Alternative splicing-dependent T218 phosphoregulation controls SR45.1 nuclear organization and salt stress responses in Arabidopsis.

key messageAlternative splicing creates an SR45.1-specifi c phosphoregulatory region in which threonine 218 (T218) contributesto nuclear speckle organization, RNA regulatory responses, and salt tolerance in Arabidopsis. Alternative splicing expands protein and functional diversity in plants, yet the mechanistic basis by which closely related splice isoforms acquire distinct biological functions remains poorly understood. In our previous work, we showed that AS of the Arabidopsis splicing factor SR45 generates two isoforms with contrasting roles in salt stress response, where SR45.1, but not SR45.2, restores salt tolerance in the sr45 mutant background. The molecular basis underlying this isoform-specific functional divergence remained unclear. Here, we investigated the role of two conserved phosphosites, threonine 218 (T218) and serine 219 (S219), within the unique C-terminal region of SR45.1, a sequence absent from SR45.2. Site-directed mutagenesis was used to generate phospho-disruptive SR45.1 variants, which were then expressed in the sr45 mutant background. Functional analyses revealed that substitution of T218, but not S219, abolished the salt-tolerance function of SR45.1. Mechanistically, disruption of T218 altered SR45.1 nuclear speckle organization, resulting in fewer enlarged nuclear speckles compared with the numerous small speckles observed in functional lines. T218 disruption was further associated with altered AS of stress-related targets, including SOS4 and RD20, as well as reduced transcript accumulation of salt-responsive genes, including RD29A, RD29B, ADH1, and DREB2A. Together, our findings identify T218 phosphoregulation as a critical determinant of SR45.1 function, linking AS-generated isoform diversity to post-translational regulation, nuclear organization, and plant salt stress responses.