Chromatin Topology Reconfiguration Orchestrates Thermotolerant Male Fertility via <i>GhAL5</i> in Cotton

Cotton, a globally vital crop, faces severe yield losses due to heat-induced male sterility. To decipher the thermotolerance mechanisms, we conducted multi-omics analyses (3D chromatin architecture, transcriptome, and epigenome profiling) on heat-tolerant (84021) and heat-sensitive (H05) lines across critical anther developmental stages. We identified subgenome homoeologous gene expression bias linked to thermotolerance, driven by high temperature (HT)-induced dynamic chromatin topology reorganization. The sensitive line exhibited aberrant 3D structural hyperactivation during anther dehiscence, causing deleterious gene overexpression. Central to this regulation is GhAL5, an Alfin-like transcription factor modulated through chromatin loop dynamics and TAD-like boundary reorganization under heat stress. Functional studies confirmed the pivotal role of GhAL5: overexpression enhanced thermotolerance, while RNAi/CRISPR lines showed compromised heat resilience. Remarkably, GhAL5 conferred cross-species heat protection when expressed in rice. Mechanistically, GhAL5 potentially orchestrates male thermotolerance through bidirectional chromatin structure modulation. This study establishes 3D genome plasticity and chromatin remodeling as key drivers of plant thermal adaptation, proposing chromatin-aware breeding strategies for climate-resilient crops.