Genome-Wide Identification, Drought-Responsive Expression, and EAR-Mediated Regulatory Network Construction of TOPLESS Genes in Populus ussuriensis Kom.

Drought stress significantly impairs plant growth and productivity, which triggers complex adaptive responses mediated by diverse gene families. Among these, the TOPLESS (TPL)/TPL-related (TPR) family of transcriptional corepressors plays a crucial role by recruiting epigenetic modifiers through interactions with EAR motif-containing proteins. However, genome-wide studies of this corepressor family and its associated regulatory networks with EAR motif-containing repressors remain limited. This study aimed to characterize the TPL/TPR transcriptional corepressor family in <i>Populus ussuriensis Kom</i>., elucidate their regulatory networks with EAR motif-containing repressors, and validate their functional roles in drought stress adaptation. To this end, we identified 21 <i>TPL/TPR</i> genes in <i>P. ussuriensis</i> (<i>PuTPLs</i>), classified them into five subfamilies, and found they are evolutionarily conserved with <i>Arabidopsis thaliana</i> and <i>Populus trichocarpa</i>, harboring characteristic CTLH and WD40 domains. Given that TPL/TPR proteins are recruited by transcription factors containing repression motifs, we constructed a putative TPL/TP<i>R</i>-EAR motif interaction network representing a core paradigm of negative regulation. Expression profiling under drought stress showed significant upregulation of most <i>PuTPLs</i> in a tissue-specific and temporal manner. Functional validation using transgenic <i>P. ussuriensis</i> lines overexpressing five <i>PuTPLs</i> demonstrated enhanced drought tolerance, evidenced by reduced electrolyte leakage and malondialdehyde content and increased proline accumulation. Our study provides the first comprehensive genome-wide analysis of the TPL/TPR family in <i>P. ussuriensis</i>, establishes a core EAR-mediated negative regulatory network, and validates the critical role of these genes in drought stress adaptation, providing valuable resources for future mechanistic research and breeding of stress-resistant trees.