The quality of broiler meat significantly impacts consumer purchasing decisions. Fast-growing (FG) broilers, while preferred by producers for their high efficiency, often have inferior flavor compared to slow-growing (SG) broilers. Understanding the molecular mechanisms underlying meat flavor formation is crucial for improving meat quality. Previous research has highlighted the role of protein phosphorylation in meat quality, with studies showing negative correlations between protein phosphorylation and tenderness, color, and water holding capacity (WHC). Protein phosphatase magnesium/manganese-dependent 1J (PPM1J), a metal-dependent protein phosphatase, regulates various cellular processes by modulating reversible protein phosphorylation. This study aimed to investigate the role of PPM1J in meat flavor formation by integrating metabolomics and transcriptomics data from FG and SG broiler breeds. The study utilizes Guangming-2 (GM2, FG broiler) and Xinghua chicken (XH, SG broiler) to investigate differences in meat flavor and the underlying genetic mechanisms, focusing on the role of PPM1J in this process.

Extensive research has explored the relationship between protein post-translational modifications (PTMs) and meat quality. Phosphorylation, a major PTM, has been implicated in influencing meat tenderness, color, and WHC. Studies have linked protein phosphorylation in the glycolysis pathway to PSE-like meat in broilers, and AMPK phosphorylation has been correlated with meat droplet and cooking loss. The PPM phosphatase family has also been implicated in adipogenesis and fat accumulation via dephosphorylation of PPARγ. However, a comprehensive understanding of how specific phosphatases, such as PPM1J, contribute to meat flavor formation remains limited.

This study employed a multi-omics approach integrating metabolomics and transcriptomics. Two broiler breeds, GM2 (FG) and XH (SG), were selected at market age. Breast muscle samples were analyzed using H&E staining to assess myofiber cross-sectional area. UHPLC-Q-TOF-MS/MS technology was used for metabolomics analysis, identifying differential metabolites between GM2 and XH. RNA-Seq analysis was performed to identify differentially expressed genes (DEGs). Metabolome and transcriptome data were integrated to construct metabolite-gene networks, identifying PPM1J as a candidate gene with high connectivity to meat flavor-related metabolites. Lentivirus-mediated PPM1J overexpression and knockdown animal models were created. Non-target metabolomic and phosphoproteomic analyses were conducted to investigate the molecular mechanism of PPM1J's function. Specific techniques included qPCR, immunofluorescence staining, measurement of meat quality traits (shear force, WHC, IMF), and 4D-labfree labeling-based quantitative phosphoproteomic analysis. Statistical analyses involved independent and paired t-tests, Pearson's correlation analysis, and enrichment analyses (GO and KEGG).

H&E staining revealed larger myofibers in GM2 compared to XH. Metabolomics identified 254 differential metabolites between GM2 and XH, with pathways enriched in amino acid biosynthesis and neuroactive ligand-receptor interaction. Transcriptomics identified 1766 DEGs, with GO terms enriched in multicellular organismal processes and KEGG pathways in neuroactive ligand-receptor interaction. Metabolite-gene network analysis revealed PPM1J as a top candidate gene. PPM1J overexpression increased the proportion of small myofibers, while knockdown increased the proportion of large myofibers. PPM1J overexpression improved meat quality (reduced shear force, increased WHC, increased IMF), while knockdown had the opposite effect. Metabolomic analysis of PPM1J knockdown muscle revealed differential metabolites, particularly in lipids and lipid-like molecules. Phosphoproteomic analysis identified 3372 phosphosites, with significant changes in phosphopeptides associated with actin binding and muscle development. Co-enrichment analysis of differential metabolites and DAPs highlighted pathways like fructose and mannose metabolism. Specifically, PPM1J knockdown-induced phosphorylation of MYLK4, AAK1, and SYNPO2L correlated with 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine content, while DUSP27 and DD12 phosphorylation negatively correlated with 1-O-hexadecyl-2-O-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glyceryl-3-phosphorylcholine content.

This study demonstrates that PPM1J plays a significant role in regulating broiler meat quality and glycerophospholipid composition. The findings support the hypothesis that PPM1J affects meat flavor through its regulation of protein dephosphorylation. The observed changes in myofiber size, meat quality traits, and glycerophospholipid composition upon PPM1J manipulation highlight its crucial role in muscle development and meat quality. The integration of metabolomics and phosphoproteomics revealed a complex interplay of metabolites and phosphorylated proteins, providing a more comprehensive understanding of PPM1J's molecular mechanism. The specific correlations between PPM1J-regulated phosphorylation events and glycerophospholipid content suggest a direct link between PPM1J activity and intramuscular fat composition, which strongly influences meat flavor and tenderness. The study’s findings have implications for improving broiler meat quality through targeted genetic manipulation or dietary strategies.

This study establishes PPM1J as a key regulator of broiler meat quality and glycerophospholipid composition, acting via protein dephosphorylation. The integration of metabolomics, transcriptomics, and phosphoproteomics provides a comprehensive understanding of its molecular mechanism. Future research could focus on identifying the specific downstream targets of PPM1J and exploring potential strategies for manipulating PPM1J expression to enhance meat quality.

The study focused on two specific broiler breeds, limiting the generalizability of the findings to other breeds. Further studies are needed to validate these findings in a wider range of broiler types. The study primarily focused on breast muscle; investigating other muscle types might reveal additional insights into PPM1J's role. The relatively small sample size could influence the statistical power of some of the analyses.