Protective mechanisms of melatonin against selenium toxicity in Brassica napus: insights into physiological traits, thiol biosynthesis and antioxidant machinery

Abstract

Background: The ubiquitous signaling molecule melatonin (N-acetyl-5-methoxytryptamine) (MT) plays vital roles in plant development and stress tolerance. Selenium (Se) may be phytotoxic at high concentrations. Interactions between MT and Se (IV) stress in higher plants are poorly understood. The aim of this study was to evaluate the defensive roles of exogenous MT (0 μM, 50 μM, and 100 μM) against Se (IV) (0 μM, 50 μM, 100 μM, and 200 μM) stress based on the physiological and biochemical properties, thiol biosynthesis, and antioxidant system of Brassica napus plants subjected to these treatments.

Results: Se (IV) stress inhibited B. napus growth and biomass accumulation, reduced pigment content, and lowered net photosynthetic rate (P_n) and PSII photochemical efficiency (Fv/Fm) in a dose-dependent manner. All of the aforementioned responses were effectively alleviated by exogenous MT treatment. Exogenous MT mitigated oxidative damage and lipid peroxidation and protected the plasma membranes from Se toxicity by reducing Se-induced reactive oxygen species (ROS) accumulation. MT also alleviated osmotic stress by restoring foliar water and sugar levels. Relative to standalone Se treatment, the combination of MT and Se upregulated the ROS-detoxifying enzymes SOD, APX, GR, and CAT, increased proline, free amino acids, and the thiol components GSH, GSSG, GSH/GSSG, NPTs, PCs, and cys and upregulated the metabolic enzymes γ-ECS, GST, and PCS. Therefore, MT application attenuates Se-induce oxidative damage in plants. MT promotes the accumulation of chelating agents in the roots, detoxifies Se there, and impedes its further translocation to the leaves.

Conclusions: Exogenous MT improves the physiological traits, antioxidant system, and thiol ligand biosynthesis in B. napus subjected to Se stress primarily by enhancing Se detoxification and sequestration especially at the root level. Our results reveal better understanding of Se-phytotoxicity and Se-stress alleviation by the adequate supply of MT. The mechanisms of MT-induced plant tolerance to Se stress have potential implications in developing novel strategies for safe crop production in Se-rich soils.

Keywords: Antioxidants; Oilseed rape; Osmolytes; Oxidative stress; Plant growth regulator; Selenium; Thiols.

Fig. 1

Interactive effects of exogenous melatonin and selenium on photosynthesis traits. Effects of different treatments of exogenous melatonin (MT) (0 μM, 50 μM and 100 μM) and selenium (Se) (0 μM, 50 μM, 100 μM and 200 μM) on the a) Chl a (mg/g FW), b) Chl b (mg/g FW), c) carotenoids (mg/g FW), d) net photosynthetic rate (μM CO₂ m^− 2 s^− 1) and (e and f) photochemical efficiency of PSII (Fv/Fm) of fully stretched leaves of Brassica napus cv. ZS 758

Fig. 2

Interactive effects of exogenous melatonin and selenium on osmotic metabolites, reactive oxidative species, relative electrolyte leakage, and histochemical staining. Effects of different treatments of exogenous melatonin (MT) (0 μM, 50 μM and 100 μM) and selenium (Se) (0 μM, 50 μM, 100 μM and 200 μM) on the (a) soluble sugar (mg/g FW), (b) relative electrolyte leakage (%) and relative water content (%), (c) proline contents (mg/g FW) and free amino acid (mg/g FW) in the leaves, and (d) H₂O₂ (nmol mg^− 1 FW), (e) O₂^•– (nmol mg^− 1 FW), (f) MDA (nmol mg− 1 FW) contents in the leaves and roots, and root staining with (g) 3,3-diaminobenzidine (DAB) and (h) nitro-blue tetrazolium (NBT) of Brassica napus cv. ZS 758

Fig. 3

Interactive effects of exogenous melatonin and selenium on the enzyme activities and phosphate/silicon transporters. Effects of different treatments of exogenous melatonin (MT) (0 μM, 50 μM and 100 μM) and selenium (Se) (0 μM, 50 μM, 100 μM and 200 μM) on the activities of (a) superoxide dismutase (SOD), (b) catalase (CAT), (APX) ascorbate peroxidase (APX), and (d) glutathione reductase (GR) in the leaves and roots, and (e) phosphate (OsPT2)/silicon influx (Lis2) transporters in the roots of Brassica napus cv. ZS 758

Fig. 4

Interactive effects of exogenous melatonin and selenium on the biosynthesis of thiolic components and their metabolic enzymes. Effects of different treatments of exogenous melatonin (MT) (0 μM, 50 μM and 100 μM) and selenium (Se) (0 μM, 50 μM, 100 μM and 200 μM) on the (a) reduced glutathione content (GSH) (μmol g^− 1 FW), (b) oxidized glutathione content (GSSG) (μmol g^− 1 FW), (c) non-protein thiols (NPTs) (μmol g^− 1 FW), (d) phytochelatins (PCs) (μmol g^− 1 FW), (e) cysteine (Cyst) (nmol g^− 1 FW), and (f) activities of γ-glutamylcysteine synthetase (γ-ECS) (units mg^− 1 protein), (g) glutathione-S-transferase (GST) (units mg^− 1 protein) and (h) phytochelatins synthase (PCS) (nmol PC2 min^− 1 mg^− 1 protein) in the leaves and roots of Brassica napus cv. ZS 758

Fig. 5

Interactive effects of exogenous melatonin and selenium on stomatal opening. Scanning electron microscope (SEM) images of stomata showed the responses of exogenous MT on the stomatal aperture of Brassica napus leaves under Se stress. a and b showing full opening of leaves stomata under no stress conditions. c and d showed the complete closure of leaves stomata under maximum Se (200 μM) stress conditions. e and f illustrated the maximum stomatal opening at 50 μM Se + 100 μM MT than other Se + MT treatments

Fig. 6

Summary of protective mechanisms of melatonin against selenium phytotoxicity. A schematic diagram showed the mitigating effects of exogenous MT on Brassica napus L. seedlings under Se (IV) stress. Se heightened its toxicity by (I) Over-accumulating ROS that leads to chlorophyll degradation and ultimately growth reduction. (II) Induction of electrolyte leakage and lipid peroxidation reflects the damages in cellular membrane. (III) Disturbances in the synchronization of the defense system by increasing SOD and APX activities, proline, and free amino acids but declined the key enzymes (CAT and GR) and soluble sugar. (IV) Osmotic stress by lowering relative water and sugar contents. (V) An increase in the levels of thiol compounds (GSH, GSSG, NPTs, cysteine, and PCs) depicted the greater potential of Brassica napus plants to confer Se tolerance. Exogenous MT ameliorated the Se toxicity by enhancing photochemical efficiency and osmo-protection, which is linked with the enhanced plant growth and biomass production. In addition, exogenous MT induced the endogenous MT content which assist in the protective role of MT against Se-prompted ROS generation by inducing enzymes involved in AsA-GSH cycle (APX and GR), ROS-detoxifying enzymes (mainly SOD and CAT), biosynthesis of thiol components (especially GSH and phytochelatins), and the enzymes involved in thiol metabolism (γ-ECS, GST and PCS). The greater accumulation of MT and thiol components in roots suggested roots as greater site for the detoxification of Se as compared with leaves. Diagram indicates O₂^•– (superoxide), H₂O₂ (hydrogen peroxide), SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase), GR (glutathione reductase), GSH (reduced glutathione), GSSG (oxidized glutathione), RWC (relative water content), Pro (proline), WSG (water soluble sugar), FAA (free amino acids), REL (relative electrolyte leakage), MDA (melondialdehyde), NPTs (non-protein thiols), PCs (phytochelatins), cyst (cysteine), γ-ECS (gamma-glutamylcysteine synthase), GST (glutathione-S-transferase) and PCS (phytochelatins synthase)