Alternative splicing acts as a strategic switch for heat shock network diversification under escalating heat stress.

Alternative splicing (AS) is a pivotal post-transcriptional mechanism for plant thermal adaptation, yet its dynamics in heat-tolerant woody species remain poorly understood. Here, we map the temperature-resolved AS landscape of the highly thermotolerant woody plant Rhododendron hainanense by combining Iso-Seq with RNA-seq. Transcriptomic profiling revealed a graded regulatory architecture: moderate heat (37 °C) elicited a targeted proteostatic response, whereas severe heat (42 °C) provoked a massive transcriptome expansion. Notably, this global AS surge coincided with a widespread downregulation of core splicing factors, a correlation suggesting that a more permissive splicing environment may contribute to the rapid generation of diverse stress-responsive isoforms. The heat shock protein (HSP) family emerged as the principal hub of this AS-associated diversification; the core chaperone genes ClpB1, HSP83, and HSP70 generated the greatest transcript complexity, with HSP83 alone producing 29 distinct AS transcripts that resolved into three temperature-dependent expression groups. Within the heat shock factor network, HSFA2 showed the strongest and most sustained induction, and DAP-seq identified in vitro HSFA2 binding at the promoters of ClpB1, HSP83, and HSP70, supporting these core chaperones as putative direct DNA-binding targets. Functional complementation in Arabidopsis further showed that three structurally distinct HSFA2 splice variants each restored thermotolerance to the hsfa2 mutant and consistently induced expression of the core ATP-dependent chaperone arm, suggesting that this central chaperone-induction capacity is retained across the tested splice variants in this heterologous system. Together, these findings provide a comprehensive atlas of temperature-modulated AS in a thermotolerant woody plant and support a model in which splicing-mediated diversification of the HSP chaperone network contributes to heat adaptation, offering post-transcriptional insights for breeding climate-resilient crops.