From annotation to regulation: Iso-seq reveals novel isoforms and alternative splicing features underlying salinity...

Soil salinity is a major environmental constraint that limits plant growth. While transcriptional reprogramming is central to salt stress responses, evidence indicates that alternative splicing (AS) provides an additional regulatory layer that modulates gene function without necessarily altering overall transcript abundance. However, the extent of salt responsive transcript diversity and its functional relevance remain incompletely understood, particularly at the isoform level. Here, we investigated the role of AS in Arabidopsis thaliana root responses to salinity by combining physiological, molecular, and integrative transcriptomic approaches. Perturbation of splicing using herboxidiene (GEX1A) resulted in enhanced sensitivity to salt, accompanied by altered ABA and cytokinin signaling outputs in the roots. Moreover, to characterize transcriptome complexity under salinity, we generated the first Iso-seq dataset for Arabidopsis roots under salinity. We revealed extensive isoform diversity and splicing defined novelty, driven mostly by intron retention and splice site variation. Integration of these data with a comprehensive meta-analysis of public short-read RNA-seq datasets identified a preliminary set of genes recurrently regulated by AS across tissues; in roots under salinity these genes displayed elevated isoform complexity. Differential splicing analyses based on Iso-seq further revealed that responsive events are enriched for exon skipping and show temporal variation between early and later timepoints. Experimental validation confirmed salt-responsive AS events in candidate genes with relatively modest gene-level expression changes, and phenotypic analyses implicated SR30 and MPK18 in regulating root architecture responses to salinity. Together, our results support a role for AS as a component of plant salinity responses, associated with extensive transcript diversity, modulation of hormone signaling outputs, and root developmental responses.