B3 Superfamily in Cucumber (Cucumis sativus L.): Identification, Evolution, Expression Patterns, and Function in Glandular Trichome Development

The B3 transcription factor superfamily, crucial for plant growth and stress adaptation, remains poorly characterized in cucumber (<i>Cucumis sativus</i>), a globally important vegetable crop. Here, we conducted the first genome-wide identification of 52 B3 superfamily genes in cucumber, classifying them into LAV, ARF, RAV, and REM subfamilies through integrated phylogenetic and structural analyses. These genes exhibited conserved B3 domains with lineage-specific motif architectures and diverse exon-intron organizations, particularly within the structurally divergent REM subfamily. Collinearity analysis revealed segmental duplication as a key driver of family expansion, notably between syntenic REM clusters on chromosomes 2 (<i>CsREM5-7</i>) and 6 (<i>CsREM18-20</i>). Promoter <i>cis</i>-element profiling identified enrichment in hormone-responsive and stress adaptation motifs, suggesting functional diversification in signaling pathways. Furthermore, tissue-specific expression divergence was observed across 10 organs, with ARF members displaying broad regulatory roles and REM genes showing apical meristem enrichment. Strikingly, <i>CsRAV8</i> exhibited glandular trichome-specific expression, a novel finding, given <i>Arabidopsis</i> RAVs' lack of trichome-related functions. Spatial validation via in situ hybridization localized <i>CsRAV8</i> transcripts to trichome glandular head cells. Functional investigation using virus-induced gene silencing (VIGS) demonstrated that <i>CsRAV8</i> suppression caused significant glandular trichome shriveling, implicating its role in maintaining glandular cavity integrity. This study provides the first comprehensive genomic inventory of B3 transcription factors in cucumber, providing evolutionary insights and functional frameworks for future functional genomics studies.