Exogenous Spermidine Enhances Drought Resistance of Mango Seedlings by Regulating Physiological and Biochemical Metabolism

Abstract

Drought stress is a major environmental factor that adversely affects plant growth and development. Spermidine (SPD), a polyamine, plays a critical role in plant defense mechanisms against drought stress. PEG was used to simulate osmotic stress, which mimics drought conditions under controlled environments. This study investigated the effects of exogenous spermidine (SPD) on the physiological and biochemical responses of mango plants under drought stress and explored its potential mitigation mechanisms. Two-year-old ‘Renong 1’ mango seedlings were subjected to drought stress induced by polyethylene glycol (PEG 6000) at concentrations of 5%, 15%, and 25%, simulating mild, moderate, and severe drought conditions, respectively. Plants were subsequently treated with 1 mmol/L spermidine. After PEG 6000 treatment and spermidine application for 3 days, the leaf morphology, relative chlorophyll content, malondialdehyde (MDA) levels, antioxidant enzyme activities (superoxide dismutase [SOD], peroxidase [POD], catalase [CAT]), and osmotic regulators (proline, soluble sugars, and soluble proteins) were analyzed. The results demonstrated that drought stress caused leaf chlorosis, desiccation, reduced relative chlorophyll content, elevated MDA levels (indicating lipid peroxidation), enhanced antioxidant enzyme activities, increased proline and soluble sugar accumulation for osmotic regulation, and decreased soluble protein content. Exogenous spermidine treatment significantly alleviated drought-induced damage by reducing leaf chlorosis, delaying relative chlorophyll degradation (by 20.0–25.7% under moderate drought and 14.1–19.1% under severe drought), and decreasing MDA levels (by 4.8–9.5% under moderate drought and 0.8–23.7% under severe drought). Furthermore, spermidine enhanced antioxidant enzyme activities (e.g., SOD activity increased by 24.9–37.4% and POD by 74.0–104.0% under moderate drought), regulated osmotic substance accumulation (e.g., proline decreased by 21%, 26%, and 24% under mild, moderate, and severe drought, respectively), and mitigated the reduction in soluble protein content (by 6.6% under moderate drought and 10.3% under severe drought). In conclusion, exogenous spermidine mitigates drought-induced damage in mango by preserving photosynthetic capacity, enhancing the antioxidant defense system, and modulating osmotic balance. These results showed that SPD could significantly improve plant vigor or survival rate under stress. It provides a theoretical basis for water-saving cultivation of mango, improving the stress resistance of mango varieties and the application of spermidine in tropical fruit production.

1. Introduction

Mango (Mangifera indica L.) is a significant tropical economic crop, with global production reaching approximately 50 million tons in 2020, ranking third after bananas and coconuts [1,2]. Native to South Asia, which accounts for half of the world’s mango production, the species exhibits the highest varietal diversity globally [3]. This diversity is attributed to multiple domestication events and hybridization processes [4]. Mangoes typically grow in regions with annual precipitation ranging from 1500 to 2600 mm. While they thrive in tropical climates, they also adapt well to semi-arid subtropical Mediterranean regions, where average temperatures range between 27 °C and 36 °C [5,6,7,8,9]. In these areas, advanced agricultural practices, particularly irrigation management, are employed to enhance the quality and yield of commercial mango crops [10]. Recognized as one of the top five tropical fruits worldwide, mangoes are often referred to as the “King of Tropical Fruits” [11]. They are widely cultivated in tropical and southern subtropical regions, such as Hainan, Yunnan and Panzhihua of Sichuan province in China, where they play a crucial role in supporting the local agricultural economy [12].

The projected increase in drought frequency due to climate change [13] is anticipated to adversely affect mango production, necessitating greater reliance on irrigation. This presents a significant challenge for the mango industry in China. Although tropical regions of China experience relatively high annual precipitation, seasonal droughts remain prevalent. The dry season is characterized by evaporation rates that exceed precipitation levels in Yunnan and Panzhihua, Sichuan, especially. Such conditions often lead to substantial fluctuations in soil moisture content, which negatively impact the photosynthetic efficiency of mango leaves, as well as fruit development and quality [12,14]. Research has shown that drought stress significantly reduces the relative water content, relative chlorophyll levels, and photosynthetic capacity of mango leaves [15,16]. Furthermore, it inhibited both longitudinal and transverse fruit growth, thereby limiting the accumulation of essential nutrients such as soluble solids and vitamin C [12]. These effects collectively result in lower yields and reduced market value.

Polyamines (PAs) are small nitrogen-containing compounds essential for plant stress responses [17]. Among these, spermidine (SPD) serves as a critical intermediate in polyamine metabolism and plays a key role in mitigating abiotic stress-induced damage in plants. This protective effect is mediated through the regulation of antioxidant enzyme activities (e.g., superoxide dismutase [SOD] and peroxidase [POD]), maintenance of cell membrane integrity, and promotion of osmotic regulator accumulation (e.g., proline and soluble sugars) [18]. Previous studies have shown that exogenous application of spermidine significantly improved the photosynthetic efficiency and antioxidant capacity of maize (Zea mays) seedlings under drought stress. Additionally, it alleviated drought-induced physiological and biochemical disruptions in blueberry (Vaccinium sp.) leaves by modulating osmotic balance and reactive oxygen species (ROS) metabolism [19].

In recent years, to meet the growing demand, the mango planting area needs to be expanded. In the southwest region of China, mangoes are prone to water stress during spring and winter, and are susceptible to environmental changes during the seedling stage. Based on the positive effect of PAs on plants mentioned above, the induction of water stress tolerance by the use of exogenous polyamines continues to be an objective of great interest. In addition, the production of ROS in plants under normal growth conditions is low. However, in response to drought, ROS are drastically increased in plants disturbing the normal balance of O₂⁻, OH and H₂O₂ in the intracellular environment by inhibition of carbon dioxide (CO₂) assimilation, coupled with the changes in photosystem activities and photosynthetic transport capacity under drought stress results in accelerated production of ROS [17].

Therefore, this study aimed to determine whether exogenous SPD was involved in drought resistance of Mangifera indica L. The effects of Exogenous Spermidine on membrane lipid peroxidation, antioxidant enzyme activities, and photosynthetic parameters of mango leaves were studied using mango ‘Renong No.1’ as test material and PEG 6000-induced osmotic stress to simulate drought conditions in a mango orchard, and the physiological mechanism of exogenous spermidine alleviating drought stress was discussed. The results provide a theoretical basis for the water-saving cultivation of mango, disaster prevention and mitigation strategies, the development of stress-resistant varieties and the application of spermidine in the cultivation of tropical fruit trees.

2. Materials and Methods

2.1. Plant Material and Treatments

In this study, two-year-old mango plants of the ‘Renong No.1’ variety (The main variety promoted in China, with high yield, excellent fruit quality and disease resistance, but susceptible to drought stress) were selected as the experimental materials. These plants were uniform in size and height and exhibited intact structures, devoid of pests, diseases, mechanical injuries, and exposure to pesticides. Three months before the experiment commenced, the plants were relocated to a greenhouse at Guangdong Ocean University, to ensure controlled environmental conditions. The plants were regularly irrigated with Hoagland’s solution. The greenhouse maintained a temperature range of 25–30 °C, humidity levels of approximately 70–80%, and a consistent photoperiod of 12 h.

After 60-day period, the seedlings displaying consistent morphology and growth were identified. Soil was removed from the nutrient bags, the roots were thoroughly cleaned, and the seedlings were transferred to perforated foam boards. Subsequently, foam boards were placed in plastic baskets containing Hoagland nutrient solution, ensuring daily aeration of the hydroponic solution. Following a one-week recovery period in 1/2 Hoagland nutrient solution, the seedlings were randomly assigned to polyethylene glycol (PEG6000) solutions with different osmotic potentials prepared using nutrient solution, and treated continuously until the end of the experiment to induce drought stress of different intensities and durations. Healthy and uniform mango seedlings were divided into the following eight treatment groups (

Table 1. Treatment of each treatment group.

Group	Treatments
5%	Mild drought stress (50 g/L PEG)
15%	Moderate drought stress (150 g/L PEG)
25%	Severe drought stress (250 g/L PEG)
CK	CK
5% + SPD	Mild drought stress (50 g/L PEG) + Spermidine (1 mmol/L)
15% + SPD	Moderate drought stress (150 g/L PEG) + Spermidine (1 mmol/L)
25% + SPD	Severe drought stress (250 g/L PEG) + Spermidine (1 mmol/L)
CK + SPD	CK + Spermidine (1 mmol/L)

), with each treatment repeated three times, leaves from three plants were pooled per replicate.

2.2. Osmotic Potential

According to standard methods in the field of plant physiology, the osmotic potential (PSI s) of PEG6000 (polyethylene glycol 6000) solution is usually calculated using the classical formula proposed by Michel and Kaufmann [20]. This formula is derived through experimental data fitting and is widely recognized as a benchmark method in drought stress simulation research.

The calculation formula is as follows (Unit: MPa):

Ψs = −[(1.18 × 10⁻²) × C − (1.18 × 10⁻⁴) × C² + (2.67 × 10⁻⁷) × C³] × T

Parameter description:

C: The mass concentration of PEG 6000 solution (unit: g/kg H₂O) needs to be converted to grams of solute per kilogram of water

T: Absolute temperature of the experimental system (unit: K, Kelvin). Calculation method: T = 273 + °C (experimental temperature)

Key points and precautions for use:

• Applicable range of concentration: The formula is applicable to a concentration range of 50–250 g/kg H₂O (i.e., 5–25% w/w). Accuracy decreases when it exceeds this range.
• The importance of temperature correction: The formula contains a temperature correction factor (T), so it must be accurately calculated based on the actual cultivation temperature. For example, at 25 °C, T = 273 + 25 = 298 K.
• Unit conversion example (taking 20% concentration and 25 °C as an example):

C = 200 g/kg H₂O, T = 298 K

Ψs = −[(1.18 × 10⁻² × 200) − (1.18 × 10⁻⁴ × 200²) + (2.67 × 10⁻⁷ × 200³)] × 298
≈ −[2.36 − 0.472 + 0.02136] × 298                  
≈ −1.909 × 298                            

2.3. Application of Treatments

During the experimental intervention period, starting from day 1, 1 mmol/L spermidine was applied using a spray bottle at 17:30 every other day until the leaf surface was fully moistened, avoiding drips (Appr 20 mL per plant). The untreated control group was sprayed with the same dose of distilled water to achieve the same level of wetness. (Table 1) [21]. Samples were gathered at 9:00 on days 0, 24 h, 48 h, and 72 h of the trial. Three replicates were collected for each treatment at each time point. Fully expanded and mature leaves from each seedling were chosen, gently washed with tap water to eliminate surface impurities, rinsed 2–3 times with distilled water, and dried by blotting excess moisture with absorbent paper. Subsequently, all samples were promptly frozen in liquid nitrogen and preserved in a −80 °C ultra-low temperature freezer for the assessment of physiological parameters.

2.4. Leaf Phenotype Measurement

At 9 a.m. on Day 0, 24 h, 48 h, and 72 h, leaves exhibiting consistent growth conditions and uniform size and morphology were selected from each group for photography.

2.5. Determination of the Relative Chlorophyll Content in Leaves

A Minolta SPAD-502 chlorophyll meter (Konica Minolta, Tokyo, Japan) was utilized in this study.

2.6. Determination of Antioxidant Enzyme Activities

Antioxidant enzyme assays were performed essentially according to Dhindsa et al. [22]. The activity of superoxide (SOD) was assayed by inhibiting the photochemical reduction in nitroblue tetrazolium (NBT) at 560 nm. One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction. POD activity is often determined by the guaiacol method. Peroxidase activity was determined by measuring the absorbance change at a wavelength of 470 nm. In the calculation of enzyme activity, the increase in absorbance A₄₇₀ value per minute by 0.1 is one enzyme activity unit (U) [23].

Catalase (CAT) activity was assessed using ultraviolet absorption. Three 10 mL test tubes were used for the assay. To each tube, 0.1 mL of the respective components was added sequentially. Following preheating at 25 °C, the crude enzyme solution, 1.5 mL of pH 7.8 phosphate buffer, and 1.0 mL of distilled water were successively introduced into each tube. Subsequently, 0.3 mL of 0.1 mol/L H₂O₂ was added to the timing commenced immediately after each addition, and the absorbance at 240 nm was promptly measured after transferring the contents into a quartz cuvette. Absorbance readings were recorded every minute for 4 min. Upon completion of the measurements for all three tubes, the enzyme activity was calculated. Each experimental group was analyzed in triplicate [24].

Calculation outcome: CAT (U/(g·min)) = ${\displaystyle \frac{\mathsf{\Delta }{\mathrm{A}}_{240}\times {\mathrm{V}}_{\mathrm{t}}}{0.1\times {\mathrm{V}}_1\times \mathrm{t}\times \mathrm{F}\mathrm{W}}}$

In the equation above, $\mathsf{\Delta }{\mathrm{A}}_{240}$ represents the change in absorbance per minute.

V_t—Total volume of crude enzyme extract (mL);

V₁—Volume of crude enzyme solution for determination (mL);

FW—Fresh weight of sample (g);

0.1—Enzyme activity unit (U) for every 0.1 decrease in A240;

t—Time from adding hydrogen peroxide to the last reading (min).

2.7. Determination of Malondialdehyde (MDA) Content

Lipid peroxidation was measured by estimating MDA according to Heath and Packer [25] using thiobarbituric acid (TBA) as the reactive material. Fresh leaf samples (0.5 g) were homogenized in 3 mL 5% (w/v) trichloroacetic acid (TCA) and centrifuged at 12,000× g for 20 min. One milliliter supernatant was mixed with 4 mL 20% TCA containing 0.5% TBA and heated at 95 °C for 30 min followed by immediate cooling on ice. The cooled solution was centrifuged again at 11,500× g for 12 min. MDA content was calculated from the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155 m/(M·cm).

2.8. Determination of Soluble Sugar Content

0.2 g of fresh leaves was taken and combined with 10 mL of distilled water. The mixture was subjected to a 30 min boiling water bath, followed by dilution of the filtrate to a 50 mL volumetric flask. 1 mL of the filtrate was taken, 1 mL of distilled water, 0.5 mL of anthrone reagent, and 5 mL of concentrated sulfuric acid were added to it. The resulting solution was placed in a boiling water bath for 1 min, allowed to cool, and then subjected to colorimetric analysis at 630 nm. The soluble sugar content was determined using the methodology outlined by Hu [26].

2.9. Determination of Proline Content

The determination of proline content referred to the sulfosalicylic acid extraction method [27]. 0.2 g of fresh leaves was weighed and 5 ml of sulfosalicylic acid was added. It was soaked in a boiling water bath for 10 min, cooled to room temperature, filtered, 2 ml of the filtrate was taken, 2 ml of glacial acetic acid was added, and 3 ml of acidic indene copper was added to the boiling water bath for 60 min. After cooling, 5 ml of toluene was added and shaken thoroughly. The upper layer of toluene solution was taken and the color was compared at 520 mm. The proline content (μg/g FW) was calculated using the formula: (C × V/a)/W, where C represents the amount of μg obtained from the standard curve, V is the total volume of the extraction solution in ml, W is the sample weight in grams, and a is the volume aspirated during determination in ml.

2.10. Determination of Soluble Protein Content

The Coomassie Brilliant Blue G-250 method [28] involves the following steps: 0.2 g of fresh leaves are taken and combined with 1 ml of phosphoric acid buffer solution. The mixture was then ground in an ice bath, followed by the addition of 4 mL of phosphoric acid buffer solution. Subsequently, the sample was centrifuged at a low temperature (−4 °C) for 20 min. Next, 0.5 mL of the supernatant was collected and mixed with 5 mL of Coomassie Brilliant Blue staining solution. After allowing the mixture to stand for 2 min, the optical density was immediately measured at 595 nm. A standard curve was prepared using bovine serum protein. The protein concentration of the sample was determined based on a standard curve, and the soluble protein content was calculated as mg/g fresh weight.

2.11. Statistical Analysis

The data were subjected to one-way analysis of variance (ANOVA) and the mean differences were compared by LSD test using the statistical software SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Different letters indicate significant differences between treatments at a significance level of p value less than 0.05. Data represented in the table and figures are means ± standard deviations (SD) of three replicates for each treatment.

3. Results

3.1. Effect of Exogenous Spermidine on Leaf Phenotypes of Mango Under Different Drought Stresses

Over a 72 h period, distinct changes in leaf color were observed across different groups, as illustrated in Figure 1A–E. Mild drought stress did not result in significant alterations in leaf color. During the initial 24 h of the experiment, the control group exhibited minimal changes in both leaf color and morphology. From the 48th hour onwards, leaves in the moderately drought-stressed group began to transition from green to yellow at the edges. Compared to the untreated group, noticeable variations in leaf color were observed only 72 h following spermidine application. Under severe drought conditions, yellowing of the leaf edges was apparent by the 48th hour, with subsequent drying and curling by the 72nd hour. Importantly, the change in leaf color following spermidine application was less pronounced than in the untreated group. These trends were consistently observed in all mango seedlings after 72 h, the significant enhancement of leaf greening under moderate and severe drought stress facilitated by spermidine application.

3.2. Impact of Exogenous Spermidine on Relative Chlorophyll Content in Mango Leaves Across Varying Drought Conditions

Figure 2 depicted a gradual decline in the relative chlorophyll content of mango leaves over time, after 72 h of drought treatment, the SPAD in the control group decreased from 57.5 to 51.5. Moderate drought and severe drought decreased by 13.6% and 29.0%, respectively, compared to the control group. However, compared with drought without spermidine, the SPAD of the moderate drought and severe drought treatment groups increased by 12.1% and 27.0%, respectively.

3.3. Impact of Exogenous Spermidine on Malondialdehyde Levels in Mango Leaves Under Varying Drought Conditions

Malondialdehyde (MDA) is a byproduct that accumulates during the lipid peroxidation of cell membranes, with its levels increasing over time and in response to heightened stress conditions. As illustrated in Figure 3, after 72 h of drought treatment, the MDA content in the control group increased from 18.9 μmol/g FW to 24.3 μmol/g FW. Mild drought, moderate drought, and severe drought increased by 0.7%, 25.3% and 42.9%, respectively, compared to the control group (Figure 3). The application of SPD had no significant effect on the MDA content of the control plants. However, compared with drought without spermidine, the MDA content of the mild drought, moderate drought, and severe drought treatment groups decreased by 9.4%, 6.9%, and 19.0%, respectively.

3.4. Impact of Exogenous Spermidine on Superoxide Dismutase (SOD) Activity in Mango Leaves Under Varying Drought Conditions

As a key enzyme for clearing ROS, the activity of SOD in mango leaves gradually increases with the increase in stress time during drought. After 72 h of drought treatment, the SOD activity in the control group increased from 78 μmol/g FW to 84 μmol/g FW. Mild drought, moderate drought, and severe drought increased by 13.5%, 25.2% and 34.0%, respectively, compared to the control group (Figure 4a). The application of SPD had no significant effect on the SOD activity of the control plants. However, compared with drought without spermidine, the SOD activity of the mild drought, moderate drought, and severe drought treatment groups increased by 1.8%, 21.1%, and 21.8%, respectively. POD can catalyze the reduction of H₂O₂ by using various electron donors such as phenolic compounds, lignin precursors, auxin, and secondary metabolites [29,30]. The POD activity of mango leaves gradually induces during the drought period. After 72 h of drought treatment, compared with the control group, mild drought, moderate drought, and severe drought increased by 2.0%, 22.0% and 41.7%, respectively (Figure 4b). The application of SPD had no significant effect on the SOD activity of the control plants. However, compared with drought without spermidine, the SOD activity of the mild drought, moderate drought, and severe drought treatment groups increased by 20.3%, 25.3%, and 15.2%, respectively.

3.5. Impact of Exogenous Spermidine on Catalase Activity in Mango Leaves Under Varying Drought Conditions

Catalase (CAT) activity exhibited a progressive increase under all stress conditions, with a marked rise followed by a subsequent decline under severe drought stress. After 72 h of drought treatment, the CAT activity in the control group increased from 18.9 u/(g·min) to 25.7 u/(g·min) Mild drought, moderate drought, and severe drought increased by 14.6%, 89.3% and 112.9%, respectively, compared to the control group (Figure 5). However, compared with drought without spermidine, the CAT activity of the mild drought, moderate drought, and severe drought treatment groups increased by 32.2%, 7.9%, and 7.9%, respectively.

3.6. Effect of Exogenous Spermidine on Proline Content of Mango Leaves Under Different Drought Stresses

Proline, an amino acid, accumulates in response to osmotic stress and serves as a critical osmotic regulator. Figure 6 illustrated the proline content in mango plants subjected to polyethylene glycol (PEG)-induced osmotic stress. The results reveal a consistent, time-dependent increase in proline levels across all PEG concentrations. Specifically, compared to the initial time, after 72 h of mild, moderate, and severe drought treatment, the proline content increased by 1.75 times, 2.28 times, and 2.82 times, respectively. Compared with drought without spermidine, the proline content of the mild drought, moderate drought, and severe drought treatment groups decreased by 62.5%, 56.7%, and 58.5%, respectively. Additionally, a rapid rise in proline content was observed within the first 24 h, with the magnitude of accumulation correlating directly with both the intensity and duration of stress.

3.7. Impact of Exogenous Spermidine on Soluble Sugar Levels in Mango Leaves Under Varying Drought Conditions

Soluble sugars function as both metabolic intermediates and critical regulators of osmotic stress in plant tissues. As shown in Figure 7, soluble sugar levels progressively increased under escalating drought stress conditions. However, the application of spermidine did not significantly affect the soluble sugar levels of plants under mild drought stress. After 72 h of stress, compared with the control group, the soluble sugar content increased by 22.8% and 44.7% under moderate and severe drought conditions, respectively. Compared to drought without spermidine, the soluble sugar content of the mild drought, moderate drought, and severe drought treatment groups decreased by 7.5%, 5.47%, and 10.5%, respectively.

3.8. Impact of Exogenous Spermidine on Soluble Protein Levels in Mango Leaves Under Varying Drought Conditions

Soluble cellular proteins play a critical role in plant metabolism, functioning as key regulators and facilitators of metabolic processes. Fluctuations in protein levels reflect changes in the synthesis and metabolic capacity of plants. In mango seedlings subjected to varying concentrations of polyethylene glycol (PEG) treatment (Figure 8), a decline in soluble protein content was observed following the cessation of stress, with the reduction becoming more pronounced at higher stress levels. After 72 h of drought treatment, the soluble protein content in the control group decreased from 5.6 mg/g to 5.0 mg/g. Moderate drought and severe drought decreased by 12.3% and 27.9%, respectively, compared to the control group. However, compared with drought without spermidine, the soluble protein content of the moderate drought and severe drought treatment groups increased by 20.8%, 6.9%, and 29.0%, respectively.

4. Discussion

Drought stress poses a major threat to plant growth and productivity by disrupting physiological and biochemical processes, including water relations, photosynthesis, and redox balance. In this study, exogenous spermidine (SPD) application effectively alleviated drought-induced damage in Mangifera indica L., consistent with findings in Ilex verticillata [31] and Achillea millefolium [32]. The observed improvements in photosynthetic pigments, and antioxidant capacity suggest that SPD enhances drought tolerance in Mangifera indica L. through multiple interconnected mechanisms.

SPAD decreased under drought in Mangifera indica L., likely due to chloroplast membrane damage and reduced pigment synthesis. SPD treatment, preserved pigment levels, consistent with results in Achillea millefolium where 1.5 μM SPD maximized chlorophyll retention [32]. Similarly, in Ilex verticillata, SPD increased chlorophyll content and net photosynthetic rate (Pn) under drought by protecting photosystem II (PSII) function [33]. Chlorophyll fluorescence parameters (Fv/Fm, Y(II), ETR) in Mangifera indica L. further supported this, as SPD alleviated the drought-induced decline in PSII efficiency, mirroring findings in Ilex verticillata [31] and other species [34,35,36].

Drought stress triggered excessive accumulation of reactive oxygen species (ROS) in Mangifera indica L., leading to lipid peroxidation and membrane injury, as indicated by increased malondialdehyde (MDA) levels. SPD treatment significantly reduced MDA content, consistent with observations in Ilex verticillata [31] and Achillea millefolium [32], where SPD suppressed MDA accumulation by enhancing antioxidant enzyme activity. In Mangifera indica L., SPD upregulated the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), particularly under severe drought. This aligns with Ilex verticillata, where SPD increased SOD and POD activities under moderate stress [31], and in Achillea millefolium, where 3 μM SPD maximized POD and CAT activities [32].

Osmotic adjustment is a key adaptive response to drought in Mangifera indica L., involving the accumulation of compatible solutes such as proline and soluble sugars. Drought increased proline and soluble sugar contents, and SPD further enhanced this accumulation. This is consistent with Achillea millefolium, where SPD boosted proline and soluble sugar levels under stress [32], and with Ilex verticillata, where SPD regulated soluble sugar content to maintain osmotic balance [31].

Notably, drought tolerance and SPD efficacy varied among species. For example, the native Ilex verticillata exhibited higher drought resistance than its varieties [31].

Similarly, in the study of Eremochloa ophiuroides, Lu Zedong et al. [37] found that SPD alleviates drought by regulating antioxidant enzymes and osmotic substances, but its optimal concentration (0.5 mM) was different from that of Achyranthes cuspidata (1.5–3 μM), highlighting species-specific SPD dose–response. In the southern Chinese yew (a variety of southern Chinese yew), Wen Jiakang et al. [38] pointed out that SPD rehydration can accelerate photosynthetic recovery indicating its drought recovery. These differences may be related to plant morphology, habitat adaptation, and endogenous polyamine metabolism.

5. Future Perspectives

While this study demonstrates SPD’s potential in enhancing drought tolerance in Mangifera indica L., several questions remain. The molecular mechanisms underlying SPD-mediated regulation of stress-responsive genes warrant investigation. Additionally, long-term field trials are needed to validate SPD efficacy under natural drought conditions, Especially for the discovery by Zhang Meiwei and others that SPD can improve grain plumpness and yield related physiological indicators [39], it indicates agricultural applicability.

Notably, the use of PEG 6000 to simulate drought in this study has limitations. PEG-induced osmotic stress differs from natural drought in soil, as it may alter root-soil interactions, nutrient availability, and oxygen diffusion, potentially leading to secondary stress (e.g., hypoxia). Future studies could combine PEG with soil-based drought simulations or field trials to better reflect real-world conditions. Exploring SPD interactions with other phytohormones (e.g., ABA, GA) and microbial communities could also provide insights into cross-regulatory networks governing drought responses in Mangifera indica L.

In conclusion, exogenous SPD enhances drought tolerance in Mangifera indica L. by improving photosynthetic efficiency, antioxidant defense, and osmotic adjustment. These results contribute to our understanding of polyamine-mediated stress adaptation and provide practical insights for sustainable agriculture in water-limited environments.

6. Conclusions

In this study, a variety of plant physiology and biochemistry techniques were employed to investigate the effects of spermidine on mango seedlings under drought stress. The results indicated that spermidine application maintained higher relative water content in mango leaves during drought, thereby preventing leaf chlorosis. Moreover, exogenous spermidine partially mitigated the reduction in relative chlorophyll content under drought conditions, resulting in a better preservation of photosynthetic rates compared to untreated leaves under similar stress.

During drought stress, mango leaf cell membranes exhibited increased lipid peroxidation, leading to elevated malondialdehyde levels. Concurrently, the activity of protective enzymes in the leaves significantly increased (p < 0.05), maintaining an effective defense system against reactive oxygen species. This system, comprising superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), efficiently scavenged reactive oxygen radicals, reduced membrane lipid peroxidation, and enhanced drought resistance. Additionally, exogenous spermidine boosted intracellular antioxidant enzyme activity, facilitating the removal of excess peroxides within the cell.

In response to water deficiency, plants accumulate osmoregulatory substances to lower intracellular water potential, aiding in water uptake from the environment. Under drought stress, levels of proline and soluble sugars increased proportionally with the intensity and duration of the stress, with proline accumulating more rapidly than soluble sugars. Conversely, soluble protein levels decreased. As soil water content decreased, proline and soluble sugar content gradually increased, while soluble protein levels continued to decline. Furthermore, exogenous polyamines alleviated drought stress in mango plants by reducing water requirements and moderately influencing soluble sugar content.

The application of spermidine can significantly enhance the growth and development of mango plants under water stress conditions. While existing research primarily focuses on physiological experiments showing the impact of exogenous spermidine on mango seedlings’ responses to drought stress by altering specific substance levels, the underlying molecular mechanisms remain unexplored. Molecular investigations into the effects of exogenous spermidine will provide a more detailed understanding of its specific functions, offering substantial theoretical and practical implications for advancing the understanding and application of spermidine in plant biology.