Melatonin Positively Regulates Both Dark- and Age-Induced Leaf Senescence by Reducing ROS Accumulation and Modulating Abscisic Acid and Auxin Biosynthesis in Cucumber Plants

Abstract

Melatonin (MT), as a signaling molecule, plays a vital role in regulating leaf senescence in plants. This study aimed to verify the antioxidant roles of MT in delaying dark- or age-induced leaf senescence of cucumber plants. The results showed that endogenous MT responds to darkness and overexpression of CsASMT, the key gene of MT synthesis, and delays leaf senescence stimulated by darkness, as manifested by significantly lower malonaldehyde (MDA) and reactive oxygen species (ROS) contents as well as higher activities and gene expression of antioxidant enzymes compared to the control. Moreover, MT suppressed both age- or dark-induced leaf senescence of cucumber, as evidenced by a decrease in senescence-related gene SAG20 and cell-death-related gene PDCD expression and ROS content and an increase in antioxidant capacity and chlorophyll biosynthesis compared with the H₂O-treated seedlings. Meanwhile, the suppression of age-induced leaf senescence by melatonin was also reflected by the reduction in abscisic acid (ABA) biosynthesis and signaling pathways as well as the promotion of auxin (IAA) biosynthesis and signaling pathways in cucumber plants in the solar greenhouse. Combining the results of the two separate experiments, we demonstrated that MT acts as a powerful antioxidant to alleviate leaf senescence by activating the antioxidant system and IAA synthesis and signaling while inhibiting ABA synthesis and signaling in cucumber plants.

Keywords: abscisic acid; antioxidant system; auxin; cucumber; leaf senescence; melatonin.

Figure 1

The variation of endogenous MT (A) and mRNA expression of TDC (B), T5H (C), SNAT (D), and ASMT (E) in leaves of cucumber plants under darkness. The two-leaf stage cucumber seedlings were treated with dark condition (day/night temperature: 25 °C/18 °C). The 2nd leaf samples were taken at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–d indicate that mean values are significantly different among samples (p < 0.05).

Figure 2

The effect of CsASMT transient overexpression on senescence of leaves under dark condition. (A) The phenotype of cotyledons; (B) MDA content; (C) soluble protein content. The CsASMT transient overexpression plants with two cotyledons were displaced into growth chambers and treated with dark condition (day/night temperature: 25 °C/18 °C). The cotyledons were collected for the determination of MDA and soluble protein contents at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–f indicate that mean values are significantly different among samples (p < 0.05).

Figure 3

The effect of CsASMT transient overexpression on ROS content of leaves under dark condition. (A) H₂O₂ content; (B) O₂⁻ content; (C) DAB and NBT staining. The CsASMT transient overexpression plants with two cotyledons were displaced into growth chambers and treated with dark condition (day/night temperature: 25 °C/18 °C). The cotyledons were collected for ROS content determination at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–f indicate that mean values are significantly different among samples (p < 0.05).

Figure 4

The effect of CsASMT transient overexpression on the activities and relative mRNA expressions of antioxidant enzyme in cucumber leaves under dark condition. (A) SOD activity; (B) the relative expression of SOD; (C) CAT activity; (D) the relative expression of CAT. The CsASMT transient overexpression plants with two cotyledons were displaced into growth chambers and treated with dark condition (day/night temperature: 25 °C/18 °C). The cotyledons were collected for the determination of antioxidant enzyme activity and related mRNA at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–e indicate that mean values are significantly different among samples (p < 0.05).

Figure 5

The effect of exogenous MT on MDA and protein contents in different age leaves of cucumber plants in a solar greenhouse. (A) Phenotype of leaves at different ages; (B) MDA content; (C) soluble protein content. At the three-leaf stage, the new leaf at the top was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days. Meanwhile, cucumber plants were sprayed with 100 μmol·L⁻¹ MT every 7 days, and plants treated with H₂O were the control. The different leaf-age leaves were sampled for MDA and protein content determination. All values shown are mean ± SD (n = 3). a–f indicate that mean values are significantly different among samples (p < 0.05).

Figure 6

The effect of exogenous MT on chlorophyll metabolism in different age leaves of cucumber plants in a solar greenhouse. (A) Chlorophyll a content; (B) chlorophyll b content; (C) PAO activity; (D) PPH activity; (E) MgCH activity; (F) FeCH activity. At the three-leaf stage, the new leaf at the top was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days. Meanwhile, cucumber plants were sprayed with 100 μmol·L⁻¹ MT every 7 days, and plants treated with H₂O were the control. The different leaf-age leaves were sampled for the determination of chlorophyll contents and relative enzyme activities. All values shown are mean ± SD (n = 3). a–g indicate that mean values are significantly different among samples (p < 0.05).

Figure 7

The effect of exogenous MT on cell-death- and senescence-related gene mRNA abundance in different age leaves of cucumber plants in a solar greenhouse. (A) The stain of programmed cell death; (B) PDCD mRNA abundance; (C) SAG20 mRNA abundance. At the three-leaf stage, the new leaf at the top was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days. Meanwhile, cucumber plants were sprayed with 100 μmol·L⁻¹ MT every 7 days, and plants treated with H₂O were the control. The different leaf-age leaves were sampled for trypan blue staining and gene mRNA abundance determination. All values shown are mean ± SD (n = 3). a–e indicate that mean values are significantly different among samples (p < 0.05).

Figure 8

The effect of exogenous MT on IAA-/ABA-related gene mRNA abundance in different age leaves of cucumber plants in a solar greenhouse. (A) ARF1 mRNA abundance; (B) YUCCA6 mRNA abundance; (C) ABI5 mRNA abundance; (D) NCED mRNA abundance. At the three-leaf stage, the new leaf at the top was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days. Meanwhile, cucumber plants were sprayed with 100 μmol L⁻¹ MT every 7 days, and plants treated with H₂O were the control. The different leaf-age leaves were sampled for gene mRNA abundance determination. All values shown are mean ± SD (n = 3). a–g indicate that mean values are significantly different among samples (p < 0.05).

Figure 9

The effect of exogenous MT on ROS content in senescent leaves of cucumber plants induced by age or darkness. (A) H₂O₂ content in different age leaves; (B), O₂⁻ content in different age leaves; (C) inverted microscope imaging of H₂O₂ in different age leaves; (D) inverted microscope imaging of O₂⁻ in different age leaves (E) H₂O₂ content in leaves under darkness; (F) O₂⁻ content in leaves under darkness; (G) inverted microscope imaging of H₂O₂ in leaves under darkness; (H) inverted microscope imaging of O₂⁻ in leaves under darkness. At the two-leaf stage, the cucumber seedlings were planted in a solar greenhouse or started to be treated with MT. At the three-leaf stage, the new leaf at the top of cucumber plants was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days in a solar greenhouse; plants were sprayed with 100 μmol·L⁻¹ MT every 7 days, and plants treated with H₂O were the control. Meanwhile, the seedlings were treated with H₂O and 100 μmol·L⁻¹ MT, respectively, 2 times, and then seedlings were displaced into growth chambers and treated with dark condition (day/night temperature: 25 °C/18 °C). The 2nd leaf samples were taken at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–e indicate that mean values are significantly different among samples (p < 0.05).

Figure 10

The effect of exogenous MT on antioxidant enzyme activity in senescent leaves of cucumber plants induced by age or darkness. (A–C) SOD, CAT, and APX activities in different age leaves; (D–F) SOD, CAT, and APX activities in leaves under darkness. At the two-leaf stage, the cucumber seedlings were planted in a solar greenhouse or started to be treated with MT. At the three-leaf stage, the new leaf at the top of cucumber plants was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days in a solar greenhouse; plants were sprayed with 100 μmol·L⁻¹ MT every 7 days, and plants treated with H₂O were the control. Meanwhile, the seedlings were treated with H₂O and 100 μmol·L⁻¹ MT, respectively, 2 times, and then seedlings were displaced into growth chambers and treated with dark condition (day/night temperature: 25 °C/18 °C). The 2nd leaf samples were taken at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–g indicate that mean values are significantly different among samples (p < 0.05).

Figure 11

The effect of exogenous MT on the mRNA abundance of antioxidant enzyme genes in senescent leaves of cucumber plants induced by age or darkness. (A–C) SOD, CAT, and APX mRNA abundance in different age leaves; (D–F) SOD, CAT, and APX mRNA abundance in leaves under darkness. At the two-leaf stage, the cucumber seedlings were planted in a solar greenhouse or started to be treated with MT. At the three-leaf stage, the new leaf at the top of cucumber plants was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days in a solar greenhouse; plants were sprayed with 100 μmol·L⁻¹ MT every 7 days, and plants treated with H₂O were the control. Meanwhile, the seedlings were treated with H₂O and 100 μmol·L⁻¹ MT, respectively, 2 times, and then seedlings were displaced into growth chambers and treated with dark condition (day/night temperature: 25 °C/18 °C). The 2nd leaf samples were taken at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–i indicate that mean values are significantly different among samples (p < 0.05).

Figure 12

The effect of exogenous MT on the ASA and GSH contents in senescent leaves of cucumber plants induced by age or darkness. (A,B) ASA and GSH contents in different age leaves; (C,D) ASA and GSH contents in leaves under darkness. At the two-leaf stage, the cucumber seedlings were planted in a solar greenhouse or started to be treated with MT. At the three-leaf stage, the new leaf at the top of cucumber plants was labeled 0 days and then labeled at 7, 14, 21, 28, 35, and 42 days in a solar greenhouse; plants were sprayed with 100 μmol·L⁻¹ MT every 7 days, and plants treated with H₂O were the control. Meanwhile, the seedlings were treated with H₂O and 100 μmol·L⁻¹ MT, respectively, 2 times, and then seedlings were displaced into growth chambers and treated with dark condition (day/night temperature: 25 °C/18 °C). The 2nd leaf samples were taken at 0, 1, 3, and 5 days. All values shown are mean ± SD (n = 3). a–f indicate that mean values are significantly different among samples (p < 0.05).