Integrated Transcriptome and Metabolome Analysis Revealed the Molecular Mechanisms of Cold Stress in Japonica Rice at the Booting Stage

Japonica rice is susceptible to cold stress at the booting stage, yet the systematic molecular mechanisms underlying varietal disparities in cold tolerance at this stage remain poorly understood. To fill this research gap, cold-tolerant LG1934 (V3) and cold-sensitive KD8 (V6) were subjected to low-temperature treatment (15 °C) for 0 h (T1), 72 h (T3), and 120 h (T5) at the booting stage, followed by analyses of agronomic traits, antioxidant physiology, metabolome, transcriptome, and weighted gene co-expression network analysis (WGCNA). Phenotypic results showed that low temperature was the main driver of differences in panicle length, seed setting rate, and grain weight between the two varieties, with V3 exhibiting significantly stronger cold tolerance. Under cold stress, V3 maintained higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), accompanied by lower O2− accumulation and higher contents of malondialdehyde (MDA), H2O2, and proline compared to V6. Metabolomic analysis identified 56 differential accumulated metabolites (DAMs), with amino acids and their derivatives (notably L-aspartic acid) as key contributors. RNA-seq analysis identified 472 common differentially expressed genes (DEGs) that were enriched in alanine, aspartate, and glutamate metabolism, with 20 transcription factors (TFs) from TCP, WRKY, and bHLH families screened. The WGCNA revealed nine DEM-correlated modules, with orange and pink modules positively associated with L-aspartic acid. Eleven core TFs were identified, among which OsPCF5 acted as a hub regulator that activated OsASN1 transcription to promote L-aspartate biosynthesis, enhancing ROS scavenging and cold tolerance. This study systematically demonstrated the cold tolerance molecular network in japonica rice at the booting stage, highlighting the antioxidant system and L-aspartate-mediated pathway, and the core genes provided valuable resources for cold-tolerance breeding.