Evolutionary and functional analysis of ARF and Aux/IAA gene families reveals their roles in sugar metabolism and watermelon domestication

Auxin signaling, mediated by auxin response factors (ARFs) and Aux/IAA proteins, is central to plant development; however, its role in regulating fruit quality traits-particularly soluble sugar and organic acid metabolism-remains largely unexplored in Cucurbitaceae crops. To address this gap, we integrated comparative genomics, metabolomics, and transcriptomics to elucidate the evolutionary conservation and functional specialization of ARF and Aux/IAA gene families in cucurbit fruit quality regulation. We identified 157 ARF and 275 Aux/IAA genes across eight cucurbit species, revealing extensive evolutionary conservation under predominant purifying selection (Ka/Ks < 0.6). Despite this constraint, domestication-driven functional divergence was evident: comparative metabolomic and population genomic analyses of wild watermelon ancestors ( Citrullus amarus and C. mucosospermus ) and cultivated accessions showed that glucose and fructose contents increased sharply during the C. amarus to C. mucosospermus transition, whereas sucrose accumulation rose later during the shift from seed-type to landrace watermelons. Four candidate genes ( ClaARF4 , ClaARF15 , ClaIAA4 , and ClaIAA22 ) exhibited significant allelic differentiation during this early sugar-accumulation transition and remained stably fixed in subsequent domestication stages, directly linking evolutionary signatures with functional outcomes. Exogenous auxin treatment increased glucose and fructose by 23–35% while reducing malate by 18%, confirming that auxin signaling reprograms carbon partitioning toward hexose accumulation. Transcriptome profiling revealed that most genes peaked during early fruit development (10–22 days after pollination), coinciding with metabolic shifts in sugar and organic acid accumulation. Promoter analysis uncovered conserved cis-regulatory elements associated with fruit development across watermelon, cucumber, and melon, suggesting shared regulatory logic. Collectively, this study provides the first integrated evidence that ARF and Aux/IAA gene families coordinate fruit quality metabolism in cucurbits through evolutionarily conserved regulatory architecture with lineage-specific functional adaptations. These findings establish a molecular framework for cucurbit quality improvement, offering candidate targets for marker-assisted selection or genome editing to enhance fruit sweetness and flavor.