Melon fruit aroma depends on the balance between ester biosynthesis and hydrolysis, yet the carboxylesterase (CXE) family responsible for hydrolysis remains uncharacterized in this crop. Here, a genome-wide identification of the CXE gene family in melon revealed 22 CmCXE genes. Phylogenetic analysis revealed that Group III members cluster with functionally validated ester-hydrolyzing CXEs from peach, tomato, and strawberry, and their expression peaks during the climacteric and post-climacteric stages—the same window when melon aroma volatiles accumulate. We further functionally characterized CmCXE7, a cold-responsive member, through subcellular localization and heterologous overexpression in tomato. CmCXE7 localized predominantly to the plasma membrane with a weak nuclear signal. Non-targeted metabolomics revealed 776 differentially accumulated metabolites (DAMs) based on VIP > 1 and p < 0.05, with 11 of 14 high-magnitude ester-related changes being downregulated. These results identify Group III genes as candidate regulators of melon ester metabolism and identify CmCXE7 as a cold-responsive CXE candidate that may participate in ester metabolism. The potential relevance of CmCXE7 to fruit ripening and postharvest storage warrants further investigation, providing a candidate gene for future functional studies and molecular breeding of melon aroma quality.

Background and Objectives: Codon usage bias (CUB), the non-uniform usage of synonymous codons, is prevalent in plant chloroplast genomes and plays important roles in gene expression and genome evolution. However, systematic comparisons of CUB patterns across genera in the Cucurbitaceae family remain limited. This study aimed to characterize CUB patterns and identify their driving forces in the chloroplast genomes of six Cucurbitaceae species. Methods: We analyzed the complete chloroplast genomes of six Cucurbitaceae species, watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (Cucumis sativus), pumpkin (Cucurbita moschata), wax gourd (Benincasa hispida), and bitter gourd (Momordica charantia). CUB patterns were assessed using ENC-plot, neutrality plot, and PR2-plot analyses, all implemented through a custom, reproducible Python-based workflow. Results: The overall codon usage bias was weak across all six species, with a clear preference for A/U-ending codons. Candidate preferred codons (hereafter referred to as optimal codons for brevity) identified independently within each species were largely shared across the six species, although species-specific codons were also detected. Combined ENC-plot, neutrality plot, and PR2-plot analyses suggest that natural selection, rather than mutation pressure, is the dominant force shaping CUB in these genomes, although these approaches provide indirect evidence. Conclusions: These findings provide a reference for codon optimization of exogenous genes in chloroplast genetic engineering of Cucurbitaceae crops. The Python-based workflow developed in this study also offers a transparent, reproducible alternative to conventional CUB analysis approaches.