PPM1J regulates meat quality feature and glycerophospholipids composition in broiler by modulating protein dephosphorylation

Poultry meat is the most consumed meat globally, with broilers favored for efficiency but often facing meat quality issues. Fast-growing (FG) broilers have higher yields but generally poorer flavor than slow-growing (SG) broilers, making meat quality a key consumer concern. Protein post-translational modifications, particularly phosphorylation, are implicated in meat quality traits such as tenderness, color, and water-holding capacity (WHC). Phosphorylation in glycolysis has been associated with PSE-like meat in broilers, and AMPK phosphorylation correlates with AMP/ATP ratio and water loss. PPM1J, a metal-dependent Ser/Thr phosphatase, regulates diverse cellular processes via reversible protein phosphorylation and has been linked to metabolic regulation. This study aimed to integrate metabolomics and transcriptomics to identify genetic regulators of meat flavor differences between FG Guangming-2 (GM2) and SG Xinghua (XH) broilers, prioritize candidate genes via metabolite–gene networks, and functionally test the top candidate (PPM1J) using gain- and loss-of-function models combined with metabolomics and phosphoproteomics to elucidate mechanisms affecting meat quality and glycerophospholipid composition.

Prior work indicates proteomics is valuable for uncovering determinants of meat quality, with protein post-translational modifications, especially phosphorylation, playing central roles. Phosphorylation has been reported to negatively affect tenderness, color, and WHC; glycolytic protein phosphorylation contributes to PSE-like meat in broilers; and AMPK phosphorylation associates with energy status and drip/cooking loss. Members of the PPM phosphatase family regulate metabolism and disease by dephosphorylating targets (e.g., PPARγ), suggesting phosphatases can influence adipogenesis and fat deposition. These findings motivated investigation of phosphatase-mediated control of meat quality and flavor-related metabolites, leading to selection of PPM1J as a candidate regulator.

Study design: Compare breast muscle from fast-growing GM2 and slow-growing XH broilers at market age using integrated metabolomics (UHPLC-Q-TOF-MS/MS) and transcriptomics (RNA-Seq) to build metabolite–gene networks and identify candidate regulators of meat flavor. Functionally validate PPM1J via lentiviral overexpression (Lv-PPM1J) and knockdown (Lv-shPPM1J) in vivo, followed by assessment of muscle traits, meat-quality parameters, non-targeted metabolomics, and phosphoproteomics. Animals and treatments: GM2 and XH broilers sourced from commercial suppliers. For functional assays, 14-week-old XH chickens were randomly assigned (n=15/group) to Lv-PPM1J vs Lv-NC or Lv-shPPM1J vs Lv-shNC. Intramuscular injections (10^7 TU) into breast muscle were administered at 14, 15, and 16 weeks; tissues collected at 17 weeks following standard euthanasia protocols. Ethics approval: South China Agricultural University (2021c008). Cell culture: Chicken primary myoblasts (CPMs) isolated from E11 leg muscle, cultured in RPMI-1640 + 20% FBS. Transfections used Lipofectamine 3000. Gene manipulation: Full-length PPM1J cloned into pLVX-mCMV-ZsGreen-IRES-Puro for overexpression. siRNAs against PPM1J designed; shRNA cassettes cloned into pLVX-shRNA2-Puro for stable knockdown. qPCR verified modulation in CPMs and in vivo. Histology and myogenesis: H&E staining to assess myofiber cross-sectional area (CSA). Myoblast differentiation assessed by MyHC immunofluorescence; myotube area quantified. Meat-quality measurements: Shear force via C-LM4 tenderness meter; WHC via 24 h drip loss; intramuscular fat (IMF) by Soxhlet extraction (Soxtec). RNA-Seq and analysis: RNA extracted with TRIzol; libraries prepared (ABclonal mRNA-seq kit) and sequenced on Illumina NovaSeq 6000 (150 bp PE). DEGs identified; GO and KEGG enrichment via clusterProfiler. Metabolomics: UHPLC (Agilent 1290) coupled to TripleTOF 6600; HILIC separation on ACQUITY BEH Amide column. ESI source: Gas1/2 60 psi, CUR 30 psi, 600 °C, ISVF ±5500 V. MS1 m/z 60–1000 (0.20 s), IDA MS/MS m/z 25–1000 (0.05 s), CE 35±15 eV, DP ±60 V. OPLS-DA models constructed. Differential metabolites: VIP>1, P<0.05; for breed comparison, additional FC>1.5 or <0.67. KEGG pathway and differential abundance analyses performed. Phosphoproteomics: Protein extraction (4% SDS, 100 mM Tris-HCl, 1 mM DTT), tryptic digestion by FASP. Phosphopeptide enrichment with Fe-NTA kit; LC-MS/MS on timsTOF Pro coupled to NanoElute; C18 column (25 cm, 75 μm, 1.9 μm). Acquisition over m/z 100–1700 with PASEF MS/MS cycles. Identification/quantitation by MaxQuant against Gallus gallus database; variable mods: Ox(M), Acetyl (N-term), Phospho (S/T/Y); fixed: Carbamidomethyl (C); FDR 1%. Differential phosphopeptides (DAPs): FC>2 or <0.5, P<0.05. Statistics: ≥3 biological replicates; mean±SEM. Independent or paired t-tests as appropriate; Pearson correlations with |R|>0.80 (unless otherwise specified) and P<0.05 considered significant. Visualization with GraphPad Prism.

- Breed differences: GM2 breast muscle showed a higher proportion of large myofibers (>1000 μm²) than XH. Metabolomics identified 254 differential metabolites between GM2-B and XH-B (84 up, 170 down in GM2-B); 77 pathways (184 metabolites) enriched (notably amino acid biosynthesis, aminoacyl-tRNA biosynthesis). RNA-Seq revealed 1766 DEGs (851 up, 915 down). - Meat flavor-related network: Integrated correlation analysis (|R|>0.8, P<0.05) produced 2538 positive and 3647 negative metabolite–gene connections. A focused meat flavor network comprised 20 connections among 8 genes and 6 metabolites; top genes by connectivity: PPM1J, ELN, CCDC92B. In GM2-B, several amino acids/nucleotides with bitter/sour taste (e.g., methionine, glycine, histidine, leucine, phenylalanine, tyrosine), hypoxanthine, inosine, and IMP were upregulated; lysine decreased. - PPM1J expression and function: PPM1J was differentially expressed and highly expressed in breast and leg muscle and heart. Overexpression in vivo increased the proportion of small myofibers (≤1000 μm²), upregulated atrophy-related genes ATROGIN1 and MURF1, reduced myotube formation; knockdown had opposite effects (increased large myofibers, enhanced differentiation). Meat-quality traits improved with PPM1J overexpression (lower shear force; higher WHC and IMF), while knockdown increased shear force and decreased WHC and IMF. - Metabolomics after PPM1J knockdown: 117 differential metabolites (75 down, 42 up). Major classes: lipids and lipid-like molecules (25.89%), organic acids/derivatives (20.54%), benzenoids (15%). Meat flavor-related metabolites altered: 1-stearoyl-2-docosahexaenoyl-PC, glycine, hypoxanthine increased; 1-O-hexadecyl-2-O-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glyceryl-3-phosphorylcholine and anserine decreased. Enriched pathways included ABC transporters, branched-chain amino acid biosynthesis, starch and sucrose metabolism. - Phosphoproteomics after PPM1J knockdown: Identified 3372 phosphosites on 2903 phosphopeptides from 1279 phosphoproteins; site distribution: Ser 79.84%, Thr 18.36%, Tyr 1.79%. Detected 33 DAPs (18 up, 15 down); 61 phosphopeptides specific to knockdown and 46 specific to control. Upregulated phosphopeptides included muscle proteins (TTN, OBSCN, TNNT3). HSP family members (HSPB1, HSPD1) and RICTOR were specifically phosphorylated upon knockdown. GO/KEGG enrichment implicated sarcomere/contractile fiber components and pathways such as vascular smooth muscle contraction, MAPK signaling, and tight junction. - Integrated phosphoproteome–metabolome: Co-enrichment in carbohydrate and core metabolic pathways (fructose/mannose, galactose, methane metabolism). Correlations linked glycerophospholipids to specific phosphoproteins: MYLK4, AAK1, and SYNPO2L phosphorylation positively correlated with 1-stearoyl-2-docosahexaenoyl-PC; DUSP27 and DDI2 phosphorylation negatively correlated with 1-O-hexadecyl-2-O-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glyceryl-3-phosphorylcholine. Findings support that PPM1J regulates glycerophospholipid composition and meat-quality traits via protein dephosphorylation.

The study addresses the need to understand molecular determinants of meat flavor and quality differences between fast- and slow-growing broilers. The integrated omics approach pinpointed PPM1J as a key gene connected to flavor-related metabolites. Functional perturbation experiments demonstrated that PPM1J influences muscle fiber characteristics and meat-quality attributes (tenderness, WHC, IMF). Knockdown and overexpression experiments, combined with metabolomics and phosphoproteomics, support a mechanism wherein PPM1J modulates phosphorylation states of muscle and signaling proteins, which in turn alters glycerophospholipid composition and flavor-related metabolites. Phosphorylation of HSPs and RICTOR upon PPM1J knockdown is consistent with known roles of these proteins in stress responses and mTOR signaling that can affect postmortem muscle proteostasis and tenderness. The observed correlations between glycerophospholipid species and phosphorylation changes in MYLK4, AAK1, SYNPO2L, DUSP27, and DDI2 further suggest that PPM1J-dependent dephosphorylation fine-tunes lipid metabolism and membrane composition, impacting juiciness and flavor precursors. Overall, the findings link a specific phosphatase to meat-quality phenotypes through coordinated control of protein phosphorylation and metabolism.

PPM1J emerged as a top connectivity gene in meat flavor-related metabolite–gene networks differentiating FG and SG broilers. It is highly expressed in muscle and regulates muscle development and key meat-quality traits (tenderness, WHC, IMF). Multi-omics analyses indicate that PPM1J governs glycerophospholipid composition and flavor-related metabolites by catalyzing protein dephosphorylation and altering phosphorylation of proteins involved in muscle structure and signaling. This work provides mechanistic insight into how protein phosphatases influence meat quality and establishes PPM1J as a candidate target for improving broiler meat characteristics. Future studies could define direct substrates of PPM1J in muscle, validate causality between specific phosphorylation events and lipid remodeling, and assess the feasibility of selective modulation of PPM1J activity in breeding or management strategies.