Genome-Wide Identification of LBD Transcription Factors Revealed the Essential Role of ClLBD2 in Root Development in Watermelon (Citrullus lanatus)

The Lateral Organ Boundaries Domain (LBD) gene family encodes plant-specific transcription factors that play pivotal roles in growth, development, and stress responses. However, a comprehensive genome-wide analysis of the LBD family in watermelon (Citrullus lanatus) has not been conducted until now. In this study, we identified 39 ClLBD genes using the latest watermelon reference genome and systematically analyzed the function of ClLBD2 in root development. These ClLBDs are unevenly distributed across 10 chromosomes except Chr4. Evolutionary analysis grouped the gene family members into six subgroups: Class I (a–e) and Class II. Physicochemical properties and gene structure analysis showed that the ClLBD protein members are tightly conserved. In the promoter regions of ClLBD genes, we identified abundant cis-acting regulatory elements related to abiotic stress and hormone responses. Through RNA-seq analysis from a cucurbit database, we found that several ClLBD genes showed high relative expression in roots, with ClLBD2 being the most highly expressed. Since its subfamily includes AtLBD25, a known root development-related gene, we hypothesized that ClLBD2 might be involved in root development. To validate this, ClLBD2-edited roots were generated using the CRISPR-Cas9 system and Agrobacterium rhizogenes-mediated transformation. Compared to the wild type, the ClLBD2 edited roots exhibited significant reduction in taproot length and lateral root numbers, indicating that ClLBD2 may regulate root development. This study provides the first comprehensive analysis of the LBD gene family in watermelon, offering valuable insights for evolutionary and further functional studies of ClLBD genes.