Unraveling the Regulation of Lipid Metabolism in Walnut (Juglans regia L.) Seeds Based on Metabolic Enzyme Activity and Transcriptomics

Abstract Walnuts ( Juglans regia L.) represents an economically important tree species, known for its woody oilseed. The synthesis of oil in their kernels involves complex physiological and biochemical changes. Elucidating changes in the expression profiles of genes associated with oil synthesis and accumulation, as well as in the activities of key enzymes involved in the oil synthesis pathway during walnut kernel development is of great significance for improving walnut oil content. In this study, the main walnut variety ‘Wen185’ ( Juglans regia ‘Wen185’) cultivated in Xinjiang, China, was used as the experimental material to analyze the crude fat content, activities of oil metabolism-related enzymes and transcriptomic profile during walnut kernell development. Crude fat accumulation and enzyme activity analyses revealed that the growth trend of crude fat content in walnut kernels showed was S-shaped. The activities of acetyl-CoA carboxylase (ACCase), fatty acid synthase (FAS), and acyltransferase (AAT) during the key seasons were synchronized with oil accumulation, and the greatest increase was observed between the early and middle stages of oil conversion. The transcriptome analysis revealed that differentially expressed genes were significantly enriched in the fatty acid metabolism and fatty acid biosynthesis pathways. Based on the enriched metabolic pathways and metabolic pathways and weighted gene co-expression network analysis (WGCNA) results, genes associated with oil synthesis and accumulation, including ACC-CTα , ACC-BCCP , KASII , SAD , FATA , LACS6 , and DGAT, were identified. Their expression was significantly upregulated from the early to the late stages of oil conversion, aligning with the trends in enzyme activity and oil content. Therefore, efficient oil accumulation in walnut kernels results from an integrated regulatory network of genes encoding key enzymes involved in the oil synthesis pathway. Specifically, the upregulated expression of genes such as ACC-CTα , ACC-BCCP , KASII , and DGAT, which encode ACCase, FAS, or AAT, likely enhances the activities of these key metabolic enzymes, thereby driving efficient oil synthesis and accumulation. These findings contribute to the development of genetic improvement strategies to achieve higher oil content in walnut kernels and provide new insights into the molecular mechanisms governing oil production during walnut kernel development.