Melatonin Suppressed the Heat Stress-Induced Damage in Wheat Seedlings by Modulating the Antioxidant Machinery

Abstract

Melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic signaling molecule that plays a crucial role in the regulation of various environmental stresses, including heat stress (HS). In this study, a 100 μM melatonin (MT) pretreatment followed by exposure to heat stress for different time periods was found to efficiently reduce oxidative stress by preventing the over-accumulation of hydrogen peroxide (H₂O₂), lowering the lipid peroxidation content (malondialdehyde (MDA) content), and increasing proline (Pro) biosynthesis. Moreover, the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were increased substantially in MT-pretreated wheat seedlings. The presence of MT significantly improved the heat tolerance of wheat seedlings by modulating their antioxidant defense system, activating the ascorbate-glutathione (AsA-GSH) cycle comprising ascorbate peroxidase (APX), and increasing glutathione reductase (GR) activities. It also held the photosynthetic machinery stable by increasing the chlorophyll content. Enhancement in the endogenous MT contents was also observed in the MT+HS-treated plants. Furthermore, the expression of reactive oxygen species (ROS)-related genes TaSOD, TaPOD, and TaCAT, and anti-stress responsive genes, such as TaMYB80, TaWRKY26, and TaWRKY39, was also induced in MT-treated seedlings. Due to these notable changes, an improvement in stress resistance was observed in MT-treated seedlings compared with control. Taken together, our findings suggest that MT can play a key role in boosting the stress tolerance of plants by modulating the antioxidant defense system and regulating the transcription of stress-responsive genes.

Keywords: antioxidant enzymes; melatonin; reactive oxygen species; wheat (Triticum aestivum L).

Figure 1

Effects of melatonin (MT; 100 μM) on malondialdehyde (MDA) content in leaves of wheat seedlings in the presence or absence of high temperature (42 °C). CK: control; HS: heat stress (42 °C); MT: melatonin (100 μM); MT + HS: melatonin (100 μM) + heat stress (42 °C). Data are means of three biological replicates (n = 3). Different letters indicate significant differences at p < 0.05 (ANOVA and Tukey HSD test); means ± SD.

Figure 2

Effects of MT (100 μM) on (A) H₂O₂ and (B) proline content in leaves of wheat seedlings in the presence or absence of high temperature (42 °C). CK: control; HS: heat stress (42 °C); MT: melatonin; MT + HS: melatonin (100 μM) + heat stress (42 °C). Data are means of three biological replicates (n = 3). Different letters indicate significant differences at p < 0.05 (ANOVA and Tukey HSD test); means ± SD.

Figure 3

Effects of MT (100 μM) on antioxidant enzymes activity. (A) Catalase (CAT), (B) peroxidase (POD), (C) superoxide dismutase (SOD), and (D) ascorbate peroxidase (APX) in leaves of wheat seedlings in the presence or absence of high temperature (42 °C). CK: control; HS: heat stress (42 °C); MT: melatonin; MT + HS: melatonin (100 μM) + heat stress (42 °C). Data are means of three biological replicates (n = 3). Different letters indicate significant differences at p < 0.05 (ANOVA and Tukey HSD test); means ± SD.

Figure 4

Effects of MT (100 μM) on glutathione reductase (GR) activity in leaves of wheat seedlings in the presence or absence of high temperature (42 °C). CK: control; HS: heat stress (42 °C); MT: melatonin (100 μM); MT+ HS: melatonin (100 μM) + HS (42 °C). Data are means of three biological replicates (n = 3). Different letters indicate significant differences at p < 0.05 (ANOVA and Tukey HSD test); means ± SD.

Figure 5

Evaluation of endogenous MT contents in (A) leaf and (B) root. CK: control; HS: heat stress (42 °C); MT: melatonin (100 μM); MT + HS: melatonin (100 μM) + HS (42 °C). The 10-day-old wheat seedlings were used to quantify the endogenous melatonin content. Different letters indicate significant differences at p < 0.05 (ANOVA and Tukey HSD test); means ± SD.

Figure 6

Effects of MT (100 μM) on chlorophyll contents. (A) Chlorophyll (Chl) a, (B) chlorophyll (Chl) b, and (C) carotenoid contents in leaves of wheat seedlings in the presence or absence of high temperature (42 °C). CK: control; HS: heat stress (42 °C); MT: melatonin (100 μM); MT + HS: melatonin (100 μM) + heat stress (42 °C). Data are means of three biological replicates (n = 3). Different letters indicate significant differences at p < 0.05 (ANOVA and Tukey HSD test); means ± SD.

Figure 7

Effects of MT (100 μM) on antioxidant related genes. (A) TaPOD, (B) TaSOD, (C) TaCAT, and stress-specific genes (D) TaWRKY26, (E). TaMYB80, and (F); TaWRKY39 in leaves of wheat seedlings in the presence or absence of high temperature (42 °C). CK: control; HS: heat stress (42 °C); MT: melatonin (100 μM); MT + HS: melatonin (100 μM) + heat stress (42 °C). Data are means of three biological replicates (n = 3). Different letters indicate significant differences at p < 0.05 (ANOVA and Tukey HSD test); means ± SD.

Figure 8

(1) The biotic and abiotic stresses triggered the production of ROS. (2) The increase of ROS level generates oxidative stress. (3) ROS (especially O₂⁻, H₂O₂), are capable of inducing the expression of melatonin biosynthesis genes (TDC, T5H, SNAT, and COMT, ASMT). (4) An increase in melatonin levels occurs as a result of the biosynthesis of endogenous melatonin. This response can be stimulated or reinforced by exogenous melatonin. (5) Melatonin, through interaction with its receptor (R = CAND2/PMTR1), induces the expression of several enzymes such as (6) RbOH and SOD, increasing O₂⁻ and H₂O₂ levels, among others (7) As a result, ROS levels are controlled by Antioxidative enzymes (CAT, APX, GPX, PRX, AsA GSH) and (8) also regulated by the direst action of melatonin (and its by-products) through their scavenging action.