Genome-Wide Identification of TPL/TPR Gene Family in Ten Cotton Species and Function Analysis of GhTPL3 Involved in Salt Stress Response

Background/Objectives: The TOPLESS (TPL) and TOPLESS-related (TPR) proteins represent a highly conserved class of transcriptional co-repressors in plants, playing pivotal roles in modulating growth, development, and stress responses through the repression of key transcriptional regulators. However, a comprehensive genome-wide analysis of the TPL/TPR gene family and its involvement in stress responses remains unexplored in cotton. Methods: In this study, 60 TPL/TPR genes were identified from the genomes of ten Gossypium species via bioinformatics approaches, and their protein physicochemical properties, gene structures, phylogenetic relationships, cis-regulatory elements, and expression profiles were characterized. Results: Chromosomal localization and collinearity analyses revealed that segmental duplication events have contributed to the expansion of the TPL/TPR gene family. Further examination of exon–intron architectures and conserved motifs highlighted strong evolutionary conservation within each TPL/TPR subgroup. Expression profiling demonstrated that TPL/TPR genes exhibit tissue-specific expression patterns, with particularly high transcript abundance in floral organs (e.g., petals and stigmas). Cis-element analysis suggested their potential involvement in multiple stress-responsive pathways. Notably, GhTPL3 showed high constitutive expression across various tissues and under stress conditions, with the most pronounced up-regulation under salt stress. Functional validation via Virus-Induced Gene Silencing (VIGS) confirmed that GhTPL3 silencing significantly impairs cotton salt stress tolerance, underscoring its critical role in abiotic stress adaptation. Conclusions: Our findings provide novel insights into the functional diversification and regulatory mechanisms of the TPL/TPR family in cotton, offering a valuable genetic resource for breeding stress-resilient cotton varieties.