Structural, Physiological, and Biochemical Responses of Oreorchis patens (Lindl.) Leaves Under Cold Stress

Cold stress significantly impairs plant growth and development, making the study of cold resistance mechanisms a critical research focus. Oreorchis patens (Lindl.) exhibits strong cold hardiness, yet its molecular and physiological adaptations to cold stress remain unclear. This study utilized microscopy, physiological assays, and RNA sequencing to comprehensively investigate O. patens’s responses to cold stress. The results reveal that cold stress altered leaf anatomy, leading to irregular mesophyll cells, deformed chloroplasts, and variable epidermal thickness. Physiologically, SOD and POD activities peaked at 5 °C/−10 °C, while CAT activity declined; osmotic regulators (soluble sugars, proline) increased with decreasing temperatures. Compared to the reference plants (e.g., Erigeron canadensis, Allium fistulosum), O. patens exhibited lower SOD and POD but markedly higher CAT activities, alongside reduced MDA, soluble sugars, proline, and proteins, underscoring its distinctive tolerance strategy. Low temperature stress (≤10 °C/5 °C) significantly decreased the SPAD index; the net photosynthetic rate (Pn) initially increased and then approached zero within the temperature range from 30 °C/25 °C to 25 °C/20 °C; transpiration rate (Tr) and stomatal conductance (Gs) changed synchronously, accompanied by an increase in intercellular CO2 concentration (Ci). RNA sequencing identified 1139 cold-responsive differentially expressed genes, which were primarily enriched in flavonoid/lignin biosynthesis, jasmonic acid synthesis, and ROS scavenging pathways. qRT-PCR analysis revealed the role of secondary metabolites in O. patens response to cold stress. This study was the first to discuss the physiological, biochemical, and molecular regulatory mechanisms of O. patens resistance to cold stress, which provides foundational insights into its overwintering mechanisms and informs breeding strategies for cold-hardy horticultural crops in northern China.