsis thaliana</i> significantly enhances salt tolerance at germination and seedling stages. Notably, under 175 mM NaCl stress, the transgenic lines exhibited a superior root system architecture, with primary root length and lateral root numbers being approximately 1.5-fold higher than those of the wild type. Furthermore, homologous overexpression in pear calli confirmed that <i>PbeDof9.1</i> mitigates oxidative damage by boosting the activities of peroxidase (POD) and catalase (CAT) to scavenge reactive oxygen species (ROS), thereby reducing malondialdehyde (MDA) accumulation. Collectively, this study characterizes the <i>PbeDof</i> family and establishes <i>PbeDof9.1</i> as a key candidate gene for the genetic improvement of salt tolerance in pear rootstocks.