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. 2020 Feb 8;9(2):220.
doi: 10.3390/plants9020220.

Melatonin Application Improves Salt Tolerance of Alfalfa ( Medicago sativa L.) by Enhancing Antioxidant Capacity

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Free PMC article

Melatonin Application Improves Salt Tolerance of Alfalfa ( Medicago sativa L.) by Enhancing Antioxidant Capacity

Huifang Cen et al. Plants (Basel). .
Free PMC article

Abstract

Alfalfa (Medicago sativa L.) is an important and widely cultivated forage grass. The productivity and forage quality of alfalfa are severely affected by salt stress. Melatonin is a bioactive molecule with versatile physiological functions and plays important roles in response to various biotic and abiotic stresses. Melatonin has been proven efficient in improving alfalfa drought and waterlogging tolerance in recent studies. In our reports, we applied melatonin exogenously to explore the effects of melatonin on alfalfa growth and salt resistance. The results demonstrated that melatonin application promoted alfalfa seed germination and seedling growth, and reduced oxidative damage under salt stress. Further application research found that melatonin alleviated salt injury in alfalfa plants under salt stress. The electrolyte leakage, malondialdehyde (MDA) content and H2O2 content were significantly reduced, and the activities of catalase (CAT), peroxidase (POD), and Cu/Zn superoxide dismutase (Cu/Zn-SOD) were increased with melatonin pretreatment compared to control plants under salt stress with the upregulation of genes related to melatonin and antioxidant enzymes biosynthesis. Melatonin was also involved in reducing Na+ accumulation in alfalfa plants. Our study indicates that melatonin plays a primary role as an antioxidant in scavenging H2O2 and enhancing activities of antioxidant enzymes to improve the salt tolerance of alfalfa plants.

Keywords: alfalfa; antioxidants; melatonin; salt tolerance.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Germination status of alfalfa seeds under various concentrations of salt and melatonin (MT). (A), Alfalfa seeds germinated under salt stress with various concentrations of NaCl (0, 100, 150, 200 and 250 mM) for 7 days; (B), Seedlings after being germinated under different concentrations of NaCl (0, 100, 150, 200 and 250 mM) for 7 days; (C), Germination rates of alfalfa seeds germinated under various concentrations of NaCl (0, 100, 150, 200 and 250 mM) for 7 days; (D), Germination rate of alfalfa seeds pretreated with various concentrations of MT (0, 1, 10, 50, 100 and 500 µM) and germinated under normal conditions for 7 days; (E), Seedlings germinated under normal conditions for 7 days with different concentrations of MT (0, 100, 150, 200 and 250 mM) pretreatment; (F), Root length of alfalfa seedlings after being germinated under normal conditions for 7 days with several concentrations of MT (0, 10 and 50 µM) pretreated; (G), Alfalfa seeds germinated under salt stress (200 mM NaCl) and salt stress with MT pretreatment (50 µM MT + 200 mM NaCl) for 4 days; (H), Germination potential of alfalfa seeds with MT (0, 10 and 50 µM) pretreated and germinated under salt stress with 200 mM NaCl for 4 days; (I), Seedlings germinated under 200 mM NaCl condition for 7 days with MT (0, 1, 10, 50, 100 and 500 µM) pretreatment, scale bar, 1 cm. Data are represented as means ± SE (n = 3), and bars with different letters indicate the differences between these different treatment groups according to ANOVA analysis (p < 0.05).
Figure 2
Figure 2
Germination rate (A), root length (B), fresh weight (C) and root/shoot ratio (D) of alfalfa seedlings pretreated with various concentrations of MT (0, 1, 10, 50, 100 and 500 µM) and germinated under 200 mM NaCl for 7 days. Data are represented as means ± SE (n = 3), and bars with different letters indicate the differences between these different treatment groups according to ANOVA analysis (p < 0.05).
Figure 3
Figure 3
The mitigated effects of various concentrations of MT (0, 1, 10, 50, 100 and 500 µM) on electrolyte leakage (A), malondialdehyde (MDA) content (B), H2O2 content (C) and the enzyme activities of peroxidase (POD) (D), catalase (CAT) (E), Cu/Zn superoxide dismutase (Cu/Zn-SOD) (F), and T-SOD (G) of alfalfa seedlings under 200 mM NaCl salinity condition. Data are represented as means ± SE (n = 3), and bars with different letters indicate the differences between these different treatment groups according to ANOVA analysis (p < 0.05).
Figure 4
Figure 4
Effects of MT (50 µM) pretreatment on one-month-old alfalfa plants exposed to salt stress (200 mM NaCl) for 15 days. A, B, Plants sprayed with MT (50 µM) or water for 7 days (A) and subsequently subjected to salt stress with 200 mM NaCl or normal conditions for 15 days (B). Plants from left to right: plants grown under normal conditions, plants pretreated with MT and grown under normal conditions, plants subjected to salt stress, and plants pretreated with MT and then subjected to salt stress; C–E, Effects of MT pretreatment on the MDA content (C), electrolyte leakage (D) and H2O2 content (E) of alfalfa plants before and after salinity treatment or under normal conditions. Data are represented as means ± SE (n = 3), and bars with different letters indicate the differences between these four different groups according to ANOVA analysis (p < 0.05).
Figure 5
Figure 5
The activities of antioxidative enzymes and the Na+, K+ contents of alfalfa plants under normal or salt stress (200 mM NaCl) conditions. A–D, the activities of CAT (A), POD (B), Cu/Zn-SOD (C) and glutathione peroxidase (GSH-PX) (D) in different groups of plants before and after salt stress treatment; E–G, Na+ content (E), K+ content (F), and the K+/Na+ ratio (G) in the shoots and roots of one-month-old alfalfa plants pretreated with MT and exposed to salt stress with 200 mM NaCl for 15 days. Data are represented as means ± SE (n = 3), and bars with different letters indicate the differences between these four different groups according to ANOVA analysis (p < 0.05).
Figure 6
Figure 6
Relative expression level of several selected genes in the melatonin biosynthesis pathway and genes encoding antioxidative enzymes involved in reactive oxygen species (ROS) metabolism A–C, relative expression level of tryptophan decarboxylase (TDC) (A), serotonin N-acetyltransferase (SNAT) (B), and N-acetylserotonin methyltransferase (ASMT) (C) in the melatonin biosynthesis pathway in different treated plants before and after 200 mM NaCl treatment for 15 days; D–F, relative expression level of Cu/Zn-SOD (D), CAT (E), and ascorbate peroxidase (APX) (F) in leaves of alfalfa plants before and after 200 mM NaCl treatment for 15 days. Data are represented as means ± SE (n = 3), and bars with different letters indicate the differences between these four different groups according to ANOVA analysis (p < 0.05).

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