Comparative Transcriptomics and Metabolomics Analysis Revealed the Mechanism of Exogenous Salicylic Acid Improving the Cold Tolerance of Walnut.

Walnut (Juglans regia) is an economically significant woody oil tree species widely cultivated in China. However, its production is increasingly threatened by extreme low-temperature events, such as unseasonal frosts and late-spring cold. Salicylic acid (SA) is a key phytohormone known to enhance cold tolerance in plants, yet its underlying mechanism in walnut remains unclear. In this study, we present the first integrated analysis combining physiological measurements, transcriptomics, and metabolomics to investigate how exogenous SA improves cold tolerance in walnut leaves. Our results showed that SA treatment significantly increased the accumulation of soluble sugars, chlorophyll, and proline, enhanced peroxidase (POD) activity, and reduced malondialdehyde (MDA) levels under cold stress. Multi-omics analysis revealed that SA modulated the expression of genes involved in multiple hormone signaling pathways, including those of SA, auxin, jasmonic acid, and abscisic acid, and altered corresponding hormone levels. Notably, carbohydrate metabolism emerged as a central pathway mediating SA-induced cold adaptation. Weighted gene co-expression network analysis (WGCNA) further identified several core candidate genes, such as JrTGA, JrPP2C, JrTPS, and JrBAM, which may play key roles in this process. Collectively, this study provides the first multi-omics perspective on the regulatory network underlying SA-enhanced cold tolerance in walnut. These findings offer both a theoretical and technical foundation for applying SA in cold-resistant walnut cultivation and contribute to the development of stress-resilient production strategies.