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Plants have developed a variety of defense mechanisms that allow them to recognize and respond to specific stressors by triggering complex signaling networks that cause appropriate biochemical changes to overcome the stress. In this study, we subjected in vitro grown N. nervosa plants to PEG-induced dehydration stress for 1 day (1DPT), 3 days (3DPT) and 6 days (6DPT). Our study investigated antioxidant enzyme activities, including catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD), unveiling dynamic responses to PEG-induced water stress. CAT levels increased initially (1DPT) but declined with prolonged treatment; while POX activity significantly increased at 3DPT and 6DPT; and SOD, particularly the Mn-SOD3 isoform, demonstrated a substantial increase, emphasizing its role in the enzymatic free-radical scavenging activity. Furthermore, examination of the phenolic acid content revealed that rosmarinic acid (RA) was the predominant phenolic compound, followed by chlorogenic acid (CHLA), while ferulic acid (FA) and caffeic acid (CAFFA) were present in lower concentrations. Notably, PEG-induced dehydration significantly boosted RA content in N. nervosa plants at 3DPT. This increase highlights the plant’s response to oxidative stress conditions and its role in non-enzymatic antioxidant defense mechanisms. These findings significantly contribute to our comprehension of N. nervosa’s adaptive strategies under PEG-induced dehydration stress, offering valuable insights into plant stress physiology within industrial and agricultural contexts. Full article