Genome-Wide Characterization of the Cinnamyl Alcohol Dehydrogenase (CAD) Gene Family and Expression Profiling of Candidate ClCAD3 Gene Associated with Lignin Biosynthesis in Watermelon

Abstract

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in lignin monomer biosynthesis and is crucial for plant cell-wall lignification. However, the CAD gene family and its role in watermelon rind lignification are poorly understood. In this study, we performed genome-wide bioinformatics analysis to identify the CAD gene family in watermelon. We identified predicted gene expression in two contrasting cultivars [WRH (hard rind, high lignin) and WRS (soft rind, low lignin)]. A total of seven CAD-like genes (ClCAD1–ClCAD7) were identified on three chromosomes (1, 2, and 5), all encoding full- or near-full-length proteins with CAD-related domains. Genomic collinearity revealed one segmental duplication (ClCAD3–ClCAD4) in watermelon; however, the comparative genomes of Arabidopsis and melon identified five and eight homologous gene pairs, respectively. Phylogenetic analysis indicated that ClCAD-like genes are more closely related to melon than to Arabidopsis. ClCAD3 and ClCAD4 proteins were grouped with AtCAD4 and AtCAD5 from Arabidopsis, which are important for lignin biosynthesis. Promoter analysis predicted elements responsive to jasmonic acid, abscisic acid, cytokinin, light, and stress. Subcellular localization analysis in the epidermal cells of Nicotiana benthamiana leaves was consistent with a cytosolic distribution for ClCAD3. STRING analysis predicted functional associations of ClCAD3 with proteins involved in aldehyde metabolism, branched-chain amino acid biosynthesis, and basal carbon metabolism, including ClALDH1/2, ClALS, Cl2HACL, and ClKBA1. qRT-PCR analysis of roots, stems, leaves, and fruit rind (21 days after pollination, DAP), as well as rind at 1, 14, and 28 DAP, indicated that ClCAD3, ClALS, and ClALDH2 are expressed at higher levels in WRH than in WRS. ClCAD3 transcript abundance in roots, leaves, and fruit rind was consistently higher in WRH. Together, these findings clarify the evolutionary features and organ-level expression of the CAD-like gene family in watermelon and identify ClCAD3 as a candidate gene associated with lignin biosynthesis. The results provide genetic insights into candidate genes potentially involved in lignification-associated rind hardness and a theoretical foundation for improving rind texture in watermelon fruit through modern molecular breeding approaches.