Transcriptome and lipidome analyses reveal membrane lipid remodeling in root tissues of Cannabis sativa L. exposed to cold stress.

To construct a regulatory network model of the cold stress response in hemp roots, we used "Longdama 9" as the experimental material and integrated phenotypic, physiological, biochemical, lipidomic, and transcriptomic analyses to examine root responses to cold stress at 4°C. After 7 d of cold treatment, root growth was significantly inhibited, with root volume and fresh weight decreasing by 24% and 28%, respectively. Cold stress also induced membrane damage, as indicated by a 2.9-fold increase in relative electrical conductivity and a 3.8-fold increase in malondialdehyde (MDA) content. In response to cold-induced damage, osmotic regulators (soluble sugars, soluble proteins, and proline) accumulated, and the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), increased. Lipidomic analysis revealed that cold stress reduced phosphatidylcholine (PC) content by 25% and increased phosphatidic acid (PA) content by 26%. For galactolipids, digalactosyldiacylglycerol (DGDG) content increased by 27%, whereas monogalactosyldiacylglycerol (MGDG) content decreased by 46%. Triacylglycerol (TAG) content increased by 69%. Furthermore, the PC/phosphatidylethanolamine (PE) and (phosphatidylinositol (PI) + PC)/(PA + PE) ratios decreased, whereas the DGDG/MGDG ratio increased. These changes may contribute to maintaining membrane homeostasis under cold stress. Transcriptomic analysis identified 5786 differentially expressed genes (DEGs), including 730 associated with lipid metabolism pathways. Genes encoding glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), diacylglycerol acyltransferase (DGAT), phospholipase Dα (PLDα), and fatty acid desaturase FAD4, FAD6, and FAD8 were upregulated, suggesting enhanced lipid synthesis, PC degradation, and fatty acid desaturation. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays further revealed that the zinc finger transcription factor CsZincF directly bound to the CsDGAT2 promoter and activated its transcription. These results provide experimental evidence for a transcriptional regulatory module associated with cold-induced TAG accumulation in industrial hemp. Collectively, hemp roots adapt to cold stress through coordinated transcriptional regulation, which promotes lipid remodeling while enhancing antioxidant defense and osmotic adjustment. These findings provide insights into the molecular basis of cold tolerance in hemp roots and may facilitate the breeding of cold-tolerant hemp cultivars.