Thermal priming enhances heat tolerance in alfalfa (Medicago sativa L.) through activation of multiple metabolic pathways

Background: Elevated environmental temperatures disrupt plant physiological homeostasis, imposing thermal stress that severely compromises growth and development. While thermal priming - a brief exposure to sublethal high temperature has been shown to enhance subsequent heat stress tolerance in plants, the underlying molecular mechanisms remain poorly characterized. In this study, we employed an integrated physiological, transcriptomic and metabolomic approach to investigate how thermal priming [37℃ for 2 h (P1) followed by 43℃ for 2 h (P2), designated P3] improves heat tolerance in alfalfa ( Medicago sativa L.) compared to unprimed controls (UP) exposed directly to 43℃. Results: Physiological analyses revealed that thermal priming significantly enhanced lodging resistance while increasing superoxide dismutase (SOD) and catalase (CAT) activities and reducing malondialdehyde (MDA) accumulation, indicative of improved oxidative stress management. Transcriptome profiling identified 1,217 upregulated genes in primed plants (P3 vs UP), with 50.2% being activated during the initial priming phase (P1). Cluster analysis demonstrated stage-specific pathway activation: brassinosteroid (BR) signaling, spliceosome activity, glutathione metabolism and fatty acid metabolism pathways were rapidly induced during early priming (P1), while phenylpropanoid biosynthesis was activated later during the second phase (P2). Metabolomic analyses provided further mechanistic insights, showing that thermal priming triggered significant lignin accumulation in stems, enhanced activity of the ascorbate-glutathione (AsA-GSH) cycle with increased antioxidant levels, and elevated content of unsaturated fatty acids including erucic acid, linolenic acid and oleic acid, suggesting membrane lipid remodeling. Conclusions: Our findings demonstrate that thermal priming establishes a multi-faceted defense system in alfalfa through BR-mediated signaling. This coordinated response involves activation of the AsA-GSH cycle for reactive oxygen species (ROS) scavenging, upregulation of phenylpropanoid biosynthesis for structural reinforcement through lignin deposition, accumulation of unsaturated fatty acids to maintain membrane stability, and enhancement of spliceosome activity to ensure proper processing of heat-responsive transcripts. The sequential activation of these pathways during the priming phases creates a 'stress memory' that prepares plants for subsequent heat challenges. These insights advance our understanding of thermal priming mechanisms and provide potential targets for improving crop heat tolerance through molecular breeding strategies.