Organ- and temperature-dependent transcriptome diversity in tomato (Solanum lycopersicum) explored by Iso-seq single-molecule sequencing

• Iso-seq across five tomato organs exposed to different temperatures revealed an additional set of approximately 89,000 new splice variants. • Splicing profile regarding transcripts and splicing events is predominantly organ- and temperature-dependent. • Transcription and splicing target largely distinct gene sets involved in distinct biological processes. • Alternative splicing of HSFA6b yields two protein isoforms, with HsfA6b-I acting as an activator and HSFA6b-II as a putative repressor. Reorganization of gene expression is a key mechanism underlying plant responses to heat stress (HS). While the transcriptional regulation of gene expression under HS has been extensively studied, the contribution of alternative splicing (AS) remains less understood, largely due to the limitations of short-read RNA sequencing in capturing splice variant diversity. To overcome this, we employed long-read RNA-Seq using PacBio Iso-Seq in tomato, a model crop for HS studies. Transcriptomes were analyzed across five vegetative and reproductive organs subjected to a series of HS conditions. Iso-Seq data revealed an additional set of more than 89,000 unique transcripts to existing long-read datasets. Combined with short-read RNA-Seq, our analysis uncovered both global as well as organ- and temperature-specific qualitative and quantitative changes in splicing and revealed that 55% of tomato genes undergo AS. Leaves showed the highest number of alternatively spliced genes, while immature green fruits (IG) showed the lowest. Based on the response to temperature changes, roots displayed the highest number of differentially alternatively spliced (DAS) genes. Gene Ontology analysis indicated that DAS genes are enriched in biological processes uniquely associated with specific organs and temperature conditions. We observed a low overlap between DAS and differentially expressed genes (DEGs), suggesting that AS and transcriptional regulation act as largely independent layers in HS response. On the example of heat stress transcription factors (HSFs), we demonstrate that AS can result in unproductive splice variants likely targeted for degradation, or in the production of protein isoforms with distinct functions. We further identify a novel AS event in HSFA6b, generating isoforms with potential activator and repressor functions. Altogether, our findings establish AS as an organ- and temperature-specific regulatory layer shaping heat stress responses, revealed by long-read transcriptome profiling.