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

<b>Background/Objectives</b>: 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 <i>Citrus sinensis</i> for improved nutrition and stress resilience. <b>Methods</b>: SnRK, PP2C, and PYL were identified by integrated HMMER-blastp-CDD pipeline in the <i>Citrus</i> 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. <b>Results</b>: We identified 26 <i>CsSnRKs</i> (1 <i>SnRK1</i>, 7 <i>SnRK2</i>, 18 <i>SnRK3</i>), 57 <i>CsPP2C</i>s, and 7 <i>CsPYL</i>s in <i>Citrus sinensis</i>. 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 (<i>CsSnRK1</i>/<i>3.5</i>/<i>3.6</i>/<i>3.16</i>, <i>CsPP2C14</i>/<i>15</i>/<i>33</i>/<i>35</i>/<i>38</i>/<i>40</i>/<i>43</i>/<i>56</i>, and <i>CsPYL6</i>), biosynthetic genes (<i>CsPSY1</i>, <i>CsZISO</i>, and <i>CsZDS</i>), and candidate transcription factors. Network analysis predicted that <i>CsSnRK</i>s, <i>CsPP2C</i>s, and <i>CsPYL</i>s regulate phytoene-derived carotenoid biosynthesis. <b>Conclusions</b>: 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.