| Summary: | The impact of drought stress induced by polyethylene glycol (PEG) on morphological, physiological, (bio)chemical, and biological characteristics of <i>Thymus lotocephalus</i> López and Morales shoot cultures have been investigated, as well as the potential of iron oxide nanoparticles, salicylic acid, and methyl jasmonate (MeJA) as alleviating drought stress agents. Results showed that PEG caused oxidative stress in a dose-dependent manner, raising H<sub>2</sub>O<sub>2</sub> levels and reducing shoots’ growth, photosynthetic pigment contents, and phenolic compounds production, especially phenolic acids, including the major compound rosmarinic acid. Moreover, Fourier Transform Infrared Spectra analysis revealed that PEG treatment caused changes in shoots’ composition, enhancing terpenoids biosynthesis. PEG also decreased the biological activities (antioxidant, anti-tyrosinase, and photoprotective) of the eco-friendly extracts obtained with a Natural Deep Eutectic Solvent. MeJA was the most efficient agent in protecting cells from oxidative damage caused by drought, by improving the biosynthesis of phenolics, like methyl 6-O-galloyl-β-D-glucopyranoside and salvianolic acids, as well as improving the extracts’ antioxidant activity. Altogether, the obtained results demonstrated a negative impact of PEG on <i>T. lotocephalus</i> shoots and an effective role of MeJA as a mitigating agent of drought stress. Additionally, extracts showed a good potential to be used in the cosmetics industry as skincare products.
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