Genome-Wide Isoform Switching Reveals SR45-Mediated Splicing Control of Arabidopsis Leaf Senescence

Leaf senescence is the final, programmed stage of leaf development, marked by nutrient remobilization and tightly regulated molecular events. Although alternative splicing has emerged as a major regulator of plant development, its role in isoform switching during leaf aging remains poorly understood. To address this, we conducted a genome-wide analysis of isoform switching in Arabidopsis, leveraging publicly available RNA-seq data from mature (16-day-old) and senescent (30-day-old) leaves, analyzed with the IsoformSwitchAnalyzeR package. Between these two developmental stages, we identified 269 genes exhibiting 377 significant isoform switches collectively predicted to alter protein localization, coding potential, and transcript stability. Experimental validation confirmed predicted switching at the PUS3 (Pseudouridine Synthase 3) locus, with sequence analysis revealing an age-dependent shift from mitochondrial-targeted to cytoplasmic isoforms. Gene Ontology enrichment analysis of switching genes revealed 82 significant terms, prominently associated with metabolism, gene expression, developmental regulation, and stress responses. Interestingly, we found nearly one-third of switching genes to overlap with known targets of the splicing factor SR45, with enrichment in pathways related to nucleotide and amino acid metabolism, energy production, and developmental processes. Correspondingly, dark-induced senescence assays revealed accelerated senescence in the sr45 mutant, confirming SR45's role in regulating leaf aging. Specific complementation of SR45's two isoforms revealed contrasting functions, with SR45.1 restoring normal senescence timing while SR45.2 failed to complement. Taken together, our findings demonstrate that differential isoform usage, orchestrated by specific splicing regulators, plays a critical role in leaf aging. This insight opens new avenues for manipulating senescence and engineering stay-green traits in crops.