A Comprehensive Analysis of the Alternative Splicing Co-Factor U2AF65B Gene Family Reveals Its Role in Stress Responses and Root Development

U2AF65, a 65 kDa splicing co-factor, promotes spliceosome assembly. Although its role in alternative splicing (AS) is known, the function of U2AF65B (the large subunit of U2AF65) remains unclear. Therefore, we systematically identified and analyzed the U2AF65B gene family across 36 plant species, revealing 103 putative members with conserved structures and functions. Phylogenetic analysis divided the genes into two clades and five subgroups, indicating evolutionary divergence. Gene structure and conserved motif analyses showed that most <i>U2AF65B</i> genes have complex structures and shared similar motifs. Homology modeling and amino acid conservation analyses revealed significant conservation in U2AF65B amino acid sequences, particularly in Groups D and E. <i>Cis</i>-acting element analysis indicated that <i>U2AF65B</i> genes respond to various stimuli, supported by expression analysis under different stress conditions. Subcellular localization predictions indicated that U2AF65B proteins primarily localize in the nucleus and the cytoplasm. Alternative splicing (AS) profile analysis showed that the AS frequency likely varies between species. Functional analysis of the <i>AtU2AF65B</i> mutant in <i>Arabidopsis</i> revealed that AtU2AF65B function loss enhances root elongation and attenuates ABA-dependent germination suppression, indicating negatively regulated seedling growth and development. These findings provide insights into the evolutionary history, molecular mechanisms, and functional roles of the U2AF65B gene family in plants.