Recent reports have uncovered the multifunctional role of melatonin in plant physiological responses under optimal and suboptimal environmental conditions. In this study, we explored whether melatonin pretreatment could provoke priming effects in alfalfa (Medicago sativa L.) plants subsequently exposed to prolonged drought stress (7 days), by withholding watering. Results revealed that the rhizospheric application of melatonin (10 μmol L ) remarkably enhanced the drought tolerance of alfalfa plants, as evidenced by the observed plant tolerant phenotype, as well as by the higher levels of chlorophyll fluorescence and stomatal conductance, compared with nontreated drought-stressed plants. In addition, lower levels of lipid peroxidation (MDA content) as well as of both H O and NO contents in primed compared with nonprimed stressed plants suggest that melatonin pretreatment resulted in the systemic mitigation of drought-induced nitro-oxidative stress. Nitro-oxidative homeostasis was achieved by melatonin through the regulation of reactive oxygen (SOD, GR, CAT, APX) and nitrogen species (NR, NADHde) metabolic enzymes at the enzymatic and/or transcript level. Moreover, melatonin pretreatment resulted in the limitation of cellular redox disruption through the regulation of the mRNA levels of antioxidant and redox-related components (ADH, AOX, GST7, GST17), as well via osmoprotection through the regulation of proline homeostasis, at both the enzymatic (P5CS) and gene expression level (P5CS, P5CR). Overall, novel results highlight the importance of melatonin as a promising priming agent for the enhancement of plant tolerance to drought conditions through the regulation of nitro-oxidative and osmoprotective homeostasis.

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Pharmaceutically active compounds (PhACs) have been recently shown to exert phytotoxic effects. The present study explores the uptake, systemic translocation, and abiotic stress responses and detoxification mechanisms induced by the exposure of alfalfa plants grown in sand under greenhouse conditions to four common, individually applied PhACs (10μgL(-1)) (diclofenac, sulfamethoxazole, trimethoprim, 17a-ethinylestradiol) and their mixture. Stress physiology markers (lipid peroxidation, proline, H2O2 and NO content, antioxidant activity assays) and gene expression levels of key plant detoxification components (including glutathione S-transferases, GST7, GST17; superoxide dismutases, CuZnSOD, FeSOD; proton pump, H(+)-ATP, and cytochrome c oxidase, CytcOx), were evaluated. PhACs were detected in significantly higher concentrations in roots compared with leaves. Stress related effects, manifested via membrane lipid peroxidation and oxidative burst, were local (roots) rather than systemic (leaves), and exacerbated when the tested PhACs were applied in mixture. Systemic accumulation of H2O2 in leaves suggests its involvement in signal transduction and detoxification responses. Increased antioxidant enzymatic activities, as well as upregulated transcript levels of GST7, GST17, H(+)-ATPase and CytcOx, propose their role in the detoxification of the selected PhACs in plants. The current findings provide novel biochemical and molecular evidence highlighting the studied PhACs as an emerging abiotic stress factor, and point the need for further research on wastewater flows under natural agricultural environments.