Abstract Background: Melatonin is a multi-functional molecule widely employed in order to mitigate abiotic stress factors, in general and salt stress in particular. Even though previous reports revealed that melatonin could exhibit roles in promoting seed germination and protecting plants during various developmental stages of several plant species under salt stress, no reports are available with respect to the regulatory acts of melatonin on the physiological and biochemical status as well as the expression levels of defense- and secondary metabolism-related related transcripts in bitter melon subjected to the salt stress. Results: Herewith the present study, we performed a comprehensive analysis of the physiological and ion balance, antioxidant system, as well as transcript analysis of defense-related genes (WRKY1, SOS1, PM H+-ATPase, SKOR, Mc5PTase7, and SOAR1) and secondary metabolism-related gene expression (MAP30, α-MMC, polypeptide-P, and PAL) in salt-stressed bitter melon (Momordica charantia L.) plants in response to melatonin treatment. In this regard, different levels of melatonin (0, 75 and 150 µM) were applied to mitigate salinity stress (0, 50 and 100 mM NaCl) in bitter melon. Accordingly, present findings revealed that 100 mM salinity stress decreased growth and photosynthesis parameters (SPAD, Fv/Fo, Y(II)), RWC, and some nutrient elements (K+, Ca2+, and P), while it increased Y(NO), Y(NPQ), proline, Na+, Cl−, H2O2, MDA, antioxidant enzyme activity, and lead to the induction of the examined genes. However, prsiming with 150 µM melatonin increased SPAD, Fv/Fo, Y(II)), RWC, and K+, Ca2+, and P concentration while decreased Y(NO), Y(NPQ), Na+, Cl−, H2O2, and MDA under salt stress. In addition, the antioxidant system and gene expression levels were increased by melatonin (150 µM). Conclusions: Overall, it can be postulated that the application of melatonin (150 µM) has effective roles in alleviating the adverse impacts of salinity through critical modifi
