Multi-omics-Based Construction of ncRNA-Gene-Metabolite Networks Provides New Insights Into Metabolic Regulation Under Salt Stress in Rice.

Rice (Oryza sativa L.), one of the most vital staple crops globally, suffers severe yield losses due to metabolic dysregulation under salt stress. However, the systemic mechanisms by which non-coding RNAs (ncRNAs) coordinately regulate metabolic reprogramming remain elusive, and the genotype-specific regulatory networks in salt-tolerant cultivars are poorly characterized. To address this, we performed metabolomic analysis using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) across different rice varieties under salt stress, identifying 327 metabolites, with the most notable fluctuations observed in lipids, polyamines, and phenolamides. The salt-tolerant variety Pokkali exhibited 51.96% and 31.37% fewer differentially accumulated metabolites (DAMs) in the shoots and roots respectively, compared to the salt-sensitive variety Nipponbare (NIP), which explains its superior salt-tolerant phenotype from a metabolic homeostasis perspective. Transcriptome profiling revealed 18,597 differentially expressed genes (DEGs), with 70.8% showing genotype-specific expression patterns. Pokkali-specific DEGs were markedly enriched in salt-responsive pathways, including reactive nitrogen species scavenging and ion compartmentalization. By integrating long non-coding RNA (lncRNA) and microRNA (miRNA) sequencing data, we constructed a four-tiered regulatory network comprising 6,201 DEGs, 458 miRNAs, 970 DElncRNAs, and 177 metabolites. In the regulatory network, Osa-miR408-3p was identified as a negative regulator of Os03 g0709300 expression. Network analysis revealed that 21 polyamine and phenolamides biosynthesis-related genes were co-regulated by eight miRNAs, each forming a feedback loop with 2-11 lncRNAs. This study constructed a four-way cascade of "lncRNA-miRNA-mRNA-metabolite", and proposed a new concept of ncRNA-mediated "network regulation instead of single-gene effect".