The involvement of hydrogen sulphide in melatonin-induced tolerance to arsenic toxicity in pepper (Capsicum annuum L.) plants by regulating sequestration and subcellular distribution of arsenic, and antioxidant defense system

Melatonin (MT) and hydrogen sulphide (H₂S) are recognised as vital biomolecules actively taking part in plant defence systems as free radical scavengers and antioxidants against a myriad of biotic and abiotic stressors. However, it has been yet unknown in plants subjected to arsenic (As) toxicity whether or not H₂S interacts with MT to regulate endogenous antioxidant defence system. Prior to beginning As stress (As–S) treatments, MT (0.10 mM) was applied externally to plants daily for three days. AsS was then started for two weeks with As(V) (0.1 mM as Na₂HAsO₄·7H₂O). The treatment of As reduced plant biomass (24.4%) and chlorophyll a (51.7%), chlorophyll b (25.9%), while it increased subcellular As in roots and leaves, levels of glutathione (GSH), hydrogen peroxide (H₂O₂), malondialdehyde (MDA), methylglyoxal (MG), H₂S and phytochelatins (PCs) in pepper plants. In As-stressed pepper plants, the application of MT increased plant biomass (16.3%), chlorophyll a (52.7%), chlorophyll b (28.2%), antioxidant enzymes’ activities, and H₂S accumulation, while it lowered the concentrations of MDA and H₂O₂. In As-treated plants, GSH and phytochelatins (PCs) were increased by MT by regulating As sequestration to make it harmless. The addition of MT increased As accumulation in the vacuoles of roots and caused the soluble fraction of As in vacuoles to become less toxic to vital organelles. MT-induced tolerance to As stress was further enhanced using NaHS, a source of H₂S. Hypotaurine (0.1 mM HT), a H₂S scavenger, was applied to the control and As-stressed plants together with MT and MT + NaHS to determine whether H₂S was implicated in MT-induced increased As–S tolerance. By reducing H₂S generation in pepper plants, HT counteracted the beneficial effects of MT, whereas the addition of NaHS to MT + HT restored the negative effects of HT, proving that H₂S is necessary for the pepper plants As-stress tolerance caused by MT.