Multi-omics integration revealed the molecular responses and key candidate genes underlying cold tolerance in Elymus...

The perennial grass Elymus nutans, native to the Qinghai-Tibet Plateau, exhibits exceptional cold tolerance. To understand its underlying mechanisms, we integrated physiological, transcriptomic, and proteomic profiling under cold stress. Our results revealed a distinct two-phase response strategy to cold stress. The early phase (0-24 h) featured rapid Ca²⁺ signaling, redox-related transcriptional reprogramming, and increased membrane permeability. The late phase (36-72 h) shifted toward primary metabolic regulation and the translation of protective proteins. Notably, a prominent time lag (temporal delay) occurred between transcript and protein accumulation. Mechanistically, transcriptomic and proteomic signatures suggested a potential energy trade-off, characterized by the extensive downregulation of photosynthetic components concurrent with the mobilization of photoprotective and carbohydrate metabolic networks. Simultaneously, defense capacity was fortified via enhanced proline, phenylpropanoid, and sustained ascorbate-glutathione pathways. Network analyses identified EnP5CS2 and EnMDHAR4 as key functional candidate genes associated with proline accumulation and redox homeostasis. Heterologous expression in yeast further indicated their basic biochemical competence in enhancing cold tolerance. Collectively, these findings provide multi-omics insights into resource reallocation and adaptive strategies employed by alpine extremophytes in response to cold stress, offering valuable genetic targets for breeding climate-resilient forage and crop.