Comprehensive Analysis of RNA Expression and Isoform Diversity Reveals Molecular Mechanisms in Plant Responses to Diverse Thermal Conditions

Abstract Elevated temperatures, including warm ambient temperature (WAT) and high ambient temperature (HAT), profoundly influence plant growth and development. While WAT promotes thermomorphogenesis and stimulates growth, HAT induces stress responses that can inhibit growth. Using Arabidopsis thaliana , we uncovered both shared and distinct molecular mechanisms underlying plant responses to these temperature conditions. We demonstrated that both WAT and HAT activate genes associated with the DNA damage response (DDR), but they differ in timing and intensity. While WAT encourages progression into the S phase of the cell cycle and enhances cell division, HAT induces G2/M arrest, indicating cell cycle inhibition caused by DNA damage accumulation and impaired DDR proteins. Notably, alternative splicing (AS), particularly intron retention (IR), emerged as a pivotal regulatory layer under heat stress. Prolonged high temperatures uniquely enriched IR events in genes involved in DNA repair and chromatin organization. Integrated analyses of AS and transcriptional changes revealed coordinated regulation, with IR events dominating genes that exhibit simultaneous upregulation. These findings highlight AS as an essential regulatory mechanism in plant responses to heat stress. Conserved responses to elevated temperatures in tobacco and tomato further suggest the presence of shared adaptive pathways across plant species. Our results provide novel insights into plant adaptation to fluctuating temperatures and highlight opportunities for engineering heat-resilient crops to mitigate the impacts of climate change.