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. 2025 Nov 7;25:1521. doi: 10.1186/s12870-025-07389-7

Exogenous melatonin mediates physiological and photosynthetic response mechanisms of maize cultivars under waterlogging stress

Ling Wang 1,2, Penghui Li 2,✉, Ying Li 2, Zhao Zhang 4, Ruiying Li 1,2, Hejing Tang 4, Zhandong Liu 1,2,3,✉
PMCID: PMC12595772  PMID: 41204187

Abstract

Background

Global warming has led to increased rainfall and waterlogging in some areas, which poses a threat to global food security.

Results

The results showed that the negative effects of different waterlogging stresses on different maize were mitigated by spraying exogenous melatonin, which maximally increased POD activity in C 7− 2 maize leaves, SOD activity in ZD 958 maize leaves and CAT activity in Z 58 maize leaves, which in turn reduced MDA in ZD 958 maize leaves and proline content in Z 58 maize leaves, increased antioxidant enzyme activities, and slowed down the damage of the stress on the plant body. Moreover, exogenous melatonin maximally increased chlorophyll b in C 7− 2 maize leaves, chlorophyll and chlorophyll a content of Z 58 maize leaves under CF2 stress, which indicated that melatonin could alleviate the integrity of chloroplast membranes by stress. Meanwhile, exogenous melatonin also increased the stomatal conductance, interstitial carbon dioxide concentration, and transpiration rate of ZD 958, and raised the net photosynthetic rate of C 7 − 2, which enhanced photosynthesis, increased the content of carbohydrates, and directly provided an energy source for the maize growth and metabolism, providing energy source and maintaining cell activity.

Conclusions

In conclusion, this study deeply explored the different regulatory effects of melatonin on maize varieties with different genetic backgrounds, showing that waterlogging stress had the least effect on Z 58 maize while melatonin had the best effect on alleviating the damage of ZD 958 under waterlogging stress, followed by Z 58, and lastly C 7 − 2, which deepened the understanding of the molecular mechanism of melatonin in alleviating adversity stress, and provided an opportunity to investigate the mechanism of melatonin action and to optimise its application in agricultural production.

Keywords: Exogenous melatonin, Waterlogging stress, Photosynthetic characteristics, Carbohydrates, Maize

Background

Maize is one of the most important cereal crops globally and plays an important role in feeding the population [1, 2]. It is estimated that more than 10% of the world’s agricultural land receives the threat of inundation and waterlogging, which frequently occurs during the rainy season and in severe cases can lead to an 80% reduction in maize production, seriously threatening global food security [3].

Due to the prolonged exposure of roots to low oxygen environment under waterlogging stress, which inhibits root vitality and fails to deliver nutrients and micronutrients to the leaves, with the continuation of waterlogging time, the above ground leaf organs will gradually yellow and wither from the bottom to the top until they fall off [4], which affects the normal growth and development of the plant [5]. In addition, one of the triggers affecting the normal growth process under waterlogging stress is the production of excessive reactive oxygen species (ROS) and malondialdehyde (MDA), which indirectly leads to damage to the plasma membrane of the cell, resulting in the breakdown of proteins and macromolecules. At the same time, activation of antioxidant enzyme system defence mechanism increases peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) enzyme activities for maintaining normal growth of plants and these changes are directly related to the waterlogging tolerance of plants [6–8]. Studies have shown that photosynthesis is the main pathway for energy and organic matter synthesis in plants, and under waterlogging stress, plant roots are covered with water and subjected to hypoxic stress, which closes the stomata and insufficient gas exchange, leading to a decrease in the efficiency of photosynthesis and a reduction in chlorophyll content [9–12]. Similarly, carbohydrate formation and transport are directly related to photosynthesis, and under waterlogging stress, plants provide osmoprotection to plant cells by down-regulating the activity and expression of relevant starch synthase enzymes affecting starch accumulation [13]. In addition to starch, saccharides can also act as osmoprotectants involved in plant biological responses, while reducing the accumulation of saccharides under waterlogging stress. It has been shown that waterlogging stress reduces the accumulation of soluble sugars and soluble proteins, which is consistent with the reduced expression of sucrose metabolism and protein metabolism genes [14, 15].

Therefore, here is an urgent need to increase the waterlogging tolerance of maize to improve plant yield and meet the human demand for food. Spraying exogenous plant growth regulators is considered as a promising strategy to enhance crop resistance to waterlogging stress [16, 17]. Melatonin is a multifunctional molecule present in individual organisms that plays an important role in maintaining normal plant growth [18, 19]. Studies have shown that under adversity stress, plants are able to sense stress signals to promote melatonin synthesis, which in turn improves antioxidant enzyme activity and mitigates the oxidative damage caused by adversity stress to plants, thus improving their waterlogging tolerance [20].

Foliar spraying of 10µM melatonin under waterlogging stress has been reported to reduce cell death in soybean roots, protect seedling growth and development, improve phenotypic status under waterlogging stress [21], alleviate the effects of waterlogging stress on photosynthesis in sorghum seedlings, increase antioxidant enzyme activities, and improve plant resistance by reducing MDA and H2O2 content [22]. In addition, it can increase the activities of antioxidant enzymes and nitrogen metabolism enzymes of Malus hupehensis, reduce the accumulation of ROS under waterlogging stress and regulate osmotic substances and carbohydrate content [23]. Exogenous melatonin can also regulate stomatal size in maize leaves, increase leaf chlorophyll content and net photosynthetic rate (Pn), improving maize growth [24]. It can be seen that melatonin has different effects on the improvement of waterlogging tolerance in different plant varieties, which may be related to the differences in physiological metabolism and genetic background among varieties [25].

In the Huanghuai region of China, substantial rainfall occurs annually from June to August. Studies have indicated an increasing trend of waterlogging disasters during the emergence-to-anthesis stages of maize over the past 50 years [26, 27], leading to severe lodging and ultimately impacting yield [28]. Current research predominantly focuses on the temporal dimension of waterlogging stress, with insufficient attention to flooding depth gradients [29]. Concomitantly, the genetic background-dependent regulatory mechanisms of exogenous melatonin remain unclear, particularly regarding cultivar-specific signaling cascades. Therefore, this study aims to compare and analyze the physiological, biochemical, and photosynthetic changes in maize varieties with different waterlogging tolerance levels under various waterlogging depths after the application of melatonin. The goal is to elucidate the potential mechanisms through which melatonin enhances plant waterlogging tolerance and to offer a theoretical basis for using exogenous melatonin to improve resistance to waterlogging.

Measurements and methods

Experimental site description

The experiment was conducted in enclosed pit measurement at the Xinxiang Comprehensive Experimental Base of the Chinese Academy of Agricultural Sciences. The base is located in a warm temperate continental monsoon climate zone (35°14’ N, 113°76’ E, 74 m above sea level), with an average annual temperature of 14 °C, 2399 h of annual sunshine, and a mean annual precipitation of 582mm, 70–80% of which occurs between June and October. The soil type of the test site was loam (tidal soil), with a bulk density of 1.51 g cm−3 and a field capacity of 20.5%. At the start of the experiment, the 0–20 cm soil layer had the following characteristics: Organicmatter 18.19 g kg−1, Total nitrogen 1.28 g kg−1, Alkaline dissolved nitrogen 95.88 mg kg−1, Available phosphorus 16.24 g kg−1, Readily available potassium 249.83 mg kg−1, Bulk density 1.50 g cm−3, and pH 8.58.

Experimental design

The maize cultivars employed in the experiment included the dominant hybrid “Zhengdan 958” (ZD 958) and its parental lines “Chang 7 − 2” (C 7 − 2) and “Zheng 58” (Z 58) [30, 31]. These cultivars were selected based on their comparable hierarchical physiological traits. The seeds are obtained from Institute of Water-saving Agriculture and Irrigation Technology in Arid and Semi-arid Areas, Chinese Academy of Agricultural Sciences, Xinxiang City, Henan Province, China. In each lysimeter, 30 plants were grown per cultivar. At the five-leaf stage, waterlogging treatments were initiated. Light waterlogging (CF1) and severe waterlogging (CF2) were maintained by keeping a water layer of 3–5 cm and 9–11 cm, respectively, above the soil surface. Starting from the beginning of waterlogging, deionized water (WL) or 100 µM/L melatonin (WL + MT) was sprayed every two days until runoff. This waterlogging and spray treatment continued for 9 days [32, 33]. The lysimeters were irrigated using a lower limit control method with a fixed water supply: when soil moisture reached 75% of field capacity, 30 mm of water was applied using a water meter. After each treatment was completed, the drainage valves in the lysimeters were opened to drain excess water from the soil.

Measurement of plant growth parameters

Following the completion of the stress treatments, three maize plants of uniform growth were randomly selected from each treatment. Plant height was measured using a measuring tape. Additionally, selected plants were cut at the root base, and then placed in a 105°C oven for 30 min for enzyme inactivation. Subsequently, the plants were dried in a 72°C oven for 72 h, after which plant dry weight was measured using an electronic balance (XS105, METTLER TOLEDO, USA) [34].

Leaf physiological parameters

Following the completion of the stress treatments, the third fully expanded leaf from the top of each plant was harvested, divided into 5 ml centrifuge tubes, immediately frozen in liquid nitrogen, and then transferred to a −80 °C freezer for storage until analysis.

SOD activity was determined using the nitroblue tetrazolium (NBT) method, based on its NBT reduction capability [35]. POD activity was measured using the guaiacol method, based on the reaction of H2O2 with guaiacol [36]. CAT activity was measured using a spectrophotometric method based on the decomposition of H2O2 [37]. Soluble sugar content was determined using the anthrone method, based on the reaction between soluble sugars and anthrone [38]. Soluble protein content was measured using the Bradford method, based on the binding of soluble proteins to Cu+ ions under alkaline conditions [39]. Proline content was measured using an acidic ninhydrin solution, based on the principle of proline extraction with sulfosalicylic acid (SA) [40]. Malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) method, based on the reaction of MDA with TBA. Glucose and fructose contents were determined using a spectrophotometric method, based on the principle that sucrose is hydrolyzed into glucose and fructose under acidic conditions, and then fructose reacts with resorcinol to form a colored product [41]. Starch content was determined using the anthrone colorimetric method, according to the method described by Weng et al. [42]. Chlorophyll, chlorophyll a, and chlorophyll b contents were determined using the ethanol extraction method [43].

Measurement of gas exchange parameters

Three maize plants of uniform growth were selected from each treatment. On the 5th day of the stress treatment, between 9:00 and 11:00 AM, gas exchange parameters were measured using a Li-6400 portable photosynthesis system (LI-COR, USA) on the central portion of the second fully expanded leaf from the top of each plant. These parameters included net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and interstitial carbon dioxide concentration (Ci). Measurements were performed using a red-blue Light source. The photosynthetic photon flux density in the leaf chamber was set to 1500mol (CO2) m−2s−1, the airflow rate was 500 µmols−1, and the reference CO2 concentration was 400 µmol mol−1. According to the nearby meteorological station, the test day recorded a maximum temperature of 35.33 °C, a minimum of 23.45 °C, a mean temperature of 29.47 °C, and a relative Humidity of 72.79%.

Data analysis

Data were systematically organized and meticulously analyzed utilizing Excel 2021 (Microsoft Corporation, Redmond, WA, USA). Data were systematically organized and meticulously analyzed utilizing Excel 2021 (Microsoft Corporation, Redmond, WA, USA). Physiological parameters underwent one-way analysis of variance (ANOVA) coupled with Fisher’s least significant difference (LSD) post-hoc testing for multiple comparisons of treatment effects. Additionally, a general linear model (GLM) was constructed to quantitatively assess the interactive effects of stress treatment and melatonin application on Maize physiological parameters. All statistical procedures were executed in SPSS 18.0 (IBM Inc., Chicago, IL, USA). Duncan’s pairwise comparison test served to ascertain significant variations at the 5% confidence level (p < 0.05). Notably, different lowercase letters designate statistically significant differences, whereas identical lowercase letters signify non-significant differences. Furthermore, the figures were skillfully plotted and Pearson correlation analysis was performed with Origin 2021 (Origin Lab Corporation, Northampton, USA).

Result

Effect of exogenous melatonin on biomass under waterlogging stress

The growth of the three varieties of maize was significantly affected under waterlogging stress (Fig. 1A-D). From the figure we were able to observe that leaf yellowing was more severe in different varieties of maize at increased waterlogging stress. The melatonin treatment significantly reduced leaf yellowing compared to no melatonin spray, suggesting that melatonin has a positive effect in mitigating waterlogging-induced leaf yellowing. The biomass of maize showed a decreasing trend with increasing waterlogging (Fig. 1E-J), with the minimum magnitude reducing plant height and dry weight of ZD 958 by 13.11% and 34.65% under mild waterlogging stress, respectively, and reducing plant height of C 7 − 2 and dry weight of ZD 958 by 16.55% and 31.63% under severe waterlogging stress, respectively. In addition, melatonin treatment significantly increased plant height and dry weight in all the varieties, melatonin was able to increase plant height of C 7 − 2 and dry weight of ZD 958 by 14.96% and 15.56%, respectively, under mild waterlogging stress and melatonin was able to increase plant height of Z 58 and dry weight of ZD 958 by 13.39% and 22.24%, respectively. Moreover, the effects of stress types (S), different spraying methods (SM) and the interaction effects of stress types and different spraying methods (S×SM) on dry weight in maize were also significant. The stress types (S) and different spraying methods (SM) were detected as significant for maize of plant height (Fig. 1).

Fig. 1.

Fig. 1

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Effect of deionized water (A) and exogenous melatonin(C) on phenotype of ZD 958, C 7 − 2, and Z 58 maize under CF1 stress. Effect of deionized water (B) and exogenous melatonin(D) on phenotype of ZD 958, C 7 − 2, and Z 58 maize under CF2 stress. Effects of deionized water and exogenous melatonin on Plant height(E-G) and Dry weight(H-J) in ZD 958, C 7 − 2, and Z 58 maize under different waterlogging condition. The part indicated by the white arrow represents the area where the leaf changes. The data is depicted as the mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 based on the Least Significant Difference (LSD) test. ns indicates no significant difference ANOVA analysis is used for S, SM and, S×SM. S: Stress types, SM: different spraying methods. * p < 0.05, ** p < 0.01 and *** p < 0.001

Effect of exogenous melatonin on antioxidant enzyme activity under waterlogging stress

Effect of melatonin on antioxidant enzyme activities of different maize cultivars under varying degrees of waterlogging stress (Fig. 2). The results showed that POD activity in Z 58 leaves, SOD and CAT activities in ZD 958 leaves were most significantly increased under CF1 stress by 81.82%, 83.60% and 6.45%, respectively, compared to CK (Fig. 2C, D and, G). The maximum increase in POD activity in Z 58 leaves, SOD and CAT activities in ZD 958 leaves under CF2 stress was 236.5%, 39.08% and 9.53%, respectively (Fig. 2C, D and, G). Under CK conditions, exogenous melatonin application showed no significant impact on POD activity in three varieties of maize, but significantly increased activities of SOD and CAT in C 7− 2, and activity of SOD in Z 58 (Fig. 2A-I). Compared with CK conditions, melatonin sprayed under CF1 stress increased CAT activities in ZD 958 and C 7 − 2 leaves by 3.2% and 16.26%, respectively, and SOD and CAT activities in Z 58 leaves by 8.51% and 12.48%, respectively, but significantly reduced POD activity in Z 58 by 19.20%. These results suggest that melatonin may have a protective effect on the growth of maize under waterlogging conditions by enhancing antioxidant enzyme activities. Exogenous melatonin significantly increased the activities of POD by 18.10% and 20.81% and SOD by 28.76% and 21.63% in ZD 958 and C 7 − 2 leaves, respectively, under CF2 stress, while the activity of POD in Z 58 leaves followed the same trend as that of CF1 stress, decreased by 25.20%. Additionally, the effects of stress types (S), different spraying methods (SM) and the interaction effects of stress types and different spraying methods (S×SM) on SOD and CAT activities in ZD 958, POD and SOD activities in Z 58 were also significant (Fig. 2).

Fig. 2.

Fig. 2

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Effect of deionized water and exogenous melatonin on activity of POD (A-C), SOD (D-F), and CAT (G-I) in ZD 958, C 7 − 2, and Z 58 maize under different waterlogging condition. The data is depicted as the mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 based on the Least Significant Difference (LSD) test. ns indicates no significant difference ANOVA analysis is used for S, SM and, S×SM. S: Stress types, SM: different spraying methods. * p < 0.05, ** p < 0.01 and *** p < 0.001

Effect of exogenous melatonin on osmotic regulatory substance content under waterlogging stress

Effect of exogenous melatonin on contents of MDA and proline under waterlogging stress

Waterlogging stress increased MDA and proline content in maize leaves (Fig. 3). Compared to the CK, under CF1 stress, modest increases were observed in content of MDA and proline in ZD 958, by 17.80% and 32.00%, respectively (Fig. 3A and D). Under CF2 stress, slight increases were observed in content of proline in C 7 − 2 and content of MDA in Z 58, by 8.77% and 12.13%, respectively (Fig. 3C and E). Under CK conditions, melatonin treatment had no significant effect on MDA and proline contents, whereas exogenous melatonin under CF1 stress significantly reduced 17.80% proline content in ZD 958 leaves and 30.64% and 13.91% proline and 14.85% and 31.17% MDA in C 7 − 2 and Z 58 leaves, respectively. In addition, exogenous melatonin significantly reduced proline content by 17.86% and 43.18%, MDA content by 28.01% and 15.17% in ZD 958 and Z 58 leaves, respectively, and proline content by 18.52% in C 7 − 2 under CF2 stress. These results suggest that melatonin may have a positive effect on plant cell membranes under waterlogging conditions by reducing MDA and proline contents. Also, the effects of stress types (S), different spraying methods (SM) and the interaction effects of stress types and different spraying methods (S×SM) on MDA and proline contents in ZD 958, proline content in Z 58 were also significant (Fig. 3).

Fig. 3.

Fig. 3

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Effect of deionized water and exogenous melatonin on contents of MDA(A-C) and Proline(D-F) in ZD 958, C 7 − 2, and Z 58 maize under different waterlogging condition. The data is depicted as the mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 based on the Least Significant Difference (LSD) test. ns indicates no significant difference ANOVA analysis is used for S, SM and, S×SM. S: Stress types, SM: different spraying methods. * p < 0.05, ** p < 0.01 and *** p < 0.001

Effect of exogenous melatonin on contents of soluble sugars and soluble proteins under waterlogging stress

Waterlogging stress resulted in decrease contents of soluble sugar and soluble protein in maize leaves (Fig. 4). Compared to the CK, under CF1 stress, the slightest decrease was observed in content of soluble protein and soluble Sugar in C 7− 2, by 45.06% and 13.30%, respectively (Fig. 4B and E). Compared to the CK, under CF2 stress, the slight decrease was observed in soluble sugar content in Z 58 and soluble protein content in C 7 − 2, by 57.77% and 30.80%, respectively (Fig. 4C and E). No significant differences were observed in content of soluble sugar among ZD 958, C 7 − 2, and Z 58 under different waterlogging stress conditions (Fig. 4D-F). However, melatonin significantly increased soluble sugars content in ZD 958 leaves by 40.54 and 50.54% under CF1 and CF2 conditions, respectively, whereas the effect on soluble sugars content in C 7− 2 and Z 58 leaves varied depending on the degree of water stress, under CF1 and CF2 stress, respectively. Under CF1 and CF2 stress significantly increased soluble sugars in C 7− 2 and Z 58 leaves as 52.49% and 44.52%, respectively. For soluble proteins content, melatonin significantly increased soluble proteins by 28.52% and 25.32% in C 7 − 2 and 41.66% and 25.23% in Z 58 leaves under CF1 and CF2 conditions, respectively, while melatonin significantly increased soluble proteins content by 66.18% in ZD 958 under CF2 conditions. Besides, the effects of stress types (S), different spraying methods (SM) and the interaction effects of stress types and different spraying methods (S×SM) on soluble sugar and soluble protein contents in ZD 958, soluble sugar content in C 7− 2 and soluble protein content in Z58 were also significant (Fig. 4).

Fig. 4.

Fig. 4

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Effect of deionized water and exogenous melatonin on contents of soluble sugars (A-C) and soluble proteins (D-F) in ZD 958, C 7 − 2, and Z 58 maize under different waterlogging condition. The data is depicted as the mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 based on the Least Significant Difference (LSD) test. ns indicates no significant difference ANOVA analysis is used for S, SM and, S×SM. S: Stress types, SM: different spraying methods. * p < 0.05, ** p < 0.01 and *** p < 0.001

Effect of exogenous melatonin on contents of starch, fructose, and sucrose under waterlogging stress

Waterlogging stress resulted in a decrease in contents of starch, fructose, and sucrose in maize leaves (Fig. 5). Compared to the CK, the modal decreases were observed in contents of starch and sucrose in C 7− 2, and in content of fructose in ZD 958, by 28.43%, 58.31%, and 46.13%, respectively under CF1 stress (Fig. 5B, D and, H). Compared to the CK, the minimal decreases were observed in content of starch in Z 58, content of fructose in ZD 958, and content of sucrose in C 7− 2, by 44.90%, 47.03%, and 63.4%, respectively under CF2 stress (Fig. 5C, D and, H). Under the CK conditions, exogenous melatonin application failed to affect content of starch, fructose, or sucrose in leaves (Fig. 5A-I). In addition, melatonin treatment had no significant effect on the content of soluble sugars and soluble proteins under CK conditions. However, melatonin significantly increased the content of fructose in the leaves of ZD 958, C 7 − 2 and Z 58 by 20.80%, 60.63% and 44.61%, respectively, under CF1 conditions. Under CF2 conditions, melatonin significantly increased the content of fructose in the leaves of ZD 958, C 7 − 2 and Z 58 leaves with sucrose content of 45.15%, 19.72% and 77.70%, respectively, and melatonin significantly increased starch content in ZD 958 and Z 58 leaves under CF1 stress with 34.26% and 48.60%, respectively, and it is noteworthy that melatonin significantly reduced starch content in C 7 − 2 maize under CF1 conditions with 24.38% and increased starch content by 29.17% under CF2 condition. Likewise, the effects of stress types (S) on starch, fructose and sucrose contents in maize were also significant (Fig. 5).

Fig. 5.

Fig. 5

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Effect of deionized water and exogenous melatonin on contents of Starch (A-C), Fructose(D-F), and Sucrose(G-I) in ZD 958, C 7 − 2, and Z 58 maize under different waterlogging condition. The data is depicted as the mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 based on the Least Significant Difference (LSD) test. ns indicates no significant difference ANOVA analysis is used for S, SM and, S×SM. S: Stress types, SM: different spraying methods. * p < 0.05, ** p < 0.01 and *** p < 0.001

Effect of exogenous melatonin on photosynthetic characteristics under waterlogging stress

Effect of exogenous melatonin on contents of chlorophyll, chlorophyll a, and chlorophyll b under waterlogging stress

Waterlogging stress resulted in a decrease in contents of chlorophyll, chlorophyll a, and chlorophyll b in maize leaves (Fig. 6). Compared to the CK, C 7 − 2 observed a limited 13.75% decrease in chlorophyll content, while the modest decreases in chlorophyll a and chlorophyll b contents were observed in ZD 958, by 14.41%, and 13.9%, respectively under CF1 stress (Fig. 6B, D and, G). Besides, modest decreases were observed in content of chlorophyll in C 7 − 2, chlorophyll a in ZD 958, and chlorophyll b in ZD 958, by 13.75%, 14.41%, and 17.61%, respectively under CF2 stress (Fig. 6B, D and, G). Melatonin treatment did not significantly affect the content of different types of chlorophyll under CK conditions in all three varieties of maize. However, melatonin significantly increased the total chlorophyll content of C 7− 2 leaves by 11.43%, 33.46% and Z 58 leaves by 8.13%, 82.04% and chlorophyll a by 34.14%, 19.63% and 6.65%, 84.14%, respectively, under CF1 and CF2 conditions. However, the effect of chlorophyll b content in C 7 − 2 and Z 58 leaves varied depending on the degree of water stress. Under CF1 stress chlorophyll b content in C 7 − 2 leaves was significantly reduced by 27.63% and under CF2 chlorophyll b content in C 7 − 2 and Z 58 leaves was significantly increased by 93.29% and 76.12%, respectively. This suggests that melatonin helps to maintain the photosynthetic pigment content of maize leaves under waterlogging conditions, thus providing protection against photosynthesis. In addition, the effects of stress types (S), different spraying methods (SM) and the interaction effects of stress types and different spraying methods (S×SM) on chlorophyll, chlorophyll a and chlorophyll b contents in maize were also significant (Fig. 6).

Fig. 6.

Fig. 6

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Effect of deionized water and exogenous melatonin on contents of chlorophyll (A-C), chlorophyll a (D-F), and chlorophyll b (G-I) in ZD 958, C 7 − 2, and Z 58 maize under different waterlogging condition. The data is depicted as the mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 based on the Least Significant Difference (LSD) test. ns indicates no significant difference ANOVA analysis is used for S, SM and, S×SM. S: Stress types, SM: different spraying methods. * p < 0.05, ** p < 0.01 and *** p < 0.001

Effect of exogenous melatonin on photosynthetic parameters under waterlogging stress

Waterlogging stress resulted in a decrease in maize leaf photosynthetic parameters (net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr)) (Fig. 7). Compared to the CK, under CF1 stress, modest decreases were observed in Pn and Tr in ZD 958, by 24.35% and 44.34%, respectively, and in Gs and Ci in Z 58, by 42.18% and 42.59%, respectively (Fig. 7A, F, I and, J). Compared to the CK, under CF2 stress, compared to CK, modest decreases were observed in Pn and Ci in Z 58, by 41.23% and 60.72%, respectively, and in Gs and Tr in ZD 958, by 59.78% and 45.88%, respectively (Fig. 7C, D, I and, J). Among the three varieties, melatonin treatment had no significant effect on Pn, Gs, Ci and Tr under CK conditions (Fig. 7A-L). However, melatonin treatment significantly increased these photosynthetic parameters under CF1 and CF2 conditions, and this effect was especially significant under CF2 conditions. In particular, in ZD 958, melatonin treatment significantly increased Gs, Ci and Tr by 81.42%, 33.25%, and 61.96%, respectively, under CF2 conditions, which suggests that melatonin improves the photosynthetic efficiency of maize under waterlogging conditions by improving stomatal function and enhancing Pn. In addition, the effects of stress types (S), different spraying methods (SM) and the interaction effects of stress types and different spraying methods (S×SM) on Pn, Gs, Ci and, Tr in maize were also significant (Fig. 7).

Fig. 7.

Fig. 7

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Effect of deionized water and exogenous melatonin on leaves of Pn (A-C), Gs (D-F), Ci (G-I), and Tr (J-L) in ZD958, C 7 − 2, and Z 58 maize under different waterlogging condition. The data is depicted as the mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 based on the Least Significant Difference (LSD) test. ns indicates no significant difference ANOVA analysis is used for S, SM and, S×SM. S: Stress types, SM: different spraying methods. * p < 0.05, ** p < 0.01 and *** p < 0.001

Correlation analysis

Principal component analysis of physiological and photosynthetic parameters under stress with exogenous melatonin

Principal Component Analysis (PCA) was performed on physiological and photosynthetic parameters of ZD 958, C 7 − 2, and Z 58 under different waterlogging stress conditions to compare the effects of different treatments and the relationships among the parameters (Fig. 8A-C). The loadings for PC1 were 65.2%, 66.9%, and 62.8% for ZD 958, C 7 − 2, and Z 58, respectively, while the loadings for PC2 were 20.2%, 13.7%, and 13.8%, respectively. For ZD 958, Pn, Gs, Ci, Tr, soluble sugar, soluble proline, chlorophyll, fructose, sucrose, and starch contributed more to PC1, whereas SOD, MDA, CAT, POD, and proline contributed more to PC2. For C 7 − 2, SOD, Pn, Gs, Ci, Tr, soluble sugar, soluble proline, chlorophyll, fructose, sucrose, and starch contributed more to PC1, while MDA, POD, and proline contributed more to PC2. For Z 58, SOD, CAT, POD, Pn, Gs, Ci, Tr, soluble sugar, soluble proline, chlorophyll, fructose, sucrose, and starch contributed more to PC1, whereas MDA and proline contributed more to PC2.

Fig. 8.

Fig. 8

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Principal Component Analysis (PCA) of physiological indices and photosynthetic parameters of ZD 958 (A), C 7 − 2 (B), and Z 58 (C) maize under waterlogging stress. A plot generated using Origin software displays the data, illustrating the segregation of samples based on waterlogging treatment through the two principal components: PC1 and PC2. Black dot A represents the application with exogenous melatonin under CK, red dot B represents the application with deionized water under CK, green dot C represents the application with exogenous melatonin under CF1 stress, dark blue dot D represents the application with deionized water under CF1 stress, purple dot represents the application with exogenous melatonin under CF2 stress, and yellow dot represents the application with deionized water under CF2 stress.; MDA: Malondialdehyde; POD: Peroxidase; CAT: Catalase; SOD: Superoxide dismutase; Pn: net photosynthetic; Gs: stomatal conductance; Tr: transpiration rate; Ci: intracellular CO2 concentration

Correlation analysis of physiological and photosynthetic parameters under waterlogging stress with exogenous melatonin

Different maize cultivars showed complex and diverse patterns of correlation between physiological indicators and photosynthetic parameters. Correlation analyses were performed between photosynthetic parameters and leaf physiological indicators under waterlogging stress (Fig. 9A-C). Among the three varieties, the content of MDA in the leaves of ZD 958 and C 7 − 2 showed negative correlation with some photosynthetic parameters. The antioxidant enzyme system showed some degree of negative correlation with photosynthetic parameters, whereas POD activity was positively correlated with photosynthetic parameters in Z 58 variety. As for carbohydrate indexes, soluble sugars, starch, fructose and sucrose in ZD 958 leaves showed positive correlation with photosynthetic parameters, but were relatively weak compared with C 7 − 2 and Z 58. The pattern of correlation between physiological indices and photosynthetic parameters changed significantly after melatonin spraying (Fig. 9D-F), and the positive correlation coefficients between antioxidant enzyme activities and photosynthetic parameters in C 7 − 2 leaves increased significantly. While in ZD 958 and Z 58 the opposite was true, in addition the content of MDA in the leaves showed significantly lower negative correlation with some of the photosynthetic parameters.

Fig. 9.

Fig. 9

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Correlation analysis of physiological parameters and photosynthetic characteristics of ZD 958, C 7 − 2, and Z 58 maize sprayed with deionized water (A-C) and exogenous melatonin (D-F) under waterlogging stress. MDA: Malondialdehyde; POD: Peroxidase; CAT: Catalase; SOD: Superoxide dismutase; Pn: net photosynthetic; Gs: stomatal conductance; Tr: transpiration rate; Ci: intracellular CO2 concentration. * p < 0.05, ** p < 0.01 and *** p < 0.001

Subsequently, we explored the relationship between Pn and physiological indices in maize leaves of different maize cultivars sprayed with 0 and 100 µM/L melatonin treatments under drowning and waterlogging stress by path analysis. For ZD 958 (Fig. 10 A and D), melatonin treatment significantly increased the positive effect of MDA content on SOD activity, while enhancing the negative effect of SOD activity and POD activity on Pn. In addition, melatonin treatment significantly increased the positive effect of starch on Pn and further enhanced the positive effect on chlorophyll content. For the C 7 − 2 variety (Fig. 11B and E), melatonin treatment significantly increased the positive effect of MDA content on POD activity and the negative and positive effects of CAT and chlorophyll on Pn, respectively. In addition, it increased the positive effect of Pn on starch. For Z 58 varieties (Fig. 11 C and F), melatonin treatment significantly increased the positive effect of POD activity on Pn and the negative effect of CAT activity and MDA content on Pn, in addition to the positive effect of starch on Pn.

Fig. 10.

Fig. 10

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Pathway analysis of net photosynthetic rate and physiological characteristics in ZD 958, C 7 − 2, and Z 58 maize treated with deionized water (A-C) and exogenous melatonin (D-F). MDA: Malondialdehyde; POD: Peroxidase; CAT: Catalase; SOD: Superoxide dismutase; Pn: net photosynthetic; Gs: stomatal conductance; Tr: transpiration rate; Ci: intracellular CO2 concentration. * p < 0.05, ** p < 0.01 and *** p < 0.001

Fig. 11.

Fig. 11

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Schematic diagram of the proposed mechanism by which melatonin regulates plant responses under waterlogging stress. Black arrows indicate stimulation, flat-head arrows indicate inhibition, red arrows indicate an increase, and blue arrows indicate a decrease. Waterlogging stress induces the accumulation of harmful substances, which leads to chlorosis and inhibits plant growth and development. Melatonin enhances antioxidant enzyme activity, which in turn suppresses the accumulation of harmful substances. This process subsequently promotes chlorophyll content, leading to an increase in photosynthetic activity. Ultimately, this resulted in increased carbohydrate content, leading to greater dry matter accumulation and yield. In summary, melatonin plays a crucial role in regulating the interplay between leaf physiology and photosynthesis, which is essential for enhancing plant tolerance to waterlogging

Discussion

Waterlogging stress is one of the major abiotic constraints on crop growth and productivity. Investigating the response and adaptive mechanisms of maize to waterlogging stress is of great significance for enhancing crop resilience and ensuring food security [44].

Firstly, due to the submergence of plant roots in water, the isolation of oxygen, which prompted the plant to be unable to absorb nutrients, resulting in the gradual yellowing and wilting of leaves from the bottom to the top (Fig. 1A and B), reducing the height of maize plants, and showing a trend of reduction with the deepening of the degree of waterlogging (Fig. 1E-G). In terms of maize growth indexes, melatonin effectively improved plant height and dry matter of maize under waterlogging stress [45–47]. This is consistent with the study that post-melatonin slowed down leaf wilting and increased plant height and dry matter in Prunus persica and chrysanthemum under drowning and waterlogging stress [48, 49]. Further delving into the effects of melatonin on the antioxidant defence system of maize (Fig. 2), we found that melatonin was able to increase antioxidant enzyme activities, scavenging the excessive ROS that anaerobic respiration would cause to accumulate in maize leaves, and alleviating the key role in reducing the damage of oxidative stress on cell membranes [50]. This is consistent with the fact that melatonin spraying all increased the activities of SOD, POD and CAT activities and reduced the ROS content in apple and kiwifruit, which restored plant growth and development to some extent [51]. It is noteworthy that the POD activity of Z 58 was reduced after spraying melatonin under different degrees of waterlogging stress, but the POD activity remained the highest compared with the other two varieties, whereas the action of melatonin was mainly used to increase the SOD and CAT activities (Fig. 2C). Correlation analysis showed that melatonin reduced the positive correlation of POD on Pn in Z 58 leaves, and further pathway analysis revealed that POD and CAT activities were negatively and positively affecting the Pn, respectively, suggesting that melatonin was able to regulate different antioxidant enzyme activities in different maize varieties in a way that ultimately improved maize resistance.

In addition, chlorophyll, as an important pigment in photosynthesis, plays a key role in absorbing and transferring light energy. In contrast, waterlogging stress leads to a decrease in chlorophyll content of maize leaves, with a decreasing trend with the intensification of waterlogging stress (Fig. 6), which is attributed to the fact that waterlogging leads to the accumulation of MDA in the leaves (Fig. 3). Studies have shown that melatonin favours the maintenance of water homeostasis in chloroplasts and repairs the chloroplast structure, stimulating the chlorophyll biosynthesis pathway [52, 53], which is in agreement with our findings.

In terms of photosynthetic properties, photosynthesis is an important process in providing energy and material to the plant itself [54]. Generally, the effect of waterlogging stress on photosynthesis is to limit stomatal opening, whereas the causes of the decrease in Pn under waterlogging stress include Gs by the simultaneous decrease in Ci and Gs, and non-stomatal limitation by the tendency of Ci to increase in the cytosol [55]. In our study it was found that waterlogging stress decreased Gs and Ci in maize leaves (Fig. 9D-I), which resulted would photo-oxidative stress, causing damage to photosynthetic organelles. When exogenous melatonin was sprayed, it increased Gs and Pn, suggesting that melatonin promotes stomatal opening under waterlogging stress, which is conducive to the maintenance of higher photosynthetic capacity in maize. Our results are consistent with Ma et al. [56] and Su et al. [57] It was also shown that melatonin inhibits the expression of chlorophyll degradation genes and delays plant yellowing, which is one of the key reasons for the ability to promote leaf photosynthesis [54]. These results are consistent with previous studies, Suggesting that melatonin is able to alleviate the inhibitory effect of waterlogging stress on photosynthesis by improving photosynthetic characteristics. In addition, by PCA and pathway analysis, we found that melatonin treatment significantly improved the positive correlation between chlorophyll content and photosynthetic rate in ZD 958 and C 7 − 2 after waterlogging stress, which further confirmed the role of melatonin in improving photosynthetic efficiency.

In addition, the regulatory role of melatonin on carbohydrate metabolism in maize under waterlogging stress is an important finding of this study (Fig. 5). The glucose ultimately synthesised by photosynthesis is the basic raw material for the synthesis of other carbons, so it is conceivable that the impediment of photosynthesis under waterlogging stress must affect carbohydrate metabolism [58]. It has also been shown that maize under waterlogging stress reduces sucrose converting enzyme and synthase activity, resulting in lower soluble sugar, sucrose and starch content [59]. Melatonin positively regulates the transcript levels of genes involved in carbohydrate accumulation and sucrose phloem transport and regulates plant growth. In our study, we found that spraying melatonin alleviated the reduction of carbohydrates, suggesting that melatonin promotes starch and sugar metabolism, influences osmoregulation and photosynthesis, and improves plant resistance to waterlogging. It is worth noting that starch was significantly reduced in C 7− 2 leaves under CF1 stress, and we speculate that the compensatory effect of exogenous melatonin may not be sufficient to fully offset the decrease in starch content due to waterlogging stress, or the C 7− 2 cultivar may be more reliant on other metabolic pathways to maintain starch synthesis under waterlogging stress, and exogenous melatonin supplementation failed to activate the metabolic pathways effectively.

The PCA analysis showed that different maize cultivars formed distinct groupings under different waterlogging stresses under melatonin treatment, indicating that melatonin treatment significantly altered the physiological status of maize (Fig. 8). In addition, ZD 958 contributed mainly to carbohydrates, while C 7− 2 and Z 58 contributed mainly to carbohydrates and light and parameters. Correlation analysis revealed that, under melatonin treatment, the positive association between carbohydrate content and photosynthetic parameters was strengthened in the leaves of both ZD958 and C7-2. However, the correlation between antioxidant enzyme activity and photosynthetic parameters showed opposing trends—either increasing or decreasing—yet remained negative. Interestingly, compared with C7-2, melatonin more effectively restored photosynthetic parameters in ZD958 leaves, whereas antioxidant enzyme activity displayed an inverse pattern. Thus, the physiological responses to exogenous melatonin differ between these maize genotypes(Fig. 9). Further pathway analyses provided us with a molecular mechanism framework for melatonin regulation of maize waterlogging tolerance on the one hand, melatonin directly activates the antioxidant enzyme system to alleviate cellular damage by oxidative stress. On the other hand, melatonin maintains the stability of the intracellular environment by regulating the accumulation of osmoregulatory substances and carbohydrates, and at the same time, melatonin also improves the photosynthetic performance to provide more energy and material support (Fig. 10)

Conclusions

In Summary, this study systematically assessed the effects of melatonin on the physiological and photosynthetic characteristics of different maize cultivars under waterlogging stress. It was revealed that it significantly enhanced the waterlogging tolerance of maize through a multidimensional adaptive mechanism that synergistically regulated the activity of antioxidant enzyme system, osmoregulatory substance balance, photosynthesis and carbohydrate metabolic pathways. This study combined various analytical methods, such as principal component analysis, correlation analysis and path analysis, to reveal the effects of melatonin on the physiological and photosynthetic characteristics of maize from different perspectives. Notably, this study deeply explored the different regulatory effects of melatonin on maize varieties with different genetic backgrounds, showing that melatonin had the best effect on alleviating the damage of ZD 958 under waterlogging stress, followed by Z 58, and lastly C 7 − 2, which deepened the understanding of the molecular mechanism of melatonin in alleviating adversity stress, and provided an opportunity to investigate the mechanism of melatonin action and to optimise its application in agricultural production. In the future, we can further focus on the analysis of melatonin signal transduction pathway and the development of variety-specific response markers, in order to promote the translation of the results to the smart agriculture scenario.

Acknowledgements

Not applicable.

Authors’ contributions

Penghui Li and Zhandong Liu conceived and designed the experiments. Ling Wang and Penghui Li carried out the experiments, analyzed the data, and prepared and revised the manuscript. Ling Wang, Ruiying Li and Hejing Tang participated in some experiments in the study. Penghui Li, Ying Li, Zhao Zhang, and Zhandong Liu edited and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Central Public-Interest Scientific Institution Basal Research Fund (FIRI2023-12, FIRI2024-12), the Xinjiang Uygur Autonomous Region Tianchi Talent Introduction Plan (2024); the China Agriculture Research System (CARS-02-18); the National Public-interested Scientific Institution Based Research Fund of China [IFI2024-21]; the Agricultural Science and Technology Innovation Program [ASTIP]; and the Key R&D Program of the Xinjiang Uygur Autonomous Region [2023B02040-2]; Nature foundation of Henan Province (252300421693).

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

This article does not involve any studies with human participants or animals. The collection of plant materials complies with all applicable institutional, national, and international guidelines and regulations.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Penghui Li, Email: lipenghui@caas.cn.

Zhandong Liu, Email: liuzhandong@caas.cn.

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