Effects of Exogenous Melatonin on Methyl Viologen-Mediated Oxidative Stress in Apple Leaf

Abstract

Oxidative stress is a major source of damage of plants exposed to adverse environments. We examined the effect of exogenous melatonin (MT) in limiting of oxidative stress caused by methyl viologen (MV; paraquatin) in apple leaves (Malus domestica Borkh.). When detached leaves were pre-treated with melatonin, their level of stress tolerance increased. Under MV treatment, melatonin effectively alleviated the decrease in chlorophyll concentrations and maximum potential Photosystem II efficiency while also mitigating membrane damage and lipid peroxidation when compared with control leaves that were sprayed only with water prior to the stress experiment. The melatonin-treated leaves also showed higher activities and transcripts of antioxidant enzymes superoxide dismutase, peroxidase, and catalase. In addition, the expression of genes for those enzymes was upregulated. Melatonin-synthesis genes MdTDC1, MdT5H4, MdAANAT2, and MdASMT1 were also upregulated under oxidative stress in leaves but that expression was suppressed in response to 1 mM melatonin pretreatment during the MV treatments. Therefore, we conclude that exogenous melatonin mitigates the detrimental effects of oxidative stress, perhaps by slowing the decline in chlorophyll concentrations, moderating membrane damage and lipid peroxidation, increasing the activities of antioxidant enzymes, and changing the expression of genes for melatonin synthesis.

Keywords: Malus; melatonin; methyl viologen; oxidative stress; reactive oxygen species.

Figure 1

Effect of exogenous melatonin (MT) on apple leaf phenotype at 48 h after MV treatment. Fully mature leaves were collected from plants pre-treated with water only (A); or with MT concentrations of 0.01 mM (B); 0.1 mM (C); 1 mM (D); or 10 mM (E).

Figure 2

Effect of exogenous melatonin on damage index at 48 h after MV treatment for apple leaves pre-treated with 0 mM, 0.01 mM, 0.1 mM, 1 mM, or 10 mM melatonin. Data represent means ± SD of 15 replicate samples. *, significant difference (p < 0.05) between plants pre-treated with and without melatonin, based on Tukey’s multiple range test.

Figure 3

Effect of exogenous melatonin on F_v/F_m (A); total chlorophyll concentration (B); and leaf relative electrolyte leakage (C) at 48 h after MV treatment for apple leaves pre-treated with 0 mM, 0.01 mM, 0.1 mM, 1 mM, or 10 mM melatonin. Values are means of 3 replicates ± SD. Different letters indicate means are significantly different (p < 0.05), based on ANOVA and Tukey’s test.

Figure 4

Effect of 1 mM melatonin on levels of H₂O₂ (A) and MDA (B) in apple leaves during MV treatment. Values are means of 3 replicates ± SD. Different letters indicate means are significantly different (p < 0.05), based on ANOVA and Tukey’s test.

Figure 5

Effect of 1 mM melatonin on activity of main antioxidative enzymes in apple leaves during MV treatment: SOD (A); CAT (B); POD (C); and APX (D). Values are means of 3 replicates ± SD. Different letters indicate means are significantly different (p < 0.05), based on ANOVA and Tukey’s test.

Figure 6

Effect of 1 mM melatonin on relative expression of pheide A oxygenase(PAO) (A); CAT (B); POD (C); cAPX (D); cGR (E); MDHAR (F); and DHAR (G) in apple leaves during MV treatment. Total RNA was isolated from samples taken at different time points, converted to cDNA, and subjected to real-time RT-PCR. Expression levels were calculated relative to expression of Malus EF-1α mRNA. Values are means of 3 replicates ± SD. Different letters indicate means are significantly different (p < 0.05), based on ANOVA and Tukey’s test.

Figure 7

Effect of 1 mM melatonin on relative expression of melatonin synthesis genes—MdTDC1 (A); MdT5H4 (B); MdAANAT2 (C); and MdASMT1 (D)—in apple leaves during MV treatment. Total RNA was isolated from samples taken at different time points, converted to cDNA, and subjected to qRT-PCR. Expression levels were calculated relative to expression of Malus EF-1α mRNA. Values are means of 3 replicates ± SD. Different letters indicate means are significantly different (p < 0.05), based on ANOVA and Tukey’s test.