Regional Variation in Mulberry Leaf Metabolites: A Combined Metabolomic and Environmental Analysis of Biosynthetic Drivers

<b>Background:</b><i>Morus alba</i> L. (family Moraceae) is widely cultivated across the world and is well-known for its medicinal and nutritional value, especially its leaves. This study investigates the regional variation in mulberry leaf metabolites, focusing on alkaloids and flavonoids, and explores the influence of climatic and environmental factors on their biosynthesis using an integrated metabolomic and environmental analysis. Mulberry leaves, known for their medicinal and nutritional value, were collected from six regions across China, including Sichuan, Xinjiang, and Tibet. <b>Methods:</b> Untargeted metabolomics via UHPLC-MS was conducted. Differential metabolites were identified through multivariate analysis and annotated using the KEGG database. Redundancy analysis was used to link metabolite profiles with climatic data. <b>Results:</b> Mulberry leaves from six Chinese regions showed significant variation in total flavonoid content (TFC), total polyphenol content (TPC), and 1-Deoxynojirmycin (DNJ), with Tibet having the highest TFC and TPC, and Panzhihua the highest DNJ. Metabolomic analysis identified 3794 metabolites, revealing distinct regional clustering. A total of 79 differential metabolites were identified, which are enriched in pathways such as galactose metabolism and phenylalanine biosynthesis. Environmental factors, especially bio3, bio10, bio2, bio5, and bio20, strongly influenced metabolite profiles. <b>Conclusions:</b> The biosynthesis and accumulation of secondary metabolites in mulberry leaves are significantly influenced by region-specific environmental factors, particularly temperature, precipitation, and light. The identified differential metabolites are mainly enriched in galactose metabolism, arginine, and proline metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. These pathways are closely associated with plant stress responses and the synthesis of secondary metabolites. The pronounced regional differences in metabolite profiles underscore the critical role of environmental factors in determining the chemical composition of mulberry leaves. This research provides valuable insights into the influence of climatic factors affecting the chemical composition of plants. It lays a theoretical foundation for the quality assessment and grading of mulberry leaves, providing scientific guidance for their targeted cultivation and utilization.