Divergent residues influence PHYTOENE SYNTHASE isozyme interactions with GGPPS and the resulting enzyme activities in peach

Carotenoids serve critical biological functions through their essential contributions to organismal survival and health. As a widely consumed fruit species, peach (Prunus persica) provides humans with valuable carotenoid sources, and these compounds also substantially enhance the aromatic properties of peach fruits. The synthesis of phytoene, catalyzed by phytoene synthase (PSY), constitutes a key rate-limiting step in carotenoid biosynthesis. In this study, we investigated PSY isoforms in peaches, uncovering their tissue-specific expression patterns and identifying functional divergence among these isoforms through transgenic approaches using peach callus systems. By integrating protein structural analyses, Yeast Two-Hybrid assays, and engineered bacterial systems, we demonstrated that the differential interaction strengths between PSY1/PSY2 and geranylgeranyl pyrophosphate synthase (GGPPS) are likely the primary drivers of their distinct functional activities. Site-directed mutagenesis of the PSY-GGPPS interaction interface further confirmed a significant positive correlation between the strength of this interaction and the functional activity of PSY. Our findings suggest that the functional differences among peach PSY isoforms may arise from the cumulative effects of divergent residues between PSY1 and PSY2 on the PSY-GGPPS interactions. Notably, we identified a key individual residue that substantially influences the PSY-GGPPS interaction and the functional activity of PSY. This study provides insights into the molecular mechanisms driving the functional differentiation of PSY isoforms and highlights the potential for engineering PSYs rationally to develop crops with enhanced carotenoid content.