Integrated physiological, transcriptomic and metabolomic analysis reveals differential cold response in wheat seedlings across varieties
Cold stress is a major environmental constraint limiting wheat productivity worldwide. Although numerous cold-responsive pathways have been identified, the molecular basis of differential cold tolerance among genetically related wheat lines remains poorly understood. In this study, two wheat sibling lines derived from a single progeny plant of the same parental cross, Luyan951 (cold-tolerant) and Luyan955 (cold-sensitive), were employed to investigate the regulatory mechanisms of cold adaptation through integrated physiological, transcriptomic, and metabolomic analyses. Physiological assays revealed that Luyan951 exhibited markedly enhanced cold tolerance, with a survival rate of 52.67% following cold treatment compared with 20.67% in Luyan955. This enhanced tolerance was accompanied by 1.90–2.41-fold greater increases in antioxidant enzyme activities (SOD, CAT, and POD) and 1.84–4.50-fold greater accumulation of proline and soluble sugars relative to Luyan955, along with substantially lower MDA accumulation. Transcriptomic and metabolomic analyses identified phenylpropanoid biosynthesis and jasmonic acid (JA) signaling as key pathways associated with cold adaptation. Compared with Luyan955, cultivar Luyan951 exhibited stronger activation of these pathways under cold stress. Key genes involved in phenylpropanoid biosynthesis ( CAD , and 4CL ) and JA signaling ( JAZ , MYC2 ) were significantly upregulated in Luyan951, as confirmed by qRT-PCR. Bioinformatic analyses further suggested that AP2/ERF transcription factors may act as upstream regulators of these pathways. Furthermore, subcellular localization and transcriptional activation experiments confirmed the nuclear localization and transactivation function of three AP2/ERF genes ( TraesCS5D02G318400 , TraesCS6A02G381000 , TraesCS6D02G366100 ). Our findings indicate that the phenylpropanoid biosynthesis pathway plays a significant role in the cold tolerance of wheat, and together with the jasmonic acid signaling pathway, it forms a crucial regulatory network. This network promotes the scavenging of reactive oxygen species, maintains osmotic homeostasis, and stabilizes metabolism under low-temperature stress. Integrated analyses further suggest that this network may be coordinated by upstream ERF transcription factors. These findings provide comprehensive insights into the molecular mechanisms of wheat cold adaptation and offer valuable candidate genes and pathways for the genetic improvement of cold tolerance in wheat.