Abstract
ABRE BINDING FACTOR 4 (ABF4) is a pivotal regulatory gene in the abscisic acid (ABA) signaling pathway, and changes in its expression levels can modulate the plant's stress resistance. To further explore the specific regulatory mechanisms of alternative splicing (AS) in the ABA signaling pathway and to identify new breakthroughs for breeding high stress-resistant varieties of Brassica napus, we identified 17 homologous genes of ABF4 in the genome. Utilizing bioinformatics techniques, we analyzed their motifs, conserved domains, and cis-acting elements of their promoters. Through transcriptome data from the stress-tolerant dwarf strain ndf2 and its parental line 3529, we uncovered a significantly differentially expressed ABF4 gene, which we named BnABF4L. Subsequently, we analyzed the AS events of BnABF4L under normal growth conditions and different abiotic stresses, as well as the impact of different transcript variants' 5' untranslated region (5'UTR) on gene translation. BnABF4L undergoes alternative 3' splice site (A3SS) selection to produce three transcripts (V1-V3) with divergent 5'UTRs. While V1 translation is suppressed by upstream ORFs (uORFs), V2/V3 exhibit enhanced translational efficiency. Under stress, ndf2 shifts splicing toward V3, circumventing uORF-mediated repression to upregulate stress-adapted isoforms. We validated the inhibitory effect of upstream open reading frames (uORFs) on protein-coding open reading frame (pORFs) and, based on the collective experimental results, proposed the flexible regulatory mechanism of AS events of BnABF4L in response to stress. Our findings provide new insights for future studies on stress resistance in rapeseed as well as for research on the regulation of alternative splicing mechanisms in the ABA signaling pathway.