Integrated multi-omics reveals the molecular basis of protein storage in developing oat grains

Oats (Avena sativa L.) are a nutritionally important cereal crop, but the molecular mechanisms regulating protein and amino acid accumulation during grain development remain poorly understood. Therefore, in this study, two oat varieties (HZ and MC) with significantly different grain protein contents were used as experimental materials. For the grains of these two varieties at different developmental stages, we conducted grain protein content determination, widely targeted metabolite detection, and transcriptome sequencing. Integrated transcriptomic and metabolomic analyses of two oat varieties (high-protein HZ and low-protein MC) revealed dynamic changes during grain filling. Key findings include: (1) The 10–20 days after flowering (DAF) period was critical for protein accumulation, with HZ showing higher expression of genes involved in carbon skeleton generation (e.g., isocitrate dehydrogenase, malate dehydrogenase) and amino acid biosynthesis (e.g., glutamate decarboxylase); (2) Weighted gene coexpression network analysis identified modules strongly correlated with protein content, including the blue module (r = 0.92, p < 0.01); (3) KEGG enrichment highlighted central roles for glycolysis, TCA cycle, and amino acid metabolism pathways; (4) A regulatory network for protein and amino acid accumulation was constructed, identifying 157 candidate genes and 7 metabolites. Four key genes (GLA, PFK, PGK, TIM) and a transcription factor (AP2/ERF-ERF ) were identified as critical regulators; (5) qRT-PCR validated 10 candidate genes, with expression patterns consistent with RNA-Seq data (R² = 0.72–0.85). This study elucidates the metabolic and transcriptional networks underlying protein accumulation in oat grains, providing targets for molecular breeding to enhance nutritional quality.