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

Melatonin-Induced Enhancement of Drought Tolerance in Okra: A Detailed Analysis of Physiological, Biochemical, and Metabolic Adaptations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Melatonin-Induced Enhancement of Drought Tolerance in Okra: A Detailed Analysis of Physiological, Biochemical, and Metabolic Adaptations Gopal Aswathi, Veerasamy Ravichandran, Dhashnamurthi Vijayalakshmi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4967383/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 6 You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction 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 (O 2 •− ) 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 (O 2 •− ) and hydrogen peroxide (H 2 O 2 ) (Jahan et al. 2020 ). It also enhances Arabidopsis' tolerance to high light levels by scavenging both H 2 O 2 and O 2 •− (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 H 2 O 2 , O 2 •− 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. Planting material and source of Melatonin 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 CO 2 m − 2 s − 1 ), transpiration rate (E, mmol H 2 O m − 2 s − 1 ), and stomatal conductance (C, mol H 2 O 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 − F 0 )/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 (C 1 ) 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 (C 2 ) was determined. The membrane stability index was determined using percent conductivity, as given by the formula MSI (%) = 1- C 1 / C 2 x 100 (3) Where C 1 = Initial electrical conductivity C 2 = 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 KH 2 PO 4 and K 2 HPO 4 (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 H 2 O 2 . 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 (K 2 HPO 4 , KH 2 PO 4 ) and H 2 O 2 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 H 2 O 2 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). Results 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 CO 2 m − 2 s − 1 ), transpiration rate (5.52 mmol H 2 O m − 2 s − 1 ) and stomatal conductance (0.47 mol H 2 O 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 CO 2 m − 2 s − 1 ), transpiration rate (8.27 mmol H 2 O m − 2 s − 1 ) and stomatal conductance (0.90 mol H 2 O 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, r 2 = 0.36), photosynthetic rate (A, r 2 = 0.05), chlorophyll fluorescence (Fv/Fm, r 2 = 0.11), membrane stability index (MSI, r 2 = 0.15), chlorophyll content (SPAD, r 2 = 0.15), ascorbate peroxidase activity (APX, r 2 = 0.86) and catalase activity (CAT, r 2 = 0.92). However, the relative water content (RWC, r 2 = -0.07), malondialdehyde content (MDA, r 2 = -0.10), transpiration rate (E, r 2 = -0.018) and stomatal conductance (C, r 2 = -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 ). Discussion 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 (H 2 O 2 ) 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 H 2 O 2 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 CO 2 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 CO 2 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 ). Conclusion 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. Abbreviations MT Melatonin ST Seed treatment FS Foliar spray RWC Relative water content MDA Melondialdehyde MSI Membrane stability index APX Ascorbate peroxidase CAT Catalase A photosynthetic rate E transpiration rate C stomatal conductance P Proline SPAD Chlorophyll index Declarations 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 References Abid M, Ali S, Qi LK, Zahoor R, Tian Z, Jiang D, Snider JL, Dai T (2018) Physiological and biochemical changes during drought and recovery periods at tillering and jointing stages in wheat ( Triticum aestivum L.). Sci. Rep. 8 (1):4615 Ahmad S, Kamran M, Ding R, Meng X, Wang H, Ahmad I, Fahad S, Han Q (2019) Exogenous melatonin confers drought stress by promoting plant growth, photosynthetic capacity and antioxidant defense system of maize seedlings. PeerJ 7:e7793 Altaf MA, Shahid R, Altaf MM, Kumar R, Naz S, Kumar A, Alam P, Tiwari RK, Lal MK, Ahmad P (2022) Melatonin: first-line soldier in tomato under abiotic stress current and future perspective. Plant Physiol Biochem 185:188-197 Arnao MB, Hernández-Ruiz J (2019) Melatonin: a new plant hormone and/or a plant master regulator? Trends Plant Sci. 24 (1):38-48 Atkin OK, Macherel D (2009) The crucial role of plant mitochondria in orchestrating drought tolerance. Ann. Bot. 103 (4):581-597 Ayub Q, Hussain I, Naveed K, Ali S, Mehmood A, Khan M, Haq N, Shehzad Q (2021) Responses of different okra ( Abelmoschus esculentus ) cultivars to water deficit conditions. J. Hortic. Sci. 16 (1):53-63 Bates LS, Waldren R, Teare I (1973) Rapid determination of free proline for water-stress studies. Plant and soil 39:205-207 Bulgari R, Franzoni G, Ferrante A (2019) Biostimulants application in horticultural crops under abiotic stress conditions. Agronomy 9 (6):306 Cakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant physiol. 98 (4):1222-1227 Campos CN, Ávila RG, de Souza KRD, Azevedo LM, Alves JD (2019) Melatonin reduces oxidative stress and promotes drought tolerance in young Coffea arabica L. plants. Agric. Water Manag. 211:37-47 Chen Q, Qi W-b, Reiter RJ, Wei W, Wang B-m (2009) Exogenously applied melatonin stimulates root growth and raises endogenous indoleacetic acid in roots of etiolated seedlings of Brassica juncea. J.Plant Physiol. 166 (3):324-328 Chen Y, Li R, Ge J, Liu J, Wang W, Xu M, Zhang R, Hussain S, Wei H, Dai Q (2021) Exogenous melatonin confers enhanced salinity tolerance in rice by blocking the ROS burst and improving Na+/K+ homeostasis. Environ. Exp. Bot. 189:104530 Cui G, Zhao X, Liu S, Sun F, Zhang C, Xi Y (2017) Beneficial effects of melatonin in overcoming drought stress in wheat seedlings. Plant Physiol Biochem 118:138-149 Dai L, Li J, Harmens H, Zheng X, Zhang C (2020) Melatonin enhances drought resistance by regulating leaf stomatal behaviour, root growth and catalase activity in two contrasting rapeseed ( Brassica napus L.) genotypes. Plant Physiol Biochem 149:86-95 Dawood MG, Sadak MS (2007) Physiological response of canola plants ( Brassica napus L.) to tryptophan or benzyladenine. Lucrari stiintifice 50 (9):198-207 Dhankher OP, Foyer CH (2018) Climate resilient crops for improving global food security and safety. vol 41. Wiley Online Library, Ding F, Wang G, Wang M, Zhang S (2018) Exogenous melatonin improves tolerance to water deficit by promoting cuticle formation in tomato plants. Molecules 23 (7):1605 Dubbels R, Reiter R, Klenke E, Goebel A, Schnakenberg E, Ehlers C, Schiwara H, Schloot W (1995) Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography‐mass spectrometry. J. Pineal Res. 18 (1):28-31 Fàbregas N, Fernie AR (2019) The metabolic response to drought. J. Exp. Bot. 70 (4):1077-1085 Gao W, Feng Z, Bai Q, He J, Wang Y (2019) Melatonin-mediated regulation of growth and antioxidant capacity in salt-tolerant naked oat under salt stress. Int. J. Mol. Sci. 20 (5):1176 Gao W, Zhang Y, Feng Z, Bai Q, He J, Wang Y (2018) Effects of melatonin on antioxidant capacity in naked oat seedlings under drought stress. Molecules 23 (7):1580 Gemede HF, Ratta N, Haki GD, Woldegiorgis AZ, Beyene F (2015) Nutritional quality and health benefits of okra (Abelmoschus esculentus): A review. J Food Process Technol 6 (458):2 Gogoi A, Tripathi B (2019) 42% India’s land area under drought, worsening farm distress in election year. India Spend González L, González-Vilar M (2001) Determination of relative water content. In: Handbook of plant ecophysiology techniques. Springer, pp 207-212 Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604-611 Huang B, Chen Y-E, Zhao Y-Q, Ding C-B, Liao J-Q, Hu C, Zhou L-J, Zhang Z-W, Yuan S, Yuan M (2019) Exogenous melatonin alleviates oxidative damages and protects photosystem II in maize seedlings under drought stress. Front. Plant Sci. 10:677 Imran M, Aaqil Khan M, Shahzad R, Bilal S, Khan M, Yun B-W, Khan AL, Lee I-J (2021) Melatonin ameliorates thermotolerance in soybean seedling through balancing redox homeostasis and modulating antioxidant defense, phytohormones and polyamines biosynthesis. Molecules 26 (17):5116 Indiastat (2023) Area, production and productivity of okra in india (1987-1988 and 1991-1992 to 2023-2024- 2nd advance estimates). Jahan MS, Guo S, Baloch AR, Sun J, Shu S, Wang Y, Ahammed GJ, Kabir K, Roy R (2020) Melatonin alleviates nickel phytotoxicity by improving photosynthesis, secondary metabolism and oxidative stress tolerance in tomato seedlings. Ecotoxicol. Environ. Saf. 197:110593 Jorge TF, António C (2018) Plant metabolomics in a changing world: metabolite responses to abiotic stress combinations. Plant Abiotic Stress Responses Clim Chang Karaca P, Cekic FÖ (2019) Exogenous melatonin-stimulated defense responses in tomato plants treated with polyethylene glycol. Int. J. Veg. Sci. 25 (6):601-609 Khan Z, Jan R, Asif S, Farooq M, Jang Y-H, Kim E-G, Kim N, Kim K-M (2024) Exogenous melatonin induces salt and drought stress tolerance in rice by promoting plant growth and defense system. Sci. Rep. 14 (1):1214 Lee H, Calvin K, Dasgupta D, Krinner G, Mukherji A, Thorne P, Trisos C, Romero J, Aldunce P, Barret K (2023) IPCC, 2023: Climate Change 2023: Synthesis Report, Summary for Policymakers. Contribution of Working Groups I, II and III to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, H. Lee and J. Romero (eds.)]. IPCC, Geneva, Switzerland. Li H, Chang J, Chen H, Wang Z, Gu X, Wei C, Zhang Y, Ma J, Yang J, Zhang X (2017) Exogenous melatonin confers salt stress tolerance to watermelon by improving photosynthesis and redox homeostasis. Front. Plant Sci. 8:295 Li J, Zeng L, Cheng Y, Lu G, Fu G, Ma H, Liu Q, Zhang X, Zou X, Li C (2018) Exogenous melatonin alleviates damage from drought stress in Brassica napus L.(rapeseed) seedlings. Acta Physiol. Plant. 40:1-11 Li Z, Su X, Chen Y, Fan X, He L, Guo J, Wang Y, Yang Q (2021) Melatonin improves drought resistance in maize seedlings by enhancing the antioxidant system and regulating abscisic acid metabolism to maintain stomatal opening under PEG-induced drought. J. Plant Biol. 64:299-312 Liang B, Ma C, Zhang Z, Wei Z, Gao T, Zhao Q, Ma F, Li C (2018) Long-term exogenous application of melatonin improves nutrient uptake fluxes in apple plants under moderate drought stress. Environ. Exp. Bot. 155:650-661 Liang G, Liu J, Zhang J, Guo J (2020) Effects of drought stress on photosynthetic and physiological parameters of tomato. J. Am. Soc. 145 (1):12-17 Liu D, Wu L, Naeem MS, Liu H, Deng X, Xu L, Zhang F, Zhou W (2013) 5-Aminolevulinic acid enhances photosynthetic gas exchange, chlorophyll fluorescence and antioxidant system in oilseed rape under drought stress. Acta Physiol. Plant. 35:2747-2759 Mathur S, Tomar RS, Jajoo A (2019) Arbuscular mycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress. Photosynth. Res. 139:227-238 Mbagwu J, Adesipe F (1987) Response of three okra ( Abelmoschus esculentus L. Moench) cultivars to irrigation at specific growth stages. Sci. Hortic. 31 (1-2):35-43 Meloni DA, Oliva MA, Martinez CA, Cambraia J (2003) Photosynthesis and activity of superoxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. Environ. Exp. Bot. 49 (1):69-76 Meng JF, Xu TF, Wang ZZ, Fang YL, Xi ZM, Zhang ZW (2014) The ameliorative effects of exogenous melatonin on grape cuttings under water‐deficient stress: antioxidant metabolites, leaf anatomy, and chloroplast morphology. J. Pineal Res. 57 (2):200-212 Mkhabela SS, Shimelis H, Gerrano AS, Mashilo J (2022) Phenotypic and genotypic divergence in okra [ Abelmoschus esculentus (L.) Moench] and implications for drought tolerance breeding: A review. S. Afr. J. Bot. 145:56-64 Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22 (5):867-880 Nalina M, Saroja S, Chakravarthi M, Rajkumar R, Radhakrishnan B, Chandrashekara K (2021) Water deficit-induced oxidative stress and differential response in antioxidant enzymes of tolerant and susceptible tea cultivars under field condition. Acta Physiol. Plant. 43:1-17 Oliveira-Spolaor B, Chiari-Bertoli S, Silva-Sukert D, Sala HR, Picoli de Oliveira BF, de Freitas ÍR, Lima-Moro A (2022) Exogenous melatonin induces tolerance to drought stress damage in seedlings and soybean plants. Chil. J. Agric. Res. 82 (4):515-526 Premachandra G, Saneoka H, Kanaya M, Ogata S (1991) Cell membrane stability and leaf surface wax content as affected by increasing water deficits in maize. J. Exp. Bot. 42 (2):167-171 Rai V (2002) Role of amino acids in plant responses to stresses. Biol. Plant. 45 (4):481-487 Raza A, Mubarik MS, Sharif R, Habib M, Jabeen W, Zhang C, Chen H, Chen ZH, Siddique KH, Zhuang W (2023) Developing drought‐smart, ready‐to‐grow future crops. The Plant Genome 16 (1):e20279 Razi K, Muneer S (2021) Drought stress-induced physiological mechanisms, signaling pathways and molecular response of chloroplasts in common vegetable crops. Crit. Rev. Biotechnol. 41 (5):669-691 Reyes F, Gosme M, Wolz KJ, Lecomte I, Dupraz C (2021) Alley cropping mitigates the impacts of climate change on a wheat crop in a Mediterranean environment: a biophysical model-based assessment. Agriculture 11 (4):356 Sairam R (1994) Effects of homobrassinolide application on plant metabolism and grain yield under irrigated and moisture-stress conditions of two wheat varieties. Plant Growth Regul. 14:173-181 Sharma A, Shahzad B, Rehman A, Bhardwaj R, Landi M, Zheng B (2019) Response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules 24 (13):2452 Sharma A, Wang J, Xu D, Tao S, Chong S, Yan D, Li Z, Yuan H, Zheng B (2020) Melatonin regulates the functional components of photosynthesis, antioxidant system, gene expression, and metabolic pathways to induce drought resistance in grafted Carya cathayensis plants. Sci. Total Environ. 713:136675 Sheshadri S, Nishanth M, Yamine V, Simon B (2018) Effect of Melatonin on the stability and expression of reference genes in Catharanthus roseus. Sci. Rep. 8 (1):2222 Silva S, Santos C, Serodio J, Silva AM, Dias MC (2018) Physiological performance of drought-stressed olive plants when exposed to a combined heat–UV-B shock and after stress relief. Functional Plant Biology 45 (12):1233-1240 Sun Y-D, Guo D-L, Yang S-D, Zhang H-C, Wang L-L, Yu Y-H (2020) Melatonin treatment improves the shelf-life and postharvest quality of table grape (Vitis labrusca L. cv.‘Fengzao’). J. Berry Res. 10 (4):665-676 Talaat IM, Bekheta M, Mahgoub MH (2005) Physiological response of periwinkle plants (Catharanthus roseus L.) to tryptophan and putrescine. Int J Agric Biol 7 (2):210-213 Talaat NB (2015) Effective microorganisms improve growth performance and modulate the ROS-scavenging system in common bean (Phaseolus vulgaris L.) plants exposed to salinity stress. Journal of Plant Growth Regul. 34:35-46 Talaat NB (2023) Role of phytohormones in regulating abiotic stresses in wheat. In: Abiotic Stresses in Wheat. Elsevier, pp 111-130 Turk H, Erdal S, Genisel M, Atici O, Demir Y, Yanmis D (2014) The regulatory effect of melatonin on physiological, biochemical and molecular parameters in cold-stressed wheat seedlings. Plant Growth Regul. 74:139-152 Velikova V, Tsonev T, Tattini M, Arena C, Krumova S, Koleva D, Peeva V, Stojchev S, Todinova S, Izzo LG (2018) Physiological and structural adjustments of two ecotypes of Platanus orientalis L. from different habitats in response to drought and re-watering. Conserv. Physiol. 6 (1):coy073 Wang F, Wan C, Wu W, Yang S, Chen X (2024) Exogenous melatonin (MT) enhances salt tolerance of okra ( Abelmoschus esculentus L.) plants by regulating proline, photosynthesis, ion homeostasis and ROS pathways. Vegetos 37 (1):224-238 Wang K, Xing Q, Ahammed GJ, Zhou J (2022) Functions and prospects of melatonin in plant growth, yield, and quality. J. Exp. Bot. 73 (17):5928-5946 YAMADA Y, KAKIBUCHI K, KOZUKI A, ISHIDA Y, IZUMORI K, TAJIMA S, AKIMITSU K, OHKOUCHI T, KASAI F (2014) Effects of the rare sugars D-psicose and D-tagatose on the sugar content and incidence of blossom end rot in tomato grown hydroponically with salinity treatment. Environmental Control in Biology 52 (3):155-160 Yang S-J, Huang B, Zhao Y-Q, Hu D, Chen T, Ding C-B, Chen Y-E, Yuan S, Yuan M (2021) Melatonin enhanced the tolerance of Arabidopsis thaliana to high light through improving anti-oxidative system and photosynthesis. Front. Plant Sci. 12:752584 Ye J, Wang S, Deng X, Yin L, Xiong B, Wang X (2016) Melatonin increased maize (Zea mays L.) seedling drought tolerance by alleviating drought-induced photosynthetic inhibition and oxidative damage. Acta Physiol. Plant. 38 (2):48 Zhang N, Zhang H-J, Sun Q-Q, Cao Y-Y, Li X, Zhao B, Wu P, Guo Y-D (2017) Proteomic analysis reveals a role of melatonin in promoting cucumber seed germination under high salinity by regulating energy production. Sci. Rep. 7 (1):503 Zhang Q, Gao F, Zhang S, Sun W, Li Z (2019) Prophylactic use of exogenous melatonin and melatonin receptor agonists to improve sleep and delirium in the intensive care units: a systematic review and meta-analysis of randomized controlled trials. Sleep and Breathing 23:1059-1070 Zhao C, Guo H, Wang J, Wang Y, Zhang R (2021) Melatonin enhances drought tolerance by regulating leaf stomatal behavior, carbon and nitrogen metabolism, and related gene expression in maize plants. Front. Plant Sci. 12:779382 Zhong C, Cao X, Bai Z, Zhang J, Zhu L, Huang J, Jin Q (2018) Nitrogen metabolism correlates with the acclimation of photosynthesis to short-term water stress in rice (Oryza sativa L.). Plant Physiol. Biochem. 125:52-62 Zhou R, Kan X, Chen J, Hua H, Li Y, Ren J, Feng K, Liu H, Deng D, Yin Z (2019) Drought-induced changes in photosynthetic electron transport in maize probed by prompt fluorescence, delayed fluorescence, P700 and cyclic electron flow signals. Environ. Exp. Bot. 158:51-62 Supplementary Files SupplementaryTablePGR.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major revisions 13 Nov, 2024 Reviewers agreed at journal 15 Sep, 2024 Reviewers invited by journal 05 Sep, 2024 Editor invited by journal 29 Aug, 2024 Editor assigned by journal 26 Aug, 2024 First submitted to journal 23 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4967383","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":350241169,"identity":"45fe8ee5-733d-4701-8bd4-2cddb1ebb3d7","order_by":0,"name":"Gopal Aswathi","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Gopal","middleName":"","lastName":"Aswathi","suffix":""},{"id":350241170,"identity":"0a0ae26f-f8c6-4862-9f70-c29f942fa48a","order_by":1,"name":"Veerasamy Ravichandran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYJCCAw8YGOTsjzcAmQYWRGpJYGAwZjhzAKRFgkhrgFoSGW4kgJhEaDFvP2N4ILHtcALjzOdXN/wokGDgb+9OwKtF5kyOAVBLWh6zdE7ZzR6gwyTOnN2AV4sEQ1oCUItNMZt0TtoNHqAWA4lcAlr4n4G0SCT2SJ5Ju/mHKC0SyQdAtiTOkGA/dps4WyQeHziQcC7N2IAnh+22jIEED2G/8Cc2f/hQdljOgP34s5tv/tjI8bf34teCBHgMwCSxykGA/QEpqkfBKBgFo2AEAQCALUi79s+PWQAAAABJRU5ErkJggg==","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Veerasamy","middleName":"","lastName":"Ravichandran","suffix":""},{"id":350241171,"identity":"a1d9a140-4861-4b7f-8f72-b6aed3ace342","order_by":2,"name":"Dhashnamurthi Vijayalakshmi","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dhashnamurthi","middleName":"","lastName":"Vijayalakshmi","suffix":""},{"id":350241172,"identity":"b21415f3-bfa4-4314-a009-af03198126c6","order_by":3,"name":"Alagarsamy Senthil","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Alagarsamy","middleName":"","lastName":"Senthil","suffix":""},{"id":350241173,"identity":"a977c54f-42e0-436e-b3b9-2c6a32df7254","order_by":4,"name":"Loganathan Arul","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Loganathan","middleName":"","lastName":"Arul","suffix":""},{"id":350241174,"identity":"69ee0500-e949-46f8-af86-cdcbef4d1823","order_by":5,"name":"Sengodan Radhamani","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sengodan","middleName":"","lastName":"Radhamani","suffix":""},{"id":350241175,"identity":"6cf75601-4db8-4280-a6fb-36b564f69e34","order_by":6,"name":"Ramasamy Jagadeeswaran","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ramasamy","middleName":"","lastName":"Jagadeeswaran","suffix":""},{"id":350241176,"identity":"2deee33f-b6f4-4598-9b01-5bbe8549cc32","order_by":7,"name":"Mottaiyan Pitchaimuthu","email":"","orcid":"","institution":"Indian Institute of Horticultural Research","correspondingAuthor":false,"prefix":"","firstName":"Mottaiyan","middleName":"","lastName":"Pitchaimuthu","suffix":""}],"badges":[],"createdAt":"2024-08-24 05:45:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4967383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4967383/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66239749,"identity":"7750e42c-4ba8-4656-baa7-b126f7a7cab8","added_by":"auto","created_at":"2024-10-09 06:22:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39346,"visible":true,"origin":"","legend":"\u003cp\u003eSoil moisture content under drought stress for a time period of ten days\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/26fbfbeb2b782e74df4cede5.png"},{"id":66239370,"identity":"c5ce8de2-78ac-4125-992b-125bc98c992a","added_by":"auto","created_at":"2024-10-09 06:14:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79383,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of melatonin on relative water content (RWC) under drought stress in okra. AC: Absolute Control, C: Control (Drought), MT-ST: Seed treatment of 100µM melatonin, MT-FS: Foliar spray of 100µM melatonin, MT- ST+FS: Combined effect of seed treatment and foliar spray of 100µM melatonin. Data in the figure are expressed as mean ± SE. Mean values followed by the same letter do not differ significantly at \u003cem\u003eP\u003c/em\u003e ≤ 0.05 by LSD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/3d432778899e82d06fc410a1.png"},{"id":66240584,"identity":"8930e4de-d305-47db-8102-3a996c0d4e02","added_by":"auto","created_at":"2024-10-09 06:30:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127715,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of melatonin on A) malondialdehyde content (MDA) and B) membrane stability index (MSI) under drought stress in okra. AC: Absolute Control, C: Control (Drought), MT-ST: Seed treatment of 100µM melatonin, MT-FS: Foliar spray of 100µM melatonin, MT- ST+FS: Combined effect of seed treatment and foliar spray of 100µM melatonin. Data in the figure are expressed as mean ± SE. Mean values followed by the same letter do not differ significantly at \u003cem\u003eP\u003c/em\u003e ≤ 0.05 by LSD.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/9b40f921a60c4742ef1fd77a.png"},{"id":66239756,"identity":"65b304ee-7ddb-4ad2-91fb-6e1f979a7fff","added_by":"auto","created_at":"2024-10-09 06:22:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69952,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of melatonin on proline content under drought stress in okra. AC: Absolute Control, C: Control (Drought), MT-ST: Seed treatment of 100µM melatonin, MT-FS: Foliar spray of 100µM melatonin, MT- ST+FS: Combined effect of seed treatment and foliar spray of 100µM melatonin. Data in the figure are expressed as mean ± SE. Mean values followed by the same letter do not differ significantly at \u003cem\u003eP\u003c/em\u003e ≤ 0.05 by LSD.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/a0e0fc83e8d23eb75eaf3ce8.png"},{"id":66239379,"identity":"c9cf2768-9509-4927-854d-156f8f949caf","added_by":"auto","created_at":"2024-10-09 06:14:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":120381,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of melatonin on A) Photosynthetic rate, B) Transpiration rate and C) Stomatal conductance under drought stress in okra. AC: Absolute Control, C: Control (Drought), MT-ST: Seed treatment of 100µM melatonin, MT-FS: Foliar spray of 100µM melatonin, MT- ST+FS: Combined effect of seed treatment and foliar spray of 100µM melatonin. Data in the figure are expressed as mean ± SE. Mean values followed by the same letter do not differ significantly at \u003cem\u003eP\u003c/em\u003e ≤ 0.05 by LSD.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/42394437077ddb228aae0b82.png"},{"id":66239755,"identity":"213d226e-baa0-47ec-9117-fe1df221d2d1","added_by":"auto","created_at":"2024-10-09 06:22:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105261,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of melatonin on A) Chlorophyll fluorescence and B) SPAD values under drought stress in okra. AC: Absolute Control, C: Control (Drought), MT-ST: Seed treatment of 100µM melatonin, MT-FS: Foliar spray of 100µM melatonin, MT- ST+FS: Combined effect of seed treatment and foliar spray of 100µM melatonin. Data in the figure are expressed as mean ± SE. Mean values followed by the same letter do not differ significantly at \u003cem\u003eP\u003c/em\u003e ≤ 0.05 by LSD.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/8bc9ce3a053fa936d6ad85ce.png"},{"id":66239750,"identity":"d0d71d9f-13e5-4732-9142-45ce0484e6eb","added_by":"auto","created_at":"2024-10-09 06:22:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104822,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of melatonin on A) Ascorbate peroxidase and B) Catalase under drought stress in okra. AC: Absolute Control, C: Control (Drought), MT-ST: Seed treatment of 100µM melatonin, MT-FS: Foliar spray of 100µM melatonin, MT- ST+FS: Combined effect of seed treatment and foliar spray of 100µM melatonin. Data in the figure are expressed as mean ± SE. Mean values followed by the same letter do not differ significantly at \u003cem\u003eP\u003c/em\u003e≤ 0.05 by LSD.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/eb58c6d8891f60d6b0d4c7b2.png"},{"id":66239754,"identity":"2ec046e0-4f03-4ca4-a50c-3520569d24dd","added_by":"auto","created_at":"2024-10-09 06:22:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":141698,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component biplot of okra subjected to melatonin treatment under drought condition. Variables are Proline, photosynthetic rate (Pn), chlorophyll fluorescence (fvfm), membrane stability index (msi), chlorophyll content (spad), ascorbate peroxidase activity (apx), catalase activity (cat), relative water content (rwc), malondialdehyde content (mda), transpiration rate (T) and stomatal conductance (gs). Treatments include T1 (Absolute control), T2 (Control), T3 (MT-ST), T4 (MT-FS) and T5 (MT-ST+FS). V1 (CO 4), V2 (Arka Anamika), V3 (Arka Abhay), V4 (Arka Nikita).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/33ceb9e98791350e1bf86dc1.png"},{"id":66239374,"identity":"0ef8d1bb-09d3-4b87-8b03-a45b07f80725","added_by":"auto","created_at":"2024-10-09 06:14:19","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":199015,"visible":true,"origin":"","legend":"\u003cp\u003ePearson correlation analysis between different parameters and okra plant subjected to melatonin treatments under drought stress. Variables include MT- Melatonin, RWC- Relative water content, MDA- Malondialdehyde content, P- Proline, A- Photosynthetic rate, E- Transpiration rate, C- Stomatal conductance, Fv/Fm- Chlorophyll fluorescence, MSI- Membrane stability index, SPAD- Chlorophyll content, APX- Ascorbate peroxidase, CAT- Catalase.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/0912c0e91a2e704ec9ba2125.png"},{"id":66239752,"identity":"b7875c73-abd5-4eb7-882b-2af6a6a13d33","added_by":"auto","created_at":"2024-10-09 06:22:19","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":544672,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map analysis of different bioactive compounds subjected to drought stress (Drought control, DC) and melatonin application as seed treatment and foliar spray (MT-ST+FS).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/cd0a62504ddfca34b8d31888.png"},{"id":66239373,"identity":"80276e20-fac5-4c53-a8b8-d719bb089886","added_by":"auto","created_at":"2024-10-09 06:14:19","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":225054,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the mechanism underlying drought tolerance in okra \u0026nbsp;subjected to exogenous melatonin application\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/9463940636c059db385872ba.png"},{"id":66240811,"identity":"71e44a80-ea63-4cbd-adae-89432bb2e257","added_by":"auto","created_at":"2024-10-09 06:38:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2304001,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/a7b77102-43f5-4841-8d97-dab5c304db88.pdf"},{"id":66239381,"identity":"63d3fb82-b22d-4615-bad5-80b35398ae9b","added_by":"auto","created_at":"2024-10-09 06:14:20","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17237,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablePGR.docx","url":"https://assets-eu.researchsquare.com/files/rs-4967383/v1/1bea082cc949786d078ec429.docx"}],"financialInterests":"","formattedTitle":"Melatonin-Induced Enhancement of Drought Tolerance in Okra: A Detailed Analysis of Physiological, Biochemical, and Metabolic Adaptations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClimate change and the rising frequency of extreme weather events present substantial risks to global crop yields and their stability (Reyes et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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 \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEach vegetable crop responds uniquely to drought, depending on its severity and the plant's growth stages. Okra (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e [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. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In India, okra cultivation spans an area of 554 lakh hectares, yielding an annual production of 7252\u0026nbsp;million metric tons for the 2023\u0026ndash;2024 period (Indiastat \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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\u0026ndash;100%, notably during flowering and pod-filling stages (Mbagwu and Adesipe \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). 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. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Drought stress resulted in reduced leaf water status, chlorophyll content, carotenoid levels, and total protein (\u0026micro;g/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. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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 \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Razi and Muneer \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMelatonin (N-acetyl-5-methoxytryptamine) was identified as a notable bioactive compound in vascular plants in 1995 (Dubbels et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Initially noted for its potent antioxidant properties, melatonin has since been recognized for its diverse roles in plant development (Sheshadri et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It contributes to various physiological processes, including seed germination (Zhang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), root elongation (Arnao and Hern\u0026aacute;ndez-Ruiz \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), photosynthesis enhancement (Li et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and leaf senescence regulation (Wang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As a plant hormone, melatonin plays a crucial role in growth regulation and developmental pathways (Arnao and Hern\u0026aacute;ndez-Ruiz \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is present in multiple plant tissues, including seeds, roots, leaves, and fruits (Zhang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Research has extensively examined melatonin's potential to improve plant growth and regulatory processes (Sun et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMelatonin 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 (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) 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 (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (Jahan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It also enhances Arabidopsis' tolerance to high light levels by scavenging both H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e (Yang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and MDA, thereby mitigating cell membrane disruption and enhancing photosynthetic efficiency (Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Imran et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExogenous melatonin significantly boosted the performance of antioxidant enzymes and improved the yield of soybean plants (Oliveira-Spolaor et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent findings suggests that external application of melatonin enhances salt tolerance in okra by increasing proline accumulation and the K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e 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. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). 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.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Planting material and source of Melatonin","content":"\u003cp\u003eOkra 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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Melatonin was sourced from Sigma-Aldrich Pvt. Ltd. A 100 \u0026micro;M 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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysio-chemical properties of soil\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil properties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRange\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTexture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRed loam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrical conductivity (ds m-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic carbon (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvailable Nitrogen (Kg ha-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e247.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvailable phosphorous (Kg ha-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvailable potassium (Kg ha-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e541.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth condition and treatment imposition\u003c/h2\u003e \u003cp\u003eSeeds were surface sterilized with 75% ethanol followed by seed treatment with 100 \u0026micro;M melatonin. Based on laboratory screening, 100 \u0026micro;M 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 100\u0026micro;M melatonin (MT - ST), Drought and foliar spray of 100 \u0026micro;M melatonin (MT - FS), and drought stress with combined effect of seed treatment and foliar spray of 100\u0026micro;M melatonin (MT - ST\u0026thinsp;+\u0026thinsp;FS). Drought stress was induced naturally by ceasing irrigation, with daily monitoring of moisture levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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 100\u0026micro;M 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhotosynthetic measurements\u003c/h2\u003e \u003cp\u003eGas 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, \u0026micro;mol CO\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), transpiration rate (E, mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and stomatal conductance (C, mol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Chlorophyll fluorescence was measured using a chlorophyll fluorometer (Heinz Walz, Germany), with leaves dark-adapted for 30 min prior to measurement.\u003c/p\u003e \u003cp\u003eMaximum quantum yield of PS II (Fv/Fm) was computed as\u003c/p\u003e \u003cp\u003eFv/Fm = (Fm\u0026thinsp;\u0026minus;\u0026thinsp;F\u003csub\u003e0\u003c/sub\u003e)/Fm (1)\u003c/p\u003e \u003cp\u003eChlorophyll index was determined using a SPAD meter (Minolta), based on the ratio of light transmitted at wavelengths of 650 nm and 940 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRelative water content (RWC)\u003c/h2\u003e \u003cp\u003eFresh 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\u0026deg;C for 24 hours to determine their dry weight.\u003c/p\u003e \u003cp\u003eRelative Water Content (RWC) was computed using the formula proposed by (Gonz\u0026aacute;lez and Gonz\u0026aacute;lez-Vilar \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRWC (%) = (FW- TW)/ (TW- DW) x 100 (2) FW\u0026thinsp;=\u0026thinsp;Fresh weight, TW\u0026thinsp;=\u0026thinsp;Turgid weight, DW\u0026thinsp;=\u0026thinsp;Dry weight\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMembrane stability index (MSI)\u003c/h2\u003e \u003cp\u003eMSI was determined following the method by (Premachandra et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), modified by (Sairam \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). 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\u0026deg;C. Electrical conductivity (C\u003csub\u003e1\u003c/sub\u003e) was measured after cooling. After adding water once more, the leaf discs were incubated for 10 min at 100\u0026deg;C in a water bath before their final electrical conductivity (C\u003csub\u003e2\u003c/sub\u003e) was determined.\u003c/p\u003e \u003cp\u003eThe membrane stability index was determined using percent conductivity, as given by the formula\u003c/p\u003e \u003cp\u003eMSI (%)\u0026thinsp;=\u0026thinsp;1- C\u003csub\u003e1\u003c/sub\u003e/ C\u003csub\u003e2\u003c/sub\u003e x 100 (3) Where C\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Initial electrical conductivity C\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Final electrical conductivity\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProline content\u003c/h2\u003e \u003cp\u003eProline levels were quantified according to the method described by (Bates et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde content\u003c/h2\u003e \u003cp\u003eMDA levels were determined as per the protocol outlined by (Hodges et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). 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\u0026deg;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\u0026deg;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 \u0026micro;mol MDA g\u003csup\u003e-1\u003c/sup\u003e FW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant Enzyme Assays\u003c/h2\u003e \u003cp\u003eFresh 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\u0026deg;C for 20 min, and the resulting extraction buffer was used for enzyme analysis.\u003c/p\u003e \u003cp\u003eFor 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\u0026deg;C, the resulting aliqout was mixed with reaction buffer containing 2 mL of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (pH 7.3). Absorbance readings were taken at 290 nm with 30 s intervals for 3 min (extinction coefficient\u0026thinsp;=\u0026thinsp;2.8 mM\u0026thinsp;\u0026minus;\u0026thinsp;1 cm\u0026thinsp;\u0026minus;\u0026thinsp;1) (Nakano and Asada \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1981\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCAT activity was assessed using a 50 mM phosphate buffer containing 100 \u0026micro;L of enzyme extract and 15 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. 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 (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 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\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, for the determination of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity (Cakmak and Marschner \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe 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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;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).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChanges in RWC of drought stressed okra hybrids under varying melatonin treatments\u003c/h2\u003e \u003cp\u003eThe 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 \u0026micro;M melatonin as seed treatment and foliar spray (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The interaction between the hybrids and the treatments showed a significant effect (F\u003csub\u003e12,60\u003c/sub\u003e = 3.65, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMDA and MSI under different melatonin treatment combinations\u003c/h2\u003e \u003cp\u003eThe results reveals that plants experiencing water deficit showed increase in malondialdehyde content (120.77 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e FW) compared to absolute control. Application of 100\u0026micro;M of melatonin ( MT- ST\u0026thinsp;+\u0026thinsp;FS) reduced the MDA content by 48%. Among hybrids, CO 4 accumulated more MDA (98.27 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e FW) and Arka Abhay the least (62.19 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Interaction effect between hybrids and treatments are significant (F\u003csub\u003e12,60\u003c/sub\u003e = 5.07, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Table S2).\u003c/p\u003e \u003cp\u003eAmong 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 100\u0026micro;M of melatonin as seed treatment and foliar spray enhanced the membrane stability index by 26% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChanges in proline content across different melatonin treatments\u003c/h2\u003e \u003cp\u003eWhen 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\u0026thinsp;+\u0026thinsp;FS) of 100\u0026micro;M melatonin (5.80 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e) and the drought stressed plants (5.42 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e) accumulated more proline. Among hybrids, Arka Anamika recorded higher proline content (6.05 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e) and Arka Nikita showed the least value (3.44 \u0026micro;mol g\u003csup\u003e-1\u003c/sup\u003e). Application of 100\u0026micro;M of melatonin as seed treatment and foliar spray enhanced the proline levels in Arka Anamika (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The interaction effect between treatment and hybrids are significant (F\u003csub\u003e12,60\u003c/sub\u003e = 147.92, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGas exchange indicators\u003c/h2\u003e \u003cp\u003eGas 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 \u0026micro;mol CO\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), transpiration rate (5.52 mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and stomatal conductance (0.47 mol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) whereas the combined application of 100\u0026micro;M of melatonin as seed treatment and foliar spray showed an increment in photosynthetic rate (12.72 \u0026micro;mol CO\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), transpiration rate (8.27 mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and stomatal conductance (0.90 mol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Among hybrids, Arka Nikita outperformed other hybrids in photosynthetic rate, transpiration rate and stomatal conductance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interaction effect between hybrids and treatments are significant (F\u003csub\u003e12,60\u003c/sub\u003e = 5.47, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; F\u003csub\u003e12,60\u003c/sub\u003e = 7.52, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; F\u003csub\u003e12,60\u003c/sub\u003e = 30.75, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFv/Fm and SPAD\u003c/h2\u003e \u003cp\u003ePlants 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 100\u0026micro;M 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 100\u0026micro;M of melatonin as seed treatment and foliar spray (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAscorbate peroxidase (APX) and catalase (CAT) activity\u003c/h2\u003e \u003cp\u003eEnzymatic 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 100\u0026micro;M of melatonin (MT- ST\u0026thinsp;+\u0026thinsp;FS) by 36.46% and 43.42% in case of ascorbate peroxidase and catalase. Among the hybrids, Arka Abhay (2.52 Ug\u003csup\u003e-1\u003c/sup\u003e FW), Arka Nikita (2.47 Ug\u003csup\u003e-1\u003c/sup\u003e FW) and Arka Anamika (2.44 Ug\u003csup\u003e-1\u003c/sup\u003e FW) exhibited almost similar ascorbate peroxidase activity, whereas Arka Nikita (510.70 Ug\u003csup\u003e-1\u003c/sup\u003e (FW)) recorded higher catalase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The interaction effects between treatment and hybrids are significant (F\u003csub\u003e12,60\u003c/sub\u003e =0.849, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; F\u003csub\u003e12,60\u003c/sub\u003e = 0.22, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis (PCA)\u003c/h2\u003e \u003cp\u003ePrincipal 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Supplementary Table S3\u0026amp;S4). Two principal components exhibited eigenvalues\u0026thinsp;\u0026gt;\u0026thinsp;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\u003csup\u003e◦\u003c/sup\u003e) 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 100\u0026micro;M 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation analysis\u003c/h2\u003e \u003cp\u003ePearson correlation analysis was carried out to check the effect of melatonin treatments on physiological and biochemical parameters in okra which include proline (P, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.36), photosynthetic rate (A, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.05), chlorophyll fluorescence (Fv/Fm, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.11), membrane stability index (MSI, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.15), chlorophyll content (SPAD, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.15), ascorbate peroxidase activity (APX, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.86) and catalase activity (CAT, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.92). However, the relative water content (RWC, r\u003csup\u003e2\u003c/sup\u003e= -0.07), malondialdehyde content (MDA, r\u003csup\u003e2\u003c/sup\u003e= -0.10), transpiration rate (E, r\u003csup\u003e2\u003c/sup\u003e= -0.018) and stomatal conductance (C, r\u003csup\u003e2\u003c/sup\u003e= -0.26) were negatively correlated with the melatonin concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMetabolite profiling of bioactive compounds detected in water deficit stressed and melatonin treated plants\u003c/h2\u003e \u003cp\u003eThe effect of application of melatonin to drought stressed (MT\u0026thinsp;+\u0026thinsp;ST\u0026thinsp;+\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAbiotic 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. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Raza et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eRelative 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. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, a combination of seed treatment and foliar spray of 100 \u0026micro;M 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. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similar findings by (Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and (Dai et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) 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. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These results align with (Turk et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), 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. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and (Talaat \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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 (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) 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 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production and lipid peroxidation. Additionally, MT is suggested to stabilize biological membranes under stress by maintaining optimal fluidity (Arnao and Hern\u0026aacute;ndez-Ruiz \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and regulating the expression of lipid peroxidase genes (Gao et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhotosynthesis, 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. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 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. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In response to drought stress, plants typically exhibit stomatal closure, resulting in reduced stomatal conductance and consequent limitations on photosynthesis (Meloni et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Melatonin mitigates chlorophyll degradation under drought stress conditions, thereby enhancing photosynthesis, transpiration, and stomatal conductance (Liang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Karaca and Cekic \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Melatonin helps maintain cellular turgor, enhancing stomatal opening and conductance (Meng et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This increased conductance improves water and CO\u003csub\u003e2\u003c/sub\u003e movement, ultimately favouring photosynthesis in melatonin-treated plants (Cui et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChlorophyll fluorescence serves as a crucial indicator for assessing the photosynthetic capacity and energy conversion efficiency of Photosystem II (PSII) in plants (Mathur et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Numerous studies suggests that prolonged water deficit induces photoinhibition in the reaction center of Photosystem II (PSII) in plants (Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 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. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This was attributed to the restricted diffusion of ambient CO\u003csub\u003e2\u003c/sub\u003e to the carboxylation sites, resulting in a relative surplus of light energy and electron sinks, thereby causing photoinhibition or photooxidation (Atkin and Macherel \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Exogenous application of melatonin enhanced photosynthetic efficiency and protected the maize plant from photoinhibition (Zhao et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The application of melatonin exerts a protective effect on chlorophyll, thereby reducing damage to the photosynthetic apparatus (Campos et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApplication 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. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gao et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Foliar application of melatonin mitigates oxidative impairement in corn seedlings (Ahmad et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Under water stress, plants develop adaptation strategy, such as ROS detoxifying enzymes to prevent free radical bursts and maintain ROS balance (Abid et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Imran et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Melatonin enhances the transcript levels and activities of antioxidant enzymes (Arnao and Hern\u0026aacute;ndez-Ruiz \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 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. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMetabolomic 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 Ant\u0026oacute;nio \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, the osmolytes buildup aids in regulating ROS levels, supplies energy to manage stress, facilitates repair mechanism, and supports continued growth (Silva et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; F\u0026agrave;bregas and Fernie \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 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. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) 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. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). 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. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) 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 \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Additionally, tryptophan enhances various biochemical attributes by regulating plant growth and divergence and improving nutrient and water uptake efficiency (Talaat et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Dawood and Sadak \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn 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.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese 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.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMelatonin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSeed treatment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFoliar spray\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRWC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRelative water content\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMelondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMembrane stability index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAscorbate peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephotosynthetic rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etranspiration rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estomatal conductance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChlorophyll index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express thanks and gratitude to Tamil Nadu Agricultural University, Coimbatore \u0026ndash; 641 003, Tamil Nadu for providing facilities to carry out the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, methodology, review and editing: Gopal Aswathi, Veerasamy Ravichandran, Dhashnamurthi Vijayalakshmi,\u0026nbsp;Alagarsamy Senthil, Loganathan Arul, Sengodan Radhamani, Ramasamy Jagadeeswaran and\u0026nbsp;Mottaiyan Pitchaimuthu;\u0026nbsp;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\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING:\u0026nbsp;\u003c/strong\u003eNo funding was received to assist with the preparation of this manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST:\u0026nbsp;\u003c/strong\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPLIANCE WITH ETHICAL STANDARDS:\u0026nbsp;\u003c/strong\u003eThe research work does not involve people as research object\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbid M, Ali S, Qi LK, Zahoor R, Tian Z, Jiang D, Snider JL, Dai T (2018) Physiological and biochemical changes during drought and recovery periods at tillering and jointing stages in wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.). Sci. Rep. 8 (1):4615\u003c/li\u003e\n\u003cli\u003eAhmad S, Kamran M, Ding R, Meng X, Wang H, Ahmad I, Fahad S, Han Q (2019) Exogenous melatonin confers drought stress by promoting plant growth, photosynthetic capacity and antioxidant defense system of maize seedlings. PeerJ 7:e7793\u003c/li\u003e\n\u003cli\u003eAltaf MA, Shahid R, Altaf MM, Kumar R, Naz S, Kumar A, Alam P, Tiwari RK, Lal MK, Ahmad P (2022) Melatonin: first-line soldier in tomato under abiotic stress current and future perspective. Plant Physiol Biochem 185:188-197\u003c/li\u003e\n\u003cli\u003eArnao MB, Hern\u0026aacute;ndez-Ruiz J (2019) Melatonin: a new plant hormone and/or a plant master regulator? Trends Plant Sci. 24 (1):38-48\u003c/li\u003e\n\u003cli\u003eAtkin OK, Macherel D (2009) The crucial role of plant mitochondria in orchestrating drought tolerance. Ann. Bot. 103 (4):581-597\u003c/li\u003e\n\u003cli\u003eAyub Q, Hussain I, Naveed K, Ali S, Mehmood A, Khan M, Haq N, Shehzad Q (2021) Responses of different okra (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e) cultivars to water deficit conditions. J. Hortic. Sci. 16 (1):53-63\u003c/li\u003e\n\u003cli\u003eBates LS, Waldren R, Teare I (1973) Rapid determination of free proline for water-stress studies. Plant and soil 39:205-207\u003c/li\u003e\n\u003cli\u003eBulgari R, Franzoni G, Ferrante A (2019) Biostimulants application in horticultural crops under abiotic stress conditions. Agronomy 9 (6):306\u003c/li\u003e\n\u003cli\u003eCakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant physiol. 98 (4):1222-1227\u003c/li\u003e\n\u003cli\u003eCampos CN, \u0026Aacute;vila RG, de Souza KRD, Azevedo LM, Alves JD (2019) Melatonin reduces oxidative stress and promotes drought tolerance in young Coffea arabica L. plants. Agric. Water Manag. 211:37-47\u003c/li\u003e\n\u003cli\u003eChen Q, Qi W-b, Reiter RJ, Wei W, Wang B-m (2009) Exogenously applied melatonin stimulates root growth and raises endogenous indoleacetic acid in roots of etiolated seedlings of Brassica juncea. J.Plant Physiol. 166 (3):324-328\u003c/li\u003e\n\u003cli\u003eChen Y, Li R, Ge J, Liu J, Wang W, Xu M, Zhang R, Hussain S, Wei H, Dai Q (2021) Exogenous melatonin confers enhanced salinity tolerance in rice by blocking the ROS burst and improving Na+/K+ homeostasis. Environ. Exp. Bot. 189:104530\u003c/li\u003e\n\u003cli\u003eCui G, Zhao X, Liu S, Sun F, Zhang C, Xi Y (2017) Beneficial effects of melatonin in overcoming drought stress in wheat seedlings. Plant Physiol Biochem 118:138-149\u003c/li\u003e\n\u003cli\u003eDai L, Li J, Harmens H, Zheng X, Zhang C (2020) Melatonin enhances drought resistance by regulating leaf stomatal behaviour, root growth and catalase activity in two contrasting rapeseed (\u003cem\u003eBrassica napus\u003c/em\u003e L.) genotypes. Plant Physiol Biochem 149:86-95\u003c/li\u003e\n\u003cli\u003eDawood MG, Sadak MS (2007) Physiological response of canola plants (\u003cem\u003eBrassica napus\u003c/em\u003e L.) to tryptophan or benzyladenine. Lucrari stiintifice 50 (9):198-207\u003c/li\u003e\n\u003cli\u003eDhankher OP, Foyer CH (2018) Climate resilient crops for improving global food security and safety. vol 41. Wiley Online Library, \u003c/li\u003e\n\u003cli\u003eDing F, Wang G, Wang M, Zhang S (2018) Exogenous melatonin improves tolerance to water deficit by promoting cuticle formation in tomato plants. Molecules 23 (7):1605\u003c/li\u003e\n\u003cli\u003eDubbels R, Reiter R, Klenke E, Goebel A, Schnakenberg E, Ehlers C, Schiwara H, Schloot W (1995) Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography‐mass spectrometry. J. Pineal Res. 18 (1):28-31\u003c/li\u003e\n\u003cli\u003eF\u0026agrave;bregas N, Fernie AR (2019) The metabolic response to drought. J. Exp. Bot. 70 (4):1077-1085\u003c/li\u003e\n\u003cli\u003eGao W, Feng Z, Bai Q, He J, Wang Y (2019) Melatonin-mediated regulation of growth and antioxidant capacity in salt-tolerant naked oat under salt stress. Int. J. Mol. Sci. 20 (5):1176\u003c/li\u003e\n\u003cli\u003eGao W, Zhang Y, Feng Z, Bai Q, He J, Wang Y (2018) Effects of melatonin on antioxidant capacity in naked oat seedlings under drought stress. Molecules 23 (7):1580\u003c/li\u003e\n\u003cli\u003eGemede HF, Ratta N, Haki GD, Woldegiorgis AZ, Beyene F (2015) Nutritional quality and health benefits of okra (Abelmoschus esculentus): A review. J Food Process Technol 6 (458):2\u003c/li\u003e\n\u003cli\u003eGogoi A, Tripathi B (2019) 42% India\u0026rsquo;s land area under drought, worsening farm distress in election year. India Spend\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez L, Gonz\u0026aacute;lez-Vilar M (2001) Determination of relative water content. In: Handbook of plant ecophysiology techniques. Springer, pp 207-212\u003c/li\u003e\n\u003cli\u003eHodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604-611\u003c/li\u003e\n\u003cli\u003eHuang B, Chen Y-E, Zhao Y-Q, Ding C-B, Liao J-Q, Hu C, Zhou L-J, Zhang Z-W, Yuan S, Yuan M (2019) Exogenous melatonin alleviates oxidative damages and protects photosystem II in maize seedlings under drought stress. Front. Plant Sci. 10:677\u003c/li\u003e\n\u003cli\u003eImran M, Aaqil Khan M, Shahzad R, Bilal S, Khan M, Yun B-W, Khan AL, Lee I-J (2021) Melatonin ameliorates thermotolerance in soybean seedling through balancing redox homeostasis and modulating antioxidant defense, phytohormones and polyamines biosynthesis. Molecules 26 (17):5116\u003c/li\u003e\n\u003cli\u003eIndiastat (2023) Area, production and productivity of okra in india (1987-1988 and 1991-1992 to 2023-2024- 2nd advance estimates). \u003c/li\u003e\n\u003cli\u003eJahan MS, Guo S, Baloch AR, Sun J, Shu S, Wang Y, Ahammed GJ, Kabir K, Roy R (2020) Melatonin alleviates nickel phytotoxicity by improving photosynthesis, secondary metabolism and oxidative stress tolerance in tomato seedlings. Ecotoxicol. Environ. Saf. 197:110593\u003c/li\u003e\n\u003cli\u003eJorge TF, Ant\u0026oacute;nio C (2018) Plant metabolomics in a changing world: metabolite responses to abiotic stress combinations. Plant Abiotic Stress Responses Clim Chang\u003c/li\u003e\n\u003cli\u003eKaraca P, Cekic F\u0026Ouml; (2019) Exogenous melatonin-stimulated defense responses in tomato plants treated with polyethylene glycol. Int. J. Veg. Sci. 25 (6):601-609\u003c/li\u003e\n\u003cli\u003eKhan Z, Jan R, Asif S, Farooq M, Jang Y-H, Kim E-G, Kim N, Kim K-M (2024) Exogenous melatonin induces salt and drought stress tolerance in rice by promoting plant growth and defense system. Sci. Rep. 14 (1):1214\u003c/li\u003e\n\u003cli\u003eLee H, Calvin K, Dasgupta D, Krinner G, Mukherji A, Thorne P, Trisos C, Romero J, Aldunce P, Barret K (2023) IPCC, 2023: Climate Change 2023: Synthesis Report, Summary for Policymakers. Contribution of Working Groups I, II and III to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, H. Lee and J. Romero (eds.)]. IPCC, Geneva, Switzerland. \u003c/li\u003e\n\u003cli\u003eLi H, Chang J, Chen H, Wang Z, Gu X, Wei C, Zhang Y, Ma J, Yang J, Zhang X (2017) Exogenous melatonin confers salt stress tolerance to watermelon by improving photosynthesis and redox homeostasis. Front. Plant Sci. 8:295\u003c/li\u003e\n\u003cli\u003eLi J, Zeng L, Cheng Y, Lu G, Fu G, Ma H, Liu Q, Zhang X, Zou X, Li C (2018) Exogenous melatonin alleviates damage from drought stress in Brassica napus L.(rapeseed) seedlings. Acta Physiol. Plant. 40:1-11\u003c/li\u003e\n\u003cli\u003eLi Z, Su X, Chen Y, Fan X, He L, Guo J, Wang Y, Yang Q (2021) Melatonin improves drought resistance in maize seedlings by enhancing the antioxidant system and regulating abscisic acid metabolism to maintain stomatal opening under PEG-induced drought. J. Plant Biol. 64:299-312\u003c/li\u003e\n\u003cli\u003eLiang B, Ma C, Zhang Z, Wei Z, Gao T, Zhao Q, Ma F, Li C (2018) Long-term exogenous application of melatonin improves nutrient uptake fluxes in apple plants under moderate drought stress. Environ. Exp. Bot. 155:650-661\u003c/li\u003e\n\u003cli\u003eLiang G, Liu J, Zhang J, Guo J (2020) Effects of drought stress on photosynthetic and physiological parameters of tomato. J. Am. Soc. 145 (1):12-17\u003c/li\u003e\n\u003cli\u003eLiu D, Wu L, Naeem MS, Liu H, Deng X, Xu L, Zhang F, Zhou W (2013) 5-Aminolevulinic acid enhances photosynthetic gas exchange, chlorophyll fluorescence and antioxidant system in oilseed rape under drought stress. Acta Physiol. Plant. 35:2747-2759\u003c/li\u003e\n\u003cli\u003eMathur S, Tomar RS, Jajoo A (2019) Arbuscular mycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress. Photosynth. Res. 139:227-238\u003c/li\u003e\n\u003cli\u003eMbagwu J, Adesipe F (1987) Response of three okra (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e L. Moench) cultivars to irrigation at specific growth stages. Sci. Hortic. 31 (1-2):35-43\u003c/li\u003e\n\u003cli\u003eMeloni DA, Oliva MA, Martinez CA, Cambraia J (2003) Photosynthesis and activity of superoxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. Environ. Exp. Bot. 49 (1):69-76\u003c/li\u003e\n\u003cli\u003eMeng JF, Xu TF, Wang ZZ, Fang YL, Xi ZM, Zhang ZW (2014) The ameliorative effects of exogenous melatonin on grape cuttings under water‐deficient stress: antioxidant metabolites, leaf anatomy, and chloroplast morphology. J. Pineal Res. 57 (2):200-212\u003c/li\u003e\n\u003cli\u003eMkhabela SS, Shimelis H, Gerrano AS, Mashilo J (2022) Phenotypic and genotypic divergence in okra [\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e (L.) Moench] and implications for drought tolerance breeding: A review. S. Afr. J. Bot. 145:56-64\u003c/li\u003e\n\u003cli\u003eNakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22 (5):867-880\u003c/li\u003e\n\u003cli\u003eNalina M, Saroja S, Chakravarthi M, Rajkumar R, Radhakrishnan B, Chandrashekara K (2021) Water deficit-induced oxidative stress and differential response in antioxidant enzymes of tolerant and susceptible tea cultivars under field condition. Acta Physiol. Plant. 43:1-17\u003c/li\u003e\n\u003cli\u003eOliveira-Spolaor B, Chiari-Bertoli S, Silva-Sukert D, Sala HR, Picoli de Oliveira BF, de Freitas \u0026Iacute;R, Lima-Moro A (2022) Exogenous melatonin induces tolerance to drought stress damage in seedlings and soybean plants. Chil. J. Agric. Res. 82 (4):515-526\u003c/li\u003e\n\u003cli\u003ePremachandra G, Saneoka H, Kanaya M, Ogata S (1991) Cell membrane stability and leaf surface wax content as affected by increasing water deficits in maize. J. Exp. Bot. 42 (2):167-171\u003c/li\u003e\n\u003cli\u003eRai V (2002) Role of amino acids in plant responses to stresses. Biol. Plant. 45 (4):481-487\u003c/li\u003e\n\u003cli\u003eRaza A, Mubarik MS, Sharif R, Habib M, Jabeen W, Zhang C, Chen H, Chen ZH, Siddique KH, Zhuang W (2023) Developing drought‐smart, ready‐to‐grow future crops. The Plant Genome 16 (1):e20279\u003c/li\u003e\n\u003cli\u003eRazi K, Muneer S (2021) Drought stress-induced physiological mechanisms, signaling pathways and molecular response of chloroplasts in common vegetable crops. Crit. Rev. Biotechnol. 41 (5):669-691\u003c/li\u003e\n\u003cli\u003eReyes F, Gosme M, Wolz KJ, Lecomte I, Dupraz C (2021) Alley cropping mitigates the impacts of climate change on a wheat crop in a Mediterranean environment: a biophysical model-based assessment. Agriculture 11 (4):356\u003c/li\u003e\n\u003cli\u003eSairam R (1994) Effects of homobrassinolide application on plant metabolism and grain yield under irrigated and moisture-stress conditions of two wheat varieties. Plant Growth Regul. 14:173-181\u003c/li\u003e\n\u003cli\u003eSharma A, Shahzad B, Rehman A, Bhardwaj R, Landi M, Zheng B (2019) Response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules 24 (13):2452\u003c/li\u003e\n\u003cli\u003eSharma A, Wang J, Xu D, Tao S, Chong S, Yan D, Li Z, Yuan H, Zheng B (2020) Melatonin regulates the functional components of photosynthesis, antioxidant system, gene expression, and metabolic pathways to induce drought resistance in grafted Carya cathayensis plants. Sci. Total Environ. 713:136675\u003c/li\u003e\n\u003cli\u003eSheshadri S, Nishanth M, Yamine V, Simon B (2018) Effect of Melatonin on the stability and expression of reference genes in Catharanthus roseus. Sci. Rep. 8 (1):2222\u003c/li\u003e\n\u003cli\u003eSilva S, Santos C, Serodio J, Silva AM, Dias MC (2018) Physiological performance of drought-stressed olive plants when exposed to a combined heat\u0026ndash;UV-B shock and after stress relief. Functional Plant Biology 45 (12):1233-1240\u003c/li\u003e\n\u003cli\u003eSun Y-D, Guo D-L, Yang S-D, Zhang H-C, Wang L-L, Yu Y-H (2020) Melatonin treatment improves the shelf-life and postharvest quality of table grape (Vitis labrusca L. cv.\u0026lsquo;Fengzao\u0026rsquo;). J. Berry Res. 10 (4):665-676\u003c/li\u003e\n\u003cli\u003eTalaat IM, Bekheta M, Mahgoub MH (2005) Physiological response of periwinkle plants (Catharanthus roseus L.) to tryptophan and putrescine. Int J Agric Biol 7 (2):210-213\u003c/li\u003e\n\u003cli\u003eTalaat NB (2015) Effective microorganisms improve growth performance and modulate the ROS-scavenging system in common bean (Phaseolus vulgaris L.) plants exposed to salinity stress. Journal of Plant Growth Regul. 34:35-46\u003c/li\u003e\n\u003cli\u003eTalaat NB (2023) Role of phytohormones in regulating abiotic stresses in wheat. In: Abiotic Stresses in Wheat. Elsevier, pp 111-130\u003c/li\u003e\n\u003cli\u003eTurk H, Erdal S, Genisel M, Atici O, Demir Y, Yanmis D (2014) The regulatory effect of melatonin on physiological, biochemical and molecular parameters in cold-stressed wheat seedlings. Plant Growth Regul. 74:139-152\u003c/li\u003e\n\u003cli\u003eVelikova V, Tsonev T, Tattini M, Arena C, Krumova S, Koleva D, Peeva V, Stojchev S, Todinova S, Izzo LG (2018) Physiological and structural adjustments of two ecotypes of \u003cem\u003ePlatanus orientalis\u003c/em\u003e L. from different habitats in response to drought and re-watering. Conserv. Physiol. 6 (1):coy073\u003c/li\u003e\n\u003cli\u003eWang F, Wan C, Wu W, Yang S, Chen X (2024) Exogenous melatonin (MT) enhances salt tolerance of okra (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e L.) plants by regulating proline, photosynthesis, ion homeostasis and ROS pathways. Vegetos 37 (1):224-238\u003c/li\u003e\n\u003cli\u003eWang K, Xing Q, Ahammed GJ, Zhou J (2022) Functions and prospects of melatonin in plant growth, yield, and quality. J. Exp. Bot. 73 (17):5928-5946\u003c/li\u003e\n\u003cli\u003eYAMADA Y, KAKIBUCHI K, KOZUKI A, ISHIDA Y, IZUMORI K, TAJIMA S, AKIMITSU K, OHKOUCHI T, KASAI F (2014) Effects of the rare sugars D-psicose and D-tagatose on the sugar content and incidence of blossom end rot in tomato grown hydroponically with salinity treatment. Environmental Control in Biology 52 (3):155-160\u003c/li\u003e\n\u003cli\u003eYang S-J, Huang B, Zhao Y-Q, Hu D, Chen T, Ding C-B, Chen Y-E, Yuan S, Yuan M (2021) Melatonin enhanced the tolerance of Arabidopsis thaliana to high light through improving anti-oxidative system and photosynthesis. Front. Plant Sci. 12:752584\u003c/li\u003e\n\u003cli\u003eYe J, Wang S, Deng X, Yin L, Xiong B, Wang X (2016) Melatonin increased maize (Zea mays L.) seedling drought tolerance by alleviating drought-induced photosynthetic inhibition and oxidative damage. Acta Physiol. Plant. 38 (2):48\u003c/li\u003e\n\u003cli\u003eZhang N, Zhang H-J, Sun Q-Q, Cao Y-Y, Li X, Zhao B, Wu P, Guo Y-D (2017) Proteomic analysis reveals a role of melatonin in promoting cucumber seed germination under high salinity by regulating energy production. Sci. Rep. 7 (1):503\u003c/li\u003e\n\u003cli\u003eZhang Q, Gao F, Zhang S, Sun W, Li Z (2019) Prophylactic use of exogenous melatonin and melatonin receptor agonists to improve sleep and delirium in the intensive care units: a systematic review and meta-analysis of randomized controlled trials. Sleep and Breathing 23:1059-1070\u003c/li\u003e\n\u003cli\u003eZhao C, Guo H, Wang J, Wang Y, Zhang R (2021) Melatonin enhances drought tolerance by regulating leaf stomatal behavior, carbon and nitrogen metabolism, and related gene expression in maize plants. Front. Plant Sci. 12:779382\u003c/li\u003e\n\u003cli\u003eZhong C, Cao X, Bai Z, Zhang J, Zhu L, Huang J, Jin Q (2018) Nitrogen metabolism correlates with the acclimation of photosynthesis to short-term water stress in rice (Oryza sativa L.). Plant Physiol. Biochem. 125:52-62\u003c/li\u003e\n\u003cli\u003eZhou R, Kan X, Chen J, Hua H, Li Y, Ren J, Feng K, Liu H, Deng D, Yin Z (2019) Drought-induced changes in photosynthetic electron transport in maize probed by prompt fluorescence, delayed fluorescence, P700 and cyclic electron flow signals. Environ. Exp. Bot. 158:51-62\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"melatonin, drought, okra, antioxidants, metabolites","lastPublishedDoi":"10.21203/rs.3.rs-4967383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4967383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs climate change intensifies, drought stress presents a critical challenge for horticultural crops like okra (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e). 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 \u0026micro;M melatonin (MT- ST), Drought and foliar spray of 100 \u0026micro;M melatonin (MT- FS), and drought stress with combined effect of seed treatment and foliar spray of 100 \u0026micro;M melatonin (MT - ST\u0026thinsp;+\u0026thinsp;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 \u0026micro;M melatonin via seed treatment and foliar spray enhances drought tolerance in okra, suggesting its potential to enhance crop resilience under water-deficit conditions.\u003c/p\u003e","manuscriptTitle":"Melatonin-Induced Enhancement of Drought Tolerance in Okra: A Detailed Analysis of Physiological, Biochemical, and Metabolic Adaptations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-09 06:14:14","doi":"10.21203/rs.3.rs-4967383/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-11-13T08:12:54+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-09-15T05:04:53+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-06T00:35:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2024-08-29T05:47:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-26T12:27:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2024-08-24T01:44:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6f1d2527-b83d-4beb-a1ea-143558ac08fe","owner":[],"postedDate":"October 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2024-11-13T13:13:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-09 06:14:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4967383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4967383","identity":"rs-4967383","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}