Genomic and functional characterization of the cinnamic acid 4-hydroxylase gene family reveals candidate genes involved in rind texture modulation in watermelon

Rind hardness is a significant horticultural trait that directly determines sensory quality and postharvest shelf life. Lignin deposition in the stone cell layer is the primary biochemical determinant of fruit rind, yet the genetic regulatory basis of lignin biosynthesis in watermelon has been studied less. Cinnamic acid 4-hydroxylase (C4H) is a rate-limiting enzyme in the phenylpropanoid metabolic cycle, essential for lignin biosynthesis in plants and the formation of robust fruit rinds. However, the in-depth genetic regulatory basis of rind hardness in many fruits has not been investigated. Herein, we conducted the first genome-wide bioinformatics analysis to identify the characterization of the C4H gene family and to explore its potential functions through physiological and molecular experiments in watermelon. The retrieved results exposed a total of 4 ClC4H members, and phylogenetic analysis classified them into two closely associated branches: Class I and Class II. The gene promoter analysis revealed that the upstream region of the ClC4H gene not only contains a large number of essential core elements for basic transcription but also is densely enriched with cis-acting elements that respond to light, various hormones (such as ABA, auxin, etc.), and non-biological stresses, forming a complex transcriptional regulatory network. Transcriptomic and physiological analysis showed that candidate ClC4H1 and ClC4H2 genes exhibited elevated expression during early fruit rind development, which tended to coincide with higher endogenous lignin content. The predicted protein-protein interaction and molecular docking suggested that ClC4H1 might form an interaction network with ClPAL and ClCCR2-like to participate in lignin synthesis. The RT-qPCR-based relative expressions of the candidate genes also validated the differentiated lignin biosynthesis at different developmental stages of rind texture in contrasted watermelon materials; however, subcellular localization confirmed that ClC4H1 is located in the endoplasmic reticulum. These findings provide important candidate genes and a theoretical basis for using molecular breeding techniques to improve the texture of watermelon fruit.