Transcriptomic and metabolomic insights into high-temperature acclimation mechanisms of Anoectochilus roxburghii

Anoectochilus roxburghii (A. roxburghii) is a valuable herb in traditional Chinese medicine, valued for its medicinal properties. Wild A. roxburghii is rare and requires specific environmental conditions, making artificial cultivation challenging. High-temperature acclimation in plants – physiological and biochemical adjustments to tolerate elevated temperatures – has practical applications in agriculture. Our previous study showed that high-temperature acclimation enhances the heat tolerance of the A. roxburghii accession ‘Meihuashan’, though the molecular basis remains unclear. In this study, we conducted transcriptomic and metabolomic analyses on control (CK), 3-day high-temperature acclimated (T1, 35°C), and 4-day high-temperature acclimated (T2, 35°C) ‘Meihuashan’ plants. RNA sequencing identified 79,086 unigenes and 12,404 protein-coding genes. Gene ontology and pathway enrichment analyses revealed that metabolic pathways were significantly enriched in the high-temperature response. Subsequent untargeted metabolomic analysis detected 1014 metabolites in the high-temperature treatment group, with 157 differentially expressed metabolites. Orthogonal partial least square-discriminant analysis revealed differentially expressed metabolites associated with flavone, phenylpropanoid, and alkaloid biosynthesis, along with amino acid, lipid, and carbohydrate metabolism. Integrated analysis further highlighted the central role of the carbon fixation pathway, in which key enzyme-encoding genes (e.g. PCK1, GOT2, ALDOA, TPI1) and their corresponding metabolites exhibited significant co-variation under high-temperature stress. These findings suggest that metabolic changes involving amino acids, lipids, and carbohydrates may play a key role in the high-temperature acclimation of ‘Meihuashan’.