Drought is a misfortune for agriculture and human beings. The annual crop yield reduction caused by drought exceeds the sum of all pathogens. As one of the gatekeepers of China’s “granary”, rice is the most important to reveal the key drought tolerance factors in rice. Rice seedlings of Nipponbare (Oryza sativa L. ssp. Japonica) were subjected to simulated drought stress, and their root systems were analyzed for the non-targeted metabolome and strand-specific transcriptome. We found that both DEGs and metabolites were enriched in purine metabolism, and allantoin accumulated significantly in roots under drought stress. However, few studies on drought tolerance of exogenous allantoin in rice have been reported. We aimed to further determine whether allantoin can improve the drought tolerance of rice. Under the treatment of exogenous allantoin at different concentrations, the drought resistant metabolites of plants accumulated significantly, including proline and soluble sugar, and reactive oxygen species (ROS) decreased and reached a significant level in 100 μmol L⁻¹. To this end, a follow-up study was identified in 100 μmol L⁻¹ exogenous allantoin and found that exogenous allantoin improved the drought resistance of rice. At the gene level, under allantoin drought treatment, we found that genes of scavenge reactive oxygen species were significantly expressed, including peroxidase (POD), catalase (CATA), ascorbate peroxidase 8 (APX8) and respiratory burst oxidase homolog protein F (RbohF). This indicates that plants treated by allantoin have better ability to scavenge reactive oxygen species to resist drought. Alternative splicing analysis revealed a total of 427 differentially expressed alternative splicing events across 320 genes. The analysis of splicing factors showed that gene alternative splicing could be divided into many different subgroups and play a regulatory role in many aspects. Through further analysis, we restated the key genes and enzymes in the allantoin synthesis and catabolism pathway, and found that the expression of synthetase and hydrolase showed a downward trend. The pathway of uric acid to allantoin is completed by uric acid oxidase (UOX). To find out the key transcription factors that regulate the expression of this gene, we identified two highly related transcription factors OsERF059 and ONAC007 through correlation analysis. They may be the key for allantoin to enhance the drought resistance of rice.

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 U2AF65B 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. Cis-acting element analysis indicated that U2AF65B 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 AtU2AF65B mutant in Arabidopsis 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.