Physiological and broadly targeted metabolomic analyses of barley (Hordeum vulgare L.) in response to low-temperature stress.

Barley, as an important grain crop, often suffers from low-temperature stress during growth and development, which constitutes a significant impact on the yield and quality of barley. Therefore, an in-depth study of the metabolic response of barley under low-temperature stress is of great significance to improve the cold tolerance of barley. In this study, metabolites in barley leaves under different times of low-temperature stress were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), together with physiological data analysis. Result: Low-temperature stress decreased Pn, Gs, Tr, and SPAD in barley leaves, leading to an increase in ROS content, and a total of 800 metabolites were identified by metabolome analysis, belonging to amino acids and their derivatives, phenolic acids, nucleotides, and their derivatives, flavonoids, coumarins, alkaloids, organic acids, and free fatty acids. A total of 92, 91, 40, and 101 significantly different metabolites were identified at 0 h-vs-12 h, 0 h-vs-48 h, 12 h-vs-48 h, and 0 h-vs-Re24h, which were mainly involved in metabolic pathways, biosynthesis of secondary metabolites, ABCs and other metabolites. These differential metabolites were mainly involved in Metabolic pathways, Biosynthesis of secondary metabolites, ABC transporters, Biosynthesis of amino acids, Phenylpropanoid biosynthesis, Tryptophan metabolism, Flavonoid biosynthesis, Glycine, serine and threonine metabolism, Histidine metabolism, and Linoleic acid metabolism. Among them, the main up-regulated metabolites under low-temperature stress were 4-Hydroxyacetophenone, O-Acetylserine, Sinapoylagmatine, and Sinapoylputrescine, and the main down-regulated metabolites were Catechin gallate, D-Melezitose and Epigallocatechin-3-gallate, and the pathways with the highest enrichment of differential metabolites were Glycine, serine and threonine metabolism and Linoleic acid metabolism. Conclusion: Through the comprehensive analysis of physiological and metabolomic data, we initially revealed the metabolic network of barley under low-temperature stress and identified the key metabolites and metabolic pathways related to cold resistance. This study not only provides a new perspective on the molecular mechanism of cold resistance in barley but also provides an important theoretical basis for breeding barley for cold resistance. In the future, we will continue to study the regulatory mechanisms of these key metabolites and metabolic pathways, to produce new barley varieties with stronger cold resistance through genetic engineering and molecular breeding.