Melatonin-Induced Enhancement of Drought Tolerance in Okra: A Detailed Analysis of Physiological, Biochemical, and Metabolic Adaptations

doi:10.21203/rs.3.rs-4967383/v1

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Melatonin-Induced Enhancement of Drought Tolerance in Okra: A Detailed Analysis of Physiological, Biochemical, and Metabolic Adaptations

https://doi.org/10.21203/rs.3.rs-4967383/v1

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As climate change intensifies, drought stress presents a critical challenge for horticultural crops like okra (Abelmoschus esculentus). The effectiveness of melatonin in reducing drought stress is investigated in this study. The treatments include: Absolute control (fully irrigated), control (Drought D), Drought and seed treatment with 100 µM melatonin (MT- ST), Drought and foliar spray of 100 µM melatonin (MT- FS), and drought stress with combined effect of seed treatment and foliar spray of 100 µM melatonin (MT - ST + FS). Physiological parameters such as photosynthetic rate, stomatal conductance, transpiration rate, Fv/Fm ratio, and chlorophyll content values were evaluated, alongside biochemical parameters including malondialdehyde, proline content, membrane stability index and antioxidant enzyme activities such as catalase and peroxidase were quantified. Melatonin supplemented as seed treatment and foliar spray enhanced both physiological and biochemical parameters including antioxidant activity compared to drought control (D). Metabolite profiling identified bioactive compounds (mainly carbohydrates and amino acids) contributing to drought tolerance in okra. The results highlights that application of 100 µM melatonin via seed treatment and foliar spray enhances drought tolerance in okra, suggesting its potential to enhance crop resilience under water-deficit conditions.

melatonin

drought

okra

antioxidants

metabolites

Climate change and the rising frequency of extreme weather events present substantial risks to global crop yields and their stability (Reyes et al. 2021). It is predicted that sustained global warming will worsen the variability of the global water cycle, the global monsoon's precipitation, extreme wet and dry weather, and climate events and seasons (Lee et al. 2023). As climate change advances, understanding its effects on various vegetable crops is vital. Certain crops may exhibit greater resilience to specific climatic conditions, necessitating strategic adaptation measures. Additionally, alterations in precipitation and temperature can influence pest and disease life cycles, impacting crop yield and quality. Drought is a complex stressor that primarily arises from reduced rainfall and prolonged dry spells (Gogoi and Tripathi 2019).

Each vegetable crop responds uniquely to drought, depending on its severity and the plant's growth stages. Okra (Abelmoschus esculentus [L.] Moench), a significant crop grown for its fruits, vegetables, and seed oil, flourishes in tropical, subtropical, arid, and semi-arid climates. Vegetable crops are pivotal for human nutrition, providing essential nutrients in daily diets. Consumption of okra has been linked to health advantages, such as reducing sugar levels in blood, lowering lipid levels, and mitigating constipation (Gemede et al. 2015). In India, okra cultivation spans an area of 554 lakh hectares, yielding an annual production of 7252 million metric tons for the 2023–2024 period (Indiastat 2023). Nonetheless, okra cultivation is notably vulnerable to the effects of climate change, directly impacting its yield. Abiotic stress, particularly drought, causes okra yield losses ranging from 30–100%, notably during flowering and pod-filling stages (Mbagwu and Adesipe 1987). Yield reductions depend on cultivar variation and the phenological stage of drought occurrence. Drought stress caused reductions in stomatal conductance (gs), transpiration rate (T), net photosynthetic rate (A), maximum photochemical efficiency (Fv/Fm), actual photochemical efficiency of PSII (φ PSII), photochemical quenching (qP), and electron transport rate (ETR) in various okra varieties (Mkhabela et al. 2022). Drought stress resulted in reduced leaf water status, chlorophyll content, carotenoid levels, and total protein (µg/g) at a drought level of 25% field capacity (FC). Conversely, levels of malondialdehyde (MDA) and proline content along with the activities of catalase (CAT) and peroxidase (POD) increased (Ayub et al. 2021). Enhancing the tolerance of vegetables to drought necessitates a multifaceted approach, integrating novel cultivation techniques and external regulatory technologies to enhance the growth and development of vegetables globally (Dhankher and Foyer 2018; Razi and Muneer 2021).

Melatonin (N-acetyl-5-methoxytryptamine) was identified as a notable bioactive compound in vascular plants in 1995 (Dubbels et al. 1995). Initially noted for its potent antioxidant properties, melatonin has since been recognized for its diverse roles in plant development (Sheshadri et al. 2018). It contributes to various physiological processes, including seed germination (Zhang et al. 2017), root elongation (Arnao and Hernández-Ruiz 2019), photosynthesis enhancement (Li et al. 2017), and leaf senescence regulation (Wang et al. 2022). As a plant hormone, melatonin plays a crucial role in growth regulation and developmental pathways (Arnao and Hernández-Ruiz 2019). It is present in multiple plant tissues, including seeds, roots, leaves, and fruits (Zhang et al. 2017). Research has extensively examined melatonin's potential to improve plant growth and regulatory processes (Sun et al. 2020).

Melatonin is an effective antioxidant compound known for its reactive oxygen species (ROS) detoxification properties. It enhances plant resilience to various stresses by neutralizing free radical species through single-electron and hydrogen transfer mechanisms. It effectively scavenges ROS such as superoxide anions (O₂^•−) and hydroxyl radicals (OḤ), reactive nitrogen species (RNS) including nitric oxide, nitrogen dioxide radicals, azide radicals, and peroxynitrite radicals, as well as other oxidative agents. Melatonin reduces oxidative damage induced by nickel in both tomato leaves and roots by inhibiting the production of superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂) (Jahan et al. 2020). It also enhances Arabidopsis' tolerance to high light levels by scavenging both H₂O₂ and O₂^•− (Yang et al. 2021). Moreover, exposure to various environmental stresses triggers the biosynthesis of endogenous melatonin upon the application of exogenous melatonin. This process effectively suppresses the accumulation of H₂O₂, O₂^•− and MDA, thereby mitigating cell membrane disruption and enhancing photosynthetic efficiency (Chen et al. 2021; Imran et al. 2021). Hence, Melatonin and its metabolites directly scavenge ROS and RNS in stressed plants. Melatonin contributes to the removal of ROS and enhances the activity of antioxidant enzymes, mitigating the impact of water stress (Zhang et al. 2019).

Exogenous melatonin significantly boosted the performance of antioxidant enzymes and improved the yield of soybean plants (Oliveira-Spolaor et al. 2022). Recent findings suggests that external application of melatonin enhances salt tolerance in okra by increasing proline accumulation and the K⁺/Na⁺ ratio. This regulation aids in maintaining water equilibrium and ionic stability, preserves membrane stability and the photosynthetic process, and activates the reactive oxygen species detoxification system (Wang et al. 2024). Based on the above findings, comprehensive studies are needed to uncover stress signaling pathways and mechanisms for maintaining homeostasis under abiotic stress. Limited research exists on the stress resilience of important but underutilized vegetable crop like okra. The present study aims at unravelling the potential of melatonin in counteracting the detrimental effect of drought stress in okra by modulating the physiological and biochemical attributes including antioxidant systems.

Okra hybrid seeds CO 4, Arka Anamika, Arka Abhay and Arka Nikita sourced from Tamil Nadu Agricultural University, Coimbatore, Kerala Agricultural University, Thrissur, and Indian Institute of Horticultural Research, Bangalore respectively. The soil mixture consisted of red soil, clay, and farmyard manure in a 2:1:1 ratio. The soil physio-chemical properties of soil are represented in Table 1. Melatonin was sourced from Sigma-Aldrich Pvt. Ltd. A 100 µM solution was prepared by dissolving the required amount of melatonin in 99.9% ethanol and then adjusting the final volume with distilled water. Tween 20 was incorporated to enhance absorption efficiency.

Table 1

Physio-chemical properties of soil
Soil properties	Range
Texture	Red loam
pH	6.42
Electrical conductivity (ds m-1)	0.08
Organic carbon (%)	1.32
Available Nitrogen (Kg ha-1)	247.52
Available phosphorous (Kg ha-1)	20.74
Available potassium (Kg ha-1)	541.6

Plant growth condition and treatment imposition

Seeds were surface sterilized with 75% ethanol followed by seed treatment with 100 µM melatonin. Based on laboratory screening, 100 µM melatonin was identified as the most effective concentration of melatonin for enhancing drought tolerance in okra (unpublished data). This concentration underwent further evaluation in pot culture experiment. Seeds were germinated and grown in pots under ambient condition during the period of July to September 2022 at the Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore, India. Melatonin-treated and untreated seeds were sown at a depth of approximately half an inch for both control and stress treatments. Each pot held two plants, which were regularly irrigated and managed according to standard crop management practices until flowering. Upon the onset of flower initiation, the pots were relocated to a controlled greenhouse setting at Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore. A total of five treatments with four replication were used in the study. The treatments include: absolute control (fully irrigated), control (Drought D), Drought and seed treatment with 100µM melatonin (MT - ST), Drought and foliar spray of 100 µM melatonin (MT - FS), and drought stress with combined effect of seed treatment and foliar spray of 100µM melatonin (MT - ST + FS). Drought stress was induced naturally by ceasing irrigation, with daily monitoring of moisture levels (Fig. 1). The stress period spanned for ten days (from the 31st to the 42th day) during the flowering stage. Five days after the initiation of stress, plants were treated with a spray of 100µM melatonin, while the remaining pots continued to receive irrigation. At the termination of the stress period, leaf samples that received the treatments were taken to analyze various biochemical parameters.

Photosynthetic measurements

Gas exchange parameters were measured on fully expanded uppermost leaves during a bright sunny day from 9:00 to 11:30 AM using the CI-340 handheld photosynthesis system (CID BioSciences Inc). Recorded parameters included photosynthetic rate (A, µmol CO₂ m^− 2 s^− 1), transpiration rate (E, mmol H₂O m^− 2 s^− 1), and stomatal conductance (C, mol H₂O m^− 2 s^− 1). Chlorophyll fluorescence was measured using a chlorophyll fluorometer (Heinz Walz, Germany), with leaves dark-adapted for 30 min prior to measurement.

Maximum quantum yield of PS II (Fv/Fm) was computed as

Fv/Fm = (Fm − F₀)/Fm (1)

Chlorophyll index was determined using a SPAD meter (Minolta), based on the ratio of light transmitted at wavelengths of 650 nm and 940 nm.

Relative water content (RWC)

Fresh samples of most juvenile and fully extended leaves were collected, cut into 1 cm pieces, and weighed (0.5 g). These samples were then immersed in water for 4 hours, after which their turgid weight was recorded. Following hydration, the samples were dried in an oven at 80°C for 24 hours to determine their dry weight.

Relative Water Content (RWC) was computed using the formula proposed by (González and González-Vilar 2001).

RWC (%) = (FW- TW)/ (TW- DW) x 100 (2) FW = Fresh weight, TW = Turgid weight, DW = Dry weight

Membrane stability index (MSI)

MSI was determined following the method by (Premachandra et al. 1991), modified by (Sairam 1994). One gram of fresh leaf material was cut into discs, cleaned, and put in glass test tubes along with a blank. To immerse the discs, 10 ml of deionized water were poured to each tube. For 30 min, the tubes were incubated in a water bath at 45°C. Electrical conductivity (C₁) was measured after cooling. After adding water once more, the leaf discs were incubated for 10 min at 100°C in a water bath before their final electrical conductivity (C₂) was determined.

The membrane stability index was determined using percent conductivity, as given by the formula

MSI (%) = 1- C₁/ C₂ x 100 (3) Where C₁ = Initial electrical conductivity C₂ = Final electrical conductivity

Proline content

Proline levels were quantified according to the method described by (Bates et al. 1973). Fresh leaf samples (0.1 g) were homogenized in 3% aqueous sulfosalicylic acid and centrifuged at 5000 rpm for 10 min. The supernatant was mixed with acid ninhydrin, glacial acetic acid, and 2 mL of enzyme extract, and the mixture was heated in a hot water bath for 1 hour, then cooled on an ice bath. Proline was extracted with 4 mL of toluene and vigorously mixed, vortexed for 10 sec. The absorbance of the resultant solution was measured at 520 nm, with toluene serving as the blank reference.

Malondialdehyde content

MDA levels were determined as per the protocol outlined by (Hodges et al. 1999). Frozen leaf samples weighing 0.1 g each were pulverized in 1 mL of 0.1% trichloroacetic acid (TCA). The resulting homogenate underwent centrifugation at 4°C for 30 min at 15000 rpm. To the supernatant, 4 mL of 20% trichloroacetic acid containing 0.5% thiobarbituric acid was added and heated at 95°C for 15 min, then immediate cooling on ice. The MDA concentration (nmol/ml) of the filtrate was measured at 532 nm and expressed as µmol MDA g^-1 FW.

Antioxidant Enzyme Assays

Fresh leaf samples (100 mg) were homogenized using 50 mM sodium phosphate buffer along with 0.05% Triton X-100, 2% polyvinylpyrrolidone (PVP), and 1 mM EDTA for enzyme extraction in APX and CAT assays. The extract was subjected to centrifugation at 18,000 rpm at 4°C for 20 min, and the resulting extraction buffer was used for enzyme analysis.

For the determination of APX activity, 0.3 g of tissue was homogenized in 3 mL of extraction buffer followed by addition of 1 mL of 5 mM ascorbate. After centrifugation at 10,000 rpm for 20 min at 4°C, the resulting aliqout was mixed with reaction buffer containing 2 mL of KH₂PO₄ and K₂HPO₄ (pH 7.3). Absorbance readings were taken at 290 nm with 30 s intervals for 3 min (extinction coefficient = 2.8 mM − 1 cm − 1) (Nakano and Asada 1981).

CAT activity was assessed using a 50 mM phosphate buffer containing 100 µL of enzyme extract and 15 mM H₂O₂. Plant tissue samples (approximately 0.2 g) were homogenized in an extraction buffer. After centrifugation at 10,000 rpm for 20 min, the supernatant was combined with a reaction buffer (K₂HPO₄, KH₂PO₄) and H₂O₂ as the enzyme substrate. Absorbance was monitored at 240 nm for 3 min at 30 s intervals, using an extinction coefficient of 39.4 mM^− 1 cm^− 1, for the determination of H₂O₂ scavenging activity (Cakmak and Marschner 1992).

Statistical analysis

The study utilized a Factorial Completely Randomized Design (FCRD) with four replications. Data collected for various traits were statistically analyzed using SPSS software (version 16.0) via a two-way Analysis of Variance (ANOVA). To assess differences among group means, the Least Significant Difference (LSD) test was applied, and the Critical Difference (CD) was calculated at a significance level of 0.05 (P ≤ 0.05). Graphs were produced using GraphPad Prism (version 8.2.0). Principal component analysis and correlation were carried out using R software (version 4.3.1).

Changes in RWC of drought stressed okra hybrids under varying melatonin treatments

The study represents a significant variation in the relative water content (RWC) of okra hybrids CO 4, Arka Anamika, Arka Abhay, and Arka Nikita under different treatments. The highest RWC, 74.65%, was achieved with a combined treatment of 100 µM melatonin as seed treatment and foliar spray (Fig. 2). The interaction between the hybrids and the treatments showed a significant effect (F_12,60 = 3.65, P = 0.001) (Table S1).

MDA and MSI under different melatonin treatment combinations

The results reveals that plants experiencing water deficit showed increase in malondialdehyde content (120.77 µmol g^-1 FW) compared to absolute control. Application of 100µM of melatonin ( MT- ST + FS) reduced the MDA content by 48%. Among hybrids, CO 4 accumulated more MDA (98.27 µmol g^-1 FW) and Arka Abhay the least (62.19 µmol g^-1 FW) (Fig. 3). Interaction effect between hybrids and treatments are significant (F_12,60 = 5.07, P = 0.001) (Table S2).

Among hybrids, Arka Abhay exhibited higher membrane stability index (69.22%) and CO 4 recorded the least (61.71%). Membrane stability index reduced significantly in case of drought stressed treatment (53.20%). Application of 100µM of melatonin as seed treatment and foliar spray enhanced the membrane stability index by 26% (Fig. 3).

Changes in proline content across different melatonin treatments

When plants face drought stress, the accumulation of proline is a key indicator of their response. In comparison to absolute control, the plants that received a treatment combination (MT- ST + FS) of 100µM melatonin (5.80 µmol g^-1) and the drought stressed plants (5.42 µmol g^-1) accumulated more proline. Among hybrids, Arka Anamika recorded higher proline content (6.05 µmol g^-1) and Arka Nikita showed the least value (3.44 µmol g^-1). Application of 100µM of melatonin as seed treatment and foliar spray enhanced the proline levels in Arka Anamika (Fig. 4). The interaction effect between treatment and hybrids are significant (F_12,60 = 147.92, P = 0.001) (Table S2).

Gas exchange indicators

Gas exchange parameters such as photosynthetic rate, stomatal conductance and transpiration rate were significantly influenced by treatments and their interaction. Among the treatments, drought stressed plants recorded a decrease in photosynthetic rate (8.41 µmol CO₂ m^− 2 s^− 1), transpiration rate (5.52 mmol H₂O m^− 2 s^− 1) and stomatal conductance (0.47 mol H₂O m^− 2 s^− 1) whereas the combined application of 100µM of melatonin as seed treatment and foliar spray showed an increment in photosynthetic rate (12.72 µmol CO₂ m^− 2 s^− 1), transpiration rate (8.27 mmol H₂O m^− 2 s^− 1) and stomatal conductance (0.90 mol H₂O m^− 2 s^− 1). Among hybrids, Arka Nikita outperformed other hybrids in photosynthetic rate, transpiration rate and stomatal conductance (Fig. 5). Interaction effect between hybrids and treatments are significant (F_12,60 = 5.47, P = 0.001; F_12,60 = 7.52, P = 0.001; F_12,60 = 30.75, P = 0.001) (Table S1).

Fv/Fm and SPAD

Plants exposed to drought stress showed reduced Fv/Fm ratio (0.71) over absolute control (0.80). Melatonin treatment significantly increased Fv/Fm ratio. Treatment effects are significant. Among the treatments, application of 100µM of melatonin as seed treatment and foliar spray showed higher Fv/Fm ratio (0.79) in comparison to other treatments. The chlorophyll content (SPAD values) declined significantly in case of drought stressed plants (47.31) compared to absolute control (55.35). Application of melatonin as seed treatment and foliar spray improved chlorophyll content (54.13). All hybrids performed better when supplemented with 100µM of melatonin as seed treatment and foliar spray (Fig. 6).

Ascorbate peroxidase (APX) and catalase (CAT) activity

Enzymatic antioxidant activity such as APX and CAT increased significantly under drought stress compared to absolute control by 23.47% and 25.31% respectively. The enzyme activity was further enhanced on application of 100µM of melatonin (MT- ST + FS) by 36.46% and 43.42% in case of ascorbate peroxidase and catalase. Among the hybrids, Arka Abhay (2.52 Ug^-1 FW), Arka Nikita (2.47 Ug^-1 FW) and Arka Anamika (2.44 Ug^-1 FW) exhibited almost similar ascorbate peroxidase activity, whereas Arka Nikita (510.70 Ug^-1 (FW)) recorded higher catalase activity (Fig. 7). The interaction effects between treatment and hybrids are significant (F_12,60 =0.849, P = 0.001; F_12,60 = 0.22, P = 0.001) (Table S2).

Principal component analysis (PCA)

Principal component biplot based on PCA analysis were used to indicate the relationships among okra hybrids for the parameters (msi, spad, Fv/Fm, T, Pn, rwc, gs, apx, cat, proline and mda) under melatonin treatment (Fig. 8; Supplementary Table S3&S4). Two principal components exhibited eigenvalues > 1 and accounted for 82.56% of total phenotypic variation. The parameters Fv/Fm, spad, msi, T, Pn, rwc and gs were positively correlated with PC1, which accounted for 64.1% of the total variation, whereas PC2 was positively correlated with proline, apx and cat, while mda negatively correlated with PC2, which accounted for 18.4% of the total variation. Traits presented by parallel vectors or those close to each other revealed a strong positive association (msi, spad, Fv/Fm, T, Pn and rwc) whereas those located nearly opposite (at 180^◦) showed a highly negative association (msi and mda). Treatment combination namely, V1T5, V2T5, V3T5, V4T5, V2T4, V3T4, V4T4 are clustered together based close correlation with msi, T, spad and Fv/Fm. V1T1, V2T1, V3T1, V4T1 and are grouped based on positive correlation with gs and rwc. V1T2, V2T2, V3T2 and V4T2 are grouped based on close association with mda. V1T3, V2T3, V3T3 and V4T3 are grouped based on apx, cat and proline. The biplot is a clear indicative that hybrids namely CO 4 (V1), Arka Anamika (V2), Arka Abhay (V3) and Arka Nikita (V4) treated with 100µM of melatonin as seed treatment and foliar spray exhibited positive correlation with all variables except for mda, suggesting the ameliorative role of melatonin in combating drought stress by enhancing physiological and biochemical parameters.

Correlation analysis

Pearson correlation analysis was carried out to check the effect of melatonin treatments on physiological and biochemical parameters in okra which include proline (P, r² = 0.36), photosynthetic rate (A, r² = 0.05), chlorophyll fluorescence (Fv/Fm, r² = 0.11), membrane stability index (MSI, r² = 0.15), chlorophyll content (SPAD, r² = 0.15), ascorbate peroxidase activity (APX, r² = 0.86) and catalase activity (CAT, r² = 0.92). However, the relative water content (RWC, r²= -0.07), malondialdehyde content (MDA, r²= -0.10), transpiration rate (E, r²= -0.018) and stomatal conductance (C, r²= -0.26) were negatively correlated with the melatonin concentration (Fig. 9). A strong positive correlation (dark green circle) was observed between A and E, indicating that higher photosynthetic rates are associated with higher transpiration rates. This is expected as both processes are regulated by stomatal opening. C showed a strong positive correlation with both A and E, underscoring the role of stomata in facilitating both gas exchange for photosynthesis and water vapour loss through transpiration. Moderate positive correlations were found between SPAD and A, E, and C, suggesting that higher chlorophyll content, indicative of healthy and productive plants, is associated with increased photosynthetic and transpiration activities. A strong negative correlation between RWC and MDA suggests that higher water content within the plant tissues is linked to reduced oxidative stress. This relationship is crucial for maintaining cellular integrity and function under stress conditions. A strong positive correlation was noted between Fv/Fm and MSI, indicating that efficient functioning of photosystem II is associated with enhanced membrane stability. This relationship highlights the interdependence of photosynthetic efficiency and cellular integrity. MT displayed a high positive correlation with CAT, suggesting that higher melatonin levels are associated with increased catalase activity. Melatonin is known for its role in mitigating oxidative stress, and this correlation underscores its protective effects in plants. Proline exhibited low correlations with other variables, indicating it might be an independent stress indicator.

Metabolite profiling of bioactive compounds detected in water deficit stressed and melatonin treated plants

The effect of application of melatonin to drought stressed (MT + ST + FS) okra plants with that of drought control (DC) plants were analysed through metabolite profiling (GC-MS/MS). Based on the percentage of peak area both treatments accumulated metabolites in varying degrees. The drought control (DC) treatment showed higher probability of the compounds mainly psicose (10.86%), D- Glucose (10.64%), glyceryl-glycoside (4.47%), Allose (4.25%), Scyllo-inositol (4.16%) whereas melatonin treated sample exhibited the presence of the following compounds, sucrose (2.01%), proline (2.33%), L- Tryptophan (2.56%), D- Altrose (4.12%), D- Talose (4.02%), Myo-inositol (6.07%), psicose (9.03%) and D- Glucose (10.73%). Other bioactive compounds were almost comparable in both the treatment combinations (Fig. 10).

Abiotic stress, which encompasses environmental factors such as dry spell, salt stress, thermal extremes, free radical stress, and metal-induced toxicity, poses a significant challenge to crop productivity worldwide (Bulgari et al. 2019; Raza et al. 2023). In vegetable crops, there are critical growth stages where irrigation is crucial to prevent substantial yield loss and quality deterioration. For okra, a vegetable prized for its nutritional quality, the flowering and pod formation stages are particularly sensitive to water availability. Insufficient irrigation during these periods can markedly reduce yield and hinder fiber development.

Relative Water Content is used as a metric to assess the hydration status of plants, and its regulation is linked to the plant's adaptation to drought stress (Liang et al. 2020). In the present study, a combination of seed treatment and foliar spray of 100 µM melatonin resulted in enhanced RWC. The reduction in Relative Water Content after 14 days of drought stress was alleviated by melatonin application. Drought stress significantly lowers leaf relative water content in tomato plants, resulting in reduced water transport from roots to stems, mesophyll flaccidity, reduced leaf water status and reduced soil moisture (Altaf et al. 2022). Similar findings by (Huang et al. 2019) and (Dai et al. 2020) suggest that melatonin enhances photosynthesis. The decline in RWC may be linked to decreased plant growth factors. Melatonin pre-treatment significantly improved water status in rice under salinity and drought stress (Khan et al. 2024). These results align with (Turk et al. 2014), indicating that melatonin likely modulates stomatal behaviour, thus regulating stomatal opening and closure to reduce water loss. Exogenous melatonin application increased the total leaf cuticular wax load and the expression of certain wax biosynthetic genes in tomato plants under water deficit conditions, thereby effectively reducing water loss in plants (Ding et al. 2018).

The study reveals that okra plants subjected to water stress exhibit oxidative damage, indicated by elevated malondialdehyde (MDA) levels and a decreased membrane stability index (MSI), in alignment with the findings of (Nalina et al. 2021) and (Talaat 2015, 2023). Interestingly, our experiment showed a significant reduction in MDA accumulation in drought stressed plants treated with melatonin. The higher MSI and lower electrolyte leakage (EL) values observed in these treated plants can be attributed to reduced MDA and hydrogen peroxide (H₂O₂) production. Multiple studies have reported that MT-treated plants under stress conditions show lower MDA and EL values and higher MSI levels. The external application of MT appears to alleviate water stress-induced cellular damage and preserve membrane integrity by reducing H₂O₂ production and lipid peroxidation. Additionally, MT is suggested to stabilize biological membranes under stress by maintaining optimal fluidity (Arnao and Hernández-Ruiz 2019) and regulating the expression of lipid peroxidase genes (Gao et al. 2019).

Photosynthesis, the fundamental process for harnessing light energy to produce carbohydrates, is integral to plant growth. Nevertheless, photosynthesis is particularly vulnerable to drought stress, with water deficiency markedly suppressing photosynthetic activity in numerous plant species (Velikova et al. 2018; Zhou et al. 2019; Sharma et al. 2020). In general terms, the decrease in photosynthetic activity is limited by reduced CO₂ diffusion to the chloroplasts, resulting from the closure of stomata (Liu et al. 2013; Ye et al. 2016). In response to drought stress, plants typically exhibit stomatal closure, resulting in reduced stomatal conductance and consequent limitations on photosynthesis (Meloni et al. 2003; Liu et al. 2013). Melatonin mitigates chlorophyll degradation under drought stress conditions, thereby enhancing photosynthesis, transpiration, and stomatal conductance (Liang et al. 2018; Karaca and Cekic 2019). Melatonin helps maintain cellular turgor, enhancing stomatal opening and conductance (Meng et al. 2014). This increased conductance improves water and CO₂ movement, ultimately favouring photosynthesis in melatonin-treated plants (Cui et al. 2017).

Chlorophyll fluorescence serves as a crucial indicator for assessing the photosynthetic capacity and energy conversion efficiency of Photosystem II (PSII) in plants (Mathur et al. 2019). Numerous studies suggests that prolonged water deficit induces photoinhibition in the reaction center of Photosystem II (PSII) in plants (Huang et al. 2019; Zhou et al. 2019). In line with these findings, a significant reduction in Fv/Fm was observed in drought-stressed maize seedlings, indicating that drought stress caused substantial damage to the PSII complexes (Zhao et al. 2021). This was attributed to the restricted diffusion of ambient CO₂ to the carboxylation sites, resulting in a relative surplus of light energy and electron sinks, thereby causing photoinhibition or photooxidation (Atkin and Macherel 2009; Zhong et al. 2018). Exogenous application of melatonin enhanced photosynthetic efficiency and protected the maize plant from photoinhibition (Zhao et al. 2021). The application of melatonin exerts a protective effect on chlorophyll, thereby reducing damage to the photosynthetic apparatus (Campos et al. 2019; Li et al. 2021).

Application of exogenous melatonin enhances the activity of key antioxidant enzymes, such as catalase and ascorbate peroxidase, which play a critical role in shielding plants from oxidative stress caused by reactive oxygen intermediates. This melatonin-induced enhancement facilitates plant survival under drought stress conditions. Numerous studies have demonstrated that melatonin effectively promotes plant survival and growth by strengthening ROS scavenging mechanisms under various abiotic stress conditions (Sharma et al. 2020; Gao et al. 2018). Foliar application of melatonin mitigates oxidative impairement in corn seedlings (Ahmad et al. 2019). Under water stress, plants develop adaptation strategy, such as ROS detoxifying enzymes to prevent free radical bursts and maintain ROS balance (Abid et al. 2018; Imran et al. 2021; Li et al. 2018). Melatonin enhances the transcript levels and activities of antioxidant enzymes (Arnao and Hernández-Ruiz 2019). The activity of these antioxidant enzymes is linked to the regulation of key genes encoding them, ensuring cellular redox balance under stressful conditions (Sharma et al. 2020).

Metabolomic assessment have revealed the retention of various osmoregulatory solutes, including carbohydrates such as glucose and sucrose, polyols like sorbitol, and amino acids such as proline, lysine, L-tryptophan, leucine, and tyrosine. These osmolytes are crucial for reducing osmotic gradient and maintaining cellular pressure by promoting water uptake. They also play a significant role in stabilizing cellular membranes, enzymes, and proteins (Jorge and António 2018; Sharma et al. 2019). Additionally, the osmolytes buildup aids in regulating ROS levels, supplies energy to manage stress, facilitates repair mechanism, and supports continued growth (Silva et al. 2018; Fàbregas and Fernie 2019). Present study reported the presence of certain carbohydrates, including sucrose, psicose, talose and myo- inositol in case of drought stressed okra plants subjected to melatonin treatments. Similar findings were reported by (Sharma et al. 2019) wherein the following compounds (sucrose, tagatose, psicose, glucoheptose, allose, talose, cellobiose and sugar alcohol inositol) were shown to exhibit a sharp increase in response to salinity. D-psicose affects plants by increasing sugar content while preserving leaf water content. One potential mechanism for this effect involves osmotic adjustment, where the leaf enhances its water absorption capacity by elevating the soluble content within its cells (YAMADA et al. 2014). L-tryptophan (Try) is a well-recognized amino acid essential for plant growth, functioning effectively under both normal environmental conditions and various abiotic stresses. In the present metabolite profiling, L-tryptophan levels were higher in case of plants treated with melatonin which is in accordance to the findings of (Chen et al. 2009) that in higher plants, L-tryptophan functions as a physiological precursor to melatonin. As an osmolyte, L-tryptophan plays a crucial role in facilitating the transport of nutrient ions, modulating stomatal aperture, and mitigating the adverse effects of heavy metals (Rai 2002). Additionally, tryptophan enhances various biochemical attributes by regulating plant growth and divergence and improving nutrient and water uptake efficiency (Talaat et al. 2005; Dawood and Sadak 2007).

In conclusion, the combined treatment of melatonin seed treatment and foliar spray proved to be an effective strategy in mitigating drought stress in okra. This integrated approach significantly enhanced photosynthetic parameters and antioxidant activity while reducing lipidperoxidation and maintaining membrane stability index (Fig. 11).

These improvements highlight the potential of melatonin applications to bolster drought tolerance in okra, thereby promoting better growth and productivity under water-limited conditions. Future research should explore the underlying mechanisms of melatonin's protective effects and investigate its efficacy across different okra varieties and environmental conditions. Additionally, studies should aim to optimize application methods and dosages to maximize the benefits of melatonin treatments in agricultural practices.

Melatonin

Seed treatment

Foliar spray

RWC

Relative water content

MDA

Melondialdehyde

MSI

Membrane stability index

APX

Ascorbate peroxidase

CAT

Catalase

photosynthetic rate

transpiration rate

stomatal conductance

Proline

SPAD

Chlorophyll index

ACKNOWLEDGEMENTS

The authors would like to express thanks and gratitude to Tamil Nadu Agricultural University, Coimbatore – 641 003, Tamil Nadu for providing facilities to carry out the research.

AUTHORS’ CONTRIBUTIONS

Conceptualization, methodology, review and editing: Gopal Aswathi, Veerasamy Ravichandran, Dhashnamurthi Vijayalakshmi, Alagarsamy Senthil, Loganathan Arul, Sengodan Radhamani, Ramasamy Jagadeeswaran and Mottaiyan Pitchaimuthu; Visualization, software: Gopal Aswathi; Validation: Gopal Aswathi, Veerasamy Ravichandran and Dhashnamurthi Vijayalakshmi; Formal analysis: Gopal Aswathi; Investigation, data curation, original draft: Gopal Aswathi and Veerasamy Ravichandran; Supervision: Veerasamy Ravichandran

FUNDING: No funding was received to assist with the preparation of this manuscript

DATA AVAILABILITY STATEMENT: Not applicable

CONFLICT OF INTEREST: The authors have no competing interests to declare that are relevant to the content of this article.

COMPLIANCE WITH ETHICAL STANDARDS: The research work does not involve people as research object

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Melatonin-Induced Enhancement of Drought Tolerance in Okra: A Detailed Analysis of Physiological, Biochemical, and Metabolic Adaptations

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Abstract

Figures

Introduction

Planting material and source of Melatonin

Plant growth condition and treatment imposition

Photosynthetic measurements

Relative water content (RWC)

Membrane stability index (MSI)

Proline content

Malondialdehyde content

Antioxidant Enzyme Assays

Statistical analysis

Results

Changes in RWC of drought stressed okra hybrids under varying melatonin treatments

MDA and MSI under different melatonin treatment combinations

Changes in proline content across different melatonin treatments

Gas exchange indicators

Fv/Fm and SPAD

Ascorbate peroxidase (APX) and catalase (CAT) activity

Principal component analysis (PCA)

Correlation analysis

Metabolite profiling of bioactive compounds detected in water deficit stressed and melatonin treated plants

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1