Effect of exogenous melatonin on growth and antioxidant system of pumpkin seedlings under waterlogging stress

Abstract

Melatonin regulates defense responses in plants under environmental stress. This study aimed to explore the impact of exogenous melatonin on the phenotype and physiology of 'BM1' pumpkin seedlings subjected to waterlogging stress. Waterlogging stress was induced following foliar spraying of melatonin at various concentrations (CK, 0, 10, 100, 200, and 300 μmol·L^-1). The growth parameters, malondialdehyde (MDA) content, antioxidant enzyme activity, osmoregulatory substance levels, and other physiological indicators were assessed to elucidate the physiological mechanisms underlying the role of exogenous melatonin in mitigating waterlogging stress in pumpkin seedlings. The results indicate that pumpkin seedlings exhibit waterlogging symptoms, such as leaf wilting, water loss, edge chlorosis, and fading, under waterlogging stress conditions. Various growth indicators of the seedlings, including plant height, stem diameter, root length, fresh and dry weight, and leaf chlorophyll content, were significantly reduced. Moreover, the MDA content in leaves and roots increased significantly, along with elevated activities of superoxide dismutase, catalase, peroxidase, and soluble protein contents. When different concentrations of melatonin were sprayed on the leaves post waterlogging stress treatment, pumpkin seedlings showed varying degrees of recovery, with the 100 μmol·L^-1 treatment displaying the best growth status and plant morphological phenotypes. There were no significant differences compared to the control group. Seedling growth indicators, chlorophyll content, root activity, antioxidant enzyme activities, soluble protein content, and osmotic adjustment substance content all increased to varying degrees with increasing melatonin concentration, peaking at 100 μmol·L^-1. Melatonin also reduced membrane damage caused by oxidative stress and alleviated osmotic imbalance. Exogenous melatonin enhanced the activities of antioxidant enzymes and systems involved in scavenging reactive oxygen species, with 100 μmol·L^-1 as the optimal concentration. These findings underscore the crucial role of exogenous melatonin in alleviating waterlogging stress in pumpkins. The findings of this study offer a theoretical framework and technical assistance for cultivating waterlogging-resistant pumpkins in practical settings. Additionally, it establishes a theoretical groundwork for the molecular breeding of pumpkins with increased tolerance to waterlogging.

Keywords: Growth characteristics; Melatonin; Physiological characteristics of stress resistance; Pumpkin; Waterlogging stress.

Figure 1. Growth index (root length, plant height) of pumpkin seedlings under waterlogging stress.

Control 1: not waterlogging (CK), Control 2: waterlogging + 0 µM melatonin (W + 0 MT), Control 3: waterlogging + 10 µM melatonin (W + 10 MT), Control 4: waterlogging + 100 µM melatonin (W + 100 MT) Control 5: waterlogging + 200 µM melatonin (W + 200 MT), Control 6: waterlogging + 300 µM melatonin (W + 300 MT), Data are presented as mean ± SD of three measurements (n = 3, biological replicates). Different lowercase letters (a–f) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test).

Figure 2. Growth index (stem thickness) of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–d) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 3. Growth index (fresh and dry weight) of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–f) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test).The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 4. Plant morphology of pumpkin seedlings under waterlogging stress.

The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 5. Root vitality of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–e) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 6. Chlorophyll contents of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–d) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 7. Malondialdehyde content of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–e) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 8. Activity of antioxidant enzymes (SOD) of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–e) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 9. Activity of antioxidant enzymes (POD) of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–e) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 10. Activity of antioxidant enzymes (CAT) of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–f) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 11. Soluble protein content of pumpkin seedlings under waterlogging stress.

Data are presented as mean ± SD (n = 3, biological replicates). Different lowercase letters (a–f) in each column indicate significant differences at p ≤ 0.05 (least significant difference (LSD) test). The abbreviations of treatment names are the same as those described in Fig. 1.

Figure 12. Pumpkin response to waterlogging stress.