The Molecular and Metabolic Mechanisms Underlying the Citrine/Yellow Coloration in the Peels of the Shiranui Mandarin Citrus Mutant

Key pigments and genes associated with peel color variation between Shiranui Mandarin and its Citrine Mutant were investigated. Combining liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis detected carotenoids in the peel of Shiranui mandarin and its mutant, Citrine Shiranui. Transcriptomic analysis revealed genes enriched in the carotenoid biosynthesis and chlorophyll degradation pathways. Weighted Gene Coexpression Network Analysis (WGCNA) predicted potential regulatory transcription factors. Dual-Luciferase Reporter Assays (LUC) demonstrated the regulatory relationships between the predicted transcription factors and key functional genes involved in carotenoid biosynthesis and chlorophyll degradation. During fruit ripening, the accumulation of β-cryptoxanthin was significantly lower in Citrine Shiranui. In fully ripened fruit, β-cryptoxanthin accounted for 43.62% of the peel pigments in Shiranui, compared to only 8.21% in Citrine Shiranui. Additionally, while chlorophyll degradation occurred during ripening in both varieties, Citrine Shiranui retained higher chlorophyll level. Notably, the expression patterns of CitBCH1 and CitNCY1 were associated with reduced β-cryptoxanthin synthesis and delayed chlorophyll degradation in Citrine Shiranui. LUC further demonstrated that CitBCH1 and CitNCY1 were positively regulated by an ethylene-responsive transcription factor (CitERF5) and an ABA-responsive element-binding factor (CitABF2), respectively. In summary, the citrine/yellow peel color of the Citrine Shiranui mutant primarily resulted from reduced β-cryptoxanthin accumulation due to down-regulation of CitBCH1, while the retention of chlorophyll, linked to lower CitNCY1 expression, might also contribute modestly to the phenotype.