Decreased Protein Abundance of Lycopene β-Cyclase Contributes to Red Flesh in Domesticated Watermelon

Red-fleshed watermelons (Citrullus lanatus) that accumulate lycopene in their flesh cells have been selected and domesticated from their pale-fleshed ancestors. However, the molecular basis of this trait remains poorly understood. Using map-based cloning and transgenic analysis, we identified a lycopene β-cyclase (ClLCYB) gene that controls the flesh color of watermelon. Downregulation of ClLCYB caused the flesh color to change from pale yellow to red, and ClLCYB overexpression in the red-fleshed line caused the flesh color to change to orange. Analysis of ClLCYB single-nucleotide polymorphisms (SNPs) using 211 watermelon accessions with different flesh colors revealed that two missense mutations between three haplotypes (ClLCYBred, ClLCYBwhite, and ClLCYByellow) were selected and largely fixed in domesticated watermelon. Proteins derived from these three ClLCYB haplotypes were localized in plastids to catalyze the conversion of lycopene to β-carotene and showed similar catalytic abilities. We revealed that ClLCYB protein abundance, instead of ClLCYB transcript level, was negatively correlated with lycopene accumulation. Different amounts of ClLCYB protein degradation among the ClLCYB haplotypes were found in ClLCYB-transgenic Arabidopsis thaliana lines. After treatment with the proteasome inhibitor MG132, the concentration of ClLCYBred increased noticeably compared to other ClLCYB proteins. These results indicate that natural missense mutations within ClLCYB influence ClLCYB protein abundance and have contributed to the development of red flesh color in domesticated watermelon.