SnRK-PP2C-PYL Gene Families in Citrus sinensis: Genomic Characterization and Regulatory Roles in Carotenoid Metabolism

Background/Objectives: Carotenoids in citrus are vital nutritional compounds and precursors of the stress hormone abscisic acid (ABA). SNF1-related kinases (SnRKs)-key regulators of plant stress signaling that phosphorylate is targeting proteins for post-transcriptional regulation-mediate ABA signaling through its subfamily SnRK2-phosphatase type-2C (PP2C)-PYR1-LIKE (PYL) cascades. This study aims to identify the SnRK-PP2C-PYL family members and decipher their underlying post-transcriptional regulatory mechanisms which control carotenoid metabolism in Citrus sinensis for improved nutrition and stress resilience. Methods: SnRK, PP2C, and PYL were identified by integrated HMMER-blastp-CDD pipeline in the Citrus genome. Using two carotenoid-divergent cultivars, 'Newhall' (yellow) and 'Cara Cara' (red, hyperaccumulating linear carotenoids), we conducted spatiotemporal expression profiling and integrated transcriptomic and metabolomic data via Weighted Gene Co-expression Network Analysis (WGCNA) to identify modules correlated with accumulation. Results: We identified 26 CsSnRKs (1 SnRK1, 7 SnRK2, 18 SnRK3), 57 CsPP2Cs, and 7 CsPYLs in Citrus sinensis. Despite a >26-fold difference in linear carotenoids, structural gene expression was similar among cultivars, strongly implicating post-transcriptional control. WGCNA identified a key turquoise module highly correlated with linear carotenoid content. This module contained phosphorylation-related genes (CsSnRK1/3.5/3.6/3.16, CsPP2C14/15/33/35/38/40/43/56, and CsPYL6), biosynthetic genes (CsPSY1, CsZISO, and CsZDS), and candidate transcription factors. Network analysis predicted that CsSnRKs, CsPP2Cs, and CsPYLs regulate phytoene-derived carotenoid biosynthesis. Conclusions: We propose a novel phosphorylation-mediated post-transcriptional regulatory network in carotenoid accumulation. This mechanism bridges ABA signaling and metabolic adaptation, providing crucial molecular targets for engineering nutrient-dense and climate-resilient citrus varieties.