CsSSU I differentially modulates plastidial prenyltransferases linked to coloration and aroma in sweet orange (Citrus sinensis L.)

Coloration (carotenoids) and aroma (volatile terpenoids) are major determinants of citrus fruit quality, but their coordinated improvement is challenging because carotenoid and volatile-terpenoid biosynthesis draw on a shared isoprenoid precursor network. Here, we used chimeric fruits of the sweet orange (Citrus sinensis L.) bud mutant “Zongcheng” to investigate this metabolic coordination. Metabolomic analyses revealed coordinated accumulation of carotenoids and volatile terpenoids in the brown peel sectors. We systematically characterized the sweet orange trans-prenyltransferase (TPT) family and identified CsGGPPS4, CsGGPPS6, and CsGGPPS8 as plastid-localized enzymes catalyze geranylgeranyl diphosphate (GGPP) biosynthesis. Comparative transcript profiling, biochemical assays, and in vivo functional analyses revealed distinct functions among these enzymes. Expression profiling and in vivo experiments demonstrated that CsGGPPS6 and CsGGPPS8 promote carotenoid accumulation, whereas CsGGPPS4 exerts only minor effects under the tested conditions. Notably, CsGGPPS6 exhibited substrate-ratio-dependent product specificity, producing predominantly farnesyl diphosphate (FPP) under equimolar substrate conditions but both FPP and GGPP when isopentenyl diphosphate (IPP) was enriched; its manipulation in vivo also altered sesquiterpenoids accumulation. CsSSU I directly interacted with CsGGPPS6 and CsGGPPS8 and differentially modulated their catalytic properties: association with CsGGPPS6 favored FPP formation and restricted further elongation to GGPP, whilst association with CsGGPPS8 enabled geranyl diphosphate (GPP) production and enhances GGPP production. Manipulation of CsSSU I in vivo coordinately affected monoterpenoids, sesquiterpenoids, and carotenoids accumulation. Collectively, these results establish that CsSSU I acts as a protein-binding modulator that remodels product output of plastid-localized TPT enzymes, and indicate that this regulatory network orchestrates terpenoid-derived pigmentation and aroma traits in citrus.