An SR45-dependent exonic splicing enhancer is essential for regulating alternative splicing in Arabidopsis

Alternative splicing (AS) is a critical gene regulatory mechanism that underpins developmental plasticity and environmental adaptation in eukaryotes. Although numerous cis-elements and trans-factors governing AS have been characterized in animals, their functional roles in plants remain largely unexplored. We previously identified the Arabidopsis sot5 mutant, which exhibits leaf virescence due to a mutation in the conserved 5 splice site (5ss) of intron 7. Here, through characterization of a suppressor of sot5 (E61), we found that E61 harbors a G to A mutation in exon 8 of the SOT5 gene. Interestingly, this mutation creates a synonymous codon but disrupts the exonic splicing enhancer (ESE) motif (AAGAAGA), thereby reducing the splicing efficiency of the adjacent intron 7. Retention of this intron produces a functional protein that rescues the sot5 virescent phenotype. We confirmed the causal role of this ESE by using a cytidine base editor (CBE) to convert the ESE sequence to AAGAAAA or AAAAAAA in sot5. The edited plants phenocopied the suppressor E61. Genetic analyses revealed that mutations of SR45 encoding an RNA-binding protein but not its paralog SR45a similarly rescues the sot5 phenotype by impairing intron 7 splicing. Furthermore, we demonstrated that SR45 directly binds to the ESE motif in vitro, and that mutating the proximal ESEs in intron 2 of another SR45 target, GPDHC1 (Glycerol-3-Phosphate Dehydrogenase C1), recapitulates the splicing defect observed in the sr45 mutant. These findings establish that SR45 specifically recognizes AAG-repeat ESEs to enhance the splicing of adjacent weak introns, revealing a potent strategy for fine-tuning gene expression through targeted editing of splicing regulatory elements for crop improvement.